CONSEJ O SUPERIOR DE INVESTIGACIONES CIENTÍFICAS

TRABAJOS DEL INSTITUTO CAJAL TOMO LXXIX C ON T I N U A C I ÓN D E L A “ R E V I S T A T R I M E S T R A L M I C R OGR Á F I C A ” F U N D A D A P OR

S. RAM ÓN Y CAJAL

2nd INTERNATIONAL MEETING

STEROIDS AND NERVOUS SYSTEM TORINO, Italy, Villa Gualino February 22 - 26, 2003

ABSTRACTS OF INVITED LECTURES AND FREE CONTRIBUTIONS

G.C. Panzica and S. Gotti, editors MADRID – 2 0 0 3

Organizers Roberto C. Melcangi GianCarlo Panzica

(Milano, Italy) (Torino, Italy)

International Scientific Committee Jacques Balthazart Luis M. García-Segura Allan E. Herbison Roberto C. Melcangi GianCarlo Panzica Phyllis Wise

Belgium Spain New Zealand Italy Italy USA

Local Organizing Committee Aldo Fasolo Mariarita Galbiati Valerio Magnaghi Roberto C. Melcangi GianCarlo Panzica Carla Viglietti Panzica

Conference organized with the support of Università degli Studi di Torino Università degli Studi di Milano Dipartimento di Anatomia, Farmacologia e Medicina Legale Fondazione Cavalieri Ottolenghi, Torino Centro Rita Levi Montalcini, Torino Center of Excellence on Neurodegenerative diseases, Milano SERONO foundation IBRO National Science Foundation Regione Piemonte Provincia di Torino Comune di Torino Celbio, Italy Leica, Italy

Visit us at our WWW site http://www.dafml.unito.it/anatomy/panzica/neurosteroids03/ The Abstracts published here were reproced directly from author’s original text with little or no alteration. The Editors take no responsibility for their content.

Contents • Action of Environmental Estrogens on Behaviorally Relevant Neural Circuits

5

• The Role of Neuroactive Steroids in Healthy Ageing: Therapeutical Perspectives

17

• Plenary Lecture: Kelly M.J.

33

• Non Classical Mechanisms of Action

37

• Glucocorticoids and Mineralcorticoids: Synthesis, Mechanism of Action and Effect

53

• Pathological Correlations and New Tools in Therapeutical Approaches

63

• Plenary Lecture: McCarthy M.

75

• Glial Cells as a Target for Steroids

79

• Steroid Regulation of Reproduction

93

• Plenary Lecture: Arnold A.

107

• Behavioural Effects

111

• Posters’ exhibition

125

SATURDAY, 22th February 2003 09.00 – 13.00 Satellite Symposium: Action of Environmental Estrogens on Behaviorally Relevant Neural Circuits

Satellite Symposium: Action of Environmental Estrogens on Behaviorally Relevant Neural Circuits (Chairs: Gahr M., Amsterdam & Panzica G.C., Torino) • Di Lorenzo D., Mussi P., Villa R., Biasiotto G., Belloli S., Ruggeri G., Apostoli P., Raviscioni M., Ciana P. and Maggi A. (Milano, Italy, EU), In vivo imaging of estrogen receptor activity • Gahr M. (Amsterdam, Holland, EU) Estrogenic effects of Alkylphenols on brain differentiation • Ottinger M.A., Hazelton J., Wu J., Ruscio M., Thompson N., Quinn M. and Abdelnabi M. (College Park, MD, USA) Assessing the Consequences of Dietary Methoxychlor: Neuroendocrine and Behavioral Measures • Santucci D., Branchi I., De Acetis G. and Alleva E. (Roma, Italy, EU) Neurobehavioural effects of perinatal exposure to PCB in developing CD-1 mice • Halldin K., Axelsson J. and Brunström B. (Uppsala, Sweden, EU) Effects of endocrine modulators on sexual differentiation and reproductive function in japanese quail • Palanza P. (Parma, Italy, EU) Impact of environmental estrogens on sexually dimorphic behavioral systems in mice

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

IN VIVO IMAGING OF ESTROGEN RECEPTOR ACTIVITY Di Lorenzo D., Mussi P., Villa R., Biasiotto G., Belloli S., Ruggeri G., Apostoli P., Raviscioni M., Ciana P. and Maggi A. Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20129 MILAN, Italy; fax: ++39.02.50318290; [email protected]

We have recently generated a transgenic mouse engineered to express a luciferase reporter driver by the activated estrogen receptors. The mouse has been fully characterized to demonstrate that the expression of the reporter is ubiquitous and that the reporter in all the tissue containing the estrogen receptors is responsive to natural or synthetic estrogens (1,2). The mouse represent a very interesting model for the study of systemic activation of estrogen receptors in animal exposed to environmental pollutants endowed of endocrine disrupter activity (3) and is a particular interesting model because the activity of compounds can be studies by immunohistochemistry, enzymatic assay and by in vivo imaging. We have recently carried out a study aimed at assessing whether our transgenic mouse was suitable to study the activity of endocrine disrupters. First we investigated the tissue specific effects of DDT isomers in adult and suckling newborn mice. The DDT isomers p,p’-DDT [1,1,1-trichloro-2,2-bis(pchlorophenyl) ethane] and o,p’-DDT [1,1,1trichloro-2(p-chlorophenyl)-2-(ochlorophenyl) ethane] were specifically selected as a weak and a strong estrogen, respectively. In adult male mice p,p’-DDT induced luciferase activity in liver, brain, thymus and prostate, but not in heart and lung. The effect of p,p’DDT was dose-dependent, maximal at 16 hours after subcutaneous treatment and completely blocked by the estrogen receptor antagonist ICI 182,780. In all the organs analysed, but the liver, administration of o,p’-DDT showed a pattern of luciferase induction superimposable to that of its isomer p,p’-DDT. In liver o,p’-DDT significantly decreased basal luciferase activity and blocked the reporter induction by 17ßestradiol. These data lead to hypothesize that a modulation of ER activity may be involved in the toxic effects of DDT demonstrated by epidemiological and experimental studies. Luciferase activity was also studied in four days old mice lactating from a mother injected with either p,p’-DDT or o,p’-DDT. Both isomers induced a 2 fold increase in the newborn brain. Opposite effect was observed in liver where p,p’-DDT increased and o,p’-DDT decreased luciferase, thus indicating that these compounds modulate ER activity in adult and newborn tissues with a similar mechanism. These results suggested that the ERE-tkLUC mouse is a suitable tool to functionally assess the tissue specificity of estrogenic/antiestrogenic compounds in adult as well as in suckling mice. We are now pursuing our studies with a systematic analysis of the activity of endocrine disrupters in the mouse also by in vivo imaging by using a CDD camera. The results of these studies will be presented. Reference List 1. 2. 3.

Ciana P., Di Luccio G., Belcredito S., Pollio G., Vegeto E., Tatangelo L., Tiveron C. and Maggi A. Engineering of a mouse for the in vivo profiling of estrogen receptor activity, Mol Endocrinol, (2001) 15(7): 110411132. Ciana P., Raviscioni M., Vegeto E., Mussi P., Que I., Parker M.Lowik C., and Maggi A. In vivo imaging of transcriptionally active oestrogen receptor, in Press. DiLorenzo D., Villa R., Biasiotto G., Belloli S., Ruggeri G., Alberini A., Apostoli P., Raviscion M., Ciana P., and Maggi A. Isomer-specific activity of ddt with estrogen receptor in adult and suckling mice. Endocrinology,(2002) 143(12):4544-4551

7

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ESTROGENIC EFFECTS OF ALKYLPHENOLS ON BRAIN DIFFERENTIATION Gahr M. Department of Developmental Neurobiology, Faculty of Biology, Vrije Universiteit Amsterdam, 1081 De Boelelaan, 1081 HV Amsterdam, The Netherlands. Email: [email protected]. Xenoestrogens might interfere with the organizational effects of endogenous hormones and induce permanent organizational abnormalities in the brain during development and hence in adult behaviour and reproduction such as inducing masculinization in females or de-masculinization in males. Similarly, in adulthood they may induce abnormal responses or normal responses at the wrong time, although these would be acute and usually reversible. The effects of natural estrogens or their mimetic xenoestrogens require, however, the uptake of xenoestrogens in relevant target tissues such as the pituitary and defined areas of the brain. These uptake sites might involve brain areas that express estrogen receptors, suggesting genomic action of xenoestrogens, and areas not expressing estrogen receptors, suggesting non-genomic action of xenoestrogens. Thus the three key sensitive points to be investigated are (1.) the sites and amount of xenoestrogen binding in-vivo, (2.) the behaviour of animals treated as juveniles or adults in relation to the binding sites in the brain, and (3.) the neural effects of xenobiotics in their brain target areas. These points are studied using the estrogen-dependent differentiation of the neural vocal control system of a bird, the zebra finch. As xenoestrogens we focused on Alkylphenols, which are accumulated in the food chain, and which have been shown to be weakly estrogenic in-vitro or in-vivo on peripheral tissues. In the zebra finch, a direct link can be made between the degree of sexual differentiation of brain vocal control areas and singing, a courtship behaviour. Only males sing and develop brain vocal control areas. Females develop these areas and song behaviour only after perinatal estrogen treatment. Further, the distribution of estrogen receptor alpha and beta in the brain of the zebra finch is well known. (1.) The binding sites of tritium-labeled Nonylphenol were mapped in the brain of the zebra finch using in-vivo autoradiographic techniques. This showed that Nonylphenol binding brain areas do, in general, not contain any estrogen receptors. Further, binding in brain areas such as the hippocampus, which contains low levels of estrogen receptors, was not nuclear but appeared to be to the cellular membrane. The binding of 3H-nonylphenol could be blocked by Nonylphenol and Octylphenol, suggesting a common binding site for both Alkylphenols, which is neither the estrogen receptor alpha nor beta. The nature of this binding site will be discussed. (2). Juvenile female zebra finches treated with Alkylphenols during the postnatal period, in which estrogens are able to masculinize the vocal behaviour, had mixed results. Some of these animals were able to produce songs or song-like vocalisations as adults while others did not. However, normal females never produce such

8

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

vocalisations. This result is similar to the effects of the estrogen 17β-estradiol on the development of female vocal behaviours. (3.) Octylphenol treatment of juvenile females induced a partial masculization of the vocal control nucleus RA (nucleus robustus archistriatalis), which is part of the song producing neural pathway of birds. In all females, including those that did not sing, the main effects of Octylphenol were on the volume of the RA and on the neuron density within RA. Again these results are similar to those of postnatal treatments of female zebra finches with the estrogen 17 β -estradiol. We suggest that Alkylphenols have specific binding sites in the brain and induce estrogenic effects during brain development. This estrogenic effect does not involve classical estrogen receptor dependent mechanisms. Further, this work points out that invitro studies using simple organisms such as estrogen receptor expressing cell-lines or yeast to characterise the estrogenic potency of xenoestrogens are not informative concerning their in-vivo potency and mode of action.

9

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ASSESSING THE CONSEQUENCES OF DIETARY NEUROENDOCRINE AND BEHAVIORAL MEASURES

METHOXYCHLOR:

Ottinger M.A., Hazelton J., Wu J., Ruscio M., Thompson N., Quinn M. and Abdelnabi M. Department of Animal and Avian Sciences, University of Maryland College Park, Maryland 20742; [email protected] Endocrine disrupting chemicals (EDCs) include pesticides, herbicides, and other chemicals that interact with endocrine systems. Many of the EDCs mimic estrogens and interact with estradiol receptors. Previous studies have shown that the male Japanese quail is exquisitely sensitive to the effects of exogenous estradiol during sexual differentiation of neural systems that regulate reproductive behavior. In addition, the hormonal basis for sexual differentiation in avian species differs from mammals, thereby making mammalian screens potentially inadequate for assessing EDC impact on avian species. Further, Bobwhite quail has been the species of choice for assessing effects of toxic compound exposure in avian species. Unfortunately, there are few data on neuroendocrine regulation of reproduction in Bobwhite quail. Conversely, a great deal is known about neuroendocrine regulation of reproduction in Japanese quail, but few data have been collected on effects of EDCs on neuroendocrine systems in quail. Initial studies showed that embryonic exposure to either methoxychlor (MXC), a widely used pesticide or vinclozolin, a fungicide resulted in impaired sexual behavior in the adult male Japanese quail [1, 2]. The long-term effects on behavior were accompanied by altered hypothalamic catecholamine and GnRH-I levels. In a subsequent study, Japanese quail were exposed to MXC via the diet (0, 0.5, or 5ppm) in a 2 generation paradigm, with MXC exposure beginning in proven pairs (P1), continuing in their offspring (F1), and observation of their offspring (F2), which were raised on control diet. These dietary levels are field relevant and below regulatory limits for exposure. Results showed effects of dietary exposure on reproductive endocrine, neuroendocrine, and behavioral end points. Some traditional toxicological end points, including fertility, hatching success, and 14 day viability did not show discernable effects, suggesting that these variables are not sufficiently sensitive to detect EDC effects. MXC exposure affected male sexual behavior, hypothalamic catecholamines, and plasma steroid hormones. Moreover, MXC exposure had reproductive consequences observable at both the lower and higher doses of MXC. In a current study, Bobwhite and Japanese quail were simultaneously exposed to dietary MXC (MXC; 0, 5ppm, and 10ppm) over 2 generations. The parent generation (P) was raised under short photoperiod, paired, and transferred to long days (16L:8D), with initiation of treatment. Basic measures of health and reproduction were monitored, including feed intake, egg production, fertility, and offspring viability. Chicks (F1) were raised on the same diet as their parents and observed for sexual maturation, reproductive behavior and endocrine end points. Results showed species differences in maturation, with Bobwhite quail requiring 3-4 weeks longer to achieve sexual maturity. Neither species showed effects of 5ppm MXC on egg production, fertility, body weight, feed intake, or chick viability. However, Japanese quail fed MXC matured more slowly, suggesting that the treatment interfered with activation of reproduction. A separation test was used in

10

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

young chicks to assess motivation to rejoin siblings. Moreover, we observed that some pairs from both species appeared less affected by MXC exposure, suggesting variability in individual sensitivities to the chemical insult. These studies verify the efficacy of neuroendocrine and behavioral measures for providing reliable indicators of embyronic EDC exposure. These measures are particularly relevant for consideration in assessing the longterm impact of EDCs on birds because they are sexually dimorphic and organized under the influence of steroid hormones during embryonic development.

Supported by EPA #R826134010 (Star Grant), NSF #9817024, and EPA R-2877801 (MAO).

Reference List [1] Ottinger et al., 2001; Horm. Behav. 40: 234-247; [2] McGary et al., 2001; Environ. Toxicol. Chem. 20, 2487-2493.

11

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NEUROBEHAVIOURAL EFFECTS OF PERINATAL EXPOSURE TO PCB IN DEVELOPING CD-1 MICE Santucci D., Branchi I., De Acetis G. and Alleva E. Behavioural Pathophysiology Section, Laboratorio di Fisiopatologia di Organo e di Sistema, Istituto Superiore di Sanita', Viale Regina Elena 299, 00161 Roma, ITALY [email protected] Polychlorinated biphenyls (PCBs) are persistent and highly lipophilic environmental contaminants. They readily accumulate in biological tissues and have long been implicated as neurotoxicant chemicals in humans and wildlife. In particular, in utero and lactational exposure to PCB has been associated with cognitive and motor-reflex dysfunction as well as with changes in selected items of sexual behaviour. Aim of this preliminary study was to evaluate short-, medium-, and long-term neurobehavioural effect of early postnatal exposure to PCB54 and PCB77, the two congeners being chosen on the basis of their different structure-activity. Neonatal mice were exposed to 0.8 and 8 mg/Kg of PCB 54 or PCB 77 during the first week of life (subcutaneously, postnatal days 3 and 5), and somatic and neurobehavioural development were scored according to a modified Fox's scale. Ultrasonic vocalization pattern, homing performance, open-field activity and social and aggressive interaction as well as testosterone and ChAT activity in adult male were also assessed. PCB exposed mice were slightly hyperactive at weaning, showing some differences in behavioural patterns during social encounter, in absence of major somatic or neurobehavioural alteration in the pre-weaning stage. Moreover, both compounds clearly affect aggressive behaviour of adult mice, PCB treated mice being significantly less aggressive. The low dose of PCB 77 resulted the most effective in reducing intermale agonistic items.

12

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EFFECTS OF ENDOCRINE MODULATORS ON SEXUAL DIFFERENTIATION AND REPRODUCTIVE FUNCTION IN JAPANESE QUAIL Halldin K., Axelsson J. and Brunström B. Department of Evolutionary biology, Environmental Toxicology, Uppsala University, Norbyvägen 18A, SE-752 36 Uppsala, SWEDEN, E-mail: [email protected], Fax: +46 18 518843. Chemical disruption of the endocrine system in various animal species and humans is currently attracting considerable interest and several in vitro and in vivo tests are employed to elucidate effects of these chemicals. However, avian models are not yet widely used in studying endocrine disturbances. Sexual behavior of male Japanese quail (Coturnix japonica) is permanently demasculinized by estrogen treatment during the critical period of sexual differentiation [1,2]. In our lab, we have investigated the usefulness of alterations in sexual behavior and reproductive organs in adult male Japanese quail as endpoints following embryonic exposure to estrogen-like chemicals. In our initial studies [5], we focused on determining whether male sexual behavior in Japanese quail is a suitable endpoint for studying long-term effects of exposure during sexual differentiation. Most previous studies have used relatively high concentrations of estrogen to induce behavioral sex reversal in genetically male quail. To be able to evaluate the potency of estrogen-like chemicals several orders of magnitudes less potent than estradiol, we needed to establish dose–response relationships and to find the lowest doses causing these effects. Apart from sexual behavior we also studied plasma testosterone concentration, testis morphology and cloacal gland size. The results showed that demasculinization of male quail by diethylstilbestrol and ethinylestradiol was induced at doses about 1,000 times lower than those usually employed and that a dose–response relationship was present. Altered sexual behavior was the most sensitive of the variables studied and we concluded that this endpoint is useful for studying effects of endocrine modulating chemicals. Other studies in our lab have included testing the environmental contaminants bisphenol A (BPA), tetrabromobisphenol A (TBBPA), and o,p´-DDT for estrogen-like properties that result in alterations similar to those seen after exposure to the two synthetic estrogens [6,7]. The endpoints studied in males were generally the same as in the initial paper. We did not find any significant effects by BPA and TBBPA on the variables studied, whereas o,p´-DDT caused profound effects on sexual behavior, cloacal gland area, and plasma testosterone concentration in males. The binding affinities of Erα and ERβ to certain ligands among phytoestrogens and estrogenic chemicals have been shown to differ. Knowledge about the expression of the ERs during embryonic development may explain potential differences in potency of estrogens and estrogen-like chemicals to induce behavioral demasculinization as compared to other disturbances. We investigated the localization of ERα and ERβ mRNA in hypothalamic and limbic structures known to play prominent roles in male sexual behavior [8]. Brains from adults and embryos were studied by in situ hybridization. Relatively high expression of ERβ mRNA in comparison with ERα mRNA was found in both adults and at the two embryonic stages investigated. Sex differences were indicated for ERβ in the 13

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

nucleus taeniae of adults, an area suggested to be implicated in male sexual behavior. The bed nucleus striae terminalis expressed very low levels of ERα. In the medial preoptic nucleus, both mRNAs were expressed, albeit with differences in density. At E17, the localization was similar to that in adults, but the relative intensity of ERα signal as compared to ERβ was lower than in adults. This study provided novel data on localization in embryos and confirmed previously published studies in adults [3,4]. One of the main findings was that only ERβ was detected at E9, whereas the brain areas included were devoid of signal for ERα mRNA. This could indicate an important role for ERβ during brain differentiation, alternatively ERα is beginning to be expressed when brain demasculinization starts. To conclude, the Japanese quail is well suited as a model animal for studying various long-term effects after embryonic exposure to estrogenic compounds. The wide range of effects provides several variables that can be measured to detect effects caused by chemicals with estrogenic properties. Hence, the suggestion from OECD that the Japanese quail be used as a species of choice for testing endocrine disrupting chemicals seems well founded. The functional significance of ERs located in the investigated brain areas needs to be studied further. The effects on sexual differentiation of agonists and antagonists specific to each of the two receptors are of interest, for elucidating both basic mechanisms and effects of endocrine modulating chemicals.

Reference List [1] Adkins EK. Embryonic exposure to an antiestrogen masculinizes behavior of female quail. Physiol Behav 17:357-359 (1976). [2] Balthazart J, De Clerck A, Foidart A. Behavioral demasculinization of female quail is induced by estrogens: studies with the new aromatase inhibitor, R76713. Horm Behav 26:179-203 (1992). [3] Balthazart J, Gahr M, Surlemont C. Distribution of estrogen receptors in the brain of the Japanese quail: an immunocytochemical study. Brain Res 501:205-14(1989). [4] Foidart A, Lakaye B, Grisar T, Ball GF, Balthazart J. Estrogen receptor-beta in quail: cloning, tissue expression and neuroanatomical distribution. J Neurobiol 40:327-42.(1999). [5] Halldin, K., Berg, C., Brandt, I., and Brunström, B. (1999). Sexual Behavior in Japanese Quail as a Test Endpoint for Endocrine Disruption: Effects of in ovo Exposure to Ethinylestradiol and Diethylstilbestrol. Environ Health Perspect 107:861-866. [6] Halldin, K., Berg, C., Bergman, Å., Brandt, I., and Brunström, B. (2001). Distribution of Bisphenol A and Tetrabromobisphenol A in quail eggs, embryos and laying birds and studies on reproduction variables in adults following in ovo exposure. Arch Toxicol 75:597-603. [7] Halldin, K., Holm, L., Ridderstråle, Y., and Brunström, B. Reproductive disturbances in male and female quail after in ovo exposure to the environmental pollutant o,p´-DDT. Arch Toxicol DOI 10.1007/s00204-002-0417-8[8] Halldin, K., Axelsson, J., Holmgren, C., Brunström, B. Localization of Estrogen Receptor-a and -b mRNA in the Brain of Embryonic and Adult Japanese Quail. Abstract in this volume.

14

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 IMPACT OF ENVIRONMENTAL ESTROGENS ON BEHAVIORAL SYSTEMS IN MICE

SEXUALLY

DIMORPHIC

Palanza P. Dipartimento di Biologia Evolutiva e Funzionale, Universita’ degli Studi di Parma, viale delle Scienze 11/a, 43100 – Parma (I). [email protected]; FAX: 0521-905657.

According the traditional theory, the development of sexual dimorphisms in behavior and cognitive function and of sexual dimorphic brain areas in vertebrates depends mostly on the epigenetic action of gonadal hormones, androgens and estrogens. Besides reproductive behaviors, specific behavioral differences between males and females M y include differences in aggression, learning, infant play, exploration, activity level, food intake and preference, novelty seeking, emotional behavior and many more (Goy and McEwan 1980). In the conceptual frame of evolutionary theory, sex-differences in behavior are thought to reflect adaptive differences of behavioral strategies in coping as resulting from sexual selection (Darwin 1871). In this contest, particular attention should be addressed to possible effects of exposure to a large class of man-made compounds present in the environment named “endocrine disrupters”, which are able to interfere with the functioning of the endocrine system in vertebrates by binding to hormone receptors, such as the intracellular receptors for estradiol and testosterone, and alter development when exposure occurs during critical developmental periods (e.g., fetal life). Such interference can range from a disruptive effect to a modulatory effect, depending on the mode of action of the substance, its concentration in the organism and, primarily, the timing of exposure. In this paper I present our ethological investigations of the effects of maternal exposure to different estrogenic endocrine disruptors, at concentrations within the range of human exposure and not patently teratogenic, on behavioral responses of male and female house mice (Mus musculus domesticus) at different developmental stages. Pregnant female mice were trained to spontaneously drink daily doses of corn oil with or without the estrogenic plastic derivative, bisphenol A (BPA 10 microg/Kg), or the estrogenic insecticide methoxychlor MXC (20-2000 microg/Kg bw) from gestation day 11 to 17 (prenatal exposure) or from gestation day 11 to postpartum day 8 (perinatal exposure). Their offspring were examined at different ages in a series of behavioral tests. The main results indicate that maternal exposure to the estrogenic chemicals affected: 1) behavioral responses to novelty at periadolescence; 2) exploration and activity in a free-exploratory open field as adults; 3) exploration in the elevated plus maze; 4) spatial learning; 5) social interaction with an unfamiliar same-sex opponent in a neutral arena. In all these different experimental settings, while a sex difference was observed in the control group, exposure to BPA or to some MXC doses decreased the sexual dimorphism of several behavioral responses of males and females. These effects were mostly due to higher sensitivity of the female offsping to the effects of the maternal exposure to the estrogenic chemicals. Furthermore, we examined the possible changes in the dopaminergic systems in the nigrostriatal area of the brain in male and female mice prenatally exposed to MXC; female mice prenatally exposed to MXC showed a decrease in dopamine (D1-like) receptor density in the nucleus accumbens and olfactory tubercle, at the same doses causing the behavioral effects. Reproductive success and parental interactions were also assessed in pairs of maternally exposed mice housed in artificial territories, in order to evaluate possible long-term effects of environmental estrogens. 15

SATURDAY, 22th February 14.30 - 18.00 Satellite Symposium: The Role of Neuroactive Steroids in Healthy Ageing: Therapeutical Perspectives

Satellite Symposium: The Role of Neuroactive Steroids in Healthy Ageing: Therapeutical Perspectives (Chairs: Melcangi R.C., Milano & Schumacher M., Bicêtre) • Baulieu E.E. (Bicêtre, France, EU) Basic and clinical aspects of the aging process: human brain and steroids • Schumacher M., Ibanez C., Weil-Engerer S., Liere P., Robert F., Guennoun R., Gago N., Baulieu E.E., and Akwa Y. (Bicêtre, France, EU) Biosynthesis of progesterone in the nervous system: trophic and protective effects • Ibanez C., Shields S.A., El-Etr M., Baulieu E.-E., Schumacher M., Li W.-W. and Franklin R.J.M. (Cambridge, UK, EU) Progesterone and CNS remyelination • Melcangi R.C., Azcoitia I., Ballabio M., Cavarretta I., Gonzalez L.C., Leonelli E., Magnaghi V., Veiga S. and Garcia-Segura L.M. (Milano, Italy, EU) Neuroactive steroids influence peripheral myelination • Garcia-Segura L.M., Veiga S., Méndez P., García-Ovejero D., DonCarlos L.L. and Azcoitia I (Madrid, Spain, EU) Neuroprotection by steroids in the central nervous system • Mayo W. (Bordeaux, France, EU) Individual differences in cognitive ageing: implication of neurosteroids • Lambert J.J., Belelli D., Callachan H., Harney S.A., Peden D., and Vardy A. (Dundee, UK, EU) The interaction of neurosteroids with recombinant and synaptic GABAA receptors

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

BASIC AND CLINICAL ASPECTS OF THE AGING PROCESS : HUMAN BRAIN AND STEROIDS Baulieu E.E. INSERM U488, 80, rue du Général Leclerc, 94276 Le Kremlin Bicêtre, France; Tel : 33 1 49 59 18 82 – Fax : 33 1 49 59 92 03 – e.mail : [email protected] This introductory paper will recall some of the main parameters related to aging in human beings, especially at the brain level. The increase of lifespan observed in the second part of the 20th century and continuing currently is unique in the history of humanity: not only numerically, but also qualitatively mankind will be changed and the main functions of people will be different of what they have been in the past. The capacity to ensure cognitive activities, metabolic controls, adaptation and reaction to social life depends largely on the pathophysiological state of the brain. I shall recall some of the mechanisms involved in brain aging, trying to dissect what is dependent on gene activities and environmental parameters. The implication of hormones, and more specifically steroids including those produced in the nervous system (neurosteroids) will be discussed. The border separating "physiological" aging and pathological events (mainly of vascular or neurodegenerative origin) will be explored. From animal experiments some concepts, such as the functional/anatomical reversibility of brain deficits, will be reported and their possible transfer to the human situation briefly discussed. I shall try to present some suggestions of new approaches dealing with novel diagnostic tools, intervention epidemiology, and clinical trials.

References List 1. Dani S.U., Hori A., Walter G.F. (eds). Principles of neural aging. Elsevier, 1997. 2. Finch C.E. Robine J.M., Christen Y. (eds). Brain and longevity. Springer, 2003. 3. Baulieu E.E., Robel P., Schumacher M. (eds). Neurosteroids : a new regulatory function in the nervous system. Humana Press, 1999. 4. Akwa Y., Ladurelle N., Covey D.F., and Baulieu E.E. The synthetic enantiomer of pregnenolone sulfate is very active on memory in rats and mice, even more so than its physiological neurosteroid counterpart : Distinct mechanisms ? Proc Natl Acad Sci USA, 98, 14033-14037, 2001.

19

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

BIOSYNTHESIS OF PROGESTERONE IN THE NERVOUS SYSTEM: TROPHIC AND PROTECTIVE EFFECTS Schumacher M., Ibanez C., Weil-Engerer S., Liere P., Robert F., Guennoun R., Gago N., Baulieu E.E. and Akwa Y. INSERM U 488, 80, rue du Général Leclerc, 94276 Bicêtre, France. Tel : 33-1.49. 59.18.95 ; Fax : 33-1.45.21.19.40 ; Email : [email protected] Steroid hormones are taking an increasingly important place in the field of research on aging. This is in part prompted by a series of new concepts concerning the aging process of the brain as well as the functions of steroids in the nervous system. Classically, the discipline of neuroendocrinology dealed with the regulation of reproductive functions and stress responses by gonadal and adrenal steroid hormones. This has completely changed over the past years with the appreciation that steroids, by means of a variety of mechanisms, regulate many important neuronal and glial functions throughout the nervous system. In addition, some of them, which have been named "neurosteroids", can be synthesized within the central (CNS) and peripheral (PNS) nervous systems by neurons and glial cells [1]. When considering the role of steroids in the nervous system, it is indeed important to keep in mind that both steroid hormones produced by the endocrine glands and locally synthesized neurosteroids contribute to the pool of steroids present in the brain and in peripheral nerves. Thus, age-related changes in the circulating levels of steroids may not necessarily reflect changes of their levels within different parts of the nervous system. We know in fact very little concerning the possible role of neurosteroids in the aging nervous system. Our appreciation of the aging nervous system has also changed very much. The idea of massive and widepread neuron death during normal aging has been challenged and it is now widely accepted that age-related changes in the brain are more subtle, involving changes in cell functions, shrinkage of neuronal cell bodies, decreased density of neurites, reduced number of synapses as well as the damage and loss of white matter. Most importantly, recent research has shown that the aging nervous system retains capacity for regeneration, which means that the treatment of age-dependent dysfunctions becomes possible. Work presented in this symposium provides evidence that age-associated memory deficits and myelin abnormalities, which correlate with reduced levels of steroids or with reduced activity of steroid metabolizing enzymes, can be reversed by the administration of steroids. Progesterone is an example of a pleiotropic steroid which can be synthesized within the CNS and PNS by neurons and glial cells. In Schwann cells, expression and activity of the 3β-hydroxysteroid dehydrogenase (3β-HSD), which converts pregnenolone to progesterone, are induced by a diffusible neuronal factor [6]. We have recently investigated the synthesis of PROG at three stages of maturation of the oligodendroglial lineage, namely, PSA-NCAM+ preprogenitors, oligodendrocyte progenitors and fully differentiated oligodendrocytes. Only the preprogenitors and progenitors, but not the mature oligodendrocytes, expressed the 3β-HSD and synthesized [3H]progesterone when incubated in the presence of [3H]pregnenolone [2]. Progesterone plays an important role in

20

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

myelination. This was first shown in the regenerating mouse sciatic nerve and in explant cultures of rat dorsal root ganglia [4, 7]. New perspectives for elucidating the role of steroids in the aging nervous system are offered by a new technology, based on the analysis steroids by gas chromatography/mass spectrometry (GC/MS). This very sensitive microassay allows for the first time to simultaneously identify and quantify femtomolar concentrations of several steroids within individual small nervous tissue samples [5]. By using GC/MS, we have analyzed developmental changes of progesterone and its precursor pregnenolone in the male rat hippocampus, a brain region critically involved in memory processes. Their concentrations were elevated the day of birth and were significantly lower at postnatal days 7 and 14 and in adults. Concomitantly, 3β-HSD mRNA levels were found to be decreased in the hippocampus with progressing age. A detailed analysis by in situ hybridization emulsion autoradiography revealed that the 3βHSD is mainly expressed by pyramidal neurons and that silver grain density per cell decreases with progressing age [3]. To investigate the physiopathological significance of brain steroids in Alzheimer's disease (AD), we have measured their concentrations by GC/MS in individual brain regions of AD patients and of aged non-demented controls, including the hippocampus, amygdala, frontal cortex, striatum, hypothalamus and cerebellum. Eleven patients of the median age of 86 years (minimum: 75.6 ; maximum: 91.5) were selected for the study. Exclusion criteria were a postmortem delay beyond 24 hours, steroid or benzodiazepine administration during the month before death and prolonged hypoxemia at the time preceeding death. A general trend toward decreased levels of all steroids was observed within all the brain regions in AD patients. A significant negative correlation was found between levels of β-amyloid peptides and those of conjugated pregnenolone in the striatum and cerebellum, and between the levels of phosphorylated tau proteins and conjugated dehydroepiandosterone in the hypothalamus [8]. References List 1. Baulieu EE, Robel P, Schumacher M (1999) Neurosteroids. A new regulatory function in the nervous system. Totowa, New Jersey: Humana Press, pp. 378. 2. Gago N, Akwa Y, Sananes N, Guennoun R, Baulieu EE, El E, Schumacher M (2001) Progesterone and the oligodendroglial lineage: stage-dependent biosynthesis and metabolism. Glia 36: 295-308. 3. Ibanez C, Guennoun R, Liere P, Eychenne B, Pianos A, El-Etr M, Baulieu EE, Schumacher M (2003) Developmental expression of genes involved in neurosteroidogenesis : 3β-hydroxysteroiddehydrogenase /∆5-∆4 isomerase in the rat brain. Endocrinology, in press. 4. Koenig H, Schumacher M, Ferzaz B, Do-Thi AN, Ressouches A, Guennoun R, Jung-Testas I, Robel P, Akwa Y, Baulieu EE (1995) Progesterone synthesis and myelin formation by Schwann cells. Science 268: 1500-1503. 5. Liere P, Akwa Y, Weill E, Eychenne B, Pianos A, Robel P, Sjövall J, Schumacher M, Baulieu EE (2000) Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl 739: 301-312. 6. Robert F, Guennoun R, Desarnaud F, Do-Thi A, Benmessahel Y, Baulieu EE, Schumacher M (2001) Synthesis of progesterone in Schwann cells: regulation by sensory neurons. Eur J Neurosci 13: 916924. 7. Schumacher M, Guennoun R, Mercier G, Desarnaud F, Lacor P, Benavides J, Ferzaz B, Robert F, Baulieu EE (2001) Progesterone synthesis and myelin formation in peripheral nerves. Brain Res Rev 37: 343-359. 8. Weill-Engerer S, David JP, Sazdovitch V, Liere P, Eychenne B, Pianos A, Schumacher M, Delacourte A, Baulieu EE, Akwa Y (2002) Neurosteroid quantification in human brain regions : comparison between Alzheimer's and non-demented patients. J Clin Endocrinol Metab 87: 5138-5143.

21

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PROGESTERONE AND CNS REMYELINATION Ibanez C., Shields S.A., El-Etr M., Baulieu E.-E., Schumacher M., Li W.-W.* and Franklin R.J.M. * INSERM U488, 80 rue du Général Leclerc, 94276 Le Kremlin Bicêtre-Cedex, France & *Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK; [email protected] Although remyelination can occur as a spontaneous regenerative process following demyelination of CNS axons, there are many circumstances where it fails such as chronic multiple sclerosis, leaving axons demyelinated and vulnerable to atrophy (2). Agents that affect the efficiency of CNS remyelination may therefore have a role in MS therapy. Progesterone regulates the expression of myelin basic protein (MBP) and CNPase (5) genes by mature oligodendrocytes (5;8), which also express the receptor for this steroid (4). Thus, progesterone might also be able to regulate myelin sheath formation during CNS remyelination. However, progesterone also has anti-inflammatory actions (1), and given the importance of the inflammatory response for efficient remyelination of toxin-induced demyelination (6), one could also hypothesize that progesterone might have inhibitory effects on remyelination. We have been examining the effects of systemically administered progesterone on CNS remyelination using a toxin-induced model in which the rate of remyelination is age-dependent (3;7). The rapid remyelination in young adult rats allowed an assessment of potential adverse effects of progesterone while the slow remyelination in older adult rats allowed an assessment of its potentially beneficial effects. There was no significant difference in the rate of remyelination between young control and treated animals. However, a modest but significant increase in the extent of oligodendrocyte remyelination in response to progesterone (and a comparable significant decrease in the proportion of axons that remained demyelinated) was observed in older rats five weeks after lesion induction. We also found a significant increase in the proportion of Schwann cell remyelinated axons between 3 and 5 weeks after lesion induction that was not apparent in the control animals. These results indicate that progesterone does not inhibit CNS remyelination and that it has a positive modulating effect on oligodendrocyte remyelination in circumstances where it is occurring sub-optimally.

(This study has been carried out with financial support from the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6CT-2000-00179)

References List 1. Drew, P. D. And J. A. Chavis. 2000. Female Sex Steroids: Effects Upon Microglial Cell Activation. J Neuroimmunol 111: 77-85. 2. Franklin, R. J. M. 2002. Why Does Remyelination Fail In Multiple Sclerosis? Nat.Rev.Neurosci. 3: 705-714. 3. Franklin, R. J. M., C. Zhao, And F. J. Sim. 2002. Ageing And Cns Remyelination. Neuroreport 13: 923-928. 4. Jung-Testas, I. And E. E. Baulieu. 1998. Steroid Hormone Receptors And Steroid Action In Rat Glial Cells Of The Central And Peripheral Nervous System. J Steroid Biochem.Mol Biol 65: 243251.

22

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 5. 6. 7. 8.

Jung-Testas, I., M. Schumacher, P. Robel, And E. E. Baulieu. 1996. The Neurosteroid Progesterone Increases The Expression Of Myelin Proteins (Mbp And Cnpase) In Rat Oligodendrocytes In Primary Culture. Cell Mol Neurobiol 16: 439-443. Kotter, M. R., A. Setzu, F. J. Sim, N. Van Rooijen, And R. J. M. Franklin. 2001. Macrophage Depletion Impairs Oligodendrocyte Remyelination Following Lysolecithin-Induced Demyelination. Glia 35: 204-212. Sim, F. J., C. Zhao, J. Penderis, And R. J. M. Franklin. 2002. The Age-Related Decrease In Cns Remyelination Efficiency Is Attributable To An Impairment Of Both Oligodendrocyte Progenitor Recruitment And Differentiation. J.Neurosci. 22: 2451-2459. Verdi, J. M. And A. T. Campagnoni. 1990. Translational Regulation By Steroids. Identification Of A Steroid Modulatory Element In The 5'-Untranslated Region Of The Myelin Basic Protein Messenger Rna. J Biol Chem 265: 20314-20320.

23

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NEUROACTIVE STEROIDS INFLUENCE PERIPHERAL MYELINATION Melcangi R.C.*, Azcoitia I.°, Ballabio M.*, Cavarretta I.*, Gonzalez L.C.*, Leonelli E.*, Magnaghi V.*, Veiga S.* and Garcia-Segura L.M.# *Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milano, Italy, Tel. +39-02-50318238, Fax: +39-02-50318204, Email: [email protected] # Instituto Cajal, C.S.I.C., 28002 Madrid, Spain. °Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, E28040 Madrid, Spain. The process of aging deeply influences morphological and functional parameters of the peripheral nerves. Our observations performed on the rat sciatic nerves have indicated that the deterioration of myelin occurring in the peripheral nerves during aging may be explained by the fall of the levels of the major peripheral myelin proteins [e.g., glycoprotein Po (Po) and peripheral myelin protein 22 (PMP22)] [1,2]. We have then demonstrated that neuroactive steroids, like for instance progesterone (P) and its physiological 5alpha-reduced derivative, dihydroprogesterone (DHP), are able to stimulate the low levels of Po present in the sciatic nerve of aged male rats [1,2]. On the contrary, the levels of PMP22 are only stimulated by the 3alpha-5alpha-reduced derivative of P, tetrahydroprogesterone (THP) [1,2]. It is important to highlight that similar effects also occur in other experimental in vivo (e.g., intact and transected sciatic nerve of adult male rats) and in vitro (rat Schwann cell cultures) models [3-5]. Po and PMP22 play an important physiological role for the maintenance of the multilamellar structure of PNS myelin [2] and consequently we have evaluated the effect of P and its neuroactive derivatives, DHP and THP, on the morphological alterations of myelinated fibers in the sciatic nerve of 22-24 month-old male rats [6]. The sciatic nerves of untreated old male rats, show a general disorganization and a significant reduction in the density of myelinated fibers, compared to nerves from 3 months-old male rats. The effect of aging is particularly evident in myelinated fibers of small caliber (< 5 µm in diameter). In addition, the sciatic nerves of aged rats show a significant increase in the number of fibers with myelin infoldings in the axoplasm and in the number of fibers with irregular shapes. Treatments of aged male rats with P, DHP and THP result in a significant increase in the number of myelinated fibers of small caliber, a significant reduction in the frequency of myelin abnormalities and a significant increase in the g ratio of small myelinated fibers. Moreover, P treatment significantly reduces the frequency of myelinated fibers with irregular shapes. In conclusion, the present data indicate that neuroactive steroids as P, DHP and THP, are able to reduce aging-associated morphological abnormalities of myelin and aging-associated myelin fiber loss in the sciatic nerve.

(This study has been carried out with financial support from the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6CT-2000-00179)

24

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 Reference List 1. Melcangi, R.C., Magnaghi, V., Cavarretta, I., Martini, L. and Piva, F., Age-induced decrease of glycoprotein Po and Myelin Basic Protein gene expression in the rat sciatic nerve. Repair by steroid derivatives, Neuroscience, 85 (1998) 569-578. 2. Melcangi, R.C., Magnaghi, V. and Martini, L., Aging in peripheral nerves: regulation of myelin protein genes by steroid hormones, Prog. Neurobiol., 60 (2000) 291-308. 3. Melcangi, R.C., Magnaghi, V., Cavarretta, I., Zucchi, I., Bovolin, P., D'Urso, D. and Martini, L., Progesterone derivatives are able to influence peripheral myelin protein 22 and Po gene expression: possible mechanisms of action, J. Neurosci. Res., 56 (1999) 349-357. 4. Melcangi, R.C., Magnaghi, V., Galbiati, M., Ghelarducci, B, Sebastiani, L. and Martini, L., The action of steroid hormones on peripheral myelin proteins: a possible new tool for the rebuilding of myelin? J. Neurocytol., 29 (2000) 327-339. 5. Magnaghi, V., Cavarretta, I., Galbiati, M., Martini, L. and Melcangi, R.C., Neuroactive steroids and peripheral myelin proteins. Brain Res Rev 37 (2001) 360-371. 6. Azcoitia, I., Leonelli, E., Magnaghi, V., Veiga, S., Garcia-Segura, L.M. and Melcangi, R.C., Progesterone and its derivatives dihydroprogesterone and tetrahydroprogesterone reduce myelin fiber morphological abnormalities and myelin fiber loss in the sciatic nerve of aged rats, Neurobiol. Aging (2003), in press.

25

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NEUROPROTECTION BY STEROIDS IN THE CENTRAL NERVOUS SYSTEM Garcia-Segura L.M.1, Veiga S.1, Méndez P.1, García-Ovejero D.1, DonCarlos L.L.2 and Azcoitia I.3 1

Instituto Cajal, C.S.I.C., Avenida Doctor Arce 37, E-28002 Madrid, Spain. E-mail: [email protected]; Fax: 34-915854754; 2Department of Cell Biology, Neurobiology and Anatomy, Loyola University Chicago, Stritch School of Medicine, Maywood IL 60153, USA; 3Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, E-28040 Madrid, Spain. There is now evidence that sex steroids, in addition to their participation in neuroendocrine regulation and sexual behavior, have neuroprotective properties. Estradiol protects hippocampal hilar neurons against the neurotoxic effect of kainic acid (KA) in both male and female rats. Administration of KA, as well as other forms of neurodegenerative stimuli, induces the expression of both the enzyme aromatase and estrogen receptor alpha in reactive astrocytes in the hilus. This indicates that astroglia react to brain lesions by producing estradiol and increasing their sensitivity to the steroid. Intracerebroventricular infusion of an estrogen receptor antagonist (ICI 182,780) blocks the neuroprotective effect of estradiol. Furthermore, genetic or pharmacological inhibition of brain aromatase results in increased degeneration of hilar neurons after the administration of KA. These findings strongly suggest that local formation and local action of estradiol in the brain are neuroprotective. Precursors of estradiol that are synthesized in the central nervous system and periphery are also neuroprotective. To assess whether the neuroprotective effects of the estrogen pre-cursors pregnenolone, dehydroepiandrosterone (DHEA) and testosterone are dependent on their conversion to estradiol, the aromatase inhibitor fadrozole was administered to gonadectomized adult male rats using osmotic minipumps. Pregnenolone, DHEA and testosterone protected hilar neurons against KA and fadrozole blocked the neuroprotective effect of these steroids. This finding suggests that estradiol synthesis by aromatase mediates the neuroprotective effects of pregnenolone, DHEA and testosterone against excitotoxin-induced neuronal death in the hippocampus. The mechanism of action of estradiol as a neuroprotectant is currently being explored. Insulin like growth factor-I (IGF-I), a trophic factor for neurons and glia, interacts with estrogen in the promotion of neuronal survival. Both systemic administration of estradiol, as well as intracerebroventricular infusion of IGF-I, prevent hilar neuronal loss induced by KA in adult ovariectomized rats. To determine the role of IGF-I receptor in the neuroprotective effect of estrogen, a specific IGF-I receptor antagonist, the peptide JB1, was infused into the lateral cerebral ventricle. The neuroprotective action of estradiol was abolished under these conditions. This finding indicates that IGF-I receptor is necessary for the neuroprotective effect of estradiol in this experimental model. Furthermore, the neuroprotective effect of IGF-I was blocked by infusion of the estrogen receptor antagonist ICI 182,780 into the lateral cerebral ventricle, indicating that activation of estrogen receptors is also necessary for the neuroprotective effect of IGF-I. These findings suggest

26

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

that co-activation of both estrogen receptors and IGF-I receptor is necessary for neuroprotection of hilar hippocampal neurons from KA toxicity. To explore the possible mechanisms involved in the interaction of estradiol and IGF-I in neuroprotection, we examined whether there is an interaction of estrogen receptor and IGF-I receptor signaling in the brain. Analysis of the distribution of these receptors in the rat brain by confocal microscopy revealed that most neurons expressing IGF-I receptor also express estrogen receptors alpha or beta. In addition, reactive astrocytes in the hilus coexpress estrogen receptor alpha and IGF-I receptor. This finding indicates that interactions of the signaling pathways of estrogen receptors and IGF-I receptor are possible at the cellular level in the brain, in neurons as well as in glial cells. Furthermore, systemic estradiol administration results in the transient activation of IGF-I receptor signaling as well as in a transient coimmunoprecipitation of the IGF-I receptor with estrogen receptor alpha in the brain of adult ovariectomized rats. Estradiol treatment also results in an enhanced coimmunoprecipitation of estrogen receptor alpha with p85 subunit of phosphatidylinositol 3-kinase, as well as an enhanced coimmunoprecipitation of p85 with insulin receptor substrate-1. The interaction with the IGF-I receptor is specific for the alpha form of the estrogen receptor and is also induced by intracerebroventricular injection of IGF-I. These hormonal actions may be part of the mechanism by which estradiol activates IGF-I receptor signaling pathways in the brain and may explain the interdependence of estrogen receptors and the IGF-I receptor in neuroprotection and other neural events. In summary, these findings indicate that local estradiol formation in the brain may be involved in the neuroprotective effects of some steroids, such as pregnenolone, DHEA and testosterone. Furthermore, the neuroprotective effects of estradiol depend on estrogen receptors and on interactions between estrogen receptors and IGF-I receptors.

This study has been supported by the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6-CT-2000-00179.

27

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

INDIVIDUAL DIFFERENCES IN COGNITIVE AGEING: IMPLICATION OF NEUROSTEROIDS Mayo W. INSERM U259, Institut François Magendie, Rue Camille Saint-Saens, 33077 Bordeaux Cedex, France; Tel : (33) 5 57 57 36 65; Fax : (33) 5 5696 68 93; e-mail: [email protected] In humans and animals individual differences in aging of cognitive functions are classically reported. Some old individuals exhibit performances similar to those of young subjects while others are severely impaired. In senescent animals we have previously demonstrated a significant correlation between the cognitive performance and the cerebral concentration of a neurosteroid, the pregnenolone sulphate (PREG-S). Indeed, rats with memory impairments exhibited low PREG-S concentrations compared to animals with correct memory performance. Furthermore these memory deficits can be reversed by intracerebral infusions of PREG-S (9). Neurosteroids are a subclass of steroids that can be synthesized in the central nervous system independently of peripheral sources. The most abundant of these neurosteroids in humans are the dehydroepiandrosterone and its sulfate (DHEA-S). In rodents, one of the major neurosteroid with properties similar to those of DHEA-S is pregnenolone sulphate (PREG-S). Several neurosteroids influence cognitive functions and particularly memory processes (5-6). Indeed, systemic or intracerebral administration of these neurosteroids, like pregnenolone (PREG) or its sulphate ester (PREG-S), enhances memory in young and old rodents. However, the neurobiological mechanisms underlying these effects still remained unknown. Neurochemically, PREG-S act as a negative modulator of the type A γaminobutyric acid receptor (GABAA) and also as a positive modulator of the N-methyl-Daspartate receptor (NMDA) subtype of glutamate receptor. Neurotransmitter systems modulated by this neurosteroid were unknown until our recent report of an enhancement of acetylcholine (ACh) release in basolateral amygdala, cortex and hippocampus induced by intracerebroventricular or intracerebral administrations of PREG-S (1-2). Central ACh neurotransmission is known to be involved in the regulation of memory processes and is affected in normal ageing and severely altered in human neurodegenerative pathologies like Alzheimer’s disease. In the central nervous system, ACh neurotransmission is also involved in the modulation of sleep-wakefulness cycle. Indeed, basal forebrain ACh neurones modulate cortical activity during sleep and lesions of these neurones suppress paradoxical sleep. PREG-S infused at the level of ACh cell bodies (nucleus basalis magnocellularis) induces a dramatic increase of paradoxical sleep in young animals (1-2). Relationships between paradoxical sleep and memory are well documented in the literature particularly in old animals in which the spatial memory performance positively correlates with the basal amounts of paradoxical sleep. Ageing related cognitive dysfunction, particularly those observed in Alzheimer’s disease, have been related to alterations of mechanisms underlying cerebral plasticity. Amongst these mechanisms, neurogenesis has been extensively studied recently. Neurogenesis takes place in the subgranular cell layer of the gyrus dentatus, and the new-

28

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

born cells differentiate into granule neurones, which add to the pre-existing granule neurones. Ageing is accompanied by a decline in neuronal progenitor proliferation in the dentate gyrus (4). This age-related decrease in neurogenesis can be reversed by pharmacological manipulations (7). As studies suggest a relationship between neurogenesis and cognitive functions (3), we have studied the modulation of cell proliferation and survival of new-born cells by neurosteroids. Our data suggest that PREG-S central infusions dramatically increase neurogenesis, this effect could be related to the negative modulator properties of this steroid at the GABAA receptor level, indeed it can be blocked by the GABA agonist muscimol. Furthermore an infusion of a neurosteroid with GABA positive action like Allopregnanolone decreases neurogenesis. Taken together these data suggest that neurosteroids can influence cognitive processes, particularly in senescent subjects, through a modulation of ACh neurotransmission associated with paradoxical sleep modifications; furthermore our recent data suggest a critical role for neurosteroids in the modulation of cerebral plasticity, mainly in hippocampal neurogenesis.

Reference list 1. Darnaudéry M., Bouyer J.J, Pallarés M., Le Moal M. and Mayo W., The promnesic neurosteroid pregnenolone sulfate increases paradoxical sleep in rats. Brain Res., 818 (1999) 492-498. 2. Darnaudéry M., Pallarés M., Bouyer J.J., Le Moal M. and Mayo W., Infusion of neurosteroids into the rat nucleus basalis affects paradoxical sleep in accordance with their memory modulatory properties. Neuroscience, 92 (1999) 583-588. 3. Gould, E., Beylin, A., Tanapat, P., Reeves, A. and Shors, T.J. Learning enhances adult neurogenesis in the hippocampal formation. Nature Neurosci., 2 (1999) 260-265. 4. Kuhn HG, Dickinson-Anson H and Gage FH. Neurogenesis in the dentate gyrus of the adult rat : agerelated decrease of neuronal progenitor proliferation. J. Neurosci., 16 (1996) 2027-2033. 5. Mayo W., Dellu, F., Robel, P., Cherkaoui, J., Le Moal, M., Baulieu, E.E. and Simon, H., Infusion of neurosteroids into the nucleus basalis magnocellularis affects cognitive processes in the rat, Brain Res., 607 (1993) 324-328. 6. Mayo W., Vallée M., Darnaudéry M. and Le Moal M., Neurosteroids : Behavioral studies. In Neurosteroids : a new regulatory function in the nervous system. E-E Baulieu, P. Robel, M. Schumacher (Eds.), The Humana Press Inc, 1999, pp 317-335. 7. Montaron MF, Petry KG, Rodriguez JJ, Marinelly M, Aurousseau C, Rougon G, Le Moal M and Abrous DN. Increase in neurogenesis but not PSA-NCAM expression in aged rats after adrenalectomy. Eur. J. Neurosci., 11 (1999) 1479-1485. 8. Pallares, M., Darnaudery, M., Day, J., Le Moal, M. and Mayo W., The neurosteroid pregnenolone sulfate infused into the nucleus basalis increases both acetylcholine release in the frontal cortex or amygdala and spatial memory, Neuroscience, 87 (1998) 551-558. 9. Vallée, M., Mayo W., Darnaudery, M., Corpechot, C., Young, J., Koehl, M., Le Moal, M., Baulieu, E.E., Robel, P. and Simon, H., Neurosteroids: deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus, Proc. Natl. Acad. Sci. U.S A., 94 (1997) 14865-14870.

29

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

THE INTERACTION OF NEUROSTEROIDS WITH RECOMBINANT AND SYNAPTIC GABAA RECEPTORS Lambert J.J., Belelli D., Callachan H., Harney S.A., Peden D. and Vardy A. Neurosciences Institute, Department of Pharmacology and Neuroscience, Univerisity of Dundee, Ninewells Hospital and Medical School, Dundee, DD1 9SY U.K. e-mail: [email protected] fax: 0044-1382-667120 Certain neurosteroids such as the progesterone metabolites 5α- or 5β-pregnan-3α-ol20-one (5α3α, 5β3α respectively) are potent allosteric modulators of the GABAA receptor [1]. Some of the pharmacological properties of the GABAA receptor are dependent upon the subunit composition of the receptor. The influence of subunit composition of the human GABAA receptor upon the enhancement of GABAA-evoked responses by 5α3α has been examined using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. The parameters quantified are i) the steroid EC50- the concentration of steroid evoking a half maximal potentiation of the peak inward current response to GABA (applied at a concentration which produces a response 10% of the GABA maximum- EC10) ii) the steroid Emax- the maximal degree enhancement of the peak inward current response to GABA (at EC10) expressed as a percentage of the response to a maximal concentration of GABA. Steroid potency (EC50) was modestly affected by the α-isoform present within the ternary complex αXβ1γ2L (x = 1-6) particularly at physiological concentrations of the neurosteroid. The β- isoform (α6βYγ2L; y = 1-3) does not influence the GABA-modulatory effect of the neurosteroid. The presence of a γ-subunit is essential for the positive allosteric effect of benzodiazepines such as flunitrazepam. By contrast, the EC50 of 5α3α is only modestly influenced by the omission of the γ2 subunit (α1β1γ2L vs α1β1) and indeed the maximal effect is favoured by the binary complex. However, the γ- isoform (α1β1γz; z = 1-3) does influence the GABAmodulatory effect of the neurosteroid with the γ1 and γ2 -containing receptors being the least and most sensitive to the action of 5α3α respectively. Incorporation of the ε subunit (α1β1ε) suppressed neurosteroid modulation, whereas the presence of the δ subunit (α4β3δ) enhanced the actions of 5α3α [2]. To determine the effects of 5β3α on GABAA-ergic inhibitory synaptic transmission the whole cell patch clamp technique was used on the rat (18-22 day old; either sex) hippocampal slice preparation. Bicuculline-sensitive miniature inhibitory postsynaptic currents (mIPSCs) were recorded from CA1 pyramidal and dentate granule cells. In CA1 pyramidal neurones physiological concentrations of 5β3α (10-30nM) prolonged mIPSC decay whereas dentate gyrus granule neurones were relatively less sensitive to the neurosteroid with 30nM 5α3α having no effect. This differential sensitivity to the neurosteroid may reflect differences in synaptic GABAA receptor subunit composition, or may be due to the phosphorylation status of the receptor or associated proteins. These possibilities will be considered.

30

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 Acknowledgements Supported by the Commission of THE European Communities, RTD Programme “Quality of Life and Management of Living Resources” QLK6-CT-2000-00179 and by the MRC. D.B. is an MRC Senior Fellow.

Reference List 1. J.J. Lambert, D. Belelli, C. Hill-Venning, , J.A. Peters. Neurosteroids and GABAA receptor function, Trends Pharmacol. Sci. 16 (1995) 295-303. 2. D.Belelli, A. Casula, A. Ling, J.J. Lambert. The influence of subunit composition on the interaction of neurosteroids with GABAA receptors. Neuropharmacology 43 (2002) 651-661.

31

SUNDAY, 23th February 12.00 - 13.00 Plenary Lecture: Kelly M.J. (Portland, OR, USA)

International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 11-14 2001

ESTROGEN MODULATION OF G PROTEIN-COUPLED ACTIVATION OF POTASSIUM CHANNELS IN THE CNS

RECEPTOR

ρ nnekleiv O.K. Kelly M.J., Qiu J., Wagner E.J. and Rρ Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239-3098, USA, [email protected], FAX: 503-494-4352 Estrogen rapidly alters the excitability of hypothalamic neurons that are involved in regulating numerous homeostatic functions including reproduction, stress responses, feeding and motivated behaviors. Some of the neurons include neurosecretory neurons such as gonadotropin-releasing hormone (GnRH) and dopamine neurons, and local circuitry neurons such as proopiomelanocortin (POMC) and gamma-aminobutyric acid (GABA) neurons. We have elucidated several rapid signaling pathways through which estrogen alters synaptic responses in these hypothalamic neurons. We have examined the modulation by estrogen of the coupling of various receptor systems to inwardly rectifying and small conductance, Ca2+-activated K + (SK) channels using intracellular sharp-electrode and whole-cell recording techniques in hypothalamic slices from ovariectomized female guinea pigs. 17beta-estradiol (E2) rapidly uncouples mu-opioid and GABAB receptors from G protein-gated inwardly rectifying K+ (GIRK) channels in POMC and dopamine neurons as manifested by a reduction in the potency of mu-opioid and GABAB receptor agonists to activate GIRK channels in these neurons. These effects are mimicked by the selective estrogen receptor modulators raloxifene and 4OH-tamoxifen, the membrane impermeable E2-BSA, but not by 17alpha-estradiol. Furthermore, ICI 182,780 antagonizes the effects of E2. In addition, protein kinase A (PKA), protein kinase C (PKC) and phospholipase C inhibitors block the actions of E2. Conversely, E2 enhances the efficacy of alpha1adrenergic receptor agonists to inhibit apamin-sensitive SK currents in GABAergic neurons, and does so in both a rapid and sustained fashion. Finally, we have observed a direct, E2-induced hyperpolarization of GnRH neurons via activation of inwardly rectifying K + channels. Therefore, these findings indicate a richly complex yet coordinated steroid modulation of K + channel activity in hypothalamic (POMC, dopamine, GABA, GnRH) neurons that are involved in regulating numerous homeostatic functions.

(Supported by PHS grants NS 38809, 35944 and DA 05158)

35

SUNDAY, 23th February 14.30 - 18.00 Symposium: Non Classical Mechanisms of Action

Symposium: Non Classical Mechanisms of action (Chairs: Lambert. J.J., Dundee & McCarthy. M., Baltimore) • Beyer C. and Küppers E. (Ulm, Germany, EU) The developing midbrain: a model to probe mechanisms of nonclassical estrogen action • Brussaard A.B. (Amsterdam, Holland, EU) Conditional regulation of neurosteroid sensitivity of GABAA receptors in oxytocin neurons during female reproductive cycle • Tasker J.G., Di S. and Malcher-Lopes R. (New Orleans, LO, USA) Non-genomic glucocorticoid actions in the hypothalamic paraventricular nucleus • Cascio C., Guarneri R., Russo D., De Leo G., Guarneri M., Piccoli F. and Guarneri P. (Palermo, Italy, EU) Neurosteroids in the retina: neurodegenerative and neuroprotective agents in retinal degeneration • Frye C.A. (Albany, NY, USA) Fluoxetine’s effects on sexual function may involve allopregnanolone in the ventral tegmental area • Marin R., Guerra B., Morales A., Díaz M. and Alonso R. (Sta Cruz de Tenerife, Spain, EU) An ICI 182,780-sensitive, membrane-related estrogen receptor contributes to estrogenic neuroprotective actions against amyloid-beta toxicity • Broekhoven F. van, Droogleever Fortuyn, H.A., Bäckström T., Span P.N., and Verkes R.J. (Nijmegen, Holland, EU) Effects of PhD examination stress on allopregnanolone and cortisol plasma levels and peripheral benzodiazepine receptor density

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

THE DEVELOPING MIDBRAIN: A MODEL TO PROBE MECHANISMS OF NONCLASSICAL ESTROGEN ACTION Beyer C. and Küppers E. Department Anatomy and Cell Biology, University of Ulm, 89069 Ulm, Germany, Fax 0049-731-5023217, E-mail [email protected] Estrogen plays an important role during mammalian brain development and is involved in the regulation of crucial steps of cell differentiation and network formation [1]. This general statement also applies to the developing nigrostriatal system which comprises the midbrain dopaminergic cell groups within the substantia nigra, ventral tegmental area, and the retrorubral field together with their corresponding GABAergic target neurons in the striatal complex [2]. In the past, we have extensively studied the role of estrogen in the developing midbrain. Both types of nuclear estrogen receptors (alpha/beta) are present in developing midbrain cells. Moreover, the estrogen-synthesizing enzyme aromatase is transiently expressed in the perinatal midbrain. This raises the questions which cell types are targets of estrogen and which physiological functions are regulated by estrogen. Interestingly, we found not only dopaminergic neurons to be estrogen-responsive but also GABAergic neurons and even astroglial cells. Using different experimental approaches and models including in utero treatment and primary embryonic cell cultures, we were able to demonstrate that estrogen is critically involved in the regulation of midbrain tyrosine hydroxylase expression, the key enzyme in dopamine synthesis [3]. This effect is classically transmitted and requires the activation of nuclear estrogen receptors, i.e. is sensitive to several pharmacological inhibitors such as ICI 182,780 and tamoxifen. In addition, we observed that estrogen also controls the expression of the neurotrophin brainderived neurotrophic factor (BDNF) [4] and glial cell line-derived neurotrophic factor (GDNF) [5] which are pivotal regulators of dopamine neuron development. In contrast to tyrosine hydroxylase, the expression of BDNF and GDNF seems to be regulated independently of nuclear estrogen receptor action. Subsequent studies have revealed that this effect depends on nonclassical estrogen signaling. In particular, we could demonstrate that estrogen governs the release of calcium from intracellular stores via phospholipase C activation and IP3 formation [6]. In a series of pharmacological studies, we were then able to demonstrate that estrogen is capable to promote a complex scenario of downstream physiological intracellular cell responses including the stimulation of the cAMP/PKA pathway in dopamine neurons [7], the MAP-kinase cascade in astrocytes [8], and the PI3kinase signaling system in neurons, presumably GABAergic cells [9]. At present, we have only rudimentary information about the cell functions which are regulated by the different signal transduction pathways in these cell types. Preliminary experimental data suggest that estrogen affects cell survival in the midbrain by the promotion of the PI3-kinase pathway in neurons and by regulating glutamate metabolism in astrocytes through the MAP-kinase signaling system. Our recent experimental work is concerned with the understanding of the initiation of rapid nonclassical estrogen mechanisms and its cell-type specific regulation. Consistent with data from the literature, we found that the estrogen receptor-alpha is, in addition to its nuclear position, also localized adjacent to the plasma membrane. From immunprecipitation studies which show several proteins assembled with

39

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

the “membrane” estrogen receptor-alpha, we assume the a membrane-associated receptor complex (with estrogen receptor-alpha in its core) is responsible for mediating rapid nonclassical estrogen signaling. In conclusion, estrogen is an important regulator of midbrain cell survival, development, and function. Estrogen appears to act by a multitude of signaling mechanisms to ensure its biological functions. This diversity involves direct interactions with target cells, i.e. through nuclear receptors and/or membrane-associated receptors. Additionally, estrogen affects developmental processes by the control of the spatio-temporal expression of growth factors in astrocytes and other cell types which, in turn, are essential for cellular differentiation. Supported by the Deutsche Forschungsgemeinschaft, the DAAD, and the European Community.

Reference List 1. C. Beyer, Estrogen and the developing mammalian brain, Anat. Embryol. 199 (1999) 379-390. 2. C. Beyer, C. Pilgrim, I. Reisert, Dopamine content and metabolism in mesencephalic and diencephalic cell cultures: sex differences and effects of sex steroids, J. Neurosci. 11 (1991) 13251333. 3. T. Ivanova, M. Karolczak, C. Beyer, Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain, J. Neurobiol. (in press). 4. T. Ivanova, E. Küppers, J. Engele, C. Beyer, Estrogen stimulates brain-derived neurotrophic factor expression in embryonic mouse midbrain neurons through a membrane-mediated and calciumdependent mechanism, J. Neurosci. Res. 66 (2001) 221-230. 5. T. Ivanova, M. Karolczak, C. Beyer, Estradiol stimulates GDNF expression in developing hypothalamic neurons, Endocrinology 143 (2002) 3175-3178 6. C. Beyer, H. Raab, Nongenomic effects of oestrogen: embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores, Eur. J. Neurosci. 10 (1998) 255-262. 7. C. Beyer, M. Karolczak, Estrogenic stimulation of neurite growth in midbrain dopaminergic neurons depends on cAMP/protein kinase A signalling, J. Neurosci. Res. 59 (2000) 197-116. 8. T. Ivanova, M. Karolczak, C. Beyer, Estrogen stimulates the mitogen-activated protein kinase pathway in midbrain astroglia, Brain Res. 889 (2001) 264-269. 9. T. Ivanova, P. Mendez, L.M. Garcia-Segura, C. Beyer, Rapid stimulation of the PI3-kinase/Akt signalling pathway in developing midbrain neurones by oestrogen, J. Neuroendocrinol. 14 (2002) 7379.

40

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

CONDITIONAL REGULATION OF NEUROSTEROID SENSITIVITY OF GABAA RECEPTORS IN OXYTOCIN NEURONS DURING FEMALE REPRODUCTIVE CYCLE Brussaard A.B. Department of Experimental Neurophysiology, Institute for Neuroscience Research & Centre for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam. E-mail: [email protected]

During pregnancy allopregnanolone, a progesterone-metabolite, by direct allosteric interaction, potentiates the GABAA receptors of rat oxytocin neurons in the supraoptic nucleus (SON). This non-genomic steroid feedback contributes to an increased inhibition of oxytocin neurons during pregnancy. After parturition, these receptors become temporarily resistant to allopregnanolone feedback, thereby leading to a robust disinhibition of oxytocin neurons required for lactation. We wanted to investigate what mechanism underlies the endogenous switching between different modes of GABAA receptor sensitivity to allosteric modulation in these neurons. We found that the neurosteroid resistance after parturition is transiently reversible and not due to a structural subunit switching of GABAA receptors, known to occur in these cells around parturition. Instead constitutive oxytocin release within the SON after parturition was found to activate autoreceptors thereby shifting the balance between endogenous phosphatase and PKC activity. The extent to which transient regulation of oxytocin release occurs and displays neuroplasticity around parturition will be addressed.

41

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NON-GENOMIC GLUCOCORTICOID ACTIONS IN THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS Tasker J.G., Di S. and Malcher-Lopes R. Division of Neurobiology, Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana, 70118, USA, email: [email protected], fax: (504) 865-6785.

The hypothalamic paraventricular nucleus (PVN) is a complex brain structure comprised of three classes of neurons, magnocellular neuroendocrine cells that secrete oxytocin and vasopressin from the posterior pituitary, parvocellular neuroendocrine cells that secrete releasing hormones into the portal system to control anterior pituitary hormone secretion, and parvocellular preautonomic neurons that control autonomic outflow via descending projections to the brainstem and spinal cord. In response to physiological and psychological stress, the hypothalamic-pituitaryadrenal (HPA) neuroendocrine axis is activated primarily by stimulation of the parvocellular corticotropin releasing hormone (CRH) neurons in the PVN and release of CRH into the portal blood. This leads to secretion of adrenocorticotropin (ACTH) from the anterior pituitary gland, which in turn causes secretion of corticosteroids from the adrenal glands into the systemic bloodstream. In addition to its role in the stress response, the HPA axis also has been implicated in affective disorders such as depression, panic disorder and anorexia nervosa, and in neurodegenerative diseases such as Alzheimer's disease. Glucocorticoids released from the adrenal cortex feed back onto the HPA axis to decrease PVN CRH and vasopressin expression and secretion. This negative feedback regulation of the HPA axis occurs acutely via a rapid inhibition of CRH release, and more tonically by a down-regulation of CRH and vasopressin synthesis in PVN parvocellular neurons. Glucocorticoid feedback appears to be mediated by actions at different central targets, either indirectly via actions on the hippocampus or directly at the levels of the hypothalamus and pituitary [1]. Glucocorticoids have been found to inhibit the activity of hippocampal CA1 neurons via genomic regulation of a Ca2+-activated K + conductance [2]. However, the acute inhibition of CRH release by glucocorticoid feedback is probably not mediated by a relay through the hippocampus since lesion of the ventral hippocampus and subiculum does not abolish the negative feedback effects of corticosteroids on HPA activity [3]. In addition to the genomic glucocorticoid actions mediated by the classical intracellular corticosteroid receptors, studies suggest that glucocorticoids may also act through G protein-coupled membrane receptors to influence transmembrane currents [4-7]. We postulated that glucocorticoids exert a direct inhibitory feedback effect on the cells responsible for activating the HPA axis during stress, the PVN CRH neurons. We studied the rapid effects of glucocorticoids on the electrical activity of rat PVN neurons using whole-cell voltage-clamp recordings in hypothalamic slices. Parvocellular and magnocellular neurons of the PVN were identified on the basis of their electrophysiological properties [8] and by post hoc single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis. Our findings provide support for glucocorticoid activation of membrane receptors that are independent of the classical type I and type II corticosteroid receptors and inhibitory actions mediated by the release of a retrograde messenger. Thus, 35 min bath application of dexamethasone (DEX, 0.1-1 µM) or corticosterone (1 µM), but

42

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

not the steroid precursor cholesterol (10 µM) or the inactive steroid isopregnanolone (10 µM), caused a dose-dependent decrease (35-45%) in the frequency of glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in both PVN parvocellular and magnocellular neurons. Additionally, magnocellular, but not parvocellular neurons responded to glucocorticoids with an increase (~40%) in the frequency of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs). The effect was not blocked when DEX was conjugated to bovine serum albumin (10 µM), whereas intracellular DEX application (1 µM) had no effect on mEPSCs, implicating a membrane glucocorticoid receptor. The intracellular type I and type II corticosteroid receptor antagonists, spironolactone (10 µM) and RU 486 (10 µM), respectively, failed to block the effect of DEX on mEPSCs, consistent with actions at a membrane receptor. Interestingly, the DEX effect on mEPSCs was blocked by intracellular application via the patch pipette of a Gprotein antagonist, GDP-beta-S (0.5-1 µM), indicating a postsynaptic signaling mechanism and suggesting the involvement of a retrograde messenger to inhibit presynaptic glutamate release. The glucocorticoid effect on mEPSCs was mimmicked by a cannabinoid receptor agonist, WIN 55,212-2 (0.5-1 µM, 36% reduction in mEPSC frequency), and was blocked by the type I cannabinoid receptor antagonists, AM251 and AM281 (1 µM, n=4), implicating an endocannabinoid as the retrograde messenger in the glucocorticoid response. Single-cell RT-PCR analysis of recorded PVN neurons indicated that several subpopulations of parvocellular and magnocellular neurons, such as CRH, thyrotropin releasing hormone, oxytocin and vasopressin neurons, responded to glucocorticoids in this manner. Thus, glucocorticoids exert a rapid inhibitory effect on PVN neurons by suppressing glutamate release and, in some cases, facilitating GABA release. The glucocorticoid actions are mediated by activation of postsynaptic membrane receptors and a G protein signaling mechanism in PVN neurons that leads to the release of a retrograde endocannabinoid messenger. These inhibitory actions of glucocorticoids and the glucocorticoidendocannabinoid interface may represent the cellular mechanism of the fast inhibitory feedback regulation of PVN neuroendocrine cells by corticosteroids. Supported by NIH grant NS/DK 39099. Reference List 1. J.P. Herman, C.M. Prewitt, W.E. Cullinan WE, Neuronal circuit regulation of the hypothalamopituitary-adrenocortical stress axis. Crit Rev Neurobiol 10 (1996) 371-394. 2. M. Joëls, E.R. de Kloet, Effects of glucocorticoids and norepinephrine on the excitability in the hippocampus. Science 245 (1989) 1502-1505. 3. J.P. Herman, W.E. Cullinan, Neurocircuitry of stress: central control of the hypothalamo-pituitaryadrenocortical axis. Trends Neurosci 20 (1997) 78-84. 4. S.Y. Hua, Y.Z. Chen, Membrane receptor-mediated electrophysiological effects of glucocorticoid on mammalian neurons. Endocrinology 124 (1989) 687-691. 5. M. Orchinik, T.F. Murray, F.L. Moore, A corticosteroid receptor in neuronal membranes. Science 252 (1991) 1848-1851. 6. M. Orchinik, T.F. Murray, P.H. Franklin, F.L. Moore, Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes. Proc. Natl. Acad. Sci. USA 89 (1992) 3830-3834. 7. J.M. ffrench-Mullen JM, Cortisol inhibition of calcium currents in guinea pig hippocampal CA1 neurons via G-protein-coupled activation of protein kinase C. J Neurosci. 15 (1995) 903-911. 8. J.L. Luther, S.S. Daftary, C. Boudaba, G.C. Gould, K. Cs. Halmos, J.G. Tasker, Neurosecretory and non-neurosecretory parvocellular neurons of the hypothalamic paraventricular nucleus express distinct electrophysiological properties. J Neuroendocrinol (2002) (in press)

43

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NEUROSTEROIDS IN THE RETINA: NEURODEGENERATIVE NEUROPROTECTIVE AGENTS IN RETINAL DEGENERATION

AND

Cascio C.1, Guarneri R.1, Russo D.1, De Leo G.2, Guarneri M.3, Piccoli F.4 and Guarneri P.1 1

Istituto di Biomedicina e Immunologia Molecolare, I.B.I.M - C.N.R, Via Ugo La Malfa 153, 90146 – Palermo, Italy, Fax: 39-091-6809548; e-mail: [email protected]; 2Dip. Biopatol. e Met. Biomed.,4Ist. Neuropsich.,3Fac. Med. Chir., Univ. Pa, Italy. A close relationship now exists between neurosteroids and/or neuroactive steroids and neurodegenerative events leading to neuronal cell death [11,7,8,3,4,12]. Decreased levels of DHEA during aging make neurons more vulnerable to be damaged [2]; DHEA and DHEAS are effective neuroprotective agents [see 12]. Sex steroid hormones, progesterone and estrogen, may influence the outcome of ischemic and traumatic injury in female and male brain, and differently promote reduction in the consequences of the injury cascade [13,15]. In peripheral nervous system, a local synthesis of progesterone shows to have a role in regenerating processes of injured peripheral nerves [12]. Conversely, increased levels of glucocorticoid or PS, as in stress or during an excitotoxic insult [1,11,7,8], is deleterious for neurons and lead t o higher vulnerability to injury and functional impairments. Our studies have been especially focused on the role served by neurosteroids in excitotoxic retinal cell death as neurodegenerative or neuroprotective agents. Excitotoxicity is regarded as an important mechanism in the pathogenesis of ocular diseases such as glaucoma, ischemia/hypoxic attack, and optic neuropathy [9], as well as in several brain neurodegenerative disorders [10]. Using the retinal excitotoxic paradigm in which a mixed pattern of apoptosis and necrosis coexist as a result of a brief exposure to NMDA or other neurotoxins, thus reproducing ischemic attacks, we have pointed out that pregnenolone sulfate, the most abundant neurosteroid in brain and retina [1,6], acts as a neurotoxic agent with agonist actions at NMDA receptors, whilst DHEA, DHEAS, PROG, and 17β-E2, which are also synthesized by the retina [6], and 3α-hydroxy-5β-pregnan-20-one sulfate (3α5βS) function as neuroprotective agents. The role of PS in excitotoxicity is worth considering especially in light of the evidence that activation of NMDA receptors induces PS synthesis before retinal death occurs, and blockade of the synthesis with aminoglutethimide, an inhibitor of cholesterol conversion into pregnenolone and pregnenolone sulfate, attenuates excitotoxicity. A brief pulse to PS as well as to NMDA triggers an apoptotic pathway characterized by a cycloheximide-sensitive program, ROS generation and lipid peroxidation, and affecting the inner nuclear layer first and then the ganglion cell layer. NMDA receptor-mediated toxicity of PS is unequivocally provided by the same effect of PS and NMDA in causing a predominant activation, in response to cytochrome c relase, of a caspase-3-dependent pathway which also requires caspase-2 activation and a late stage of cytochrome c release for the amplification of death signal in peculiar retinal cells. PS-induced retinal toxicity is effectively prevented by the neurosteroid 3α5βS which properly acts as NMDA receptor antagonist [14], as well as by 17βE2 which inhibited the caspase cascade by blocking cytochrome c release and arresting ROS and lipid peroxide production and DNA fragmentation. The neuroprotective role of 17β-E2 is intriguing and still under investigation. Actions of 17β-E2 are known to imply estrogen receptor-mediated or receptor-independent mechanisms, the latter including the ability of 17βE2 to have antioxidant properties, directly inhibit NMDA receptors, rapidly release Ca2+ from intracellular stores, block Ca2+-channel entry and stabilize mitochondrial function [5,15]. In our paradigm, the high efficacy of 17β-E2 when used at the concentration of 10µM appears t o account for a rapid action of the steroid. However, the complete neuroprotection promoted by 17β-E2 but not by caspase inhibitors blocking only the commitment to death of a peculiar set of retinal cells, might outline an additional mode of the steroid action. Indeed, our recent results appear to suggest a role of 17β-E2 in the modulation of BDNF expression. After exposure of the retina to PS, changes in phosphorylation of ERK1/2 and Akt/PKB kinases

44

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 may be prevented by the addition of BDNF, thus suggesting alterations in BDNF-mediated survival pathway. The overall findings indicate the retina as a suitable CNS structure to define the functional role of neurosteroidogenesis inside neural systems and to find out their actions as neurodegenerative and neuroprotective agents, the definition of which could contribute t o identify potential treatment strategies in that measures to control PS or 17β-E2 production could potentiate or prevent neurodegeneration during an excitotoxic insult. In this context, it is worth to consider the role played by cholesterol as a precursor of steroids/neurosteroids and important dispatcher in neurodegenerative processes in that it may interact with β-amyloid. We have observed that dysfunction in architectural structure of cytoskeleton usually occurring in neurodegenerative disorders including Alzheimer disease, affects basal and cAMP/PKA stimulated transformation of cholesterol into pregnenolone by altering cholesterol distribution, thus suggesting possible mechanisms through which steroid and/or neurosteroids may interfere with neuropathology.

Reference list 1. Baulieu EE, Robel P, Vatier O, Haug A, Le Goascogne C, Bourreau E. Neurosteroids: pregnenolone and dehydroepiandrosterone in rat brain. In Receptor-Receptor interaction: A new Intramembrane Integrative Mechanism (Fuxe K and Agnati LF, eds.), Basingstone: MacMillan (1987) pp. 89-104.. 2. Cardounel A, Regelson W, Kalimi M. Dehydroepiandrosterone protects hippocampal neurons against neutoxin-induced cell death: mechanism of action. Soc. Experimental Biol. Med., 222 (1999)145-149. 3. Cascio C, Guarneri R, Russo D, De Leo G, Guarneri M, Piccoli F, Guarneri P. Pregnenolone sulfate, a naturally-occurring excitotoxin involved in delayed retinal cell death.J. Neurochem.74 (2000) 23802391. 4. Cascio C, Guarneri R, Russo D, De Leo G, Guarneri M, Piccoli F, Guarneri P. A caspase-3-dependent pathway is predominantly activated by the excitotoxin pregnenolone sulphate and requires early and late cytochrome c release and cell-specific caspase-2 activation in the retinal cell death.J. Neurochem. (2002) in press. 5. Garcia-Segura L.M., Azcoitia I., DonCarlos L.L. Neuroprotection by estradiol. Prog. Neurobiol. 63 (2001) 29-60. 6. Guarneri P., Guarneri R., Cascio C., Pavasant P., Piccoli F., and Papadopoulos V. (1994). Neurosteroidogenesis in rat retina. J. Neurochem. 63, 86-96. 7. Guarneri P, Russo D, Cascio C, De Leo G, Piccoli F, Guarneri R. Induction of neurosteroid synthesis by NMDA receptors in isolated rat retina: a potential early event in excitotoxicity. Eur. J. Neurosci. 10(1998)17521763. 8. Guarneri P, Russo D, Cascio C, De Leo G, Piccoli T, Sciuto V, Piccoli F, Guarneri R. Pregnenolone sulfate modulates NMDA receptors, inducing and potentiating acute excitotoxicity in isolated retina. J. Neurosci. Res. 54 (1998) 787-797. 9. Lev S. Molecular aspects of retinal degenerative diseases. Cell Mol Neurobiol.2 (2001)575-89. 10. Lipton SA and Rosenberg PA. Excitatory amino acids as a final common pathway for neurological disorders. N. Engl. J. Med. 330 (1994) 613-622. 11. McEwen BS Protective and damaging effects of stress mediators. N. Engl. J. Med. 338 (1998)171179. 12. Schumacher M, Akwa Y, Guennoun R, Robert F, Labombarda F, Désarnaud F, Robel P, De Nicola A, Baulieu EE. Steroid synthesis and metabolism in the nervous system: trophic and protective effects. J. Neurocytology 29 (2000) 307-326. 13. Stein DG. Brain damage, sex hormones and recovery: a new role for progesterone and estrogen? Trends Neurosci. 24 (2001)386-391. 14. Weaver CE, Wu FS, Gibbs TT, Farb DH. Pregnenolone sulfate exacerbates NMDA-induced death of hippocampal neurons. Brain Res. 803 (1998) 129-136. 15. Wise PM, Dubal DB, Wilson ME, Rau SW, Bottner M. Minireview: neuroprotective effects of estrogen-new insights into mechanisms of action. Endocrinology 142 (2001) 963-973.

45

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 FLUOXETINE’S EFFECTS ON SEXUAL FUNCTION MAY INVOLVE ALLOPREGNANOLONE IN THE VENTRAL TEGMENTAL AREA

Frye C.A. The University at Albany-SUNY, Behavioral Neuroendocrinology Laboratory, Departments of Psychology and Biological Sciences, and The Center For Neuroscience Research, 1400 Washington Avenue Albany, NY, 12222 USA. [email protected]. 518-442-4867.

Although fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has beneficial effects for treatment of depression and anxiety disorders, it can lead to sexual dysfunction and decreased medication compliance [1]. SSRIs effects on sexual function of people, or in animal models, has received little attention. We have recently demonstrated that systemic fluoxetine produces robust decrements in sexual responses of hamsters that can be overcome by co-administration of a phosphodiesterase-5 inhibitor, zapinast [3]. Progesterone and its metabolite, the neurosteroid, 5[AC1]α-pregnan-3α-ol-20-one (3α ,5α-THP) mediate the duration of sexual receptivity in rodents through actions in the midbrain Vental Tegmental Area (VTA) [2]. Preclinical and clinical studies indicate that fluoxetine can modulate neurosteroid synthesis [4]. Fluoxetine’s effects on 3α,5α-THP levels depend upon the administration regimen used [5-6]. Together these data suggest that fluoxetine may influence sexual behavior of rodents by altering 3α,5α-THP levels. Experiments investigated the effects and mechanisms of systemic and intraVTA fluoxetine on sexual function of rats. We tested the hypothesis that fluoxetine’s effects on sexual behavior were in part due to altering 3α,5α-THP levels in the midbrain VTA. In Experiment 1, ovariectomized (ovx) rats were estradiol (EB)-primed (5 µg SC) at hours 0 and 24, administered fluoxetine (20 mg/kg; IP) or vehicle (saline; IP) at hour 44 and tested for sexual behavior with a stimulus male immediately following drug administration. Acute fluoxetine significantly decreased the incidence (lordosis quotients; LQs) and intensity (lordosis ratings; LRs) of lordosis compared to vehicle. See Table 1. In Experiment 2, ovx rats were administered fluoxetine (10 mg/kg; IP) for 15 days, EB-primed on days 16 and 17, and tested for sexual behavior on day 18. Following chronic administration of fluoxetine, EB-primed rats had significantly lower LQs and LRs than did vehicle-administered EB-primed rats. See Table 1. In Experiment 3, levels of 3α,5α-THP in the midbrain of rats administered acute fluoxetine, chronic fluoxetine, or vehicle were measured and found to be significantly lower in rats administered acute or chronic fluoxetine compared to vehicle. See Table 1. In Experiment 4, ovx rats with unilateral cannula to the VTA were primed with EB (5 µg) at hrs 0 and 24, and received SC P (0 or 100 µg) at hr 44. Rats were infused with fluoxetine (0 or 3.6 µM) at hr 47.5, and tested for sexual behavior at hr 48. Infusions of fluoxetine (3.6 µM) to the VTA significantly increased LQs and LRs compared with vehicle to the VTA. See Table 1. In Experiment 5, EB + P (0 or 100 µg)-primed rats with unilateral cannula to the VTA were infused with fluoxetine (0 or 3.6 µM) at hr 47.5 and tested for sexual behavior at hr 48 and tissues were collected for later measurement of midbrain 3α,5α-THP. Fluoxetine infusions aimed at the VTA increased lordosis and midbrain 3α,5α-THP concentrations, compared to vehicle infused rats. See Table 1. In Experiment 6, ovx rats with unilateral cannula to the VTA were EB-primed (5 µg SC) at hour 0 and 24, administered fluoxetine (20 mg/kg; IP) or vehicle (saline; IP) at hour 46

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

44, and infused to the VTA with either 3α,5α-THP (100 ng), fluoxetine (3.6 µM) or vehicle and immediately tested for sexual behavior with a stimulus male. Infusions of 3α,5α-THP or fluoxetine to the VTA significantly increased lordosis compared to vehicle administration. The hypothesis that fluoxetine’s effects on sexual behavior were due in part to altering 3α,5α-THP levels in the midbrain VTA was supported. Systemic administration of fluoxetine, either acutely or chronically, decreased the incidence and intensity of lordosis of female rats and decreased levels of 3α,5α-THP in the midbrain. Infusions of 3.6 µM of fluoxetine to the VTA significantly increased the incidence and intensity of lordosis and elevated levels of midbrain 3α,5α-THP compared to rats that received vehicle. Notably, infusions of 3α,5α-THP or fluoxetine to the VTA reinstated lordosis of rats that had decreased sexual receptivity due to acute fluoxetine administration. Thus, these data extend previous findings that sexual dysfunction can be induced by acute or chronic treatment with SSRIs to suggest that deficits in sexual function may be related to decreased levels 3α,5α-THP in the midbrain VTA. Acknowledgment: Supported by NSF (98-96263)

Reference List [1] R. Balon, Fluoxetine and sexual dysfunction, JAMA 273 (1995) 1489-1490. [2] C.A. Frye, The role of neurosteroids and non-genomic effects of progestins and androgens in mediating sexual receptivity of rodents, Brain Res. Rev. 37 (2001) 201-222. [3] C.A. Frye, M.E. Rhodes, Zaprinast, a phosphodiesterase 5 inhibitor, overcomes sexual dysfunction produced by fluoxetine, a selective serotonin reuptake inhibitor in hamsters, Neuropsychopharmacol (in press). [4] L.D. Griffin, S.H. Mellon, Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes, Proc. Natl. Acad. Sci. USA 96 (1996) 13512-13517. [5]M. Serra, M.G. Pisu, M. Muggironi, V. Parodo, G. Papi, R. Sari, L. Dazzi, F. Spiga, R.H. Purdy, G. Biggio G, Opposite effects of short- versus long-term administration of fluoxetine on the concentrations of neuroactive steroids in rat plasma and brain, Psychopharmacol. 158 (2001) 48-54. [6] D.P. Uzunova, T.B. Cooper, E. Costa, A. Guidotti, Fluoxetine-elicited changes in brain neurosteroid content measured by negative ion mass fragmentography. Proc. Natl. Acad. Sci. USA 93 (1996) 12599-12604.

Table 1: Summary of Data From Mean + Standard Error of the Mean Experiments 1-5 Lordosis Lordosis Ratings Midbrain Quotients % 3a,5a-THP (ng/g) Acute Fluoxetine 47.9 + 10.1 0.7 + 0.2 1.2 + 0.2 Acute Vehicle 84.1 + 3.3 1.8 + 0.2 3.2 + 0.6 Chronic Fluoxetine 76.6 + 12.1 1.5 + 0.8 1.8 + 0.3 Chronic Vehicle 98.3 + 1.6 2.4 + 0.1 Intra-VTA Fluoxetine EB 54.1 + 8.0 1.0 + 0.2 3.8 + 0.7 EB + P 75.8 + 6.1 1.8 + 0.2 6.8 + 1.1 Intra-VTA Vehicle EB 38.8 + 8.9 0.6 + 0.2 2.4 + 0.4 EB + P 43.7 + 7.6 0.8 + 0.2 5.7 + 0.6

47

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

AN ICI 182,780-SENSITIVE, MEMBRANE-RELATED ESTROGEN RECEPTOR CONTRIBUTES TO ESTROGENIC NEUROPROTECTIVE ACTIONS AGAINST AMYLOID-BETA TOXICITY Marin R.*, Guerra B.*, Morales A.*, Díaz M.+ and Alonso R.* *Laboratory of Cellular Neurobiology, Department of Physiology, University of La Laguna, School of Medicine, 38071 Sta. Cruz de Tenerife; Tenerife, Spain. Tel.: +34 922 319411; E-mail: [email protected]; +Laboratory of Animal Physiology, Department of Animal Biology, Faculty of Biology, 38206 Sta. Cruz de Tenerife, Tenerife, Spain. Estrogen contributes to brain preservation against a variety of toxic situations. One of the consequences of the dramatic decline of estrogen levels occurring after menopause may be an increasing risk of developing Alzheimer’s disease (AD), a degenerative pathology of the nervous system characterized by a progressive loss of memory and cognitive functions. A prominent feature of AD is the presence of extracellular neuritic plaques mainly formed by amyloid-β peptide (Aβ) that contribute to dysfunction in septal cholinergic circuits. Estradiol-mediated prevention of cell degeneration has been reported in cellular paradigms of AD neurotoxicity, describing different mechanisms of action of this hormone to palliate injury [1]. However, the importance of estrogen receptors (ERs) together with the potential mechanisms of action of these proteins in prevention of brain injury are still largely unknown. Here we present recent evidences demonstrating that estrogen may exert neuroprotection against Aβ-induced toxicity by modulation of alternative membranerelated pathways. These studies have been performed in murine septal SN56 cells that show cholinergic, peptidergic and nitrergic properties [2], respond to Aβ-related toxicity and constitutively express ERs which show transcriptional activity [3]. Using the trypan blue exclusion method, we found that cell death provoked by the 1-40 residue of Aβ (Aβ140) was significantly reduced (60%) by 15 minutes exposure to estradiol. These results were reproduced with the membrane impermeable estradiol coupled to horseradish peroxidase. Palliation of cell death was blocked in the presence of the ER antagonist ICI 182,780 as well as by MC-20 polyclonal antibody directed to the vicinity of the ligand binding domain of ER-alpha. Confocal microscopy observations on unpermeabilized SN56 cells exposed to MC-20 antibody have revealed immunostaining at the plasma membrane level. Membrane-related immunolocalization of ER has been confirmed by western blot analysis on purified SN56 cell membrane fractions. Using two conjugated forms of the steroid, E-HRP and E-BSA-FITC, we demonstrated that SN56 cells contain surface binding sites for E2, and that binding of both conjugates was blocked by pre-incubation with either E2 or ICI 182,780. MC-20 antibody also competed with E-BSA-FITC-binding. We propose that, in this model, estrogen-mediated cell protection requires the participation of a putative membrane form of ER that may share some structural homologies with ERα.

48

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 This work was supported by grant SAF200-3614-C03-01/02. Partial financial support was from Lilly S.A. and Astra-Zeneca.

Reference List [1] L.M. Garcia-Segura, I. Azcoitia, L.L. DonCarlos, Neuroprotection by estradiol, Prog. Neurobiol. 63 (2001) 29-60. [2] J.R. Martinez-Morales, I. Lopez-Coviella, J.G. Hernández-Jimenez, A.R. Bello, G. Hernández, J.K. Blusztajn, R. Alonso, Sex steroids modulate LHRH secretion in a cholinergic cell line form the basal forebrain, Neuroscience 103 (2001) 1027-1033. [3] R. Marin, B. Guerra, R. Alonso, The amount of estrogen receptor a increases after heat shock in a cholinergic cell line from the basal forebrain, Neuroscience 107 (2001) 447-454.

49

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

EFFECTS OF PhD EXAMINATION STRESS ON ALLOPREGNANOLONE AND CORTISOL PLASMA LEVELS AND PERIPHERAL BENZODIAZEPINE RECEPTOR DENSITY Broekhoven F. van1, Droogleever Fortuyn H.A.1, Bäckström T.3, Span P.N.2 and Verkes R.J.1 1

University Medical Centre Nijmegen, Departments of 1Psychiatry and 2Chemical Endocrinology 3 Umeå University Hospital, Department of Obstetrics and Gynecology, Umeå, Sweden University Medical Centre Nijmegen, Department of Psychiatry, Unit for Clinical Psychopharmacology and Neuropsychiatry, Internal postal code 331, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands; email: [email protected]; Fax: +31 24 3540561 The peripheral benzodiazepine receptor (PBR) is located at the outer mitochondrial membrane, and plays a role in the translocation of cholesterol from the outer to the inner mitochondrial membrane, which is the rate limiting step for the synthesis of (neuro)steroids. Allopregnanolone is a metabolite of progesterone and a potent positive allosteric modulator of the gamma-aminobutyric acid receptor type A (GABAA receptor). Both PBR and allopregnanolone increase following acute stress. Until now, the relation between PBR density and allopregnanolone plasma levels following acute stress in humans has not been investigated. In the present study, we investigated the possible increase in allopregnanolone concentration following acute stress in humans. Furthermore, we examined our hypothesis that in acute stress, the increase in plasma allopregnanolone concentration is correlated with the increase in PBR density. PhD examination was considered to be a stressful event and was therefore used as a model of acute stress. PBR density (Bmax) was measured by use of [3H]PK 11195 binding to platelets according to a slight modification of the method of Gavish et al. [1]. Bmax and plasma allopregnanolone levels were assessed in 15 PhD students four weeks before the PhD examination (T1), 45 minutes before the examination (T2), during the examination (T3), and four weeks after the graduation (T4). Stress was measured by changes in plasma cortisol, blood pressure (BP), heart rate (HR), and in the scores on the Zung Self-Rating Anxiety Scale and in the state scores on the Spielberger State-Trait Anxiety Inventory (STAI). Differences in means at the different time points and intraindividual correlations were statistically tested using mixed model analyses of variance (ANOVA) with subjects as random factor and time points as fixed factor. Post-hoc comparisons between time points were tested with paired t-tests. Bmax, allopregnanolone, cortisol, systolic BP, diastolic BP, and HR reached the highest levels during the examination (T3). Results of Zung and STAI, which were not scored at T3, were highest immediately before PhD examination (T2). Bmax was significantly correlated with allopregnanolone plasma concentrations (B=3.3x10-5; 95% C.I. [5.7x10-6, 6.0x10-5] p=0.02). This is the first study to show that, in acute stress, the increase in plasma allopregnanolone concentration was correlated with an increase in PBR density in blood

50

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

platelets. The fact that during the PhD examination, plasma cortisol concentration, BP, and HR reached their highest scores, supported our assumption that PhD examination was a valid model for acute stress. This study replicated previous findings that PBR density in platelets significantly increases in examination evoked acute stress [2].

Reference List 1. M. Gavish, A. Weizman, L. Karp, S. Tyano, Z. Tanne, Decreased peripheral benzodiazepine binding sites in platelets of neuroleptic-treated schizophrenics, Eur.J.Pharmacol. 121 (1986) 275-279. 2. L. Karp, A. Weizman, S. Tyano, M. Gavish, Examination stress, platelet peripheral benzodiazepine binding sites, and plasma hormone levels, Life Sci. 44 (1989) 1077-1082.

51

TUESDAY, 24th February 08.30 - 11.00 Symposium: Glucocorticoids and Mineralcorticoids: Synthesis, Mechanism of Action and Effects

Symposium: Glucocorticoids and Mineralcorticoids: Synthesis, Mechanism of Action and Effects (Chairs: Piva F. & Riva M.) • Holmes M.C., Yau J.L., Kotelevtsev Y., Mullins J.J. and Seckl J.R. (Edinburgh, UK, EU) 11ß-hydroxysteroid dehydrogenases in the brain: two enzymes, two roles

• Moore F.L. (Corvallis, OR, USA) Structure and function of a membrane corticosteroid receptor

• Gass P. (Heidelberg, Germany, EU) Corticosteroid receptor transgenic mice: are they depressed or not?

• Holsboer F. (Munich, Germany, EU) The corticosteroid receptor hypothesis of depression

• Joëls M., Verkuyl M. and van Riel E. (Amsterdam, Holland, EU) Hippocampal function after chronic stress

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

11ß-HYDROXYSTEROID DEHYDROGENASES IN THE BRAIN: TWO ENZYMES, TWO ROLES Holmes M.C., Yau J.L., Kotelevtsev Y., Mullins J.J. and Seckl J.R. Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. E-mail: [email protected]; Fax: 44-131-651-1085 Glucocorticoids have a plethora of effects, influencing many systems of the body including the brain. Most neural pathways are modified by glucocorticoids, as target genes include neurotransmitter synthesis enzymes, receptors as well as enzymes involved in calcium activation and ion channels (eg K+ channels). High levels of glucocorticoids are deleterious to the homeostasis of the body, causing abnormalities in development through to potentiation of cognitive deficiencies seen in aging. Normally glucocorticoid levels are strictly controlled by a negative feedback action of glucocorticoids on the hypothalamopituitary adrenal (HPA) axis; impairments in this regulation modify lifetime levels of glucocorticoids generating maladaptive effects in the brain. Central glucocorticoid effects are not only dependent on plasma glucocorticoid levels, but also levels of CBG and corticosteroid receptors, GR (glucocorticoid receptor) and M R (mineralocorticoid receptor). Finally, it has been shown that pre-receptor metabolism by the glucocorticoid metabolising enzymes, 11ß-hydroxysteroid dehydrogenases (11ßHSDs), also play a crucial role in modifying glucocorticoid action in a tissue-dependent manner giving a further layer of complexity and specificity. There are 2 isozymes, 11ßHSD-1 and –2, which in most cases, carry out the reverse reaction. In our laboratory we have produced mice with a targeted disruption in the 11ß-HSD1 as well as 11ß-HSD2 genes. These mice have been used to elucidate the role of each enzyme in glucocorticoid modulation of the brain at a molecular, cellular and behavioural level. 11ß-HSD-1, although a reversible enzyme in vitro, generally acts as a reductase, regenerating active glucocorticoids from their inactive 11-keto derivatives in vivo. It is highly expressed in most regions of the brain, producing higher cellular levels of active glucocorticoid than levels suggested by plasma concentrations. It is also important to note that 11ß-HSD1 is also highly expressed in many peripheral organs, the liver being particularly key as a potential secondary (extra- adrenal) source of generation of active glucocorticoids that may reach the general circulation. Mice lacking 11ß-HSD1 (11ß-HSD1 KO) activity show modified HPA regulation. Under basal conditions, nadir plasma ACTH and corticosterone levels are significantly elevated in 11ß-HSD1 KO mice resulting in hypertrophied adrenals. In contrast, tissue levels of corticosterone in brain extracts were reduced. 11ß-HSD1 KO mice also showed an exaggerated corticosterone response to restraint stress compared to WT mice and an impairment in the negative feedback regulation of the HPA axis. Hence, loss of 11ß-HSD1 causes a subtle change in HPA activity, most likely due to a reduction in the central cellular glucocorticoid signalling. The consequences of decreased cellular corticosterone levels could potentially have profound effects over the lifetime of the animal, which may be particularly crucial in the aging process. Chronically elevated glucocorticoid levels through life have been shown to increase

55

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

risk of cognitive impairment with aging. In keeping with this theory, we have found 11ßHSD1 KO mice are less impaired than WT mice in a spatial memory task. 11ß-HSD2 acts exclusively as a dehydrogenase, inactivating glucocorticoids prior to corticosteroid receptor activation. It is very highly expressed in the kidney and vasculature where it protects MR from illicit activation by glucocorticoids. There is very little expression of 11ß-HSD2 in adult rat brain, being restricted to small nuclei involved in central regulation of blood pressure (NTS) and salt appetite (SCO, amygdala). The mouse exhibits an even more discrete expression than the rat. As in the kidney, the role of 11ßHSD2 activity in these regions is to allow aldosterone activation of MR by inactivating the promiscuous glucocorticoids. However, it is in perinatal development that 11ß-HSD2 activity has a profound effect in the brain. Glucocorticoids are known to inhibit neuronal proliferation, migration, differentiation and cell migration. Furthermore, rats exposed to elevated glucocorticoids from stress or exogenous administration appear to be ‘programmed’ through life to have altered HPA activity and regulation and demonstrate a more anxious phenotype. The fetus is normally protected from glucocorticoid overexposure by 11ß-HSD2 expression in the placenta, and as a second protective barrier, 11ß-HSD2 is also expressed in the brain of the fetus and neonate. When the neurones are mature, 11ß-HSD2 expression is switched off leaving only the discrete pattern described above. Interestingly, we hypothesize the role of 11ß-HSD2 in development is to protect neurones from effects mediated by GR, rather than MR. To assess the developmental role of 11ß-HSD2 we have looked in 11ß-HSD2 KO mice. These mice have impaired neonatal growth, with the cerebellar growth being particularly affected. As adults they exhibit an abnormal behavioural phenotype consistent with glucocorticoid programming. In conclusion, both 11ß-HSD1 and 2 act to modify glucocorticoid action in the brain at different stages in life.

56

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

STRUCTURE AND RECEPTOR

FUNCTION

OF

A MEMBRANE CORTICOSTEROID

Moore F.L. Department of Zoology, Oregon State University, Corvallis, OR 97331 USA [email protected], FAX: 541 737-0501

Email:

The neuroendocrine mechanisms that control rapid behavioral responses to acute stress are highly conserved among vertebrates. Behavioral studies in an amphibian model, the roughskin newt (Taricha granulosa), demonstrate that acute stress triggers the secretion of corticosterone and that elevated corticosterone concentrations cause rapid changes in reproductive behaviors and neuronal activity. These responses to corticosterone occur within minutes and are mediated by non-genomic mechanisms [5] that involve a membrane-associated corticosteroid receptor (mCR) in the G-protein coupled receptor superfamily [6, 7]. Partial purification of the solubilized mCR protein using sequential chromatography schemes revealed a putative receptor protein with an apparent mass of 63 kDa [2, 3]. This finding was confirmed using two independent systems--a differentialdisplay CORT-Sepharose affinity chromatography that visualized the protein on 2-D SDS-PAGE and a photoaffinity-labeling system that visualized the protein with western blot methodology and anti-CORT antiserum [1]. Pharmacological characterization of mCR found that not only is the [3H]-CORT binding site highly selective for corticosterone and cortisol, but it also recognizes a subset of kappa opioid receptor-selective ligands. These opioid ligands interact directly (not allosterically) by competing for the same binding pocket [4]. These binding data are consistent with behavioral and physiological studies showing that corticosterone and specific kappa opioid ligands trigger similar responses. The research project is currently at the stage where an opioid-like receptor with promising characteristics has been cloned from brain cDNA and is being evaluated using transient expression strategy.

Reference List [1] Evans, S.J. and Moore, F.L., Nonradioactive photoaffinity labeling of steroid receptors using western blot detection system, Methods Mol Biol, 176 (2001) 261-72. [2] Evans, S.J., Moore, F.L. and Murray, T.F., Solubilization and pharmacological characterization of a glucocorticoid membrane receptor from an amphibian brain, J Steroid Biochem Mol Biol, 67 (1998) 1-8. [3] Evans, S.J., Murray, T.F. and Moore, F.L., Partial purification and biochemical characterization of a membrane glucocorticoid receptor from an amphibian brain, J Steroid Biochem Mol Biol, 72 (2000) 209-21. [4] Evans, S.J., Searcy, B.T. and Moore, F.L., A subset of kappa opioid ligands bind to the membrane glucocorticoid receptor in an amphibian brain, Endocrinology, 141 (2000) 2294-300. [5] Moore, F.L. and Evans, S.J., Steroid hormones use non-genomic mechanisms to control brain functions and behaviors: A review of evidence, Brain Behav Evol 54 (1999) 41-50. [6] Orchinik, M., Murray, T.F., Franklin, P.H. and Moore, F.L., Guanyl nucleotides modulate binding to steroid receptors in neuronal membranes., Proc Natl Acad Sci USA, 89 (1992) 3830-3834. [7] Orchinik, M., Murray, T.F. and Moore, F.L., A corticosteroid receptor in neuronal membranes, Science, 252 (1991) 1848-1851.

57

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

CORTICOSTEROID RECEPTOR TRANSGENIC MICE: ARE THEY DEPRESSED OR NOT? Gass P. Central Institute of Mental Health Mannheim, University of Heidelberg, Germany [email protected] Impaired corticosteroid receptor signaling is a key mechanism in the pathogenesis of stress-related psychiatric disorders such as depression and anxiety. Since in vivo expression and functional studies of corticosteroid receptors are not feasible in the human central nervous system, such analyses have to be done in animal models. Transgenic mice with mutations of corticosteroid receptors are promising tools which allow to investigate the role of these proteins in the pathogenesis of symptoms characteristic for depression and anxiety. This talk summarizes the neuroendocrinological and behavioral findings that have been obtained in six different mouse strains with specific mutations that influence the expression or the function of the glucocorticoid or the mineralocorticoid receptor. The analyses of these mice helped to define molecular concepts of how corticosteroid receptors regulate the activity of the hypothalamic-pituitary-adrenal (HPA) system. Furthermore, some of the mutant mice with altered glucocorticoid receptor expression exhibited characteristic changes in behavioral tests for anxiety and despair. Using high throughput technologies for the identification of genes regulated by GR and MR in brain areas responsible for these stress-related symptoms will yield potential new drug-targets. However, so far none of the mouse strains investigated could be viewed as an animal model of a specific psychiatric disease defined by common diagnostic criteria. The lack of a full depressive syndrome in these mice may result from compensatory developmental mechanisms, because the targeted manipulations in corticosteroid receptor expression are already effective during development. Therefore, the next generation of corticosteroid receptor transgenic animals must be based on conditional and/or reversible mutagenesis.

58

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

THE CORTICOSTEROID RECEPTOR HYPOTHESIS OF DEPRESSION Holsboer F. Max Planck Institute of Psychiatry, Kraepelinstrasse 10, 80804 Munich, Germany, [email protected] Many patients with severe depression show peripheral signs of increased hypothalamicpituitary adrenocortical (HPA) activity. After it was discovered that the central driving force causing HPA-hyperdrive is hypersecretion of Corticotropin-Releasing Hormone (CRH) and Vasopressin (AVP) many studies were conducted that converged in the view that disturbance of the central regulation of these neuropeptides is a causally relevant mechanism among all stress-related clinical conditions, even if the peripheral signs i.e. of stress, i.e. hypercortisolism is not present. Some more recent discoveries substantiate this view: (1) It was found that CRH is elevated in various brain areas of depressives (including the prefrontal cortex) and many studies agreed that CRH conveys behavioral responses reminiscent of depression (increased anxiety, psychomotor retardation, sleep disturbance, cognitive changes, loss of sexual activity, decreased food intake etc.). The question which of the CRH receptors mediates these effects could be clarified using mouse mutants, where the CRH-type1 receptor (CRHR1) was deleted in the limbic system (sparing the hypothalamus) and the prefrontal cortex. These mice had reduced anxiety-like behavior. Also rats with increased innate anxiety responded to CRHR1 knock-down and CRHR1 antagonists with decreased anxiety-like behavior. Finally, a clinical study using a CRHR1 antagonist provided preliminary results, supporting the view that these drugs may be beneficial to treat depression. (2) A rat model which as a consequence of selective breeding over 50 generations showed high innate anxiety, has increased AVP in the hypothalamus. When treated with an antidepressant (paroxetin) the AVP-levels return to baseline. These rats show the same neuroendocrine test results as depressives: When subjected to the dexamethasone/CRH-test patients as well as these rats show elevated ACTH and cortisol (corticosterone in rats) secretion. After successful antidepressant treatment the neuroendocrine test results become indistinguishable between patients and controls, as well as between high anxiety rats and low anxiety rats. These findings point to vasopressin as a major causative factor in depression and several pharmaceutical industries now explore the potential of AVP receptor antagonists for that indication. (3) Corticosteroid receptor signaling itself is also a possible area of interest to identify structures which either predispose to depression and/or which are targets for interventions. Up to now preliminary evidence exists that a glucocorticoid receptor antagonist (mifeprestone) which also blocks progesterone receptors is beneficial in psychotic depression. Further, all antidepressants tested so far, increase glucocorticoid receptor

59

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

function. Yet it is unclear by which mechanism they achieve this. From molecular biology cues are derived pointing to a role of chaperones, i.e. proteins that are involved in corticosteroid receptor folding, determining the affinity of steroid binding and the function of ligand-activated receptors on regulatory DNA sequences. Taken together, the corticosteroid receptor hypothesis pulls together various set points of stress-hormone regulation and has already resulted in a number of innovative drug discovery strategies.

60

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

HIPPOCAMPAL FUNCTION AFTER CHRONIC STRESS Joëls M., Verkuyl M. and van Riel E. Swammerdam Institute for Life Sciences, section Neurobiology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands; [email protected]; fax: 00-3120-5257709 Hyperactivity of the hypothalamo-pituitary-adrenal (HPA) axis is often observed in patients with major depression. Clinical studies support the notion that HPA hyperactivity is not merely associated with but even causal to the onset of clinical symptoms. Two important questions in this respect are: 1) Which molecular and cellular processes in the brain can contribute to or exacerbate HPA-axis hyperactivity? 2) How can hyperactivity of the HPA-axis lead to attenuation of the central serotonergic system, supposedly a key factor in the onset of clinical symptoms in depression? We approached these questions in an animal model for chronic stress, which is associated with HPA-axis hyperactivity. More specifically, to address the first question in this model, we focused on electrical properties and inputs to parvocellular neurons in the paraventricular nucleus (PVN) of the hypothalamus, i.e. cells that play a pivotal role in the regulation of HPA-axis activity. The second question was approached by studying the serotonin responsiveness of pyramidal neurons in the hippocampus, an area critically involved in mood disorders. Male Wistar rats (around 150 grams at the start of the experiment) were exposed to unpredictable stressors, twice daily for 21 days. These stressors included: immobilization (at room temperature or 4°), swim stress (at room temperature or 4°), shaking, isolation and crowding. Control rats were handled daily. One day after the last stressor rats were decapitated in the morning, under rest. Plasma corticosterone levels at that time were low and slightly though not significantly elevated in chronically stressed versus control rats. Gain in body weight and thymus weight were attenuated, while adrenal weight was increased in stressed compared to control rats. These data indicate that the stressed rats had indeed experienced a prolonged period of HPA-axis hyperactivity, prior to the experiment. Brain slices containing the PVN or hippocampus were prepared and used for in vitro electrophysiological experiments. A limited number of slices was frozen or fixed, for later in situ hybridization or immunocytochemistry. Paraventricular neurons in the PVN were recorded in the patch clamp whole cell configuration. After recording, the cellular content including RNA was aspirated; RNA was linearly amplified and hybridized with clones of interest on a slot blot. Pyramidal neurons in the CA1 hippocampal area were recorded with microelectrodes, allowing investigation of their response to exogenously applied serotonin (5-hydroxytryptamine; 5-HT). Parvocellular neurons in the PVN were selected under visual control, based on their location and fusiform shape of the cellbody. From each neuron we monitored the input resistance, membrane capacitance and properties of the evoked inhibitory postsynaptic potential (IPSC) and miniature (m) IPSC, an index for the spontaneous GABAergic inhibitory tone. The GABAergic input reflects the transsynaptic inhibition exerted by limbic areas as well as local inhibition by hypothalamic interneurons projecting to the PVN parvocellular neurons. It appeared that chronic stress did not change the input resistance or

61

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

membrane capacitance of thus identified parvocellular neurons. However, marked differences were observed with respect to the mIPSC properties. In chronically stressed rats, mIPSC frequency was significantly reduced compared to the control group. Neither the mIPSC amplitude nor the kinetic properties were significantly affected by chronic stress. This suggests pre- rather than postsynaptic changes in GABAergic input after chronic stress. Paired pulse responsiveness of evoked IPSCs was not changed after chronic stress, indicating that the release probability in the synapses involved was unaltered. Together these data point to a reduction in GABAergic inhibitory synapses onto parvocellular PVN cells after chronic stress. In agreement with this reduced GABAergic projection, the amplitude of the evoked IPSC was significantly reduced after chronic stress. All in all, the present data support that chronic stress leads to synaptic reorganization in the PVN, reducing the number of GABAergic inhibitory synaptic contacts. This process may contribute to and/or exacerbate the dysinhibited HPA-axis seen after periods of chronic stress. What are the consequences of HPA-axis hyperactivity for 5-HT responsiveness in the hippocampus? Brief perfusion of hippocampal slices with 10 mM 5-HT results in an initial hyperpolarization of the membrane accompanied by a decreased resistance. This response is mediated by 5-HT1A receptors. It was found that the 5-HT evoked hyperpolarization is significantly decreased in CA1 cells from animals earlier exposed to chronic unpredictable stress compared to the controls. This effect reflected a difference between the two groups under conditions that corticosteroid levels are low. The reduced responsiveness to 5-HT was not accompanied by changes in 5-HT receptor mRNA expression, as shown by in situ hybridization in tissue from the same animals. Also, basal cell properties (resting membrane potential and input resistance) were comparable for the stressed and control groups. Earlier it was shown in non-stressed animals that rises in corticosteroid level are associated with an enhanced response to 5-HT. A comparable (percentual) enhancement was also observed in CA1 cells from chronically stressed rats, although the resulting response was still smaller than seen in slices from control rats treated with a high concentration of corticosterone. These data indicate that there is no resistance to glucocorticoid-receptor activation, even after a prolonged period of unpredictable stress. In conclusion, chronic hyperactivity of the HPA-axis results in an overall suppression of the 5-HT responsiveness in the CA1 hippocampal area. It is presently unresolved whether this is caused by the changes in corticosteroid or central corticotropin releasing hormone levels associated with chronic stress. Interestingly, comparable reduction of 5-HT responses was earlier observed in animals that received high doses of exogenously administered corticosterone, which on the one hand results in high corticosteroid levels but on the other hand largely suppresses the endogenous HPA-axis activity. Since the main commonality in these two studies is the strong elevation in corticosterone level, it is tempting to conclude that prolonged over-exposure to corticosteroids rather than other factors causes attenuation of central serotonergic responses.

62

TUESDAY, 25th February 2003 08.30 - 12.00 Symposium: Pathological Correlations and New Tools in Therapeutical Approaches

Symposium: Pathological Correlations and New Tools in Therapeutical Approaches (Chairs: Garcia-Segura L.M., Madrid & Mensah G., Strasbourg Cedex ) •

Hurd Y.L., Östlund H. and Keller E. (Stockholm, Sweden, EU) Estrogen receptor gene expression in relation to neuropsychiatric disorders

•

Stoffel-Wagner B. (Bonn, Germany, EU) Neurosteroid metabolism in the human brain and its clinical implications

•

Simpkins J.W., Yang S.-H. and Liu R. (Fort Worth, TX, USA) The use of estrogens and related compounds in the treatment of damage from cerebral ischemia

•

Murphy D.G.M. and Cutter W. (London, UK, EU) The effect of oestrogen on brain ageing

•

Bäckström T. (Umea, Sweden, EU) Pathogenesis in menstrual cycle linked CNSdisorders

•

Merchenthale I. (Collegeville, PA, USA) Neuroprotection by estrogen in global and focal ischemia

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ESTROGEN RECEPTOR GENE NEUROPSYCHIATRIC DISORDERS

EXPRESSION

IN

RELATION

TO

Hurd Y.L.1, Östlund H1. and Keller E.2 Karolinska institute, 1department of clinical neuroscience, psychiatry section, stockholm, sweden. 2department of forensic medicine, semmelweis university, hungary. Email: [email protected]. Fax:468-346563 There is now compelling evidence for an involvement of estrogen in the regulation of mood and cognitive functions. Serum estrogen levels have been shown to play an important role in the expression of psychiatric and neurological disorders such as depression, schizophrenia, and Alzheimer's disease. For example, low levels of serum estrogens are often associated with affective disorders such as premenstrual syndrome, postnatal depression, and post-menopausal depression [1,4]. In schizophrenia, estrogen is also hypothesized to have a protective effect and to account for the gender differences in onset and symptomotology that have been reported for the disease (see [2,7]). We have characterized the distribution of the two known estrogen receptor (ER) subtypes (alpha and beta) in the human brain and showed a preferential limbic-related expression pattern for these transcripts [5,6,8]. The ER-alpha mRNA dominates in the amygdala and hypothalamus, suggesting a main role of the ER-alpha in estrogen modulation of autonomic and neuroendocrine functions as well as emotional interpretation and processing. In contrast, the hippocampal formation, entorhinal cortex, and thalamus appear to be ER-beta dominant areas, suggesting a putative role for ER-beta in cognition, nonemotional memory, and motor functions. In addition to differential anatomical localization, estradiol differentially modulates the ER-alpha and ER-beta mRNA expression in limbic brain regions. Estradiol has also been shown to regulate different components of the serotonin (5-HT) system that has been strongly implicated in the pathophysiology of affective disorders. We have previously examined the possible link between estrogen and 5HT in depression by using an experimental genetic animal model of depression, the Flinder sensitive line (FSL) rats [9]. FSL animals showed altered 5-HT receptor (5-HT1A and 5HT2A) mRNA levels in discrete brain regions; many of the abnormalities are reversed by estradiol treatment [9]. Another biogenic amine that has been widely implicated in depression disorders is norepinephrine (NE). The NE system is, similar to the serotoninergic neuronal pathways, a target for antidepressant drugs and has an abundant forebrain innervation to various mesocorticolimbic structures. We have recently observed that NE neurons in the human locus coeruleus express moderate levels of both ER transcripts. The possibility for estrogen to regulate locus coeruleus function has been documented. For example, it has been observed that estrogen differentially regulates the expression of the tyrosine hydroxylase gene in the locus coeruleus of males and females mice [3]. Results from our preliminary experiments have revealed that the ER-beta mRNA is decreased in suicide subjects, a cause of death that is highly linked to affective disorder. Follow-up studies are currently underway with a much larger population of suicide victims (n = 31) and control subjects (n = 19) to validate these brainstem changes in the ER gene in relation to affective disorder. In addition to the estrogen receptor interactions with the biogenic amine system, our studies

65

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

have shown that there is a strong overlap of the ER-alpha mRNA in the human amygdala with neurons expressing the opioid neuropeptide prodynorphin. Dynorphins are linked to negative mood states which provides an interesting potential for estrogen to regulate mood via interactions with these amygdala dynorphin cells. Overall, the discrete anatomical organization of the ER mRNAs in the human brain provide evidence as to the specific neuronal populations in which the nuclear receptor actions of ERs could modulate mood and thus underlie the neuropathology of psychiatric disorders such as depression.

References List [1] Fink, G., Sumner, B.E.H., Rosie, R., Grace, O. and Quinn, J.P., Estrogen control of central neurotransmission: Effect on mood, mental state and memory, Cell. Molec. Neurobiol., 16 (1996) 325-344. [2] Stevens, J.R., Schizophrenia: reproductive hormones and the brain, Am J Psychiatry, 159 (2002) 7139. [3] Thanky, N.R., Son, J.H. and Herbison, A.E., Sex differences in the regulation of tyrosine hydroxylase gene transcription by estrogen in the locus coeruleus of TH9-LacZ transgenic mice, Brain Res Mol Brain Res, 104 (2002) 220-6. [4] Vliet, E.L. and Hutcheson Davis, V.L., New perspectives on the relationship of hormone changes to affective disorders in perimenopause, NAACOG´s clinical issues, 2 (1991) 453-471. [5] Österlund, M., Kuiper, G.G.J.M., Gustafsson, J.-Å. and Hurd, Y.L., Differential distribution and regulation of estrogen receptor-a and -b mRNA within the female rat brain, Mol. Brain Res., 54 (1998) 175-180. [6] Österlund, M.K., Gustafsson, J.Å., Keller, E. and Hurd, Y.L., Estrogen receptor b mRNA expression within the human forebrain: distinct expression pattern to the estrogen receptor a mRNA, J. Clinical Endocrinology & Metabolism, 85 (2000) 3840-3846. [7] Österlund, M.K. and Hurd, Y.L., Estrogen receptors in the human forebrain and the relation to neuropsychiatric disorders, Prog. Neurobiol., 64 (2001) 251-267. [8] Österlund, M.K., Keller, E. and Hurd, Y.L., The human amygdaloid complex is characterized by high expression of the estrogen receptor a mRNA, Neuroscience, 95 (2000) 333-342. [9] Österlund, M.K., Overstreet, D.H. and Hurd, Y.L., The Flinder’s sensitive rat line, a genetic model of depression, show abnormal serotonin receptor mRNA expression in the brain that is reversed by 17-b estradiol, Molec. Brain Res., 74 (1999) 158-166.

66

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

NEUROSTEROID METABOLISM IN THE HUMAN BRAIN AND ITS CLINICAL IMPLICATIONS Stoffel-Wagner B. Department of Clinical Biochemistry, University of Bonn, Sigmund-Freud-Str. 25, D53127 Bonn, Germany, [email protected], Fax: +49-228-2875789 This paper summarizes the current knowledge of the biosynthesis of neurosteroids in the human brain, the enzymes mediating these reactions, their localization and the putative effects of neurosteroids. Molecular biological and biochemical studies have now firmly established the presence of the steroidogenic enzymes cytochrome P450SCC, aromatase, 5αreductase, 3α-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase in human brain [1-10]. Their presence in the cerebral cortex and in the subcortical white matter indicates that various cell types, either neurons or glial cells, are involved in the biosynthesis of neurosteroids and neuroactive steroids in the brain. However, the (patho)physiological significance of these findings remains to be elucidated. Steroid hormone effects on the brain have typically been associated with gene regulation via intracellular steroid receptors. In contrast to reproductive and neuroendocrine actions of steroids via these intracellular receptors, which regulate transcriptionally directed changes in protein synthesis, modulatory actions on the GABA receptor system can rapidly alter the excitability of neurons. The functions attributed to specific neurosteroids include modulation of GABAA, N-methyl-D-aspartate (NMDA), nicotinic, muscarinic, serotonin (5-HT3), kainate, glycine and sigma receptors, neuroprotection and induction of neurite outgrowth, dendritic spines and synaptogenesis. The potential anaesthetic properties of neurosteroids have already been suggested in 1941. The observation that epileptic seizures in cycling women are less frequent in the luteal phase, when circulating levels of progesterone are high, appears to be associated with cyclical variations in the metabolism of progesterone to allopregnanolone in the brain. Progesterone and 3α-reduced neuroactive steroids have potent anticonvulsant effects. Synthetic derivates of neuroactive steroids are under investigation for treatment of epilepsy disorders. Neuroactive steroids may also be involved in physiological conditions where fluctuations of the hormonal balance occur. For example, increased fatigue during pregnancy may be a consequence of higher concentrations of progesterone and GABA agonistic 3αreduced neuroactive steroids like 3α,5α-THP, whereas a rapid decline in these substances may lead to the premenstrual syndrome or post partum depression. Moreover, fluctuations in neuroactive steroid concentrations may in part contribute to the increased risk of developing psychiatric diseases in women at the perimenstrual phase, during pregnancy and the post partum period and around menopause. In conclusion, molecular biological and biochemical studies have now firmly established that several key enzymes of steroidogenesis, namely cytochrome P450SCC, aromatase, 5α-reductase, 3α-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase, are present in human brain. We still do not know whether and how the steroidogenic enzymes are involved in the pathophysiology of the nervous system. First clinical investigations in humans produced evidence for an involvement of neuroactive 67

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

steroids in conditions such as fatigue during pregnancy, premenstrual syndrome, post partum depression, catamenial epilepsy and depressive disorders. Better knowledge of the biochemical pathways of neurosteroidogenesis and their actions on the brain seems to open new perspectives in the understanding of the physiology of the human brain as well as in the pharmacological treatment of its disturbances.

Reference List [1] S. Beyenburg, B. Stoffel-Wagner, M. Watzka, I. Blümcke, J. Bauer, J. Schramm, F. Bidlingmaier, C.E. Elger, Expression Of Cytochrome P450scc Mrna In The Hippocampus Of Patients With Temporal Lobe Epilepsy, Neuroreport. 10 (1999) 3067-3070. [2] S. Steckelbroeck, D. Heidrich, B. Stoffel-Wagner, V.H.J. Hans, J. Schramm, F. Bidlingmaier, D. Klingmüller, Characterization Of Aromatase Cytochrome P450 Activity In The Human Temporal Lobe, J Clin Endocrinol Metab. 84 (1999) 2795-2801. [3] S. Steckelbroeck, B. Stoffel-Wagner, R. Reichelt, J. Schramm, F. Bidlingmaier, L. Siekmann, D. Klingmüller, Characterization Of 17b-Hydroxysteroid Dehydrogenase Activity In Brain Tissue: Testosterone Formation In The Human Temporal Lobe, J Neuroendocrinol. 11 (1999) 457-464. [4] S. Steckelbroeck, M. Watzka, R. Reichelt, V.H.J. Hans, B. Stoffel-Wagner, D.D. Heidrich, J. Schramm, F. Bidlingmaier, D. Klingmüller, Characterization Of The 5a-Reductase-3a-Hydroxysteroid Dehydrogenase Complex In The Human Brain, J Clin Endocrinol Metab. 86 (2001) 1324-1331. [5] B. Stoffel-Wagner, S. Beyenburg, M. Watzka, I. Blümcke, J. Bauer, J. Schramm, F. Bidlingmaier, C.E: Elger, Expression Of 5α -Reductase And 3α -Hydroxisteroid Oxidoreductase In The Hippocampus Of Patients With Chronic Temporal Lobe Epilepsy, Epilepsia. 41 (2000) 140-147. [6] B. Stoffel-Wagner, M. Watzka, J. Schramm, F. Bidlingmaier, D. Klingmüller, Expression Of Cyp19 (Aromatase) Mrna In Different Areas Of The Human Brain, J Steroid Biochem Molec Biol. 70 (1999) 237-241. [7] B. Stoffel-Wagner, M. Watzka, S. Steckelbroeck, J. Schramm, F. Bidlingmaier, D. Klingmüller, Expression Of 17β-Hydroxysteroid Dehydrogenase Types 1, 2, 3 And 4 In The Human Temporal Lobe, J Endocrinol. 160 (1999) 119-126. [8] B. Stoffel-Wagner, M. Watzka, S. Steckelbroeck, R. Schwaab, J. Schramm, F. Bidlingmaier, D. Klingmüller, Expression Of Cyp19 (Aromatase) Mrna In The Human Temporal Lobe, Biochem Biophys Res Commun. 244 (1998) 768-771. [9] B. Stoffel-Wagner, M. Watzka, S. Steckelbroeck, L. Wickert, J. Schramm, G. Romalo, D. Klingmüller, H.U. Schweikert, Expression Of 5α-Reductase In The Human Temporal Lobe Of Children And Adults, J Clin Endocrinol Metab. 83 (1998) 3636-3642. [10] M. Watzka, F. Bidlingmaier, J. Schramm, D. Klingmüller, B. Stoffel-Wagner, Sex- And AgeSpecific Differences In Human Brain Cyp11a1 Mrna Expression, J Neuroendocrinol. 11 (1999) 901905.

68

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

THE USE OF ESTROGENS AND RELATED COMPOUNDS IN THE TREATMENT OF DAMAGE FROM CEREBRAL ISCHEMIA Simpkins J.W., Yang S.-H. and Liu R. Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107 USA [email protected] There are 750,000 new cases of stroke each year in the U.S. and brain damage from stroke leads to high health care costs and disabilities. Needed, but currently not available, are therapies that can be administered prior to, during or following cerebral ischemia that reduce or eliminate neuronal damage from stroke. To address this issue, we began to assess the neuroprotective effects of estrogens and related compounds in stroke neuroprotection to determine if these compounds had potential for clinical application. First, we demonstrated that 17 β-estradiol (E2) pretreatment exerted potent neuroprotection of the cerebral cortex over a wide dose range and pretreatment interval. Thereafter, we assessed the ability of a variety of non-feminizing estrogens to protect brain tissue from stroke. We observed that pretreatment with 17 α-estradiol; the complete enantiomer of E2 (ENT-E2); 2-adamantylestrone; and the enantiomer of 17-desoxyestradiol, were as effective as E2 in pretreatment protection from stroke damage. These data suggest that non-estrogen receptor mechanisms are involved in brain neuroprotection under our treatment conditions. We then determined if the observed E2 protection could be extended to times after the onset of the cerebral ischemic event. Using a formulation of E2 that rapidly delivers the steroid, a necessary condition for acute therapy of an ongoing stroke, we demonstrated that 100µg E2/kg could protect brain tissue for up to 3 hours after the onset of the stroke. To determine if this therapeutic window could be extended with higher doses of the steroid, we conducted a dose-response assessment of E2 when administered at 6 hours after the onset of the ischemic event. While the effectiveness of the 100 µg E2/kg was reduced at this time interval, higher doses of E2 were effective. E2, at doses of 500 and 1000 µg/kg, reduced infarct volume by over 50% even with this 6 hour delay in treatment. Collectively, these data indicate that estrogens could prove to be useful therapies in preventing brain damage from strokes.

(Supported by NIH Grant AG 10485, U.S. Army Grant DAMD 17-19-1-9473 and MitoKor, Inc.)

69

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

THE EFFECT OF OESTROGEN ON BRAIN AGEING Murphy D.G.M. and Cutter W. PO 50, Institute of Psychiatry, [email protected]

Decrespigny Park,

London SE5 8AF,

UK.

Background. The biological basis of human brain aging is poorly understood and there are few proven treatments for age-related brain disease. Recently, however, it has been suggested that HRT may have a ‘neuroprotective’ effect. Some studies report no beneficial effect of HRT on cognitive function in healthy older women though most show a significant benefit t o memory (1) and/or a general cognitive enhancement. There is perhaps greatest evidence for a positive effect on verbal memory. Moreover, HRT may reduce the risk and delay the onset of developing Alzheimer's disease (AD) (2). However, recent randomized trials suggest no beneficial effect of long term oral HRT on cognitive function in women who already have a diagnosis of AD (3). Although one study (using oestradiol patches) reported a significant benefit (4). Thus there is significant evidence that HRT improves cognitive function in postmenopausal women, and reduces a womans’ risk for developing AD but there is less evidence that HRT is an effective treatment for established AD. The mechanisms by which HRT exerts these effects are unknown, and are most likely multimodal. One explanation for the beneficial effect of HRT on brain may be that it reduces age-related differences in brain systems which are crucial to higher cognitive function, and which are also implicated in AD and aging (e.g. the cholinergic system). I will present recent work from our laboratory investigating the effect of oestrogen on brain aging. Also, I will present evidence that the action of oestrogen occurs in the same brain regions which are affected by gene products coded for by X chromosome CGG triplet repeats. Ageing of the serotonergic system. The serotonergic system is implicated in mood and memory. We recently reported that HRT reduces the effect of aging on responsivity of the serotonergic system, using neuroendocrine tests (5). However, these are only an indirect measure of brain function, and may be confounded by the effect of oestrogen on pituitary function. Ageing of the cholinergic system. Cholinergic neurotransmission is crucial for cognitive function, and it is widely accepted that cholinergic dysfunction/loss of cholinergic neurons is in part responsible for age-related cognitive decline and the cognitive impairments seen in AD. Amongst the most biologically plausible explanations for HRT use enhancing memory are modulatory effects on the cholinergic system and in brain regions affected by healthy aging and AD (e.g. hippocampus and association neocortex) (6). Thus we tested the hypothesis that use of long-term HRT in postmenopausal women enhances cholinergic responsivity in a crosssectional pilot study of 30 postmenopausal women (7). We found that cholinergic responsivity was significantly higher in HRT treated women than in women who had never received HRT. Moreover, within long-term ERT users there was a significant positive correlation between enhanced cholinergic neurotransmission and longer duration of oestrogen exposure. This observation is consistent with results from a recent SPET study demonstrating increased survival or plasticity of cholinergic synaptic terminal with increasing years of ERT use in healthy postmenopausal women (8). These findings suggest that oestrogen is involved in the physiological regulation of cholinergic projections. Effect of oestrogen on human brain anatomy. Healthy brain aging in humans is associated with loss of gray and white matter volume. Estrogens reduce oxidative stress and increase neuronal survival in vitro. However, nobody has investigated if estrogen replacement therapy (ERT) modulates age-related differences in regional grey matter volume. Thus we used magnetic resonance imaging and voxel-based morphometry to examine the effects of longterm ERT on age-related differences in (a) normalized lobar brain volumes and (b) regional gray and white matter volumes. We included 61 healthy women; 23 young, 19 postmenopausal

70

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 long-term ERT users and 19 postmenopausal ERT never-users. ERT users did not differ significantly from never-users in age, duration of menopause, general intelligence, mnemonic function or apolipoprotein E allele frequency. Compared to young women, both ERT users and never-users had significantly smaller normalized volumes of whole brain and left and right frontal lobes. However, ERT users did not differ significantly from never-users in lobar brain volumes. Compared to young women and ERT users, never-users had significantly less gray matter bilaterally in orbitofrontal cortices and cerebellum, right inferior frontal and precentral cortices, and left paracentral cortex. Effect of Oestrogen on neuronal intergrity. Normal aging is associated with breakdown in neuronal cell membranes. We recently reported that women who take long-term HRT have significantly less age-related neuronal membrane breakdown than women who are HRT naïve (9). I will present preliminary data from a 5yr follow up in these same women demonstrating that differences in cell membrane death in women off HRT preceded cell death. Also women on HRT have significantly less cell death in parietal lobe and hippocampal regions, as compared to women who are HRT naïve, at 5yr follow up. Conclusion ERT modulates age-related differences in serotonergic and cholinergic neurotransmission; regional gray matter volume; membrane turnover, and neuronal death. These actions most likely partially explain reports that HRT improves cognitive function in women Reference List 1. Maki PM, et al. Enhanced verbal memory in nondemented elderly women receiving hormonereplacement therapy. Am J Psychiatry. 2001. 158: 227-233. 2. Yaffe K, et al. Estrogen therapy in postmenopausal women; effects on cognitive function and dementia. JAMA. 1998. 279: 688-695. 3. Mulnard RA, et al. Estrogen replacement therapy for treatment of mild to moderate Alzheimer disease: A randomized controlled trial. JAMA. 2000. 283: 1007-1015. 4. Asthana S, et al. High-dose estradiol improves cognition for women with AD: results of a randomized study. Neurology. 2001. 57(4): 605-612. 5. Van Amelsvoort T, et al. Prolactin response to d-fenfluramine in postmenopausal women on and off ERT: comparison with young women. Psychoneuroendocrinology. 2001. 26(5): 493-502. 6. McEwen BS, et al. Ovarian steroids and the brain: implications for cognition and aging. Ovarian steroids and the brain: implications for cognition and aging. Neurology. 1997. 48(5 Suppl 7): S8S15. 7. Van Amelsvoort T, et al. Effects of long-term estrogen replacement therapy on growth hormone respone to pyridostigmine in healthy postmenopausal women. Psychoneuroendocrinology. 2002 - in press. 8. Smith YR, et al. Effects of long-term hormone therapy on cholinergic synaptic concentrations in healthy postmenopausal women. Journal of Clinical Endocrinology & Metabolism. 2001. 86(2): 679-84. 9. Robertson D, et al. Effects of Estrogen replacement therapy on human brain aging; an in vivo 1H MRS study. Neurology. 2001. 57: 2114-2117.

71

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PATHOGENESIS IN MENSTRUAL CYCLE LINKED CNS-DISORDERS Bäckström T. Department of Obstetrics & Gynecology, Umeå University, Umea, Sweden [email protected] The action of 3alpha-hydroxy-5alpha/beta- pregnane-20-one steroids on the GABAA receptor is well characterized and understood. Animal and human studies show that these substances have a bimodal effect. In high concentrations GABAA modulators like barbiturates, benzodiazepins, alcohol and allopregnanolone are CNS depressants, anesthetic, antiepileptic and anxiolytic. In low concentrations usually reached endogenously, they can induce adverse emotional reactions in up to 30% of individuals. A bimodal effect has also been noted of different dosages of medroxyprogesterone and natural progesterone in postmenopausal women. Adverse mood symptoms are induced by progestagens but women feel better on a higher dosage compared to a lower dosage. It is also known that 3alpha-hydroxy-5alpha/beta-pregnane-steroids can induce tolerance to themselves and other similar substances after exposure, and that abstinence effects occur at withdrawal of the 3alpha-hydroxy-5alpha/beta-pregnane-steroids. Menstrual cycle linked disorders can be understood in the concept that they are caused by the action of endogenously produced 3alpha-hydroxy-5alpha/beta-pregnane-steroids through three possible mechanisms: a) direct action, b) tolerance induction, and c) withdrawal effect. Examples of symptoms and disorders caused by the direct action of 3alpha-hydroxy5alpha/beta-pregnane-steroids are sedation, tiredness, memory disturbance, learning disturbance, disturbance of motor function, clumsiness, increased appetite and food cravings, worsening of Petit Mal epilepsy, negative mood as tension, irritability and depression the cardinal symptoms in the premenstrual dysphoric disorder (PMDD). Continuous and long exposure to 3alpha-hydroxy-5alpha/beta-pregnane-steroids seems to cause tolerance and women with PMDD are less sensitive to GABAA modulators indicating a malfunctioning of the GABAA receptor system. Women with PMDD have signs of a tolerance during the luteal phase with the symptoms stress sensitivity, concentration difficulties, clumsiness, loss of impulse control (irritability) and depression. A continuous exposure to 3alpha-hydroxy-5alpha/beta-pregnane-steroids results in a withdrawal effect when the exposure is ended. This phenomenon occurs e.g. at post partum and during menstruation when the production of 3alpha-hydroxy-5alpha/betapregnane-steroids by the corpus luteum or placenta is interrupted. A condition that is explained by this withdrawal/ abstinence phenomenon is “catamenial epilepsy” where patients have an epileptic focus in the cerebral cortex and where a worsening occurs at the withdrawal period during menstruation. Other examples are menstrual related migraine and stress related migraine and mood changes post partum. Similar phenomenons are noted during stress. As a response to stress, the adrenals produce 3alpha-hydroxy-5alpha/beta-pregnane-steroids. These steroids may, depending on duration of production and concentrations, induce a) direct action, b) tolerance, and c) have withdrawal effect. This work was supported by an EU Regional fund Objective 1 grant.

72

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

NEUROPROTECTION BY ESTROGEN IN GLOBAL AND FOCAL ISCHEMIA Merchenthale I. Wyeth Research, Women’s Health Research Institute, 500 Arcola Rd. Collegeville, PA 19426, USA; [email protected] Estrogen is believed to protect against brain injury, neurodegeneration and cognitive decline. Two rodent models, the rat and mouse focal ischemia models representing cerebrovascular stroke and the gerbil global ischemia model representing acute heart attack, were used to test the hypothesis that estrogen protects against neuronal cell death caused by ischemia. Large numbers of estrogen receptors (ERs) are present in the CA2-3 regions of the rat and mouse hippocampus. In contrast, our in vivo binding studies with 125I-estrogen in gerbils revealed the presence of nuclear estrogen binding sites in CA1 neurons, but not in CA2-3 regions as in rats and mice. Because of the characteristic blood supply of the gerbil, the gerbil ischemia model (two vessel occlusion) was used to evaluate the neuroprotective actions of estrogen in the hippocampus. Estrogen not only prevented but protected against ischemic insult. Analysis of neurogranin mRNA, a marker of viability of hippocampal neurons, with in situ hybridization, revealed that estrogen treatment resulted in a complete protection in the CA1 regions not only when administered before, but when given two hours after the insult. Together, these observations demonstrate that estrogen protects from ischemic injury in both the focal and global ischemia models by acting primarily via classical nuclear receptors. To identify the ER subtype mediating the protective effect of estrogen in focal ischemia, ovariectomized ERαKO, ERβKO and wild type mice were treated with physiological doses of estrogen prior to or simultaneously with the insult. These experiments clearly established that the ERα subtype is the critical ER mediating neuroprotection in focal ischemia.

73

TUESDAY, 25th February 12.00 - 13.00 Plenary Lecture: McCarthy M.(Baltimore, MD, USA)

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ESTRADIOL MODULATION OF ASTROCYTES AND THE ESTABLISHMENT OF SEX DIFFERENCES IN THE BRAIN. McCarthy M.M. and Amateau S.K. Department of Physiology and Program in Neuroscience, University of Maryland, Baltimore, 655 W. Baltimore St., Baltimore MD. 21201 USA [email protected], FAX 410-706-8341 Astrocytes are the most numerous and enigmatic subtype of glia, a broad categorization applied to all non-neuronal cells in the brain other than endothelium. Glia includes reactive and protoplasmic astrocytes, oligodendrocytes and microglia. Neuronalglial interactions within the central nervous system (CNS) begin during early stages of fetal development and extend throughout the life span, with glia playing different roles at different stages. During neurogenesis and early development, radial glia provide scaffolding for migrating neurons and can give rise to neurons themselves. More often, the radial glia serve as the precursors to mature astrocytes, one category of which is the protoplasmic astrocyte. A hallmark of astrocyte maturation is increased stellation and synthesis of GFAP, an intermediate filament protein which is readily visible by immunocytochemistry. Using GFAP as a marker of astrocyte morphology, Garcia-Segura and colleagues [5] have established the ability of astrocytes to respond to gonadal steroid hormones with changes in morphology in the hypothalamic arucate nucleus. We have found that astrocytes in the arcuate nucleus and preoptic area (POA) are also targets of steroid-mediated sexual differentiation in that they permanently differentiate during a perinatal sensitive period. Astrocytes in males exhibit a higher degree of stellation as well as having more and longer primary processes. This differentiation is mediated by estradiol in both brain regions [2,8,9]. We have further investigated the mechanisms by which estradiol differentiates astrocytes, and modulates neuronal morphology. Our findings elucidate three general principles: 1) neuron-to-astrocyte-to-neuron communication is central to steroid-induced differentiation, 2) the precise cellular mechanism is distinct for each brain region and 3) the correlation between astrocyte complexity and neuronal morphology is not consistent across brain region. Neuron-to-Astrocyte-to-Neuron Communication in the Arcuate Nucleus. Arcuate astrocytes in neonatal males exhibit a greater degree of stellation and longer processes than those observed in females. Converging evidence indicates that estradiol acting at the alpha variant of the estradiol receptor is the primary event in this differentiation [7]. This would suggest the astrocyte as the primary target for estradiol action. However our attempts to localize estradiol receptors within astrocytes failed [9], leaving the question of whether astrocytes in the arcuate are capable of responding directly to estradiol unresolved. This question became superfluous subsequent to our discovery that a purely neuronal factor, GABA, was the transducer of estradiol-mediated astrocyte differentiation. On the day of birth, levels of the amino acid transmitter, GABA, and its rate-limiting enzyme, GAD, are greater in males versus females in several hypothalamic regions including the arcuate nucleus [3,4]. Preventing the increase in males or mimicking GABA action in females modulates astrocytes accordingly, i.e. the stellation of astrocytes in males is reduced by blocking GABA action whereas administering GABA agonists to females induces astrocyte differentiation [10]. This leads us to conclude that estradiol increases GABA synthesis in neurons which then gains access to the extracellular space (not necessarily via synaptic transmission) and acts on astrocytes to induce differentiation. Astrocytes then feedback on neurons to suppress the formation of dendritic spine synapses [11]. The mechanism by which astrocytes communicate back to neurons in this brain region remains unknown but may involve a physical interaction similar to the phasic synaptic remodeling observed in the adult arcuate nucleus [6]. Neuron-to-astrocyte-to-neuron Communication in the Medial Preoptic Nucleus. The sexual dimorphism of astrocytes in the mPOA is essentially identical to that seen in the arcuate nucles in that male astrocytes exhibit greater stellation with longer and more numerous processes [2]. Surprisingly, however, a primary aspect of sexually differentiation in neuronal morphology, the density of dendritic spines, is opposite to that 77

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

in the arcuate. In this brain region, males have more dendritic spines than females. The increase in spines on male neurons appears to originate with the release of a signal from the astrocytes, that signal being glutamate. However control of the release of glutamate by the astrocytes begins with estradiol action in the neurons and involves the prostaglandin PGE2. The rate limiting enzyme in PGE2 synthesis is COX-2, which is found predominantly in neurons. Estradiol treatment of females selectively increases PGE2 synthesis in the POA, having no effect on the levels of 6 related prostanoids. When females are treated with either estradiol or PGE2 there is a doubling of dendritic spines in the POA, but not in the hippocampus. The estradiol-induced increase in spines is prevented by pretreatment with a COX-2 inhibitor and is mimicked by exogenous glutamate. More importantly, both the PGE2 and estradiol-induced increases in dendritic spines are blocked by pretreatment with an AMPA receptor antagonist, implicating this glutamate receptor subtype as the final mediator of neuronal morphology. From this evidence we have constructed a working model in which in the developing male preoptic area, estradiol acts on neurons to induce PGE2 synthesis. The prostaglandin is then released by the neurons and acts on the astrocytes to release glutamate, which then acts on the neurons to increase dendritic spine formation [1]. The preoptic area is a brain region central to expression of male sexual behavior, making this a logical behavioral endpoint to assess for functional significance of prostaglandin action during development. Treatment of newborn males with a COX-2 inhibitor dramatically impaired performance of male sexual behavior in adulthood. Even more striking was the induction of high levels of male sexual behavior in females treated with PGE2 as neonates and given exogenous testosterone as adults. These behavioral results establish the functional significance of prostaglandin signaling during perinatal development to masculinize the brain. Reference List [1] Amateau, S.K. and McCarthy, M.M., A novel mechanism of dendritic spine plasticity involving estradiol induction of prostglandin-E2, J. Neurosci., 22 (2002) 8586-8596. [2] Amateau, S.K. and McCarthy, M.M., Sexual Differentiation of Astrocyte Morphology in the Developing Rat Preoptic Area., J. Neuroendo., 14 (2002) 904-910. [3] Davis, A.M., Grattan, D.R., Selmanoff, M. and McCarthy, M.M., Sex differences in glutamic acid decarboxylase mRNA in neonatal rat brain: implications for sexual differentiation, Horm. Behav., 30 (1996) 538-552. [4] Davis, A.M., Ward, S.C., Selmanoff, M., Herbison, A.E. and McCarthy, M.M., Developmental sex differences in amino acid neurotransmitter levels in hypothalamic and limbic areas of rat brain., Neuroscience, 90 (1999) 1471-1482. [5] Garcia-Segura, L.M., Cardona-Gomez, G.P, Trejo, J.L, Fernandez-Galaz, M.C., Chowen, J.A., Glial cells are involved in organizational and activational effects of sex hormones in the brain. In A. Matsumoto (Ed.), Sexual Differentiation of the Brain, CRC Press, Boca Ratan, 2000, pp. 83-93. [6] García-Segura, L.M., Chowen, J.A., Parducz, A. and Naftolin, F., Gonadal hormones as promoters of structural synaptic plasticity: Cellular mechanisms, Prog Neurobiol, 44 (1994) 279-307. [7] McCarthy, M.M., Amateau, S.K. and Mong, J.A., Steroid modulation of astrocytes in the neonatal brain: Implications for adult reproductive function, Bio. Reprod., 67 (2002) 691-698. [8] Mong, J.A., Glaser, E. and McCarthy, M.M., Gonadal steroids promote glial differentiation and alter neuronal morphology in the developing hypothalamus in a regionally specific manner, J. Neurosci., 19 (1999) 1464-1472. [9] Mong, J.A., McCarthy, M.M., Steroid-induced developmental plasticity in hypothalamic astrocytes: Implications for synaptic patterning, J. Neurobiol., 40 (1999) 602-619. [10] Mong, J.A., Nunez, J.L. and McCarthy, M.M., GABA mediates steroid-induced astrocyte differentiation in the neonatal rat hypothalamus, J. Neuroendo., 14 (2002) 1-16. [11] Mong, J.A., Roberts, R.C., Kelly, J.J., McCarthy, M.M., Gonadal steroids reduce the density of axospinous synapses in the developing rat arcuate nucleus: An electron microscopy analysis., J. Comp. Neurol., 432 (2001) 259-267.

78

TUESDAY, 25th February 2003 14.30 - 19.00 Symposium: Glial Cells as a Target for Steroids

Symposium: Glial Cells as a Target for Steroids (Chairs: Melcangi M.C., Milano & Schumacher M., Bicetre) •

Azcoitia I., Veiga S., Sierra A., Méndez P. and Garcia-Segura L.M. (Madrid, Spain, EU) Neuroactive steroids and neuroprotection

•

Glaser M., Rodriguez-Waitkus P. M., Ng B.K., Lafollette A., Zhu T.S. and Conrad H.E. (Urbana, IL, U.S.A). Steroid hormone signaling between schwann cells and neurons that regulates the rate of myelin synthesis

•

De Nicola A.F., Labombarda F. Gonzalez S., Gonzalez Deniselle M.C., Guennoun, R. and Schumacher M. (Buenos Aires, Argentina) Steroid effects on glial cells: detrimental or protective for spinal cord function?

•

Carrer H.F., Cambiasso M.J., Brito V. and Gorosito S. (Córdoba, Argentina) Neurotrophic factors and estradiol interact to control axogenic growth in hypothalamic neurons

•

Drew P.D, Chavis J.A., and Bhatt R. (Little Rock, AK, USA) Sex steroid regulation of microglial cell activation: relevance to multiple sclerosis

•

Giachino C, Galbiati M, Fasolo A, Peretto P, Melcangi RC (Torino, Italy, EU) Effects of progesterone derivatives, dihydroprogesterone and tetrahydroprogesterone, on newly formed cells and glial tubes of the subependymal layer

•

Nichols N.R. (Melbourne, Australia) Ndrg2, a novel gene regulated by adrenal steroids and antidepressants, is highly expressed in astrocytes

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

NEUROACTIVE STEROIDS AND NEUROPROTECTION Azcoitia I.1,Veiga S.2, Sierra A.2, Méndez P.2 and Garcia-Segura L.M.2 1

Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, E28040 Madrid SPAIN and 2Instituto Cajal, E-28002 Madrid SPAIN. [email protected] In addition to function as a sex hormone, estradiol as well as some of its precursors have widespread effects through the brain, both during development and in adult life. The role of estradiol as a neuroprotective agent has been reported in different animal models of brain injury, during aging and in cultures. Additionally it has been also suggested that gonadal hormones act against Alzheimer’s and Parkinson’s diseases and in head trauma and stroke. The brain itself is a source of sex steroids and aromatase, the enzyme that catalyzes the transformation of testosterone into estrogens is expressed during development and in certain areas of the adult brain, such as the amygdaloid complex. Furthermore aromatase is expressed de novo in the brain after excitotoxicity, mechanical lesion or vasogenic edema. In all these cases the expression of the enzyme is a local phenomenon, restricted to the lesion area, being the astrocytes the neural cells exhibiting the highest immunoreactivity. On the other hand, hippocampal postnatal astrocytes grown in cultures exhibit a basal aromatase expression, that is manifold increased after stressing conditions like serum deprivation, or treatment with excitotoxins or pro-inflammatory interleukines. In mixed neuron and glia hippocampal embryonic cultures, a robust aromatase immunoreactivity is also detected in some neurons, both in cell body and in processes, and thus matching the estrogen receptor distribution. The importance of aromatase induction in neuroprotection can be deduced by the fact that the genetic and pharmacological blockage of the enzyme increases neurodegeneration. Aromatase knock out mice showed significant neuronal loss in the hippocampal formation after the systemic injection of excitotoxins in a dose which has no adverse effect in control littermates. In addition, the peripheral or intracerebral administration of the aromatase inhibitor fadrozole enhances neurotoxicity in male rats, and this effect is counterbalanced by the administration of estradiol. Consistently with these results, aromatase inhibition in hippocampal cultures has also a negative impact. When fadrozole is added to the medium, the extension of neurites is significantly reduced, a effect overridden by the simultaneous administration of estradiol. Moreover, fadrozole enhances the LDH release to the medium, a effect paralleled with the increase in the number of neuronal apoptotic nuclei. Neuronal death is more evident after the administration of excitotoxins and again, the treatment with estradiol abrogates the deleterious effect of fadrozole. All these observations suggest that the local induction of aromatase and estrogen formation in brain after injury are neuroprotective.

This study has been supported by the Commission of the European Communities, specific RTD programme “Quality of Live and Management of Living Resources”, QLK6-CT-2000-00179.

81

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

STEROID HORMONE SIGNALING BETWEEN SCHWANN CELLS AND NEURONS THAT REGULATES THE RATE OF MYELIN SYNTHESIS Glaser M., Rodriguez-Waitkus P.M., Ng B.K., LaFollette A., Zhu T.S. and Conrad H.E.

Department of Biochemistry and the Neuroscience Program, University of Illinois, Urbana, IL 61801, U. S. A., e-mail: [email protected], FAX: 1-217-244-5858 There is extensive signaling and cross talk between myelin-forming cells and neurons to control the various stages of myelin formation, e. g., the stages of cell proliferation, migration, and differentiation. Of particular interest to our laboratory are the steroid hormones and growth factors that regulate the initiation and biosynthetic rate of myelin synthesis. The factors involved in regulating the different developmental stages are primarily being studied in co-cultures of Schwann cells and dorsal root ganglia neurons. A method has been developed to measure the rate of myelin synthesis by using fluorescence digital imaging microscopy to follow the incorporation of a fluorescently labeled lipid precursor into myelin. This method allows the continuous monitoring and a quantitative measurement of the time and rate of myelin synthesis. Progesterone and other steroid hormones accelerated the time required to initiate myelin synthesis as well as increasing the peak rate of myelin synthesis. The mRNAs for the enzymes that make progesterone were induced at the onset of myelin synthesis. One effect of progesterone was to bind to the progesterone receptor in neurons and cause it to translocate to the nucleus. This caused changes in the expression of a number of genes that may be important in preparing the axons for myelination. An isoform of platelet-derived growth factor (PDGF) A-chain was made by neurons and also appears to be important in regulating myelin synthesis by Schwann cells. When the signaling by this isoform of PDGF was interrupted by heparin treatment there was a significant reduction of progesterone receptor immunostaining in neurons. Thus, this isoform of PDGF may act together with progesterone to regulate the rate of myelin synthesis.

82

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

STEROID EFFECTS ON GLIAL CELLS: DETRIMENTAL OR PROTECTIVE FOR SPINAL CORD FUNCTION? De Nicola A.F.*,# , Labombarda F.*, Gonzalez S.*, Gonzalez Deniselle M.C.*, Guennoun, R.** and Schumacher M.** *Laboratory of Neuroendocrine Biochemistry, Instituto de Biología y Medicina Experimental, and Dept. of Human Biochemistry, Faculty of Medicine, University of Buenos Aires, Argentina, and **INSERM U488, Kremlin-Bicetre, France. #Instituto de Biologia y Medicina Experimental, Obligado 2490, 1428 Buenos Aires, Argentina. E-mail : [email protected]. FAX : +54-11—4786-2564. Repair of damage and recovery of function are fundamental endeavors for recuperation of patients with spinal cord injury. Steroid hormones offer promising therapeutic perspectives, since they showed beneficial and neuroprotective effects on damaged neurons. Expression of classical receptors for estrogens, androgens, and adrenocortical steroids in spinal cord neurons and/or glial cells support direct steroid effects on cell types populating this tissue. The growing list of neuroprotectant steroids also incorporated progesterone (PROG), which showed regenerative and myelinating properties following injury of the peripheral and central nervous system [5, 6]. In rats with complete spinal cord transection (TRX), neuronal deafferentiation reduced the levels of the α3 subunit mRNA of the Na,K-ATPase and choline acetyltransferase (ChAT), while the mRNA of the growth-associated protein GAP-43 was upregulated. In vivo PROG treatment during 72 h restored levels of the sodium pump mRNA and ChAT to normal, whereas grain density of GAP-43 mRNA was further enhanced [1]. These responses were interpreted as restorative and beneficial for neuronal function. To complement the neuronal effects, we studied the response of glial cells to PROG. Using an antibody specific for the B isoform of the PROG receptor (PR), we were able to show PR immunopositive cells in white and gray matter resembling astrocytres and oligodendrocytes [2]. Modulation of astrocyte responses was determined after treating spinal cord transected rats with 4 mg/kg/day PROG for 3 days. Two astrocyte proteins: NADPH-diaphorase, an enzyme with nitric oxide synthase activity, and glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity were determined [3]. The proteins were studied at three levels of the spinal cord from rats with total TRX at T10 : above (T5 level), below (L1 level) and caudal to the lesion (L3 level). Equivalent regions were dissected in controls. The number and area of NADPH-diaphorase active or GFAP immunoreactive astrocytes/0.1 mm2 in white matter (lateral funiculus) or gray matter (Lamina IX) was measured by computerized image analysis. In controls, PROG increased the number of GFAP-immunoreactive astrocytes in gray and white matter at all levels of the spinal cord, while astrocyte area also increased in white matter throughout and in gray matter at the T5 region. In control rats PROG did not change NADPH-diaphorase activity. In rats with TRX and not receiving hormone, a general up-regulation of the number and area of GFAP-positive astrocytes was found at all levels of the spinal cord. In rats with TRX, PROG did not change the already high GFAP-expression. In the TRX group, instead, PROG increased the number and area of NADPH-diaphorase active astrocytes in white

83

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

and gray matter immediately above and below, but not caudal to the lesion. Thus, the response to PROG was conditioned by environmental factors, in that NADPH-diaphorase was hormonally modulated in cells reacting to trauma, whereas up-regulation of GFAP by PROG was produced in resting astrocytes from non-injured animals. Oligodendrocytes, the myelinating cells of the spinal cord, are also profoundly affected after spinal cord injury. Prevalent oligodendrocyte apoptosis and demyelination are common features around the lesioned site [4]. Employing an antibody reacting against myelin basic protein (MBP) and immunocytochemical techniques, we determined by computerized image analysis the MBP staining intensity of the corticospinal tract (CST) and the dorsal ascending funiculus (DAT). MBP staining caudal to the lesion for the descending CST represented axons undergoing wallerian degeneration, whereas those in DAT the response of proximal axons. After TRX, both MBP staining in CST and DAT showed a 50% reduction respect of control, sham-operated animals. PROG treatment of rats with TRX recovered MBP staining intensity to control levels. The response seemed region-specific, since MBP staining in the white matter ventral funiculus was neither reduced after TRX nor modified by PROG treatment of the lesioned animals. Therefore, in rats with spinal cord TRX, PROG induced an up-regulation of glial cell parameters including astrocyte NADPH-diaphorase and oligodendrocyte MBP staining. In terms of beneficial or detrimental consequences, these PROG effects may be supportive of neuronal recuperation. As further evidence, we recently observed that motoneuron chromatolysis originating in rats with spinal cord TRX, was considerably prevented when rats received an intensive course of PROG treatment. Thus, PROG effect on glial cells goes in parallel with morphological and biochemical enhancement of damaged motoneurons.

Reference List [1] F. Labombarda, F., S. Gonzalez, M.C. Gonzalez Deniselle, R. Guennoun, M. Schumacher, A.F. De Nicola, Cellular basis for progesterone neuroprotection in the injured spinal cord. J. Neurotrauma 19 (2002) 343-355. [2] F. Labombarda, R. Guennoun, S. Gonzalez, P. Roig, A. Lima, M. Schumacher, A.F. De Nicola, Immunocytochemical evidence for a progesterone receptor in neurons and glial cells of the rat spinal cord. Neurosci. Lett. 288 (2000) 29-32. [3] F. Labombarda, S. Gonzalez, P. Roig, A. Lima, R. Guennoun, M. Schumacher, A.F. De Nicola, Modulation of NADPH-diaphorase and glial fibrillary acidic protein by progesterone in astrocytes from normal and injured rat spinal cord. J. Steroid Biochem. Mol. Biol. 73 (2000) 159-169. [4] D.M. McTigue, P. Wei, B.T. Stokes, Proliferation of NG2-positive cells and altered oligodendrocyte numbers in the contused rat spinal cord, J. Neurosci. 21 (2001) 3392-3400. [5] M. Schumacher, I. Akwa, R. Guennoun, F. Robert, F.Labombarda, F. Desarnaud, P. Robel, A.F. De Nicola, E.E. Baulieu, Steroid synthesis and metabolism in the nervous system: trophic and protective effects. J. Neurocytology 29 (2000) 307-326 . [6] D.G. Stein, Z.L. Fulop, Progesterone and recovery after traumatic brain injury: an overview, Neuroscientist 4 (1998) 435-442.

84

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

NEUROTROPHIC FACTORS AND ESTRADIOL INTERACT TO CONTROL AXOGENIC GROWTH IN HYPOTHALAMIC NEURONS Carrer H.F., Cambiasso M.J., Brito V. and Gorosito S. Instituto de Investigación Médica M. y M. Ferreyra, INIMEC-CONICET, Casilla de Correo 389, 5000 Córdoba, Argentina; [email protected]; 54(351)4695163 Previous work from our laboratory has shown that in cultures of hypothalamic neurons taken from gestation day 16 (GD16) embryos, treatment of sexually segregated cultures with 17-β-estradiol (E2) induces axon growth in neurons from males but not from females [1]. This response to estrogen is contingent upon co-culture with heterotopic glia (mostly astrocytes) from a target region (amygdala) harvested from same sex fetuses, whereas in the presence of homotopic glia or in cultures without glia, E2 had no effect [2]. It was concluded that the axogenic effect of E2 depends on interaction between neurons and glia from a target region and that neurons from fetal male donors appear to mature earlier than neurons from females, a differentiated response that takes place prior to divergent exposure to gonadal secretions. The effects of target and non-target glia-conditioned media (CM ) on the E2-induced growth of neuronal processes of hypothalamic neurons obtained from sexually segregated fetal donors were also studied. Estrogen added to media conditioned by target glia modified the number of primary neurites and the growth of axons of hypothalamic neurons of males but not of females [3]. Neither the Type III steroidal receptor blocker tamoxifen nor Type I antiestrogen ICI 182,780 prevented the axogenic effects of the hormone. Estradiol made membrane-impermeable by conjugation to a protein of high molecular weight (E2BSA) preserved its axogenic capacity, suggesting the possibility of a membrane effect responsible for the action of E2 [4]. Western blot analysis of the tyrosine kinase type A (TrkA), type B (TrkB), type C (TrkC) and insulin like growth factor (IGF-I R) receptors in extracts from homogenates of cultured hypothalamic neurons showed that in cultures of male derived neurons grown with E2 and CM from target glia, the amounts of TrkB and IGF-I Rβ increased notably [3]. Densitometric quantification showed that these cultures had more TrkB than cultures with CM alone or E2 alone. On the contrary, in cultures of female derived neurons, the presence of CM alone induced maximal levels of TrkB, which were not further increased by E2; female derived neurons in all conditions did not contain IGF-I Rβ. Levels of TrkC were not modified by any experimental condition in male or female derived cultures and TrkA was not found in the homogenates. To investigate whether the axogenic response in males depends on the upregulation of TrkB, we analyzed neuritic growth and neuronal polarization in cultures treated with an antisense oligonucleotide against TrkB mRNA [5]. In cultures without E2, treatment with 7.5 or 10 µM antisense reduced TrkB levels and the percentage of neurons showing an identifiable axon; number and length of minor processes were increased. In cultures treated with 5 µM antisense, morphometric parameters were normal although total TrkB levels were reduced. The same dose prevented the E2-dependent increase of TrkB levels and suppressed the axogenic effect of E2. Treatment with sense oligonucleotide did not affect TrkB levels or axonal growth in cultures with or without E2. These results indicate that TrkB is necessary for normal neuronal growth and maturation and further suggest that an increase in TrkB is necessary for E2 to exert its axogenic effect in male derived neurons. 85

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

We also assessed whether TrkB signaling is affected by E2. We have analyzed the activation of the extracellular signal-regulated kinase (Erk), which is a point of convergence for distinct neurite outgrowth signaling. Addition of 10 nM E2 resulted in the phosphorylation of Erk1/2 at 60 min after hormone exposure. Inhibition of MAPK signaling with MAPK inhibitor UO126 (25-50 µM) blocks the axogenic effect of E2. This findings suggest that estrogen signaling may interact with MAPK signaling involved in the promotion of axon growth. These results are integrated in a model for the confluent interaction of estrogen and neurotrophic factors released by glia that may contribute to the sexual differentiation of the brain [6].

Supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina (CONICET), Agencia Córdoba Ciencia, Agencia Nacional de Promoción Científica y Tecnológica and the European Commission.

Reference List 1. Diaz,H., Lorenzo,A., Carrer,H.F., and Caceres,A., Time lapse study of neurite growth in hypothalamic dissociated neurons in culture - sex differences and estrogen effects, J. Neurosci. Res. 33 (1992) 266-281. 2. Cambiasso,M.J., Diaz,H., Caceres,A., and Carrer,H.F., Neuritogenic effect of estradiol on rat ventromedial hypothalamic neurons co-cultured with homotopic or heterotopic glia, J. Neurosci. Res. 42 (1995) 700-709. 3. Cambiasso,M.J., Colombo,J.A., and Carrer,H.F. Differential effect of oestradiol and astrogliaconditioned media on the growth of hypothalamic neurons from male and female rat brains. Eur. J. Neurosci. 12 (2000) 2291-2298. 4. Cambiasso,M.J. and Carrer,H.F. Nongenomic mechanism mediates estradiol stimulation of axon growth in male rat hypothalamic neurons in vitro. J. Neurosci. Res. 66 (2001) 475-481. 5. Brito, V.I., Carrer, H.F. and Cambiasso, M.J. Inhibition of TrkB synthesis blocks axogenic effect of estradiol on hypothalamic neurons in vitro, unpublished 6. Carrer H.F. and Cambiasso M.J. Sexual differentiation of the brain brain: genes, estrogen and neurotrophic factors, Cell. Molec. Neurobiol, 2002 in press.

86

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

SEX STEROID REGULATION OF MICROGLIAL RELEVANCE TO MULTIPLE SCLEROSIS

CELL

ACTIVATION:

Drew P.D., Chavis J.A. and Bhatt R. University of Arkansas for Medical Sciences, Department of Anatomy and NeurobiologySlot 510, 4301 W. Markham Street, Little Rock, AR 72205 USA, [email protected], FAX: (501) 686-6382 Multiple sclerosis occurs more frequently in females than males. However, the mechanisms resulting in gender differences in multiple sclerosis are unknown. Several studies have suggested that sex steroids influence the development and severity of multiple sclerosis. For example, pregnancy influences multiple sclerosis symptomology, with remission in the third trimester of gestation, followed by exacerbation in the post-partum period. In addition, oral contraceptives containing female sex steroids have been associated with a lower risk of developing multiple sclerosis, and decreased disability. Experimental autoimmune encephalomyelitis (EAE) is an animal autoimmune disorder characterized by central nervous system (CNS) inflammation and demyelination, and remittent paralysis; features consistent with multiple sclerosis. Recent studies have suggested that female sex steroids may modulate EAE, at least in part, through effects upon T-cells. For example, investigators have demonstrated that not only are female mice more susceptible than males to EAE, but also that T-cells from female mice produce more severe disease when adoptively transferred into recipients. In addition, estrogens and testosterone have been demonstrated to repress EAE. Sex steroids also shift T-cells toward a Th2 phenotype in vitro, and cytokines produced by Th2 cells generally suppress EAE. Collectively, these studies suggest that female sex steroids modulate EAE, at least in part, through effects upon T-cell phenotype. In addition to autoreactive T-cells, activated microglia participate in pathology associated with multiple sclerosis. However, the effect of sex steroids upon activation of microglia has not been extensively investigated. Microglia are resident CNS cells that function in host defense. These cells may serve as antigen presenting cells, and can be phagocytic. Upon CNS injury or inflammation, microglia become activated, resulting in increased proliferation and altered morphology. Activated microglia also produce a variety of cytokines including TNF-alpha, as well as increased MHC class II and nitric oxide (NO). Although molecules including NO and TNF-alpha are toxic to pathogens, these agents can also be toxic to CNS cells including myelin-producing oligodendrocytes, which are compromised in the course of multiple sclerosis. These molecules also may be toxic to neurons, and thus may contribute to axonal degeneration characteristic of multiple sclerosis. Thus, agents which inhibit NO and TNF-alpha synthesis may be effective in the treatment of multiple sclerosis. We have demonstrated that the female sex steroids estriol, beta-estradiol, and progesterone inhibit lipopolysachharide (LPS) induction of NO production by primary rat microglia and by the mouse N9 microglial cell line. These hormones act by inhibiting the production of inducible nitric oxide synthase (iNOS) which catalyses the synthesis of NO. Estriol likely inhibits iNOS gene expression since the hormone blocks LPS induction of iNOS RNA levels. The pro-inflammatory cytokines IFN-gamma and TNF-alpha are

87

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

believed to be important modulators of multiple sclerosis. Here, we demonstrate that estrogens and progesterone also inhibit NO production by microglial cells activated in response to these cytokines. Activated microglia elicit TNF-alpha and IL-1beta in addition to NO, and we further demonstrate that estrogens and progesterone repress production of these cytokines by microglial cells. Estriol and progesterone, at concentrations consistent with late pregnancy, inhibit NO and TNF-alpha production by activated microglia, suggesting that hormone inhibition of microglial cell activation may contribute to the decreased severity of multiple sclerosis symptoms commonly associated with pregnancy. In addition, estrogens and progesterone inhibit IL-12 production by microglia, suggesting that the hormones may alter T-cell phenotype. Finally, estrogens and progesterone were demonstrated to inhibit hydrogen peroxide mediated toxicity of CG4 oligodendrocyte cells. This suggests that these steroids may protect oligodendrocytes which are compromised in multiple sclerosis

Acknowledgements: Supported by grant RG 3198A1 from the National Multiple Sclerosis Society, USA.

88

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EFFECTS OF PROGESTERONE DERIVATIVES, DIHYDROPROGESTERONE AND TETRAHYDROPROGESTERONE, ON NEWLY FORMED CELLS AND GLIAL TUBES OF THE SUBEPENDYMAL LAYER Giachino C.1, Galbiati M.2, Fasolo A.1, Peretto P.1 and Melcangi R.C.2 1

Department of Animal and Human Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy. [email protected] 2

Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20133 Milan, Italy.

In specific areas of the adult mammalian central nervous system, like for instance the subependymal layer (SEL) of the rostral forebrain and the dentate gyrus of the hippocampus, neurogenesis is protracted virtually through whole life [1,3,4]. The SEL provides a continuous supply of newborn cells that migrate to the olfactory bulb (OB) where they differentiate into interneurons [3,4]. These newly formed cells reach the OB after long tangential migration within a channel system (glial tubes) formed by protoplasmic astrocytes [7]. Several studies have shown that steroid hormones can act on adult hippocampal neurogenesis by modulating cell proliferation [2,10]. Conversely, a direct effect of steroids on SEL neurogenesis has not been demonstrated. In this work we studied the possible effects of progesterone (P) and/or its neuroactive metabolites dihydroprogesterone (DHP) and tetrahydroprogesterone (THP) on the two cellular components of the SEL (i.e., proliferating/migrating neuroblasts and protoplasmic astrocytes) in adult male rat. DHP and THP can be synthesizes directly by the central nervous system starting from P [5]. P, DHP or THP were administered by intraventricular injection and after 2 days the SEL was analysed by immunohistochemistry with antiGFAP and anti-vimentin antibodies, to label the glial compartment, and anti-PSA-NCAM antibody, to label the migrating neuroblasts. Moreover, the newly formed cells were identified by using intraventricular injections of 5-bromo-2’-deoxyuridine (BrdU) detected immunohistochemically. Our results demonstrate that DHP and THP, but not P treatments, drastically decrease the number of BrdU-labeled cells within the SEL. Moreover, THP, DHP, and to a little extent P, modify the structural organization of the SEL astrocytes (glial tubes) and decrease GFAP-immunoreactivity mainly in the SEL glial compartment. The reduction of newly formed cells within the SEL after THP and DHP treatments could be explained considering the distribution and types of P, THP and DHP receptors. So far no evidences in literature demonstrate a direct action of P and its neuroactive derivatives on neuroblasts proliferation. However, a recent work [9] demonstrated that neuronal progenitors of the neonatal rat subventricular zone (SVZ), namely the SEL precursor region, express functional GABAA receptors, suggesting that THP may act on SEL proliferation by activating GABAA. Considering the modifications induced in the glial tubes by THP and DHP treatments, an effect of neuroactive steroids on glial cells is not surprising since they express both classical (i.e., progesterone receptor) and non classical (i.e., GABAA) steroid receptors and consequently this cellular compartment can be a target for these molecules

89

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

[6]. Future experiments will be requested to explain if these receptors are involved in such effects. Altogether, the present results indicate that neuroactive derivatives of P (i.e., DHP and THP) exert direct effects on both neuroblasts and astrocytes of the SEL, suggesting that the steroidal environment of the brain, besides modulating adult hippocampal neurogenesis, may also play an important role in SEL neurogenesis. This work was supported by grants from MURST and Compagnia di San Paolo

Reference List [1] H.A. Cameron, C.S. Woolley, B.S. McEwen, E. Gould, Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat, Neuroscience 56 (1993) 337-344. [2] E. Gould, H.A. Cameron, D.C. Daniels, C.S. Woolley, B.S. McEwen, Adrenal hormones suppress cell division in the adult rat dentate gyrus, J. Neurosci. 12 (1992) 3642-3650. [3] C. Lois, A. Alvarez-Buylla, Long-distance neuronal migration in the adult mammalian brain, Science 264 (1994) 1145-1148. [4] M.B. Luskin, Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone, Neuron 11 (1993) 173-189. [5] R.C. Melcangi, V. Magnaghi, M. Galbiati, L. Martini, Formation and effects of neuroactive steroids in the central and peripheral nervous system, International Rev. Neurobiol. 46 (2001) 145-176. [6] R.C. Melcangi, V. Magnaghi, M. Galbiati, L. Martini, Glial cells: a target for steroid hormones. Prog. Brain Res. 132 (2001) 31-40. [7] P. Peretto, A. Merighi, A. Fasolo, L. Bonfanti, The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain, Brain Res. Bull. 49 (1999) 221243. [8] M.T. Smith, V. Pencea, Z. Wang, M.B. Luskin, T.R. Insel, Increased number of BrdU-labeled neurons in the rostral migratory stream of the estrous prairie vole, Horm. Behav. 39 (2001) 11-21. [9] R.R. Stewart, G.J. Hoge, T. Zigova, M.B. Luskin, Neural progenitor cells of the neonatal rat anterior subventricular zone express functional GABA(A) receptors, J. Neurobiol. 50 (2002) 305-322. [10] P. Tanapat, N.B. Hastings, A.J. Reeves, E. Gould, Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat, J. Neurosci. 19 (1999) 5792-5801.

90

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

NDRG2, A NOVEL GENE REGULATED BY ADRENAL STEROIDS AND ANTIDEPRESSANTS, IS HIGHLY EXPRESSED IN ASTROCYTES Nichols N.R. Department of Physiology and Monash Institute of Neurological Diseases, Monash University, Wellington Road, 3800, Melbourne, Australia. Fax +61-3-9905-2547 e-mail: [email protected] Previously, we cloned mRNAs from the hippocampus that were increased or decreased in response to glucocorticoid treatment (10 mg corticosterone /day for 3 days) of adrenalectomised rats [1]. Several glial mRNAs were isolated that were regulated by glucocorticoids in opposite directions. Glial fibrillary acidic protein (GFAP), a marker of astrocytes, is under negative regulation by corticosterone. Furthermore, GFAP dramatically increased in the subgranular zone of the dentate gyrus after adrenalectomy; this effect was dependent on the presence of dying granule neurons in response to removal of trophic adrenal steroids [2]. Based on these data, we proposed that GFAP down regulation is an adaptive effect of glucocorticoids, which returns astrocytes to a normal state after their reactivity in response to insults [1,3]. From the same hippocampal cDNA library, we cloned an unknown gene (CR62) as an increased response to glucocorticoids and subsequently showed that it was expressed in cultured astrocytes. Additional sequence analysis now shows homology of CR62 to Ndrg2, a member of a new gene family, the Nmyc downstream-regulated gene (NDRG) family. Recently, functional genomics was used to determine that a mutation in NDRG1 was the cause of an autosomal recessive peripheral neuropathy predominantly found in the Roma Gypsy community in Europe [4]. The functions of NDRG proteins are of major interest because of their expression in the developing and adult nervous system and suggested roles in neural differentiation synapse formation and axonal survival. Sequence alignments indicate that Ndrg2 and Ndrg4 belong to a separate subfamily and both of these genes are highly expressed in adult human and rat brain [5]. Another group reported that Ndrg2 transcripts in rat frontal cortex were down regulated by chronic antidepressant or electroconvulsive shock treatment, another effective therapy for depression [6]. High levels of glucocorticoids accompany many cases of depression and can damage the brain resulting in loss of neuronal connections, reduced neurogenesis and mental deficits. Antidepressants may alleviate symptoms of depression by reversing the effects of glucocorticoids [7]. Our data show that Ndrg2 mRNA is up regulated by glucocorticoids, in the opposite direction to that of antidepressants. Furthermore, glucocorticoids decrease neurogenesis in the adult dentate gyrus and antidepressants increase neurogenesis after several weeks, a timeframe that corresponds to the length of treatment required for their effectiveness in reducing symptoms of depression. Therefore, regulation of Ndrg2 may be a molecular correlate of morphological and functional changes in the hippocampus (and possibly other brain regions) that are related to depression. Since glucocorticoids increased Ndrg2 mRNA in cultured astrocytes, we examined its cellular localisation and regulation in adult brain by in situ hybridisation. Male Fischer 344 rats (2-3 months) were sham-operated or adrenalectomised with or without corticosterone

91

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

replacement (200 µg/ml) in drinking water. Brain sections were hybridised with radiolabeled cRNA probes and quantified by grain counting after emulsion autoradiography. Ndrg2 mRNA is highly expressed throughout the brain section in a pattern similar to GFAP mRNA, except it is more prevalent in gray matter than in white matter. In contrast to GFAP, Ndrg2 mRNA in the subgranular zone of the dentate gyrus is decreased after adrenalectomy and restored to sham-operated levels by corticosterone replacement. Increased expression of Ndrg2 mRNA in the subgranular zone of the dentate gyrus by glucocorticoids suggests that it could be involved in inhibition of neurogenesis or in cell differentiation. The high expression of Ndrg2 in astrocytes and the down regulation of this gene by antidepressants strongly implicate glia as the target of these therapeutic drugs in the brain [8]. Furthermore, since antidepressants may alleviate symptoms of depression by reversing the effects of glucocorticoids, our findings suggest that further studies on Ndrg2 regulation and function could contribute to understanding of the pathogenesis and treatment of depression.

Reference List 1. N. R. Nichols, C.E. Finch, Gene products of corticosteroid action in hippocampus, Ann. N.Y. Acad. Sci. 746 (1994) 145-154. 2. N. Bye, N.R. Nichols, Adrenalectomy-induced apoptosis and glial responsiveness during aging, Neuroreport 9 (1998) 1179-1184. 3. N.R. Nichols, Glial responses to steroids as markers of brain aging, J. Neurobiol. 40 (1999) 585601. 4. L. Kalaydjieva, D. Gresham, R. Gooding, L. Heather, F. Baas, R. de Jonge, K. Blechschmidt, D. Angelicheva, D. Chandler, P. Worsley, A. Rosenthal, R.H.M. King, P.K. Thomas, N-myc downstream-regulated gene 1 is mutated in hereditary Motor and Sensory Neuropathy-Lom. Am. J. Hum. Genet. 67 (2000) 47-58. 5. X. Qu, Y. Zhai, H. Wei, C. Zhang, G. Xing, Y. Yu, F. He, Characterisation and expression of three novel differentiation-related genes belonging to the human NDRG gene family. Mol. Cell. Biochem. 229 (2002) 35-44. 6. K. Takahashi, M. Yamada, M. Hirano, G. Nishioka, K. Kudo, K. Kamijima, K. Momose, M. Yamada, Expression of two novel splice variants for putative rat NDR2 gene after chronic treatment with antidepressant and ECT. Soc. Neurosci. Abst. #218.17 (2001). 7. B. Czeh, T. Michaelis, T. Watanabe, J. Frahm, G. de Biurrun, M. van Kampen, A. Batolomucci, E. Fuchs, Stress-induced changes in cerebral metabolites, hippocampal volume, and cell proliferation are prevented by antidepressant treatment with tianeptine. Proc. Natl. Acad. Sci. 98 (2001) 12796-12801. 8. D.R. Cotter, C.M. Pariante, I.P. Everall, Glial cell abnormalities in major psychiatric disorders: the evidence and implications. Brain Res. Bull. 55 (2001) 585-595.

92

WEDNESDAY, 26th February 2003 08.30 - 12.00 Symposium: Steroid Regulation of Reproduction

Symposium: Steroid Regulation of Reproduction (Chairs: Fasolo A., Torino & Herbison A., Cambridge) •

Moenter S.M., DeFazio R.A., Straume M. and Nunemaker C.S. (Charlottesville, VI, USA) Steroid regulation of GnRH neurons

•

Ábrahám I.M., Han S.-K., Todman M. and Herbison A.E. (Cambridge, UK, EU) Rapid effects of estrogen on GnRH neurons

•

Bourguignon J-P., Matagne V., Gérard A. and Lebrethon M-C. (Liège, Belgium, EU). Estrogens and GnRH secretion in vitro throughout development

•

Sisk C. (East Lansing, MI, USA) Pubertal maturation of the brain and reproductive behavior: a recasting of behavioral potential

•

Etgen A.M. (Bronx, NY, USA) Functional interactions between estrogen and insulin-like growth factor-I in the regulation of hypothalamic α1-adrenoceptor signaling and female reproductive function

•

Galbiati M., Martini L. and Melcangi R.C. (Milano, Italy, EU) Steroid hormones and growth factors act in an integrated manner at the levels of hypothalamic astrocytes: a role for neuroendocrine control of reproduction

•

Thiéry J.-C. and Malpaux B. (Nouzilly, France, EU) Seasonal modulation of the passage of sex steroids from peripheral blood to brain in sheep.

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

STEROID REGULATION OF GnRH NEURONS Moenter S.M., DeFazio R.A., Straume M. and Nunemaker C.S. Departments of Medicine and Cell Biology, University of Virginia, PO BOX 800578, Charlottesville, VA 22908, USA; FAX 434-982-0088; [email protected] GnRH neurons form the final common pathway for regulating fertility. Estradiol feedback controls GnRH release, but the cellular mechanisms are unknown. To study GnRH neurons directly, we use transgenic mice with green fluorescent protein targeted to these cells, permitting identification for electrophysiological studies. In the studies presented here, we used a model for estradiol negative feedback. Specifically, adult females were ovariectomized (OVX) and some received an estradiol capsule at the time of surgery (OVX+E). A week later, brain slices were prepared and GFP-GnRH neurons targeted for recording. LH was reduced in OVX+E mice, confirming this as a model for negative feedback in terms of the net output of the reproductive neuroendocrine system. Targeted single-unit extracellular recordings were used to examine the firing rate of GnRH neurons (1). This approach provides an electrophysiological measure of the integrated response of the GnRH neuron to a treatment. GnRH neurons from OVX+E mice were more quiescent than those from OVX mice, consistent with the reduction in LH levels seen in OVX+E animals. In longer recordings, it was possible to assess the effect of estradiol on the pattern of firing. Estradiol more than doubled the time between episodes of increased firing rate as detected by the Cluster7 pulse detection algorithm. As these episodes of increased firing rate occur with a period that resembles the frequency of LH release in rodents, it is tempting to speculate these events are causally related. In addition to changes in firing rate that occur on the same time scale as would be predicted for GnRH release, higher frequency rhythms have been observed these neurons, for example in calcium oscillations (2) and in firing rate of isolated cells (3). The data from the above study were thus subjected to spectral analysis via fast Fourier transform to identify rhythms with different periods, particularly those that have a shorter period than that of the episodic firing patterns previously observed (1). The rhythms identified in this manner were arbitrarily grouped by period: bursts (period <100 sec), clusters (period 1001000 sec) or episodes (period >1000 sec) (4). Bursts were observed in ~90% of GnRH neurons and consisted of trains of action currents (the currents during action potentials). The lower frequency changes in firing rate (episodes and clusters) were generated by changing the spacing between bursts and not other characteristics of bursts such as duration or the number of action currents per burst. This suggests that in GnRH neurons bursts represent the fundamental unit of activity from which lower frequency rhythms are constructed. Consistent with this, estradiol did not alter burst characteristics, but rather changed the patterning of inter-burst intervals to increase the period of the low-frequency episode rhythm. This suggests an estradiol-sensitive, low-frequency rhythm alters the patterning of a fundamental unit of activity to change ultimately GnRH pulse frequency. To bring about the changes in interburst-interval described above, we hypothesize estradiol alters conductances in GnRH neurons. Because voltage-gated potassium channels determine the pattern of activity and response to synaptic inputs in many neurons, we tested if estradiol altered potassium currents in these cells (5). Whole-cell voltage-clamp

95

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

studies revealed estradiol affected the amplitude, decay time and the voltage dependence of both inactivation and activation of A-type potassium currents in GnRH neurons, and also altered a slowly inactivating current, IK. Blockade of the protein kinases PKA, PKC and PKG reversed the effects of estradiol on current amplitude and decay time, but not on voltage dependence. Estradiol did not affect IA or IK in neurosecretory neurons of the paraventricular nucleus, suggesting this action is at least in part specific to GnRH neurons. Consistent with the changes in IA, estradiol increased excitability in GnRH neurons, decreasing latency for action potential generation. Of interest, acute treatment of guinea pig GnRH neurons with estradiol causes hyperpolarization, likely via activation of a potassium conductance (6). Potassium channels are thus one target for estradiol regulation of GnRH neurons and this regulation is in part due to changes in phosphorylation of potassium channel components. Together these data show multiple targets for estradiol feedback regulation of GnRH neurons and suggest adjusting the balance of these and other steroid actions may determine if the net feedback effect on hormone release is negative or positive.

Supported by NIH HD 41469 and HD 34860 and NICHD/NIH cooperative agreement U54 HD28934 as part of the Specialized Cooperative Centers Program in Reproduction Research.

References List 1. Nunemaker CS, DeFazio RA and Moenter SM 2002 Estradiol-sensitive afferents modulate longterm episodic firing patterns of gonadotropin-releasing hormone neurons. Endocrinology 143:22842292. 2. Terasawa E, Schanhofer WK, Keen KL, Luchansky L 1999 Intracellular Ca(2+) oscillations in luteinizing hormone-releasing hormone neurons derived from the embryonic olfactory placode of the rhesus monkey. J Neurosci 19:5898-909 3. Kuehl-Kovarik MC, Pouliot WA, Halterman GL, Handa RJ, Dudek FE, Partin KM 2002 Episodic bursting activity and response to excitatory amino acids in acutely dissociated gonadotropin-releasing hormone neurons genetically targeted with green fluorescent protein J Neurosci 22: 2313-2322 4. Nunemaker CS, DeFazio RA, Straume M, Moenter SM 2003 Gonadotropin-releasing hormone neurons generate interacting rhythms in multiple time domains. Endocrinology , in press, March 2003. 5. DeFazio RA, Moenter SM 2002 Estradiol feedback alters potassium currents and firing properties of central neurons controlling reproduction. Mol Endocrinol 2255-2265. 6. Lagrange AH, Ronnekleiv OK Kelly MJ 1995 Estradiol-17 beta and mu-opioid peptides rapidly hyperpolarize GnRH neurons: a cellular mechanism of negative feedback? Endocrinology 136: 23412344

96

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

RAPID EFFECTS OF ESTROGEN ON GNRH NEURONS Ábrahám I.M.1,3, Han S.-K.1,2, Todman M.1 and Herbison A.E.1,2 1

Laboratory of Neuroendocrinology, Babraham Institute, Cambridge, UK, 2Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, Dunedin, New Zealand, 3Neurobiology Research Group, HAS-Eötvös Loránd University, Pázmány Péter st. 1c, 1117, Budapest, Hungary, e-mail: [email protected], FAX:36-1-3812182 Estrogen, as a classical feedback molecule, is a pivotal regulator of GnRH neuron gene expression and secretory activity. Although estrogen is primarily thought to alter neuronal genomic mechanisms via intracellular estrogen receptors (ERs: ERα and ERβ) it also exerts poorly understood rapid non-genomic effects on neurons by altering membrane excitability and consequently, signal transduction pathways. One transcription factor well established to be regulated in a rapid manner by these second messenger systems is the cAMP response element-binding protein (CREB). Neuronal membrane potential changes, intracellular calcium increase or enhanced cAMP concentration can all activate phosphorylation of CREB, which is critical for CREB to regulate the transcription of genes. In present experiments we have characterized the rapid estrogen effect on membrane excitability in vitro and on CREB phosphorylation of GnRH neurons in vivo. In order to eliminate endogenous estrogens, all experiments were performed on ovariectomized (OVX) adult female mice. We have investigated the effect of estrogen on GnRH neuron excitability by undertaking gramicidin-perforated patch recordings of GnRH-GFP neurons in the acute brain slice preparation. A new line of GnRH-GFP mice was produced in which a mutated GFP, selected for its low cytotoxicity, was employed. In these experimental conditions, estrogen administered for 10-15min depolarized approximately 40% of GnRH neurons. In some cases this was followed by a prolonged period of hyperpolarization. These responses were also observed in the presence of tetrodotoxin indicating that they occurred directly at the GnRH neuron. In order to evaluate whether rapid estrogen actions may also occur in vivo we have characterized time, concentration and ER dependence of estrogen effect on the CREB phosphorylation in GnRH neurons of wild type and ERα or ERβ knock out (ERαKO or ERβKO) animals using dual-labelling immunocytochemistry. We have determined the expression of CREB and phosphorylated-CREB (pCREB) in GnRH neurons at different time points after injection of vehicle or different concentrations of estrogen to mice. Whereas estrogen had no effect on the numbers of GnRH neurons expressing CREB, an increase in pCREB expression was detected in GnRH neurons 15 min following estrogen administration and the levels remained elevated at 4h. The administration of 1 and 10 µg doses of estrogen evoked a clear dose-dependent elevation in pCREB expression. In ERαKO animals, estrogen significantly increased the pCREB-immunoreactivity in GnRH neurons but this effect was blocked completely in ERβKO animals. In a further series of investigations in vitro experiments were performed used the acute brain slice preparation derived from OVX mice. The ability of estrogen to induce 97

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

CREB phosphorylation in GnRH neurons was retained in vitro and also found to exist following the addition of tetrodotoxin, again indicating that estrogen acts directly upon GnRH neurons to phosphorylate CREB. In contrast the membrane impermeant estrogenBSA conjugate failed to phosphorylate CREB in GnRH neurons suggesting that estrogen must pass through the cell membrane to alter intracellular signalling within the GnRH cells. Taken together these observations demonstrate that estrogen exerts rapid, direct actions upon the GnRH neuronal phenotype in vitro and in vivo, and that this is critically dependent upon ERβ. In addition, these studies provide evidence for a functional ER in the GnRH neuron and show that ERβ is involved in non-genomic estrogen signaling within the brain.

Work supported by the BBSRC and EU

98

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ESTROGENS AND DEVELOPMENT

GnRH

SECRETION

IN

VITRO

THROUGHOUT

Bourguignon J.-P., Matagne V., Gérard A. and Lebrethon M.-C. Developmental Neuroendocrinology Unit, Center for cellular and Molecular Neurobiology Research (CNCM), University of Liège, CHU, Sart-Tilman, B-4000 Liège, Belgium. e-mail: [email protected] Fax: 00 32 4 366 7246 Estrogens have prominent negative feedback effects on the pituitary gonadotropin secretion while positive feedback effects are likely mediated in the hypothalamus. Using hypothalamic explants of 50-day-old male rats in a static incubation system, 17betaestradiol (estradiol, 10-7M) increased the glutamate-evoked GnRH secretory response (143±14% of controls, mean±SD). This effect was dose-dependent and involved the kainate receptor subtype and estrogen receptors. Testosterone (T, 10-7M) was as effective as estradiol in increasing the GnRH secretory response evoked by 10-2M of glutamate (176±43%) or kainate (150±28%) whereas T had no effect on the NMA-evoked GnRH secretion (10-2 M, 99±7%). T effect occurred similarly in both sexes and in the adult and immature hypothalamus since T increased the GnRH secretory response evoked by kainate similarly using explants from 5-day-old male and female rats (131±9% and 138±10%, respectively). Aromatase was involved in T effect since the aromatase inhibitor R76713 (10-5M) prevented T-induced increase of the GnRH secretory response (104±11%). In addition, 5alpha-DHT (10-7M), a non aromatizable androgen had no effect on the glutamate-evoked GnRH secretory response. Since estradiol effect occurred within 7.5 to 15 min, we hypothesized that this rapid effect was mediated by intracellular second messengers such as protein kinases. Inhibitors of protein kinase A, C and MAPK were used and each of them prevented the estradiol increase of glutamate-evoked GnRH secretion. An increase in intracellular calcium level could activate several protein kinases and induce GnRH release. Preliminary experiments indicate that nifedipine, a L-type calcium channel blocker, resulted in reduction of the glutamate-evoked GnRH secretion and prevented the estradiol effect. Since the studied retrochiasmatic explants (RCH) were found to contain virtually no GnRH cell bodies, hypothalamic explants including the preoptic area (RCH-POA) were studied as well as median eminence explants in order to localize the estradiol effects. Estradiol increased the glutamate-evoked GnRH secretion using both the RCH-POA explants (159±14%) and the median eminence explants (170±10%) similarly as using RCH explants. Spontaneous pulsatile GnRH secretion can be observed in vitro using RCH explants and displays a developmental decrease in interpulse interval between 5 and 25 days (males: 90±1min and 40±5min; females: 90±1min and 35±4min, respectively, mean±SD). Incubation of female hypothalamic explants with estradiol (10-7M) resulted in reduction of the GnRH interpulse interval at 5 and 15 days. Using male explants, a slight but significant effect was seen at 15 days only. Using explants obtained at 25 and 50 days in both sexes, estradiol had no effect. A possible estradiol-mediated effect of aromatized testosterone on pulsatile GnRH secretion was studied. Using female explants, testosterone (10-7M) caused a reduction of the GnRH interpulse interval at 5 and 15 days as well as at 15 days using

99

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

male explants. These effects were not different from those obtained using estradiol and they were prevented using R76713 (10-5M), an aromatase inhibitor, indicating aromatase involvement in the testosterone-induced acceleration of pulsatile GnRH secretion. We hypothesized that these sexually dimorphic effects of E2 on GnRH interpulse interval resulted from neonatal brain imprinting by sex steroids. Using hypothalamic explants from neonatally androgenized female rats studied at 5 and 15 days, E2 was not able to decrease the GnRH interpulse interval. Conversely, using hypothalamic explants from male rats treated prenatally with the aromatase inhibitor ATD and studied at 5 and 15 days, estradiol was able to reduce the GnRH interpulse interval. It is concluded that both estradiol and testosterone can stimulate GnRH secretion in vitro in an aromatase-dependent manner through a kainate-receptor involving mechanisms. Two different effects could coexist: (i) the GnRH interpulse interval is increased preferentially in the immature female rat retrochiasmatic hypothalamus depending on perinatal sex steroid imprinting. In contrast, (ii) an estradiol stimulation of the glutamate-evoked secretion occurs in the median eminence and is age- and gender-independent. The physiological relevance of these two different effects remains to be elucidated.

This study was supported by : ARC (99/04-241), FRSM (3.4515.01), BSGPE, the Faculty of Medicine at the University of Liege

100

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

PUBERTAL MATURATION OF THE BRAIN AND REPRODUCTIVE BEHAVIOR: A RECASTING OF BEHAVIORAL POTENTIAL Sisk C. Neuroscience Program, 108 Giltner Hall, Michigan State University East Lansing, M I 48824, USA; fax (517) 432-2744; e-mail: [email protected] The classical view of steroid-dependent organization of brain and behavior holds that steroids act during an early critical period of development to cause permanent changes in nervous system structure, which in turn determines adult responses to steroids in adulthood. Almost 20 years ago, Breedlove and Arnold revised the definition of organizational effects by pointing out that steroid hormones can induce permanent changes in the adult brain. John Paul Scott laid the theoretical groundwork for multiple critical periods for the progressive organization of the nervous system, and noted that critical periods are most likely to occur during periods of rapid developmental change. A two-stage model is proposed for steroid-dependent development and maturation of male social behaviors: perinatal masculinization of neural circuits, followed by peripubertal organization of these circuits, during which behavioral responsiveness to gonadal hormones in adulthood is enhanced. This hypothesis is based on experiments in Syrian hamsters that show that adult-typical reproductive behavior is not activated by testosterone prior to puberty, and that the absence of gonadal hormones during the normal time of puberty results in long-lasting compromises in behavioral responses to testosterone. These experiments provide evidence that neural circuits underlying behavior are organized by steroid hormones during puberty. Whether puberty is a critical period for these organizational effects remains to be empirically determined.

101

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

FUNCTIONAL INTERACTIONS BETWEEN ESTROGEN AND INSULIN-LIKE GROWTH FACTOR-I IN THE REGULATION OF HYPOTHALAMIC α1ADRENOCEPTOR SIGNALING AND FEMALE REPRODUCTIVE FUNCTION Etgen A.M. Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 USA. Email: [email protected]. Fax: 718-430-8654 The ovarian hormone estradiol (E2) and insulin-like growth factor-I (IGF-I) interact in the CNS to regulate neuroendocrine function and synaptic remodeling. Previously, our laboratory demonstrated that treatment of ovariectomized (OVX) rats with doses of E2 that are sufficient to prime female reproductive behavior (lordosis) induces α1Badrenoceptor expression in the preoptic area (POA) and hypothalamus (HYP)[5,7]. This adrenergic receptor subtype is thought to mediate norepinephrine facilitation of both lordosis behavior and the preovulatory luteinizing hormone (LH) surge (see [3,4]). We also showed that behaviorally effective E2 treatments promote IGF-I enhancement of α1adrenoceptor signal transduction, as evidenced by an increase in α1-adrenergic potentiation of cAMP accumulation in HYP and POA slices [8]. Therefore, we examined the hypothesis that E2-dependent aspects of female reproductive function, including LH release, lordosis behavior and α1B-adrenoceptor expression and function in the POA and HYP, require concurrent activation of brain IGF-I receptors (IGF-IRs) in female rats. OVX rats were implanted with a guide cannula aimed at the third ventricle and were treated in vivo with vehicle or E2 (2 µg of E2 benzoate) daily for two days prior to experimentation. Intracerebroventricular (icv) infusions of 4-10 µg of JB-1, a selective IGFIR antagonist, were administered every 12 hours beginning 1 hour before the first E2 injection. Animals also received progesterone 4 hours prior to behavioral testing. Administration of JB-1 during E2 priming completely blocks LH release induced by either E2 alone or E2 plus progesterone. The IGF-IR antagonist partially inhibits hormonedependent reproductive behavior as well. If JB-1 is administered only during the first or last 12 hr of estrogen treatment, it is unable to modify lordosis. Reproductive behavior is restored in JB-1-exposed animals by icv infusion of 8-bromo-cGMP, the second messenger implicated in α1-adrenergic facilitation of lordosis [2]. In addition, blockade of IGF-IRs with JB-1 during E2 priming prevents E2-induced increases in α1B-adenoceptor binding density and abolishes acute IGF-I enhancement of norepinephrine-stimulated cAMP accumulation in HYP and POA slices [9]. We are also examining possible downstream targets of E2 and IGF-I signaling that may be important for lordosis behavior. Both of these signaling molecules can activate kinases such as phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK)[1,6,10,11]. Therefore, we infused agents that block PI3K and/or MAPK activity icv into the third ventricle every 12 hours during a two-day E2 treatment as described above. Both PI3K inhibitors (wortmannin and LY294002) and MAPK inhibitors (PD98059 and U0126) partially attenuate lordosis responding when administered during estrogen priming. None of these drugs modifies lordosis if they are infused only once, during the last 12 hours of estrogen treatment. When both wortmannin and PD98059 are infused during E2 priming, lordosis behavior is completely abolished. These data document 102

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

the existence of a novel mechanism by which IGF-I participates in the remodeling of noradrenergic receptor signaling in the HYP and POA following E2 treatment. They suggest further that activation of both PI3K and MAPK by E2 and IGF-I mediates hormonal facilitation of lordosis behavior. These molecular events may help coordinate the timing of ovulation with the expression of sexual receptivity, thereby maximizing reproductive success.

Reference List [1] Cardona-Gomez, G.P., Mendez, P. and Garcia-Segura, L.M., Synergistic interaction of estradiol and insulin-like growth factor-I in the activation of PI3K/Akt signaling in the adult rat hypothalamus, Brain Res Mol Brain Res, 107 (2002) 80-8. [2] Chu, H.P. and Etgen, A.M., Ovarian hormone dependence of α1-adrenoceptor activation of the nitric oxide-cGMP pathway: relevance for hormonal facilitation of lordosis behavior, J Neurosci, 19 (1999) 7191-7. [3] Etgen, A.M., Ansonoff, M.A. and Quesada, A., Mechanisms of ovarian steroid regulation of norepinephrine receptor-mediated signal transduction in the hypothalamus: implications for female reproductive physiology, Horm Behav, 40 (2001) 169-177. [4] Etgen, A.M., Chu, H.P., Fiber, J.M., Karkanias, G.B. and Morales, J.M., Hormonal integration of neurochemical and sensory signals governing female reproductive behavior, Behav Brain Res, 105 (1999) 93-103. [5] Karkanias, G.B., Ansonoff, M.A. and Etgen, A.M., Estradiol regulation of α1b-adrenoceptor mRNA in female rat hypothalamus-preoptic area, J Neuroendocrinol, 8 (1996) 449-55. [6] Kim, B., Leventhal, P.S., Saltiel, A.R. and Feldman, E.L., Insulin-like growth factor-I-mediated neurite outgrowth in vitro requires mitogen-activated protein kinase activation, J Biol Chem, 272 (1997) 21268-73. [7] Petitti, N., Karkanias, G.B. and Etgen, A.M., Estradiol selectively regulates α1B-noradrenergic receptors in the hypothalamus and preoptic area, J Neurosci, 12 (1992) 3869-76. [8] Quesada, A. and Etgen, A.M., Insulin-like growth factor-1 regulation of α1-adrenergic receptor signaling is estradiol dependent in the preoptic area and hypothalamus of female rats, Endocrinology, 142 (2001) 599-607. [9] Quesada, A. and Etgen, A.M., Functional interactions between estrogen and insulin-like growth factor- I in the regulation of α1B-adrenoceptors and female reproductive function, J Neurosci, 22 (2002) 2401-8. [10] Singh, M., Ovarian hormones elicit phosphorylation of Akt and extracellular-signal regulated kinase in explants of the cerebral cortex, Endocrine, 14 (2001) 407-15. [11] Singh, M., Setalo, G., Jr., Guan, X., Frail, D.E. and Toran-Allerand, C.D., Estrogen-induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-α knock-out mice, J Neurosci, 20 (2000) 1694-700.

103

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

STEROID HORMONES AND GROWTH FACTORS ACT IN AN INTEGRATED MANNER AT THE LEVELS OF HYPOTHALAMIC ASTROCYTES: A ROLE FOR NEUROENDOCRINE CONTROL OF REPRODUCTION Galbiati M.*, Martini L. and Melcangi R.C.* *Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milano, Italy, Tel. +39-02-50318238, Fax: +39-02-50318204, Email: [email protected] In vitro observations have suggested that several growth factors [e.g., transforming growth factors beta 1 and beta 2 (TGFbeta 1 and TGFbeta 2), transforming growth factor alpha (TGFalpha), basic fibroblast growth factor (bFGF)], originating by glia compartment, may participate in the control of the synthesis and/or of the release of LHRH from hypothalamic neurons (see for review 1). We have also recently demonstrated that the messenger levels of TGFbeta1 and bFGF show a significant increase at hypothalamic level during the day of the rat proestrus (2). In agreement with a possible role of steroid hormones, and particulary of estrogens in inducing these effects we have also observed that estrogens are able to stimulate the gene expression of TGFbeta1 and bFGF in the hypothalamus of ovariectomized rats (2). On the basis of the in vivo observations mentioned above, we have hypothesized that steroid hormones, like estrogens, androgens and progestagens, influence the LHRH neurons by modulating in glial cells the synthesis and the release of TGFbeta1 and/or bFGF. This hypothesis is supported by our previous observations indicating that expression of TGFb1 is modulated in hypothalamic astrocytes by progesterone and its derivatives (i.e., dihydroprogesterone and tetrahydroprogesterone), while that of bFGF is modulated by didydrotestosterone (3). Moreover, other observations present in literature (4) indicate an increase, induced by estrogens, of the active and latent forms of TGFbeta1 in the medium of cultured hypothalamic astrocytes. However, no data were available in literature regarding the possible interaction between estrogens and bFGF at the hypothalamic level. To this purpose, we have evaluated whether estrogens might be able to influence the mRNA and the protein levels of bFGF in female rat hypothalamic type 1 astrocytes in culture. Moreover, we have also verified whether, TGFalpha, a growth factor sensitive to estrogens and involved in the control of LHRH-secreting neurons (5), might also modulate the expression of bFGF in these cultures. The data obtained have indicated that: 1) 17-beta estradiol is able to increase both the mRNA and protein levels of bFGF in cultured hypothalamic astrocytes; 2) TGFalpha increases the synthesis of bFGF in the same cells; 3) the effect of estrogens is blocked by the presence of an antibody raised against the TGFalpha receptor. The present observations (6) clearly indicate that estrogens are able to influence the synthesis of bFGF in hypothalamic astrocytes via the involvement of TGFalpha, which, as previously mentioned, may be released by astrocytes after estrogen treatment. These findings support the concept that steroid hormones and growth factors act in an integrated manner at the level of hypothalamic astrocytes adding a further piece of knowledge in the understanding of the mechanisms controlling LHRH neurons.

104

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

Acknowledgements The financial support of COFIN 990 61 531 87 and "FIRST - Special Project" is gratefully acknowledged.

Reference List 1. Melcangi RC, Martini L, Galbiati M 2002 Growth factors and steroid hormones: a complex interplay in the hypothalamic control of reproductive functions. Progress in Neurobiology 67:421-449. 2. Galbiati M, Magnaghi V, Martini L, Melcangi RC 2001 Transforming growth factor b1 and basic fibroblast growth factor mRNA expression is modified during the rat oestrous cycle. Journal of Neuroendocrinology 137:5605-5609. 3. Melcangi RC, Cavarretta I, Magnaghi V, Martini L, Galbiati M. 2001 Interactions between growth factors and steroids in the control of LHRH neurons. Brain Research Review 37:223-234. 4. Buchanan CD, Mahesh VB, Brann DW 2000 Estrogen-astrocyte-luteinizing hormone-releasing hormone signaling: a role for transforming growth factor b1. Biology of Reproduction 62:1710-1721. 5. Ma YJ, Berg-von der Emde K, Moholt-Siebert M, Hill DF, Ojeda SR 1994 Region specific regulation of transforming growth factor alpha (TGFa) gene expression in astrocytes of the neuroendocrine brain. Journal of Neuroscience 14:5644-5651. 6. Galbiati M, Martini L, Melcangi RC 2002 Oestrogens, via Transforming Growth Factor alpha, modulate basic Fibroblast Growth Factor synthesis in hypothalamic astrocytes: in vitro observations. Journal of Neuroendocrinology 14:829-835.

105

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

SEASONAL MODULATION OF THE PASSAGE OF SEX STEROIDS FROM PERIPHERAL BLOOD TO BRAIN IN SHEEP Thiéry J.-C. and Malpaux B. UMR INRA-CNRS-Université de Tours, Physiologie de la Reproduction et des Comportements, 37380 Nouzilly, France. [email protected] In the ewe, seasonal anestrus has been related to increased in brain responsiveness t o steroids. However, we hypothetized that the seasonal shift in brain responsiveness could also depend upon variable uptake of steroids in the brain. We previously observed (1) in ovariectomized (Ovex) ewes having a subcutaneous implant that preoptic tissular content of progesterone (P) was higher in long days (LD) than in short days (SD), following the same peripheral treatment (chronic release of physiological levels by intrauteine device (CIDR). Also, in ewes equipped with an intracerebral cannula in order to collect in vivo the cerebrospinal fluid (CSF), we have seen that injection into the carotid artery (IC) of massive doses (10 mg) of hydrosoluble, cyclodextrin-P resulted in detectable P in the CSF only during LD. In the present work performed in 3 experiments, we always used Ovex ewes, in order t o have a reliable control of the sex steroids. We checked in the first experiment whether physiological amount of P given by CIDR could generate different concentrations of P in the CSF in relation to daylength. In a second experiment, we extended the search for a photoperiodic control of brain access to oestradiol (E2), and in a third experiment, we compared the effect of photoperiod on E2 access to the brain between female and male. Experiment 1; we compared the concentration of P in blood plasma and CSF from 6 E2treated ewes bred under LD with 5 E2-treated ewes bred under SD. The plasma concentrations of P (Mean ± SEM) were 2.57 ± 0.27 ng/ml and 2.50 ± 0.33 ng/ml, while the CSF concentrations were 0.57 ± 0.29 ng/ml and 0.12 ± 0.03, for LD and SD, respectively (P<0.05). Experiment 2; we compared the E2 concentrations in the CSF between 7 ewes bred under LD and 6 ewes bred under SD, before E2 treatment, and 21 hours after receiving a 2-cm subcutaneous E2 implant and 21 hours after receiving a 4-cm additional subcutaneous E2 implant. The concentrations of E2 during the control period were not different (14.2 ± 3.7 vs 16.1 ± 4.3 pg/ml, during the first hour), but became significantly higher in LD than in SD ewes (18.3 ± 4.3 vs 13.9 ± 3.1 pg/ml) after 21 hours with a 2 cm implant and 22.4 ± 4.9 vs 10.7 ± 1.2 pg/ml after 21 hours with a 6-cm implant, respectively). Experiment 3; we compared a group of 6 ewes with a group of 5 castrated rams, both bearing a subcutaneous E2 implant. Female and males CSF and blood were sampled under LD and after 10 SD and 25 SD (when LH is inhibited), and after 70 SD in female or 55 SD in male (when LH was stimulated). In females, E2 in CSF fell from 43.3 ± 17.9 pg/ml during LD to 3.7 ± 0.7 pg/ml, 8.5 ± 3.2 and 7 ± 1.4 pg/ml after after 10 and 25 SD and after 70 SD, respectively. In male, under LD, E2 in the CSF (9.2 ± 2.6 pg/ml) was lower than in female and did not change significantly under SD (8.1 ± 2.6 pg/ml, 6.1 ± 1.5pg/ml and 7.4 ± 1.9 pg/ml for 10 SD, 25 SD and 55 SD, respectively). Results from the three experiments demonstrated an effect of daylenth on the access of sex steroids to the brain in ovariectomized ewes. This effect appears to be limited to female.

Reference List 1. Thiery J-C, Robel P, Corpechot C, Delaleu B, Malpaux B, 2000. Brain access of progesterone changes with photoperiod in ovariectomized ewes. 30th Annual Meeting of the Society for Neurosciences, 4-9 november 2000, New Orleans (USA). Vol 30, Part2 Abst.541.1.

106

WEDNESDAY, 26th February 12.00 - 13.00 Plenary Lecture: Arnold A. (Los Angeles, CA, USA)

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

THE ROLE OF SEX CHROMOSOMES IN SEXUAL DIFFERENTIATION OF THE BRAIN AND POSSIBLE INTERACTIONS WITH GONADAL STEROIDS Arnold A.P. University of California, Los Angeles, Department of Physiological Science, Laboratory of Neuroendocrinology of the Brain Research Institute, 641 Charles Young Drive South, Los Angeles CA 90095-1606 USA, [email protected], FAX: 310-825-8081 A male child is born with brain cells that express Y chromosome genes not present in females. A female child may have a higher dose, relative to a male, of some X chromosome genes expressed in each brain cell. Do these differences in gene expression induce sex differences in brain function? The dominant theory of mammalian and avian sexual differentiation holds that sex differences in neural phenotype are attributed exclusively to the action of gonadal secretions during early periods of development, not to the direct action of sex chromosome genes in brain cells themselves. We have found evidence in mice and songbirds that the genetic sex of brain cells contributes to sex differences in phenotype, thus undermining an exclusively gonadal hormonal theory of brain sexual differentiation. We have compared the neural and behavior phenotype of mice in which the testis determination gene Sry is deleted from the Y chromosome (creating the “Y minus” chromosome Y-) and moved via transgenic insertion onto an autosome, so that testis determination is inherited independently from the sex chromosomes[3]. Four genotypes are produced: XY- females (defined by the presence of ovaries), XX females, XY-Sry males (possessing the Y- chromosome plus Sry transgene), and XXSry males. By comparing animals with the same gonadal phenotype but different genetic sex of cells (XXSry males vs. XY-Sry males; XX females vs. XY- females), we were able to ask whether the complement of sex chromosomes influences brain phenotype. We measured numerous behavioral and neural phenotypes that are known to be sexually dimorphic in rodents. Most of these phenotypes were different in males and females but equivalent in comparisons of XXSry vs. XY-Sry males and XX vs. XY- females. Thus, these specific sexual dimorphisms are attributable to the action of gonadal steroid hormones, not to differences in the complement of sex chromosomes. Some phenotypes showed sex chromosome effects, however. The density of vasopressin fibers in the lateral septum was more masculine in XY-Sry males than in XXSry males, and more masculine in XY- females than in XX females. Thus the sex chromosomes influence sexual differentiation of this trait via mechanisms that are probably not hormonal. We also reinvestigated the sex difference in phenotype of mesencephalic neurons cultured from rodent embryos. Reisert, Pilgrim, Beyer and colleagues reported sex differences in the dopamine phenotype of these cultures. Because the cells are harvested before the onset of large sex differences in the plasma levels of gonadal steroids, the sex differences in vitro were attributed to the genetic sex of the cells rather than to the action of gonadal steroids. We prepared primary cell cultures from embryonic day 14 mouse embryos, and found that XY cultures (either XY-Sry or XY-), irrespective of gonadal sex, possessed more dopamine neurons than XX cultures (either XXSry or XX) [2]. Thus, the genetic sex of the cells is the primary determinant of the sex difference in these neurons in vitro. This finding raises the important questions of whether the genetic sex of dopamine neurons influences their development in vivo, and if XY and XX dopamine systems are more susceptible to the action of neurotoxins that contribute to Parkinson’s and other diseases. Transcripts of eight specific Y chromosome genes are expressed in mouse brain and are thus candidates for direct Y effects on the brain [6]. These include Usp9y, Ube1y, Smcy, Eif2s3y, Uty, Dby, Sry, and Sts. mRNAs encoding some X genes are expressed at a higher level in the brain of adult females than in males. The differences between XX and XY cells are attributable the action of Y genes or dosage differences in X genes. In the zebra finch (Taeniopygia guttata), males sing a courtship song that females cannot sing because the male’s neural song circuit is much larger than the female’s. Estrogen treatment of hatchling females causes partial masculinization of the song system. The estrogenic masculinization appears to require an androgen-dependent step, because coadministration of estradiol and the androgen receptor blocker flutamide prevents estrogen-

109

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 induced masculinization of females [4]. No steroid manipulation of males has prevented masculine neural development in vivo. Thus, steroids play a role in sexual differentiation, but the evidence leaves room for other factors. When genetic female zebra finch embryos are given fadrozole, an aromatase inhibitor, ovarian development is blocked and testes develop bilaterally. Genetic females with large amounts of testicular tissue possess a feminine song system, raising doubts that testicular secretions are responsible for triggering sexual differentiation [5]. We have analyzed the brain of a rare bilateral gynandromorphic finch [1]. This bird had male plumage and a testis on the right side of its body, and female plumage and an ovary on the left. Male birds are homogametic (ZZ) and females heterogametic (ZW). W genes were expressed in the brain predominantly on the left side, and a Z gene was expressed at higher level on the right, a pattern compatible with a ZZ genotype on the right and ZW genotype on the left. In the brain, nucleus HVC was 82% larger on the right side than on the left, a lateral difference far outside the normal range. The asymmetry of masculinization of HVC, plumage and gonads are all attributable to differences in genetic sex between the two sides. Thus, the genetic sex of brain cells plays a role in their sexual differentiation. Because both sides of the brain were more masculine than in females, hormones synthesized on the male side (gonads or brain) also contributed to sexual differentiation. Our current model of sexual differentiation of the song system is that genes on the Z or W chromosomes trigger sex differences in expression of genes such as steroid receptor coregulators or steroid synthetic enzymes in the brain, which in turn cause sexual differentiation.

These studies were supported by NIH grants MH59268 and DC00217.

Reference List 1. Agate,R.J., Grisham,W., Wade,J., Mann,S., Wingfield,J., Schanen,C., Palotie,A., and Arnold,A.P., Neural not gonadal origin of brain sex differences in a gynandromorphic finch, submitted, (2003). 2. Carruth,L.L., Reisert,I., and Arnold,A.P., Sex chromosome genes directly affect brain sexual differentiation, Nat. Neurosci., 5 (2002) 933-934. 3. De Vries,G.J., Rissman,E.F., Simerly,R.B., Yang,L.Y., Scordalakes,E.M., Auger,C.J., Swain,A., Lovell-Badge,R., Burgoyne,P.S., and Arnold,A.P., A model system for study of sex chromosome effects on sexually dimorphic neural and behavioral traits, J. Neurosci., 22 (2002) 9005-9014. 4. Grisham,W., Lee,J., McCormick,M.E., Yang-Stayner,K., and Arnold,A.P., Antiandrogen blocks estrogen-induced masculinization of the song system in female zebra finches, J. Neurobiol., 51 (2002) 1-8. 5. Wade,J. and Arnold,A.P., Functional testicular tissue does not masculinize development of the zebra finch song system, Proc. Natl. Acad. Sci. U. S. A., 93 (1996) 5264-5268. 6. Xu,J., Burgoyne,P.S., and Arnold,A.P., Sex differences in sex chromosome gene expression in mouse brain, Hum. Mol. Genet., 11 (2002) 1409-1419.

110

WEDNESDAY, 26th February 2003 14.30 - 18.00 Symposium: Behavioural Effects

Symposium: Behavioural Effects (Chairs: Balthazart J., Liege & Panzica G.C., Torino) •

Ball G.F., Sartor J.J., Castelino C.B. and Maney D.L. (Baltimore, MD, USA) Androgens, song behavior and seasonal neuroplasticity: where and how?

•

Blaustein J.D. (Amherst, MA, USA) Progestin receptors: neuronal integrators of hormonal and afferent stimulation

•

Swann J.M. (Bethlehem, PA, USA) hormonal regulation of behavior: a tale of two sexes

•

Hines M. (London, UK, EU) Sex steroids and human behavior: prenatal androgen levels influence gender-typical behavior in childhood

•

Bakker J., Honda S., Harada N. and Balthazart J. (Liege, Belgium, EU) The aromatase knockout mouse provides new evidence that estradiol is required during development in females for a normal expression of socio-sexual behaviors in adulthood

•

Evrard H.C. and Balthazart J. (Liège, Belgium, EU) Long term and short term aromatase inhibitions result in slow and rapid alteration of pain

•

Aloisi A.M., Ceccarelli I. and Fiorenzani P. (Siena, Italy, EU) Gonadectomy affects hormonal, behavioural and neuronal responses to repetitive nociceptive stimulation in male rats.

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ANDROGENS, SONG WHERE AND HOW?

BEHAVIOR AND SEASONAL NEUROPLASTICITY:

Ball G.F., Sartor J.J., Castelino C.B. and Maney D.L. Department of Psychological and Brain Sciences, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218-2686 USA email: [email protected]; Fax: +1 410-5166008 Seasonal changes in the brain of songbirds represent a dramatic example of naturally occurring neuroplasticity [10]. In male songbirds that breed seasonally, the volumes of several telencephalic nuclei that control song behavior are significantly larger in the spring than in the fall. These increases in volume are correlated with high rates of singing and high concentrations of testosterone in the plasma. Several song nuclei express either androgen or estrogen receptors, therefore it is possible that testosterone acting via estrogenic or androgenic metabolites regulates song behavior by modulating the morphology of these song control nuclei. The causal links among these variables have not been established. Dissociations among high concentrations of testosterone, enlarged song nuclei and high rates of singing behavior have been observed [3]. New findings have suggested alternative views as to how steroid hormones might modulate the expression of song behavior and neuroplasticity. It is known that changes in daylength promote increases in plasma testosterone (T) and singing behavior that are correlated with changes in the volumes of song control nuclei such as HVc [3]. T treatment increases the volume of HVc in part by inducing the expression of brain-derived neurotrophic factor (BDNF) so that T could directly affect the volume of HVc and consequently increase singing rate [5,7]. Alternatively, T could directly affect the amount of time the bird spends singing, leading to an activity-dependent change in BDNF expression and consequently an increase in HVc volume [1]. We tested this idea using different manipulations of song output in European starlings (Sturnus vulgaris). We first suppressed song behaviorally using dominance hierarchies in starlings housed in semi-natural settings and showed that song suppression in subordinate males attenuates the long day-induced HVc growth. We then irreversibly devocalized castrated males by lesioning the syrinx. Birds subsequently received either T-filled or control (C) empty implants. Devocalized +T males had smaller HVc volumes than sham operated+T males such that they were similar to sham+C males. Devocalized+C males had smaller HVc volumes than all other groups. With the use of a non-isotopic in situ hybridization procedure for the localization of the mRNA for BDNF, we found a significance decrease in the devocalized birds in the area within HVc defined by BDNF message expression. Another emerging concept is that T may stimulate song behavior and/or seasonal neuroplasticity in the song control system by acting in brain regions outside of the telencephalic song control system such as in the preoptic area or in catecholamine cell groups in the brainstem that project either directly or indirectly to these nuclei[3]. Thus T effects on neuroplasticity in the song system may be indirect in that behavioral activity stimulated by T acting in sites that promote male sexual behavior could in turn promote morphological changes in the song system. For example, the preoptic area is a well-known site for the regulation of male reproductive behaviors. Lesions to the preoptic area in starlings blocks male-typical courtship behaviors including song [8] and the volume of this area varies seasonally and is correlated with seasonal changes in song behavior [9]. Catecholaminergic cell groups in the brainstem also play an important role in the regulation of motivated behavior, including reproductive behavior. In songbirds, some of these cell groups project to telencephalic song nuclei [2] and express sex steroid hormone receptors [6], implicating them in the seasonal regulation of song. In a study designed to assess the role of these cell groups in song behavior, free-living male song sparrows (Melospiza melodia) were subjected to simulated territorial intrusion (STI), which stimulates territorial singing. The resulting fos-like immunoreactivity (FLI) was quantified in two dopaminergic regions of the brainstem that project to the song nuclei HVc-and robust nucleus of the archistriatum (RA): the area ventralis of Tsai (AVT) and the midbrain central gray (GCt). Males subjected to STI

113

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 exhibited more FLI in both of these regions than control males. In addition, FLI in both nuclei was correlated positively with the number of songs sung in response to STI. The number of flights directed at the intruder was correlated with FLI in AVT but not GCt. These results suggest a role for AVT and GCt, and thus possibly catecholamines, in the regulation of territorial behavior in songbirds. Brain nuclei in the anterior forebrain pathway of the song control system are not needed for song production but are involved in the maintenance of song and they exhibit remarkable changes in immediate early gene (IEG) induction that are modulated by social context [4]. In zebra finches (Taeniopygia guttata) it has been found that when song is directed at females there is a low level of induction of the IEG zenk in the song nucleus area X while when the song is undirected the induction is quite high[4]. We administered the noradrenergic neurotoxin DSP4 to male zebra finches prior to singing either directed or undirected song and found that males treated with DSP4 singing song directed at females had high levels of zenk protein expression characteristic of males singing undirected song. These data implicate the noradrenergic system in regulating social context effects on song system activity. In summary recent findings indicate that the expression of song behavior itself influences seasonal neuroplasticity in the avian song control system and that steroid hormone action in nuclei that project to telencephalic areas specialized for song is important in activating song behavior and associated neuroplasticity.

Reference List [1] Alvarez-Borda, B. and Nottebohm, F., Gonads and singing play separate, additive roles in new neuron recruiment., Journal of Neuroscience, 22 (2002) 8684-8690. [2] Appeltants, D., Absil, P., Balthazart, J. and Ball, G.F., Identification of the origin of catecholaminergic inputs to HVc in canaries by retrograde tract tracing combined with tyrosine hydroxylase immunocytochemistry, J.Chem.Neuroanat., 18 (2000) 117-133. [3] Ball, G.F., Riters, L.V. and Balthazart, J., Neuroendocrinology of song behavior and avian brain plasticity: Multiple sites of action of sex steroid hormones, Frontiers in Neuroendocrinology, 23 (2002) 137-178. [4] Jarvis, E.D., Scharff, C., Grossman, M.R., Ramos, J.A. and Nottebohm, F., For whom the bird sings: context-dependent gene expression, Neuron, 21 (1998) 775-788. [5] Li, X.C., Jarvis, E.D., Alvarez-Borda, B., Lim, D.A. and Nottebohm, F., A relationship between behavior, neurotrophin expression, and new neuron survival, Proceedings of the National Academy of Sciences of the United States of America, 97 (2000) 8584-8589. [6] Maney, D.L., Benard, D.J. and Ball, G.F., Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brain, Neuroscience Letters, 31 (2001) 189-192. [7] Rasika, S., Alvarez-Buylla, A. and Nottebohm, F., BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain, Neuron, 22 (1999) 53-62. [8] Riters, L.V. and Ball, G.F., Lesions to the medial preoptic area affect singing in the male European starling (Sturnus vulgaris), Horm.Behav., 36 (1999) 276-286. [9] Riters, L.V., Eens, M., Pinxten, R., Duffy, D.L., Balthazart, J. and Ball, G.F., Seasonal changes in courtship song and the medial preoptic area in male European starlings (Sturnus vulgaris), Horm.Behav., 38 (2000) 250-261. [10] Tramontin, A.D. and Brenowitz, E.A., Seasonal plasticity in the adult brain, TINS, 23 (2000) 251258.

114

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

PROGESTIN RECEPTORS: NEURONAL INTEGRATORS OF HORMONAL AND AFFERENT STIMULATION Blaustein J.D. Center for Neuroendocrine Studies, 135 Hicks Way, University of Massachusetts, Amherst, MA 01003-9271, USA, [email protected] Fax: 1.413.545.0769 There is general agreement that steroid hormones act through neuronal steroid hormone receptors functioning as transcriptional regulators, as well as by other mechanisms (e.g., membrane receptors), to induce the full expression of female sexual behaviors in rats and other rodent species. The dependence of progesterone-facilitated sexual behavior on progestin receptors (PRs) has been shown by the use of progesterone antagonists, antisense oligonucleotides to PR mRNA, and PR gene-disrupted mice. Generally, when PR levels are elevated, animals show behavioral response to progesterone, and when they are reduced, either by interference with the receptor, by lack of estradiol, or by down-regulation by progesterone itself, animals are hyposensitive or unresponsive to progesterone. While originally believed that neuronal steroid hormone receptors require binding to cognate ligand for activation, more recent evidence suggests that the receptors can be activated indirectly by other compounds, such as neurotransmitters and growth factors, acting through their own membrane receptors and intracellular signaling pathways. For example, intracerebroventricular infusion of a D1/D5 -specific, dopamine receptor agonist substitutes for progesterone in facilitating sexual behavior in estradiol-primed rats. As with progesterone facilitation of sexual behavior, progesterone antagonists, antisense oligonucleotides directed at the PR mRNA, or PR gene disruption block this, suggesting that dopamine agonists facilitate sexual behavior by ligand-independent activation of PRs. Ligand-independent activation of neuronal PRs is not limited to dopamine; facilitation of sexual receptivity by a PR-dependent process has been observed for a variety of other compounds. In order to develop a model with which to study ligand-independent activation of PRs by physiological/behavioral factors, we turned to mating-enhancement of sexual receptivity. When estradiol-treated, ovariectomized rats, which show little or no sexual receptivity, are repeatedly exposed to males (e.g., for 15 min at a time followed by 15 min away for the male), their sexual receptivity increases over the course of a few hours. Treatment with progestin antagonists before exposure to the males completely blocks this response to mating stimulation by male rats, although it does not block the lordosis response to all types of stimulation. This suggests that ligand independent activation of PRs is involved in the process by which mating-stimulation enhances sexual behavior. The fact that mating-enhancement does not occur in rats in which vaginocervical stimulation (VCS) is prevented, suggests that specific genitosensory stimulation (GSS) activates PRs thereby enhancing sexual receptivity. However, because VCS only partially substitutes for mating with a male rat, it is likely that other stimuli are involved as well. Most importantly, these results suggest that neuronal PRs can undergo ligand independent activation in vivo by a physiologically relevant stimulus, as well as pharmacologically by a D1 dopamine agonist. In other experiments, the possibility is being studied that progestin

115

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

receptors are involved in the process by which mating stimulation by the male leads to abbreviation of the period of sexual receptivity. In a study of a GSS-induced neuronal response that is apparently mediated by ligand independent activation of PRs, we found that injection of a progesterone antagonist an hour before GSS inhibits stimulation-induced Fos expression in the medial preoptic area, bed nucleus of stria terminalis and parts of the ventromedial hypothalamus, but not other areas studied. Many of the neurons that respond to GSS with Fos expression coexpress PRs. In the rostral medial preoptic area, a progestin antagonist specifically blocks Fos expression in PR-containing cells, suggesting that the Fos expression in response to afferent input is gated by the PR in some neurons. Thus, although not necessarily the only site involved, the rostral medial preoptic area is likely to be one of the sites at which GSS can activate PRs by ligand independent activation. Interestingly, Fos expression in response to odors of male rats is not coexpressed with PRs, and it is not blocked by a progesterone antagonist, so the phenomenon is specific to particular types of stimulation. Consistent with the hypothesis that dopamine is a critical factor that is released in response to mating-related stimulation, which then results in ligand independent activation of PRs, work by others suggests that mating-related stimuli induce the release of dopamine in particular forebrain areas. GSS also induces phosphorylation of the protein phosphatase-1 inhibitor, DARPP-32, which in turn has been reported to activate PRs. Although noradrenergic and dopaminergic neurons are each involved in various effects of GSS on the brain, GSS-induced Fos is completely blocked by a D1/D5 dopamine receptor antagonist in all forebrain areas studied, suggesting a critical role for dopamine and D1/D5 receptors in GSS-induced activation of PRs. Preliminary data suggest that PR-containing neurons in some forebrain areas coexpress D5 dopamine receptors. Therefore, ligand independent activation of neuronal PRs with consequent behavioral effects can be induced by elements of the social environment as well as by pharmacological means. Besides demonstrating a novel mechanism by which the social environment influences behavior, these results are relevant to other work in neuroendocrinology. It is possible that in some experiments, which have evaluated the effects of neurotransmitters on progesterone-influenced neuronal and behavioral responses, drugs may be inducing ligand independent activation of PRs with consequent PR-dependent changes in behavior and physiology.

(Supported by Senior Scientist Award MH 01312 and research grants NS 19327 and MH 56187, all from the National Institutes of Health).

116

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

HORMONAL REGULATION OF BEHAVIOR: A TALE OF TWO SEXES Swann J.M. Department of Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem Pa, 18015, USA, [email protected], Fax:1-610-758-4004 A wide variety of behaviors are differentially expressed in males and females. While the type and degree of differentiation varies with the species most animals show marked sex differences in mating behavior. Laboratory studies suggest that these differences arise from presence or absence of gonadal steroids (particularly testosterone and its metabolites) during development. Female rodents treated with gonadal steroids at birth show male typical behavior in adulthood (masculinization) while males castrated after birth fail to show male typical behaviors in adulthood (demasculinization). Thus, in rodents neonatal exposure to gonadal steroids is critical for the expression of male typical behavior in adulthood. Despite the wealth of data that supporting the role of steroids in the regulation of behavior the neural mechanisms that underlie sexual differentiation are poorly understood. A number of sexually dimorphic neural parameters have been identified. However, the significance of sex differences in neural morphology has not been determined primarily because the role of these sexually dimorphic structures in the regulation of male sex behavior is poorly defined. In contrast to the rat and mouse the pathway that regulates male sex behavior in the hamster is well documented and highly conserved. Mating behavior in the male hamsters is initiated by the detection of pheromones from females. Pheromones are perceived by chemosensory systems that project to the medial nucleus of the amygdala (Me), the bed nucleus of the stria terminalis (BNST). Both the Me and BNST project to the magnocellular medial preoptic nucleus (MPN mag). The integrity of the MPN mag is essential for mating in hamsters. Destruction of this nucleus eliminates copulation in male hamsters. Moreover several lines of evidence suggest that the MPN mag regulates behavior by integrating pheromonal and hormonal signals. We have shown that castration reduces pheromonal stimulation of the MPN mag. Testosterone treatment restores this effect in males but not in females suggesting that sex differences in input to the MPN mag underlie behavioral responses to sexually relevant pheromones. Our recent data indicate that the number and density of neurons in the MPN mag are sexually dimorphic in the hamster. Taken together our data indicate that sex differences in the cellular organization of the MPN mag. The present study correlates behavior and neuronal morphology in adult hamsters treated with neonataly with gonadal steroids and their antagonists to determine the role of neural number and function in the regulation of behavior.

117

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

SEX STEROIDS AND HUMAN BEHAVIOR: PRENATAL ANDROGEN LEVELS INFLUENCE GENDER-TYPICAL BEHAVIOR IN CHILDHOOD Hines M. Behavioural Neuroendocrinology Research Unit, Department of Psychology, City University, Northampton Square, London, UK, EC1V 0HB. [email protected]. Fax: 44 (0) 20 7040 8947. Gonadal steroids, particularly androgens, exert powerful influences on development of the mammalian brain and behavior. These influences are seen in numerous species ranging from rodents to non-human primates, and involve not only reproductive behaviors, but also other behaviors that show sex differences, meaning that they differ on the average for males and females of the species. In both rats and rhesus macaques, the behaviors influenced by hormones include rough-and-tumble play in juvenile animals. In both species, males show more rough-and-tumble play than females, and females treated with androgen prenatally show increased rough-and-tumble behavior [1]. Similar influences on human neural and behavioral development have been harder to establish, largely because experimental manipulation of hormones during human development is unethical. Most of the available information has come from so-called “experiments of nature”, where individuals experience unusual hormone environments during early development for other reasons, for example because they suffer a genetic disorder or because their mothers were prescribed hormones during pregnancy for medical reasons. Females exposed to higher than normal levels of androgen prenatally, because of the genetic disorder, congenital adrenal hyperplasia (CAH), show increased male-typical play as children. They are more likely than either unaffected female relatives, or matched controls, to play with toys typically chosen by boys (e.g., cars, trucks, guns) and less likely to play with toys typically chosen by girls (e.g., dolls, cosmetics). They also show increased preferences for males as playmates, and for male-preferred activities, such as rough, active, outdoor play [2]. These effects have been seen in a number of studies in a variety of countries, including the U.S.A., Canada, Germany, Sweden and the U.K. The findings could suggest that androgen alters basic processes of neural development in a manner similar to that seen in other mammals, and that androgen-induced neural effects underlie the changes in childhood play behavior. However, those skeptical of this conclusion point out that girls with CAH typically are born with virilized genitalia (e.g., fused labia and an enlarged clitoris) and that this could lead to alterations in the socialcognitive mechanisms thought to underlie childhood gender role behavior. Parents reinforce girls and boys differently for playing with toys like dolls, and cognitive processes involved in the development of gender identity lead children to adopt behaviors that they are told are appropriate for their sex [3]. From these perspectives, girls with CAH may be socialized differently or may develop a weaker female gender identity compared to other girls, and this, rather than a hormonal influence on the developing brain, could result in more maletypical childhood behavior [4]. Here I present two studies that argue against social-cognitive explanations of associations between prenatal androgen and alterations in childhood gender role behavior. The first relates normal variability in maternal testosterone during pregnancy to childhood

118

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

play behavior [5], and the second demonstrates sex differences in preferences for children’s toys in a species of non-human primate [6]. Study 1 used data for 679 children from the Avon Longitudinal Study of Parents and Children (ALSPAC), a longitudinal, population sample of children born in a geographically defined region (Avon, England) during a specified time period (April 1, 1991 to December 21, 1992). All pregnant women in the geographical area were notified about the study, and approximately 90% participated, representing 13,998 pregnancies and 14,138 offspring. When children were 3 1/2 years of age, a parent, usually the mother, completed the Preschool Activities Inventory, a standardized questionnaire assessing children’s sex-typed toy, playmate and activity preferences. Based on these scores, we selected six groups of children for further study: 1.128 highly masculine boys; 2.113 highly masculine girls; 3.112 highly feminine boys; 4.118 highly feminine girls; 5.102 randomly selected boys; and 6.106 randomly-selected girls. Testosterone was then measured in maternal blood samples that had been taken during pregnancy (M, SD = 16, 8 weeks gestation). Among girls, but not boys, maternal testosterone during pregnancy related linearly to gender role behavior (p < .001). In study 2, we introduced boy-prefered toys (a car, a ball), girl-prefered toys (a doll, a cooking pot) and neutral toys (a book, a stuffed dog) into enclosures housing a total of 44 male and 44 female vervet monkeys (cercopithecus aethiops sabaeus). The animals were videotaped while the toys were in their enclosures and simultaneous audiotapes described the identifying markings on the animals as they interacted with the toys. Tapes were scored subsequently by raters who did not know the sex of individual animals. Male monkeys spent more time than females with the boy-prefered toys (p < .05), female monkeys spent more time than males with the girl-prefered toys (p < .005), and there were no sex differences in contact with the neutral toys. None of the girls in Study 1 was born with virilized genitalia. Thus, this mechanism could not account for associations between their behavior and maternal testosterone levels during pregnancy. Similarly, the animals in Study 2 had no prior experience with sex-typed toys. Thus, social-cognitive mechanisms thought to explain the acquisition of sex-typed toy preferences in children cannot explain the sex-typed toy preferences seen in these nonhuman primates. Taken together, results of these two studies reinforce the conclusion that androgen is one of the factors influencing children’s acquisition of sex-typed behavior.

Reference List 1. R.W. Goy, B.S. McEwen, Sexual Differentiation of the Brain, MIT Press, Cambridge, MA, 1980. 2. M. Hines, Sexual differentiation of human brain and behavior, in: D.W. Pfaff, A.P.Arnold, A.M. Etgen, S.E. Fahrbach, R.T. Rubin (Eds.), Hormones, Brain and Behavior, Academic Press, New York, 2002, pp. 425-462. 3. S. Golombok, R. Fivush, Gender Development, Cambridge University Press, Cambridge, U.K., 1994. 4. A. Fausto-Sterling, Myths of Gender, Basic Books, New York, 1992. 5. M. Hines, S. Golombok, J. Rust, K.J. Johnston, J. Golding, the ALSPAC Study Team, Testosterone during pregnancy and gender role behavior of pre-school children: A longitudinal, population study, Child Development 73 (2002) 1678-1687. 6. G.M. Alexander, M. Hines, Sex differences in responses to children’s toys in non-human primates (cercopithecus aethiops sabaeus), Evolution and Human Behavior 23 (2002) 467-479.

119

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

THE AROMATASE KNOCKOUT MOUSE PROVIDES NEW EVIDENCE THAT ESTRADIOL IS REQUIRED DURING DEVELOPMENT IN FEMALES FOR A NORMAL EXPRESSION OF SOCIO-SEXUAL BEHAVIORS IN ADULTHOOD Bakker J.1, Honda S.2, Harada N.2 and Balthazart J.1 1

Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Liege, B 4020 Liege, Belgium ;2Division of Molecular Genetics, Fujita Health University, Toyoake, Aichi, Japan. The classic dogma of sexual differentiation is that testosterone secreted by the testes promotes the development of a male brain whereas a female brain develops in the absence of any sex steroid action. This assumption has been based on the finding that male rats castrated at birth showed female-typical levels of lordosis when treated with estradiol and progesterone in adulthood suggesting that the neural mechanisms that control later femaletypical sexual behavior normally develop perinatally in females without the need for any sex steroid stimulation [1, 2]. In addition, the ovaries have been shown to be functionally quiescent during perinatal development thereby leaving little evidence for an active contribution of ovarian secretions to female neural development. However, the possible importance of estrogens in the development of the female brain was first suggested by Toran-Allerand [3] who found that estradiol promoted neurite outgrowth from fetal hypothalamic explants of both sexes. Further support for an active role of estradiol in female neural development stems from reports that neonatal administration of anti-estrogen reduced the later ability of ovariectomized female rats to show lordosis in response to estradiol and progesterone [4] and that prenatal treatment with the aromatase blocker, ATD, reduced the ability of adult treatment with low doses of estradiol to activate sexual behavior in ovariectomized female ferrets [5]. The recent introduction of the aromatase knockout (ArKO) mouse [6] which is deficient in aromatase activity due to a targeted mutation in the CYP19 gene allowed us to readdress the hypothesis that the normal femaletypical differentiation of brain and behavior requires perinatal exposure to estradiol [7]. Female mice of three different genotypes, i.e. wild type (WT), heterozygous (HET), and homozygous (ArKO), were ovariectomized in adulthood and subsequently tested for odor preferences (choice: intact male versus estrous female) in a Y-maze. When treated with testosterone, ArKO females spent significantly less time sniffing odors (both volatile and non-volatile) from either male or female stimuli compared to WT and HET females. These behavioral differences could have reflected differences in activation of olfactory investigation by estradiol since testosterone treatment presumably generated higher levels of estradiol biosynthesis in WT and HET than in ArKO females. Therefore, to distinguish between activational and organizational effects of estradiol on olfactory investigation, the same ovariectomized female subjects were tested again for odor preferences after receiving estradiol treatment for several weeks in adulthood. The deficits in olfactory investigation of volatile odors by ArKO females persisted after estradiol treatment, suggesting that the normal, female-typical differentiation of the capacity of the main olfactory system to respond to volatile odors may depend on the presence of estrogenic stimulation at some earlier point during development. By contrast, estradiol-treated ArKO females spent as

120

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

much time as WT and HET females investigating soiled bedding of gonadally intact males and estrous females. These findings suggest that the accessory olfactory system is functional in ArKO females. The display of lordosis in reponse to mounts from a stimulus male was severely impaired in ARKO females that were treated with estradiol and progesterone. The same hormone treatment was, however, effective in inducing lordosis behavior in WT and HET females. It seems unlikely that the deficit in lordotic responsiveness seen in ArKO females resulted somehow from a defeminizing action of testosterone exposure during development. ArKO females that received either testosterone or testosterone + estradiol treatment showed significantly lower levels of male-typical sexual behavior (mounts and intromission-like movements) in tests with an estrous female than either WT or HET controls. Such an outcome would not have occurred had ArKO females been exposed perinatally to high levels of testosterone capable of masculinizing their coital capacity. In summary, it seems most likely that the reduced lordotic potential of ArKO females resulted from a perinatal deprivation of estrogenic stimulation that disrupted the normal development of the neural mechanisms controlling the expression of this behavior in adulthood. The neural mechanisms affected may include the main olfactory system in that ArKO females also showed severe impairments in olfactory investigation of volatile body odors from conspecifics. Thus, the concept that the female brain develops in the absence of any hormonal stimulation should be reconsidered.

References List 1. Grady KL, Phoenix CH, Young WC. Role of the developing rat testis in differentiation of the neural tissues mediating mating behavior. J Comp Physiol Psychol 1965; 59: 176-182. 2. Feder HH, Whalen RE. Feminine behavior in neonatally castrated and estrogen-treated male rats. Science 1965; 147: 306-307. 3. Toran-Allerand CD. Sex steroids and the development of the newborn mouse hypothalamus and preoptic area in vivo: implications for sexual differentiation. Brain Res 1976; 106: 407-412. 4. Döhler KD, Hancke JL, Srivastava SS, Hofman C, Shrine JE, Gorski RA. Participation of estrogens in female sexual differentiation of the brain: neuroanatomical, neuroendocrine, and behavioral evidence. In De Vries GJ, De Bruin JPC, Uylings HBM, Corner MA, eds. Progress in Brain Research, vol 61, Sex differences in the brain: the relation between structure and function. Amsterdam: Elsevier, 1984: 99-117. 5. Baum MJ, Tobet SA. Effect of prenatal exposure to aromatase inhibitor, testosterone, or antiandrogen on the development of feminine sexual behavior in ferrets of both sexes. Physiol Behav 1986; 37: 111-118. 6. Honda S, Harada N, Ito S, Takagi Y, Maeda S. Disruption of sexual behavior in male aromatasedeficient mice lacking exons 1 and 2 of the Cyp19 gene. Biochem Biophysical Res Communications 1998; 252: 445-449. 7. Bakker J, Honda S, Harada N, Balthazart J. The aromatase knockout mouse provides new evidence that estradiol is required during development in the female for the expression of socio-sexual behaviors in adulthood. J Neurosci 2002; 22: 9104-9112.

121

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

LONG TERM AND SHORT TERM AROMATASE INHIBITIONS RESULT IN SLOW AND RAPID ALTERATION OF PAIN Evrard H.C. and Balthazart J. Center for Cellular and Molecular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Liège, 17 Place Declour, B-4020 Liège, Belgium; email: [email protected]; Fax: 32-4-366.59.70 The enzyme aromatase catalyses the conversion of androgens into estrogens in various tissues including the brain (i.e. mainly the hypothalamus). Recently, we observed that, besides the hypothalamus, aromatase immunoreactivity is present in numerous neuronal structures (perikarya and/or fibers) in most sensory nuclei of the hindbrain and in the sensory (dorsal) horns of the spinal cord in Japanese quail and rats [2-4]. Interestingly, in rats and, to a lesser extent, in quail, these areas contain nuclear estrogen receptors and their activity is controlled by estrogens at least in part through relatively slow, presumably genomic, mechanisms [1, 6, 9]. These anatomical data raise the possibility that estrogens produced locally could control sensitivity by a local action in the dorsal horns of the spinal cord and/or in brain sensory nuclei. Through a focus on the spinal cord We thus initiated a research program to assess the impact of spinal aromatization on pain. In the present communication, we describe a first set of results suggesting the existence of slow and rapid controls of pain by aromatization. Pain tolerance was quantified in quail by a hot water test in which a foot of the bird is immerged in a 53°C water-bath and the experimenter records the latency of foot withdrawal in seconds [5]. In a first set of experiments, castrated male quail were implanted subcutaneously with capsules that were empty (CX), filled with 17 beta estradiol (E2; CX+E2) or testosterone (T; CX+T). Two weeks later, latency of withdrawal was measured in each bird and mean latency of each group was compared to a mean latency (baseline) previously recorded in intact males (i.e. sexually mature males exposed to a long day photoperiod and having a high plasma concentration of T) [5]. CX birds displayed a significantly longer latency (16.0 ± 2.5 s) than intact ones (3.8 ± 0,7 s). E2 and T similarly restored the baseline latency (CX+E2 = 5.8 ± 1.9 s; CX+T = 4.2 ± 0.7 s). To assess whether this similarity was due to a conversion of T into E2 by aromatase, all birds received a daily i.p. injection of VOROZOLE (non-steroidal aromatase inhibitor; VOR; 3 mg/kg) or its vehicle (VHC) for 10 days and were tested 24 hours after the last injection. VOROZOLE significantly increased latency of withdrawal in CX+T (VOR: 12.7 ± 1.5 s; VHC: 3.05 ± 0.3) but had no effect in CX+E2 (VOR: 3.3 ± 0.5; VHC: 2.7 ± 0.4). After about 15 days, the effect of VOROZOLE disappeared. A subsequent 10 days treatment with TAMOXIFEN (estrogen receptor antagonist; TAM; 16 mg/kg) significantly blocked the effect of both E2 (CX+E2, TAM: 16.6 ± 3.4; VHC: 3.3 ± 0.3 s) and T (CX+T, TAM: 15.8 ± 3.4 s; VHC: 4.2 ± 1.7 s). Again, this effect disappeared only after about 15 days. Besides their relatively slow effects on transcription (hours to days), estrogens rapidly activate intracellular signal transduction cascades through their binding to membrane receptors (seconds to minutes) [7]. In a second set of experiments, we assessed whether a tonic inhibition of spinal aromatase is able to rapidly influence pain latencies in intact male quail. Birds were implanted with a permanent cannula in the spinal cord at the level where

122

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

the foot nerves enter in the dorsal horn. Microinjections through this type of cannula have been shown to induce only local effects around the site of injection [8]. One week after surgery, birds were submitted to the hot water test just before they received a microinjection of VOROZOLE (15 µg/5µl) or its vehicle in the spinal cord. They were tested again 1, 5 and 30 minutes after the injection. One minute after injection, VOROZOLE significantly increased the foot withdrawal latency (VOR: 12.2 ± 2.9 s; VHC: 2.8 ± 0.7 s). This effect further increased after 5 minutes (VOR: 16.1 ± 2.2 s; VHC: 4.5 ± 2.2 s) and totally disappeared after 30 minutes (VOR: 1.9 ± 0.3 s; VHC: 1.8 ± 0.5 s) contrary to what had been observed for the daily i.p. injection. A treatment with 1,4,6androstatriene-dione-3,17 (ATD, steroidal aromatase inhibitor; 15 µg/5µl) produced similar effects. Taken together, the present data suggest that estrogens could control pain, and thus possibly other sensory modalities, through two different mechanisms. On the one hand, E2 may exert long-term effects on pain processes through the activation of nuclear receptors. Accordingly, a repeated treatment with VOROZOLE or TAMOXIFEN may disrupt these transcriptional effects of E2 that require then a relatively long period to recover from the pharmacological treatments. Our previous immunocytochemical data in quail suggest that these effects could depend, at least in part, on the spinal aromatization of androgens into estrogens and on the activation of nuclear estrogens receptors found in the vicinity of aromatase in the spinal dorsal horns. On the other hand, pain processes in intact birds may also be subjected to rapid changes driven by estrogens produced in a tonic fashion locally in the spinal cord. This would explain why a brief inhibition of spinal aromatase resulting in a rapid diminution of the local estrogen concentration could disrupt within minutes the pain control mechanisms. The effects of estrogens within one minute after their application are now known to involve the activation of intracellular transduction cascades and could thus depend on the binding of estrogens to membrane rather than nuclear receptors [7]. The mechanisms through which spinal aromatase and estrogens influence pain should be clarified in future studies. Supported by : MH50388, ARC99/04-241, FRFC 2.4555.01.

Reference list: 1. H.C. Evrard, Localization of estrogen receptors in the sensory and motor areas of the spinal cord in Japanese quail (Coturnix japonica), J. Neuroendocrinol., (2002) in press. 2. H. Evrard, M. Baillien, A. Foidart, P. Absil, N. Harada, J. Balthazart, Localization and controls of aromatase in the quail spinal cord, J. Comp. Neurol., 423 (2000) 552-64. 3. H. Evrard, J. Balthazart, Localization of estrogen-synthase and estrogen receptor immunoreactive cells in the descending nucleus of the trigeminal nerve in Japanese quail, Eur. J. Neurosci. 12 suppl 11 (2000) 145. 4. H.C. Evrard, J. Balthazart, Localization of estrogen-synthase (aromatase) in the rat spinal cord, Abst. Proc. 31st Ann. Meet. Soc. Neurosci. (2001). 5. H.C. Evrard, J. Balthazart, The assessment of nociceptive and non-nociceptive skin sensitivity in the Japanese quail (Coturnix japonica), J. Neurosci. Methods, 116 (2002) 135-46. 6. R.B. Fillingim, Sex, Gender, and Pain, IASP Press, Seattle, (2000). 7. M.J. Kelly, O.K. Ronnekleiv, Rapid membrane effects of estrogen in the central nervous system, in Hormones, Brain and Behavior, D.W. Pfaff, Elsevier Science: New York (2002) 361-380. 8. R. Nadeson, C.S. Goodchild, Antinociceptive role of 5-HT1A receptors in rat spinal cord, Br. J. Anaesth., 88 (2002) 679-84. 9. P.J. Shughrue, M.V. Lane, I. Merchenthaler, Comparative distribution of estrogen receptor-a and -b mRNA in the rat central nervous system, J. Comp. Neurol., 388 (1997) 507-525.

123

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

GONADECTOMY AFFECTS HORMONAL, BEHAVIOURAL AND NEURONAL RESPONSES TO REPETITIVE NOCICEPTIVE STIMULATION IN MALE RATS Aloisi A.M., Ceccarelli I. and Fiorenzani P. Department of Physiology, Section of Neuroscience and Applied Physiology, University of Siena, Via Aldo Moro, 2; 53100 Siena, Italy e-mail: [email protected]; Phone +390577234103, Fax+390577234037 In humans and experimental animals, testosterone and estradiol are the main gonadal hormones in males and females, respectively. Sex differences have repeatedly been observed in chronic pain syndromes in both humans and animals with females showing an higher incidence; it is likely that the gonadal hormones are responsible for these differences. To examine the role of male gonadal hormones on repetitive nociceptive stimulation, we studied male rats, half of them gonadectomized (GDX) and half intact (INT). Starting from the third week after surgery, they were subjected to the formalin test once a week for three weeks (50 microliters 5% formalin injected s.c. in the dorsum of the hind paw: right, left and right). Formalin induced behavioural responses (licking duration, flexion duration and paw-jerk frequency) were recorded and analysed for each of the three tests. One week after the last test, animals were perfused, and the brains were removed and processed for c-Fos immunohistochemistry. For all three behavioural responses, two-way analyses of variance showed significant differences between GDX and INT animals depending on the test considered (first, second or third). Indeed, although the GDX and INT groups showed a similar amount of formalininduced behavioural responses during the first behavioral test; during the second and third sessions pain responses showed a sort of adaptation in INT animals, not present in the GDX ones, resulting in lower levels of pain responses during the third test in INT animals with respect to GDX animals. Regarding c-Fos immunohistochemistry of the arcuate nucleus of the hypothalamus and the paraventricular nucleus of the thalamus, we found a higher number of c-Fos labelled cells in formalin-treated GDX animals than INT ones. Plasma corticosterone levels determined at the end of the tests were higher in GDX animals than INT ones. Plasma Testosterone levels were drastically decreased by gonadectomy. In conclusion, these data suggest that male gonadal hormones play a key role in inhibiting the behavioural and neuronal (c-Fos) responses to repeated nociceptive stimulation. This support the hypothesis that the lower incidence of chronic pain syndromes in males could be due to the presence of these hormones.

124

MONDAY, 24th February 14.00 - 17.00 Posters’ Exhibition

Posters’ Exhibition: Non Classical Mechanisms of Action • Birzniece V., Zhu D., Lindblad C., Turkmen S., Johansson I.-M., Wahlström G.a and Bäckström T. (Umeå, Sweden, EU) Acute allopregnanolone tolerance • Cascio C., Guarneri R., Russo D., Guarneri M., Piccoli F. and Guarneri P. (Palermo, Italy, EU) Retinal steroidogenic machinery: expression and activity of P450scc, P450c17, 3βHSD, P450aromatase • Díaz M., Morales M., Ropero A.B., Nadal A. and Alonso A. (Tenerife, Spain) Acetylcholine-induced calcium signals in Gt1-7 cells are acutely modulated by 17β-estradiol via a membrane-related mechanism • Haage D., Druzin M., Johansson S. (Umeå, Sweden, EU) Presynaptic action of the neurosteroid allopregnanolone • Herd M.B. and Belelli D. (Dundee, UK, EU) Modulation Of GABAA receptor mediated synaptic transmission by neurosteroids and benzodiazepines in the rat central amygdala • Hill M., Havlíková H. and Parízek A. (Prague, Czech Republic) Steroid GABA modulators in human serum, formation and transport between maternal and fetal compartment • Milman A., Weizman R., Peter Y., Paz L., Schreiber S., Gavish M. and Pick C.G. (Tel Aviv, Israel) Effect of the pregnane-related GABA-active steroid alphaxalone on mice performance in the staircase test • Mitchell E.A. And Belelli D. (Dundee, U.K, EU) Analgesic neurosteroids and inhibitory synaptic transmission in the spinal cord dorsal horn • Petralia S.M. and Frye C.A. (Albany, NY, USA) progestins in the ventral tegmental area may mediate lordosis, in part, by actions at dopamine type 1 receptors, cAMP and DARPP-32 • Rosellini R.A., Rhodes M.E, Svare B. and Frye C.A. (Albany, NY, USA) Testosterone’s hedonic effects may involve metabolism to 3α-diol and actions at GABAA receptors in the nucleus accumbens • Shi D. and. Tasker J.G (Tulane, Louisiana, USA) Rapid glucocorticoid effects on synaptic activity in hypothalamic magnocellular neurons • Strömberg J., Lundgren P. and Bäckström T. (Umeå, Sweden, EU) progesterone metabolites interact with the GABA-mediated chloride flux in opposite directions • Vardy A.W. and Lambert J.J. (Dundee, UK, EU) Neurosteroid modulation of cortical synaptic GABAA receptors: a putative role for protein kinase C • Wang M., Zorumski C.F., Mennerick S. and Bäckström T. (Umeå, Sweden, EU) Recent developments in structure-activity relationships for steroid modulator of GABAA receptors: 3beta-hydroxypregnane steroids are pregnenolone sulfate-like GABAA-receptor antagonists • Wong C.G.T., Wang Y.T., Mielke J.G., Persad V. and Snead III O.C. (Toronto, Canada) Progesterone increases cell surface GABAB receptors by inhibiting endocytosis

• Zhu D., Wang M.D., Bäckström T. and Wahlström G. (Umeå, Sweden, EU) Evaluation and comparison of the pharmacokinetic and pharmacodynamic properties of allopregnanolone and pregnanolone at induction of anaesthesia in the male rat

2nd International Meeting STEROIDS AND NERVOUS SYSTEM

Villa Gualino, TORINO, Italy. February 22-26 2003 ACUTE ALLOPREGNANOLONE TOLERANCE Birzniece V., Zhu D., Lindblad C., Turkmen S., Johansson I.-M., Wahlström G. a and Bäckström T. Department of Clinical Science, Obstetrics and Gynecology, a Department of Pharmacology, Umeå University Hospital, SE-901 85 Umeå, Sweden; [email protected], fax 09013 09 53.

Allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) acts as a positive modulator of the GABAA receptor and has anxiolytic, hypnotic and anticonvulsant effects. In women, there are situations when tolerance to prolonged progesterone (allopregnanolone) exposure can occur (e.g., the luteal phase of the menstrual cycle or pregnancy). During chronic treatment with other GABAA receptor active substances tolerance gradually develops, resulting in hypofunction of the brain GABA system. The aim was to detect if acute tolerance develops to allopregnanolone and if changed expression of any GABAA receptor subunit is involved in tolerance development. Allopregnanolone (3 mg/ml in 10% 2hydroxypropyl-beta-cyclodextrin) was infused intravenously 4 mg/kg/min in male Sprague-Dawley rats. By continuous EEG recording the amount of allopregnanolone needed to reach the criterion “silent second” (SS – burst suppression for 1 second or more) was identified. After different time intervals (first SS, 30 min or 90 min of anesthesia) the last infusion period to SS was followed by decapitation and samples from blood, different brain regions, muscle and fat tissue were analyzed for allopregnanolone content. In situ hybridization was performed for the GABAA receptor subunits α1, α2, α4, α5, α6, β2 and δ in different brain regions, analysis was by grain counting over individual neurons. The dose of allopregnanolone needed to maintain anesthesia was significantly higher (p<0.001) in the time period 65-85 min (0.98±0.04 mg/kg/min) compared with the period 10-30 min (0.67±0.03 mg/kg/min), meaning development of acute tolerance. Allopregnanolone concentrations were increased in the 90 min group in serum, hippocampus and midbrain regions. In animals with 90 min of anaesthesia an increase in the GABAA β2 subunit mRNA was detected in the CA3 and DG regions of the dorsal hippocampus, compare with the 30 min group. For the α4 subunit, a decrease in mRNA expression was detected in the VPM nucleus of thalamus in the 90 min group, compared with first SS group. The individual increases in the dose of allopregnanolone (mg/kg/min) needed to retain the silent second from the period 10-30 min of anaesthesia to the period 65-85 min is negatively correlated with the mRNA expression of the GABAA receptor α4 subunit in the VPM of thalamus. No significant changes in mRNA expression between the different groups for the GABAA receptor subunits α1, α2, α4, α5 and δ were found in the dorsal hippocampus or for the α4 subunit in the ventral hippocampus and dorsal raphe region. Neither were any changes detected for α1, α2, α5, β2 and δ subunit mRNA in the thalamus, hypothalamus, cortical regions, nor β2 in the amygdala, or α4 and α6 in the cerebellum. However, further analysis of data are performed. Changes in the GABAA β2 subunit mRNA expression in the dorsal hippocampus and α4 subunit mRNA expression in the VPM of thalamus might be important in acute allopregnanolone tolerance development. This work was supported by an EU Regional fund Objective 1 grant.

127

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

RETINAL STEROIDOGENIC MACHINERY: EXPRESSION AND ACTIVITY OF βHSD, P450AROMATASE P450SCC, P450C17, 3β Cascio C.1, Guarneri R.1, Russo D.1, Guarneri M.2, Piccoli F.2 and Guarneri P.1 1

Istituto di Biomedicina e Immunologia Molecolare, I.B.I.M - C.N.R, Via Ugo La Malfa 153, 90146 – Palermo, Italy, Fax: 39-091-6809548; e-mail: [email protected]; 2Istituto di Neuropsichiatria, Facoltà di Medicina e Chirurgia, Università di Palermo, Italy.

These last two decades have been characterized by several discoveries that have strengthened the importance of interaction between steroids and the nervous system. There are now clear evidence proving that the nervous system possesses the complete enzymatic machinery for the synthesis of steroids termed neurosteroids, and showing implications of steroids and/or neurosteroids in stress, seizures, anxiety, unipolar and postpartum depression, memory and cognition, in peripheral nerve regeneration and in neurodegenerative events [1-3, 6, 8]. Especially, the characterization of steroidogenic enzymes in several structures of CNS has allowed to identify CNS as a real steroidogenic site, other than a target for steroids, with a high degree of complexity in the expression and activities of enzymes, similarly reported in endocrine tissues [1, 10, 11]. Interestingly, steroid enzymes have been found to be differently distributed among brain regions, thus suggesting a close relationship between steroid product and specialization of brain area function [7]. In this context, we have been involved in studying the characterization of synthesis and function of steroids in the retina which is a readily accessible CNS structure to the in vitro and in vivo experimental manipulation. The retinal explant, in fact, maintains the integrity of membrane surrounding living cells, even after prolonged incubations, and allows to obviate any interference from blood-circulating steroid hormones. Using this model, we have previously demonstrated the synthesis from cholesterol of pregnenolone and pregnenolone sulphate and detected 17α-hydroxypregnenolone, 17αhydroxyprogesterone, DHEA, DHEAS, PROG, desoxycorticosterone, THDOC, 3αhydroxy-5α-dyhydroprogesterone, and 17β-estradiol [4, 5]. We have also found that cytochrome P450scc is mainly located at ganglion cells that carry visual information from retina to the brain [5]. Their role in visual function has been also proved in that levels of pregnenolone, DHEA and 17β-estradiol are higher in the dark than in the light, and changes in their amount and in THDOC levels are found after dark-adaptation [4]. The functional importance of these neurosteroids has been lately underscored by findings of their role in excitotoxic-mediated retinal cell death, but also in other CNS structures, as neurodegenerative (pregnenolone sulphate) and neuroprotective (DHEA/S and 17βestradiol) agents [2, 6]. In the present study, we investigated mRNA and protein expression of P450scc, P450c17, 3βHSD and P450aromatase in the rat retina. The results reveal that all mRNAs are expressed in the retina and appear to be identical to those in adrenal gland. However, the different ratio we observed in protein expression and activity of these enzymes appear to indicate complex regulatory mechanisms. The highest amount of P450scc protein and pregnenolone synthesized when compared to those of the other enzymes appears to be consistent with a constitutive upregulatory mechanism for P450scc enzyme. Thus, the retina as well as other CNS structures possesses the 128

2nd International Meeting STEROIDS AND NERVOUS SYSTEM

Villa Gualino, TORINO, Italy. February 22-26 2003 complete machinery for steroid production, the importance of which in retinal cellular environment has been in part underscored but surely it deserves important prospective for understanding the implication of neurosteroidogenesis at both physiological and pathological levels.

Reference list 1. Baulieu E.E., Robel P., Schumacher M. Neurosteroids: beginning of the story. Int Rev Neurobiol. 46 (2001)1-32. 2. Cascio C., Guarneri R., Russo D., De Leo G., Guarneri M., Piccoli F., Guarneri P. A caspase-3dependent pathway is predominantly activated by the excitotoxin pregnenolone sulphate and requires early and late cytochrome c release and cell-specific caspase-2 activation in the retinal cell death. J. Neurochem. (2002) in press. 3. Garcia-Segura L.M., Azcoitia I., and DonCarlos L.L. (2001). Neuroprotection by estradiol. Prog. Neurobiol. 63, 29-60. 4. Guarneri P. Neurosteroids in retina: synthesis and neuronal function. In Genazzani AR, Petraglia F, Purdy RH, editors. The brain: source and target for sex steroid hormones. New York, The Parthenon Publishing Group, 1996, pp. 63-81. 5. Guarneri P., Guarneri R., Cascio C., Pavasant P., Piccoli F., Papadopoulos V. Neurosteroidogenesis in rat retinas. J Neurochem 63 (1994)86-96. 6. Guarneri P., Russo D., Cascio C., De Leo G., Piccoli F., Guarneri R. Induction of neurosteroid synthesis by NMDA receptors in isolated rat retina: a potential early event in excitotoxicity. Eur J Neurosci. 10 (1998)1752-63. 7. Kohchi C., Ukena K., Tsutsui K. Age- and region-specific expressions of the messenger RNAs encoding for steroidogenic enzymes p450scc, P450c17 and 3beta-HSD in the postnatal rat brain. Brain Res 801 (1998)233-8. 8. Lapchak P.A. and Araujo D.M. Preclinical development of neurosteroids as neuroprotective agents for the treatment of neurodegenerative diseases. Int Rev Neurobiol 46 (2001)379-9. 9. Schumacher M., Akwa Y., Guennoun R., Robert F., Labombarda F., Desarnaud F., Robel P., De Nicola A.F., Baulieu E.E. Steroid synthesis and metabolism in the nervous system: trophic and protective effects. J Neurocytol 29 (2000) 307-26. 10. Stoffel-Wagner B. Neurosteroid metabolism in the human brain. Eur J Endocrinol 145 (2001) 669-79. 11. Strömstedt M. and Waterman M.R. Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain. Mol Brain Res 34 (1995)75-88.

129

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ACETYLCHOLINE-INDUCED CALCIUM SIGNALS IN GT1-7 CELLS ARE β-ESTRADIOL VIA A MEMBRANE-RELATED ACUTELY MODULATED BY 17β MECHANISM Díaz M.+, Morales M.*, Ropero A.B.†, Nadal A.† and Alonso A.* +

Laboratory of Animal Physiology, Department of Animal Biology, Faculty of Biology, 38206 Tenerife, Spain. *Laboratory of Cellular Neurobiology, Department of Physiology, University of La Laguna, School of Medicine, 38071 Tenerife, Spain. † Department of Physiology, Institute of Bioengineering, Miguel Hernández University, Alicante, Spain Correspondence to: Dr. Mario Díaz, Laboratory of Animal Physiology, Department of Animal Biology, Faculty of Biology, 38206 Tenerife, Spain. Tel.: +34 922 318343 Fax: +34 922 318311 E-mail: [email protected]

Hypothalamic LHRH neurons constitute the final common pathway for the central control of reproduction. LHRH secretion is subjected to complex regulation by several neurotransmitters. Acetylcholine is thought to be one of the neural regulators of LHRH neurons acting both through muscarinic and nicotinic receptors [1]. To date, the precise molecular mechanisms of regulation remain elusive due, in part, to the scattered pattern of LHRH cells distribution in the basal forebrain. In this sense, the GT1-7 cell line shares most of the functional characteristics of normal LHRH neurons and has been used as a valuable model to study the mechanisms of LHRH secretion [2]. Previous results obtained in our laboratory have shown that GT1-7 cells do respond to ACh with increases in intracellular calcium through activation of muscarinic receptors. Muscarinic activation triggers a transient calcium release from intracellular stores, which eventually leads to LHRH secretion. It has been solidly established that estradiol is an important feedback regulator of LHRH release [3]. Therefore, we have studied the possible modulation of acetylcholine response by estradiol (E2) in GT1-7 cells. [Ca2+]i measurements were performed in cells loaded with fluo-3 AM by confocal microscopy as described elsewhere [4]. Our results demonstrate that acetylcholine-induced calcium signals were modulated by short-term preincubation (2-3 min) with E2 (10 pM-1µM) as well as by the estradiolperoxidase conjugate (E-HRP, 10 nM). Both, the latency of the effect and the response to the membrane impermeant conjugate, suggested a membrane-mediated mechanism. The existence of specific membrane binding sites for E2 was further explored by using E-HRP and estradiol-BSA-fluorescein isothiocyanate (E-BSA-FITC) conjugates. Binding of these compounds to the plasma membrane was blocked by preincubation with E2, but not by 17β-E2, ICI182,780, tamoxifen or progesterone, indicating that the putative estrogen membrane binding site expressed in GT1-7 cells displays a pharmacological profile different from those of canonical α and β estrogen receptors [5]. Emerging evidence have revealed that acute estrogen effects may be mediated by the rapid generation of intracellular signals, often implying second messengers generation [6]. We thus investigated the possible involvement of second messengers in E2-induced responses on GT1-7 cells using different agents known to alter cyclic nucleotides metabolism, including dibutyryl cAMP (dBcAMP), forskolin (FK), 8-bromoguanosine 3’,5’ cyclic monophosphate (8-Br-cGMP) and sodium nitroprusside (SNP). Application of 8-Br-cGMP also reduced ACh-induced maximal [Ca2+]i, while neither db-cAMP, FK, or SNP exerted any effect. We conclude that physiological concentrations of E2 affect different mechanisms of ACh-induced Ca2+

130

2nd International Meeting STEROIDS AND NERVOUS SYSTEM

Villa Gualino, TORINO, Italy. February 22-26 2003 transients in GT1-7 cells in a rapid manner, in a way that is consistent with the activation of a plasma membrane receptor and the initiation of a downstream signalling cascade that might lead to cGMP production by a membrane-associated guanylyl cyclase. Also, these results are indicative of an acute mechanism by which estrogen might modulate the response of LHRH neurons to a cholinergic presynaptic input.

(Supported by SAF2001-3614-C03-01/02).

References List 1. L.Z. Krsmanovic, N. Mores, C.E. Navarro, S.A. Saeed, K.K. Arora, K.J. Catt. Muscarinic regulation of intracellular signaling and neurosecretion in gonadotropin-releasing hormone neurons, Endocrinology 139 (1998) 4037-4043. 2. P.L. Mellon, J.J. Windle, P.C. Goldsmith, C.A. Padula, J.L. Roberts, R.I. Weiner. Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis, Neuron 5 (1990) 1-10 3. D.K. Sarkar, G. Fink. Luteinizing hormone releasing factor in pituitary stalk plasma from long-term ovariectomized rats: effects of steroids, J. Endocrinol. 86 (1980) 511-524. 4. A. Nadal, J.M. Rovira, O. Laribi, T. Leon-Quinto, E. Andreu, C. Ripoll, B. Soria Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor, FASEB J. 12 (1998) 13411348. 5. G.G. Kuiper, B. Carlsson, K. Grandien, E. Enmark, J. Häggblad, S. Nilson, J.K. Gustafsson. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta, Endocrinology 138 (1997) 863-870. 6. A. Nadal, M. Díaz, M.A. Valverde. The estrogen trinity: membrana, cytosolic and nuclear effects, NIPS 16 (2001) 251-255.

131

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PRESYNAPTIC ACTION OF THE NEUROSTEROID ALLOPREGNANOLONE Haage D.a, Druzin M.a,b and Johansson S.a a

Department of Integrative Medical Biology, Section for Physiology, Umeå University, S901 87 Umeå, Sweden; [email protected] b Laboratory of Ionic Channels of Cell Membranes, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia.

In the present study the mechanism underlying the effect of the endogenous neurosteroid 3α-hydroxy-5α-pregnane-20-one (allopregnanolone) on the frequency of spontaneous GABA release was investigated by recording the inhibitory postsynaptic currents (sIPSCs) in neurons from the medial preoptic nucleus (MPN) of rat. Acutely dissociated neurons with functional adhering nerve terminals were studied by perforatedpatch recording under voltage-clamp conditions. In a normal extracellular solution with a [K+]o of 5 mM, 2.0 µM allopregnanolone caused a clear increase in sIPSC frequency. However, the effect declined rapidly with increased [K+]o which should lead to a depolarization of the presynaptic membrane but not the postsynaptic membrane which was held in the voltage-clamp mode. At high [K+]o allopregnanolone had an opposite effect and reduced the sIPSC frequency. Further, the effect of allopregnanolone was also strongly dependent on the external Cl- concentration ([Cl-]o). In a reduced [Cl-]o (40 mM, but with a standard [K+]o of 5 mM), expected to change the equilibrium-potential of Cl- to a more positive value, the effect on sIPSC frequency was larger than that in the standard [Cl-]o of 146 mM. The dependence of the effect of allopregnanolone on [K+]o was also altered by the reduction in [Cl-]o. The effect of allopregnanolone in low [Cl-]o was reversed at a [K+]o which corresponded to the eqiulibrium potential for Cl-. Finally, the GABAA receptor agonist muscimol also potentiated the sIPSC frequency. Altogether, the results suggest that allopregnanolone exerts a presynaptic effect by increasing the presynaptic Clpermeability, most likely via GABAA receptors.

132

2nd International Meeting STEROIDS AND NERVOUS SYSTEM

Villa Gualino, TORINO, Italy. February 22-26 2003 RECEPTOR MEDIATED SYNAPTIC MODULATION OF GABAA TRANSMISSION BY NEUROSTEROIDS AND BENZODIAZEPINES IN THE RAT CENTRAL AMYGDALA Herd M.B. and Belelli D. Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee, United Kingdom, DD1 9SY, E-mail: [email protected], Fax: +44 (0)1382 667120 A number of naturally occuring neurosteroids are now established as potent facilitators of GABAA receptor mediated neurotransmission, and display a variety of centrally depressant behavioural effects [1]. Thus, it is an intriguing prospect that centrally synthesised neurosteroids may act as endogenous anticonvulsant or anxiolytic agents. Recently, the central amygdala, a brain region strongly implicated in the generation of fear and anxiety, has been shown to mediate the anxiolytic action of the neurosteroid 5alphapregnan-3alpha-ol-20-one (5alpha,3alpha; [2]). However, the neurosteroid sensitivity of synaptic GABAA receptors located in the amygdala is, as yet, unknown. Using the whole cell patch clamp technique, we have investigated the effects of 5alpha,3alpha upon GABAA mediated synaptic currents in rat amygdaloid slices. At a holding potential of –60mV, in the presence of 500nM tetrodotoxin and 2mM Kynurenic acid, bicuculline sensitive miniature inhibitory postsynaptic currents (mIPSCs) were recorded from the central nucleus of the amygdala (CeA). Thus, at a temperature of 35°C, CeA mIPSCs displayed a relatively large mean peak amplitude of –94 ± 3pA and a mean charge transfer of -940 ± 36fC (n = 66). Analysis of synaptic current time course revealed a mean 10-90% rise time of 0.59 ± 0.02ms, with averaged mIPSCs decaying in a biexponential manner, resulting in a mean weighted decay time constant (tauw) of 10 ± 0.3ms. Application of a physiological concentration of 5alpha,3alpha (100nM) had no effect on mIPSC rise time, peak amplitude, charge transfer or tauw (n = 5). Likewise, 300nM 5alpha,3alpha produced no significant alteration of CeA mIPSC properties. However, high concentrations of the neurosteroid (1,3 and 10microM) produced a concentration dependent prolongation of tauw (n = 4-6). In addition, application of high concentrations of 5alpha,3alpha produced a clear enhancement in mIPSC peak amplitude in some, but not all cells, indicating that, at some CeA synapses, incomplete receptor occupancy may occur upon action potential independent neurotransmitter release. This was supported by application of the benzodiazepine agonist flunitrazepam, which, unlike high concentrations of 5alpha,3alpha does not have complicating GABAA-mimetic effects. Thus, 1µM flunitrazepam significantly enhanced the mean peak amplitude of CeA mIPSCs (mean enhancement = 29 ± 8%, n = 10). In summary, synaptic GABAA receptors in the CeA display a reduced sensitivity to neurosteroid action when compared with other brain regions such as the hippocampus (i.e the CA1 [1]). The reasons underlying the reduced sensitivity are currently being investigated. Furthermore, the relatively large peak amplitude of mIPSCs and their enhancement by flunitrazepam suggests that some CeA GABAA-ergic synapses may contain a large number of receptors which are not fully saturated upon vesicular neurostransmitter release. 133

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

Supported by: Anonymous Trust and MRC Senior Fellowship (DB)

Reference List 1. Lambert,J.J., Belelli,D., Harney,S.C., Peters,J.A., and Frenguelli,B.G., Modulation of native and recombinant GABA(A) receptors by endogenous and synthetic neuroactive steroids, Brain Res. Brain Res. Rev., 37 (2001) 68-80. 2. Akwa,Y., Purdy,R.H., Koob,G.F., and Britton,K.T., The amygdala mediates the anxiolytic-like effect of the neurosteroid allopregnanolone in rat, Behav. Brain Res., 106 (1999) 119-125.

134

2nd International Meeting STEROIDS AND NERVOUS SYSTEM

Villa Gualino, TORINO, Italy. February 22-26 2003 STEROID GABA MODULATORS IN HUMAN SERUM, FORMATION TRANSPORT BETWEEN MATERNAL AND FETAL COMPARTMENT

AND

Hill M.1, Havlíková H.1 and Parízek A.2 Institute of Endocrinology, Prague, 2Clinics of Gynecology and Obstetrics, First Medical School, Charles University, Prague, Czech Republic E-mail: [email protected]

1

The discovery of oxytocin down-regulation by allopregnanolone[1-3] as the identification of changes in GABA-receptor affinity highlight the role of neuroactive steroids in human reproduction while reversing effect of sulfation on steroid GABA-activators shows importance of their conjugates. Although the origin of 5α, 3α/β- isomers in maternal compartment was described several years ago[4], the formation of their 3α/β,5β- isomers is still doubtful. To give some new details, time profiles of pregnanolone isomers (Pals), i.e. epipregnanolone (3β-hydroxy-5β-pregnan-20-one, P3β,5β), allopregnanolone (3α-hydroxy5α-pregnan-20-one, P3α,5α), pregnanolone (3α-hydroxy-5β-pregnan-20-one, P3α,5β), isopregnanolone (3β-hydroxy-5α-pregnan-20-one, P3β,5α), and pregnenolone were followed in serum of 15 women around parturition as in corresponding samples of umbilical serum at delivery using a GC/MS[5] (Pals) or RIA[6] (Preg). The maternal blood was collected in 5 steps. The first and the second collection was performed at cervical dilatation 3 and 11 cm, respectively, while the third, fourth and fifth collection, was completed after lapses of 1 hour, 1 day and 5 days from delivery. The results were evaluated using canonical correlations. The variables were transformed to rank order values from 0 to 1 due to severe non-symmetry in the distribution as the non-constant variance. Firstly, the correlations were evaluated between first sets of variables comprising the levels of the individual Pals in the samples of maternal serum from the 2st 3nd 4rd and 5th collection and the second set including each time the levels of all Pals and pregnenolone near delivery. The time of the second collection was close to the collection of umbilical serum. Secondly, the canonical correlations were computed between sets of variables comprising the levels of the individual Pals and the corresponding set of pregnenolone levels in maternal serum, both in all stages of the experiment. No correlation was detected between 3β, 5α/β-isomers in maternal serum and the steroids in umbilical serum, while the both 3α, 5α/β- isomers in maternal serum correlated with P3α,5α in umbilical serum (Tab.1). No correlation was observed between P3α,5α and pregnenolone. P3α,5α in maternal serum negative correlated with P3α,5β in umbilical serum. P3β,5β and Preg∆ 5 (r1=0.936, Wilks λ=0.009, χ2=39.8, p<0.04) correlated within the maternal serum. The correlation as markedly lower maternal serum levels of epipregnanolone in comparison with other Pals and 3 times higher proportion of 3α/β,5β- Pals together with 5 times higher proportion of pregnenolone in the fetal compartment [7] may indicate a direct formation of epipregnanolone from pregnenolone. As documented by the coefficients for 1 st canonical variable of the second set the best correlations of P3α,5α in umbilical serum with P3α,5α and P3α,5β in maternal serum were found the 1st day after delivery probably due to rising variance during rapid changes of steroid levels near delivery. The strong correlation even 1 day after delivery indicates gradual decrease of allopregnanolone due to low rates of clearance of its precursor 5α-dihydroprogesterone (5αDHP) [8]. In conclusion, a significant correlation between fetal and maternal compartment was clearly indicated only in allopregnanolone. In the other pregnanolone isomers, as in the pregnenolone, no equivalent correlations were found. The results also show that epipregnanolone in the maternal compartment may originate from maternal pregnenolone, while the presence of pregnanolone in maternal serum is connected with the transport of 5αDHP between the compartments. The findings are of interest in connection with timing of parturition and the role of pregnanolone isomers as GABA-receptor modulators.

135

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

Eigenvalue

Canonical Correlation

Wilks λ

χ2

D.F.

Eigenvalue

Canonical Correlation

Wilks λ

χ2

D.F.

P-Value

0.703

0.838

0.103

20.4

20 0.431

1 0.951

0.975

0.013

39.1

20

0.006

2

0.529

0.727

0.347

9.5

12 0.658

2 0.582

0.763

0.266

11.9

12

0.454

3

0.242

0.492

0.736

2.8

6 0.839

3 0.321

0.567

0.638

4.1

6 0.670

4

0.028

0.168

0.972

0.3

2 0.879

4 0.061

0.247

0.939

0.6

2 0.754

P-Value

st

canonical variable of the first set (umbilical) 5 P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? Preg ? -0.220

1.644

-1.020 0.404

Coefficients for 1

st

-0.369

canonical variable of the second set (maternal)

Allopregnanolone (P3 α ,5 α)

Coefficients for 1

Number

Number 1

Coefficients for 1

st

canonical variable of the first set (umbilical) 5 P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? Preg ? 0.027

1.299 -0.716 -0.082 0.039

Coefficients for 1

st

canonical variable of the second set (maternal)

11cm 1 Hour 1 Day 5 Days

11cm 1 Hour 1 Day 5 Days

-0.040

0.159

1.074

-0.126

0.006

1

0.953

0.976

0.030

31.7

20

0.047

2

0.263

0.513

0.626

4.2

12

0.979

3

0.107

0.327

0.850

1.5

6

0.962

4

0.049

0.221

0.951

0.5

2

0.798

Coefficients for 1

st

canonical variable of the first set (umbilical) 5 P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? Preg ? 0.135

0.670

-0.191 -0.108 0.453

Coefficients for 1

st

canonical variable of the second set (maternal)

Isopregnanolone (P3 β ,5 α )

Pregnanolone (P3 α ,5 β )

Epipregnanolone (P3 β ,5 β)

Table 1: Canonical correlations between time profiles of pregnanolone isomers in maternal serum and the levels of pregnanolone isomers and pregnenolone in umbilical serum at delivery.

-0.008 0.786

0.283

1

0.730

0.854

0.131

18.3

20

0.570

2

0.347

0.589

0.486

6.5

12

0.889

3

0.207

0.455

0.744

2.7

6

0.851

4

0.061

0.247

0.939

0.6

2

0.753

Coefficients for 1

st

canonical variable of the first set (umbilical) 5 P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? P3 ? ,5 ? Preg ? 0.270

0.441 -0.119 -0.168 0.557

Coefficients for 1

st

canonical variable of the second set (maternal)

11cm 1 Hour 1 Day 5 Days

11cm 1 Hour 1 Day 5 Days

-0.060 -0.091 1.175

0.324

-0.086

0.398

1.010

-0.317

References List 1. Brussaard AB, Wossink J, Lodder JC et al., Progesterone-metabolite prevents protein kinase Cdependent modulation of gamma-aminobutyric acid type A receptors in oxytocin neurons. Proc Natl Acad Sci U S A, 2000; 97: 3625-30. 2. Brussaard AB and Koksma JJ, Short-term modulation of GABAA receptor function in the adult female rat. Prog Brain Res, 2002; 139: 31-42. 3. Leng G and Russell JA, Coming to term with GABA. J Physiol (Lond), 1999; 516 (Pt 2): VI. 4. Dombroski RA, Casey ML, and MacDonald PC, 5-Alpha-dihydroprogesterone formation in human placenta from 5alpha-pregnan-3beta/alpha-ol-20-ones and 5-pregnan-3beta-yl-20-one sulfate. J Steroid Biochem Mol Biol, 1997; 63: 155-63. 5. Hill M, Parizek A, Bicikova M et al., Neuroactive steroids, their precursors, and polar conjugates during parturition and postpartum in maternal and umbilical blood: 1. Identification and simultaneous determination of pregnanolone isomers. J Steroid Biochem Mol Biol, 2000; 75: 23744. 6. Hill M, Lukac D, Lapcik O et al., Age relationships and sex differences in serum levels of pregnenolone and 17-hydroxypregnenolone in healthy subjects. Clin Chem Lab Med, 1999; 37: 43947. 7. Hill M, Bicikova M, Parizek A et al., Neuroactive steroids, their precursors and polar conjugates during parturition and postpartum in maternal blood: 2. Time profiles of pregnanolone isomers. J Steroid Biochem Mol Biol, 2001; 78: 51-7. 8. Dombroski RA, Casey ML, and MacDonald PC, The metabolic disposition of plasma 5 alphadihydroprogesterone (5 alpha-pregnane-3,20-dione) in women and men. J Clin Endocrinol Metab, 1993; 77: 944-8.

136

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EFFECT OF THE PREGNANE-RELATED GABA-ACTIVE STEROID ALPHAXALONE ON MICE PERFORMANCE IN THE STAIRCASE TEST Milman A., Weizman R., Peter Y., Paz L., Schreiber S., Gavish M. and Pick C.G. Department of Anatomy and Anthropology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv; Israel, [email protected], FAX +972-3-640-8287

We evaluated the modulatory effect of the GABA-active neurosteroid alphaxalone on the staircase test behavior of mice. The mouse staircase paradigm is a relatively simple and efficient procedure for screening anxiolytic agents. It combines step-climbing, which serves as an index of locomotor activity, and rearing, which serves as an index of anxiety and exploratory behavior. Results were compared with the benzodiazepine alprazolam, the GABAA agonist muscimol and the peripheral steroids corticosterone and progesterone. Alphaxalone and alprazolam reduced rearing activity in a dose-dependent manner, at doses that did not suppress climbing. The rearing-suppression effect of alprazolam, but not of alphaxalone, was blocked by the benzodiazepine antagonist flumazenil. No such dissociation between the effect on rearing and climbing was obtained with muscimol, and both activities were suppressed, in a flumazenil-insensitive pattern, at high doses. Corticosterone and progesterone did not affect the behavior of the mice. The lack of sensitivity of both phenobarbital and alphaxalone to flumazenil indicates that neither agents act via the benzodiazepine recognition site at the GABAA receptor complex.

137

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ANALGESIC NEUROSTEROIDS AND INHIBITORY TRANSMISSION IN THE SPINAL CORD DORSAL HORN

SYNAPTIC

Mitchell E.A. and Belelli D. University of Dundee, Department of Pharmacology and Neuroscience, Ninewells Hospital and Medical School, Dundee, Scotland, U.K., DD1 9SY, email: [email protected] Fax: +44 (0)1382 667120 Neurosteroids have emerged as potent and selective endogenous modulators of the GABAA receptor. Consistent with their ability to enhance neuronal inhibition, neurosteroids display clear anxiolytic, anticonvulsant and analgesic properties [2]. To date, research has focused on neurosteroid activity in brain regions putatively involved in their anxiolytic and anticonvulsant actions (e.g. hippocampus). However, little attention has been devoted to neurosteroid modulation of inhibitory synaptic transmission in centres of the central nervous system likely to be implicated in their analgesic properties (e.g. spinal cord). In particular, the superficial dorsal horn is a key centre for the integration and transmission of nociceptive stimuli from the periphery to the brain. Accordingly, inhibitory transmission in this region, mediated by both GABAA and glycine receptors, has a crucial role in regulating overall pain transmission [2]. Here, we have applied the whole-cell patch-clamp technique to a spinal cord slice preparation to characterise inhibitory synaptic transmission in the superficial layers (laminae I and II) of the dorsal horn of Sprague-Dawley rats (P15-P22; either sex). Both glycine and GABAA receptor-mediated miniature inhibitory post-synaptic currents (mIPSCs) were recorded from spinal neurones at 35°C, at a holding potential of –60mV and in the presence of 500nM TTX and 2mM kynurenic acid. Glycine and GABAA receptormediated mIPSCs were pharmacologically isolated using bicuculline methobromide (10 micromolar) and strychnine hydrochloride (500nM), respectively. mIPSCs were characterised with respect to peak amplitude, rise time, charge transfer and decay time course. The two populations of events could be clearly distinguished by their distinct decay kinetics. Thus, in lamina II, the majority of strychnine-sensitive, glycine receptormediated mIPSCs decay in a mono-exponential fashion with a decay time constant tau of 3.50 ± 0.22 (n=7). In contrast, the GABAA receptor-mediated mIPSC decay was considerably slower and best described by a bi-exponential function with a mean weighted decay time constant tauw of 21.9 ± 1.97 (n=3). Preliminary experiments investigating the effect of analgesic neurosteroids at inhibitory synapses in the dorsal horn, demonstrate that certain neurosteroids selectively act at GABAA receptors whereas others are active at both glycine and GABAA receptors. Acknowledgements: Supported by the MRC (DB and EAM). Reference List 1. Lambert,J.J., Belelli,D., Harney,S.C., Peters,J.A., and Frenguelli,B.G., Modulation of native and recombinant GABA(A) receptors by endogenous and synthetic neuroactive steroids, Brain Res. Brain Res. Rev., 37 (2001) 68-80. 2. Dickenson,A.H., Chapman,V., and Green,G.M., The pharmacology of excitatory and inhibitory amino acid-mediated events in the transmission and modulation of pain in the spinal cord, Gen. Pharmacol., 28 (1997) 633-638.

138

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

PROGESTINS IN THE VENTRAL TEGMENTAL AREA MAY MEDIATE LORDOSIS, IN PART, BY ACTIONS AT DOPAMINE TYPE 1 RECEPTORS, cAMP AND DARPP-32 Petralia S.M.1 and Frye C.A.1-3 The University at Albany-SUNY, Behavioral Neuroendocrinology Laboratory, 1Departments of Psychology, 2Biological Sciences, and 3Center For Neurobiology, 1400 Washington Avenue Albany, NY, 12222 USA. [email protected]. 518-442-4247. Progestins may modulate sexual behavior of female rodents through actions at dopamine (DA) type 1 receptors (D 1) in the ventral tegmental area (VTA). There are few intracellular progestin receptors [2,3], but many D 1 receptors in the VTA [9]. Activating or blocking D 1 receptors by intracerebroventricular (ICV) or intravenous infusions of agonists or antagonists respectively increases and decreases lordosis of estradiol benzoate (EB) + P-primed rats [1,4,10]. In Experiment 1, we examined whether P’s actions in the VTA to facilitate lordosis involve activation of D 1 receptors. EB-primed (10 µg) rats with bilateral guide canulae to the VTA (AP: -5.3; ML: +0.4; DV: -7.0) were infused with SCH23390 (0 or 100 ng), given systemic P (0, 50, or 100 µg; SC) 30 mins later, and 2.5 hrs later were tested for lordosis. As figure 1 shows, P-facilitated lordosis was blocked by infusions of SCH23390, but not vehicle, t o the VTA. Thus, D 1 receptors may be substrates though which P, in the VTA, modulates lordosis of. In the VTA, progestin’s actions for lordosis may be adenosine 3’,5’-monophosphate (cAMP)-dependent. D 1 receptors are coupled to adenylyl cyclase [13], such that activating them can increase cAMP [5-8]. ICV infusions of P, the D 1 agonist, SKF38393, or the cAMP analogue 8-bromo-cAMP produce maximal lordosis responses in EB-primed rats [11]. In Experiment 2, we examined whether 8-bromo-cAMP infusions to the VTA increase lordosis of rats undergoing natural estrous termination. Cycling rats, late in behavioral estrus, were pretested for lordosis, infused bilaterally with 8-bromo-cAMP (0 or 200 ng) to the VTA and posttested for lordosis 30 mins later. As figure 2 illustrates, 8-bromo-cAMP, but not vehicle infusions, to the VTA increased lordosis of rats tested at estrous termination. These findings suggest P’s actions to facilitate lordosis in the VTA may be enhanced by cAMP. However, in this experiment naturally cycling rats were utilized, therefore, we cannot discern whether 8bromo-cAMP augmented the effects of EB or P. In Experiment 3, we examined whether P facilitated lordosis is enhanced by 8-bromo-cAMP (100 ng) infusions to the VTA of ovariectomized rats. EB (5 µg) + P (0 or 100 µg; SC)-primed rats were pre-tested for lordosis, infused with 8-bromo-cAMP (0 or 100 ng) to the VTA, and post-tested 30 minutes later. As Fig 3 shows, P’s actions in the VTA are required for infusions of 8-bromo-cAMP to increase lordosis of EB-primed rats. Thus, P’s actions in the VTA may involve cAMP-dependent processes. Progestins’ actions in the VTA to facilitate lordosis may involve phosphorylation of the DA- and cAMP-regulated phosphoprotein (DARPP-32). P-mediated lordosis is attenuated in DARPP-32 knockout, but not wildtype mice and, in rats, following ICV infusions of DARPP-32 anti-sense oligonucleotides [11]. DARPP-32 has been localized to the VTA [12]. In Experiment 4, we tested whether, in the VTA, P-facilitated lordosis is dependent upon DARPP-32 phosphorylation. Rats were primed with EB (5 µg) and infused with anti-sense oligonucleotides (4 nM) against DARPP-32 mRNA or vehicle to the VTA at hrs 0 and 24. At hr 44, rats were pre-tested for lordosis, infused with P (1 µg) to the VTA, and re-tested. As Fig 4 shows, the facilitative effects of P to the VTA of EB-primed rats was attenuated by DARPP32 anti-sense, but not vehicle, infusions to the VTA. Thus, some of P’s actions in the VTA may involve phosphorylation of DARPP-32. In sum, P-dependent lordosis was attenuated by inhibiting D 1 activity, enhanced by increasing cAMP, and reduced by blocking DAPRR-32 phosphorylation. Together, these data suggest progestins, in the VTA, facilitate lordosis by initiating a signaling pathway that includes activation of D 1 receptors, up-regulation of cAMP, and enhancement of DARPP-32 phosphorylation.

139

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 Supported by NSF grants IBN 98-96263 and DBI 00-97343. Reference List [1] E.M. Apostolakis, J. Garai, C. Fox, C.L. Smith, S.J. Watson, J.H. Clark, B.W. O'Malley, Dopaminergic regulation of progesterone receptors: brain D5 dopamine receptors mediate induction of lordosis by D1-like agonists in rats, J. Neurosci. 16 (1996) 4823-4834. [2] C.A. Frye, The role of neurosteroids and non-genomic effects of progestins and androgens in mediating sexual receptivity of rodents, Brain Res. Brain Res. Rev. 37 (2001) 201-222. [3] C.A. Frye, The role of neurosteroids and nongenomic effects of progestins in the ventral tegmental area i n mediating sexual receptivity of rodents, Horm. Behav. 40 (2001) 226-233. [4] C.A. Frye, L.E. Bayon, J. Vongher, Intravenous progesterone elicits a more rapid induction of lordosis i n rats than does SKF38393, Psychobiol. 28 (2000) 99-109. [5] P. Greengard, A. Nairn, J. Girault, C. Ouimet, G. Snyder, G. Fisone, P. Allen, A. Fienberg, A. Nishi, The DARPP-32/protein phosphatase-1 cascade: a model for signal integration, Brain Res. Brain Res. Rev. 2 6 (1998) 274-284. [6] P. Greengard, P.B. Allen, A.C. Nairn, Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade, Neuron 23 (1999) 435-447. [7] H.C. Hemmings Jr, P. Greengard, H.Y. Tung, P. Cohen, DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1, Nature 310 (1984) 503-505. [8] H.C. Hemmings Jr, A.C. Nairn, P. Greengard. Protein kinases and phosphoproteins in the nervous system, Res. Publ. Assoc. Res. Nerv. Ment. Dis. 64 (1986) 47-69. [9] Q. Huang, D. Zhou, K. Chase, J.F. Gusella, N. Aronin, M. DiFiglia, Immunohistochemical localization of the D1 dopamine receptor in rat brain reveals its axonal transport, pre- and postsynaptic localization, and prevalence in the basal ganglia, limbic system, and thalamic reticular nucleus, Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 11988-11992. [10] S.K. Mani, J.M. Allen, J.H. Clark, J.D. Blaustein, B.W. O'Malley. Convergent pathways for steroid hormoneand neurotransmitter-induced rat sexual behavior, Science 265 (1994) 1246-1249. [11] S.K. Mani, A.A. Fienberg, J.P. O'Callaghan, G.L. Snyder, P.B. Allen, P.K. Dash, A.N. Moore, A.J. Mitchell, J. Bibb, P. Greengard, B.W. O'Malley, Requirement for DARPP-32 in progesterone-facilitated sexual receptivity in female rats and mice, Science 287 (2000) 1053-1056. [12] C.C. Ouimet, P.E. Miller, H.C. Hemmings Jr, S.I. Walaas, P. Greengard, DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. III. Immunocytochemical localization, J. Neurosci. 4 (1984) 111-124. [13] J.C. Stoof, J.W. Kebabian, Two dopamine receptors: biochemistry, physiology and pharmacology Life Sci. 35 (1984) 2281-2296.

*

50

25

75

Post-test

50

25

0 2 00 0 8-Bromo-cAMP (ng)

0 0 100 200 Progesterone Dosage (µg; SC)

Figure 1: LQs of EBprimed rats following VTA infusions of SCH23390 (striped) or vehicle (open) and 0, 100, or 200 µg P (SC). * indicates a significant difference (p<0.05) between SCH23390 and vehicle at that P dosage.

140

*

*

Figure 2: Pre-test (striped) and post-test (solid) LQs of naturally receptive rats given intra-VTA infusions of vehicle (left) or 8-BromocAMP (right; 100 ng). * indicates a significant difference (p<0.05) pre-and post-tests.

75

50

* Pre-test Post-test

25

0 0 100 Progesterone (µg; SC)

Figure 3: Pre (striped) and post-test (solid) LQs of ovx, EB + P (0 or 100 µg)primed rats administered 8-bromo-cAMP. * sign difference (p<0.05) between pre- and posttests.

Lordosis Quotient (+ SEM)

*

Pre-test

Lordosis Quotient (+SEM)

Lordosis Quotient (+SEM)

75

100

100

Vehicle SCH23390 (100 ng)

Lordosis Quotient (+SEM)

100

Pre-test Post-test

75

50

25

0 Saline

DARPP-32 Oligo

Figure 4: Pre (striped) and post (solid) test LQs of ovx, EB (10 µg)-primed rats with VTA infusions of P following intraVTA infusions of saline (left) or anti-sense oligos for DARPP-32 (right). *sign difference (p<0.05) between pre & posttest.

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

αTESTOSTERONE’S HEDONIC EFFECTS MAY INVOLVE METABOLISM TO 3α DIOL AND ACTIONS AT GABAA RECEPTORS IN THE NUCLEUS ACCUMBENS Rosellini R.A., Rhodes M.E, Svare B. and Frye C.A. The University at Albany – SUNY, Department of Psychology, 1400 Washington Avenue, Albany, NY 12222 USA, [email protected], (518) 442-4867 Androgens may be abused in part due to their ability to produce positive, hedonic effects [4]. Conditioned Place Preference (CPP) is used to examine hedonic effects of drugs [9]. Testosterone (T) can induce CPP [1,7]; however, its effects are variable. T’s metabolism by 5α-reductase to dihydrotestosterone (DHT) and by 3-oxidoreductase to 3α-androstanediol (3α-diol) may be necessary for its hedonic effects. Recent data from our lab suggest that 3αdiol may mediate T’s effects on CPP. First, systemic 3α-diol is more effective than T or DHT at producing CPP [5]. Second, when systemic T or DHT are administered in a time frame sufficient to allow for metabolism to 3α-diol, they are equally effective at producing a place preference, as is 3α-diol [5]. Third, the metabolism enzymes necessary for T to be metabolized to DHT and 3α-diol have been localized to the nucleus accumbens (NA) [2], a brain area important for hedonic effects of drugs. Finally, administration of T, DHT, or 3α-diol to the NA produces similar enhancement of CPP [6,8]. We investigated the effects of blocking androgen metabolism on CPP. Intact rats were administered T, DHT, or 3α-diol in time frames that we have previously shown produce CPP when paired with the conditioning chamber (T at 90 min; DHT at 180 min, 3α-diol at 30 min). T rats received vehicle or finasteride, a 5α-reductase inhibitor, which blocks DHT and 3α-diol formation. DHT rats received vehicle or indomethacin, a 3-hydroxysteroid dehydrogenase inhibitor, which blocks reduction to 3α-diol. 3α-diol rats received vehicle or indomethacin, which blocks 3α-diol’s back conversion to DHT. Co-administration of systemic T or DHT with a metabolism inhibitor (finasteride or indomethacin, respectively) decreased time spent on the originally non-preferred side of the conditioning chamber and whole brain 3α-diol levels compared to rats that were administered T, DHT, or 3α-diol, in conjunction with vehicle (see Figure 1 and Table 1). However, rats coadministered 3α-diol and indomethacin did not show a decrease in CPP or 3α-diol levels compared to those administered 3α-diol and vehicle (see Tables 1 and 2). Although there was a correspondence between 3α-diol levels and formation of CPP, this same pattern was not observed for T or DHT and CPP (see Table 1). Together these data suggest that 1) androgens can modulate formation of CPP, 2) inhibiting androgen metabolism to 3α-diol attenuates CPP, and 3) androgen regimen that increase levels of 3α-diol enhance CPP. In physiological concentrations, T and DHT bind readily to intracellular androgen receptors, but 3α-diol does not and has actions primarily through GABAA/benzodiazepine receptor complexes (GBRs) [3]. Experiment 2 investigated whether blocking or activating GBRs would influence 3α-diol-mediated CPP. Some rats received a high dosage of 3α-diol (1 mg, that induces CPP), with vehicle or the GBR antagonist, bicuculline to the NA prior t o being put in the conditioning chamber. Others were systemically administered a lower dosage of 3α-diol (1 mg/kg; that is insufficient to induce CPP) with vehicle or the GBR agonist, muscimol to the NA prior to being put in the conditioning chamber. As previously demonstrated, rats administered 1 mg of systemic 3α-diol showed an increase in time spent on the non-preferred side of the conditioning chamber on test day compared to rats that received vehicle (see Table 3). Co-administration of bicuculline, a GBR antagonist, to the NA, attenuated 3α-diol-induced CPP. Bicuculline administration alone had no effect on CPP, nor did vehicle administration alone. Rats administered the lower dose of 3α-diol (1 mg/kg) did not spend significantly more time on the non-preferred side of the chamber on test day compared to rats that received vehicle (see Table 3). However, administration of muscimol, a GBR agonist, to the NA together with this sub-threshold dose of 3α-diol, was sufficient to induce a CPP. Muscimol

141

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 alone was not sufficient to produce a CPP and was no more effective than vehicle at enhancing CPP. Thus, 3α-diol’s ability to mediate CPP may be due in part to actions at GBRs. Together data suggest that T’s effects on CPP may require metabolism to 3α-diol and its ability t o modulate CPP may be due in part to actions at GBRs in the NA. Acknowledgments: Funded by grants from the National Science Foundations (IBN 98-96262 and DBI-0097343) to CAF.

Reference List [1] G. Alexander, M. Packard, M. Hines, Testosterone has rewarding affective properties in male rats: implications for the biological basis of sexual motivation. Behav Neurosci 108 1994 424-428. [2] R. Celotti, R.C. Melcangi, L. Martini, The 5 alpha-reductase in the brain: molecular aspects and relation t o brain function. Front Neuroendocrinol. 13 1992 163-215. [3] G.R. Cunningham, D.J. Tindall, A.R. Means, Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor, Steroids 33 1979 261-276. [4] M. I. Fingerhood, J. T. Sullivan, M. Testa, D.R. Jasinski, Abuse liability of testosterone. J Psychopharmacol. 11 1997 59-63. [5] C.A. Frye, D. Park, M. Tanaka, R. Rosellini, B. Svare, The testosterone metabolite and neurosteroid 3alphaandrostanediol may mediate the effects of testosterone on conditioned place preference. Psychoneuroendocrinol. 26 2001 731-750. [6] C. Frye, M. Rhodes, R. Rosellini, B. Svare, The nucleus accumbens as a site of action for rewarding properties of testosterone and its 5alpha-reduced metabolites. Pharmacol Biochem Behav. 74 2002 119 [7] M. Packard, A. Cornell, G. Alexander, Rewarding affective properties of intra-nucleus accumbens injections of testosterone. Behav Neurosci. 111 1997 219-224. [8] R.A. Rosellini, B.B. Svare, M.E. Rhodes, C.A. Frye, The testosterone metabolite and neurosteroid 3alphaandrostanediol may mediate the effects of testosterone on conditioned place preference. Brain Res Brain Res Rev. 37 2001 162-171. [9] M. Scoles, S. Siegel, A potential role of saline trials in morphine-induced place-preference conditioning. Pharmacol Biochem Behav 25 1986 1169-1173. Table 1: Whole brain DHT levels (ng/g) of rats administered androgens and vehicle or a metabolism inhibitor. Experimental condition (androgen 1 mg SC and blocker or vehicle) Androgen T + Vehicle T + DHT + Vehicle DHT + 3α-diol + Vehicle 3α-diol + Levels Finasteride Indomethacin Indomethacin T (ng/g) DHT (ng/g) 3α-diol (ng/g)

8.8 6.2 3.2

1.7 2.3 0.6

1.4 7.1 3.2

1.3 7.1 1.1

1.3 3.1 3.5

1.3 3.2 3.7

Table 2: Time on non-preferred side of conditioning chamber (secs + SEM) on test day of rats administered androgen (1 mg SC) and vehicle or metabolism inhibitor. Experimental T+ T+ DHT + DHT + 3α-diol + 3α-diol + Condition Vehicle Finasteride Vehicle Indomethacin Vehicle Indomethacin Time on non- 699 + 143 438 + 76 972 + 172 486 + 198 1093 + 181 1075 + 180 preferred side Table 3: Time on non-preferred side of conditioning chamber (secs + SEM) on test day of rats administered diol or vehicle and/or intra-accumbens GBR antagonist or agonist or vehicle. Experimental Vehicle SC 3α- IntraSC 3α- IntraSC 3α-diol + Vehicle control accumbens Condition control accumbens diol diol Intramuscimol bicuculline accumbens bicuculline Time on non- 555 + 1226 + 477 + 456 + 239 + 54 557 + 89 + preferred side 409 389 353 436 402 17

142

SC 3αSC 3α-diol + Intraaccumbens muscimol 1356 + 333

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

RAPID GLUCOCORTICOID EFFECTS ON SYNAPTIC HYPOTHALAMIC MAGNOCELLULAR NEURONS

ACTIVITY

IN

Shi D. and Tasker J.G. Division of Neurobiology, Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana, USA [email protected] The present study focused on the rapid effect of glucocorticoids on the synaptic activity of rat hypothalamic magnocellular neurons. Magnocellualr neurons of the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) were recorded in coronal hypothalamic slices using the whole-cell patch-clamp technique. Bath application of dexamethasone (DEX, 1 µM, 3-5 min, n=25), caused a 34% decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs). The DEX effect was dosedependent and steroid specific. Corticosterone (1 µM, n=5) caused a similar decrease in the frequency of mEPSCs (28%), suggesting a glucocorticoid inhibition of glutamate release. Bath application of a DEX-BSA conjugate (10 µM, n=4) had a similar effect, whereas intracellular DEX application (1 µM) had no effect on mEPSCs, implicating a membrane glucocorticoid receptor. The intracellular type I and type II corticosteroid receptor antagonists, spironolactone (10 µM, n=6) and RU 486 (10 µM, n=7), respectively, failed to block the inhibitory effect of DEX on mEPSCs, consistent with actions at a membrane receptor. The DEX effect on mEPSCs was blocked by intracellular application of a Gprotein antagonist, GDP-beta-S (0.5-1 µM, n=5), in magnocellular neurons, which indicated a postsynaptic signaling mechanism and suggested the involvement of a retrograde messenger to inhibit presynaptic glutamate release. The glucocorticoid effect on mEPSCs was mimmicked by a cannabinoid receptor agonist, WIN 55,212-2 (0.5-1 µM, 36% reduction in mEPSC frequency, n=9), and was blocked by the type I cannabinoid receptor antagonist, AM251 (1 µM, n=4), implicating an endocannabinoid as the retrograde messenger in the glucocorticoid response. The present data indicate that the inhibitory effect of glucocorticoids on glutamate release is mediated by the activation of a membrane receptor and G protein signaling mechanism that leads to the release of an endocannabinoid, which then acts as a retrograde messenger to inhibit presynaptic glutamate release onto magnocellular neurons.

Supported by NIH grant NS/DK 39099.

143

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PROGESTERONE METABOLITES INTERACT WITH THE GABA-MEDIATED CHLORIDE FLUX IN OPPOSITE DIRECTIONS Strömberg J., Lundgren P. and Bäckström T. Umeå University, Dept of Clinical Science, Obstetrics and Gynecology, SE-901 85 UMEÅ, Sweden; [email protected] The progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one or allopregnanolone increases Gamma amino butyric acid (GABA)-mediated chloride conductance by GABAA receptors in a similar fashion as sedative drugs such as benzodiazepines and barbiturates. GABA is the major inhibitory transmitter in the central nervous system (CNS). Altered GABAergic function is associated with neurological and psychiatric disorders of humans, including Huntington’s chorea, epilepsy, alcoholism, schizophrenia, sleep disorders, and Parkinson’s disease. The GABA-system is also associated with premenstrual syndrome (PMS) by progesterone metabolites. The steroid effects are thought to be mediated by binding of steroids to specific sites on GABAA receptors. This alteration of GABA conductance might be an explanation for the mood variations during the female menstrual cycle. The 3beta isomer, 3beta-hydroxy-5alphapregnan-20-one (isoallopregnanolone) does not have these effects on GABAA receptors. Therefore we have studied the interaction between allopregnanolone and isoallopregnanolone in cortical homogenates from adult male Whistar rats. We measured the chloride uptake into micro sacs in presence of 10 µM GABA and various concentrations of the steroids. Isoallopregnanolone decreased the allopregnanolone and GABA mediated chloride uptake in a dose dependent manner. We also investigated if isoallopregnanolone interacts with GABA itself, or benzodiazepines and barbiturates. No interaction was found with either substance. These results indicate a possible role for isoallopregnanolone and structurally related compounds in the control of not desired GABAergic inhibition caused by high levels of allopregnanolone.

This work was supported by an EU Regional fund Objective 1 grant.

144

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

NEUROSTEROID MODULATION OF CORTICAL SYNAPTIC RECEPTORS: A PUTATIVE ROLE FOR PROTEIN KINASE C

GABAA

Vardy A.W. and Lambert J.J. Department of Pharmacology & Neuroscience, University of Dundee, Ninewells Hospital, Dundee, Scotland, DD1 9SY, [email protected] Tel: +44(0)1382 632161 Fax: +44(0)1382 667120 The GABAA receptor is responsible for the majority of rapid inhibitory synaptic transmission throughout the central nervous system. GABAA receptors are a therapeutic target for a wide range of compounds including general anaesthetics and benzodiazepines. However, certain steroidal compounds have been demonstrated to potentiate the action of GABA at the GABAA receptor [2]. These neurosteroids, such as 5alpha-pregnan-3alphaol-20-one (5alpha3alpha), are synthesized locally within the CNS [3] and are proposed to endogenously modulate the tone of inhibitory neurotransmission. To investigate the interaction of the neurosteroid 5alpha3alpha with synaptic GABAA receptors, the whole cell mode of the patch-clamp technique has been utilised to record miniature inhibitory post-synaptic GABA-ergic currents (mIPSCs) obtained from rat primary cultured cortical neurons. mIPSCs were recorded at 35°C and at a holding potential of -60mV in the continued presence of 0.5µM tetrodotoxin and 2mM kynurenic acid. These events have an average peak amplitude of -51.8 ± 4.3pA, a rise time of 0.49 ± 0.01ms and a mean weighted decay time constant (Tau) of 7.6 ± 0.7ms (n = 7). Upon application of 300nM 5alpha3alpha these events are significantly prolonged with an increased mean weighted Tau of 22.3 ± 3.8ms (n = 4, P<0.001). Additionally the neurosteroid produced an increase in peak amplitude of the synaptic event to -67.8 ± 8.5pA (n = 4, P<0.01). It is now apparent that phosphorylation of the GABAA receptor may affect function and recent reports suggest that the interaction of neurosteroids with the GABAA receptor may be additionally influenced [1]. Application of a protein kinase C (PKC) pseudosubstrate peptide (fragment 19-36), via the electrode into the cell interior prior to recording, appears to have little effect on the function of the synaptic GABAA receptor per se. These events, recorded following 10 minutes of peptide incubation, had a peak amplitude of -47.4 ± 2.5pA, a rise time of 0.44 ± 0.03ms and a mean weighted Tau of 7.0 ± 0.3ms (n = 9). However, application of 300nM 5alpha3alpha in the continued presence of the PKC inhibitor modestly, but significantly, enhances the effectiveness of the neurosteroid at the synapse with the mean weighted Tau increasing to 29.2 ± 2.6ms (n = 4, P<0.05) under these conditions. Thus, cultured cortical neurons appear to exhibit a high sensitivity to the neurosteroid 5alpha3alpha and this sensitivity is enhanced when protein kinase C is inhibited.

This work was supported by the Medical Research Council and the Commission of the European Communities, RTD Programme “Quality of Life and Management of Living Resources” QLK6-CT-200000179.

145

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 Reference List [1] J.J. Lambert, D. Belelli, S.C. Harney, J.A. Peters, B.G. Frenguelli, Modulation of native and recombinant GABA(A) receptors by endogenous and synthetic neuroactive steroids, Brain Res Brain Res Rev 37 (2001) 68-80. [2] J.J. Lambert, D. Belelli, C. Hill-Venning, J.A. Peters, Neurosteroids and GABAA receptor function, Trends Pharmacol. Sci. 16 (1995) 295-303. [3] A.G. Mensah-Nyagan, J-L. Do-Rego, D. Beaujean, V. Luu-The, G. Pelletier, H. Vaudry, Neurosteroids: Expression of Steroidogenic Enzymes and Regulation of Steroid Biosynthesis in the Central Nervous System, Pharmacol. Rev. 51 (1999) 63- 81.

146

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

RECENT DEVELOPMENTS IN STRUCTURE-ACTIVITY RELATIONSHIPS FOR RECEPTORS: 3BETASTEROID MODULATOR OF GABAA HYDROXYPREGNANE STEROIDS ARE PREGNENOLONE SULFATE-LIKE GABAA-RECEPTOR ANTAGONISTS Wang M., Zorumski C.F., Mennerick S. and Bäckström T. Dept. of Clinical Science, Section of Obstetrics & Gynecology, Umeå University, Umea, Sweden, Dept. of Psychiatry, Washington University School of Medicine, St. Louis, MO, USA In contrast to their endocrine actions, certain steroid hormones have been shown to rapidly alter brain excitability and to produce behavioural effects within seconds to minutes. Steroids can both positively and negatively modulate GABAergic neurotransmission. The steroid effects are thought to be mediated by binding of steroids to specific sites on GABAA - receptors (GABAA R). Endogenous neurosteroids have rapid actions on ion channels, particularly GABAA R, which are potentiated by nanomolar concentrations of 3 alpha-hydroxypregnane steroids (ex, allopregnanolone and pregnanolone). Previous evidence suggests that 3 beta-hydroxypregnane steroids may competitively antagonize potentiation induced by their 3 alpha-diastereomers. Because of the potential importance of antagonists as experimental and clinical tools, we characterized the functional effect of 3 beta-hydroxypregnane steroids. Although 3beta-hydroxypregnane steroids reduced the potentiation induced by 3 alplha-hydroxypregnane steroids, 3 betahydroxypregnane steroids acted non-competitively with respect to potentiating steroids and inhibited the largest degrees of potentiation most effectively. 3 beta-hydroxypregnane steroids also reduced potentiation by high concentrations of barbiturates. 3 betahydroxypregnane steroids are also direct, non-competitive GABAA R antagonists. 3 betahydroxypregnane steroids co-applied with GABA alone significantly inhibited responses to > 15 µM GABA. The profile of block was similar to that exhibited by pregnenolone sulphate, known blockers of GABAA R. This direct, non-competitive effect of 3betahydroxypregnane steroids was sufficient to account for the apparent antagonism of potentiating steroids. Mutated receptors exhibiting decreased sensitivity to pregnenolone sulphate block were insensitive to both the direct effects of 3 beta-hydroxypregnane steroids on GABAA R and to the reduction of potentiating steroid effects. At concentrations that had little effect on GABAergic synaptic currents, 3betahydroxypregnane steroids and low concentrations of pregnenolone sulphate significantly reversed the potentiation of synaptic currents induced by 3 alpha-hydroxypregnane steroids. We conclude that 3 beta-hydroxypregnane steroids are not direct antagonists of potentiating steroids but rather are non-competitive, likely state-dependent, blockers of GABAA R. Nevertheless, these steroids may be useful functional blockers of potentiating steroids when used at concentrations that do not affect baseline neurotransmission.

This work was supported by an EU Regional fund Objective 1 grant.

147

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PROGESTERONE INCREASES CELL SURFACE GABAB RECEPTORS BY INHIBITING ENDOCYTOSIS

Wong C.G.T.1, Wang Y.T.2, Mielke J.G.1, Persad V. and Snead III O.C.1 1

The Brain and Behaviour Program, Hospital for Sick Children, Toronto, Canada. Department of Medicine and Brain Research Center, Vancouver Hospital and University of British Columbia. [email protected] 2

Introduction and Rationale: Absence seizures are characterized by a paroxysmal loss of consciousness of abrupt onset and offset. A characteristic EEG pattern known as spike and wave discharge (SWD) is observed during these seizures. The GABAB Receptor (GABABR) is believed to mediate the events that lead to this SWD and consequently, absence seizures. Our lab has observed that the severity of atypical absence seizures vary as function of the estrus cycle in rats. Furthermore, we have made the observation that GABABR antagonist binding, also varies as a function of the estrus cycle [1, 2]. Seizures and GABABR binding both peak when progesterone is at it highest level. Additionally, injections of progesterone exacerbate absence seizures and increase maximal binding at the GABABR. This positive correlation between seizures, GABABR binding and hormone levels strongly suggest that changes in progesterone effect GABABR function, which ultimately manifests itself as alterations in seizure susceptibility. We hypothesize that fluctuations in GABABR binding during the estrus cycle are caused by hormone-induced changes in the intracellular trafficking of the GABABR, and that it is this perturbation of intracellular trafficking that ultimately mediates the severity of absence seizures. Progesterone Increases Cell Surface GABABR’s: Here, we report that treatment of cortical neuronal cultures with 80 nM progesterone resulted in a 24.6% increase in the number of cell-surface GABABR’s, as determined using a colourimetric assay (p < 0.008). This increase was rapid, being observed 15 minutes after progesterone was applied to the cultures. Classically progesterone is known to bind to intracellular nuclear receptor that exerts transcriptional changes. However, the progesterone effect in this study was nongenomic in nature as blockade of the nuclear receptor with RU486 did not prevent the increase in surface receptors. Additionally, immunoblotting revealed that there were no changes in GABABR protein expression levels in neuronal cultures after progesterone treatments. A metabolite of progesterone, allopregnanolone, has been intensively studied in epilepsy research because it is an allosteric modulator of the GABAAR. Thus, progesterone could affect GABABR’s via its allopregnanolone metabolite. However, inhibiting the formation of this metabolite, did not prevent the surface receptor increase. Therefore, progesterone and not alloprenanolone appears to exerts effect on the GABABR. Progesterone Inhibits Endocytosis: Progesterone could increase surface levels of the GABABR by increasing the insertion rate of receptors into the plasma membrane or by inhibiting the endocytosis of the receptor. We pre-labeled a sub-population of cell surface GABABR’s and observed their localization under basal conditions and after progesterone treatments. Under normal conditions, 30% of GABABR’s were internalized after 15 minutes at 37°C. However, after progesterone treatments, only 5% of receptors were internalized (p < 0.004), strongly suggesting that progesterone inhibited endocytosis of the

148

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

GABABR. This finding was observed using immunostaining of neuronal cultures, colourimetric assay and by biotynylation of surface receptors. Progesterone Increases GABABR Function: Progesterone effects on GABABR function were determined by using a cAMP assay. Activation of GABABR is known to inhibit forskolin-induced increases in cAMP and to potentiate isoproterenol-induced increases in cAMP. In this assay rat cortical brain tissue was used, in which control rats received saline injections and progesterone-treated rats received a 30mg/kg injection of progesterone. Progesterone injections increased GABABR-mediated inhibition of forskolininduced cAMP by 24%. Also, progesterone injections increased GABABR-mediated potentiation of isoproterenol-induced cAMP by 42%. Thus, progesterone treatments also potentiated GABABR function. Progesterone Offsets Agonist-Induce Internalization: Treatments of neuronal cultures with 500 µM GABA caused in a 30% decrease in cell-surface GABABR’s after 15 minutes. This result indicates that activation of GABABR’s results in a rapid internalization that effectively tones down GABABR-mediated signaling. Co-application of progesterone with GABA offset the agonist-induced internalization – cell surface numbers remained stable and GABAB-mediated signaling likely does not fall below appreciable levels. Our studies provides the first evidence that a sex steroid can modulate the intracellular trafficking of a neuroreceptor. Furthermore, our studies raise the possibility that a novel cellular mechanism underlies the hormonal cycling of GABABR binding and the pro-absence effects of progesterone.

This work was supported by Canadian Institute for Health Research (CIHR), the Heart and Stroke Foundation of Canada and the Bloorview Children’s Hospital Foundation. Reference List 1. Persad V.et. al, (2002) Steroids and Nervous System Abstract 2. Al-Dahan MI et. al, Brain Res. 640:33-9

149

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

EVALUATION AND COMPARISON OF THE PHARMACOKINETIC AND PHARMACODYNAMIC PROPERTIES OF ALLOPREGNANOLONE AND PREGNANOLONE AT INDUCTION OF ANAESTHESIA IN THE MALE RAT Zhu D.1, Wang M.D.1, Bäckström T.1 and Wahlström G.2 1

Department of Clinical Science, Section of Obstetrics and Gynaecology, University of Umeå, S-901 87 Umeå, Sweden. 2Department of Pharmacology and Clinical Neuroscience, University of Umeå, S-901 87 Umeå, Sweden. [email protected] We have evaluated and compared the pharmacokinetic and pharmacodynamic properties of allopregnanolone and pregnanolone at induction of anaesthesia in male rats. A threshold method was used, and the first burst suppression period of 1 s or more in the EEG was selected as the end-point after fairly slow infusions. An optimal dose of 4.0 mg kg–1 min–1 was noted for both steroids. Brain concentrations were low at low infusion rates, indicating that acute tolerance was not occurring. Significant positive correlations were noted between dose rate and serum concentrations of allopregnanolone (r = 0.94, P<0.001) and pregnanolone (r = 0.88, P<0.001). Such correlations were also seen in striatum, cerebellum, cortex and muscle for both steroids (P<0.01). Despite changing infusion rates, the concentrations of both steroids in brainstem, hippocampus and fat remained stable. Because no correlation between infusion rate and steroid concentration was noted in the brainstem and hippocampus, these two brain areas may be regarded as primary sites of action for allopregnanolone and pregnanolone. Pregnanolone concentrations in the brainstem and hippocampus were significantly higher than those of allopregnanolone, suggesting that allopregnanolone was more potent than pregnanolone in inducing anaesthesia.

This work was supported by an EU Regional fund Objective 1 grant.

150

Posters’ Exhibition: Glucocorticoids and Mineralcorticoids: Synthesis, Mechanism of Action and Effects •

Bellido I., Hansson A., Gómez-Luque A., Andbjer B. and Fuxe K. (Malaga, Spain, EU) The pituitary-adrenal axis strongly modulates the affinity of the 5-HT1A autoreceptors in the dorsal raphe nucleus and influences their responses to galanin in vitro

•

Catania C., Sotiropoulos I., Michaelidis T. and Almeida O.F.X. (Munich, Germany, EU) Glucocorticoid regulation of Alzheimer’s disease-related proteins

•

Chiavegatto S. and Nelson R.J. (Sao Paulo, Brazil) Elevated corticosterone concentrations in male mice lacking neuronal nitric oxide synthase

•

Constantinescu C.S. and Gottlob I. (Nottingham, UK, EU) Improvement in amblyopia after optic neuritis in the fellow eye treated with steroids suggests role of corticosteroids in nervous system plasticity

•

Hansson A.C., Metsis M., Sommer W., Strömberg I., Agnati L.F. and Fuxe K. (Stockholm, Sweden, EU) Corticosterone actions on the hippocampal BDNF expression are mediated by exon II and exon IV promoters

•

Mitsuyo T., Adachi N. And Arai T. (Ehime, Japan) Relationship between dopamine release and deleterious effects of dexamethasone in ischaemic rat brain

•

Taherian A.A.,. Vafaei A.A, Rashidy-pour A. (Semnan, Iran) The modulatory effects of glucocortoids on morphine addiction phenomena in mice

•

Vafaei A.A. and Rashidy-Pour A. (Semnan, Iran) Modulatory roles of glucocorticoid receptors on spatial memory in place avoidance learning in rats

•

Vlad A.G and Raica M. (Timisoara, Romania) The effect of steroid hormones on hippocampus and subependymal neuronal layer of the hypothalamus

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

THE PITUITARY-ADRENAL AXIS STRONGLY MODULATES THE AFFINITY OF THE 5-HT1A AUTORECEPTORS IN THE DORSAL RAPHE NUCLEUS AND INFLUENCES THEIR RESPONSES TO GALANIN IN VITRO Bellido I.1,2, Hansson A.1, Gómez-Luque A.2, Andbjer B.1 and Fuxe K.1 1

Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. [email protected] 2 Department of Pharmacology and Clinical Terapeutics. School of Medicine, Malaga, Spain. [email protected] Introduction Mood disorders are related to corticosteroid hormone and serotoninergic 5-HT 1A receptor dysfunctions [1]. Clinical studies suggest increased glucocorticoid levels and a hyperfunction of the hypothalamo-pituitary-adrenal axis activity [2, 3] as well as enhanced dorsal raphe 5-HT 1A autoreceptor function and blunted postsynaptic 5-HT 1A receptor signalling in depressed patients [4]. Acute hydrocortisone diminishes the postsynaptic 5-HT 1A receptor function in healthy human volunteers [5], and galanin reduces the dorsal raphe firing activity partly via 5-HT 1A autoreceptors [6] contributing to behavioural signs of depression in animals. An up-regulation of galanin receptors of the dorsal raphe nucleus has been reported in an experimental model of depression [7]. There seem to be no studies on the effects of adrenal steroids on the dorsal raphe 5-HT 1A receptor binding characteristics and their interactions with galanin. In the present paper the modulation of the 5-HT1A autoreceptors of the dorsal raphe by corticosterone and galanin has been studied in adrenalectomized rats. Material and Methods Adult male Sprague Dawley rats (n=12) were bilaterally adrenalectomized (ADX) under halothane anaesthesia. The sham animal group (n=6) underwent the same surgical procedure but only with exposure of the adrenal glands. All surgical procedures and hormone injections were performed between 7:00 and 9:00 h. Twenty four hours after adrenalectomy the rats were subcutaneously (1 ml/kg) injected with a single dose of corticosterone (10 mg/kg) or propylene glycol (hormone vehicle). The sham group was injected with propylene glycol. Four hours after the injections the animals were decapitated and their brains removed and frozen in isopentane (40ºC). Brains were cryostat sectioned for autoradiography with 3H-8OH-DPAT used t o characterize the 5-HT1A receptor agonist binding sites of the dorsal raphe nucleus (Bregma level –7.3 mm) in saturation experiments with or without porcine galanin (10 nM) modulation. Trunk blood was collected for analyses of serum corticosterone with RIA. Results and Discussion The 5-HT1A receptor agonist affinity of the dorsal raphe nucleus was markedly reduced (K d increase of +87.4%) in ADX versus the sham group. Acute corticosterone treatment of the ADX rats markedly increased the affinity of the 5-HT1A autoreceptors, especially versus the ADX alone group (table 1). The maximal receptor densities of the agonist binding sites (Bmax) were not significantly affected by adrenalectomy with (Bmax increase of 51.3%) or without (Bmax increase of 47.3%) acute corticosterone treatment. Porcine galanin did not modify the 5-HT 1A autoreceptor binding characteristics in the sham group nor in the ADX alone group. The acute corticosterone sensitized the dorsal raphe 5-HT 1A autoreceptors to the galanin effects, possibly caused by an increased gene expression of galanin receptors by the corticosterone treatment. Galanin increased the Bmax values of the 5-HT1A high affinity agonist binding sites (+64.07%) and reduced their affinity (K d increase of 248%). The “in vitro” modulation seen with galanin in the

152

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 present study is probably the result of an antagonistic interaction withing a Gal R / 5-HT 1A autoreceptor heteromeric complex of the dorsal raphe 5-HT nerve cell. Activation of the galanin receptor by galanin “in vitro” may result in an allosteric change of the 5-HT 1A autoreceptor after the corticosterone treatment, leading to reduced affinity and a reduced cross regulation by the GTP activated G-protein with the appearance of an increassed number of agonist binding sites in the high affinity state (increased Bmax). Thus, the early modulation by galanin is one of antagonism of 5-HT 1A autoreceptor regulating. The major result of this study is that acute corticosterone in a high dose can produce a marked increase in the affinity of the 5-HT1A autoreceptor binding sites of the dorsal raphe. This action may be caused by the activation of the large numbers of the glucocorticoid receptors found in the dorsal raphe cells leading to changes in gene expression of proteins controlling the affinity state of 5-HT 1A autoreceptors. Such an action by corticosterone can help explain its depressant action since enhancement of 5-HT1A autoreceptor function should lead to reduced 5-HT neuronal activity and thus to a depressive state.

Effects of adrenalectomy with or without acute corticosterone treatment on the 3H-8OHDPAT binding charateristics in the dorsal raphe nucelus of the rat. Treatment

Basal Bmax

Basal Kd

(fmol/mg of protein)

(nM)

Porcine Galanin Bmax

Change respect to value

(10 nM) Kd

with Change with respect to basal basal value (%)

(%) 7.71 ± 0.6 +24.5 -15.01 14.6 ± 2.8 +31.5 -10.2 (+87.4%)(*) ADX 1.35 ± 0.3 +64.07 (a) +248 (b) CORT (-82.6%) (*)(***) Data are mean+s.e.m., n = 5-6. Basal: One way ANOVA followed by Scheffé post-test.* P < 0.05 vs. sham . *** P < 0.001 vs. ADX. Galanin modulation: One way ANOVA repeated measures followed by Scheffé posttest. (a) P < 0.05 vs. basal. (b) P < 0.05 vs. basal. Sham ADX

46.7 ± 6.9 68.8 ± 11.2 (+47.3%) + 70.7 ± 8.9 (+51.3%)

Reference list 1. Holsboer F. (2000) The corticosteroid receptor hypothesis of depression. Neuropsychopharmacology 23 (5): 477-501. 2. Barden N., Reul J.M. and Holsboer F. (1995) Do antidepressants stabilize mood through actions on the hypothalamic-pituitary-adrenocortical system. Trends Neurosci 18 (1): 6-11. 3. Pariante C.M. and Miller A.H. (2002) Glucocorticoid receptors in major depression: relevance to pathophysiology and treatment. Biol Psychiatry 49: 391-404. 4. Van Praag H.M. (2001) Anxiety/agresión-driven depresión. A paradigm of functionalization and verticalization of psychiatry diagnosis. Prog Neuropsychopharmacol Biol Psychiatry 25 (4): 893-924. 5. Porter R.J., McAllister-Williams R.H., Lunn B.S. and Young A.H. (1998) 5-Hydroxytryptamine receptor function in humans is reduced by acute administration of hydrocortisone. Psychopharmacology 139 (3): 243-250. 6. Razani H., Díaz-Cabiale Z., Fuxe K. and Ögren S-V. (2000) Intraventricular galanin produces a timedependent modulation of 5-HT1A receptors in the dorsal raphe of the rat. NeuroReport 11 (18): 3943-3948 7. Bellido I., Díaz-Cabiale Z., Jiménez-Vasquez P., Andbjer B., Mathé A. and Fuxe K. (2002) Increased density of galanin binding sites in the dorsal raphe in a genetic rat model of depression. Neuroscience Letters 317: 101-105.

153

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

GLUCOCORTICOID PROTEINS

REGULATION

OF

ALZHEIMER’S

DISEASE-RELATED

Catania C., Sotiropoulos I., Michaelidis T. and Almeida O.F.X. Max Planck Institute of [email protected]

Psychiatry,

Kraepelinstrasse

2-10,

D80804

Munich,

Neuritic plaques or deposits of amyloid, resulting from proteolysis of amyloid precursor protein (APP), and neurofibrillary tangles, resulting from hyperphosphorylated filaments of the cytoskeleton protein tau, are the pathological hallmarks of Alzheizmer's disease (AD). Both APP and tau are intricately linked to neuronal function and survival but the factors influencing their biochemical modification is poorly understood. Since AD is associated with dysregulation of corticosteroid receptor number and function and adrenocortical secretion, we hypothesize that glucocorticoids might play either contributory or ameilorative roles in AD pathology. Specifically, we are examining whether glucocorticoids can alter the synthesis, processing and function of AD-relevant proteins in a variety of neural cell lines (e.g. PC12, SHSY5Y and NT2, as well as a PC12 line engineered to stably express human tau); specific differentiating factors are used to induce neuronal-like features in each of these cell lines. We have seen that specific activation of glucocorticoid receptors (GR) by dexamethasone (DEX) is followed by increased expression of certain tau phosphorylation- and comformationdependent epitopes in differentiated, but not undifferentiated, cells. Current studies are aimed to investigate whether, and if so which, downstream kinases may be involved; at present we focuss on cyclin-dependent kinase-5 (cdk5) and gylcogen synthase kinase 3 (GSK3) and importantly, we notice a complete abrogation of the DEX-induced alterations of tau phosphorylation after exposure to a series of specific kinase inhibitors. In light of possible cross-talk between products of APP metabolism and tau phosphorylation we also examining whether amyloid can potentiate the effects DEX on on tau phosphorylation. In parallel studies we have observed significant increases in the levels of distinct isoforms of APP following exposure of differentiated PC12 and NT2 cells to DEX and we are now analysing whether GR activation can lead to selective processing of APP into neurotrophic (soluble APP) vs. neurotoxic (insoluble amyloid).

Partly supported by the Deutsche Forschungsgemeinschaft (Al 311/7-1).

154

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 ELEVATED CORTICOSTERONE CONCENTRATIONS IN MALE MICE LACKING NEURONAL NITRIC OXIDE SYNTHASE

Chiavegatto S.* and Nelson R.J.# *Institute of Psychiatry and Heart Institute (InCor), School of Medicine, University of Sao Paulo, Eneas de Carvalho Aguiar, 44, 10 o floor, 05403-904, Sao Paulo, SP, Brazil -fax: (55) 11 30695022, [email protected]. #Departments of Psychology and Neuroscience, Ohio State University, Columbus, OH 43017 USA, [email protected]. Mice lacking the gene for neuronal nitric oxide (NO) synthase (nNOS-/-) exhibit a variety of abnormalities including enlargement of the stomach, hypertrophied urinary bladders, reduced parasympathetic tone, and nocturnal motor deficits. In addition, male nNOS-/- mice display dramatic increases in aggressive and sexual behavior [8]. Plasma androgen concentrations can affect the display of aggressive behavior in male mice, but testosterone concentrations in nNOS-/and wildtype (WT) mice are similar. Several studies of the brain mechanisms underlying aggression reveal that central serotonergic neuronal circuits are prominent in the regulation of aggression. We have recently shown that the excessive aggressiveness and impulsivity of male nNOS-/- mice are caused by selective decrements in 5-HT turnover and deficient 5-HT 1A and 5-HT 1B receptor function in brain regions regulating emotion, suggesting a relationship between 5-HT and NO systems in the aggressive behavior [2]. Because serotonergic mediation of social behavior, such as aggression and reproduction, may be affected by glucocorticoids, we determined the corticosterone and ACTH concentrations in the plasma of nNOS-/- and WT male adult mice. The twelve animals were isolated in their cages for at least one month before the assays. They were transported to the experimental room and weighted 3 h before decapitation (~1400 h; light/dark cycle: 0600-2000 h). Trunk blood was collected in chilled centrifuge tubes containing 50 µl of a 0.3M EDTA (pH 7.4) solution. Blood was centrifuged (1600 xg for 15 min) and the supernatant stored at -70 °C for subsequent determination of plasma hormone concentrations. Corticosterone and ACTH were determined in duplicate using a 125 I radioimmunoassay (RIA) kit (ICN Biomedicals, CA, USA). The intraassay variability for each hormone was < 10%, and in order to preclude interassay variability, analyses of all samples were conducted in a single run. Corticosterone was significantly increased in nNOS-/- (91.93 ± 12.85 ng/ml, SEM) as compared t o WT mice (20.33 ± 3.72 ng/ml) (p<0.001). The ACTH concentrations were not different (nNOS-/= 1158.94 ± 82.59 pg/ml, SEM and WT = 850.50 ± 162.24 pg/ml, p>0.05). Interestingly, adult male nNOS-/- mice displayed reduced body weight (nNOS-/- = 27.53 ± 0.71 g, SEM and WT = 31.50 ± 0.24 g, p<0.001). The nNOS positive cells are present throughout the hypothalamic-pituitary-adrenal (HPA) axis [9] suggesting a participation of NO in neuroendocrine modulation. Our data showing increased corticosterone concentrations in nNOS-/- mice are indicative of a physiological inhibitory role of NO upon corticosterone synthesis or secretion. This result does not seem to be mediated by the absence of nNOS during neurodevelopment of the nNOS-/- mice, because the administration of specific inhibitors for NO synthesis significantly increases plasma corticosterone concentrations, both in rats [1] and mice [6]. In fact, the steroidogenesis in zona fasciculata adrenal cells has been shown to be negatively modulated by NO donors and L-arginine-derived NO in rats [4,3]. Secretion of corticosterone from adrenal cortex is under the stimulatory control of ACTH. The present increase in corticosterone was not accompanied by significant increases in ACTH concentrations, suggesting a higher sensitivity of nNOS-/- mice adrenal cells to the stimulating effects of this hormone. On the other hand, the production of ACTH by the pituitary is also under the influence of corticosterone concentrations. When corticosterone increases, the production of ACTH decreases because of a negative feedback control. In the nNOS-/- mice, ACTH appears unresponsive to the higher levels of corticosterone, implying a disruption in the negative feedback control.

155

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 Isolated adult male nNOS-/- mice show reduced body weight, which is not observed when these mice are grouped-housed (previously reported [8]). This observation suggests that mice lacking nNOS display higher sensitivity to isolation-induced stress. In fact, nNOS activity and transcription are increased in brain regions mediating stress following various noxious stimuli [7, 11, 5]. Thus, it has been proposed that psychological and/or physiological stress causes NO release in the HPA axis, and the magnitude of activation in NOS enzyme activity varies, being higher in the adrenal cortex than in the anterior pituitary [10], in accordance to our results. In conclusion, our data from the nNOS-/- mice strongly confirm an inhibitory role for nNOSderived NO in corticosterone synthesis and further implicate the NO system in activation of the stress response. Acknowledgment: SC has fellowship from FAPESP 01/09079-1 (BRAZIL), and RJN has NIH grant MH 57760 (USA). Reference List 1. Adams ML, Nock B, Truong R, Cicero TJ. Nitric oxide control of steroidogenesis: endocrine effects of NG-nitro-L-arginine and comparisons to alcohol. Life Sci. 1992;50(6):PL35-40. 2. Chiavegatto S, Dawson VL, Mamounas LA, Koliatsos VE, Dawson TM, Nelson RJ. Brain serotonin dysfunction accounts for aggression in male mice lacking neuronal nitric oxide synthase. Proc Natl Acad Sci U S A. 2001;98(3):1277-81. 3. Cymeryng CB, Dada LA, Colonna C, Mendez CF, Podesta EJ. Effects of L-arginine in rat adrenal cells: involvement of nitric oxide synthase. Endocrinology. 1999;140(7):2962-7. 4. Cymeryng CB, Dada LA, Podesta EJ. Effect of nitric oxide on rat adrenal zona fasciculata steroidogenesis. J Endocrinol. 1998;158(2):197-203. 5. de Oliveira RM, Aparecida Del Bel E, Mamede-Rosa ML, Padovan CM, Deakin JF, Guimaraes FS. Expression of neuronal nitric oxide synthase mRNA in stress-related brain areas after restraint in rats. Neurosci Lett. 2000;289(2):123-6. 6. Giordano M, Vermeulen M, Trevani AS, Dran G, Andonegui G, Geffner JR. Nitric oxide synthase inhibitors enhance plasma levels of corticosterone and ACTH. Acta Physiol Scand. 1996;157(2):259-64. 7. Kishimoto J, Tsuchiya T, Emson PC, Nakayama Y. Immobilization-induced stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamic-pituitary-adrenal axis in rats. Brain Res. 1996;720(1-2):159-71. 8. Nelson RJ, Demas GE, Huang PL, Fishman MC, Dawson VL, Dawson TM, Snyder SH. Behavioural abnormalities in male mice lacking neuronal nitric oxide synthase. Nature. 1995;378(6555):383-6. 9. Nelson RJ, Kriegsfeld LJ, Dawson VL, Dawson TM. Effects of nitric oxide on neuroendocrine function and behavior. Front Neuroendocrinol. 1997;18(4):463-91. 10. Tsuchiya T, Kishimoto J, Koyama J, Ozawa T. Modulatory effect of L-NAME, a specific nitric oxide synthase (NOS) inhibitor, on stress-induced changes in plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in rats: physiological significance of stress-induced NOS activation in hypothalamicpituitary-adrenal axis. Brain Res. 1997;776(1-2):68-74. 11. Tsuchiya T, Kishimoto J, Nakayama Y. Marked increases in neuronal nitric oxide synthase (nNOS) mRNA and NADPH-diaphorase histostaining in adrenal cortex after immobilization stress in rats. Psychoneuroendocrinology. 1996;21(3):287-93.

156

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

IMPROVEMENT IN AMBLYOPIA AFTER OPTIC NEURITIS IN THE FELLOW EYE TREATED WITH STEROIDS SUGGESTS ROLE OF CORTICOSTEROIDS IN NERVOUS SYSTEM PLASTICITY Constantinescu C.S.* and Gottlob I. *Division of Clinical Neurology, University Hospital, Queen’ Medical Centre, Nottingham NG7 2UH, United Kingdom, and Department of Ophthalmology, Leicester-Warwick Medical School, Leicester University, Leicester Royal Infirmary, Leicester LE2 5WW, United Kingdom; e-mail: [email protected]

Amblyopia is a disorder of visual functions consisting of reduced visual acuity in the absence of organic disease, caused by deficient visual stimulation most commonly due to squint or refractive. Amblyopia is potentially reversible until up to the age of about eight years (critical period) and is usually treated with occlusion of the fellow eye to stimulate neural plasticity in the amblyopic eye. There is recent evidence for visual system plasticity extending beyond the critical period, supported by reports of improvement in visual acuity in the amblyopic eye following loss of vision in the contralateral eye. This suggests that the adult visual system exhibits sufficient plasticity to allow such improvement. We describe here improvement in visual acuity in three amblyopic patients after receiving intravenous high dose glucocorticoids for optic neuritis in the contralateral eye. In all cases the impovement occurred only after institution of steroids. We found in our records at least three cases with a history of optic neuritis not treated with steroids and amblyopia in the contralateral eye, which did not show any considerable improvement. However, the analysis of these cases is retrospective and some details may be missing. In all cases we present the improvement was sustained, even after the recovery from the optic neuritis. Because steroids affect neural plasticity we hypothesize that they facilitate and enhance visual improvement in amblyopia, This quality which is currently being tested in a controlled trial.

157

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

CORTICOSTERONE ACTIONS ON THE HIPPOCAMPAL BDNF EXPRESSION ARE MEDIATED BY EXON II AND EXON IV PROMOTERS Hansson A.C.1,3, Metsis M.2, Sommer W.3, Strömberg I.1, Agnati L.F.4 and Fuxe K.1 Departments of Neuroscience1, Medical Biochemistry and Biophysics2 and NEUROTEC3, Karolinska Institutet, 171 77 Stockholm, Sweden. Department of Biomedical Science, University of Modena, 41100 Modena, Italy4. E-mail: [email protected]; Tel.: +46-858589651 Fax: +46-8-58585785 The brain-derived neurotrophic factor (BDNF) contains four separate promoters upstream of the non-coding exons I to IV. While BDNF expression is strongly regulated by activated gluco- and mineralocorticoid receptors, it is presently unknown which of the four promoters mediate these effects. In order to study the corticosterone-mediated effects on BDNF expression independently from circulating hormone levels we used male adrenalectomized (ADX) rats, which are depleted of endogenous adrenocorticosteroid hormones. A time course experiment demonstrated that corticosterone (10 mg/kg, s.c.) induces a downregulation of BDNF mRNA levels in the hippocampus peaking at 4 h. This is closely followed by a decline in BDNF protein levels by approximately 20%. However, there was also a fast component of corticosterone action, demonstrated by an initial rise of 38% in hippocampal BDNF tissue concentration. Next we analyzed the corticosterone effect at peak time for reduction of BDNF mRNA levels in subregions of the dorsal hippocampus by means of in situ hybridization using specific riboprobes to exons I to IV and to the protein coding exon V. As expected, corticosterone significantly downregulated the levels of exon V transcripts in the CA1 by approximately 40% and in the dentate gyrus (DG) by about 70% vs the ADX group. The exon II transcripts were downregulated in the DG by 41% but were below detection limit in CA1. In contrast, BDNF exon IV transcripts were downregulated in all hippocampal subregions by 41% to 57%. BDNF exon I and exon III mRNA levels were apparently not affected in any region by the treatment. To further elucidate transcriptional mechanisms we analyzed the corticosteronemediated effects on phosphorylation of the cyclic AMP response element binding (pCREB) protein. Corticosterone significantly increased the levels of pCREB in DG by 15%. Thus, it seems that the promoter regions II and IV are involved in the corticosterone induced downregulation of the BDNF gene with the strongest effects in DG, where an involvement of pCREB in the hormonal response can take place. The present findings for the first time indicate a role of the exon IV promoter in the regulation of the BDNF gene expression in the rat brain.

158

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

RELATIONSHIP BETWEEN DOPAMINE RELEASE AND DELETERIOUS EFFECTS OF DEXAMETHASONE IN ISCHAEMIC RAT BRAIN Mitsuyo T., Adachi N. and Arai T. Department of Anesthesiology and Resuscitology, Ehime University School of Medicine, Shitsukawa, Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan; Telephone: +81-89-9605383;Telefax: +81-89-960-5386; E-mail address: [email protected] Glucocorticoids have been reported to aggravate ischaemic neuronal damage in both humans and experimental animals. The agents also change the activity of the central dopaminergic system. Because excess release of neurotransmitters is closely related to the outcome of ischaemic neuronal damage, the authors evaluated the effects of dexamethasone on dopaminergic release and histologic outcome. Changes in the extracellular concentrations of monoamines and their metabolites in the striatum produced by occlusion of the middle cerebral artery for 20 minutes were investigated by a microdialysis procedure and effects of intracerebroventricular administration of dexamethasone (10 µg) were evaluated in halothane-anaesthetized rats. The histologic outcome was examined 7 days after ischaemia by light microscopy. The relationship between ischaemic release of dopamine and the histologic outcome was estimated by assessing the effect of the lesion of the substantia nigra. The extracellular concentration of dopamine was not affected by the administration of dexamethasone in the non-ischaemic state. The occlusion of the middle cerebral artery produced a marked increase in the extracellular concentration of dopamine in the striatum, the peak value being 240 times that before ischaemia. The value returned to the basal level immediately after reperfusion. The preischaemic administration of dexamethasone enhanced the increase in the dopamine level during ischaemia, and the peak value in the dexamethasone group was 640% of that in the vehicle group. The value returned to the basal level 2 hours after reperfusion. After 7 days, ischaemic neuronal damage in the dexamethasone group was severe compared with that in the vehicle group. In rats receiving the substantia nigra lesion, ischaemic release of dopamine was abolished, and aggravation of ischaemic neuronal damage by dexamethasone was completely alleviated. Changes in release of dopamine may be a contributing factor in development of ischaemic neuronal damage by glucocorticoids.

159

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PERIPHERAL INJECTION OF GLUCOCORTICOIDS REDUCES ANXIETY RELATED BEHAVIOR IN MICE: AN INTERACTION WITH OPIOIDERGIC NEURONS Rashidy-Pour A., Vafaei A.A., Taherian A.A, Hejazi J. and Arab-Yarmohammadi M. Dept. of Physiology, Faculty of Medicine, University of Medical Sciences, Semnan, Iran [email protected]

40

* 30

*

Con Dex 0.3mg Dex 1mg Dex 2mg

20 10 0

*P<0.05

Fig 1: The effect of dexamethasone (0.3, 1 and 2 mg/kg, SC) on anxiety related behavior in mice. *P<0.05.

160

Persent of time spent in open arm (%)

Persent of time spent in open arm (%)

Introduction Stress initiates a cascade of biochemical and endocrine events which results in behavioral and electrophysiological effects in both animals and humans. Among these behavioral effects are modulation of learning, memory and anxiety. Since adrenal glucocorticoids are released during stress, it assumed that most behavioral effects of stress are mediated by these hormones. Extensive evidence indicate the modulatory effects of glucocorticoids on various phases of memory process [1,3,5], however, their effects on anxiety related behaviors are not clear. On the others hand, may neurotransmitter system, such as opioidergic system, have been shown to modulate the anxiety [2,4]. Therefore, in this study, we investigated the effects of dexamethasone, as a synthetic glucocorticoid, and its interaction with opioidergic system on anxiety related behavior in mice. Material And Methods Young adult male mice (20-30 g) were used in this study. A standard elevated plus-maze was used to determine anxiety level in animal. This maze consists of two open arms and two arms that are enclosed by high walls. Two behavioral measures were used: the percentage of time spent in the open arms and the ratio of open arm entries to total entries during 5 min. More entries into the open arms and more time spent in the open arms were interpreted as indicating lower levels of anxiety. Different doses of dexamethasone (0.3, 1 and 2 mg/kg, S.C) or vehicle was injected 30 min before of evaluation. Naloxone (0.5, 1 and 2 mg/kg, i.p) was injected 5 min before dexamethasone (0.3 mg/kg) administration. Results Analysis of data (the percentage of time spent in the open arms) indicated that dexamethasone at doses of 0.3 and 1, but not 2 mg/kg significantly reduced anxiety related behavior in mice (Fig.1). Same results were obtained with analysis of the ratio of open arm entries to total entries (data not shown). Pretreatment of naloxone at all tested doses (0.5, 1 and 2 mg/kg) attenuated the effects of dexamethasone on anxiety related behavior (Fig.2).

30

20

Con N0.5mg+D0.3mg N1mg+D0.3mg N2mg+D0.3mg

10

0

Fig 2: The effects of naloxone pretreatment on anti-anxiolytic effects of dexamethasone in animals.

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 Conclusion The results of this study, for first time, indicates that peripheral injection of glucocorticoids abolishes anxiety related behavior in mice. Although the underlying mechanism remain to determined, data also revealed an involvement of opioidergic system in the antianxiolytic effects of glucocortcoid. Since stress alters anxiety related behavior in animals, it can be assumed that activation of glucocorticoid system during stress may mediate the modulatory effects of stress on anxiety related behavior. Presently, We are planning to do some experiments in order to understanding the anti-anxiolytic effects of glucorticoid in animals.

References List [1] B.Roozendaal, J.L.McGaugh, Glucocorticoids receptor agonist and antagonist administration into the basolateral but not central amygdala modulates memory storage. Neurobiol. Learn. Mem. 67 (1997), 176-179. [2] D.J. Nutt, Neurobiological mechanisms in generalized anxiety disorder, J Clin Psychiatry, 62 (2001), (Suppl.11) 22-27. [3] J.L. McGaugh, LCahill, B.Roozendaal, Involvement of the amygdala in memory storage interaction with other brain systems. Proc. Natl. Acad. Sci 93 (1996), 13508-13514. [4] J.W.Tiller, N. Biddle, K.P Maguire, The dexamethasone suppression test and plasma dexamethasone in generalized anxiety disorder. Biol. Psychiatry, 23 (1998), 261-270. [5] L.Cahill, J.L. McGaugh, Mechanisms of emotional arousal and lasting declarative memory. Trends Nerurosci. 21(1998) : 294-299.

161

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 REGIONALLY-SELECTIVE UPREGULATION OF HIPPOCAMPAL GLUCOCORTICOID RECEPTORS IN RESPONSE TO CHRONIC UNAVOIDABLE STRESS IN THE RAT Robertson D.A., Beattie J.E., Reid I.C. and Balfour D.J.K. University of Dundee, Department of Psychiatry, Ninewells Hospital and Medical School, Ninewells Hospital, Dundee, DD1 9SY Scotland, U.K. e-mail [email protected]; phone: 44-1382 660111 ext 35115 fax: 44-1382 633923.

Exposure to a chronic unavoidable stress leads to an increase in hippocampal glucocorticoid receptors (GR). This effect has been implicated in the mechanisms underlying habituation to stress and impaired adaptation may be a factor in the psychopathology of depressive disorder [2]. The primary aim of this study was to investigate the time course and regional selectivity of this response. Male Sprague-Dawley rats (250g, N=6 per group) were subjected to elevated platform stress [1] for 1 hour per day for 1, 10, 20 or 30 days. Control animals were kept in their home cages, but were removed to the experimental room for the 1-hour stress period. Immediately following the final session on the platform, the animals were humanely killed and blood was taken for measurement of plasma corticosterone and brain tissue was taken for analysis of GR and mineralocorticoid receptor (MR) in the hippocampus, cerebral cortex and hypothalamus. Plasma corticosterone was measured using a radioimmunoassay. GR and MR were measured in solubilised membrane extracts using Western blot analysis. The protein content was determined using a Bio-Rad protein assay kit. The results were quantified using scanning and densitometry. One-way analysis of variance was used to detect any statistical significance. Post hoc analyses were performed using Duncan’s test. Corticosterone levels were elevated by acute, 1-day stress (from 2.0 ± 0.31 to 55.3 ± 15.80µg.100ml-1; P<0.01). Repetitive exposure to the stressor resulted in habituation of the response (10 days = 22.3 ± 3.64; 20 days = 5.5 ± 1.12; 30 days = 1.33 ± 0.25 µg.100ml-1). The density of GR in the hippocampus was significantly increased (from 80.2 ± 9.2 in unstressed rats to 181.2 ± 22.8 density units/100µg of protein; P<0.01) by 20 days exposure to the stressor. After 30 days exposure to the stressor, the density (106.7 ± 25.4 density units/100µg protein) was no longer significantly different from control. No significant differences in GR density were observed in the other brain regions investigated. MR density was not significantly influenced in any of the brain regions studied. Our findings indicate that repetitive exposure to this unavoidable stressor is associated with a regionally selective upregulation of GR density in the hippocampus. The response peaks after 20 exposures to the stressor and, thereafter, returns to levels close to control. The putative relationship between upregulation of the receptor and habituation of the plasma corticosterone response to the stressor remains to be established. Acknowledgements: Supported by the Wellcome Trust. Reference List 1. Balfour, D.J.K. Reid, A. Effects of betamethasone on the stimulation of corticosterone secretion in rats. Arch. Int Pharmacodyn. Ther.237 (1979), 67-74. 2. Holmes, M.C., French, K.L. & Seckl, J.R, Dysregulation of diurnal rhythms of serotonin 5-HT2C and corticosteroid receptor gene expression in the hippocampus with food restriction and glucocorticoids. J. Neurosci. 17(11) (1997); 4056-4065.

162

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

THE MODULATORY EFFECTS OF GLUCOCORTICOIDS ON MORPHINE ADDICTION PHENOMENA IN MICE Taherian A.A., Vafaei A.A. and Rashidy-Pour A. Dept. of Physiology, [email protected]

Faculty

of

Medicine,

Univ.

of

Med. Sci., Semnan,

Iran;

INTRODUCTION: In the long term, opioids induce the two adaptative phenomena known as tolerance and dependence whose mechanisms are considerably less well understood. Considerable evidence indicate the involvement of several neurotransmitter and hormonal system in induction of morphine tolerance and withdrawal. It is reported that the plasma level of glucocorticoids changed during morphine injection. Thus, glucocorticoids may play a role in development of morphine tolerance and withdrawal. In favour of this hypothesis, it has been shown that dexamethasone inhibits opiate withdrawal, in an in-vitro model. The mechanisms underlying this effect is not clear, but it suggested that dexamethasone could reduce opiate withdrawal by blocking the release of prostaglandin’s precursor, arachidonic acid through protein synthesis dependent-mechanisms [1,2]. The aim of this study was to evaluate the effect of dexamethasone on morphine dependency and withdrawal syndrome in mice. MATERIAL AND METHODS: Male albino mice (20-30 gr.) were used through the experiments. Morphine tolerance was induced by Marshall method [6,7]. During three days, three doses of morphine (50, 50 and 75 mg/kg) were injected in three times each day (each time one dose). Naloxone (2mg/kg) was used to induced withdrawal syndrome (WS). The number of jumping (NOJ) and the amount of weight loss (WL) were used as indexes of WS sings. Dexamethasone (Dex; 0.3, 1 and 2 mg/kg) was injected 30 min before daily administration of morphine or 30 min before induction of WS by naloxone. RESULTS: Fig. 1 shows the effect of pretrement injection of different doses of Dex on development of morphine tolerance induction (TI). Analysis of data (weight loss and number of jumping) indicated that the amount of weight loss but not NOJ in Dex-treated animals was significantly different from that of control group. Fig.2 illustrates the effect of different doses of Dex on the WS in morphine-dependent mice. Analysis of data revealed that Dex in doses of 0.3 and 1 mg/kg significantly attenuate the WS in morphine dependent mice.

Fig.1. Effects of difference doses of the dexamethasone (Dex) and vehicle on WS sign (numbers of jumping) . *P< 0.01 in comparison with control group.

163

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

Fig.2 Effects of difference doses of the dexamethasone (Dex) and vehicle on WS sign (Weight loss) . *P< 0.01 in comparison with control group. CONCLUSION: This study addressed the question of whether the modulation of glucocorticoid system can influences the morphine tolerance development and withdrawal syndrome. The data, for first time, indicate that: 1- injection of Dex, as a glucocorticoid agonist, before daily administration of morphine significantly attenuated WS, and 2- injection of Dex before induction of WS in morphine dependent animals, again, significantly diminished WS. Thus, it can be concluded that glucocorticoids may play an important role in different aspects of morphine addiction process. References List [1] A. Capes, Dexamethasone inhibition of acute opioid physical dependence in vitro is reverted by antilipoprotein-1 and mimicked by anti –type II exteracellular pla2 antibodies, Life Sci 61(1997) 127 – 134. [2] A. Capasso, Dexamethasone selective inhibition of acute opioid physical dependence in isolated tissue, J pharmacol exp 276 (2)(1997) 743 – 751. [3] G. DI chiara, and R. Alan north, Neurobiology of opiate abuse, Trends Pharmacol Sci., 13(1992) 185-193. [4] G.F. Koob, Drugs of abuse: anatomy, Pharmacology and function of reward pathways, trends Pharmacol Sci., 13 (1992) 177-184. [5] G.F. Koob, and F.E. Bloom, Cellular and molecular mechanisms of drug dependence, Science, 242(1998) 715-723. [6] J.C. Meunier, Opioid receptors, tolerance and dependence, Therapie, 47(6) (1992)492-502. [7] R.B. Rothman, A review of the role of antiopioid Peptides in morphine tolerance and dependence, Synapse 12 2 (1991)129-138.

164

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

MODULATORY ROLES OF GLUCOCORTICOID RECEPTORS ON SPATIAL MEMORY IN PLACE AVOIDANCE LEARNING IN RATS Vafaei A.A. and Rashidy-Pour A. Dept of Physiology, Faculty of Medicine, Univ. of Med. Sci, Semnan, Iran; [email protected] Introduction: Glucocorticoid hormones are released in response to stressful events influence manifold neuronal processes including learning, memory, and emotion via the glucocorticoid receptor (GR). Behavioral studies have been reported that glucocorticoids play an important role in regulation of memory process in both humans and animals [7,8]. Behavioral effects of glucocorticoids are mediated by intracellular receptors of two types, mineralcorticoid (MR, type I) a glucocorticoids (GR, type II). These receptors found in a variety of brain structures such as the amygdala, hippocampus, septum and neocortex. M R is high affinity binding sites for corticosterone and almost saturated under basal conditions, whereas GR show lower affinity for glucocorticoids and mediate responses to stress level of glucocorticoids. Increasing body of knowledge indicates that Basolateral amygdala (BLA) and Dorsal hippocampus (DH) mediate the effects of glucocorticoids on memory processing [9]. Previous studies have focused mainly on the effects of glucocorticoids on inhibitory avoidance memory, which is a form of non-spatial memory. On the others hand, the effects of glucocorticoids on memory retrieval is not clear. Therefore, in this study, we further explored the effects of glucocorticoid receptor modulation in the BLA or DH on different phases (acquisition, consolidation or retrieval) of spatial memory in place avoidance learning (PAL). Material and Methods: Adult male rats of Long-Evans strain, carrying chronically implanted cannulae aimed at the BLA (AP = - 3 mm, ML=+4.9 mm, and DV=-6.4 mm) or DH (AP= -3mm, ML=+1.5, DV=3) were used in this study. Behavioral training: PAL task. An elevated (50 cm) circular metal arena 80-cm in diameter was used. A computer controlled feeder mounted 2 m above the arena dropped 20-mg pasta pellets to random places in the arena at 10 s intervals. Over 3 days, the rats were trained in a daily 20-min session to forage for scattered food. The rat movement was tracked by a computerized tracking system. The next day avoidance training began. Rats were trained in a single 30-min session to avoid a 60-degree segment in one of the four quadrants on the stable circular arena. Whenever the rat entered the prohibited sector for >0.5 s, 50 Hz current (<0.6 mA) was delivered for 0.5 s between the implanted wire and the high impedance contact between the rat’s feet and the grounded arena floor. The shock was repeated after 3 s if the animal did not leave the prohibited area. Avoidance memory of all rats was assessed by a 30-min extinction trial 24 hours later, during which shock was never delivered. Two measures of avoidance memory are used: the time to first entry into the punished sector (T) and the number of entrance (N). Drugs injections. RU38486 (3 ng/0.6 µl, per side) as a glucocorticoid antagonist were injected bilaterally into the BLA or DH 5 min before training, immediately and 60 and 120 min after training and 5 min before of retrieval test. Control animals received same volume of vehicle at the same times.

165

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

500 400

Control Number of Entrances

Latency (Sec) to the first entry

Results: PAL task: Statistical analysis of the retention data (the time to first entry into the punished sector and Number of Entrances) showed that pre-training or posttraining injections of RU38486 (Fig. 1) into the BLA or DH significantly (P<0.01) impaired retention performance. 2) The effects of post-training injections were timedependent. Injections of GR agonist or antagonist immediately and 60 min, but not 120 min after training significantly (P<0.01) modulated spatial memory. 3) Retrieval was not affected under injections and analysis of the number of entrance data showed similar results (data not shown).

Test

300 200 100 0

60

Control Test

40 20 0 Acq Acq

BLA DH Acquisistion

0

60 120 0 60 BLA DH Consolidation

120

Acquisistion

0

60 120 0 60 120 BLA DH Consolidation

Fig 1. Effects of pre-training or post-training injections of RU38486 or vehicle into the BLA or DH on spatial memory in a PAL task. Data (A. latency to first entry and B. Number of Entrances) are expressed as a mean (SEM. *P<0.01 in comparison with vehicle group). Conclusion: 1-The major finding of the present experiment is that infusion of the drugs into the BLA or DH pre-or post-training affecting GR modulates spatial memory processing. 2-GR contributes to spatial memory modulation at least 60 min after training. 3-Involvement of GR in retrieval of spatial information is unlikely. 4- The present results also make an important contribution to a growing body of evidence that indicate the effects of glucocorticoids on memory process, particularly spatial memory.

References List [1] Cahill, L. and McGaugh, J.L., Mechanisms of emotional arousal and lasting declarative memory, Trends Neurosci. 21,294-299,1998. [2] McGaugh, J.L., Cahill, L. and Roozendaal, B., Involvement of the amygdala in memory storage: interaction with other brain systems, Proc. Natl. Acad. Sci. 93,13508-13514,1996. [3] Oitzel, M.S. and Dekloet, E.R., Selective corticosteroid antagonists modulate specific aspects of spatial orientation learning, Behave. Neurosci. 106, 62-71,1992. [4] Roozendaal, B. and McGaugh, J.L., Glucocorticoid receptor agonist and antagonist administration into the basolateral but not central amygdala modulates memory storage, Neurobiol. Learn. Mem.,67,176-179,1997. [5] Roozendaal, B. and McGaugh, J.L., Basolateral amygdala lesions block the memory enhancing effect of glucocorticoid administration in the dorsal hippocampus of rats, Eur. J. Neurosci. 9,76-83,1997. [6] Roozendaal, B., Glucocorticoids and regulation of memory consolidation, psychoneuroendocrinology, 25,213-238,2000. [7] Roozendaal, B., Basolateral amygdala lesions block glucocorticoid-induced modulation of memory for spatial learning, Behave. Neurosci. 110, 1074-1083,1996. [8] Vafaei, A.A., Rashidy-Pour, A., Burrs, J., Sharifi, M.R. and Fenton, A.A., Glucocorticoid antagonist administration into the basolateral amygdala modulates place avoidance memory, DARU, 8,30-35,2000.

166

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

THE EFFECT OF STEROID HORMONES ON HIPPOCAMPUS SUBEPENDYMAL NEURONAL LAYER OF THE HYPOTHALAMUS

AND

Vlad A.G. and Raica M. Dept. of Clin Endocrinology, Sitalul Clinic Judetean, and Dept. of Histology. UMF. Timisoara. ROMANIA. [email protected] Recently we report that in newborn mice and in human embryo six weeks aged it is a continuity between dorsal hippocampus neuronal cells and subependymal cells layer of the hypothalamus which in development disappear. The cellular components of this two region is similar, with large cells. After neonatal periode in the mouse brain this kind of cells have a particular evolution, decrease the size of cells and nuclear volume and have a lateral evolution in the hypothalamus but remain in a small amount in the “hilus” hippocampi. In present study on the newborn mice brain we develop the observation of the effect of estradiol, progesterone, hidrocortisone acetate, testosterone and cyproterone acetate on hippocampal cells and subependymal cells of hypothalamus. The results of this experiment reveal that all steroids applied induced a delayed of involution of the cellular components with large size and mantain the continuity between hippocampus and subependymal cell layer of the hypothalamus. We conclude that the steroid hormones can facilitate the development of new contact between neurons and decrease the neuronal death.

Reference List A.Vlad and M.Raica.-ICN Congress Bristol. Sept. 2002.Abstract volume.

167

Posters’ Exhibition: Pathological Correlations and New Tools in Therapeutical Approaches •

Andréen L., Bixo M., Nyberg S., Sundström-Poromaa I. and Bäckström T. (Umeå, Sweden, EU) Progesterone effects during sequential hormone replacement therapy

•

Boscaro V. and Cassone M.C. (Torino, Italy, EU) Antioxidant potentialities of 17betaestradiol in brain regions

•

Dominguez R., Jalali K. And De Lacalle S. (Los Angeles, CA, USA) Structural plasticity of the nervous system: the role of estrogen

•

Ishunina T.A. and Swaab D.F. (Kursk, Russia) Estrogen receptor immunocytochemical expression in the hypothalamus and cholinergic basal forebrain nuclei in Alzheimer's disease

•

Nobahar M. and Vafaei A.A. (Semnan, Iran) Sex hormones as a factor for incidence of cerebral stroke in reproductive age in female

•

Nuñez J.L., Alt J.J. and McCarthy M.M. (Baltimore, MD, USA) effect of sex and hormones in a model of preterm infant brain injury

•

Nyberg S., Sundström Poromaa I. and Bäckström T. (Umeå, Sweden, EU) Patients with premenstrual dysphoric disorder have a decreased sensitivity to alcohol in the luteal phase

•

Patte-Mensah C., Kappes V., Freund-Mercier M.J., Tsutsui K. and Mensah-Nyagan A.G. (Strasbourg Cedex, France, EU) Immunohistochemical localization of cytochrome p450scc in the nociceptive nervous system of rat

•

Persad V., Wong C.G.T., Cortez M.A. and Snead III O.C. (Toronto, Canada) Steroids and atypical absence seizures

•

Rhodes M.E. and Frye C.A. (Albany, NY, USA) Androgens anti-seizure effects may be due in part to actions at intracellular androgen receptors in the hippocampus

•

Ritz M.-F., Schmidt P. and Mendelowitsch A. (Basel, Switzerland, EU) Effect of 17β estradiol on the release of excitatory amino acids and energy metabolites during transient cerebral ischemia in male rats

•

Veiga S., Garcia-Segura L.M. and Azcoitia I. (Madrid, Spain, EU) Role of aromatase on neuroprotective effects of pregnenolone and dehydroepiandrosterone

•

Walf A.A. and Frye C.A. (Albany, NY, USA) Systemic and intra-amygdala administration of estrogen and progesterone to ovariectomized rats increases analgesia

•

Wihlbäck A.-C., Sundström-Poromaa I., Allard P., Mjörndal T., Spigset O. and Bäckström T. (Umeå, Sweden, EU) Influence of postmenopausal hormone replacement therapy on platelet serotonin uptake site and 5HT2A receptor binding

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

PROGESTERONE EFFECTS REPLACEMENT THERAPY

DURING

SEQUENTIAL

HORMONE

Andréen L., Bixo M., Nyberg S., Sundström-Poromaa I. and Bäckström T. Department of Obstetrics and Gynecology, Umeå University Hospital, Umeå, Sweden. [email protected] Objective: To investigate the effect on mood and the physical symptoms of two dosages of natural progesterone and placebo in postmenopausal women with and without a history of premenstrual syndrome (PMS). Study design: Postmenopausal women (n = 36) with climacteric symptoms were randomized in a placebo-controlled, double-blind, crossover study. They received 2 mg estradiol continuously during three 28-day cycles. Vaginal progesterone suppositories with 800 mg/day, 400 mg/day, or placebo were added sequentially for 14 days per cycle. Daily symptom ratings using a validated rating scale were kept. Results: Women without PMS history showed cyclicity in both negative mood and physical symptoms while on 400 mg/day of progesterone but not on the higher dose or placebo. Women without a history of PMS had more physical symptoms on progesterone treatment compared to placebo. Women with prior PMS reported no progesterone-induced symptom cyclicity. Conclusion: In women without prior PMS progesterone causes negative mood effects similar to those induced by progestogens.

This work was supported by an EU Regional fund Objective 1 grant.

169

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ANTIOXIDANT REGIONS

POTENTIALITIES

OF

17BETA-ESTRADIOL

IN

BRAIN

Boscaro V. and Cassone M.C. Dept. Anatomy, Pharmacology and Forensic Medicine - University of Turin, C.so Raffaello, 33 – 10125 TORINO (Italy), [email protected]; fax +39-011-6707688 17beta-estradiol (17beta-E2) can act as antioxidant because of the phenolic A ring of steroid, which is a potent electron donor and a free radical scavenger [2]. In the last few years, there has been a growing interest in the neuroprotective effects of estrogens and the possible beneficial effects of estrogens in neurodegenerative pathologies such as stroke, Alzheimer and Parkinson diseases [2]. Our previously studies show that 17beta-E2 can modify total antioxidant power of polymorphonuclear granulocytes, which are directly and indirectly involved in the maintenance of intercommunication between brain and immune system. So we investigated if 17beta-E2 is able to modify antioxidant power of rats discrete brain regions, such as cerebral cortex, striatum, hypothalamus, hippocampus. The experiments were performed on no stressed and stressed rats (restraint for 15 minutes, sacrifice after 0’, 15’, 24 hours). This stress model was chosen as it is able to induce oxidative stress, increasing reactive oxygen species production. Antioxidant power was measured by FRAP assay (Ferric Reducing Antioxidant Power): this is a colorimetric method, in which absorption at 593nm, measured in a 0- to 6-minutes reaction time window at room temperature, is directly correlated to antioxidants concentration [1]. The results, expressed as means ± standard errors (at least 6 rats for each groups), are statistically analysed with the t-test to evaluate the significance of differences. The obtained data underline that a concentration at least of 10microM 17beta-E2 is necessary for a significant effect. The antioxidant activity is evident only at pharmacological concentrations, but not at physiological ones (nanomolar range).

Reference List 1. I.F.F. Benzie, J.J. Strain, Ferric reducing /antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and acid ascorbic concentration, Methods Enzymol, 299 (1999) 15-27 2. K.M. Dhandapani, Brann D.W., Protective effects of estrogen and selective estrogen receptor modulators in the brain, Biol. Reprod. 67 (2002) 1379-1385

170

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

STRUCTURAL PLASTICITY OF THE NERVOUS SYSTEM: THE ROLE OF ESTROGEN Dominguez R., Jalali K. and de Lacalle S. California State University Los Angeles, Department of Biological Sciences, 5151 State University Drive, Los Angeles, CA 90032, USA; email: [email protected]; fax: (323)343-2016 Estrogen has a wide range of effects on the structure and function of a variety of brain regions, most notably regions not directly linked to reproductive functions such as the hippocampus and the basal forebrain cholinergic system. In the hippocampus estrogen has been shown to regulate dendritic density and spines [6]. In the present study we report a potent effect of estrogen on neurite outgrowth in cholinergic neurons in culture, an effect that involves activation of extracellular signal-regulated kinases (ERK). Postnatal day 1 (<24 hours) Fisher 344 pups were separated by sex and the basal forebrain primordium was dissected following a well established protocol [5]. Cells were dissociated and cultured for 2 weeks. On the 14 th day, cultures were exposed to 5 nM 17betaestradiol or vehicle for 30 minutes (for Western blot analysis of ERK activation) or 24 hours (for morphological studies using immunocytochemistry). Cultures exposed for 24 hours were fixed and stained with an antibody against the vesicular acetylcholine transporter (VAChT), a specific cholinergic protein. Activation of ERK was studied in the presence and absence of the estrogen receptor antagonist, ICI 182,780, which inhibits both the alpha and beta isoform of the estrogen receptor, and in the presence or absence of the MEK1/2 inhibitor UO126. Estrogen induced neurite outgrowth in basal forebrain cholinergic neurons in a markedly different sex-dependent manner. Morphological analysis of cholinergic neuron outgrowth was performed on digital images using NIH Image 1.62, and three parameters of VAChTimmunoreactive neurons were calculated: mean neurite length/neuron; total neurite length/neuron and total number of segments/neuron. Increases in branch length and number would indicate an increase in arborization. Using as a reference mean neurite length/neuron, untreated (control) male cultures showed on average significantly longer neurites (34 microns/neuron) than females (18 microns/neuron). After estrogen treatment, mean neurite length in males increased 2.5-fold over untreated levels, but only 1.8-fold in females. Nonetheless, the most significant finding in our study was the demonstration that female cholinergic neurons treated with estrogen significantly increased the total neurite length/neuron (4.5-fold over controls) and the number of neurites/neuron (2.3-fold over controls). By contrast, male cholinergic neurons experienced no change in total neurite length/neuron with respect to controls, and furthermore showed a significant decrease in the number of neurites/neuron (0.5-fold below control measures). To verify further whether estrogen had an effect on the outgrowth of new primary neurites, or was inducing terminal-like arborization, we analyzed total branch length/neuron and the number of branch segments/neuron. In female cultures, the length of segments increased significantly in estrogen treated compared t o controls, with primary neurite length measuring 90 microns in control cultures, and 475 microns after estrogen. Secondary, tertiary and quaternary neurites in treated cultures more than doubled their size when compared to the untreated controls. By contrast, primary neurite length in male cultures measured 175 microns in controls, and 215 microns after estrogen treatment, a non-significant effect present also in secondary, tertiary and quaternary neurites. In addition, the number of primary neurites in female cultures treated with estrogen was twice the number found in untreated controls. We were able to measure neurite arborization up to the 7th order in treated cultures, but only up to the 5th order in untreated controls. By contrast, the number of primary neurites in estrogen-treated male cultures was half of the number in untreated controls. We were unable to find branching in male cultures beyond the 4 th degree. From these data we conclude that estrogen treatment induces outgrowth in female cholinergic neurons by increasing the length and number of primary neurites, and also inducing increased branching that resembles an enlarged terminal-like arborization.

171

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 ERK1 (44 kDa) and ERK2 (42 kDa), which are encoded by different genes, are expressed at high levels in the developing and adult CNS [1, 2, 3]. In neuronal cells, they are present in cytoplasmic compartments, including dendrites, prior to their stimulation [7]. Once activated, they can phosphorylate a wide variety of substrates. Their targets can be membrane-associated proteins, such as EGF receptors, phospholipase A2, cytoskeletal proteins, including microtubule associated proteins and neurofilaments [4]. Phosphorylation of these cytoskeletal proteins by ERKs could arbitrate morphological changes that underlie plastic properties of neurons. We analyzed whether the significant plastic changes induced by estrogen on the cholinergic neurons could be also mediated via the ERK signaling pathway. We found a significant increase in the double phosphorylated form of ERK (dpERK) within 30 minutes of exposure to 5 nM estrogen in female cultures. Interestingly, this increase in dpERK levels was not blocked by the addition of ICI or by ICI alone. In male cultures, neither estrogen, ICI, nor the combination of both induced an upregulation of dpERK above control levels. When male and female cultures were treated with the ERK inhibitor UO126, the levels of dpERK dramatically decreased 0.5-fold below untreated controls. When administered at the same time, UO126 was able to block dpERK upregulation also 0.5-fold below control levels. Consistent with a recent study that has reported sex differences in the estrogen-modulated activation of MAPKs in cortical astrocytes, our results also suggest that this differential regulation may contribute to sexual dimorphisms in brain development [8]. We conclude that the activation of ERK is an important element in the mechanism of estrogen-induced morphological reorganization in the female cholinergic system. Understanding these phenomena may be useful in unraveling how cholinergic neurons can be particularly susceptible to degeneration in postmenopausal women. These results are important in the context of cholinergic neurodegenerative diseases such as Alzheimer’s disease. This work was supported by USPHS grant AG17605 to S.L. and an MBRS-RISE fellowship to R.D.

Reference List [1] Boulton, T. G., S. H. Nye, et al., ERKs: a family of protein-serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF, Cell 65 (1991) 663-75. [2] Fiore, R. S., V. E. Bayer, et al., p42 mitogen-activated protein kinase in brain: prominent localization in neuronal cell bodies and dendrites, Neuroscience 55 (1993) 463-72. [3] Flood, D. G., J. P. Finn, et al., Immunolocalization of the mitogen-activated protein kinases p42MAPK and JNK1, and their regulatory kinases MEK1 and MEK4, in adult rat central nervous system, J. Comp. Neurol. 398 (1998) 373-92. [4] Grewal, S. S., R. D. York, et al., Extracellular-signal-regulated kinase signaling in neurons, Curr. Opin. Neurobiol. 9 (1999) 544-53. [5] Hartikka, J. and F. Hefti, Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes, J. Neurosci. 8 (1988) 2967-85. [6] Leranth, C., M. Shanabrough, et al., Hormonal regulation of hippocampal spine synapse density involves subcortical mediation, Neuroscience 101 (2000) 349-56. [7] Ortiz, J., H. W. Harris, et al., Extracellular signal-regulated protein kinases (ERKs) and ERK kinase (MEK) in brain: regional distribution and regulation by chronic morphine, J. Neurosci. 15 (1995) 1285-97. [8] Zhang, L., B.-S. Li, et al., Sex-related differences in MAPKs activation in rat astrocytes: effects of estrogen on cell death. Mol. Brain Res. 103 (2002) 1-11.

172

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ESTROGEN RECEPTOR IMMUNOCYTOCHEMICAL EXPRESSION IN THE HYPOTHALAMUS AND CHOLINERGIC BASAL FOREBRAIN NUCLEI IN ALZHEIMER'S DISEASE Ishunina T.A.1,2 and Swaab D.F.1 1

Netherlands Institute for Brain Research, Amsterdam, The Netherlands Department of Histology and Embryology, Kursk State Medical University, Kursk, Russia [email protected]

2

Alzheimer's disease (AD) affects women more often than men, and women with dementia show greater memory deficits during the course of their illness. Although not without controversy, estrogen replacement therapy (ERT) has been shown to i) reduce the risk, ii) retard development and severity of AD in postmenopausal women and iii) to augment the therapeutic benefits of medications designed to treat AD. Estrogens exert their action mainly via estrogen receptors (ER) α and β. In the present study we aimed at finding out whether i) ER expression is present in the hypothalamic and cholinergic basal forebrain nuclei involved in cognition and memory, i.e. the nucleus basalis of Meynert (NBM), the vertical limb of the diagonal band of Broca (VDB), the tuberomamillary (TMN) and the medial mamillary nucleus (MMN) and in the supraoptic nucleus (SON) which remains exceptionally intact in AD, ii) whether it changes in aging and in AD patients and iii) whether these areas may be a target for estrogen replacement therapy. We have observed a remarkable rise in the number of neurons expressing nuclear ERα in the NBM, VDB, MMN, but not in the TMN or SON. For ERβ, that is present to a lesser extent and mainly in the cytoplasm, the alterations were different per area. Furthermore, nuclear ERα was more prominently increased in AD than in control men compared to AD versus control women in the MMN and SON. Neither ERα nor ERβ expression were related to neuronal metabolic activity in any of the brain areas studied. Taken together our data demonstrate that changes in ERs expression in aging and AD are region-specific and are not simply related to alterations in sex steroid levels. The cause for the unexpected increase in nuclear ERα in a number of brain areas is now under investigation.

Brain material was obtained from the Netherlands Brain Bank, Amsterdam (coordinator Dr. R. Ravid). References List Ishunina TA, Swaab DF. Increased expression of estrogen receptor α and β in the nucleus basalis of Meynert in Alzheimer's disease. Neurobiol Aging 2001; 22: 417-426.

173

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

SEX HORMONES AS A FACTOR FOR INCIDENCE OF CEREBRAL STROKE IN REPRODUCTIVE AGE IN FEMALE Nobahar M. and Vafaei A.A. Faculty of Nursing and Paramedical, University of Medical Sciences, Semnan, Iran; [email protected] INTRODUCTION The effects of sex steroids on neurological function in health and disease constitute a rich and rapidly expanding area of basic and clinical neuroscience. Stroke is a leading cause of serious disability and the third leading cause of death in the more countries. Stroke is the second leading cause of death in women after coronary heart disease. Previous study indicated that sex is a one of major risk factors for incidence of cerebral stroke. Stroke is more common in men than in women but previous evidence indicated that incidence of cerebral stroke in during of reproductive age is a more in women than men that may be sex hormone involvement in incidence of stroke. The incidence of ischemic stroke in women of childbearing age is estimated between 3.5 and 18 per 100000 per year in Western countries. This risk may be slightly increased during pregnancy, particularly during the postpartum period. To our knowledge, no data are available on the influence of pregnancy on the risk of recurrent stroke. Also cerebral thromboembolism resulting from strogen induced hypercoagulability is a likely etiology for such strokes. Estrogen increases plasma levels of fibrinogen and clotting factors VII, VIII, IX, X, and XII. The steroid also enhances platelet aggregation and suppresses antithrombin III activity and the fibrinolytic system. Elam and associates have reported an increased prevalence of mitral valve prolapse among users of oral contraceptives with ischemic stroke. Sex hormone– induced hypercoagulability is thought to play an important role in the pathogenesis of cerebral venous thrombosis complicating pregnancy, the puerperium, and use of hormonal contraceptives. Therefore in this study we investigated the prevalence and factors (Sex hormone) that may be involvement of cerebral stroke in reproductive age in female. METHODS Data were collected from all patients that they had lower than age from 45 years old and hospitalized during 10 years gradually in academic medical center (Fatemiah hospital) in Semnan, Iran. We used from questioner and checklist that include of demographic data and data regarding to all of risk factors and sign of stroke. We identified by medical record review women and men admitted for stroke from 1992 through 2002. Stroke risk factor include of prior stroke (PS), diabetes mellitus (DM), hypertension (HTN), heart disease (HD), hyperlepidemia (HL), family history (FH), smoking (S) and atrial fibrillation (AF) were documented for each subject. Stroke sign include of Headache, Hemiplegia, Aphasia, Low consciousness, Syncope, Seizure and Visual disorders were documented for each subject. RESULTS Data indicated that overall, 60% from the all cerebral strokes patient were female and 40% were male and 10% reported that have a history of stroke. The percent of risk factors

174

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

in female were 41% (hypertension), 17% (diabetes), 14% (hyperlipidemia) 27.6% (heart diseases), 0%(smoking), 10% (AF) and 21% reported a stroke in their family. But in male the percent of risk factors were 32% (hypertension), 16% (diabetes), 10% (hyperlipidemia) 5% (heart diseases), 47%(smoking), 0% (AF) and 21% reported a stroke in their family. The most common sign in women were in 55% right hemiplegia, 31% left hemiplegia. Also they have in 34/5% aphasia, 17% low consciousness level and 14% headache. Sort of cerebral stroke in women were in 69% ischemic 17.2% hemorhagic and 13.8% embolitic and 14% were died. But in men were 79% ischemic and 21% hemorhagic and 16% were died.

Fig 1: Compare between of risk factors that involvement in stroke incidence in female and male that age of those were lower than 45 years old

Fig 2: Compare between of stroke incidence in female and male that age of those were lower than 45 years old. .

CONCLUSIONS Finding above highlight the importance of this point that incidence of cerebral stroke more incidence in female than male in age of lower than of 45 years old (reproductive age). Previous study indicated that stroke is more common in men than in women but in this study we indicated that cerebral stroke during of reproductive age is a more prevalence in women than men that may be sex hormone involvement in incidence of stroke. To our knowledge, this study is the first to assess the risk of stroke incidence associated with subsequent pregnancies and levels of sex hormones in young women that afflicted of stroke. References List [1] C. Lamy, J.B. Haman, J. Coste and J.L Mas, Ischemic stroke in young women: Risk of recurrence during subsequent pregnancies, J Neurology, July 2000, 269-274. [2] C.D. Bushnell, G.P. Samsa and L.B. Goldstein, Hormone replacement therapy and ischemic stroke severity in women: A case-control study, J Neurology 56 2001, 1304-1306. [3] H.M. Schipper, Sex hormones and the nervous system, Neurology and General Medicine, 2001, 365381 [4] M. Weih, K. Kallenberg, A. Bergk, U. Dirnagl, and L. harms, K.D. Wernecke, K.M. Einhaupl, Attenuated stroke severity after prodromal TIA: A role for ischemic tolerance in the brain? J of Stroke. 30(1999), 1851-1854. [5] M. Victor, A.H. Ropper, Principles of Neurology, 7th Edition Vo1 2, 2001. [6]

W.J Weiner, C.G. Goetz, Neurology for the Non-Neurologist, 4th Edition, Lippincott Williams & Wilkins, 1999.

175

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

EFFECT OF SEX AND HORMONES IN A MODEL OF PRETERM INFANT BRAIN INJURY Nuñez J.L., Alt J.J. and McCarthy M.M. University of Maryland, Baltimore, Physiology Department, 5-014 Bressler Research Building, 655 W Baltimore Street, Baltimore, MD 21201, USA. Email: [email protected]. Fax: 410-706-8341. We have recently developed a model for preterm infant hypoxia-ischemia in which muscimol (5µg), the selective GABAA receptor agonist, administered to the postnatal day 0 and 1 rat induces significant hippocampal damage. In the neonatal rat brain, GABAA receptor activation leads to membrane depolarization and hence neuronal excitation as opposed to inhibition. We have determined that GABAA-induced cell death is due to excessive calcium influx through L-type voltage sensitive calcium channels. There has been considerable interest in the potential neuroprotective effects of estradiol in the adult brain. We investigated the effect of pretreatment with estradiol (50µg) on our model of prenatal hypoxia-ischemia in male and female rats. The number of neurons in the hippocampal formation (CA1, CA2/3 and the dentate gyrus) of control animals was compared to animals treated with muscimol alone and estradiol + muscimol treated animals. Muscimol alone increased neuron loss in the hippocampus, and this damage was significantly exacerbated by pretreatment with estradiol. A hippocampal culture paradigm was used to mirror the in vivo investigation. Intriguingly, we observed that estradiol + muscimol treated cultures displayed elevated cytotoxicity by 48 hours after treatment, but decreased cytotoxicity relative to muscimol alone treated cultures between 2 and 24 hours after treatment. In order to see if the actions of estradiol on muscimol-induced damage was via the nuclear estrogen receptor, estradiol + muscimol treated cultures were pretreated with ICI-182,780, a selective estrogen receptor antagonist. Treatment with ICI-182,780 blocked the potentiating effect of estradiol on the late time period (48-96 hours) GABAAinduced hippocampal cell loss, but failed to block the protective effect of estradiol on the early phase (2-24 hours) of cell loss. Thus, there appears to be a biphasic action of estradiol in our model of neonatal brain injury that involves non-genomic receptor mediated protection early followed by deleterious genomic effects late.

Supported by NIH grant R01 MH 52716 to MMM and SFN Minority Postdoctoral Fellowship to JLN.

176

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

PATIENTS WITH PREMENSTRUAL DYSPHORIC DISORDER DECREASED SENSITIVITY TO ALCOHOL IN THE LUTEAL PHASE

HAVE

A

Nyberg S., Sundström Poromaa I. and Bäckström T. Department of Clinical Sciences, Obstetrics and Gynecology, Umeå University, Umea, Sweden. ([email protected]) Objective: Women with premenstrual dysphoric disorder (PMDD) have previously been shown to have a reduced sensitivity to GABAergic compounds such as benzodiazepines and pregnanolone, a progesterone metabolite. We have evaluated the functional sensitivity to alcohol in thirteen women with and twelve women without premenstrual dysphoric disorder (PMDD) at two stages of the menstrual cycle, by comparing the effects of an intravenous alcohol infusion on saccadic eye velocity (SEV) and self-rated sedation. Results: The alcohol infusion produced significant changes in SEV, saccade deceleration and self-rated alcohol intoxication compared to placebo infusion. PMDD patients responded with a decreased SEV and saccade deceleration response to alcohol infusion in the luteal phase compared with the follicular phase (F(1,12) = 5.42; p < 0.05 and F(1,12) = 6.15; p < 0.05, respectively), whereas alcohol sensitivity in control subjects remained unaltered across the menstrual cycle. Self-rated sedation and alcohol intoxication scores did not differ between phases in either group. Conclusion: These findings are compatible with decreased alcohol sensitivity in brain areas controlling saccadic eye movements among PMDD patients in the late luteal phase.

This work was supported by an EU Regional fund Objective 1 grant.

177

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

IMMUNOHISTOCHEMICAL LOCALIZATION OF CYTOCHROME P450scc IN THE NOCICEPTIVE NERVOUS SYSTEM OF RAT Patte-Mensah C.1, Kappes V.1, Freund-Mercier M.J.1, Tsutsui K.2 and MensahNyagan A.G.1* 1

Laboratoire de Neurophysiologie Cellulaire et Intégrée, CNRS, UMR 7519, Université Louis Pasteur, 67084 Strasbourg Cedex, France. 2Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739, Japan. *E-Mail: [email protected]

Various studies have suggested the involvement of neurosteroids in the modulation of nociceptive transmission but anatomical and functional evidences supporting this hypothesis are almost completely unknown. As a first step toward the determination of neurosteroid importance in the modulation of pain sensation, we have investigated the presence of cytochrome P450side chain cleavage (P450scc) in the nociceptive nervous system of adult female and male rats. P450scc is a key enzyme which catalyzes the conversion of cholesterol into pregnenolone, a crucial biochemical reaction that paves the way for biosynthesis of all classes of steroids. Two different antisera were successfully used to localize P450scc-immunoreactive structures in the dorsal root ganglia (DRG), spinal cord (SC) and nociceptive supra-spinal nuclei (NSN) of female and male rats. The first antiserum was raised in rabbit against the carboxy-terminal domain of rat P450scc and the second is a polyclonal antibody against purified bovine adrenal P450scc. A strong immunoreactivity was observed in several cell bodies of DRG isolated from the cervical, thoracic, lumbar and sacral regions of SC. Double-labeling experiments revealed that P450scc-positive cell bodies located in DRG also contained type 2 microtubuleassociated protein (MAP-2), a specific neuronal marker. A moderate P450scc-immunofluorescent signal was detected throughout the white matter in all SC segments. The superficial layers (laminae I-III) in the dorsal horn (DH) of the gray matter, which play a fundamental role in the transmission and modulation of nociceptive sensory messages, were intensely labeled with both P450scc antisera. In particular, a detailed morphological study using a confocal laser scanning microscope made it possible to localize P450scc in islet-cells and stalked-cells, two major classes of neurons which are crucial for the DH circuitry conveying nociceptive informations. A dense network of P450scc-immunoreactive fibers were also observed in the DH. Cell type identification using antibodies against the glial fibrillary acidic protein ( a specific marker for astrocytes), galactocerebroside (a marker for oligodendrocytes) and MAP-2 indicated that 80 % of P450scc-positive fibers located in the DH were neuronal and 20 % corresponded to astroglial processes. A number of P450scc-containing nerve fibers extended along the rostrocaudal axis of the SC and the others were radially orientated toward the deep DH (laminae IV-VI). The immunostaining was also found in ependymal glial cells located on the periphery of the central canal. In the ventral horn, various motoneurons expressed P450scc-immunoreactivity from the cervical to sacral regions of the SC.

178

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

Three main NSN were highly stained with P450scc antibodies in adult female and male rat brain, namely, the parabrachial, raphe magnus and dorsal raphe nuclei. At high magnification using the confocal microscope, it appeared that the immunoreactive material was concentrated in organelles located in the cytoplasm and cytoplasmic extensions of P450scc neuronal populations detected in NSN. The anatomical and cellular distribution of P450scc-immunostaining was similar in nociceptive pathways of female and male animals. In both sex, an intense immunoreaction was observed in the adrenocortical zones using the two different antisera against P450scc. Preincubation of the antibodies (diluted at 1/500) with purified bovine P450scc at the concentration of 10-6 M resulted in complete disappearance of the immunostaining, an observation which confirmed the specificity of the labeling. In conclusion, this study provides the first detailed mapping of P450sccimmunoreactive elements in the nociceptive nervous system of adult rat. Our data also suggest that bioactive neurosteroids may be synthesized within the nociceptive pathways and locally regulate the activity of nerve cells and fibers involved in the processing of painful messages.

This work was supported by the Fondation pour la Recherche Médicale (FRM), Conseil Régional d'Alsace, Centre National de la Recherche Scientifique (CNRS) and Université Louis Pasteur de Strasbourg.

179

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

STEROIDS AND ATYPICAL ABSENCE SEIZURES Persad V.145, Wong C.G.T.145, Cortez M.A.2345 and Snead III O.C.12345 1

Institute of Medical Science, 2Department of Pediatrics, Faculty of Medicine, University of Toronto; 3Division of Neurology, 4The Program in Brain and Behavior; Hospital for Sick Children; 5The Bloorview Epilepsy Research Program [email protected] Treatment of Long Evans hooded rats during post-natal brain development with the cholesterol synthesis inhibitor, AY9944 (AY) results in atypical absence seizures that are frequent, recurrent, and life-long. AY-induced slow spike-and-wave discharges (SSWD) are significantly more frequent and prolonged in females than males. A time course study that examined the effects of the female estrous cycle on SSWD, Gamma-aminobutyric B Receptor (GABABR) binding and protein expression was carried out. Additionally, a pharmacological study using the hormones progesterone, 17β-estradiol, mifepristone (intracellular progesterone receptor antagonist), tamoxifen (intracellular estrogen receptor antagonist) and allopregnanolone (progesterone metabolite) was performed to examine their effects on seizures in the AY model. The data indicate that there is a significant increase of SSWD and GABABR binding seen only during the proestrus stage of the estrous cycle, the stage in which the levels of progesterone are at its highest. No changes in GABABR protein levels were observed. In addition, the administration of both progesterone and allopregnanolone exacerbated seizures, while 17β-estradiol attenuated the SSWD. In addition, co-administration of mifepristone with progesterone showed no significant change in SSWD when compared with progesterone alone. Similarly, co-administration of tamoxifen with 17β-estradiol made no significant change in SSWD compared to 17βestradiol alone, suggesting that both progesterone and 17β-estradiol are working in a manner independent of their intracellular receptors. Our data indicate that there is an important role for steroid hormones in the regulation and maintenance of AY-induced atypical absence seizures, although the exact mechanisms are yet to be determined.

180

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ANDROGENS ANTI-SEIZURE EFFECTS MAY BE DUE IN PART TO ACTIONS AT INTRACELLULAR ANDROGEN RECEPTORS IN THE HIPPOCAMPUS Rhodes M.E1. and Frye C.A.1-3 The University at Albany-SUNY, Behavioral Neuroendocrinology Laboratory, 1Departments of Psychology and 2Biological Sciences, and 3 The Center For Neuroscience Research, 1400 Washington Avenue Albany, NY, 12222 USA. [email protected]. 518-442-4867. The animal and clinical literature suggest that androgens can modulate ictal activity. Male rodents are less susceptible than are females to various types of seizures [6,8,14]. Removal of the testes, the primary endogenous source of androgens, exacerbates picrotoxin [6,13] and pentylenetetrazole (PTZ) [7] –induced seizures. Testosterone (T) replacement to gonadectomized (gdx) rodents increases seizure threshold; seizure thresholds are also greater when endogenous levels of androgens are higher. T treatment to gdx rodents reduces incidence of seizures [6]. Androgen deficiency is over-represented among men with epilepsy [11]. Administration of testenat, a synthetic androgen, decreases the rate and reduces the severity of epileptic seizures [1]. T administration to a young man with post-traumatic seizures resulted in fewer seizures compared to the number of seizures experienced prior to T [12]. The hippocampus is involved in seizure processes and is a target of androgen action. Ictal activity can result in cell death in the hippocampus of rats [3]. People with seizure disorder often have neurodegeneration in the hippocampus [10]. There are also deficits in learning and memory associated with ictal activity that may be in part due to hippocampal damage [10]. 3H T [9] and intracellular androgen receptors (ARs) [2] have been localized t o the hippocampus of rats. Administration of T to the hippocampus enhances cognitive performance in some tasks [4] and may have some role in modulating ictal activity. Although these data, together with those above, suggest that androgens can modulate ictal activity and that the hippocampus may be an important site of action for these effects, there have been few investigations of the mechanisms by which androgens may mediate seizure susceptibility. The present studies utilized an animal model of ictal activity, to investigate the effects and mechanisms of androgens to modulate seizure susceptibility. In most experiments, male, Long-Evans rats were administered vehicle or flutamide (10 mg SC or intra-hippocampal implants) two hours prior to PTZ administration (70 mg/kg, IP). Following P T Z administration, the latency and number of myoclonic seizures was recorded for 10 minutes. In Experiment 1, ictal activity of adult (~55 days old; n=18) intact male rats was compared to that of juvenile (~24 days old; n=23) intact male rats. Juvenile males had a significantly greater number of myoclonic seizures compared to adult males (see Table 1). Experiment 2 compared the ictal activity of adult (~55 days old; n=10) intact male rats to that of aged (~1 year old; n=6) intact male rats. There was a tendency for aged male rats to have a greater number of myoclonic seizures compared to adult males (see Table 1). In Experiment 3 ictal activity of gdx male rats (n=17) was compared to that of adult intact male rats (n=14). Gdx male rats had significantly increased numbers of myoclonic seizures compared to intact males (see Table 1). Experiment 4 compared the ictal activity of intact males (n=14) to that of intact males administered SC flutamide (n=16), an androgen receptor antagonist. Intact rats adminstered flutamide had more myoclonic seizures than did intact rats administered vehicle (see Table 2). Experiment 5 examined the ictal activity of gdx rats with silastic capsules of DHT that were also administered flutamide SC (n=16) compared to that of gdx (n=16) and gdx + DHT males administered vehicle (n=20). Gdx rats with DHT capsules had significantly fewer myoclonic seizures compared to gdx males. Administration of flutamide increased the number of myoclonic seizures compared to that of gdx + DHT males (see Table 2). Experiment 6 examined the effects of flutamide administered to the hippocampus of gdx + DHT rats (n=7) compared to that of gdx (n=10) and gdx + DHT rats administered vehicle (n=5). Gdx rats with DHT capsules had significantly fewer myoclonic seizures than did gdx males. Futamide to the hippocampus of gdx rats with DHT capsules significantly increased the number of myoclonic seizures compared to gdx rats with DHT capsules (see Table 2).

181

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 The present findings are consistent with our hypothesis that androgens can modulate seizure susceptibility. Endogenous hormonal milieu associated with relatively lower androgen levels (juvenile and aged males) resulted in a greater incidence of myoclonus than that seen in adult rats, which would be expected to have higher androgen levels. Further, these data suggest that androgens may have actions in part via intracellular androgen receptors in the hippocampus to modulate ictal activity. Administration of flutamide was more effective than gdx at increasing the incidence of myoclonus and intra-hippocampal administration of flutamide was able to account for the effects seen with SC administration.

ACKNOWLEDGMENT: Funded by the Whitehall (096-010) and National Science Foundations (98-96263)

Reference List [1] L.O. Badalian, P.A. Temin, K. Mukhin, Androgen treatment of sex disorders in men with epilepsy, Sov Med 4 1991 62-64. [2] E.W. Bingaman, L.M. Baeckman, J.M. Yracheta, R.J. Handa, T.S. Gray, Localization of androgen receptor within peptidergic neurons of the rat forebrain, Brain Res. Bull. 35 1994 379-382. [3] E.D. French, C. Aldinio, R .Schwarcz, Intrahippocampal kainic acid, seizures and local neuronal degeneration: relationships assessed in unanesthetized rats, Neurosci. 7 1982 2525-2536. [4] C.A. Frye, A.M. Seliga, Testosterone increases analgesia, anxiolysis, and cognitive performance of male rats. Cog. Affect. Behav. Neurosci. 1 2001 371-381. [5] A.G. Herzog, Psychoneuroendocrine aspects of temporolimbic epilepsy. Part III: Case reports, Psychosomatics. 40 1999 109-116. [6] D. Pericic, H. Manev, M. Bujas, Gonadal hormones and picrotoxin-induced convulsions in male and female rats, Brain Res. 736 1996 174-179. [7] M.E. Pesce, X. Acevedo, D. Bustamante, H.E. Miranda, G. Pinardi, Progesterone and testosterone modulate the convulsant actions of pentylenetetrazol and strychnine in mice, Pharmacol. Toxicol. 87 2000 116-119. [8] M.E. Pesce, X. Acevedo, G. Pinardi, H.F. Miranda, Gender differences in diazepam withdrawal syndrome in mice, Pharmacol. Toxicol. 75 1994 353-355. [9] M. Sar, W.E. Stumpf, Autoradiographic localization of radioactivity in the rat brain after the injection of 1,2-3H-testosterone, Endocrinol. 92 1973 251-256. [10] R. Schwarcz, M.P. Witter, Memory impairment in temporal lobe epilepsy: the role of entorhinal lesions, Epilepsy Res. 50 2002 161-177. [11] S. Schwartz-Giblin, A. Korotzer, D.W. Pfaff, Steroid hormone effects on picrotoxin-induced seizures in female and male rats, Brain Res. 476 1989 240-247. [12] M. Tan, U. Tan, Sex difference in susceptibility to epileptic seizures in rats: importance of estrous cycle, Int. J. Neurosci.108 2001 175-191. [13] J. Thomas, J.H. McLean, Castration alters susceptibility of male rats to specific seizures, Physiol Behav. 49 1991 1177-1179. [14] J. Thomas, Y.C. Yang,Allylglycine-induced seizures in male and female rats, Physiol Behav. 49 1991 1181-83. Table 1: Incidence of myoclonus for groups in Experiments 1-3. * denotes significant difference at p< 0.05, # denotes tendency 0.10

0.05. CONDITION # MYOCLONIC SEIZURES Adult Intact 3.4 + 0.7 Juvenile Intact 8.7 + 1.9* Adult Intact 2.6 + 1.0 Aged Intact 6.7 + 3.2# Adult Intact 2.9 + 0.8 GDX 7.8 + 1.6*

182

Table 2: Effects of flutamide on androgen-mediated seizures. * significant difference at p< 0.05, # tendency for difference at 0.10>p>0.05. CONDITION # MYOCLONIC SEIZURES Adult Intact + Vehicle 2.5 + 0.7 Adult Intact + SC Flutamide 4.4 + 0.8# GDX 8.3 + 1.6 GDX + DHT Capsule 4.5 + 0.5* GDX + DHT Capsule + SC Flutamide 6.9 + 1.2 GDX 7.8 + 0.6 GDX + DHT Capsule 4.2 + 0.6* GDX + Intra-hippocampal Flutamide 11.2 + 2.1 GDX + DHT Capsule + Intra13.0 + 2.6 hippocampal Flutamide

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

β-ESTRADIOL ON THE RELEASE OF EXCITATORY AMINO EFFECT OF 17β ACIDS AND ENERGY METABOLITES DURING TRANSIENT CEREBRAL ISCHEMIA IN MALE RATS Ritz M.-F., Schmidt P. and Mendelowitsch A. Neurosurgery Laboratory, University Hospital Basel, Switzerland. Marie-Franç[email protected] Introduction: Estrogen plays an important role in protecting neurons in the cerebral cortex against cerebral ischemia. Cell death after cerebral ischemia is mediated by energy depletion, release of excitatory amino acids, calcium influx into cells, and generation of free radicals. The release of excitatory neurotransmitters glutamate and aspartate contribute to the brain injury. Using the middle cerebral artery occlusion (MCAO) model in the male rat, we investigated if 17β-estradiol injection at the onset of occlusion modifies the release of energy metabolites lactate and glucose and the two excitatory neurotransmitters glutamate and aspartate. Material and Methods: A microdialysis probe (CMA/12, 4 mm membrane), inserted into the right cortex of male Wistar rats (250-300 g) anesthetized with pentobarbital (50 mg/kg i.p.), was perfused with artificial cerebrospinal fluid at a flow rate of 2 ml/min and samples were collected every 15 min 2 hours before, during, and 3 hours after MCAO. Transient cerebral ischemia was performed using the method of Longa et al (1). An intraluminal threat (3-0 blue suture) was advanced 22 mm into the right internal carotid artery and left for 90 min before its withdrawal to allow reperfusion for 24 hours. 17β-estradiol (0.8 mg/kg in 40 % ethanol) or the vehicle was injected i.v. in the femoral vein at the onset of occlusion. Brains were removed, frozen and sectioned and brain injury was evaluated by immunohistological analysis using anti-MAP-2 antibody. Extracellular amino acids levels (glutamate and aspartate) were monitored by HPLC from microdialysis samples. Lactate and glucose levels were quantified using an enzymatic YellowSpring analyser. Results: An infarct of about 240 mm3 was observed in the cortex and striatum of controls rats, whereas the infarct was reduced by 50% in 17β-estradiol-treated animals. Occlusion induced rapid peaks of glutamate and aspartate 75 min and 45 min after the onset of occlusion, respectively. During reperfusion, glutamate and aspartate levels reached their basal levels. The injection of 17β-estradiol at the onset of occlusion induced a smaller peak of these two amino acids and rapid decreases of the amino acids levels were observed after 15-35 min of ischemia. The extracellular level of glucose decreased near zero during occlusion, and increased at about 40-50% of its basal level during reperfusion. The level of lactate increased rapidly after occlusion and remained high during the analysed reperfusion period (3 hours). Treatment with 17β-estradiol did not significantly modify the variations of both energy metabolites during ischemia or reperfusion. Conclusion: These data suggest that 17β-estradiol exerts neuroprotection through modulation of excitatory amino acids release in the extracellular space during ischemia. Reference List 1. Longa, E.Z., Weinstein, P.R., Carlson, S. and Cummins, R. 1989. Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke. 20:84-91.

183

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ROLE OF AROMATASE ON NEUROPROTECTIVE PREGNENOLONE AND DEHYDROEPIANDROSTERONE

EFFECTS

OF

Veiga S.1, Garcia-Segura L.M.1 and Azcoitia I.2 1

Instituto Cajal, C.S.I.C., Avenida Doctor Arce 37, E-28002 Madrid, Spain. E-mail: [email protected]; Fax: 34-915854754; 2Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, E-28040 Madrid, Spain. Pregnenolone and dehydroepiandrosterone (DHEA) are neuroprotective steroids synthetized in the central nervous system from cholesterol. Effects of pregnenolone and DHEA may be in part mediated by their conversion to testosterone. In turn, testosterone may be converted to estradiol by the enzyme aromatase. This enzyme is induced in reactive astrocytes after different forms of neurodegenerative lesions and the local production of estradiol in the brain has been shown to be neuroprotective. In the present study it has been assessed whether the neuroprotective effect of pregnenolone and dehydroepiandrosterone may be mediated by their metabolism to estradiol. The protective effect against systemic kainic acid (7 mg/Kg b.w.) of different doses (12.5, 25, 50 and 100 mg/Kg) of pregnenolone or DHEA was assessed on hippocampal hilar neurons in gonadectomized Wistar male rats. To assess whether the neuroprotective effect of pregnenolone and DHEA was dependent on their conversion to estradiol, the aromatase inhibitor fadrozole (4.16 mg/ml) was administered using osmotic minipumps. The number of Nissl stained neurons in the hilus of the dentate gyrus of the left hippocampal formation was estimated by the optical disector method. The administration of kainic acid resulted in a significant decrease in the number of hilar neurons compared to rats injected with vehicles. Pregnenolone and DHEA showed a dose-dependent protective effect of hilar neurons against kainic acid. The administration of the aromatase inhibitor fadrozole blocked the neuroprotective effect of pregnenolone and DHEA. These findings suggest that estradiol formation by aromatase mediates neuroprotective effects of pregnenolone and DHEA against excitotoxic-induced neuronal death in the hippocampus of adult male rats.

This study has been supported by the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6-CT-2000-00179.

184

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

SYSTEMIC AND INTRA-AMYGDALA ADMINISTRATION OF ESTROGEN AND PROGESTERONE TO OVARIECTOMIZED RATS INCREASES ANALGESIA Walf A.A.1 and Frye C.A.1-3 The University at Albany-SUNY, Behavioral Neuroendocrinology Laboratory, 1Departments of Psychology and 2Biological Sciences, and 3 The Center For Neuroscience Research, 1400 Washington Avenue Albany, NY, 12222 USA. [email protected]. 518-442-4867. Steroid hormones may influence nociception of female rats. Female rats in proestrous have decreased sensitivity to thermal stimuli, as indicated by increased tailflick or pawlick latencies, compared to rats in other stages of the estrous cycle [7]. Increased nociception of ovariectomized (ovx) rats can be attenuated by systemic [1,3,6], intracerebroventricular [4,5], or intra-amygdala [8] administration of estrogen (E2) and/or progestins. Steroid hormone effects on nociception may also influence stress-induced analgesia (SIA) of female rats. Stress-induced analgesia is increased during proestrus compared to other stages of the estrous cycle [17]. Ovariectomy can reduce SIA; this effect can be reversed by E 2 or progestin administration [11,15,17]. Interestingly, circulating and central levels of E 2 and progestins increase in rats after exposure to stressors [13,14,16]. Thus, SIA may influence and be altered by circulating and central concentrations of steroid hormones. The medial amygdala may be important for actions of E 2 and/or progesterone (P) on stress-induced analgesia responses. Footshock SIA can be attenuated by lesioning of the central amygdala [9]. There are stress-induced changes in the amygdala following exposure to predator stimuli, such as greater FOS immunoreactivity [2] and dopaminergic activity [12]. Experiments tested the hypothesis that ovarian hormones may mediate antinociceptive responses of female rats following exposure to a stressor, predator odor. The extent to which systemic or intra-amygdala administration of E 2 and/or P to ovx rats can influence nociceptive responses after exposure to predator or no odor was investigated. Ovx, Long-Evans rats were administered subcutaneous (Exp. 1) or intra-amygdala (Exp. 2) vehicle, E 2, P, or E 2 + P and randomly assigned to groups that received exposure to an isolated component of fox feces, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), or the no odor condition. TMT has been shown to produce SIA in rats [10]. Rats were exposed to T M T or no odor for 10 minutes immediately preceding tailflick testing. Experiment 1: TMT exposure significantly increased tailflick latencies (7.8 + 0.5) compared to no odor exposure (5.1 + 0.5; Figure 1). E 2 (7.0 + 0.4), P (6.4 + 0.5), or E 2 + P (7.5 + 0.5) significantly increased tailflick latencies compared to vehicle (4.8 + 0.5; Figure 1) in TMT- and non-TMT exposed rats. Experiment 2: TMT exposure significantly increased tailflick latencies (7.2 + 0.7) compared to no odor exposure (4.3 + 0.5; Figure 2). E2 + P (6.6 + 0.8) to the medial amygdala significantly increased tailflick latencies compared to control implants (4.8 + 0.4; Figure 2) for rats exposed to the TMT or no odor condition. Our hypothesis that E 2 and/or P may influence pain responses following predator odor exposure in part through actions in the medial amygdala was supported. TMT exposure, and not exposure to the chamber alone, increased tailflick latencies in ovx rats. E 2, P, and E 2 + P administered systemically to ovx rats increased anti-nociception, indicated by increased tailflick latencies, compared to vehicle. Intra-amygdala E 2 + P produced similar increases in tailflick latencies. Thus, the similar effects of steroids in rats exposed or not to TMT indicates steroids can produce analgesia, in part through actions in the medial amygdala, and can have additional effects to amplify SIA produced by predator odor exposure. Acknowledgment: Supported by NSF (98-96263) and Whitehall Foundation (96-010).

185

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

Reference List [1] H.B. Bradshaw, K.J. Berkley, Estrogen replacement reverses ovariectomy-induced vaginal hyperalgesia in the rat, Maturitas, 41 (2002) 157-65. [2] R.A. Dielenberg, G.E. Hunt, I.S. McGregor, “When a rat smells a cat": the distribution of Fos immunoreactivity in rat brain following exposure to a predatory odor, Neurosci. 104 (2001) 1085-97. [3] L.J. Forman, V. Tingle, S. Estilow, J. Cater. The response to analgesia testing is affected by gonadal steroids in the rat, Life Sci. 45 (1989) 447-54. [4] C.A. Frye, J.E. Duncan. Progesterone metabolites, effective at the GABAA receptor complex, attenuate pain sensitivity in rats, Brain Res., 643 (1994) 194-203. [5] C.A. Frye, J.E. Duncan, Estradiol benzoate potentiates neuroactive steroids' effects on pain sensitivity, Pharmacol. Biochem. Behav. 53 (1996) 27-32. [6] C.A. Frye, E.H. Lacey, Progestins influence performance on cognitive tasks independent of changes in affective behavior, Psychobio. 28 (2000) 550-63. [7] C.A. Frye, S.M. Petralia, M.E. Rhodes, Estrous cycle and sex differences in performance on anxiety tasks coincide with increases in hippocampal progesterone and 3α,5α-THP, Pharmacol. Biochem. Behav. 67 (2000) 587-96. [8] C.A. Frye, A.A. Walf, Estrogen and/or progesterone systemically or to the amygdala can have anxiety, fear, and pain reducing effects in ovariectomized rats, Behav. Neurosci. (submitted). [9] F.J. Helmstetter, The amygdala is essential for the expression of conditional hypoalgesia, Behav. Neurosci. 106 (1992) 518-28. [10] G. Hotsenpiller, J.L. Williams, A synthetic predator odor (TMT) enhances conditioned analgesia and fear when paired with a benzodiazepine inverse agonist (FG-7142), Psychobio. 25 (1997) 83-8. [11] J.S. Mogil, W.F. Sternberg, B. Kest, P. Marek, J.C. Liebeskind, Sex differences in the antagonism of swim stress-induced analgesia: effects of gonadectomy and estrogen replacement, Pain 53 (1993) 17-25. [12] B.A. Morrow, A.J. Redmond, R.H. Roth, J.D. Elsworth, The predator odor, TMT, displays a unique, stress-like pattern of dopaminergic and endocrinological activation in the rat, Brain Res. 864 (2000) 146-51. [13] S. Plas-Roser, C. Aron, Stress related effects in the control of sexual receptivity and in the secretion of progesterone by the adrenals in cyclic female rats, Physiol. Behav. 27 (1981) 261-4. [14] R.H. Purdy, A.I. Morrow, P.H. Moore, S.M. Paul, Stress-induced elevations of γ-aminobutyric acid type-A receptor-active steroids in rat brain, PNAS 88 (1991) 4553-7. [15] S.M. Ryan, S.F. Maier, The estrous cycle and estrogen modulate stress-induced analgesia, Behav. Neurosci. 102 (1988) 371-80. [16] T.J. Shors, J. Pickett, G. Wood, M. Paczynski, Acute stress persistently enhances estrogen levels in the female rat, Stress 3 (1999) 163-71. [17] W.F. Sternberg, J.S. Mogil, B. Kest, G.G. Page, Y. Leong, V. Yam, J.C. Liebeskind, Neonatal testosterone exposure influences neurochemistry of non-opioid swim stress-induced analgesia in adult mice, Pain 63 (1995) 321-6.

Tail Flick Latency (+secs)

*

10 *

*

8 * * 6

*

4

*

8

*

* 6

4 2

2 0

0 P EB+P VEH EB n=11 n=11 n=12 n=12 0 TMT

VEH EB P EB+P n=9 n=12 n=12 n=12 100 TMT

Figure 1: Figure depicts data of mean (+ SEM) tailflick latency of ovx rats administered vehicle (white), E2 (striped), P (gray), or E2 + P (black) systemically. * above grouped bars denotes a significant effect of TMT exposure (100 ng) compared to no TMT exposure. * above each bar denotes a significant difference of hormone compared to vehicle.

186

Tail Flick Latency (+secs)

*

10

P EB+P VEH EB n=12 n=8 n=8 n=12 0 TMT

VEH EB P EB+P n=8 n=6 n=6 n=7 100 TMT

Figure 2: Figure depicts data of mean (+ SEM) tailflick latency of ovx rats administered vehicle (white), E2 (striped), P (gray), or E2 + P (black) to the amygdala. * above grouped bars denotes a significant effect of TMT exposure (100 ng) compared to no TMT exposure. * above each bar denotes a significant difference between E2 + P compared to vehicle.

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

INFLUENCE OF POSTMENOPAUSAL HORMONE REPLACEMENT THERAPY ON PLATELET SEROTONIN UPTAKE SITE AND 5-HT2A RECEPTOR BINDING Wihlbäck A.-C., Sundström-Poromaa I., Allard P., Mjörndal T., Spigset O. and Bäckström T. Dept. of Clinical Science/Obstetrics and Gynecology; Psychiatry; and Clinical Neuroscience and Pharmacology, University Hospital of Umeå, Umeå, Sweden; and the Department of Clinical Pharmacology, Regional and University Hospital of Trondheim, Norway. [email protected] Objective: To examine whether binding of [3H]paroxetine to the platelet serotonin transporter or binding of [3H]LSD to the platelet 5-HT2A receptor are influenced by postmenopausal estrogen/progestagen treatment. Methods: Twenty-three postmenopausal women with climacteric symptoms completed this double masked, randomized, cross-over study. The women received 2 mg estradiol (E2) continuously during four 28-day cycles. A sequential addition of 10 mg medroxyprogesterone acetate, 1 mg noretisterone acetate or placebo was given the last 14 days of each cycle. Before treatment, as well as once during the last week of each treatment blood samples were collected for the analysis of [3H]LSD and [3H]paroxetine binding. The power of the study setup was 81%. Results: Bmax or Kd for [3H]paroxetine binding did not change during estrogen or estrogen/progestagen treatment, nor did Bmax or Kd for the [3H]LSD binding change during the different treatments. However, in a subgroup of depressed patients, the decrease in Bmax for [3H]LSD binding during treatment was significantly more pronounced than in the nondepressed subgroup, P < .05. Conclusion: Estrogen treatment with or without the addition of progestagen does not affect the binding to the serotonin transporter or to the serotonergic 5-HT2A receptor in healthy postmenopausal women.

This work was supported by an EU Regional fund Objective 1 grant.

187

Posters’ Exhibition: Glial Cells as a Target for Steroids • Belcredito S., Vegeto E., Etteri S., Ciana P. And Maggi A. (Milano, Italy, EU) Reactivity of microglia in estrogen receptor-alpha null mice

• Ientile R., Campisi A., Raciti G., Caccamo D., Currò M., Cannavò G., Macaione S., Vanella A. and Avola R. (Catania, Italy, EU) Steroid-growth factor cross-talk on translutaminase, GFAP and vimentin expression during astroglial cell proliferation and differentiation in primary culture

• Magnaghi V., Ballabio M., Cavarretta I., Gonzalez L.C., Leonelli E., Veiga S. and Melcangi R.C. (Milan, Italy, EU) In vitro effects of neuroactive steroids on male and female Schwann cells: a study by RNAse protection assay

• Marchetti B, Tirolo C, Gennuso F, L’Episcopo F., Testa N, Caniglia S, and M orale M C. (Sassari, Italy, EU) oxidative stress, estrogens and neuroprotection of nigrostriatal dopaminergic system: bidirectional effects on astrocyte and microglia

• Menuet A., Pellegrini E., Anglade I., Le Guevel R., Kah O. and Pakdel F. (Rennes, France, EU) Direct cooperation between estrogen receptors and astrocyte-specific factors elicits high activity of the zebrafish brain aromatase gene.

• Ng B.K., Conrad H. E. and Glaser M. (Urbana, IL, USA) Characterization of an alternatively spliced platelet-derived growth factor variant during myelin synthesis

• Pinos H., Collado P., Rodriguez M. and Guillamón A. (Madrid, Spain, EU) Sexual dimorphism in radial glia in adult locus coeruleus in the rat

• Rodriguez-Waitkus P.M., LaFollette A., Zhu T.S., Suidan G. and Glaser M . (Urbana, IL, USA) Expression of the progesterone receptor and identification of progesterone inducible genes in Schwann cell/neuronal co-cultures

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

REACTIVITY OF MICROGLIA IN ESTROGEN RECEPTOR-ALPHA NULL MICE Belcredito S., Vegeto E., Etteri S., Ciana P. and Maggi A. Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, University of Milan, 20133-Milan, Italy. [email protected]; FAX 00390250318284 Even though the underlying mechanisms are still to be fully understood, growing evidence exists on a protective role of estrogens against neurodegeneration. Several studies have shown 17β-estradiol (E2) participates in neuron metabolic and survival pathways. We have shown that blood and brain-derived macrophages are targets for E2 action [1,2]. In the present study we used estrogen receptor alpha (ERα) and beta (ERβ)-null mice to identify the molecular mechanism of the in vivo anti-inflammatory activity of hormone in brain. Interestingly, we observed that genetic disruption of the ERα gene accounts for an increased reactivity of microglia, the resident macrophages of the brain. The spontaneous activation of microglia is observed in specific areas of the mouse brain (cingulated, parietal and rhinal cortices, hippocampus and amigdala), as revealed by the increase in complement receptor C3 expression in microglia of these brain regions. Microglia activation in the absence of exogenous stimuli is detected as ERα-null mice age, while it does not occur in wild-type or in ER beta-null littermates. In order to study the inflammatory response of the brain and the role of exogenous estrogen on this reaction, we injected lipopolysaccharide (LPS) in the brain lateral ventricle of wild-type mice and analyzed microglia activation. After 24 hours from LPS injection, microglia reactivity was observed in the hippocampus. Accordingly to results we previously obtained in rats, treatment with E2 6 hours before LPS prevents the morphological and biochemical activation of microglia. Further studies on hormone action on the LPS-induced response in the brains of ERα and ERβ-null mice will be discussed. Our preliminary results point to ERα as the molecular target of estrogen protective role in inflammation-based neurodegenerative diseases.

Reference List 1. Vegeto E. et al. (1999). Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-alpha-induced apoptosis. FASEB J. 13: 793-803 2. Vegeto E. et al. (2001) Estrogen prevents the Lipopolysaccharide-induced inflammatory response in microglia. J.Neurosci.21:1908-1818

189

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

STEROID-GROWTH FACTOR CROSS-TALK ON TRANSLUTAMINASE, GFAP AND VIMENTIN EXPRESSION DURING ASTROGLIAL CELL PROLIFERATION AND DIFFERENTIATION IN PRIMARY CULTURE Ientile R., Campisi A.2, Raciti G.2, Caccamo D., Currò M., Cannavò G., Macaione S., Vanella A.2 and Avola R.1 Dept of 1Chemical Sciences, Section of Biochemistry and Molecular Biology, University of Catania, Viale Andrea Doria 6, 90125 Catania (Italy), e-mail address: [email protected], fax number 095/336990; Dept of 2Biochemistry, Medical Chemistry and Molecular Biology, University of Catania; Dept. of Biochemical, Physiological and Nutritional Sciences, University of Messina, (Italy).

It has been previously reported that the interactions between growth factors and steroid hormones modulate and control markedly the development and the maturation of astroglial cell in culture [2]. EGF, IGF-I, Insulin (INS) and bFGF are mytogenic polypeptides that participate in neuron-glia cross-talk stimulating nerve cell proliferation and differentiation [3]. Glucocorticoids (GCs) and estrogens (E) play a pivotal role during neuronal cell differentiation in culture and influence also astroglial compartment regulating glial fibrillary acidic protein (GFAP) expression [6]. Serum deprivation is one exogenous stimulus, like GCs and E, capable to enhance spontaneous or growth factor-induced astroglial differentiation [5]. In this research we investigated the interactions between growth factors (GFs) or dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression in 25 DIV stressed astrocyte cultures differently treated. Furthermore, we studied the interaction between GCs or E and GFs on tissue transglutaminase (tTG) expression in 15 DIV astrocyte cultures treated for 12h with DEX or Extradiol (E2) alone or in combination with GFs. tTG is well known enzymatic activity involved in cell differentiation and it plays a modulatory role in apoptosis and the cell's response to stressors, depending on the type of the stimuli that provoke an increase in the transamidating activity.[8] Materials And Methods: We prepared primary astroglial cultures according to our previous experiments [1, 7]. Cultures at 19 days in vitro (DIV) were treated, as reported below, in order to explore the different behaviour of cytoskeletal protein expression. In detail: Condition 1): 24h pretreatment with bFGF, subsequent 72h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24h, after a prolonged (60h) DEX treatment. Condition 2): 36h pretreatment with DEX (with bFGF in the last 24h), followed by SFM for 60h and final addition for 24 h with GFs alone or by two. Cytoskeletal protein expression was evaluated by Western blotting and studied through computerized densitometric analysis.tTG expression was assayed according to Ientile et al. [4] Results And Discussion: Western blot analysis data showed a marked GFAP expression in cultures submitted to condition 1 and then treated for 60h with DEX alone or plus IGF-I or EGF or INS or EGF+IGF-I or IGF+INS, added in the last 24h only,

190

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

comparing results to untreated or treated controls. Under the same culture conditions, VIM expression was instead significantly reduced after IGF-I or EGF or INS addition in the last 24h of 60h DEX treatment, respect to control DEX-pretreated ones. On the contrary, referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to condition 2 and then treated in the last 24h with IGF-I or EGF or INS or EGF+INS, respect to both control untreated or treated cultures. VIM expression was instead interestingly modulated, showing a significant decrease in the last 24h IGF-I-treated cultures, while the addition for 24h of INS or INS+EGF or EGF+IGF-I (without effect when added alone), markedly and progressively raised it. These results reflect the preference of GFs in upregulating GFAP (a well known astrocyte differentiation marker) over VIM (a proliferative marker), when GCs are present, not only in physiological, but also in prolonged stressful events. Collectively, our findings evidence an interactive dialogue between GFs and GCs in our astroglial cultures, differently copretreated with DEX and bFGF, in regulating astrocytic cytoskeletal network under adverse environmental conditions. Concerning astroglial tTG expression experiments, our preliminary data, obtained by confocal laser microscopy and western blot analysis, showed that the treatment for 12h with E2 and EGF as well as DEX and IGF-I significantly increased tTG expression in 15 DIV astrocyte cultures. On the contrary, a very slight enhancement of tTG expression in 12h DEX or DEX+EGF or DEX+INS -treated astrocyte cultures at 15 DIV was observed. No significant effect on tTG expression in 12h E2+INS or E2+IGF-I-treated astrocyte cultures was found. In conclusion, these results suggest that steroid-GFs cross-talk plays a crucial role during astroglial cell maturation in serum-free stressed cultures, differently pretreated or treated with DEX or E2 and GFs. This in order to better elucidate, how at cytoskeletal and cytoplasmic level, these mitogenic, trophic and hormonal factors regulate astroglial cell differentiation in our in vitro stress model.

Supported by a grant MIUR/COFIN n° 2001054379.

References List 1. Avola R. Et Al. (2002), "Clinic. And Exper.Hypertension", 24, Nos. (7&8): 753-767 2. Avola R. Et Al., (2000), "Int. J. Devl.Neurosci.", 18: 743-763. 3. Gomes Et Al. (1999), "Braz. Med. Biol. Res." 32 (5): 619-631 Rev. 4. Ientile R. Et Al. (2002), "Neuroscience", 115: 723-729. 5. Loo T. Et Al., (1995), "J.Neurosci.Res". 42 (2): 184-191. 6. O'callaghan J. P.Et Al., (1989) "Brain Res". 494 (1): 159-161. 7. Spina-Purrello V. Et Al., (2002), "Mech. Age Dev", 123: 511-520. 8. Tucholski J., And Johnson, G. V.W. (2002), "J.Neurochem.", 81: 780-791

191

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

IN VITRO EFFECTS OF NEUROACTIVE STEROIDS ON MALE AND FEMALE SCHWANN CELLS: A STUDY BY RNase PROTECTION ASSAY Magnaghi V., Martini L., Ballabio M., Cavarretta I., Gonzalez L.C., Leonelli E., Veiga S., Melcangi R.C. Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milano, Italy, Tel. +39-02-50318238, Fax: +39-02-50318204, Email: [email protected] Schwann cells possess both classical (e.g., progesterone receptor, PR) and nonclassical (e.g., GABAA receptor) steroid receptors and consequently may represent a target for the action of neuroactive steroids [see for review 1]. Our previous observations have indicated that neuroactive steroids, like for instance progesterone (P), dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), stimulate the mRNA levels of two myelin proteins [i.e., glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22)] in cultures of rat Schwann cells [2-4]. In particular, Po is stimulated by P, DHP or THP treatment, while PMP22 is stimulated only by THP. However, since these neuroactive steroids are able to interact with different receptors (i.e., P and DHP with PR, THP with GABAA receptor), we have recently evaluated the role of PR and GABAA on the control of Po and PMP22 utilizing specific agonists and antagonists of these receptors [5]. The data obtained have indicated that, at least in rat Schwann cell cultures, the expression of Po is under the control of PR, while that of PMP22 under GABAA receptor [5]. Po and PMP22 play an important physiological role for the maintenance of the multilamellar structure of the myelin of the peripheral nervous system [see for review 6]. Consequently, on the basis of the present findings might be possible to suggest the use of neuroactive steroids as new therapeutically approaches for the rebuilding of the peripheral myelin. However, it is important to highlight that also other proteins expressed by Schwann cells are involved in the peripheral myelination, [e.g., myelin associated glicoprotein (MAG), connexin 32 kDa (Cx32) and myelin and lymphocyte protein (MAL)] [6,7]. Moreover, it is also important to point out that in our in vitro observations mentioned above we have utilized cultures of Schwann cells obtained from sciatic nerves of newborn rats without to take in consideration the animal sex. Consequently, we have now obtained sex-specific cultures of Schwann cells and by RNase protection assay (RPA), which is a profitable method that allow the simultaneous analysis of more mRNA transcripts, we have evaluated the effect of P and its derivatives on the expression of Po, PMP22, MAG, Cx32 and MAL. The results so far obtained indicate that, female Schwann cell cultures express higher levels of messengers coding for Po, PMP22 and MAG respect to those of male Schwann cell cultures. Moreover, there is also a different response to the neuroactive steroids depending on the sex, since neuroactive steroids are able to induce Po and PMP22 only in the male and not in female cultures. In conclusion, the finding that in Schwann cells the expression of myelin proteins and their control by neuroactive steroids is different in the two sexes should be taken in consideration for future experiments evaluating the role of neuroactive steroids on peripheral myelination.

192

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

(This study has been carried out with financial support from the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6-CT-2000-00179)

Reference List 1) Melcangi, R.C., Magnaghi, V., Galbiat, M. and Martini, L., Glial cells: a target for steroid hormones, Prog. Brain Res. 132 (2001) 31-40. 2) Melcangi, R.C., Magnaghi, V., Cavarretta, I., Martini, L. and Piva, F., Age-induced decrease of glycoprotein Po and Myelin Basic Protein gene expression in the rat sciatic nerve. Repair by steroid derivatives, Neuroscience, 85 (1998) 569-578. 3) Magnaghi V, Cavarretta I, Zucchi I, Susani L, Rupprecht R, Hermann B, Martini L, Melcangi RC. Po gene expression is modulated by androgens in the sciatic nerve of adult male rats. Mol Brain Res 1999; 70:36¯44. 4) Melcangi, R.C., Magnaghi, V., Cavarretta, I., Zucchi, I., Bovolin, P., D'Urso, D. and Martini, L., Progesterone derivatives are able to influence peripheral myelin protein 22 and Po gene expression: possible mechanisms of action, J. Neurosci. Res., 56 (1999) 349-357. 5) Magnaghi, V., Cavarretta, I., Galbiati, M., Martini, L. and Melcangi, R.C., Neuroactive steroids and peripheral myelin proteins. Brain Res Rev 37 (2001) 360-371. 6) Melcangi, R.C., Magnaghi, V. and Martini, L., Aging in peripheral nerves: regulation of myelin protein genes by steroid hormones, Prog. Neurobiol., 60 (2000) 291-308. 7) Broenstein, J.M., Function of tetraspan proteins in the myelin sheath, Curr.Opin. Neurobiol., 10 (2000) 552-557.

193

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

OXIDATIVE STRESS, ESTROGENS AND NEUROPROTECTION OF NIGROSTRIATAL DOPAMINERGIC SYSTEM: BIDIRECTIONAL EFFECTS ON ASTROCYTE AND MICROGLIA *°Marchetti B., *Tirolo C., *Gennuso F., L’Episcopo F.,*Testa N., *Caniglia S . and Morale M.C. *Neuropharmacology, OASI Institute for Research and Care (IRCCS) on Mental Retardation and Brain Aging, Troina (EN) 94018 Troina , Italy and °Dept. Pharmacol. Medical School, Univ of Sassari, Italy [email protected] Inflammation and oxidative stress are closely associated with the pathogenesis of degenerative neurological disorders. Indeed, oxidative stress is a common final pathway inducing apoptotic cell death secondary to different cellular insults possibly mediated by common mechanisms in both neuronal and non-neuronal cells. Glial cells are elements of the the central nervous system (CNS) that share with the neuronal, endocrine and immune cells, similar recognition and transduction capabilities, and that represent key entities during the development of the CNS, in the adult brain as well as during aging and aging-associated pathologies, such as neurodegenerative disorders (1). Astroglia mediated protection and trophic support to neurons, but like neurons are vulnerable to oxidative stress. Decreased function of glial cells as a result of oxidative stress may contribute to neurodegeneration. Although microglia can act both neurotrophically and neurotoxically, it is still unknown what determines and how microglia assume either a neurotrophic or a neurotoxic state. In this context, neuron-astrocyte-microglial interactions may play a major role in modifying glial neuroprotective and neurotoxic properties. Inducible nitric oxide synthase (iNOS)-derived NO is believed to be a prominent mediator of neuronal insult. NO can be generated by infiltrating macrophages, activated astroglial and microglial cells during CNS injury, thereby contributing to neurodegeneration. In addition, glial cells generate superoxide via NADPH-oxidase which reacts with NO to form the powerful oxidant peroxynitrites that attacks a wide range of targets and is believed to mediate most toxic effects of iNOS/NO activation. The nigrostriatal dopaminergic (DA) system is particularly vulnerable to oxidative stress-induced DA neuron cell death and estrogens have been shown to exert powerful neuroprotective effects in diferent models of DA neurotoxicity (2-4). Here we have used confocal laser microscopy coupled to flow cytometry and a number of biochemical determinations to study the role of astroglial cells in estrogen induced neuroprotection in two different model of oxidative stress-induces DA neuron death. In a first model, primary mesencephalic (E16-17) neuronal cultures were established and the protective effect of E2 investigated under serum deprivation (SD) conditions, both in the absence or the presence of hypothalamic, mesencephalic or striatal astrocytes. In a second model, E2 protective effect was assessed in lipopolysaccharide (LPS)-stimulated astrocytemicroglia cocultured with mesencephalic neurons. The generation of peroxynitrites at a single cell level in iNOS expressing astrocyte (GFAP+) and microglia (Mac-1/CD11b), using the fluorogenic dye DCFH-DA, was assessed under the different oxidative stress paradigms. Such analyses were complemented by NADPH-diaphorase histochemistry and nitrotyrosine immunocytochemistry to confirm iNOS/NO activation and nitrosative stress effect in single cells. Moreover, peroxynitrites decomposition products (nitrites/nitrates),

194

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

and cell survival vs cell death (TUNEL/Anexin V) were monitored at different timeintervals following application of the stressor. In developing mesencephalic neurons cultured alone, tyrosine hydroxylase positive neuron (TH+) survival sharply decreased in a time-dependent fashion under SD, and after 4-6 days (DIV), only few neurons survived. In these experimental conditions, different schedules of E2 application had only weak effects on DA neuron survival. By contrast, coculture with astrocytes, had a potent neurotrophic and neuroprotective effects. At a functional level, coculture with glia significantly increased the incorporation of [3H]DA. The application of E2 in this experimental model potentiated neurotrophic and neuroprotective effects of glia. Both soluble factors and cell-cell contacts were involved in the observed effects, and E2-induced increased bFGF/FGF receptor 1 (FGFR-1) signaling via mitogen activated protein kinase (MAPK) was identified as the key mechanism responsible for the potent morphogenic and functional effects. Treatment of mixed astrocyte-microglial cutures with LPS failed to protect DA neurons from oxidative stress-induced cell death. LPS exposure lead to a time- and dosedependent increase in NO production as determined by nitrites, the major decomposition product in cell supernates. The concomitant presence of the specific iNOS inhibitor, LNIL, resulted in a complete suppression of nitrite production for all the course of the experiment. Double immunofluorescent staining revealed a bright iNOS signal in both astrocytes and micoglial cells. Localization of iNOS was confirmed by NADPH-diaphorase histochemistry. Peroxynitrites formation in NO-producing astrocytes and microglia monitored by increased DCF fluorescence was significantly increased after LPS application as compared to control cultures or cultures treated concomitantly with L-NIL or peroxynitrites decomposition catalysts. E2 pretreatment of glial cultures rescued glial neuroprotective effects and suppressed nitrosative stress. A similar effect was observed upon treatment of glial cultures with the specific iNOS inhibitor, L-NIL or peroxynitrites catalysts. These findings underly the ability of E2 to trigger astrocyte-microglial-neuron interactions via multiple signaling cascades, and to act at the neuroendocrine-immune interface as a key player promoting differentiation and neuroprotection.

Reference List 1. Morale MC, Gallo F, Testa N., Caniglia S., Marletta N., Spina-Purrello, V., Avola R, Caucci F, Tomasi P, Delitala G, Barden N, and Marchetti B. Neuroendocrine-immune (NEI) circuitry from neuron-glial interactions to function: Focus on Gender and HPA-HPG interactions on early programming of the NEI system. Immunol. Cell Biol. 2001; 79:400-417. 2. Schneider LS, Finch CE. Can estrogens prevent neurodegeration?. Drugs and Aging 1997; 11: 87-95. 3. Behl C, Skutella T, Lezzoulach F, Post A, Widmann N, Newton CJ, Holsboer F. Neuroprotection against oxidative stress by estrogens: structure-activity relationship. Mol. Pharmacol 1997; 110: 535541. 4. Dubal DB, Kashon ML, Pettigrew LC, RenJM, Finkelstein SPO, Rau SW, Wise PM. Estradiol protects against ischemic injury. J. Cerbr. Blood Flow Metab 1998; 18: 1253-1258.

195

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

DIRECT COOPERATION BETWEEN ESTROGEN RECEPTORS AND ASTROCYTE-SPECIFIC FACTORS ELICITS HIGH ACTIVITY OF THE ZEBRAFISH BRAIN AROMATASE GENE. Menuet A., Pellegrini E., Anglade I., Le Guevel R., Kah O. and Pakdel F. Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, Université de Rennes 1, 35042 Rennes cedex, France ; [email protected] A growing body of evidence shows that estradiol (E2) acts as a general neurotrophic and neuroprotective factor on many brain regions throughout life. It is also believed that local aromatization of androgens into estrogens in discrete regions of the brain plays an important role for the development and functioning of the central nervous system. In mammals, the peak of aromatase activity coincides with the critical period of perinatal neurogenesis. Although in adults under normal physiological conditions, aromatase is only expressed in neurons, it was recently shown that, following brain injury, a de novo expression of aromatase takes place in reactive astrocytes. These data suggest that estrogens produced locally by aromatase could be implicated in brain repair [1]. In this respect, teleost fish are interesting models as it is known that the brain of adult fish exhibits an extremely high aromatase activity (100 to 1000 fold higher than in mammals) [2], which results from the expression of a specific brain aromatase gene (AROb or cyp19b). In addition, recent studies in fish embryos [3] or adults [4] indicate that AROb is upregulated by E2. For these reasons, we investigated the aromatase and estrogen receptors (ER) expression in fish brain and analysed the molecular mechanisms underlying the E2 regulation of the AROb gene. At first, in trout, by in situ hybridization, strong aromatase mRNA signals were obtained in cells bordering the ventricles in the telencephalon and ventral diencephalon, with the highest expression in the preoptic area and hypothalamus. We also used an antibody against fish aromatase showing that aromatase expression corresponds mainly to radial glial cells, which in fish persist during adulthood. These results agree with those obtained previously in another fish [5]. The comparison of aromatase and ER expression shows that although both messengers were detected in the same regions, no obvious coexpression in the same cells was found. However, RT-PCR experiments on primary glial cell cultures from trout brain suggest that ER could be expressed, at low level, in glial cells. In a second step, we decided to study in vitro the potential direct E2 regulation of the zebrafish AROb gene at the promoter level. We have isolated a 0.5kb fragment of the AROb promoter which contains an estrogen-responsive-element (ERE) located 315 bp upstream the transcription start site. This promoter was fused to a luciferase reporter gene which was transfected in cell lines from different origins together with zebrafish ER expression vectors [6]. Interestingly, although this reporter gene was weakly induced by ERs in a ligand-dependent manner, it becomes highly inducible only in glial cell lines. The functionality of the ERE and its synergistic effect with several proximal ERE-half-sites, present within the AROb promoter and confering the E2-stimulation, was clearly established by mutagenesis. These data together with gel mobility shift assays strongly suggest that an astrocyte specific protein complex cooperates with ER to markedly upregulate AROb gene expression.

196

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

In conclusion, these results indicate that E2 acts directly via an ER-dependent mechanism on the AROb promoter activity specifically in glial cells in vivo and in vitro. It is tempting to speculate that the high expression of aromatase in glial cells observed in vivo could be mediated by a constitutive E2 regulation. Because radial glial cells are well known for being involved in neuronal migration, the high expression of aromatase in radial cells and its direct regulation by E2 could be related to the well known ability of the fish brain to grow during adulthood. Taken together, these data point to fish as interesting models to study the role of aromatase in neurogenesis.

Supported by the CNRS, the Minister of Research and Education and the European Union.

References List 1. Garcia-Segura LM, Naftolin F, Hutchison JB, Azcoitia I, Chowen JA. 1999. Role of astroglia in estrogen regulation of synaptic plasticity and brain repair. J Neurobiol. 40:574-584. 2. Pasmanik M, Callard GV. 1985. Aromatase and 5 alpha-reductase in the teleost brain, spinal cord, and pituitary gland. Gen Comp Endocrinol 60: 244-251. 3. Kishida M, Callard GV. 2001 Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development. Endocrinology. 142:740-750. 4. Gelinas D, Pitoc GA, Callard GV. 1998. Isolation of a goldfish brain cytochrome P450 aromatase cDNA: mRNA expression during the seasonal cycle and after steroid treatment. Mol Cell Endocrinol. 138:81-93. 5. Forlano PM, Deitcher DL, Myers DA, Bass AH. 2001. Anatomical distribution and cellular basis for high levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source. J Neurosci 21:8943-8955. 6. Menuet A, Pellegrini E, Anglade I, Blaise O, Laudet V, Kah O, Pakdel F.2002. Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions. Biol Reprod. 66:1881-1892.

197

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

CHARACTERIZATION OF AN ALTERNATIVELY SPLICED PLATELET-DERIVED GROWTH FACTOR VARIANT DURING MYELIN SYNTHESIS Ng B.K., Conrad H.E. and Glaser M. Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, Illinois, 61801, USA. Email: [email protected]. Fax: 1-217-244-5858. An alternatively spliced platelet-derived growth factor (PDGF) A-chain isoform was detected in rat dorsal root ganglion (DRG) neuron/Schwann cell cocultures and shown to be involved in the regulation of myelin synthesis. RT-PCR, performed on mRNA extracts of cocultures from different stages during myelin synthesis, revealed an inverse pattern of expression for long (LF-PDGF-A) and short (SF-PDGF-A) variants of PDGF A. While the expression level of SF-PDGF-A decreased during active myelin formation, a concomitant increase in the expression level of LF-PDGF-A was observed. In situ hybridization of LFPDGF-A transcript in actively myelinating cocultures revealed LF-PDGF-A expression in neuronal cell bodies but not in Schwann cells. Furthermore, LF-PDGF-A immunoreactivity was detected along axonal surfaces and Schwann cell surfaces in direct contact with axons. Similar immunoreactivity was found in the sciatic nerves of 2-4 day-old rat pups. Immunocytochemical analysis of the PDGF alpha-receptors in cocultures has revealed immunoreactivity along Schwann cell processes in contact with axonal surfaces during active myelin synthesis. Such immunoreactivity is not seen during earlier or later stages of myelin synthesis. Cell surface expression of LF-PDGF-A was attributed to the basic moiety of the LF-PDGF-A exon 6 peptide region and its binding to heparan sulfate proteoglycans. The displacement of LF-PDGF-A from the cell surface was achieved by treating cocultures with free heparin or exon 6 peptide and was confirmed by immunocytochemical staining. Heparin or exon 6 peptide treated cocultures exhibited reduced myelin formation with a corresponding 5-fold reduction in MBP expression. LFPDGF-A added to heparin treated cocultures restored MBP expression to normal, myelinating levels. Preliminary research has also shown that neurons, when in coculture with Schwann cells and treated with heparin or the exon 6 peptide, show a significant reduction in progesterone receptor (PR) immunostaining in the nucleus. Thus, LF-PDGF-A signaling in myelin synthesis may precede the production of progesterone. Previous studies have shown that monitoring the translocation of PR from the cytosol to the nucleus of the neuronal cell body can provide a functional assay for progesterone synthesis. Progesterone has been implicated as a major steroid hormone that is synthesized and utilized by neural and glial cells during myelin synthesis. These results suggest that LF-PDGF-A plays an important role along with steroid hormones in the regulation myelin synthesis.

198

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

SEXUAL DIMORPHISM IN RADIAL GLIA IN ADULT LOCUS COERULEUS IN THE RAT Pinos H., Collado P., Rodriguez M. and Guillamón A. Dpto. Psicobiologia, Facultad Psicología UNED. Spain. [email protected] The locus coeruleus is a pontine structure that constitutes the major source of noradrenaline for the CNS. This nucleus is involved in a variety of important physiological and behaviorial funtions. The LC is a sexually dimorphic structure, showing adults female larger volume and greater number of neurones than males.In a previous work, we studied the development of sex differences in this nucleus and we found that both sexes have different patterns of develpoment, reaching the adults values in distints periods of life. The sexual dimorphism is expressed transiently during development, and is established in adult life. In previous works we reported the ontogeny of radial and matura glia using inmunocitochemical procedures with antibodies Vimentin, that is asociated with radial glia and GFAP that, detects mature glia o astrocytes. It is kwon radial glia supports neuronal migration during development but their function in adult is not well kwon. Radial glia also constitute the initial step in the maturation of astrocytes. Our previous results showed that radial glia can be detected from day 1 (D1) until day 90 (D90) and both sexes had a increment with higers values in day 7 (D7), and day 60 (D60).GFAP is detectable from day 15 (D15) in coincedence with a decrease in radial glia, until D90, being the concentration similar in both sexes. The goal of the present work is know if radial glia presents sexual dimorphism in ages in which this structure does.We use 20 Wistar rats (10 males, 10 females) ages in two groups acordding age and sex: day 35 and day 60. Vimentin was used to detect radial glia, and all cells and process was taking into account. Data was submitted a ANOVA.The result show that radial glia is sexodimorphic at D35 (F= 5,418 p<0,05) and at D60 (F=4,856 p<0,05) having males more number of process than females. Taken together this and previous result might indicate that migratory activity has not finished in prepuberal period but continues during adult life. The presence of radial glia in adulthood can contribute to establihs sex differences in this structure.

This works is supported by MCYT: BOS2000-0145 and Plan de Promoción de la Investigación de la UNED.

199

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

EXPRESSION OF THE PROGESTERONE RECEPTOR AND IDENTIFICATION OF PROGESTERONE INDUCIBLE GENES IN SCHWANN CELL/NEURONAL COCULTURES Rodriguez-Waitkus P.M., LaFollette A., Zhu T.S., Suidan G. and Glaser M. University of Illinois, Department of Biochemistry and Neuroscience Program, 600 S. Mathews Ave. Urbana, IL 61801 USA, FAX:1-217-244-5858. [email protected] The major objective of this research is to identify the progesterone signal transduction pathway, between neurons and Schwann cells, that regulate the steps in myelin formation. Progesterone was found to increase myelin synthesis in a dose dependence manner. Fluorescent lipid incorporation into newly synthesized myelin was measured for cocultures treated with different concentrations of progesterone. Interestingly, 100 nM of the steroid produced a 2.5-fold increase in the rate of myelin synthesis over the control. Progesterone was synthesized by Schwann cell/neuronal cocultures during active myelin synthesis. The mRNAs for P450scc and 3B-HSD were expressed in Schwann cells that were elongated and actively forming myelin. Immunocytochemistry and Western blots studies confirmed that P450scc is expressed in myelinating Schwann cells and that its protein levels increase during myelination. The progesterone receptor (PR) was found to be present in neurons, and it was found to translocate into the nucleus at the onset of myelin synthesis. Electron microscopy studies using co-cultures that were treated with RU-486 for 15-days after induction showed a significant decrease in the number of myelinated axons and number of lamellae. These results suggest that one effect of progesterone is to act via its classical receptor to induce gene expression. Identifying the genes induced by progesterone in Schwann cell/neuronal co-cultures and dorsal root ganglia (DRG) neurons in the presence and absence of progesterone, could elucidate the signaling mechanisms involved in the regulation of myelin synthesis. Differential Display PCR was employed to identify the changes in gene expression induced by progesterone, and several new genes are being characterized. These include: rap1b, a small GTPase protein; trophinin, a cell adhesion molecule; voltage dependent anion channel (VDAC); and PRPP-associated protein-39kd, a protein regulator of PRPP synthase activity. Expression of these genes increase in Schwann cell/neuronal cocultures during myelin synthesis and can be inhibited by addition of RU-486. Addition of progesterone to neurons shows an increase in the expression of these genes. Identification of these progesterone inducible genes could provide new insights of the signaling cascades that this steroid triggers during myelin synthesis.

200

Posters’ Exhibition: Steroid Regulation of Reproduction •

Canoine V., Fusani L., Schlinger B.A. and Hau M. (Los Angeles, CA, USA) Expression of androgen receptor, estrogen receptor, and aromatase in the brain of a tropical bird, the Spotted Antbird: MapPing and seasonal comparison

•

Cornil C.A., Seutin V., Motte P., Balthazart J. (Liège, Belgium, EU) Dopamine actions on MPOA neurons are mediated in part by alpha-noradrenergic receptors

•

Forlano P.M. and Bass A.H. (Ithaca, NY, USA) Seasonal plasticity of brain aromatase mRNA expression in glia: regulation by gonadal steroids and divergence between the sexes

•

Grattan D.R., Liu L., Augustine R.A., Davey H.W. and Bunn S.J. (Otago, New Zealand) Sex differences in hypothalamic dopamine neurons are independent of prolactin signalling through Stat5b

•

Guerriero G., Roselli C.E. And Ciarcia G. (Napoli, Italy, EU) Progesterone receptor in the frog brain: seasonal expression and hormonal dependence

•

Guerriero G., Birch L., Prins G.S., Roselli C.E. and Ciarcia G. (Napoli, Italy, EU) Progesterone receptor in the lizard: distribution and properties of progesterone binding sites in the brain

•

Hazelton J.L., Hoffman G.E., Cushing B.S., Levine K., Le W.W., Ottinger M.A. and Carter CS (Baltimore, MD, USA) Activation of the HPG axis following cohabitation in the Prairie Vole (Microtus orchrogaster)

•

Matagne V., Lebrethon M-C., Gerard A., Bourguignon J-P. (Liège, Belgium, EU) Estradiol stimulation of pulsatile gonadotropin releasing hormone (GnRH) secretion from prepubertal rat hypothalamic explants: a perinatally programmed and sexually dimorphic effect

•

Morale M.C., L’Episcopo F., Testa N.*, Caniglia S., Tirolo C., Gennuso F. and Marchetti B. (Troina, Italy, EU) Estrogen as a key morphogenic and differentiation factor for immortalized hypothalamic LHRH neurons: crosstalk between the cytoskeleton and intracellular signaling pathways

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

EXPRESSION OF ANDROGEN RECEPTOR, ESTROGEN RECEPTOR, AND AROMATASE IN THE BRAIN OF A TROPICAL BIRD, THE SPOTTED ANTBIRD: MAPPING AND SEASONAL COMPARISON Canoine V.1,2, Fusani L.2, Schlinger B.A.2 and Hau M.1 1

Department of Ecology & Evolutionary Biology, Princeton University, Princeton, NJ 08544-1003 2 Department of Physiological Science, University of California, Los Angeles, P.O. Box 951527, 621 Charles Young Drive South , Los Angeles, CA 90095-1527 Email: [email protected], Fax: +1-(310)-206-9184 In bird species breeding in temperate zones, aggressive and reproductive behaviors depend on sex steroids produced in the gonads. The few studies on tropical birds indicate that in these species sex steroid levels are low throughout the year, even when they breed and are territorial. Tropical bird species may have evolved mechanisms controlling aggressive behavior that differ from those of temperate zones birds. The neuroendocrine control of aggressive behavior in tropical birds has been widely neglected so far. We are studying the neuroendocrine control mechanisms of aggressive behavior in a tropical model species, the Spotted Antbird (Hylophylax naevioides). This suboscine passeriform species breeds seasonally (during the raining season). Although Spotted Antbirds are paired and are territorial year-round, circulating androgen levels are low throughout the year. Nevertheless territorial behavior in this species seems to be androgenand/or estrogen dependent, because blocking androgen receptors and the conversion from androgen into estrogen reduces aggressive behavior during the breeding season [1]. The question arises, how can territorial behavior be androgen-dependent when circulating androgen-levels are low? We are considering two possible explanations: 1. The sensitivity to low circulating androgen is increased by up-regulating androgen and/or estrogen receptors (AR, ER) and/or aromatase in the brain. 2. Androgen-dependent behavior might be controlled by androgen deriving from extragonadal sources such as the brain itself. The first step of our study was to map the distribution of AR, ER and aromatase in the Spotted Antbird brain, and to compare their expression across breeding and nonbreeding seasons. We collected brain tissues from male Spotted Antbirds in the breeding season (June), in the late breeding season (September) and in the nonbreeding season (January) in the rainforests of Panama. The mRNAs of AR, ER, and aromatase, were localized by means of in situ hybridization, using a protocol routinely utilized in our laboratory. For ER and aromatase, probes were prepared using zebra finch (Taeniopygia guttata) sequences. For AR, we used a sequence from another tropical suboscine, the golden-collared manakin (Manacus vitellinus). No specific labelling was detected using the sense probes. Labelled areas were compared with similar regions of the zebra finch and manakin brain. AR was found to be expressed widely in the male Spotted Antbird brain. Expression was particularly high in the rostral telencephalon along the ventricles and in the neostriatum surrounding the lobus paraolfactorius. High AR expression was also detected in the septum, the hypothalamus/preoptic area, the nucleus taeniae of the archistriatum, the

202

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

dorsal thalamus, in Purkinje cells of the cerebellum, in the nucleus intercollicularis and nucleus vestibularis. In contrast to AR, ER has a more restricted distribution. Although some ER was detected in the rostral neostriatum and hyperstriatum, expression was highest in the septum, hypothalamus/preoptic area and bed nucleus of the stria terminalis and in the nucleus taeniae. Aromatase was also widely distributed in the brain, appearing more like that seen in the oscine songbirds than in non-passeriform species. Expression was most abundant in the hyperstriatum and hippocampus, but was also present at high levels in the hypothalamus/preoptic area, nucleus taeniae, and in the caudal medial neostriatum. To our knowledge this is the first report of the distribution of AR, ER, and aromatase in the brain of a suboscine tropical bird. At present, we are comparing the level of expression of these factors between seasons. The next step will be to measure the activity of steroidogenic enzymes in the brain. These studies will help us determine if sex steroid sensitivity and/or synthesis in the brain are regulated seasonally to activate aggressive behaviors when peripheral sex steroids are low.

Supported by grants from NSF IBN-0196297 (to MH) and MH61994 (to BAS).

Reference List [1] Hau, M., Wikelski, M., Soma, K.S. and Wingfield, J.C., Testosterone and Year-Round Territorial Aggression in a Tropical Bird, General and Comparative Endocrinology, 117 (2000) 20-33.

203

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

DOPAMINE ACTIONS ON MPOA NEURONS ARE MEDIATED IN PART BY ALPHA-NORADRENERGIC RECEPTORS Cornil C.A., Seutin V., Motte P. and Balthazart J. Center for Molecular and Cellular Neurobiology, Research Group in Behavioral Neuroendocrinology, University of Liège, 17 place Delcour (Bât L1) B-4020 Liège Belgium. Email : [email protected].

The conversion of androgens into estrogens by aromatase plays a crucial role in the control of many behavioral and physiological processes such as the activation of male sexual behavior and sexual differentiation of the brain. Brain aromatase activity (AA) is controlled by a synergistic action of androgens and estrogens that modulate the transcription of the enzyme [1]. A host of arguments suggest that aromatase may also be controlled trans-synaptically by catecholamines. The medial preoptic nucleus (POM), a necessary and sufficient site for the activation of male sexual behavior by steroids that contains also the largest group of aromatase-expressing neurons, receives inputs from various dopaminergic and noradrenergic areas. Moreover, there is in this nucleus a close association between aromatase-immunoreactive cells (ARO-ir) and fibers immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. Estrogen has been shown to control catecholamines activity in a variety of animal models and estrogen receptors are colocalized with TH in several catecholaminergic areas of the avian and mammalian brain. The effects of estrogen on AA could thus be mediated through changes of the catecholaminergic activity [2]. Pharmacological studies support this notion derived originally from neuroanatomical data. Manipulations of the endogenous brain levels of catecholamines by neurotoxins indicate that noradrenaline (NE) inhibits AA while the enzyme activity is stimulated by dopamine (DA). These effects are observed several days after the pharmacological treatment suggesting that they result from changes in aromatase concentration. On another hand, a direct inhibitory effect of dopamine and of a variety of dopaminergic compounds on AA has been demonstrated during in vitro studies of AA in brain homogenates [3] and in preoptic quail explants maintained in vitro [4]. These effects may involve the modulation by catecholamines of cAMP levels in ARO-ir cells which would then affect aromatase activity as demonstrated in a variety of animal models and tissues including the brain [2]. However, to date, there is no evidence for the presence of dopaminergic receptors at the surface of aromatase cells. Together, these data clearly indicate that catecholamines can markedly affect estrogen synthesis in the brain. However the underlying mechanism(s) mediating these effects remain(s) to be identified. Do catecholamines interact with specific receptors located on the aromatase neurons or on adjacent cells? Or do they interact directly with aromatase [4]? As a first step to address these questions, we analyzed the electrophysiological effects of DA superfused on brain slices containing the medial preoptic nucleus of quail. Intracellular recordings showed that 100 µM DA hyperpolarized most POM neurons (76%) whereas few of them were depolarized (6%). Some of these intracellularly recorded cells were labeled by biocytin and subsequently immunostained for aromatase. Thus far, all double-labeled cells (n = 3) were hyperpolarized by DA.

204

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

To investigate the type of receptors mediating the DA-induced hyperpolarizations in POM neurons, the effects of selective dopaminergic antagonists were examined. Surprisingly, DA-induced hyperpolarizations were not antagonized by D1 nor by D2 receptor antagonists (SCH-23390 and sulpiride, 1µM). During extracellular recordings, the superfusion of DA had profound effects on the firing rate of POM neurons. The firing rate of most neurons (52%) was decreased by DA while the firing of 24 % of these neurons was increased by DA. Like in intracellular experiments, the D2 antagonist sulpiride (1 µM) failed to suppress the DA-induced inhibitory effects in most cells. Likewise, the D1 antagonist SCH-23390 (1 µM) also did not block firing excitations induced by DA in the some cells. Norepinephrine (NE) also affected the firing rate of POM neurons. NE decreased the firing rate of most neurons (62%), while the firing rate of a smaller population of neurons was increased by NE (11 %). When both amines were successively applied, NE mimicked the effect of DA in 83% of the cells. To test the hypothesis that DA produces its effects mainly by interaction with noradrenergic receptors, we examined the effect of noradrenergic antagonists on the responses to DA. Yohimbine (1 µM), an α2-noradrenergic receptor antagonist, suppressed the inhibitory effect of DA in most cells and prazosin (1 µM), an α1-noradrenergic antagonist, blocked the excitation produced by DA in all cells that were tested. Superfusion of either cysteine (1mM) or fusaric acid (100µM), two well-known inhibitors of dopamine-ß-hydroxylase (DBH, the enzyme converting DA into NE), failed to significantly modify the inhibitory and excitatory effects of DA suggesting that the effects of DA on the α-noradrenergic receptors are not mediated by a metabolic conversion of DA into NE. Yohimbine also shifted the inhibitory concentration-response curves for DA and NE with the same ability suggesting that both DA and NE interact with an α2receptor. Taken together, these results demonstrate that the effects of DA in the POM are mediated mainly by the activation of α2 (inhibitions) or α1 (stimulations) NE receptors. The presence of aromatase in three cells hyperpolarized by DA suggests that these receptors might be expressed at the surface of aromatase-containing neurons. These data are therefore consistent with a direct control of aromatase activity by dopamine acting through α-noradrenergic receptors located at the surface on aromatase cells. Additional studies should however be performed to determine whether the colocalization of aromatase and these dopamine receptors takes place in the majority of aromatase neurons, as suggested by the few cells studied so far. Supported by: MH50388, ARC99/04-241, FRFC 2.4555.01, FRFC 2.4542.00 and the Fonds Spéciaux pour la Recherche. C.A.C is an FNRS Research Fellow Reference List 1. Balthazart, J. and G.F. Ball, New insights into the regulation and function of brain estrogen synthase (aromatase). TINS, 21 (1998) 243-249. 2. Absil, P., et al., The control of preoptic aromatase activity by afferent inputs in Japanese quail. Brain Res Rev, 37 (2001) 38-58. 3. Baillien, M. and J. Balthazart, A direct dopaminergic control of aromatase activity in the quail preoptic area. J.Steroid Biochem.Molec.Biol., 63 (1997) 99-113. 4. Balthazart, J., M. Baillien, and G.F. Ball, Interactions between aromatase (estrogen synthase) and dopamine in the control of male sexual behavior in quail. Comp.J.Biochem.Physiol., part B 132 (2001) 37-55.

205

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

SEASONAL PLASTICITY OF BRAIN AROMATASE mRNA EXPRESSION IN GLIA: REGULATION BY GONADAL STEROIDS AND DIVERGENCE BETWEEN THE SEXES Forlano P.M. and Bass A.H. Cornell University, Department of Neurobiology and Behavior, Seeley G. Mudd Hall, Ithaca, NY 14853, USA. [email protected]; 607 254 4308

The plainfin midshipman fish, Porichthys notatus, exemplifies one of the most broadly studied neuroethological models for brain mechanisms underlying the performance of reproductive tactics among teleosts [1]. In the summer (May-August), males migrate from deep, offshore waters to excavate nests under rocks in the intertidal zone along the northwest coast of the U.S. and Canada. Nesting males produce a courtship “hum” that attracts females who then spawn once and soon return offshore. Males alone provide parental care while continuing to acoustically court females throughout the summer. During the breeding season, nesting males have high levels of circulating 11ketotestosterone (11-KT) that gradually declines with increased parental care [8]. Females have higher levels of testosterone (T) than males and low levels of estradiol (E) [4]. Midshipman [9], as other teleosts [5] express levels of brain aromatase activity several fold higher than other vertebrate groups. Using teleost-specific antibodies and midshipmanspecific probes, we determined brain aromatase (ARO) enzyme and mRNA distribution to be localized to glia in midshipman breeding adults [6]. Like other vertebrates, ARO is expressed in hypothalamic and preoptic areas, but also found along ventricular surfaces throughout the brain, as well as in and around the dimorphic sonic motor nucleus in the hindbrain. Abundance of immunoreactive cells and mRNA signal corroborate regional differences in activity levels [9]. This study investigated seasonal expression of ARO mRNA by in situ hybridization (ISH) in the brain of males and females and its relationship to their levels of circulating steroids (to be reported in [10]). Adult fish were collected from nests during the breeding season or by otter trawl in 60-120m depths offshore in September, December, February, March and April. Blood samples, morphometrics (body and gonad weight) and brains (after perfusion with 4% paraformaldehyde) were collected shortly after capture. 11-KT, T, and E were analyzed by radioimmunoassay [10] and brains and ovaries processed for ISH [6]. Females collected in the winter (December and February) have regressed ovaries, low gonadosomatic index (GSI), low circulating steroids and undetectable aromatase mRNA in the ovary. ARO expression is only evident in the ventrolateral telencephalon and preoptic area. In March and April, females undergo gonadal recrudescence; T and E levels peak [10] and ARO expression is highest throughout the brain, including the vocal hindbrain, periaqueductal midbrain, periventricular hypothalamus, and anterior preoptic area. Summer females collected from nests have circulating steroid levels equivalent to winter fish, although GSI is maximized just prior to spawning [10]. ARO expression is high in both spent and developing ovaries. ARO in the brain, however, again follows steroid levels and although it is higher than winter females, is lower than spring females. During gonadal regression in September, brain ARO is still detectable in the midbrain, periventricuar hypothalamus, and preoptic area. In another study, we verified that brain ARO mRNA in 206

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ovariectomized winter females resembles intact winter females (signal detectable only in the forebrain), while females implanted with T or E restore brain ARO levels similar to females undergoing gonadal recrudescence [Forlano and Bass, unpub]. Like females, winter males show lowest levels of brain ARO mRNA expression, only evident in the forebrain. However, unlike females, males only show a significant change in one steroid, 11-KT, which increases in the spring and remains elevated during summer courtship [4,8,10]. Brain ARO mRNA expression in males parallels the increase in androgens in spring and reaches a peak in the summer; highest levels are in the periventricular hypothalamus and preoptic areas, with comparatively less expression in the vocal hindbrain. However, mRNA expression in vocal hindbrain areas is greater in the summmer breeding season than in winter. Like females, early fall (post-spawning) shows a decrease in brain ARO with a corresponding decrease in gonadal steroids [10]. In sum, males and females show a temporal difference in seasonal changes in brain mRNA expression of ARO that closely follows changes in circulating steroid levels. The increased ARO expression in hypothalamic/preoptic areas during gonadal recrudescence in both sexes is likely involved in gonadotropin feedback. However, the maintenance of elevated levels in males into the spawning period reflects their continued courtship and spawning activity throughout the summer; by contrast, females are adapted to spawn once. Seasonal ARO expression in androgen-sensitive hindbrain vocal centers [2] may reflect changes in circulating steroids that prime circuitry necessary for reproductive-related vocal behaviors in males, while upregulation of female ARO around vocal centers may function to prevent their masculinization during periods of high circulating T [9]. In addition, locally formed neuroestrogen in females could act as a neuromodulator to enhance the phonotaxic response to the male mate call. These results are the first to demonstrate seasonal changes and regulation of ARO mRNA expression in situ in behaviorally identified (sonic) nuclei in a teleost fish. Support from NIMH predoctoral training grant (5T32MH15793) and NSF Research Grant (IBN9987341). Reference List [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

A.H. Bass, Shaping brain sexuality, Am. Sci. 84 (1996) 352-363. A.H. Bass, Alternative life history strategies and dimorphic males in an acoustic communication system, In Proceedings of the fifth international symposium on the reproductive physiology of fish, (1995) 258-260. J. Balthazart, G. Ball, New insights into the regulation and function of brain estrogen synthase (aromatase), Trends Neurosci. 21 (1998) 243-248. R.K. Brantley, J.C. Wingfield, A.H. Bass, Sex steroid levels in Porichthys notatus, a fish with alternative reproductive tactics, and a review of the hormonal bases for male dimorphism among teleost fishes. Horm. Behav. 27 (1993) 332-347. G.V. Callard, B.A Schlinger, M. Pasmanik, Nonmammalian vertebrate models in studies of brain-steroid interactions, J Exp Zool Suppl 4 (1990) 61-64. P.M. Forlano, D.L Deitcher, D.A Myers, A.H. Bass, Anatomical basis for high levels of aromatase activity i n the brain of teleost fish: aromatase enzyme and mRNA expression identify glia as source, J. Neurosci. 2 1 (2001) 8943-8955. D. Gelinas. G.A. Pitoc, G.V. Callard, Isolation of a goldfish brain cytochrome P450 aromatase cDNA: mRNA expression during the seasonal cycle and after steroid treatment, Mol. Cell. Endocrinol. 138 (1998) 81-93. R. Knapp, J.C. Wingfield, A.H. Bass, Steroid hormones and paternal care in the plainfin midshipman fish (Porichthys notatus), Horm. Behav. 35 (1999) 81-89. B.A. Schlinger, C. Greco, A.H. Bass, Aromatase activity in the hindbrain vocal control region of a teleost fish: divergence among males with alternative reproductive tactics, Proc. R. Soc. Lond. B. 266 (1999) 131136. J.A. Sisneros, P.M. Forlano, R. Knapp, A.H. Bass, Seasonal changes in steroid hormone concentrations in a substrate spawning, vocal teleost, the plainfin midshipman, in prep.

207

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

SEX DIFFERENCES IN HYPOTHALAMIC DOPAMINE NEURONS INDEPENDENT OF PROLACTIN SIGNALLING THROUGH STAT5b

ARE

Grattan D.R.1, Liu L.1, Augustine R.A.1, Davey H.W.2 and Bunn S.J.1 Centre for Neuroendocrinology, 1Department of Anatomy and Structural Biology and 2Department of Physiology, University of Otago, P.O. Box 913, Dunedin, New Zealand [email protected], fax (64)(3) 479-7254 Sex differences in the brain may arise from the permanent organisational effects of exposure to sex steroids during development, or from the exposure to a differential hormonal milieu in the adult. There are marked sex differences in the neuroendocrine mechanisms that regulate prolactin secretion from the anterior pituitary gland. Levels of prolactin in the blood are higher in females than in males, at least partially due to stimulatory actions of estrogen on prolactin gene transcription [5,6] and prolactin secretion [4] from the anterior pituitary gland. Similarly, basal activity of tuberoinfundibular dopamine (TIDA) neurons, which are involved in the tonic suppression of prolactin secretion, are approximately two fold higher in females than in males [2]. It is well established that prolactin stimulates the activity of these TIDA neurons, thereby regulating its own secretion by short-loop feedback [1]. Hence, it seems possible that the elevated TIDA neuronal activity in the female is at least partially due to the higher levels of circulating prolactin in the blood. We have recently demonstrated that the intracellular pathways mediating prolactin stimulation of TIDA neurons require the transcription factor, signal transducer and activator of transcription 5b (STAT5b) [3]. The availability of STAT5b-deficient mice allows us to investigate the hypothesis that the sex difference in TIDA neuronal activity is dependent on stimulation of these neurons by prolactin acting through STAT5b. Male and female STAT5b-deficient and wild type mice were sacrificed by CO2 inhalation. A blood sample was collected by cardiac puncture, then the brain removed and frozen on dry ice. Serum prolactin levels were measured by radioimmunoassay. Specific brain regions were microdissected using the punch technique. Samples of median eminence, which contains the nerves terminals of the TIDA neurons, were placed into 0.1 M perchloric acid, and concentrations of dopamine and its metabolite dihydroxyphenylacetic acid (DOPAC) were measured in the acid extract by high performance liquid chromatography with electrochemical detection. Samples of hypothalamic arcuate nucleus, containing the cell bodies of the TIDA neurons, were placed into lysis buffer for extraction of total RNA. Levels of tyrosine hydroxylase mRNA were quantified by real-time RTPCR using the Taqman® system. Similarly, nerve terminal (striatum) and cell body (substantia nigra) regions of nigrostriatal dopaminergic neurons were dissected from all brains, and DOPAC concentrations and tyrosine hydroxylase mRNA measured, respectively, for comparison. In both male and female STAT5b deficient mice, prolactin levels were markedly elevated compared with wild type mice. This is consistent with previous data [3], suggesting an absence of negative feedback in mice lacking STAT5b. In both genotypes,

208

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

prolactin levels were significantly higher in females than in males. DOPAC concentrations in the median eminence provide an index of activity of the TIDA neurons, and were significantly higher in females than in males. Levels of median eminence DOPAC were significantly decreased in STAT5b-deficient mice compared with wild type mice, but the sex difference in TIDA activity was retained. Levels of tyrosine hydroxylase mRNA in the arcuate nucleus were also significantly lower in STAT5b-deficient mice compared with wild type mice in both males and females. Interestingly, levels of expression of tyrosine hydroxylase in the arcuate nucleus were higher in males than females, regardless of genotype. All of these changes appeared to be specific to the TIDA neurons, as there were no gender or phenotype specific changes in activity of the nigrostriatal dopaminergic neurons. These data confirm marked gender differences in the activity of the TIDA neurons that control prolactin secretion. This sex difference persists in STAT5b-deficient mice. Since STAT5b is required for the stimulatory actions of prolactin on these neurons[3], we can conclude that the sexual dimorphism in brain function is independent of gender differences in blood levels of prolactin in the adult. It seems likely that differential exposure to gonadal steroid hormones, either during development or in adulthood, might underlie the sex difference in TIDA neuronal activity.

Reference List [1] Ben-Jonathan, N. and Hnasko, R., Dopamine as a Prolactin (PRL) Inhibitor, Endocrine Rev, 22 (2001) 724-763. [2] Demarest, K.T. and Moore, K.E., Sexual differences in the sensitivity of tuberoinfundibular dopamine neurons to the actions of prolactin, Neuroendocrinology, 33 (1981) 230-4. [3] Grattan, D.R., Xu, J.J., McLachlan, M.J., Kokay, I.C., Bunn, S.J., Hovey, R.C. and Davey, H.W., Feedback regulation of PRL secretion is mediated by the transcription factor, signal transducer, and activator of transcription 5b, Endocrinology, 142 (2001) 3935-3940. [4] Larson, G.H. and Wise, P.M., Constitutive and regulated prolactin secretion: effects of estradiol, Biol Reprod, 50 (1994) 357-62. [5] Maurer, R.A., Estradiol regulates the transcription of the prolactin gene, J Biol Chem, 257 (1982) 2133-6. [6] Shull, J.D. and Gorski, J., Estrogen stimulates prolactin gene transcription by a mechanism independent of pituitary protein synthesis, Endocrinology, 114 (1984) 1550-7.

209

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PROGESTERONE RECEPTOR IN THE FROG BRAIN: SEASONAL EXPRESSION AND HORMONAL DEPENDENCE Guerriero G.1,2, Roselli C.E.2 and Ciarcia G.1 1

Department of Zoology, "Federico II" University, Via Mezzocannone 8, 80134 Naples, (I) 2Department of Physiology and Pharmacology, O.H.S.U., Portland, OR 97201, USA. e-mail [email protected] Progesterone, like estradiol, appears to be involved in the regulation of follicular development, ovulation, and modulation of reproduction behavior. Specifically, in lower vertebrates progesterone, it has also been reported that inhibits estrogen-induced vitellogenesis. How progesterone inhibits either directly via the vitellogenesis gene or indirectly via receptor or both, is not yet known. Certainly, progesterone action requires the presence of a specific receptor [1] whereas a progesterone receptor, have been identified in the brain of different lower vertebrates [2]. Our hypothesis is progesterone brain receptor should affect the physiological function related to vitellogenesis and reproduction. In order to test this hypothesis we carried out a study on the seasonal expression of progesterone receptor (PR) in the brain of Rana esculenta. The occurrence of progesterone receptor has been detected by classical binding studies through the reproductive cycle comparing to plasma steroid seasonal variation, and following progesterone (P) and estradiol (E2) treatment. Adult female frogs of Rana esculenta were collected from the surrounding of Naples during the main phases of the reproductive cycle: early recovery (October) and later recovery (February); spawning (from March to May); post-reproductive (from June to September). During recovery, the vitellogenin is synthesized in the liver and accumulated by growing oocytes. Plasma was obtained after centrifugation of the blood at 800 g for 10’, at 4°C and stored at —20°C until use. P and E2 content in plasma samples were assayed by a RIA method. Ovaries are included in paraffin and a follicle diameter analysis was monthly measured by micrometry lents. For biochemical studies, brains were immediately dissected out and pooled into three groups of about 20 adult females each and stored in liquid nitrogen until use. For Scatchard analysis and binding specificity evaluation, aliquots of cytosol (cyt) and nuclear extract (n.e.) of brain the main phases of the reproductive cycle were treated according to the usual procedures [2]. For the steroid treatment animals were captured during the month of October, when circulating vitellogenin is low and separated into seven groups, with intact sacrificed soon after the arrival; sham operated soon after the arrival and kept in captivity for the whole duration of the experiment; intact in captivity; and ovariectomized injected with saline solution, with E2, with P, with E2 and P. Steroids were treated and added to the saline solution at the required concentration [3]. Animals were fed on meat worms ad libitum. At end of the experiment, each group consisted of twenty animals (n = 20). Animals were sacrificed the day after the last injection and brains were processed as already reported. P in plasma peaked in the later recovery and in post-reproductive phase; E2, later in the recovery and it remained at low value until the next recovery. In the later recovery, the follicles showed the maximum diameter. 3H-P binding activity was detected in both cyt and

210

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

n.e. of the female frog brain of Rana esculenta. Only one binding component was present with high affinity. The average Kd was about 10-9 nM and the seasonal Kd mean values did not show any significant variation. Competition studies show that in the cyt, P, testosterone and deoxycorticosterone all competed to the same extent. The other competitors did not compete at all. In the n.e. progesterone was the best competitor, followed by R5020. E2, corticosterone and the antagonist RU26988-5 competed poorly. Testosterone, deoxycorticosterone, 17α-hydroxyprogesterone and the antagonists 2914-R2 and RU486 were not effective. [3H]-P binding brain activity was high in all period examined except during spawning whereas no binding was detected. [3H]-P binding was high in n.e. in early recovery and low especially in the spawning. PR in the n.e. brain of Rana esculenta resulted high in the brain when the levels of circulating progesterone and E2 are low, at the beginning of the recovery period and it appear lower when the follicular growth proceeds and the P and E2 increase. Hormonal steroids treatment report effects on brain PR activity levels. Ovariectomized females treated with saline solution showed the highest levels PR brain in both cyt and n.e.. Indeed, PR levels were significantly higher in ovariectomized females than in intact females sacrificed soon after capture, in intact females kept in captivity for the whole duration of the experiment, and in the sham operated females. Treatment with E2 and/or P brought about a decrease in progesterone brain receptor levels. The data provide a seasonal evaluation of progesterone brain receptor expression of the amphibia anura Rana esculenta. The saturation binding capacity resulted very similar to those found in the amphibia urodele, Xenopus laevis [4]; Scatchard analysis revealed only one binding site for P with an average of Kd of about 10-9 M, a value similar to that one reported for PR in different tissues of other lower vertebrate [3]. The binding in the brain n.e. of Rana esculenta was specific for P; the low specificity in the brain cyt could be due to an interference exerted by other binding molecules [2]. PR fluctuates throughout the reproductive cycle, along with the plasma steroids E2 and P and PR levels result high when the levels of circulating E and P are low, as occurs at the beginning of the recovery period, and therefore vitellogenin synthesis is kept at its minimum; and it appears lower when the follicular growth proceeds and the E2 and P levels increase. The decrease of brain PR levels could depend to the increase of P and E2 and can be related to the augmentation of the vitellogenin synthesis. In fact, in Rana esculenta ovariectomy is followed by an increase of PR brain levels and these levels decrease in E and/or P brain animals injected. Thus, in Rana esculenta, the ovary seem to exert a negative regulation on PR. Supposing a P role as a suppressor of vitellogenesis during the reproductive cycle, we can hypothesize a similar physiological role in the frog, Rana esculenta for plasma P aging by progesterone brain receptor.

Reference List 1. De Mayo, F.J., Zhao, B., Takamoto, N.,.Tsai, S.Y. Ann.N.Y.Acad Sci 955 (2002) 48-59. 2. Guerriero, G. and Ciarcia, G. Brain Research Review 37 (2001) 172-177. 3. Paolucci, M., Guerriero, G., Ciarcia, G. J. Exp. Zool. 284(1999) 765-775. 4. Roy, E.J. Wilson, M.A. Kelley, D.B. J. Steroid Biochem. 19 (1983) 1571-1575.

211

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PROGESTERONE RECEPTOR IN THE LIZARD: DISTRIBUTION PROPERTIES OF PROGESTERONE BINDING SITES IN THE BRAIN

AND

Guerriero G.1-3, Birch L.2, Prins G.S.2, Roselli C.E.3 and Ciarcia G.1 1

Department of Zoology, "Federico II" University, Via Mezzocannone 8, 80134 Naples, (I) 2 Department of Urology, University of Illinois at Chicago, Chicago, IL 60612, USA 3 Department of Physiology and Pharmacology, O.H.S.U., Portland, OR 97201, USA. e-mail [email protected] Among reptiles, evidence for a central regulation and feedback action of gonadal steroids have been reported in certain lizard. The presence of progesterone target neurons, as well as their anatomical distribution, has been demonstrated in mammalian, and nonmammalian vertebrates. As known, progesterone operates, like other steroid hormones, through specific intracellular receptors which mediate hormone action in the target cell nucleus [1] . We have undertaken the present study with the aim of investigating the distribution of progesterone receptor (PR) and their binding sites properties in the brain of the lizard, Podarcis sicula. Adult female lizards of Podarcis sicula were collected from the neighborhood of Naples. For immunohistochemistry studies, animals after brief cold exposure were submitted to transcardial perfusion with saline solution and then brains were dissected out, fixed, cryoprotected and stored in liquid nitrogen until sectioning. The antiserum PR22 used to detect receptor-immunoreactive (PR-ir) cells was a monoclonal anti-PR raised against chicken PR [8] and immunohistochemistry followed the procedure reported in [3,4]. The specificity of the PR 22 antibody was tested by omission of the primary antibody, with the anti-mouse IgG, by western blotting analysis and using the lizard liver as a positive control tissue. Representative sections indicating the location of immunoreactive cells were drawn aided by a camera lucida. Nomenclature and identification of neuroanatomical areas are based on the descriptions of Smeets et al., [7], and Ten Donkelaar et al., [9]. The western followed standard procedure with only specific conditions for the anti-PR antiserum [5]. The pool samples were processed for biochemical approach , as reported in other tissue in previous works [3] in order to obtain cytosol and nuclear extract from hypothalamic and extra-hypothalamic areas. The hypothalamus were sectioned from the brain, under a stereomicroscope, using the following margins: anterior, septo-mesencephalic tract; posterior, where the third nerve enters the brain; dorsal, the level of the anterior commissure. Samples were pooled into three groups of about 20 animals each and stored in liquid nitrogen until use. For Scatchard analysis and for binding specificity evaluation, aliquots of cytosol (cyt) and nuclear extract (n.e.) were treated according to the usual procedures [3,6]. Protein concentration was determined by Lowry method. Numerical data were analyzed by a two-way ANOVA method, followed by Duncan's multiple range test. Values were expressed as means ± S.E. Our immunocytochemical results represent a first proposed progesterone receptor topographical distribution in the lizard brain Podarcis sicula. We provided evidence that the areas of PR-immunoreactive cells in the female lizard brain are the preoptic-septal 212

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

region, the hypothalamus, the amygdala, the thalamus, the tectum, the torus semicircolaris, the dorsal area of the medulla. Large numbers of immunoreactive cells were found especially in the infundibulum and in the mesencephalon. The preoptic-septal area, hypothalamus, and anterior pituitary have been demonstrated in reptiles to be involved in neuroendocrine regulation and in the control of sex behaviors [2,4]. Most of the other PR immunoreactive cells found in the brain of Podarcis sicula were localized in these same brain regions of different species, thus indicating a vertebrate-wide stable core of steroid receptor cells in neuroanatomical defined regions. Western analysis sustains the presence of one form of progesterone receptor in the lizard brain Podarcis sicula with a molecular weight of about 67 kDa, a value in good agreement with the molecular weight of progesterone receptor in other lower vertebrate tissue reported [3]. Biochemical studies demonstrated the properties of progesterone binding sites in the nuclear lizard progesterone receptor (PRn) in the hypothalamus and extrahypothalamus. 3 H-P binding activity was present in both hypothalamus and extrahypothalamus n.e., showing high affinity and low capacity for the ligand. The saturation binding capacity resulted very similar to those found in other lower vertebrates [3]; Scatchard analysis revealed only one binding site for progesterone with an average of Kd of about 10-9 M, a value similar to that one reported for PR in different tissues of other lower vertebrate [5]. The PRn in the hypothalamus and in the extrahypothalamus of Podarcis sicula was specific for progesterone. The progesterone-binding moiety does not bind the antiprogestins RU486 and the 2914R-2. Since the PR of turtle, chick, and several mammals do not bind RU486, yet the PR of the rodent and human do [5], it may be suggested that there is a change in the progesterone–binding site structure within the vertebrates. The majority of the 3H-P binding activity is ascribable to the hypothalamic area. This finding is consistent with the well-known role of the hypothalamus in the vertebrate reproductive control.

Reference List 1. De Mayo, F.J., Zhao, B., Takamoto, N.,.Tsai, S.Y. Ann.N.Y.Acad Sci 955 (2002) 48-59. 2. Etgen, . A.M. In: Encyclopedia of Reproduction (1999) pp. 1-5, Academic Press, San Diego, Eds: Knobil E. and Neill J.D. 3. Guerriero, G. and Ciarcia, G. Brain Research Review 37 (2001) 172-177. 4. Moga, M.M., Geib, B.M., Zhou, D., Prinz,G.S., Brain Res.879 (2000) 174-182. 5. Paolucci, M., Guerriero, G., Ciarcia, G. J. Exp. Zool. 284(1999) 765-775. 6. Roselli, C.E. and Snipes, C.A. J. Steroid Biochem. 19 (1983) 1571-1575. 7. Smeets, W.J.A.J., Hoogland, P.V., Iohman, A.H.M. J.Comp. Neurol. 254 ( 1986) 1-19. 8. Sullivan, W.P., Beito, T.G., Profer, J., Krco C.J. and Toft, D.O. Endocrinology 119 (1986) 15491557 9. Ten Donkelaar, H.J., Bangma, G.C., Barbas-Henry, H.A., Boer-Van Huizen, R., Wolters, J.G. In : Adv. Anat. Embryol. Cell Biol. (1987) .Vol 197, Berlin: Springer

213

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ACTIVATION OF THE HPG AXIS FOLLOWING COHABITATION IN THE PRAIRIE VOLE (MICROTUS ORCHROGASTER) Hazelton J.L., Hoffman G.E.*, Cushing B.S.†, Levine K.†, Le W.W.*, Ottinger M.A. and Carter C.S. † University of Maryland, Department of Animal and Avian Sciences, MA Ottinger Lab; 3113 Animal and Avian Sciences Bldg; College Park, MD 20742, USA; Fax no. 1 301 314 9059; [email protected] *Dept of Anatomy and Neurobiology, Univ of MD, Baltimore , MD, USA † Dept of Psychiatry, Univ of Illinois, Chicago, USA Prairie voles are monogamous rodents that form long-term pair bonds. This has been shown to be an important component in the activation of the HPG axis in this species. Female voles are induced ovulators requiring chemosensory cues from the urine of a novel male in order to reach sexual maturity. From a previous cohabitation study, areas involved in initial partner preference formation are known as well as those involved in activation of the HPG axis. The goal of this study was to examine the activation of LHRH systems in male and female prairie voles as a function of time spent cohabitating with a novel member of the opposite sex. Treatment groups were paired for 0, 1, 2, 6, and 12-hour cohabitations from randomly selected sibling caged animals 60 - 90 days of age. (n=6 for all treatments for both sexes). Following cohabitations, animals were perfused with 4% paraformaldahyde and 2.5 % Acrolein at appropriate times. Double label immunocytochemisty was used to stain tissue for LHRH and cFos antibodies. LHRH neurons are quantified as either positive or negative for presence of cfos and morphological features noted. Primary areas of analysis focus on the DBB, OVLT, MPOA and LPOA. Results point to changes associated with length of time spent in cohabitation. Neuroanatomical data will be presented.

214

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 ESTRADIOL STIMULATION OF PULSATILE GONADOTROPIN RELEASING HORMONE (GnRH) SECRETION FROM PREPUBERTAL RAT HYPOTHALAMIC EXPLANTS: A PERINATALLY PROGRAMMED AND SEXUALLY DIMORPHIC EFFECT Matagne V., Lebrethon M.-C., Gerard A. and Bourguignon J.-P. Developmental Neuroendocrinology Unit, Center for cellular and Molecular Neurobiology Research (CNCM), University of Liège, B-4000 Liège, Belgium. Fax: 00 32 4 366 2977; e-mail : [email protected]

GnRH is a key hormone stimulating the pituitary-gonadal axis through changes in amplitude and frequency of secretory episodes. Using rat hypothalamic explants, GnRH is secreted in a pulsatile manner and displays a developmental decrease in interpulse interval between 5 and 25 days (males : 90±1min and 40±5min ; females : 90±1min and 35±4min, respectively, mean±SD). Incubation of female hypothalamic explants with estradiol (107 M) resulted in reduction of the GnRH interpulse interval (p<0.0001) at 5 days (75±1min vs 90±1min) and 15 days (48±4min vs 60±1min). Using male explants, a slight but significant effect was seen at 15 days only (57±4min vs 60±2min). Using explants obtained at 25 and 50 days in both sexes, estradiol had no effect. A possible estradiol-mediated effect of aromatized testosterone on GnRH pulsatile secretion was studied. Using female explants, testosterone (10-7M) caused a reduction of the GnRH interpulse interval at 5 days (77±4min) and 15 days (47±4min) as well as at 15 days using male explants (55±4min). These effects were not different from those obtained using estradiol and they were prevented using R76713 (10-5M), an aromatase inhibitor, indicating aromatase involvement in the testosterone-induced acceleration of pulsatile GnRH secretion. We hypothesized that this sexually dimorphic effects of E2 on GnRH interpulse interval resulted from neonatal brain imprinting by sex steroids. Immature female rats were androgenized by sc injection of T (1.25mg in sesame oil) on postnatal day 1 and they were sacrified at 5 or 15 days for in vitro study of pulsatile GnRH secretion. Incubation of hypothalamic explants from androgenized female rats with E2 did not decrease the GnRH interpulse interval at 5 days (90±0min vs 85±4min, control vs E2) or 15 days (61±2min vs 58±3min, control vs E2). In contrast, E2 reduced the GnRH interpulse interval using explants from female rats injected with vehicle at 5 days (90±0min vs 75±0min, control vs E2) or 15 days (61±2min vs 46±3min, control vs E2). Conversely, using explants obtained from male rats treated prenatally with the aromatase inhibitor ATD, the GnRH interpulse interval was decreased by incubation with estradiol at 5 days (90±0min vs 81±5min, control vs E2) and 15 days (60±0min vs 48±4min, control vs E2). Using explants from male rats treated prenatally with the vehicle, E2 did not affect the GnRH interpulse interval neither at 5 days (90±0min vs 89±3min, control vs E2) nor at 15 days (60±0min vs 58±3min, cont vs E2). It is concluded that both estradiol and testosterone can accelerate the frequency of pulsatile GnRH secretion in vitro in an aromatase-dependent manner, preferentially in the immature female rat hypothalamus. Such a sexuallly dimorphic effect could be related to perinatal masculinization of the brain and provides an early evidence of the characteristic female positive feedback to sex steroids. This study was supported by : ARC (99/04-241), FRSM (3.4515.01), BSGPE, the Faculty of Medicine at the University of Liege

215

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ESTROGEN AS A KEY MORPHOGENIC AND DIFFERENTIATION FACTOR FOR IMMORTALIZED HYPOTHALAMIC LHRH NEURONS: CROSSTALK BETWEEN THE CYTOSKELETON AND INTRACELLULAR SIGNALING PATHWAYS Morale M.C.*, L’Episcopo F.*, Testa N.*, Caniglia S.*, Tirolo C.*, Gennuso F.* and Marchetti B.*° *Neuropharmacology, OASI Institute for Research and Care (IRCCS) on Mental Retardation and Brain Aging, 94018 Troina (EN), and ° Dept. Pharmacol., Univ of Sassari, Italy [email protected] Estrogen, the key ovarian hormone, acts within the central nervous system (CNS) t o modulate neuroendocrine functions, synaptic remodelling and neuronal survival through a variety of mechanisms and signaling pathways, via receptor- and non-receptor-mediated events. Estrogen represents one of the most critical factor in the regulation of LHRH neuronal activity, exerting both positive and negative effects on LHRH neurons, thus playing a pivotal role in the positive regulation of LHRH necessary for the preovulatory surge. However, the biochemical and molecular mechanisms of such regulation are still unclear. Indeed, the lack of evidence of estrogen receptor (ER) expression in LHRH neurons led to the belief that estrogen-receptive interneurons are necessary to communicate estrogen signals t o the LHRH neuronal system. In addition, since glial cells are known to harbor receptors for estradiol and progesterone, they have been suggested as potential mediators of estrogen effect on LHRH neurons. Indeed, astrocytes participate in several aspects of neuronal growth and differentiation both by providing cell-cell contacts, and by secreting neuronal growth factors. In turn neurons influence the cellular behavior of astrocytes by secreting substances in the microenvironment. We previously characterized striking neurotrophic and differentiation properties of developing glia and glial-derived growth factors on immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, in vitro, and demonstrated a key role of glial- and neuronal-derived basic fibroblast growth factor (bFGF) and FGFR receptor-1 (FGFR1) in neuronal survival and growth, in the acquisition of LHRH neuronal phenotype by the GT 1 cells as well as in secretory LHRH neuronal response (1-3). In turn, LHRH neurons exerted profound effects on astroglia differentiation and proliferation (1,2). In addition, studying a number of glial-and neuronal-derived growth factors (GFs), we demonstrated that bFGF was the most potent differentiation agent for LHRH neurons in culture, and that FGFR-1 trafficking and targeting to axons and growth cones played a pivotal role in bFGF-induced LHRH neuronal differentiation (3). Moreover, when used as a “ primer” bFGF was able to increase the responsiveness of LHRH neurons to a number of GFs,both at a morphological and functional level and sharply increased the production and release of LHRH in the culture medium (3), suggesting a potential involvement of bFGF both in the extensive synaptic remodelling of the arcuate nucleus observed during the reproductive cycle, and in the massive LHRH release which preceedes the preovulatory LH surge. Little is known on the effect of estrogen during LHRH neuron migration, survival and differentiation. T he recent identification of ER-alpha immunoreactivity, ER mRNA transcripts and [ 125 I]-estrogen binding sites in LHRH neurons of rodents, coupled to the expression of ERbeta transcript in developing physiological LHRH neurons (4-6), clearly support that this class of hormones may exert direct receptor-mediated effects. In addition, estrogen via membrane receptors can rapidly trigger a variety of second messenger signaling events, including the mobilization of intracellular calcium, production of cAMP, generation of inositol phosphate, and activation of the mitogen-activated protein (MAP) kinases, Erk-1 and Erk-2 (see 7). The aim of the present study was to investigate both direct and glial-mediated effects of 17-beta estradiol (E2), on the survival, growth and differentiation of LHRH neuron in culture. For this aim we ressorted to the GT1 LHRH neuronal cell line and used both morphological and biochemical determinations in our established developmental model of LHRH neuron differentiation, in vitro, both in the absence or the presence of hypothalamic astrocytes (2,3).We here report that E2 (10 -11-10 -10M) is an extremely potent survival and differentiation

216

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 factor for GT1 cells. Using immunocytochemistry coupled to confocal laser microscopy, striking morphological effects were observed. Thus, E2-induced a striking increase in neurite outgrowth, in neurite branching and in development of growth cone morphologies. These effects were stereospecific and involved ER activation. Confocal imaging of LHRH neurons under E2 treatment revealed that steroid hormone triggered increased expression focal adhesion kinase (Fak), a 125 kDa non-receptor protein tyrosine kinase involved in signal transduction and highly expressed during CNS development. In addition, the expression of paxillin (Pax), a tyrosine phosphorylation protein, involved in remodelling of the cytoskeleton, showed a significant increase. In particular, Fak and Pax translocated from the cytoplasm to neurite initiation sites, and colocalized at “point of contacts” with FGFR-1 immunoreactive signal. Mitogen-activated protein kinases (MAP/Erk) and Ca2+ were involved in the observed effects, since blocking Ca2+ or MAPK resulted in partial or complete suppression of E 2 morphological effects. In GT1 neuron-glial cocultures, morphological differentiation of GT1 cells was significantly amplified by E 2 treatment, as revealed by quantitative morphometric analysis of all morphological indices of neuronal differentiation. Such effects were abolished by either PD098059, or a FGFR-antagonist. In summary we identified a novel mechanism of E 2 action during LHRH neuron development involving E 2 cosignaling via RAF-PAX-associated kinases, the triggering of MAPK/Erk and FGFR-1 recruitment. The MAPK/Erk signaling cascade and autocrine-paracrine bFGF/FGFR-1 feedback loop appears also responsable for E 2 induced amplification of glial-mediated effects on LHRH neuron growth and differentiation, suggesting that a dynamic interaction between the cytoskeleton and the nucleus coordinate E 2 effects on LHRH neuron growth and differentiation.

Reference List 1. Gallo F, Morale MC, Avola R, Marchetti B. Cross-talk between luteinizing hormone-releasing hormone (LHRH) neurons and astroglial cells: developing glia release factors that accelerate neuronal differentiation and stimulate LHRH release from the GT1 cell line and LHRH neurons modulate astroglia proliferation. Endocrine J 1995; 3: 863-874. 2. Gallo F, Morale MC, Purrello V, Tirolo C, Testa N, Farinella Z, Avola R, Beaudet A, Marchetti B. Basic fibroblast growth factor (bFGF) acts on both neurons and glia to mediate the neurotrophic effects of astrocytes on LHRH neurons in culture. Synapse 2000; 36: 233-253. 3. Gallo F, Morale MC, Tirolo C, Testa N, Farinella Z, Avola R, Beaudet A, Marchetti B. Basic fibroblast growth factor (bFGF) priming increases the response of LHRH neurons to neurotrophic factors J. Neuroendocrinol 2000; 12: 941-959. 4. Herbison AE. Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr Rev 1998; 19: 302-330. 5. Hrabovosky E, Shughrue PJ, Merchenthaler I, Hajszan T, Carpenter CD, Liposits Z, Petersen SL. Detection of oestrogen receptor–beta messenger ribonucleic acid and 125I binding sites in luteinizing hormone–releasing hormone neurons of the rat brain. Endocrinology 2000; 141: 3506-3509. 6. Wong C-W, McNally C, Nickbarg E, Komm BS, Cheskis BJ. Estrogen receptor interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade. Proc Natl Acad Asci USA, 99: 14783-788.

217

• • • • • • • • • • • • • • • • • •

Posters’ Exhibition: Behavioural Effects Arteaga R. (La Habana, Cuba) The small SDN of POAH in homosexual men may be the result of the interaction of aromatised metabolites of testosterone with a very low expressed receptor type 2 o f vasopressin during preoptic development Bartolomucci A., Chirieleison A., Pederzani T., Sacerdote P., Ceresini G., Panerai A.E., Parmigiani S. and Palanza P. (Parma, Italy, EU) Social context and housing condition affects behavioral and immunoendocrine responses to psychological stress in mice Belle M.C.D., Sharp P.J. and Lea R.W. (Preston, UK, EU) Co-Localization of hypothalamic progesterone and androgen receptors and regulation by aromatase in the ring dove (Streptopelia risoria): implications for sexual behaviours Collado P., Carrillo B., Pérez-Torrero E., García-Falgueras A., Pinos H., Guillamón A. and Panzica G.C. (Madrid, Spain, EU) Role of nitric oxide in the control of maternal behavior in rats Dalla C., Bakker J., Honda S., Harada N., Antoniou K., Papadopoulou-Daifoti Z. and Balthazart J. (Liège, Belgium, EU) Behavioral and neurochemical characterization of aromatase deficient female mice. Edinger K., Walf A. and Frye C.A. (Albany, NY, USA) Androgen implants to the dorsal hippocampus can enhance anxiolysis, analgesia, and cognitive performance of male rats Freytes P. and Carrer H.F. (Córdoba, Argentina) Effect of neonatal ovariectomy on learning and synaptic transmission in the hippocampus Koshibu K. and Levitt P. (Pittsburgh, PA, USA) A Stress-associated and gender-specific conditioning deficit in waved-1 mutant mice

fear

Kudwa A.E., Gustafsson J-Å and Rissman E.F. (Charlottesville, VA, USA) Estrogen receptor α is required for dopaminergic facilitation of female sex behavior in mice Küppers E., Brito V. and Beyer C. (Ulm, Germany, EU) Expression and intracellular trafficking o f striatal dopamine d1 receptors is affected in estrogen receptor-α knockout (ERKO) mice Lariviere W.R., Ceccarelli I., Fiorenzani P. and Aloisi A.M. (Siena, Italy, EU) The effect of acute estrogen receptor antagonism on estradiol-induced increases in formalin inflammatory pain behaviour and interferon-gamma production in male rats Mele P., Serra M., Pisu M.G., Biggio G. and Eva C. (Torino, Italy, EU) NPY-Y1 receptor gene expression increases in transgenic mice amigdala following restraint stress Orso F., Sica M. Panzica G.C. and De Bortoli M. (Torino, Italy, EU) possible oestrogenic effects on AP-2 alfa expression and AP-2 alfa potential role in nnos regulation in mouse hypothalamus. Pérez-Laso C., Ortega E., Izquierdo M.A.P., Moreno N., Rivera L., Segovia S. and Del Cerro M.C.R. (Madrid, Spain, EU) Male rats adopted at birth by stressed dams display feminine behavioral patterns in induced maternal behavior Pincemy G. and Guyomarc’h C. (Rennes1, France, EU) variation of the physiological state and socials behavior in Japanese quail. Sica M., Allieri F., Plumari L., Bakker J , Honda S., Harada N., Viglietti-Panzica C., Balthazart J. and Panzica G.C. (Torino, Italy, EU) Activational effects of estradiol and dihydrotestosterone on the arginine-vasopressin immunoreactive system of male mice lacking a functional aromatase gene. Thompson N., Micevych P.and and Ottinger M.A. (College Park, Maryland, USA) Comparison o f neuroanatomical distribution of delta opioid receptor and tyrosine hydroxylase immunoreactivity i n young and old male quail Turkmen S., Birzniece V., Johansson I.M. and Backstrom T. (Umeå, Sweden, EU). Negative allopregnanolone effects on learning in the Morris watermaze test can be inhibited.

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

THE SMALL SDN OF POAH IN HOMOSEXUAL MEN MAY BE THE RESULT OF THE INTERACTION OF AROMATISED METABOLITES OF TESTOSTERONE WITH A VERY LOW EXPRESSED RECEPTOR TYPE 2 OF VASOPRESSIN DURING PREOPTIC DEVELOPMENT Arteaga R. Servicio de Oncohematología. Hospital Pediátrico Universitario Juan Manuel Márquez. Calle 31 y Avenida 78. Marianao. Ciudad de La Habana. Cuba. E mail: [email protected] Levay found that heterosexual men have an interstitial nuclei 3 (INAH 3) of the preoptic anterior hypothalamus (POAH) twice as large, and with double number of neurons than homosexual men and women. Larkin has recently arrived to similar findings in domestic sheep [1]. Hamer found a strong linkage between male homosexuality and Xq28 band. The vasopressin receptor type 2 (V2R) gene is located on Xq28. Higher vasopressinergic signal (or vasotosinergic AVT as avian homologue to mammal’s vasopressin AVP) in parvocellular hypothalamus (PH): bed nucleus of stria terminalis (Bst), lateral septum (LS), sex dymorphic nuclei (SDN) of medial preoptic area (POM) has been related to male typical sexual behavior, and lower AVT/AVP signal is related to female typical sexual behavior in vertebrates [2-5]. AVT expression is strongly induced by testosterone (T) on PH [2-4]. Male´s PH structures with high AVT/AVP expression are larger in size than female´s structures with lower AVT/AVP. V2R has been found expressed during the development of brain in mammals, but not in adult cerebrums, humans included [6,7]. T virilizating action on PH is achieved through the cytochrome P450 aromatase (ARO). Reciprocally, T seems to regulate the rate of its own aromatization by modulating the expression of ARO rather than by acting at a post transcriptional level [8]. Furthermore, T plays a physiological role in maintenance of the V2R expression [9]. Rates of mammalian brain ARO are high during perinatal development (males > females), then rapidly decline in both sexes during postnatal development with a notable decrease at puberty with relatively low ARO expression during adulthood [10]. Sexual differentiation of the human hypothalamus takes place much later than originally claimed (midpregnancy): At birth the SDN contains only some 20% of the cells found at 2 to 4 years of age. The cell number increases equally rapidly in boys and girls until 2 to 4 years of age. After that age period, a decrease in cell number takes place in girls, but not in boys. This causes the sexual differentiation of the SDN [11]. This ontogenic order seems to be similar for many vertebrates: BST of chickens, At E14, AVT expressing cells are already clearly visible both in males and females. The subsequent time-course of the sexual differentiation includes a progressive rise of cell numbers in both sexes, at D14, there is no obvious sex difference in the number of AVT mRNA expressing cells as well as in the intensity of signal in positive cells [14]. However, from D35 posthatch onward, parvocellular AVT neurons in the BST of female chickens disappear, indicating the existence at that age of clear sexual differences. Similar posthatching developmental changes have been observed also in a preliminary study on quail [13] and on canary brain, [15]. In rats, the parvocellular sexually dimorphic part of the AVP system is not detectable before the first postnatal days. In the SL of male and female rats, immunoreactive (ir) AVP fibers are first observed at D10 but the gender differences become apparent from D12 onwards [16]. It is notable that a common feature of the AVT/AVP system in the few investigated species is the delay in development of the sexually dimorphic hypothalamic part [4]. Sexual dymorphism of rat POAH occurs due to an early post natal increase of neuronal apoptosis in females but not in males, because T has a profound inhibitory effect on the incidence of apoptosis that begins at post natal ages [12]. T levels has been consistently probed as similar between hetero an homosexual men.

219

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 Homosexual sheep of Larkin study may have lower ARO ir probably because they have less SDN preoptic neurons, but not because they have less ARO concentration per neuron. If homosexual men have the same concentration and activity (per neuron) of T and ARO during INAH3 development, then the resultant of a smaller INAH 3 may probably be the consequence of a low expression of V2R, as a very decisive AVP receptor on INAH 3 neurons during their development. So, in humans, male homosexuality may be related to the inheritance of a promoter of the V2R gene on Xq28 with a pattern of low expression along INAH3 development, that produce a lower AVP signal, a higher post natal apoptosis, and a smaller INAH 3 as an androphilic cytoarchitecture. This morphology and sexual attraction functionality stay unchanged during adulthood. In adulthood V2R is not longer expressed out of the kidney. That may be why dymorphism of these hypothalamic nucleus are organizational in nature. V2R in POAH may function as a transitory developer (interacting with aromatised metabolites of T and probably with non aromatised T too) of a strong AVP signal with anti apoptotic consequences that shall avoid neuronal apoptosis when V2R expression ontogenically stops). Higher V2R during PH development might be connected to the higher perinatal expression of ARO that declines in adulthood. In females, V2R (after stopping its expression) can not protect from apoptosis the neurons of SDN hypothalamus because they lacked of T during morphogenesis. As V2R is a protein G coupled receptor, many potential mechanisms can be evaluated for this molecule to demonstrate his sexual dymorphic determinant role on hypothalamus. Reference List 1. Larkin G. et al. Homosexuality is biological, suggests gay sheep study. NewScientist.com news 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

220

service.10:51 (2002) 05 November 02. Panzica GC, Aste N, Castagna C, Viglietti-Panzica C, Balthazart J. Steroid-induced plasticity in the sexually dimorphic vasotocinergic innervation of the avian brain: behavioral implications. Brain Res Brain Res Rev 37 (2001) 178-200 Panzica G, Viglietti-Panzica C, Balthazart J. Sexual dimorphism in the neuronal circuits of the quail preoptic and limbic regions. Microsc Res Tech 54 (2001) 364-74 Jurkevich A, Grossmann R, Balthazart J, viglietti-Panzica C. Gender-related changes in the avian vasotocin system during ontogeny. Microsc Res Tech 55 (2001) 27–36 Gilmore DP. Sexual dimorphism in the central nervous system of marsupials. Int Rev Cytol 214 (2002);:193-224 Kato Y, Igarashi N, Hirasawa A, Tsujimoto G, Kobayashi M. Distribution and developmental changes i n vasopressin V2 receptor mRNA in rat brain. Differentiation 59 (1995) 163-9. Ostrowski NL, Lolait SJ, Bradley DJ, O'Carroll AM, Brownstein MJ, Young WS 3rd. Distribution of V1a and V2 vasopressin receptor messenger ribonucleic acids in rat liver, kidney, pituitary and brain. Endocrinology 131 (1992) 533-5. Aste N, Panzica GC, Viglietti-Panzica C, Harada N, Balthazart J. Distribution and effects of testosterone o n aromatase mRNA in the quail forebrain: a non-radioactive in situ hybridization study. J Chem Neuroanat. 14 (1998) 103-15. Pavo I, Varga C, Szucs M, Laszlo F, Szecsi M, Gardi J, Laszlo FA. Effects of testosterone on the rat renal medullary vasopressin receptor concentration and the antidiuretic response. Life Sci 56 (1995) 1215-22. Lephart ED, Lund TD, Horvath TL. Brain androgen and progesterone metabolizing enzymes: biosynthesis, distribution and function. Brain Res Brain Res Rev 37 (2001) 25-37 Swaab DF, Hofman MA. Sexual differentiation of the human hypothalamus: ontogeny of the sexually dimorphic nucleus of the preoptic area. Brain Res Dev Brain Res 44 (1988) 314-8. Davis EC, Popper P, Gorski RA. The role of apoptosis in sexual differentiation of the rat sexually dimorphic nucleus of the preoptic area. Brain Res 734 (1996) 10-8. Aste, N., Baiamonte, G., Grossmann, R. and Panzica, G.C., Postnatal development and sexual differentiation of quail vasotocin system, Italian J. Anatomy Embriol., 102, Suppl. (1997) 82. Jurkevich, A., Barth, S.W., Kuenzel, W.J., Kohler, A. and Grossmann, R., Development of sexually dimorphic vasotocinergic system in the bed nucleus of stria terminalis in chickens, Journal of Comparative Neurology, 408 (1999) 46-60. Voorhuis, T.A.M., De Kloet, E.R. and De Wied, D., Ontogenetic and seasonal changes in immunoreactive vasotocin in the canary brain, Developmental Brain Research, 61 (1991) 23-31. de Vries GJ, Buijs RM, Swaab DF. Ontogeny of the vasopressinergic neurons of the suprachiasmatic nucleus and their extrahypothalamic projections in the rat brain--presence of a sex difference in the lateral septum. Brain Res Aug 10; 218 (1981) 67-78

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

SOCIAL CONTEXT AND HOUSING CONDITION AFFECTS BEHAVIORAL AND IMMUNO-ENDOCRINE RESPONSES TO PSYCHOLOGICAL STRESS IN MICE Bartolomucci A.1,2, Chirieleison A.1, Pederzani T.1, Sacerdote P.3, Ceresini G.4, Panerai A.E.3, Parmigiani S.1 and Palanza P.1 1

Dip. Biologia Evolutiva e Funzionale, University of Parma, Parco area delle scienze 11/A, 43100 Parma, Italy. Email: [email protected]. Fax: 0039-0521-905657 2 Ist. Psicologia, University of Milan, Milan, Italy 3 Ist. Medicina Interna, University of Parma, Parma Italy 4 Dip. Farmacologia, Chemioterapia e Tossicologia Medica, University of Milan, Milan, Italy There is a general consensus that social relationships affect the behavior and the physiological functions of many mammalian species, including humans. A main source of stress in humans is of social nature and contributes to the development and expression of health disorders such as cardiovascular disease, ulcerative colitis, infectious disease, neoplasic disease and psychiatric disorders have all been related to psychosocial stress. However, the conventional animal models of stress appear to be quite far from real life events, either for the experimental animal model or the human counterpart. Consequently, animal models that involve a social context seem to be more appropriate as they mimic the stressful situations that an animal may meet in its everyday life in a natural habitat, and for which behavioral and neuroendocrine responses had been shaped by evolutionary processes. The present study was designed to understand how social context and social status in aggressive encounters can affect the behavior and the hormonal and immune responses of male house mice as animal model. Specifically, we examined the effects of social deprivation as compared to group housing or to chronic psychosocial stress (chronically coexisting resident/intruder dyads) on a number of behavioral and physiological responses. We analyzed the temporal dynamic of isolation response with increasing time of isolation (24h to 42 days) and whether housing conditions would affect the reaction to an acute mild stress, i.e. forced exposure to a novel environment and a free-exploratory paradigm allowing the animals the free choice between the home cage and a novel environment. Plasma Corticosterone, in vitro splenocytes proliferation and release of Interleukine-2 (IL-2), IL-4, IL-10 and Interferon-gamma in response to the mitogen Concavaline-A were assessed. Four are the main findings: (1) individually housing mice for increasing time periods did not induce per se main immuno-endocrine effects compared to a stable sibling group housing. (2) when exposed to a mild acute stress, isolated mice showed higher basal corticosterone and lower immune functions compared to males living with siblings. (3) when faced with a free choice between a novel environment and their home cage, individually housed mice showed increased exploration of the novel environment, thus suggesting a low-anxiety profile. These findings suggest that individual housing in itself does not change immune-competence and corticosterone (stress hormone) level, but does affect subsequent responses to potentially stressful situations (4) Male mice living in groups of siblings since birth did not show any immuno-endocrine alterations [1].

221

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

We designed a model in which mice are constantly exposed to an aggressive conspecific but without relevant injuries being present. To induce chronic psychosocial stress, a mice was introduced as Intruder in the cage of a Resident (living in a cage alone for 7 days) male. After 10min, the two animals were divided by means of a perforated partition allowing a continuous visual-olfactive-acoustic but not aggressive interaction. The partition was removed daily (15-21 days) allowing physical interaction for 10min, interrupted if fighting escalated. Four groups were categorized: Residents becoming Dominants (RD), Intruders becoming Dominants (InD), Residents becoming Subordinates (RS) and Intruders becoming Subordinates (InS). We measured: plasma Corticosterone; in vitro splenocytes proliferation and release of cytokines IL-2, IL-4, IL-10 and IFN-g in response to the mitogen Concavaline A; behavior in the Open Field Test. When mice are subjected to chronic psychosocial stress, a peculiar response emerged. All mice showed an increased plasma corticosterone (stress hormone) level. However, only those mice that were previously resident ("owner") in a territory but were defeated and became subordinate when an unfamiliar mouse entered the territory, showed immune impairments [2]. Our findings are of interest since they represent a first series of studies investigating a whole range of naturally occurring (group housing and isolation) as well as experimentally-induced-stressful (chronic psychosocial stress model) settings in an ethological perspective, i.e. by taking into account the evolutionary significance and the ecology of the species investigated. Our approach is relevant since in many neurobiological and biomedical studies mice are commonly housed in individual cages, a condition that, as based on our findings, induce hyperreactivity to stressors. Housing animals under particular social conditions may in fact differentially alter mice behavioral responses, as well as physiology and biochemical status. Additionally, stressful events are thought to participate in the induction of many different pathologies. Our finding that only the loss of a relevant resource (the territory for male mice) may induce a state of immune impairment (in presence of high stress hormones and hyperactivity of the cardiovascular system) may offer the opportunity to understand the role of psychological variables in determining the role of individual differences in disease vulnerability.

Reference List [1] Bartolomucci, A, Palanza, P., Sacerdote P, Ceresini, G., Chirieleison A, Panerai, A.E., Parmigiani, S. (2002) Individual housing induce altered immuno-endocrine responses to psychological stress in male mice. Psychoneuroendocrinology (in press). [2] Bartolomucci, A, Palanza, P., Gaspani, L., Limiroli, E., Panerai, A.E., Ceresini, G., Poli, M.D., Parmigiani, S. (2001) Social status in mice: behavioral endocrine and immune changes are context dependent. Physiology and Behavior, 73-3: 401-410.

222

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

CO-LOCALIZATION OF HYPOTHALAMIC PROGESTERONE AND ANDROGEN RECEPTORS AND REGULATION BY AROMATASE IN THE RING DOVE (STREPTOPELIA RISORIA): IMPLICATIONS FOR SEXUAL BEHAVIOURS Belle M.C.D., Sharp P.J.* and Lea R.W. Department of Biological Sciences, University of Central Lancashire, Preston PR1 2HE, UK. E-mail: [email protected] Fax:01772 892929. *Roslin Institute, Roslin, Midlothian, EH25 9PS, UK In the ring dove (Streptopelia risoria) aggressive courtship behaviour and the initiation of incubation behaviour is thought to be influenced by androgen and progesterone respectively [1, 2]. Progesterone administration to courting birds terminates the aggressive component of courtship but does not disrupt oestrogen-dependent nesting behaviour [2, 3], suggesting an antagonistic interaction with androgen. We have mapped cells containing androgen and progesterone receptor immunoreactivity (AR-ir and PR-ir) in the hypothalamus of both sexes to regions thought to be involved in the expression of courtship and incubation behaviours [4, 5]. Some progesterone receptors are known to be inducible by oestrogen [4], while oestrogen synthesis in the hypothalamus is dependent on neurones containing aromatase [6, 7]. The aim of this study was to determine whether there are neurones in the hypothalamus containing ARir that also contain PR-ir. The co-localization of AR and PR might explain the inhibitory effect of progesterone on aggressive courtship behaviour. Further evidence for this hypothesis was sought by determining the effect of inhibiting aromatase activity on the development of courtship and nest orientated behaviours, and correlated changes in the intensity of AR-ir and PR-ir in neurones in the hypothalamus. In the first experiment, male and female doves (20 males and 20 females) caged as isolates for at least 2 weeks before pairing, were identified as non-breeding. Birds were then randomly paired, whilst controls were maintained in isolation. Brain tissues and sex organs were collected from 15 pairs at three stages of the breeding cycle: day 3 of courtship, day 1 of incubation, and 2 days post-hatch (brooding). Birds were perfuse-fixed through the heart and brain sections processed for PR-ir and AR-ir. In the second experiment, non breeding doves were injected intramuscularly with the aromatase inhibitor, fadrozole (0.2mg/bird, 4 males and 4 females) and paired with saline injected controls (4 males and 4 females) in cages containing nest bowls and nesting material. Fadrozole or saline vehicle was administered for 3 days at 12 hour intervals. Saline-injected control males displayed significantly (90%) more aggressive courtship and nest orientated behaviours than fadrozole treated males. Saline injected control females displayed nest oriented behaviours and were sitting on the nest by day 2, while fadrozole-injected females showed none of these behaviours. Following terminal anaesthesia on day 4, birds were perfuse-fixed with Zamboni’s fixative and the brains removed for immunocytochemistry for AR, PR and P450AROM, In saline injected courting doves of both sexes nuclear AR-ir was widespread throughout the hypothalamus while the distribution of nuclear PR-ir was more restricted. PR-ir was colocalized in 70-90% of AR-ir neurones in the nucleus preopticus anterior (POA), nucleus medialis (POM), nucleus medialis, pars medianis (POMm), nucleus preopticus paraventricularis magnocellularis (PPM), nucleus hypothalami lateralis (PLH), and tuberal hypothalamus (Tu). A lower percentage of co-localisation was seen in these nuclei in doves at other stages of the breeding cycle. In both sexes, the number of PR-ir neurones did not change significantly during the breeding cycle, apart from an elevation in the number of PR-ir neurones (p<0.05) in the PPM of courting males. In contrast, nuclear PR-ir expression in the Tu changed significantly during the breeding cycle. In both sexes, a significant decrease in nuclear PR-ir cell number was seen in the ventral region of the Tu of brooding birds (females p<0.05, males p<0.001). This may be explained by a difference in regional sensitivity to oestrogen (Askew et al., 1997). PR-ir staining intensity, however, increased significantly in courting birds (p<0.001) in all regions of the hypothalamus. In courting birds, 70% of hypothalamic neurones with high nuclear fluorescent intensity for PR-ir also contained nuclear androgen receptor. Cells with low PR-ir intensity (30%) rarely co-localised with AR ir. The number and staining intensity of AR-ir

223

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 neurones throughout the hypothalamus was significantly (P<0.001) higher in courting doves than in doves taken at the other stages of the breeding cycle. There was no sex difference in the number of AR-ir neurones, but AR-ir staining intensity was significantly higher (p<0.001) in males than females at all stages of the breeding cycle. Fadrozole treatment in both sexes resulted in the disappearance of nuclear AR-ir and a significant decline in the PR-ir in the POM, POMm, and Tu (p<0.001). A significant decrease (p<0.001) in AR-ir and PR-ir co-localisation was also seen in the POA, PPM POM, POMm, PLH and Tu. A sex difference in PR-ir expression was observed in the POA and PPM (p<0.001) of fadrozole treated birds, with male showing higher number of PR-ir than females. Neurones containing AROM-ir were observed in the POM and POMm of saline injected males, and were found to co-express PR-ir (99%). This study is the first to report co-localisation of PR-ir and AR-ir in the hypothalamus of male and female ring doves, and is the first to provide immunocytochemical evidence that progesterone may inhibit androgen-dependent aggressive courtship behaviour by acting on PR to inhibit AR actions. This result is consistent with biochemical studies reporting a direct “cross-talk” between AR and PR [8]. PR-ir staining intensity measurement in courting birds suggests that inhibition of AR occurs following an increase in PR levels in neurones containing AR. It is suggested that the inhibition of androgen-dependent aggressive courtship display by progesterone occurs through an aromatisable steroid-induced PR-ir in the POM and POMm, whist the POA may be involved in progesterone facilitation of aggressive courtship display. References list [1] Lea,R.W., Clark,J.A., and Tsutsui,K., Changes in central steroid receptor expression, steroid synthesis, and dopaminergic activity related to the reproductive cycle of the ring dove, Microsc. Res. Tech., 55 (2001) 12-26. [2] Komisaruk,B.R., Effects of local brain implants of progesterone on reproductive behavior in ring doves, J. Comp Physiol Psychol., 64 (1967) 219-224. [3] Erickson,C.J., Bruder,R.H., Komisaruk,B.R., and Lehrman,D.S., Selective inhibition by progesterone of androgen-induced behavior in male ring doves (Streptopelia risoria), Endocrinology, 81 (1967) 39-44. [4] Askew,J.A., Georgiou,G.C., Sharp,P.J., and Lea,R.W., Localization of progesterone receptor in brain and pituitary of the ring dove: influence of breeding cycle and estrogen, Horm. Behav., 32 (1997) 105-113. [5] Belle,M.D. and Lea,R.W., Androgen receptor immunolocalization in brains of courting and brooding male and female ring doves (Streptopelia risoria), Gen. Comp Endocrinol., 124 (2001) 173-187. [6] Hutchison,J.B., Schumacher,M., Steimer,T., and Gahr,M., Are separable aromatase systems involved in hormonal regulation of the male brain?, J. Neurobiol., 21 (1990) 743-759. [7] Hutchison,R.E., Wozniak,A.W., and Hutchison,J.B., Regulation of female brain aromatase activity during the reproductive cycle of the dove, J. Endocrinol., 134 (1992) 385-396. [8] Yen,P.M., Liu,Y., Palvimo,J.J., Trifiro,M., Whang,J., Pinsky,L., Janne,O.A., and Chin,W.W., Mutant and wild-type androgen receptors exhibit cross-talk on androgen-, glucocorticoid-, and progesterone-mediated transcription, Mol. Endocrinol., 11 (1997) 162-171.

224

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ROLE OF NITRIC OXIDE IN THE CONTROL OF MATERNAL BEHAVIOR IN RATS Collado P., Carrillo B., Pérez-Torrero E., García-Falgueras A., Pinos H., Guillamón A. and Panzica G.C.* Departamento de Psicobiología, Universidad Nacional de Educación a Distancia, Juan del Rosal, 10, 28040-Madrid, Spain. [email protected] * Centro Rita Levi Montalcini, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Laboratorio di Neuroendocrinologia, Università di Torino, Torino, Italy. Maternal behavior in rats depends on a complex integration of behavioral patterns regulated by neurohormonal mechanisms. Several brain areas have been identified as part of the circuits implicated in the control of the display of this behavior. However, the study of the neurotransmitter systems implicated in this control is only at the beginning. Participation of dopamine in the expression of maternal behavior during early postpartum period has been demonstrated [1]. Moreover, inhibitory influence over maternal behavior has been reported either by local GABAergic inhibitory interneurons in the medial preoptic area, ventral bed nucleus of the stria terminalis and the lateral and ventrolateral regions of the midbrain periaqueductal gray [4] or by a tachkynine neuropeptide K inhibitory projection from medial amygdaloid nucleus to the ventromedial nucleus of the hypothalamus [5,7]. The results of some recent investigations have suggested that the nitric oxide (NO) might play some role in the control of maternal behavior. In fact, an increase of NADPHdiaphorase (ND) staining and nitric oxide synthase (NOS) mRNA expression occurs in last day of pregnancy and during lactation in paraventricular and supraoptic nuclei of rats [6]. The aim of the present study was to investigate the role of nitric oxide in the control of maternal behavior. During four days after delivery, lactating females were daily injected intraperitoneally 15 minutes before the behavioral test with NO precursor L-arginine (25 mg/kg/day) or 60 minutes before the behavioral test with NOS inhibitor, nitro-L-arginine methyl ester (L-NAME), (25 mg/kg/day). Females of the control groups were injected with vehicle either 15 or 60 minutes before the behavioral test. After the treatments, we have checked the nest quality and recorded the mother-litter interactions (grooming, licking, crouching and retrieval) for 10 minutes every day by means of a computer program for maternal behavior (MBR). Behavioral data were statistically analyzed by Kruskal-Wallis and Mann-Whitney U-tests. Since no differences between controls were found, data from these two groups were combined for statistical analyses. Our results (see table 1) revealed a strong significant effect of administration of NO precursor, L-arginine, on crouching behavior (p<0,007). GROUPS Control L-NAME L- Arginine

Session #1 53±11.1 32.2±72.76 238±53.12

Session #2 33.57±11.1 82.8±72.76 288±53.12

Session #3 121.86±37.5 233.8±86.2 380.6±38.13

Session #4 69.57±26.25 191.6±55.22 352.2±37.28

Table 1. Crouching time in the four sessions recorded. Data are expressed as time in seconds ± S.E.M.

225

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

Comparisons between groups reveals that L-arginine facilitates the expression of crouching (p<0,016). Moreover, this effect is maintained during the four sessions (p<0.003 for each session). The administration of NO inhibitor, L-NAME, did not statistically affect the display of crouching. Significant differences between L-NAME and L-arginine groups were found only in the first session (p<0.008, p>0,05, in the rest of the sessions). Present data suggest that NO might play some role in the expression of crouching behavior in lactating rats, but does not statistically affect other parameters, such as retrieval, licking and grooming. It might be, that, as reported for the regulation of sex behavior in male rats [3], NO is mediating in some way the dopamine/GABA activity that controls this behavior. Our previous quantitative histochemical data [2] have shown changes in ND activity in some structures implicated in the control of maternal behavior during the estral cycle, suggesting, therefore, a relationship among ND and estrogens levels. The action of NO might be, consequently, exerted at central level. However, implication of NO at the peripheral level can not be discarded at this point. Further studies should be carried out to determine it. Finally, these data are in accordance with results of other authors suggesting that the different components of maternal behavior have their own sensory and neural determinants [8].

Reference List 1. E. Byrnes, B.E. Rigero, R.S. Bridges, Dopamine antagonists during parturition disrupt maternal care and the retention of maternal behavior in rats, Pharmacol. Biochem. Behav. 73 (2002) 869-875. 2. P. Collado, H. Pinos,, C. Rodríguez, A. García-Falgueras, B. Carrillo, A. Guillamón, G.C. Panzica, NADPH-diaphorase histochemical variations throughout the estrous cycle in the bed nucleus of the accessory olfactory tract (BAOT) in the rat, 3rd Forum of European Neuroscience, 13-17, July, 2002. 3. E.M. Hull, D.S. Lorrain, J.F. Du, L. Matuszewich, L.A. Lumley, S.K. Putnam, J. Moses, Hormone-neurotransmitter interactions in the control of sexual behavior, Behav. Brain Res. 105 (1999) 105-116. 4. J.S. Lonstein, G.J. De Vries. Maternal behavior in lactating rats stimulates c-fos in glutamate decarboxylase-synthesizing neurons of the medial preoptic area, ventral bed nucleus of the stria terminalis, and ventrocaudal periaqueductal gray, Neuroscience 100 (2000) 557-568. 5. M. Numan, T.P. Sheehan, Neuroanatomical circuitry for mammalian maternal behavior. In C.C. Carter, I.I, Lederhendler, B.Kirkpatrick. The integrative neurobiology of affiliation. Annals of the New York Academy of Sciences vol. 807, 1997. 6. N. Popeski, S. Amir, B. Woodside, Changes in NADPH-d staining in the paraventricular and supraoptic nuclei during pregnancy and lactation in rats: role of ovarian steroids and oxytocin., J. Neuroendocrinol. 11 (1999) 53-61. 7. E.C. Stack, R. Balakrishnan, M.J. Numan, M. Numan, A functional neuroanatomical investigation of the role of the medial preoptic area in neural circuits regulating nmaternal behavior, Behev. Brain Res. 131 (2002) 17-36. 8. J.M. Stern, J.S. Lonstein, Neural mediation of nursing and related maternal behaviors. Prog. Brain Res. 133 (2001) 263-278.

226

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 BEHAVIORAL AND NEUROCHEMICAL CHARACTERIZATION OF AROMATASE DEFICIENT FEMALE MICE Dalla C. 1,3 , Bakker J. 1 , Honda S. 2 , Harada N. 2, Antoniou K.3, Papadopoulou-Daifoti Z. 3 and Balthazart J.1 Ctr. Cell. Molec. Neurobiol., Univ. Liège, Belgium 2Div. Mol. Genetics Fujita Health Univ., Aichi, Japan 3 Dep. of Pharmacology, Medical School Univ. of Athens, Mikras Asias 75, 11527, Athens, Greece. [email protected], fax: 0032 43665970.

1

INTRODUCTION Numerous studies have shown that estrogens can affect mood and mental state in humans. For instance, low levels of estrogens in women have been associated with the premenstrual syndrome, postnatal depression, and post-menopausal depression. One key symptom of depression in humans is a reduced motivation or drive. Estrogens act as neuromodulators on central catecholaminergic systems and may play a role in the mechanisms associated with psychiatric disorders and their treatment [2]. Aromatase-knockout mice (ArKO) are depleted of estrogens due to a targeted mutation in the aromatase gene and therefore they are a useful model to investigate the role of estrogens on the CNS. We recently found that female ArKO mice deficient in estradiol showed a reduced motivation to sniff reproductively-relevant odors from either an estrous female or intact male conspecific compared to wild-type (WT) females [1]. Therefore, in the present study, we asked whether female ArKO mice are more prone to depression due to their prolonged estrogen-deficiency. We also wanted to investigate the effect of estrogens on behavioral and neurochemical responses to stress. We used the forced swim test (FST) to investigate behavioral despair and depression in ArKO mice. Behavioral responses in the FST such as increased floating and decreased active behaviors such as swimming or struggling compared to controls are thought to be an indication of “depressive-like” symptomatology. We also investigated levels of anxiety, general activity and exploration by using the elevated plus maze test (EPT) and the openfield test (OFT). METHOD Female WT and ArKO mice were ovariectomized in adulthood and received an estradiol or an empty implant. Three weeks later all mice were subjected to the OFT and to the EPT. Mice were then divided into two groups (stressed group and control group) and were subjected to four sessions (one session every week) of the FST for 5 min in a big cylinder (diameter: 17 cm). Behavioral responses such as struggling, swimming and floating were recorded. Mice from the stressed group were killed by decapitation 10 min after the last FST. Mice from the control group were also killed after taken them directly out of their homecage. The hippocampus and hypothalamus were dissected, weighted and stored in –80 0C. The tissue was then homogenized and centrifuged and the supernatant was used for chemical determination of norepinephrine (NE), dopamine (DA) and its metabolites DOPAC and HVA with HPLC-ECD [3]. Statistical analysis: Behavioral data from OFT and EPT were analyzed by two way anova with genotype and estradiol treatment as factors. Behavioral data from FST were analyzed by repeated two way anova with genotype and estradiol treatment as factors. Neurochemical data were analyzed by three way anova with genotype, estradiol treatment and FST as factors. BEHAVIORAL RESULTS • No effect of genotype or estradiol treatment was observed in OFT and EPT. • ArKO mice struggled less and floated more than WT in every FST session. After repeated testing floating duration increased over time for all mice. There was no effect of estradiol treatment.

227

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 NEUROCHEMICAL RESULTS Hippocampus • There was a significant effect of genotype, FST, estradiol treatment, an interaction between genotype and estradiol treatment and an interaction between genotype, estradiol treatment and FST on NE levels. FST increased NE levels in ArKO, but not in WT females. However, FST did not increase NE levels in estradiol-treated ArKO females. • There was a significant effect of estradiol treatment, an interaction between genotype and estradiol treatment on DA and DOPAC levels. Basal DA and DOPAC levels were higher in ArKO compared to WT. However, DA and DOPAC levels decreased in ArKO but not WT females following estradiol treatment. • There was a significant effect of FST, an interaction between genotype and estradiol treatment and an interaction between genotype, estradiol treatment and FST on HVA levels. FST increased HVA levels in ArKO, but not in WT females. However, FST did not increase HVA levels in estradiol-treated ArKO females. Hypothalamus • There was a significant effect of estradiol treatment on DA levels, the ratio DOPAC/DA and HVA/DA. DA levels were increased in estradiol-treated subjects compared to non-treated subjects. Furthermore, the ratios DOPAC/DA and HVA/DA were decreased in estradiol-treated subjects. • There was a significant FST effect on HVA levels. FST increased HVA levels in both WT and ArKO females. DISCUSSION The behavioral data suggest that female ArKO mice exhibit normal levels of anxiety, activity and exploration, but they make fewer escape attempts and spend more time immobile in FST. It can be argued that this “depressive” symptomatology may be related to their reduced sexual motivation. Adult estrogen treatment was unable to correct the behavioral deficits observed in FST, suggesting that these deficits are due to the lack of estrogens sometime before treatment with estradiol in adulthood. The neurochemical results indicate that the dopaminergic system in the hippocampus of ArKO females is different from that of W T animals. They also seem to have a different response to FST than WT, as far as NE and HVA levels in the hippocampus are concerned. These differences in the hippocampal, catecholaminergic system of ArKO female mice could be corrected with estradiol treatment in adulthood that seems to have a “protective” effect from stress. Estradiol treatment in adulthood reverses the neurochemical findings in the hippocampus of ArKO female mice, while it was not sufficient to correct their behavioral deficits in the FST. This suggests that the neurochemical differences observed cannot be the only cause for their behavioral profile and that different neurotransmitters or brain regions are involved. This research has been supported by a Marie Curie fellowship of the EC programme “Quality of Life and Management of Living Resources” under contract number QLK6-CT-2000-60042 and fellow reference number QLK6-GH-00-60042-03. It was also supported by a grant from the Government of the French Community of Belgium (ARC 99/04-241). Reference List 1. Bakker, J., Honda, S., Harada, N. and Balthazart, J., The aromatase knockout mouse provides new evidence that estradiol is required during development in the female for the expression of socio-sexual behaviors in adulthood, J. of Neurosci, 22 (2002) 9104-9112. 2. McEwen, B. and Alves, S., Estrogen Actions in the Central Nervous System, Endocr Rev, 20 (2000) 279–307. 3. Papadopoulou-Daifotis, Z., Antoniou, K., Vamvakidis, A., Kalliteraki, I. and Varonos, D., Neurochemical changes in dopamine and serotonin turnover rate in discrete regions of rat brain after the administration of glycinergic compounds, Acta Therap, 21 (1995) 5-18.

228

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 ANDROGEN IMPLANTS TO THE DORSAL HIPPOCAMPUS CAN ENHANCE ANXIOLYSIS, ANALGESIA, AND COGNITIVE PERFORMANCE OF MALE RATS 1

Edinger K., Walf A.1-3 and Frye C.A.

The University at Albany-SUNY, Behavioral Neuroendocrinology Laboratory, 1Departments of Psychology and 2Biological Sciences, and 3The Center For Neuroscience Research, 1400 Washington Avenue Albany, NY, 12222 USA. [email protected]. 518-442-4867.

Androgens may be important for affective and cognitive performance. Clinical reports suggest androgen replacement can improve both mood and spatial and verbal memory [2-3]. Data from animal models of affect and cognition also support an important role of androgens in these processes. First, there are sex differences in both affective and cognitive behaviors among rodents. Male rats, which have higher androgen levels than do females, exhibit less anxiogenic behavior than do females and perform better on spatiallyoriented cognitive tasks [4,6]. Second, removal of the primary endogenous source of androgens, the testes, attenuates sex differences in cognitive and affective behavior. Gonadectomized (gdx) male rodents have increased anxiety behavior and decreased cognitive performance [1,7] compared to intact males. Third, androgen replacement with testosterone (T), its 5α-reduced metabolite, dihydrotestosterone (DHT), or its metabolite, 5α-androstane-3α, 17β-diol (3α-Diol), to gdx male rodents results in affective and cognitive behaviors similar to that of intact male rats [8]. The hippocampus is thought to be important for affective and cognitive processes and is a target for androgens. Systemic administration of tritiated T is localized to the hippocampus [9]. Testosterone administration to gdx rats increases neuronal excitability [10] and prevents stress-induced cell death in the dorsal hippocampus [5]. Castrated rats with silastic implants of DHT demonstrated anxiolytic, analgesic, and cognitive-enhancing compared to rats with blank silastics; however, these effects are attenuated by implants of indomethacin to the dorsal hippocampus [8]. We investigated the effects of androgens to the dorsal hippocampus on affective and cognitive performance. Four to six weeks after GDX, 32 male rats were implanted with bilateral guide cannulae to the dorsal hippocampus. One week later, rats (n=8/group) were administered T, DHT, 3α-diol or control bilateral implants (1 µg) to the dorsal hippocampus. Rats were then immediately tested in the open field, plus maze, tailflick, pawlick, and defensive freezing tasks or were trained in the inhibitory avoidance task and tested 24 hours later. Thus, rats were assigned a condition, and were implanted and tested on two occasions. Although there were no significant differences between group rats with 3α-diol to the dorsal hippocampus had more central activity in the open field than did rats with control implants or implants of T or DHT. Castrated rats with implants of 3α-diol to the dorsal hippocampal made an average of 4.2 + 2.1 entries into central squares in the open field, whereas rats with T, DHT or control implants made very few entries to the center of the open field (range of 1.1 to 1.9 entries). Peripheral or total square entries in the open field did not differ across groups. Again there were no significant differences across groups in open arm activity in the in the elevated plus maze; however, androgen did increase this measure. Rats that received T (9.3 + 6.9 secs) or 3α-diol (6.5 + 3.6 secs) spent longer on the open arms of the plus maze than did rats with DHT (0.5 + 0.5 secs) or control implants (1.7 + 1.5 secs); 229

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

however, these differences were not significant and open arm activity was very low. There were no differences between groups in the closed arm time or total arm entries. 3α-diol implants to the dorsal hippocampus significantly increased tailflick and pawlick latencies compared to control implants. Tailflick latencies were longest following 3α-diol (6.7 + 0.9 secs) compared to DHT (5.9 + 0.5 secs) or T (5.6 + 0.5 secs) or control implants (4.2 + 0.5 secs). All androgens increased latencies to lick the front- or hind-paws when placed on a hotplate. The latencies to lick the front paw were longer following T (163.3 + 14.1 secs), DHT (139.5 + 18.3 secs) or 3α-diol (149.1 + 14.7 secs) compared to control (98.9 + 18.2 secs) implants. Latencies to lick the hind paw was also greater after T (161.0 + 10.7 secs), DHT (158.1 + 14.1 secs) or 3α-diol (166.3 + 9.8 secs) compared to control (124.1 + 17.1 secs) implants. Rats with androgen implants to the dorsal hippocampus spent significantly less time freezing in response to shock than did rats with control implants. Implants of T (411.1 + 77.7 secs), DHT (488.0 + 60.7 secs), or 3α-diol (507.3 + 95.7 secs) reduced the duration of freezing compared to control (763.8 + 45.3 secs) implants to the dorsal hippocampus. In the inhibitory avoidance task, androgens increased cross-over latencies to the shock associated side of the chamber. Rats with T (157.1 + 63.9 secs), DHT (240.2 + 36.2 secs), or 3α-diol (182.0 + 72.2 secs) implants to the dorsal hippocampus had longer crossover latencies compared to rats with control (126.3 + 43.4 secs) implants to the dorsal hippocampus. These findings suggest that 3α-diol enhanced analgesia, all androgens reduced fear responses, and DHT and 3α-diol improved cognitive performance. Notably, anxiety behavior was only marginally reduced by 3α-diol in the open field, and T or 3α-diol in the plus maze. Given that rats were tested in these tasks within 15 minutes of implants, we must consider whether the lack of effects was due to insufficient androgen exposure. Experiments are underway to ascertain effects if longer androgen exposure produces more robust effects. Acknowledgment: Supported by NSF (IBN 98-96262 and DBI-0097343) to CAF. Reference List [1] A. Adler, P. Vescovo, J.K. Robinson, M.F. Kritzer, Gonadectomy in adult life increases tyrosine hydroxylase immunoreactivity in the prefrontal cortex and decreases open field activity in male rats. Neurosci. 89, (1999) 939-54. [2] M.M.Cherrier, B.D. Anawalt, K.L. Herbst, J.K. Amory, S. Craft, A. M. Matsumoto, W.J. Bremner, Cognitive effects of short-term manipulation of serum sex steroids in healthy young men. J Clin Endocrinol Metab. Jul;87 (2002) 3090-6. [3] M.A.Cooper, E.C Ritchie. Testosterone replacement therapy for anxiety. Am J Psychiatry. Nov;157 (2000) 1884. [4] C.A. Frye, Estrus-associated decrements in a water maze task are limited to acquisition. Physiol Behav. Jan;57 (1995) 5-14. [5] C.A.Frye, C.M. McCormick, Androgens are neuroprotective in the dentate gyrus of adrenalectomized female rats. Stress. May;3 (2000) 185-94.. [6] C.A.Frye, S.M.Petralia, M.E. Rhodes, Estrous cycle and sex differences in performance on anxiety tasks coincide with increases in hippocampal progesterone and 3alpha,5alpha-THP. Pharmacol Biochem Behav. Nov;67 (2000) 587-96. [7] C.A. Frye, A.M. Seliga, Testosterone increases analgesia, anxiolysis, and cognitive performance of male rats. Cogn. Affect Behav . Neurosci, 1, (2001) 37-51. [8] C.A. Frye & A.M. Seliga, Indomethacin implants to the hippocampus attenuate anxiolysis and cognitive performance if intact and dihydrotestosterone-administered male rats. Horm Behav. June; 41 (2002) 467. [9] D.W. Pfaff, Autoradiographic localization of radioactivity in rat brain after injection of tritiated sex hormones. Science. Sep 27;161 (1968) 1355-6. [10] M.D. Smith, L.S. Jones, M.A. Wilson, Sex differences in hippocampal slice excitability: role of testosterone. Neuroscience. 109 (2002) 517-30.

230

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EFFECT OF NEONATAL OVARIECTOMY ON LEARNING AND SYNAPTIC TRANSMISSION IN THE HIPPOCAMPUS Freytes P. and Carrer H.F. Instituto de Investigación Médica M. y M. Ferreyra, INIMEC-CONICET, Casilla de Correo 389, 5000 Córdoba, Argentina; [email protected]; 54(351)4695163 To investigate the possible participation of ovarian secretions on postnatal development of the hippocampus, we studied the effect of neonatal ovariectomy on an hippocampus-dependent active avoidance task and on evoked potentials in the CA1 region of the hippocampus. Two days after birth female rats were ovariectomized (0VX) or pseudo-ovariectomized and returned to their mothers. At forty days of age animals were submitted to the open field test, where exploratory activity was evaluated. OVX animals showed significantly less visits to central squares (p< 0,01) than pseudo-OVX animals. Five days later animals were trained during 5 days, ten trials per day, in an active avoidance test in a T-maze. OVX animals showed a significantly longer escape latency (p<0,002) during training. Ten days later animals were submitted to a retention test in the same maze; OVX animals needed significantly more (p<0,05) trials than pseudo-OVX animals to reach the pre-established criterium. Two to five days later the animals were sacrificed and slices from the frontal hippocampus (400µm) were obtained to record in the pyramidal layer of CA1, the extracellular field potential evoked by stimuli applied to the Schaffer collaterals. The stimulus input-output curve showed a tendency (p < 0.07) for population spike amplitude to be larger in pseudo-OVX than in OVX animals at equivalent stimulus intensities. No differences were observed in the paired pulse facilitation curve. These results suggest that hormones segregated by the neonatal ovary are essential for normal hippocampal synaptic function and learning of a spatial task in adult animals.

231

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

A STRESS-ASSOCIATED AND GENDER-SPECIFIC FEAR CONDITIONING DEFICIT IN WAVED-1 MUTANT MICE Koshibu K.* and Levitt P. *Dept Neurobiology, Univ Pittsburgh, School of Medicine, Pittsburgh, PA, 15261, USA, [email protected]; J. F. Kennedy Ctr for Research in Human Development & Dept Pharmacology, Vanderbilt Univ, Nashville, TN, 37203, USA Waved-1 (Wa-1) is an autosomal recessive, spontaneous mutant of the transforming growth factor-α (TGF α) gene[1-4], resulting in a hypomophic state of gene expression [4,5]. Where as adult male mutant mice exhibit a fear conditioning deficit, females are normal. Because the fear conditioning paradigm necessarily involves footshock-induced stress as a part of the learning process, the effect of potential differences in stress response on learning between mutant and wildtype mice is important to examine. There is evidence suggesting that TGF α may play a role in stress function. For instance, TGF α is expressed in regions mediating learning, such as the hippocampus and amygdala, as well as in the regions associated with stress, including the hypothalamus, pituitary, and adrenal gland [6,7]. In addition, male transgenic mice that over express TGF α show increased immobility during the forced swim test, which is indicative of decreased stress coping ability [8]. However, female TGF α transgenics show shortened immobility in the swim test, suggesting their improved ability to cope with stress [9]. These findings suggest that gonadal steroids and/or stress factors may modulate the learning deficit in Wa-1 in a genderspecific fashion. We examined the stress response of adult male and female Wa-1 mice using a behavioral paradigm that measured stress-induced hyperthermia. In agreement with the fear conditioning results, only the male Wa-1 mice exhibited a decreased stress response, suggesting a better stress coping ability than the wildtype controls. There were no differences between female wildtype and Wa-1 mice. However, the wildtype females showed a blunted hyperthermia stress response compared to wildtype males, although fear conditioning was approximately the same in both sexes, suggesting that there may be a different mechanism underlining stress-regulated learning between male and female wildtype mice. These results suggest that the gender-specific learning deficit observed in Wa-1 may be due, in part, to the abnormal stress response in the male mutant mice. In order to address potential molecular mechanisms of the gender specificity, we measured mRNA expression of TGF α and the epidermal growth factor receptor (EGFR) in the two key structures involved in fear conditioning, hippocampus and amygdala, and in the hypothalamus, pituitary, and adrenal glands, HPA structures involved in the stress response. There were differences across gender in the patterns of mRNA expression in HPA structures, but not for the learning-associated brain regions. The region-specific differences of TGF α and EGFR expression support the importance of the HPA axis in influencing the gender-specific phenotype in Wa-1 mice.

232

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

These findings suggest that the hypomorphic expression of TGF α may result in an altered stress homeostasis, which in turn may cause fear conditioning deficits in a genderdependent manner. Supported by: NIMH grant MH45507(PL) and NSF IGERT predoctoral fellowship DGE-9987588 (KK)

Reference List 1. Crew FAE, J Genetics, 1933, 27:95-96 2. Fowler KJ, Mann GB & Dunn AR, Genomics, 1993, 16(3): 782-784 3. Mann GB et al, Cell, 1993, 73(2): 249-261 4. Luetteke NC et al, Cell, 1993, 73(2): 263-278 5. Weickert CS & Blum M, Brain Res Dev Brain Res, 1995, 86(1-2):203-216 6. Lazar LM & Blum M, J Neurosci, 1992, 12(5): 1688-1697 7. Serrogy KB, Han VK, & Lee DC, Neurosci Lett, 1991, 125(2): 241-245 8. Hilakivi-Clarke LA et al, Brain Res, 1992, 588(1): 97-103 9. Hilakivi-Clarke LA & Goldberg R, Eur J Pharmacol, 1993, 237(1): 101-108

233

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ESTROGEN RECEPTOR α IS REQUIRED FOR DOPAMINERGIC FACILITATION OF FEMALE SEX BEHAVIOR IN MICE. Kudwa A.E. 1, Gustafsson J.-Å.3 and Rissman E.F. 1,2 1

Graduate Program in Neuroscience and 2Dept. of Biochemisty and Molecular Genetics, University of Virginia, Charlottesville, VA 22903 USA; 3Karolinska Institute, Huddinge, Sweden. In female rodents, dopaminergic facilitation of lordosis requires both unoccupied progestin receptors (PR) and D1-like dopamine receptors. In this study I asked which estrogen receptor (α or β) is needed to mediate PR induction and activate dopaminergic facilitation of female sex behavior. Heterozygous breeding pairs, possessing one functional and one disrupted copy of both the estrogen receptor α and β genes (ERα, ERβ), were used to produce the ERα/ β wild-type (WT), ERα knock-out (ERαKO), ERβKO and ERα βKO offspring used in this study. Adult females were ovariectomized and implanted with Silastic capsules containing 50µg estradiol benzoate (EB). First, lordosis in response to a solicitous male was observed over three trials following acute apomorphine administration (APO; 2µg/20g in 0.2% ascorbic acid) in the absence of progesterone. However, maximal APO-mediated enhancement of lordosis was observed only after the females were tested two additional times in the presence of progesterone (100µg) prior to a fourth, and final, APO trial. Mice lacking functional ERα (ERαKO and ERα βKO) did not display lordosis in response to APO or P injections. ER βKO females exhibited lordosis behavior equal to or greater than that observed in WT females. This finding provides evidence that dopaminergic modulation of lordosis requires ERα. Estradiol-mediated progestin receptor (PR) induction was assessed using immunocytochemistry. Although constitutive PR-immunoreactive cells were observed in the ventromedial hypothalamic nucleus of all genotypes, E2-mediated PR induction was only observed in WT and ER βKO females. In addition, social preference tests conducted 2-3 days following acute APO injections revealed that APO-treated females of all genotypes showed increased chemoinvestigatory behavior as well as a preference for the female stimulus animal. Collectively, the data implicate a role for ERα, but not ERβ, in dopamine receptormediated modulation of female sex behavior in mice.

234

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EXPRESSION AND INTRACELLULAR TRAFFICKING OF STRIATAL α DOPAMINE D1 RECEPTORS IS AFFECTED IN ESTROGEN RECEPTOR-α KNOCKOUT (ERKO) MICE Küppers E., Brito V. and Beyer C. Department Anatomy und Cell Biology, University of Ulm, D-89069 Ulm, Germany [email protected] The differentiation of the striatal anlage and in particular of GABAergic neurons is critically depending on the establishment of functional contacts with the dopaminergic projections from the substantia nigra of the midbrain. In vitro studies have provided evidence that dopamine signaling via D1 receptors is pivotal for the development of the GABAergic phenotype. In addition, estrogen which is synthesized perinatally in the striatum regulates dopamine receptor function. We have used an estrogen receptor-α knockout model (ERKO) to analyze the role of estrogen on dopamine receptor expression and availability in the striatum. Immunoblotting and RT-PCR studies revealed that dopamine D1 receptor expression is increased in (-/-) females compared to (wt) females. In contrast, the expression of DriP78 which is implicated in D1 receptor trafficking from the endoplasmic reticulum/Golgi complex to the plasma membrane is significantly depleted in (-/-) males. Confocal laser microscopy additionally reveals a shift in the membrane/cytosolic D1 receptor ratio in (-/-) males compared to (wt) male animals. Taken together, our data provide strong evidence that the disruption of estrogen signaling leads to massive disturbances of dopamine signaling in the striatum. This effect may be causally linked to the known motor activity dysfunction observed in ERKO mice.

Supported by the Medical Clinic of the University of Ulm.

235

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

THE EFFECT OF ACUTE ESTROGEN RECEPTOR ANTAGONISM ON ESTRADIOL-INDUCED INCREASES IN FORMALIN INFLAMMATORY PAIN BEHAVIOUR AND INTERFERON-GAMMA PRODUCTION IN MALE RATS Lariviere W.R., Ceccarelli I., Fiorenzani P. and Aloisi A.M. Pain and Stress Neurophysiology Laboratory, Department of Physiology, University of Siena, Via Aldo Moro, 2, San Miniato 53100 SIENA Italy, [email protected], Fax: 0577 234037 Background: Females show a greater incidence of several chronic pain syndromes and generally a greater sensitivity to experimental stimuli compared to males [4]. To study the underlying mechanisms, we previously showed that female rats show more pain behaviour in the formalin inflammatory pain test compared to male rats [1], and that centrally administered estrogens increase formalin pain responding (licking) in male rats [2]. We have also reported that there are sex differences in interferon-gamma (IFN) production by lymphocytes, and that formalin injection significantly decreases IFN production [3], which could also modulate inflammatory pain. Aim: This study examined whether acute blockade of estrogen receptors prior to administration of the formalin test is able to block the estradiol-induced changes in formalin pain responding, spontaneous behaviours, and IFN production by lymphocytes in the male rat. Methods: Male Wistar rats (240-280 g) received two days of pre-treatment with an intracerebroventricular (icv) injection of water soluble, cyclodextrine-encapsulated 17βestradiol (1 µg in 5µl; Sigma, USA) or saline vehicle. On the third day, rats received an icv injection of the non-specific estrogen receptor antagonist, ICI 182,780 (ICI; 100 nmol in 5µl). Fifteen min later, a subcutaneous injection of 5% formalin (50 µl) was given in the dorsal hind paw producing the characteristic biphasic pattern of pain behaviours lasting approximately 1 h. Pain behaviours (licking, jerking, or flexing of the hind paw) and spontaneous behaviours (rearing frequency, and duration of locomotion, sitting alert, crouching, and grooming, and internal and external line crossings in an open field) were video recorded for 60 min and scored by an experimenter blind to the treatment. Immediately after, animals were killed with an overdose of Nembutal, a blood sample was taken from the exposed abdominal aorta (for assessment of IFN production by Concanavalin-A-stimulated lymphocytes), and brain tissue was extracted for verification of cannula placement. Results: As we reported previously, estradiol pre-treatment significantly increased licking duration during the second phase of the formalin pain response. ICI treatment prior to the formalin test blocked the increase in duration of licking during the second phase in estradiol pre-treated animals, and had no effect on pain behaviours in the second phase in saline pre-treated animals. However, ICI increased the duration of paw flexing during the characteristic interphase depression in responding. ICI also reversed the estradiol-induced increase in rearing frequency, and decreased rearing frequency and increased sitting alert duration in saline pre-treated animals. Estradiol increased IFN production by lymphocytes in formalin-injected animals, and IFN production was not affected by ICI pre-treatment in estradiol nor saline pre-treated animals. 236

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

Conclusions: Estradiol induced increases in formalin-induced licking and rearing are mediated by the acute activation of centrally located estrogen receptors. ICI has effects of its own on spontaneous behaviour and pain behaviour (possibly on the central pain suppression mechanisms that contribute to the formalin interphase depression); however, these effects cannot explain the reversal of estradiol-induced changes by ICI. Immune mechanisms that may modulate inflammatory pain mechanisms are also affected by central administration of estradiol, but are not mediated by the acute activation of estrogen receptors.

Acknowledgements: This research was supported by funds from the University of Siena and by postdoctoral fellowship to W.R.L. from the Natural Sciences and Engineering Research Council of Canada.

Reference List [1] A.M. Aloisi, M.E. Albonetti, G. Carli, Sex differences in the behavioural response to persistent pain in rats, Neurosci. Lett. 179 (1994) 79-82. [2] A.M. Aloisi, I. Ceccarelli, Role of gonadal hormones in formalin-induced pain responses of male rats: modulation by estradiol and naloxone administration, Neuroscience 95 (2000) 559-66. [3] A.M. Aloisi, M. Muscettola, C. Lupo, Effects of gonadectomy and pain on interferon-gamma production in splenocytes of male and female rats, Brain Behav. Immun. 15 (2001) 266-72. [4] K.J. Berkley, Sex differences in pain, Behav. Brain Sci. 20 (1997) 371-80.

237

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NPY-Y1 RECEPTOR GENE EXPRESSION INCREASES IN TRANSGENIC MICE AMIGDALA FOLLOWING RESTRAINT STRESS Mele P.a., Serra M.b., Pisu M.G. b, Biggio G. b and Eva C. a a

Dipartimento di Anatomia, Farmacologia e Medicina Legale, Sezione di Farmacologia, Università di Torino, Via P. Giuria, 13, 10125 Torino, Italy; e-mail: [email protected]; fax: 011-3267718. bDipartimento di Biologia Sperimentale, Sezione di Neuroscienze, Università di Cagliari, Cagliari, Italy. NPY plays an important role in the regulation of both emotional behavior and stressful stimuli, similar to that elicited by positive modulators of GABAA receptor such as benzodiazepines or neuroactive steroids [1]. Previous studies demonstrate that NPY inhibits several behavioral and physiological effects of stress through the activation of the Y1 receptor subtype in the amygdala, suggesting that this neuropeptide may be an endogenous agent that buffers against the stressor-induced release of CRF [2-3]. In agreement with the antistress effects observed following central administration of NPY, a role of NPY-Y1 neurotransmission in the control of stress-related behavior is suggested by the observation that acute restraint stress, which promotes experimental anxiety, suppresses NPY mRNA and peptide levels. Moreover, repeated exposure to the same stressor once daily for ten days leads to a complete behavioral and endocrine habituation accompanied to the up-regulation amygdala NPY expression [4]. Functional and neuroanatomical studies have shown that GABA and NPY coexist in the same neurons of the amygdaloid complex and that they may functional interact in the regulation of anxious behavior [5-6]. In addition NPY and GABA may act in concert for counteracting the excitatory effects of CRF in the amygdala, and thereby to contribute to the net arousal of an organism following exposure to stress and threat [3]. Brain and plasma concentrations of neuroactive steroids are modulated by stress exposure, suggesting that neuroactive steroids may play an important role in the regulation of GABAergic transmission both in physiological and in pathological conditions [7-8]. By using a transgenic mouse line harboring the a fusion construct (Y1R/LacZ) that comprises the mouse Y1 receptor gene promoter linked to the LacZ gene we previously showed that a persistent increase in the brain concentration of neuroactive steroids during pregnancy or following pharmacological long-term treatment with progesterone or allopregnanolone induces an increase of Y1 receptor gene expression in the medial and central amygdala similar to that produced by diazepam [9-10]. In this study we have investigated the effect of a single or of repeated exposures to restraint stressor on cerebrocortical concentrations neuroactive steroids of and on Y1R/LacZ transgene expression in the medial and in the central amygdala. A single 1 h restraint induced a transient increase of neuroactive steroids concentration in the cerebral cortex that decreases 60 min after the treatment. Computerized quantitative histochemical analysis of beta-galactosidase activity demonstrated that the same treatment induces an increase of Y1R/LacZ transgene expression in the medial and in the central amygdala that was observed six hours after the end of the restraint period. No changes in the transgene expression were observed in the medial habenula (control region). The daily administration of the 5α-reductase inhibitor finasteride (25 mg/Kg/s.c) for 2 days before the exposure to

238

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

the stressor failed to prevent the increase of beta galactosidase expression suggesting that this effect was not mediated by the increase in neuroactive steroid cerebrocortical concentrations. Conversely following repeated restraint (1h/day, 10 days) neither the transgene expression, nor the cerebrocortical concentrations of neuroactive steroids were affected. Our results demonstrate that the exposure to a single restraint stress, that suppresses the endogenous amygdala NPY expression [4], also induces a compensatory up-regulation of the Y1 receptor gene expression in the same brain area. These data provide further evidence to the hypothesis that a decrease in the functional activity of the NPY-Y1 transmission in the amygdala may in part account for the anxiogenic-like action of the stressor. Our data also suggest that, although a single 1 h restraint induces a transient increase in brain neuroactive steroid concentrations, the changes in the NPY-Y1 activity induced by the stressor are mediated by molecular mechanisms independent from the interaction with the GABAergic system. The observation that repeated exposure to restraint stress fails to affect both amigdala Y1 receptor gene expression and neuroactive steroid concentration suggests that adaptive behavioral changes, such as habituation or sensitization, might occurr.

References List [1] A. Kask, J. Harro, S. von Horsten, J.P. Redrobe, Y. Dumont, R. Quirion, The neurocircuitry and receptor subtypes mediating anxiolytic-like effects of neuropeptide Y, Neurosci. Biobehav. Rev. 26 (2002) 259-283. [2] M. Heilig, G.F. Koob, R. Elkman, K.T. Britton, Corticotropin-releasing factor and neuropeptide Y: role in emotional integration, TINS 17 (1994) 80-85. [3] K.T. Britton, Y. Akwa, M.G. Spina, G.F. Koob, Neuropeptide Y blocks anxiogenic-like behavioral action of corticotropin-releasing factor in an operant conflict test and elevated plus maze, Peptides 21 (2000) 37-44. [4] A. Thorsell, M. Michalkiewicz, Y. Dumont, R. Quirion, L. Caberlotto, R. Rimondini, A.A. Mathe, M. Heilig, Behavioral insensitivity to restraint stress, absent fear suppression of behavior and impaired spatial learning in transgenic rats with hippocampal neuropeptide Y overexpression, Proc Natl Acad Sci U S A. 97 (2000) 12852-12857. [5] A. Kask, L.Rago, J. Harro, Anxiogenic-like effect of the neuropeptide Y Y1 receptor antagonist BIBP3226: antagonism with diazepam, Eur. J. Pharmacol. 317 (1996) R3–R4. [6] A. Oberto, G.C. Panzica, F. Altruda, C. Eva, GABAergic and NPY-Y1 network in the medial amygdala: a neuroanatomical basis for their functional interaction, Neuropharmacology 41 (2001) 639–642. [7] M.L. Barbaccia, G. Roscetti, F. Bolacchi A. Concas, M.C. Mostallino, R.H. Purdy, G. Biggio, Stress-induced increase in brain neuroactive steroids: antagonism by abecarnil, Pharmacol. Biochem. Behav. 54 (1996) 205-210. [8] M. Serra, M.G. Pisu, M. Littera, G. Papi, E. Sanna, F. Tuveri, L. Usala, R.H. Purdy, G. Biggio, Social isolation-induced decreases in both the abundance of neuroactive steroids and GABAA receptor function in rat brain, J. Neurochem. 75 (2000) 732–740. [9] G. Ferrara, M. Serra, F. Zammaretti, M.G. Pisu, G.C. Panzica, G. Biggio, C. Eva, Chronic treatment with progesterone and with allopregnanolone increases NPY-Y1 receptor gene expression in the medial amygdaloid nucleus of transgenic mice, J. Neurochem. 79, (2001) 417–425. [10] A. Oberto, M. Serra, M.G. Pisu, G. Biggio, C. Eva, Changes in expression of the neuropeptide Y Y1 receptor gene in the medial amygdala of transgenic mice during pregnancy and after delivery, J. Neurochem. 82 (2002) 1272-1281.

239

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

POSSIBLE OESTROGENIC EFFECTS ON AP-2 ALFA EXPRESSION AND AP-2 ALFA POTENTIAL ROLE IN nNOS REGULATION IN MOUSE HYPOTHALAMUS Orso F.1, Sica M.2, Panzica G.C.2 and De Bortoli M.1,3 1

Dept. of Animal & Human Biology, University of Turin, Italy; 2Dept. of Anatomy Pharmacology & Forensic Medicine; 3Institute of Cancer Research & Treatment, University of Turin, Italy [email protected] AP-2 transcription factors are a family of four closely related and evolutionarily conserved sequence-specific DNA-binding proteins, AP-2α, β, γ and δ. AP-2 proteins are expressed in the embryo showing spatially- and temporally-regulated patterns in different tissues (neural crest derivatives, neural, epidermal and urogenital tissues). Transcription factors AP-2 play a role in cell growth, differentiation, apoptosis, mutations or changes in precisely programmed expression patterns (5). AP-2 proteins are highly similar but their roles are specific and not redundant as it is suggested by the different phenotypes of nullmutant mice of AP-2 genes (α, β, and γ). In fact, AP-2α-/- mice die peri-natally showing dramatic defectsin development of cranio-facial structures and toraco-abdominoschisis, due to incomplete neural tube closure (9, 12); AP-2β-/- mice die since renal differentiation is not completed (7); AP-2γ-/- mice die at day 7.5 to 8.5 since all extra-embryonic tissues are malformed and a normal maternal-embryonic interface is not established (1, 11). Recently, we demonstrated that in an oestrogen-responsive model system (human breast cancer cell lines: ZR75.1 and MCF-7) AP-2α and AP-2γ expression is under oestrogen regulation, in particular 17-β-oestradiol strongly induces AP-2γ and concomitantly down-regulates AP2α expression at both mRNA and protein level. AP-2γ induction is an immediate event, as it is clearly visible 1 hour after oestrogen treatment. By examining the AP-2γ 5’UTR sequence we found two ERE sequences: one upstream noncanonicalsequence and one downstream perfect ERE sequence. In vitro assays demonstrated that both EREs are footprinted, but only the downstream ERE is able to bind rER in EMSA analysis. Functional data demonstrated that downstream ERE is probably the one responsible for AP-2γ induction. The downstream ERE shows a high conservation in human and mouse while upstream ERE is not conserved (8). Since i) 17-β-estradiol (E2) exerts a critical influence over the architecture and survival of neural cells in both central and peripheral nervous system during development; ii) it shows a considerable activity on neural cell metabolism and plasticity in the mature central nervous system; iii) AP-2α is highly represented within CNS, we decided to look at a possible oestrogen-regulation of AP-2 in adult central nervous system, with particular attention to the nuclei described as positive for oestrogen receptor α and β. Since nNOS expression is under control of steroid hormones (10, 2, 6) and nNOS positive elements are present in ERα- and ERβ- rich regions, we looked at the possible colocalization of AP-2α and nNOS in male and female mice. The presence of a potential AP2 site in the 5’-flanking region of human neuronal nitric oxide synthase gene has been demonstrated by Hall, et al. (4). We performed our study on both male (n= 4) and female (n= 4) CD1 mice of three months of age. All the animals were perfused with 4% paraformaldheyde. Brains were serially sectioned with a cryostat: the first series were

240

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

processed for AP-2α immunocytochemistry. We employed an anti-AP-2α (C-18) antibody (Santa Cruz) at the dilution of 1:6,000 and sections were immunostained by the indirect biotin-avidin system. The second series were processed for NADPH diaphorase (3). Preliminary results of this study are shown here. In male mice we observed immunoreactive elements for AP-2α in BSTMPL (bed nucleus of stria lateralis, medio posterolateral portion), in the PALM (lateral paraventricular hypothalamic nucleus, magnocellar portion), PaAP (anterior paraventricular hypothalamic nucleus, parvicellular portion) and PaPo (posterior paraventricular hypothalamic nucleus) of paraventricular nucleus and in the lateral hypothalamus (LH). In female mice, immunoreactive elements were observed in the same regions but their number was significantly lower than in male. NADPH-diaphorase histochemistry demonstrated the presence of NO producing elements in the same area where AP-2α is expressed, except than in the PaPO. The number of NADPH-diaphorase neurons was significantly lower in female mice. These results suggest a possible oestrogenic control of AP-2α in mouse hypothalamus and a possible regulation of nNOS by AP-2α.

References List 1. H.J. Auman, T. Nottoli, O. Lakiza, Q. Winger, S. Donaldson, T.Williams, Transcription factor AP2gamma is essential in the extra-embryonic lineages for early postimplantation development. Development 129 (2002) 2733-2747 2. J. Du, E.M. Hull, Effects of testosteroe on neuronal nitric oxide synthase and tyrosine hydroxylase. Brain Res. 836 (1999) 90-98 3. D.W. Ellison, N.W. Kowall, J. B. Martin, Subset of neurons characterized by the presence of NADPH-diaphorase in human substantia innominata. J Comp Neurol. 260 (1987) 233-245. 4. A.V. Hall, H. Antoniou, Y. Wang, A.H. Cheung, A.M. Arbus, S.L. Olson, W.C. Lu, C.L. Kau, P.A. Marsden, Structural organization of the human neuronal nitric oxide synthase gene (NOS1). J Biol Chem. 269 (1994) 33082-90. 5. K. Hilger-Eversheim, M. Moser, H. Schorle and R. Buettner, Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control. Gene. 260 (2000) 1-12. Review. 6. E.M., Hull, J. Du, D.S. Lorrain and L. Matuszewich, Testosterone, preoptic dopamine and copulation in male rats. Brain Res Bull. 44 (1997) 327-33 7. M. Moser, A. Pscherer, C. Roth, J. Becker, G. Mucher, K. Zerres, C. Dixkens, J. Weis, L. GuayWoodford, R. Buettner, R. Fassler, Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta. Genes Dev. 11 (1997) 1938-1948. 8. F. Orso, E.Cottone, M.D. Haselton, P.Sismondi, H.C. Hurst, M. De Bortoli , AP-2γ expression is specifically induced by oestrogens through binding of the oestrogen receptor to a 5’-UTR element. Submitted to NAR. 9. H. Schorle, P. Meier, M. Buchert, R. Jaenisch, P.J. Mitchell, Transcription factor AP-2 essential for cranial closure and craniofacial development. Nature 381 (1996) 235-238. 10. M. Sica, L. Plumari , S. Honda, N. Harada, P. Absil, C. Viglietti-Panzica, J. Balthazart, G.C. Panzica Changes in the neuronal nitric oxide synthase immunoreactive system in male mice lacking a functional aromatase gene. Hormones and Behavior (2002). 41: 490. 11. U. Werling, H. Schorle, Transcription factor gene AP-2 gamma essential for early murine development. Mol Cell Biol. 22 (2002) 3149-3156. 12. J. Zhang, S. Hagopian-Donaldson, G. Serbedzija, J. Elsemore, D. Plehn-Dujowich, A.P. McMahon, R.A. Flavell, T. Williams, Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. Nature 381 (1996) 238-241.

241

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

MALE RATS ADOPTED AT BIRTH BY STRESSED DAMS DISPLAY FEMININE BEHAVIORAL PATTERNS IN INDUCED MATERNAL BEHAVIOR Pérez-Laso C.1, Ortega E.2, Izquierdo M.A.P.1, Moreno N.1, Rivera L.1, Segovia S. 1 and Del Cerro M.C.R.1 1

Dept. Psychobiology, UNED, Ciudad Universitaria s/nº 28040 Madrid, Spain. 2Dept. Biochemistry and Molecular Biology, Medicine Faculty, Granada, Spain. Dpto. de Psicobiología, Facultad de Psicología, UNED, Ciudad Universitaria s/nº 28040 Madrid, Spain [email protected] fax 34-913986287.

Maternal behavior (MB) is a sexually dimorphic behavior. In previous studies, we have demonstrated that environmental prenatal stress (EPS) has long term behavioral and neuroendocrine effects, in the female rat. It impairs natural and induced maternal behavior and induces tonic dysfunction of the hypothalamo-pituitary-adrenal (HPA) axis and ovarian hormones, in a dose dependent way. We have also demonstrated that behavioral effects, in the female rat, can be counteracted by adoption at birth. In the present study we investigate the effects of postnatal manipulation of environment in the male rat. Repeated environmental stress during the last week of pregnancy was used as prenatal stressor and adoption at birth was used to change the early postnatal environment. Subjects: Seventy rats of the Wistar Strain (Iffacredo, Barcelona- Spain), divided into five groups: C-M and C-F (control males and females fostered by their own mothers, N=17 and 15); CN1-M (control males fostered by stressed dams, N=11); PS1-M prenatally stressed males fostered non-stressed dams (N=12) and PS-M (prenatally stressed males fostered their own stressed mothers, N=15). Animals of the two prenatally stressed groups (PS-M and PS1-M) were randomly selected between the male pups of a group of pregnant rats that were exposed to three daily stress sessions of 45 minutes each, during the last week of gestation. We used the Ward Paradigm (1972): Restrain, light (2,500 luxes) and heat (31±1º). Animals of the three groups (C-M, C-F and CN1-M) were selected between female pups of a group of mothers that were left undisturbed throughout the whole length of pregnancy. Results: We have replicated sex differences between males and females in the control groups. Both prenatally stressed groups of males (reared by their biological mother or by control dams) do not differ from control males. However, the group of control males reared by stressed dams has significantly higher scores in all the studied parameters of maternal behavior and does not differ from control female group. The percentage of males from this group (CN1-M) that became maternal and those that accomplished retrieval during the 12 days of the induced maternal behavior test is statistically similar to that obtained in the control female group. Also, a significant decrease in plasma T levels has been detected in this group of males when compared with control or with the prenatally stressed male groups. Our results evidence that early postnatal environment can influence the sexual differentiation of maternal behavior in males. This work has been supported by DGESIC BS02000-0109.

242

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

VARIATION OF THE PHYSIOLOGICAL STATE AND SOCIALS BEHAVIOR IN JAPANESE QUAIL Pincemy G. and Guyomarc’h C. UMR 6552 Ethologie Evolution Ecologie, Université de Rennes1, Avenue du Général Leclerc, 35042 Rennes, France. email : [email protected] . Fax number : (33)2 23 23 69 27 In a series of experiences, we wanted to test the impact of physiological changes, related to the somatic and sexual development, on the potential to get synchronised for individuals living in social groups. Our subject of experience is the Japanese quail (Coturnix coturnix japonica). Some similar studies were made by Domjan (1987) who worked on the impact of hormonal concentration in the proximity for males Japanese quail. Thus, the tendency for a male to get close to the female would be in close relation with the testosterone concentration. We follow more particularly the synchronization and exclusion behaviours between individuals from the juvenile states (chicks) until they get sexually mature. These observations help us to appreciate the majors changes in inter individual relationship occurring during the sexual development, and so potentially link to the physiological conditions of individuals (steroid production). This characteristics were studied for 7 social groups of 8 to 14 chicks, affiliated or not affiliated, raised in the same conditions. We show up the individuals profiles for the general activity behaviour (feeding, resting, and displacement). And we followed the proximity relationship between individuals during the 15-20 first days of life. More over, reproduction behaviour profiles (sing and sexual parade) were established when animals were sexually mature. These results allow us to think of doing other experimental protocol. The goal will be to control the consequences of an artificial variation in the testosterone concentration by castration and/or implantation, on the expression of social affinities. And this, thinking of the individual differences about the behaviour profile (individual presenting a low or a high social potential). We’ll change the internal states of birds and will control the impacts of this change on the individual capacity to get synchronised with others and thus the effects of this modification on the group cohesion.

243

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ACTIVATIONAL EFFECTS OF ESTRADIOL AND DIHYDROTESTOSTERONE ON THE ARGININE-VASOPRESSIN IMMUNOREACTIVE SYSTEM OF MALE MICE LACKING A FUNCTIONAL AROMATASE GENE Sica M.1, Allieri F.1, Plumari L.1, Bakker J.2, Honda S.3, Harada N.3, VigliettiPanzica C.1, Balthazart J.2 and Panzica G.C.1 1

Laboratory of Neuroendocrinology, Rita Levi Montalcini Centre for Brain Repair, Department of Anatomy, Pharmacology and Forensic Medicine, University of Torino, Torino, Italy. [email protected] 2 Centre for Cellular and Molecular Neurobiology, University of Liege, Lie ge, Belgium. 3 Molecular Genetics, Fujita Health University, Toyoake, Japan.

In rodents, parts of the arginine-vasopressin (AVP) neuronal system are sexually dimorphic and sensitive to sex steroids (3). For example, the bed nucleus of stria terminalis (BNST) and the medial amygdala (Me) (6) contain more AVP immunoreactive cells (AVPir) in males than in females and the projections from these nuclei to the lateral septum are denser in males than in females (1, 2). In male rodents the expression of AVP in neurons of the BSTN and Me is controlled by testosterone (T): gonadectomy inhibits mRNA expression and AVP immunoreactivity in both nuclei, whereas AVP expression is restored to the level observed in intact males by treatments with exogenous T (4). AVP transcription in these nuclei is increased in adulthood by a synergistic action of androgenic and estrogenic metabolites of T and, accordingly, androgen and estrogen receptors are present in the BNST, SL and Me. In a recent study (5) we demonstrated that the chronic lack of aromatized T metabolites, as observed in aromatase knock-out mice lacking a functional aromatase enzyme (ArKO), results in a significant decrease of AVP-ir structures in the BST, Me and SL of male mice. To determine whether this effect is due to a lack of organizational or activational effects of estrogens, we analyzed here the AVP immunoreactive system in the forebrain of adult male ArKO mice that had been treated or not from six week of age with estradiol benzoate (EB) in association with dihydrotestosterone propionate (DHTP). Five month-old wild-type and ArKO male mice that had been kept as intact controls or treated with EB and DHTP were perfused with 4% paraformaldehyde and 0.1% glutaraldehyde 48 h after they had received an i.c.v. colchicine injection. Brains were cut in the coronal plane with a cryostat at 25 µm thickness. Every fourth section was stained for AVP by immunohistochemistry using the free-floating technique. Sections were immunostained by the indirect biotin-avidin system with a commercial AVP antibody (ICN, Pharmaceuticals, CA, USA) at the dilution of 1:20,000. In WT mice, treatment with exogenous hormones did not influence the distribution of AVP-ir elements in the Me and SL. As expected, a prominent decrease of the density of AVP-ir structures was observed in the Me (p=0,0074) and SL (p=0.0026) of non-treated ArKO mice in comparison with WT, thereby confirming previous data obtained in our laboratory (5). Treatment of ArKO mice with exogenous EB+DHTP restored the density of AVP-ir structures to the level typical of WT males: there was a significant increase of the density of AVP-ir structures both in the Me (p=0.0254) and SL (p<0.0001) of steroidtreated ArKO mice by comparison with non treated subjects.

244

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

These data thus suggest that the steroid-sensitive sexually dimorphic AVP system of the mouse forebrain is controlled by activational effects of estrogenic and androgenic metabolites of T, as previously demonstrated in the rat. Moreover, they suggest that the decrease of AVP-ir structures previously observed in the forebrain of ArKO mice is probably not the result of the absence of estrogens during ontogeny. The AVP-ir system of male ArKO mice can still be activated by exogenous hormones in adulthood which clearly indicates that the decrease observed in untreated subjects is not the result of irreversible organizational effects (neuronal death due to the lack of estrogens during ontogeny) but is rather due to a lack of activation by steroids in adulthood.

References List 1. A.R. CaffË, W.F. Van Leeuween, P.G.M. Luiten. Vasopressin cells in the medial amygdala of the rat project to the lateral septum and ventral hyppocampus. J. Comp. Neurol 261(1987):237-252. 2. G.J De Vries, R.M. Buijs. The origin of the vasopressinergic and oxytociner(gic innervation of the rat brain with special reference to the lateral septum. Brain Res. 273 (1983):307-317. 3. G.J De Vries, Miller M.A. Anatomy and function of extrahypothalamic vasopressin system in the brain. Prog. Brain Res 119 (1998): 3-20. 4. M.A. Miller, J.H. Urban, D.M. Dorsa. Steroid dependency of vasopressin neurons in the bed nucleus of the stria terminalis by in situ hybridization. Endocrinology 125 (1989): 2335-2340. 5. L. Plumari, C. Viglietti-Panzica, F. Allieri, Honda S., Harada N., Absil P., J. Balthazart, G.C. Panzica. Changes In the arginine-vasopressin immunoreactive systems in male mice lacking a functional aromatase gene. Journal of Neuroendocrinology 14 (2002) 971-978 6. F.W. Van Leeuwen, A.R. Caffé, G.J. De Vries. Vasopressin cells in the bed nucleus of the stria terminalis of th rat: sex differences and the influence of androgens. Brain Res 325 (1985): 391-394.

245

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

COMPARISON OF NEUROANATOMICAL DISTRIBUTION OF DELTA OPIOID RECEPTOR AND TYROSINE HYDROXYLASE IMMUNOREACTIVITY IN YOUNG AND OLD MALE QUAIL Thompson N., Micevych P. + and Ottinger M.A. Dept. of Animal and Avian Sciences, University of Maryland, College Park, MD 20742, USA, [email protected] and + Dept. of Neurobiology, UCLA Center Health Sciences, Los Angeles, CA 90095 USA During the process of aging, the male Japanese quail shows a decrease in courtship and mating behavior that occurs prior to a discernable decline in pituitary gland and testicular function (for review, see [1]). Moreover, the loss of circulating androgen is delayed and becomes significant after the male has become reproductively senescent, with testicular regression. In fact, the Leydig cells still retain some steroidogenic capability in aged males; in spite of a loss of gonadotropin receptors [2]. The data point to the question of the mechanisms involved in the process of aging, i.e. the basis of the loss of reproductive behavior and the role of testicular androgens in the loss. It appears that neuroendocrine elements that underlie reproductive endocrine and behavioral responses become less responsive during the process of aging in the male Japanese quail. This hypothesis is based on several lines of evidence. First, males that are senescent do have decreased plasma androgen levels and increased testicular pathologies [3, 4]. However, the aged male still remained responsive to exogenous testosterone, which restored mating behavior in senescent males [1]. Middle-aged and aged males that remain sexually active have elevated plasma estradiol levels, suggesting that they produce and peripherally metabolize more steroid hormones to stimulate sexual behavior. Therefore, in the current study, we investigated potential neural substrates upon which steroid hormones act to restore sexual behavior. We also considered opioid peptide systems because there is a strong link to regulation of the GnRH-I system as described below. Enkephalin (ENK) modulates reproduction in birds and is found in areas that regulate avian gonadotropin releasing hormone –I (GnRH-I). Previous studies showed that delta opioid receptors (DOR) colocalize on the GnRH-I neuron, providing a mechanism for direct regulation of GnRH-I release in the Japanese quail. These data were supported by in vitro studies, which showed that ENK and b-endorphin inhibited GnRH-I release in vitro from parasaggittal hypothalamic slices in a dose dependent manner. Therefore, DOR, which binds both ENK and $-endorphin are likely to directly affect the GnRH-I neuron. In the current study, we focused on the catecholamine containing neurons, which are important modulators of the GnRH-I system. The relationship of catecholamine containing neurons was assessed by immunohistochemiistry for tyrosine hydroxylase (TH). TH immunoreactive (TH-ir) and DOR immunoreactive (DOR-ir) neurons were examined in selected areas of the hypothalamus and in the septal preoptic region during aging in male Japanese quail. Fixed brains were collected from young reproductive males (n=5) and old senescent males (n=5). The brains were sectioned (25µ) rostral from the preoptic-septal region (POA-SL) through to the median eminence (ME). Sections were doubly stained for

246

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

TH (TH-ir; DiaSorin) and DOR (DOR-ir; DiaSorin); TH-ir and DOR-ir were visualized using Cy2 and Cy3, respectively (AffiniPure Goat Anti-mouse or Anti- rabbit IgG; Jackson ImmunoResearch Labs, Inc). Sections were thoroughly examined under fluorescent microscopy (400X) and both systems were examined in the following areas: preoptic area (POA), medial preoptic area (POM), lateral septal region (SL), nucleus accumbens (n. accumbens), lateral hypothalamus, paraventricular nucleus, and median eminence (ME). Semi quantitative assessment was conducted in all sections; presence of immunoreactive fibers and cells were recorded for both single and doubly labeled cells. Results in young males showed single labeled cells in most regions, including the preoptic area, POM, SL, n. accumbens, lateral hypothalamus, paraventricular nucleus (PVN), and ME. Many of these regions, such as the n. accumbens contained both TH-ir and DOR-ir fibers. Mid and lateral septal regions had areas that were enriched in the number of doubly labeled neurons. In the POA, TH-ir neurons were apposed by DOR-ir fibers. In the central parts of the ME, THir predominated in the ME, with DOR-ir appearing in more ventral regions. There appeared to be relatively small changes in both TH and DOR during aging. Further, TH-ir staining appeared less intense in young active males compared to senescent males. These data are similar to our previous observations (Panzica et al, unpublished data 5]) in that there were some differences in TH-ir in the hypothalamus. However, in that study we observed marked decreases in TH-ir in some other brain regions. In this study, we did not examine those brain regions. In summary, our current observations complement previous data collected on the aging male Japanese quail. Therefore, these data support a mechanism for opioid modulation of TH input to GnRH-I neurons, especially in regions including the preoptic septal region. Further, this mechanism appears to remain intact during the process of aging, at least in regions, which contain the GnRH-I system.

Supported by EPA R826134, IBN-9817024 (MAO) & NS 39495.

Reference List 1. Ottinger, M. A. (1998). Male reproduction: testosterone, gonadotropins, and aging. In: Functional Endocrinology of Aging. Mobbs, C.V., Hof, P.R. (eds). Karger Press. Vol 29:105-126. 2. Ottinger, M.A., K. Kubakawa, M. Kikuchi, N. Thompson, and S. Ishii. (2002). Effects of exogenous testosterone on testicular LH and FSH receptors during aging. Exper. Biol. Medicine. In Press. 3. Ottinger, M.A., M. Masson and C.S. Duchala (1983). Age-related reproductive decline in the male Japanese quail. Horm. Behav. 17:197-207. 4. Gorham, S.L. and M.A. Ottinger (1986). Sertoli cell tumors in Japanese quail. J. Avian Diseases. 30:337-339.

247

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NEGATIVE ALLOPREGNANOLONE EFFECTS ON LEARNING IN THE MORRIS WATERMAZE TES T CAN BE INHIBITED Turkmen S., Birzniece V., Johansson I.M. and Backstrom T. Department of Clinical Science, Obstetrics and Gynecology. Umeå University Hospital, SE-901 85 Umeå, Sweden. [email protected] The progesterone metabolite 3alfa-OH-5alfa-pregnane-20-one (allopregnanolone) has an inhibitory effect on neuronal functions. This effect is mediated by GABAA receptor activation and exhibits a wide range of behavioural activities including anticonvulsant, anxiolytic and sedative-hypnotic effects [3,4]. There are some suggestions that allopregnanolone can impair learning and memory [2]. And, we have demonstrated that acute allopregnanolone inhibits spatial learning in the Morris water maze [1]. The purpose of this study was to evaluate the possibility to block the negative allopregnanolone effects on learning with the substance (UC1011) in the Morris Water maze test. We used adult male Wistar rats (n=46). The rats where injected (i.v) daily with either UC1011 20mg/kg (n=14), allopregnanolone 2mg/kg (n=14), allopregnanolone+UC1011 2:20 mg/kg (n=14), or vehicle 10% 2-hydroxypropyl-beta-Cyclodextrin (n=4). Animals were given four swim trials daily with maximal swimming time 120 second for each trial. The rats did swim 8 minute after the injection for 6 days and at day seven they were decapitated 8 minute after the injection. Trunk blood and brain were collected for later analysis. The performance of rats was monitored with an overhead video camera connected to an image analyser (HVS Image, Hampton, U.K.) and analysed by the water-maze software HVS Water 2020. For statistical analysis of data, we used analysis of variance (ANOVA) with repeated measures and the LSD (Least Significant Difference) post hoc test. As shown earlier allopregnanolone injection inhibits spatial learning and memory. In the watermaze, the latency to find the platform was in this group still above 80 sec. after 6 days with practise. The rats injected with the mixture of UC1011 + allopregnanolone (concentration ratio 10/1) had a lower latency time (p<0.05 day 4-6) compared with the allopregnanolone-injected group. Thus, these rats where able to learn the task. The group that only received UC1011 learned to find the platform as quickly as the control group (vehicle) and had a shorter time to finding the hidden platform then allopregnanoloneinjected rats (p<0.05 day 3-6). There was no significant difference in speed between the four groups. The novel substance UC1011 can inhibit negative allopregnanolone effects on spatial learning and memory impairment.

248

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003 latency

speed 35

120

20 β 30 Allopregnanolone Allo:20β s 25 Vehicle / 20 m c 15

100

c e s

80 60 40

10

20

5 0

0 0

1

2

3

Day

4

5

6

0

1

2

3

4

5

6

Day

Acknowledgment: This work was supported by an EU Regional fund Objective 1 grant. Reference List 1. Johansson IM. et.al. (2002) Allopregnanolone inhibits learning in the Morris watermaze. Brain Res.May 3. 934(2) 125:31 2. Ladurelle N., Baulieu EE. et.al (2000) Prolonged intracerebroventricular infusion of neurosteroids affects cognitive performances in the mouse. Brain Res.Mar10; 858(2):371-9 3. Paul SM, Purdy RH (1992) Neuroactive steroids. FASEB J 6:2311- 2322 4. Rupprecht and F. Holsboer (1999)Neuroactive steroids. TINS vol.22,no.9

249

Posters’ Exhibition: Action of Environmental Estrogens on Behaviorally Relevant Neural Circuits •

Alò R., Madeo M., Giusi G., Facciolo R.M., Carelli A. and Canonaco M. (Arcavacata di Rende (CS), Italy, EU) Influences of environmental chemical compounds occur preferentially on certain somatostatin receptor subtypes in some brain regions of teleost fishes.

•

Capone F., Branchi I., Costa L.G. and Alleva E. (Roma, ITALY, EU) Developmental exposure to a polybrominated diphenyl ether (PBDE 99) alters thyroid homeostasis and induces neurobehavioural effects in CD-1 mice

•

Colciago A., Pravettoni A., Negri-Cesi P. and Celotti F. (Milano, Italy, EU) A transplacental exposure to xenobiotics affects dimorphic differentiation of rat brain during embryogenesis

•

Cottone E., Guastalla A., Mosconi G., Polzonetti-Magni A.M., Kikuyama S. and Franzoni M.F. (Torino, Italy, EU) Effects of the xenoestrogen 4-nonylphenol on some neuroendocrine mechanisms of the urodele amphibian Triturus carnifex

•

Della Seta D., Minder I. , Dessì-Fulgheri F. and Farabollini F. (Siena, Italy, EU) Effects of exposure to bisphenol a on maternal behavior, in rats

•

Facciolo R.M., Madeo M., Alò R, Canonaco M. and Dessì-Fulgheri F. (Arcavacata di Rende (CS), Italy, EU) Neurobiological activities of the environmental estrogen bisphenol a in limbic regions of the rat occur via its interaction with the SRIF receptor subtype SST3

•

Gioiosa L., Fissore E. and Palanza P. (Parma, Italy, EU) Effect of perinatal exposure to environmental estrogens on learning ability and working memory in adult mice

•

Laviola G., Gioiosa L., Adriani W., Palanza P. (Roma, Italy, EU) D-Amphetaminerelated reinforcing effects are reduced in mice exposed prenatally to estrogenic endocrine disruptors

•

Quinn M.J.Jr., Summitt C.L. and Ottinger M.A. (Baltimore, Maryland, USA) Effects of p,p’-DDE on sexual maturation and copulatory behavior in the Japanese quail (Coturnix japonica)

•

Razzoli M., Massardi B., valsecchi P.and Palanza P. (Parma, Italy, EU) Chronic exposure to xenoestrogens interfere with pair-bonding and maternal behavior in female Mongolian gerbils.

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

INFLUENCES OF ENVIRONMENTAL CHEMICAL COMPOUNDS OCCUR PREFERENTIALLY ON CERTAIN SOMATOSTATIN RECEPTOR SUBTYPES IN SOME BRAIN REGIONS OF TELEOST FISHES Alò R., Madeo M., Giusi G., Facciolo R.M., Carelli A. and Canonaco M. Comparative Neuroanatomy Lab., Ecology Dept., University of Calabria, Ponte Pietro Bucci 87030 Arcavacata di Rende (CS), Italy. fax number: +39 984 492986 E-mail: [email protected]; Numerous studies have shown that estrogen activities, at the brain level, are mediated either genomically through binding of specific intracellular receptors located within target cells or through local membrane type of mechanisms at the cell surface. A number of environmental agents such as the environmental estrogen bisphenol A (BPA) has been shown to interact at the estrogen receptor level [5]. As a result, public and scientific interests regarding estrogen-like functions have focused their attention on both the toxic and biologically beneficial actions of these classes of xenoestrogens. In the case of BPA, this environmental estrogen is widely used in the manufacture of polycarbonate plastics, epoxy resins for lining food cans, dental sealants and as a stablizing agent in plastics such as polyvinyl chloride. These man-made chemicals, due to their leaching from the numerous reservoirs, can enter the body by ingestion or adsorption, and mimic the actions of estrogens. Due to the accumulation of these chemical compounds in marine sites plus to the commercial food resource supplied by this environment niche, attention has been directed to biological effects of BPA on marine fauna of southern Tyrrhenian sea and in particular on Coris julis, a teleost species important from an ecological point of view on the account of its “cleaning ability” of the sea floor. Despite the 1000 fold less potent activity of BPA with respect to that of estradiol, it is still able to mimic its biological actions such as vaginal cornification and growth and differentiation of the mammary gland as well as inducing tumour activity [3]. In this context, the growth hormone system and, above all, the neuropeptide somatostatin (SRIF) play a crucial role on neurosecretory functions at the encephalic level, especially in relation to tissue content, which is still an unresolved facet of BPA effects. This neuropeptide which exists in two biologically active forms i.e. the cyclic tetradecapeptide SRIF-14 and the N-terminally elongated SRIF-28, is widely distributed in the mammalian [4] brain plus in that of nonmammalian species including the fish [1]. To date, five distinct receptor subtypes (sst1-5) have been identified as a family of G protein-coupled receptors. Of these subtypes, sst2 and sst5 are distributed in the various brain regions and are considered to be, from a functional point of view, the more important subtypes, as revealed by the successful neurobiological functions especially in the presence of estrogens [8]. On the basis of the specific interactions between sex steroids and SRIF receptor subtypes due to their colocalization in some neuronal regions, we investigated the effects of BPA (80 µg/ml oil) on sst receptor levels in discrete brain regions of the Coris julis maintained in plexiglass tanks under the following laboratory conditions (temp. = 18-23°C; salinity = 37°/oo; pH = 8.44; density = 1.027). From the binding changes of [125I] Tyr1-SRIF14 (25pM) in the presence of different concentrations (100nM-100pM) of the highly specific nonpeptide agonists (L-779,976 and 251

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

L-817,818), selective for sst2 and sst5 [6], respectively, it was possible to show that high levels of the two subtypes were obtained in the presence of BPA. Fishes that were fed with BPA provided altered expession levels (p< 0.01) of sst2, especially for the high affinity type of recedptor in hypothalamic regions such as suprachiasmatic nucleus and the preoptic area. On the hand, low expression levels of the sst5 subtype, were mostly registered in extrahypothalamic regions. The differentiated variations of sst2 and sst 5 activities in hypothalamic regions under the influence of this xenoestrogen suggest that, like in mammals, this neuronal receptor system undergoes an upgrading of the hypophysiotropic neurohormonal functions, very probably during the responsive events of the gonadal-hypothalamic axis [2]. Moreover, variations of the two SRIF subtypes in other thalamic areas and in telencephalic regions also suggests probable neurotransmitter and/or neuromodulator roles of this receptor system in these encephalic sites noted for their control of locomotor and olfactive-dependent behaviors [7]. References List [1] R. Cardenas, X. Lin, M. Chavez, C. Aramburo, R.E. Peter, Characterization and distribution of somatostatin binding sites in golfish brain, Gen. Comp. Endocrinol. 117 (2000) 117-128. [2] Z. Csaba, P. Dournaud, Cellular biology of somatostatin receptors, Neuropeptides 35 (2001) 1-23. [3] C. Gupta, Reproductive malformation of the male offspring following maternal exposure to estrogenic chemicals, Proc. Soc. Exp. Biol. 224 (2000) 61-68. [4] J. H. Ives, D.L. Drewery, C.L. Thompson, Expression of the five somatostatin receptor (sst1-5) subtypes in rat pituitary somatotriphes : quantitative analysis by double-label immunofluorescence confocal microscopy, Endocrinol. 138 (2002) 4473-4476. [5] N. Olea, R. Pulgar, P. Perez, F. Olea-Serrano, A. Rivas, A. Novillo-Fertrell, V. Pedrazza, A.M. Soto, C. Sonnenschein, Estrogenicity of resin- based composites and sealants used in dentistry, Environ. Health Perpect. 104 (1996) 298-305. [6] S.P Rohrer, E.T. Birzin, R.T. Mosley, S.C. Berk, S.M. Hutchins, D.M. Shen, Y. Xiong, E.C. Hayes, R.M. Parmar, F. Foor, S.W. Mitra, S.J. Degrado, M. Shu, J.M. Klopp, S.J. Cai, A. Blake, W.W. Chan, A. Pasternak, L. Yang, A.A. Patchett, R.G. Smith, K.T. Chapman, J.M. Schaeffer, Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry, Science 28 (1998) 737-740. [7] C. Viollet, C. Vaillend, C. Videau, M.T: Bluette-Pajot, A. Ungerer, A. L’Héritier, C. Kopp, B. Potier, J.M. Billard, J. Schaeffer, R.G. Smith, S.P. Rohrer, H. Wilkinson, H. Zheng, J. Epelbaum, Involvement of sst2 somatostatin receptor in locomotr, esploratory activity an emotional reactivity in mice, Eur. J. Neurosci. 12 (2000) 3761-3770. [8] H.A. Visser-Wisselaar, C.J.C. Van Uffelen, P.M. Van Koetsveld, EG.R. Lichtenauer-Kaligis, A.M. Waaijers, P. Uitterlinder, D.M. Mooy, S.W. Lamberts, L.J. Hofland, 17-b-Estradio-dependent regulation of somatostatin receptor subtype expression in the 7315b prolactin secreting rat pituitary tumor in vitro andin vivo, Endocrinology 138 (1997) 1180-1189.

252

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

DEVELOPMENTAL EXPOSURE TO A POLYBROMINATED DIPHENYL ETHER (PBDE 99) ALTERS THYROID HOMEOSTASIS AND INDUCES NEUROBEHAVIOURAL EFFECTS IN CD-1 MICE Capone F.1,2, Branchi I.2, Costa L.G. 1,3 and Alleva E. 2 1

University of Roma "La Sapienza", Department of Pharmacology of Natural Substances and General Physiology, Roma, ITALY; 2Istituto Superiore di Sanità, Section of Behavioural Phathophysiology, Lab. of Fisiopatologia, Roma, ITALY; [email protected] 3 Department of Environmental Health, University of Washington, Seattle, WA, USA. Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in numerous consumer products. Because of their lipophilicity and persistence, PBDEs have become ubiquitous environmental contaminants, as polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT). In contrast to the well documented neurotoxicity of PCBs, almost no information is available on the neurotoxicity of PBDEs. Their levels are lower than those detected for PCBs and DDT, but over the past 25 years, while other organohalogens have decreased in concentration, PBDEs levels increased in various wildlife species, from marine freshwater and terrestrial environment. In humans PBDEs have been detected in blood, adipose tissue and breast milk. Of particular concern are the reported high levels of PBDE in human breast milk, as almost no information is available on their potentially noxious effects on developing mammals. In a first study, we investigated the effects of perinatal PBDE exposure on mouse neurobehavioural development. 2,2',4,4',5-pentabromodiphenylether (PBDE 99; 0.6, 6, and 30 mg/kg/day) was administered daily to CD-1 Swiss female by gavage from gestational day (GD) 6 to postnatal day (PND) 21. Aroclor 1254 (A1254; 6 mg/kg per day) a PCB mixture was administered following the same schedule and served as a positive control. On PND 11, the homing test revealed a trend for treated animals, particularly the A1254 group, to be more active than controls. This activity level alteration was strongly increased on PNDs 34 and 60 in an open-field arena. On PND 60, treated mice showed also an altered thigmotaxis, spending more time in the centre of the arena than controls. At adulthood, A1254 mice were still hyperactive whereas PBDE 99 groups tended to be slightly hypoactive. These findings suggested that perinatal PBDE 99 exposure disrupt specific behavioural endpoints in developing mice and that its effects are not always similar to those of A1254. In a second study, PBDE 99 (18 or 36 mg/kg per day) or A1254 (10 mg/kg per day) were administered daily to lactating CD-1 Swiss female mice from PND 1 to PND 21 by a different ooral route. This procedure, in which dams spontaneously drink corn oil containing the test compound, was utilised in order to minimise the invasive procedure of gavage administration, that may be stressful to animals and a potential confounder. On PND 26, choline acetyltransferase (ChAT) activity in three areas of the brain (hippocampus, striatum and cortex) and serum levels of total and free thyroxine were measured. Spontaneous behaviour and learning and memory performances were evaluated on PNDs 24 and 90 and on PNDs 90 and 120, respectively.

253

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

A significant decrease of circulating thyroxine levels (total and free) and a tendency to a reduction in hippocampal ChAT activity were found in both PBDE 99- and A1254treated mice, while no effect on ChAT activity was observed in striatum and cortex. No treatment-related effects were found in the behavioural testing. In particular, no behavioural differences in mouse activity in an open-field arena and in mouse performances in passive avoidance and in Morris water-maze tests were observed. These findings suggest that postnatal PBDE 99 exposure altered thyroid homeostasis and ChAT during development, but did not seem to alter motor development or cognitive behaviour. The somewhat discordant results obtained in these two studies may be due to the different developmental phases of e (pre- and postnatal vs. postnatal only), to different stress levels induced by the two administration procedures or by a combination of these two factors. Ongoing studies are addressing these issues in order to draw firmer conclusions on the potential developmental neurotoxicity of PBDE 99 in mice.

Study supported by EU, Contract N°QLK4-CT-1999-01562 (L.G.C.) and by ISS Project N°1103/RI2001/2002 Neurotrophins and neurobehavioural plasticity: animal models (E.A.)

254

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

A TRANSPLACENTAL EXPOSURE TO XENOBIOTICS AFFECTS DIMORPHIC DIFFERENTIATION OF RAT BRAIN DURING EMBRYOGENESIS Colciago A., Pravettoni A., Negri-Cesi P. and Celotti F. Dept .of Endocrinology, via Balzaretti 9, 20133 Milano, Italy; [email protected]; fax 39.2.503.18204 It is known that, during embryogenesis, estradiol arising from in situ conversion of testosterone (T) via the hypothalamic CYP-450 Aromatase (Aro), is able to influence brain masculinization in rats. Although the hypothalamic Aro peaks in a very limited period during late gestation, no data are presently available about the possible dimorphism of this peculiar expression pattern. In the same brain area, T is also activated by its 5alphareduction into 5alpha-dihydrotestosterone (DHT) via the two isoforms of 5alphaReductase (5alpha-R, type 1 and type 2); while 5alpha-R type 1 is almost a constitutive enzyme, the role of the type 2 isoform in rat brain sexual differentiation, as well as its possible dimorphism, are presently unknown. It has been described that polychlorinated biphenyls (PCBs) have the ability to induce several forms of liver microsomal CYP-450s and that a transplacental exposure to PCBs in laboratory animals is related to developmental neurotoxicity resulting in behavioral changes during adulthood. Among the widely distributed PCBs, Aroclor 1254 represents a commercial mixture of many industrial pollutants, closely related to those really present in the ecosystem. The effects of Aroclor 1254 on Aro and 5alpha-R profiles during rat embryogenesis have never been completely elucidated so far. We identified two aims: 1) to evaluate the dimorphic profile of hypothalamic metabolizing enzymes during ontogenesis and 2) to investigate the possible influence of Aroclor 1254 on these parameters. 1) rat embryos from gestational day (GD) 16 to GD20 were sex-screened for the presence of SRY gene. Since both Aro and 5alpha-R type 2 are present only in neurons, a semi-quantitative evaluation of their expression was obtained by RT-PCR co-amplification of each mRNAs with the mRNA of a specific neuronal marker (MAP2c, a cytoskeletal protein highly expressed in the embryonic/perinatal neurons). A clear-cut dimorphism in the hypothalamic Aro was observed, with a peak of expression at GD18/20 only in male embryos. 5alpha-R type 2 expression showed a progressive increase during the gestational period considered without any clear dimorphism. In order to evaluate the possible involvement of post-trascriptional mechanisms in determining the differential pattern of enzime expression, mRNA stability of Aro and 5alpha-R type 2 trascripts was analyzed studying their polyadenilation extent with a modified RT-PCR allowing for the poly(A) tail to be preserved. Preliminary results suggest that Aro poly(A) tail has different extent during gestation and between male and female, being costantly longer in males; 5alpha-R type 2 poly(A) tail seems to be quite costant among the GDs considered without any differences between sexes. 2) Aroclor 1254 was given by gavage (25 mg/kg/die) to pregnant rats from GD15 to GD19. The effect of a prenatal exposure to xenobiotics has been evaluated in the hypothalami of male GD20 embryos, since, as previously described, T metabolizing enzymes seem to be more critical for male embryos than for female: Aro and 5alpha-R type

255

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

2 expression and mRNA stability were then evaluated. The results obtained indicate that Aroclor 1254 is able to positively affect both the enzyme expression and the poliadenilation extent of both the enzymes considered. From the results obtained so far, we can conclude that 1) a clear-cut dimorphism in the expression and mRNA stability of T metabolizing enzymes has been demostrated only for Aro, suggesting that the mechanisms governing brain sex-differentiation are malespecific and take place both at transcriptional and at post-transcriptional level. 2) the developing hypothalamus of male embryos might be sensitive to prenatal PCB exposure: these compounds, modulating Aro and 5alpha-R availability could interfere with the mechanisms controlling the sexual differentiation of developing brain.

Supported by MURST giovani.

256

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EFFECTS OF THE XENOESTROGEN 4-NONYLPHENOL NEUROENDOCRINE MECHANISMS OF THE URODELE TRITURUS CARNIFEX

ON SOME AMPHIBIAN

Cottone E.1, Guastalla A.1, Mosconi G.2, Polzonetti-Magni A.M.2, Kikuyama S. 3 and Franzoni M.F.1 1

Dipartimento di Biologia Animale e dell’Uomo, Università degli Studi di Torino, via Accademia Albertina 13, 10123 Torino, Italy. E-mail: [email protected] Fax: +39 011 6704692 2 Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Università di Camerino, Camerino (MC), Italy 3 Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169-8050, Japan Recent reports have shown that different chemicals discharged into the environment are able to mimic hormonal effects on reproduction in vertebrates, man included. Due to their life style, amphibians are particularly sensitive to water pollutants and can be taken as models to investigate the effects of these environmental endocrine disruptors. In the present study, the model used was a wildlife population of Triturus carnifex (urodele amphibian) adult males. Animals were reared 4 weeks in water containing different concentrations of 4-nonylphenol (4NP), a breakdown product of detergents widely used in agricultural and industrial applications, and found as a contaminant in the aquatic environment and also in food. By analysing the concentration of plasma vitellogenin (VTG), a widely used biomarker of feminization by xenoestrogens, we found that VTG, undetectable in the control males, increased in a dose-dependent manner in males treated with 10-10 M, 10-9 M , 10-8 M and 10-7 M 4NP. Since in male frogs, kept under comparable experimental conditions, modifications in plasma gonadotropins and prolactin have been detected [4], in the present research we have investigated possible perturbations in the pituitary lactotrophs of the treated newts. In the pituitary distal lobe of these animals we observed an increase in the intensity of the immunoreactivity (IR) for PRL, associated to a wider extension of the PRLimmunolabelled area. Moreover, PRL mRNA level was also increased in the treated animals in respect to controls. It is generally recognized that aromatization to estradiol is an essential step for the full expression of testosterone action in the CNS of vertebrates and brain aromatase is concentrated in mammalian limbic regions implicated in steroid control of reproduction and sex behaviour [3, 5]. Recent immunohistochemical observations have shown that aromatase-containing neurons are present in the prosencephalon of the crested newt (Cottone et al., personal communication). The immunopositive neurons are in fact distributed in both basal telencephalon, in the amygdala and septum in particular, and preoptic area, retrochiasmatic and tuberal hypothalamus, areas well known as deeply involved in the control of amphibian reproduction. So, in the present research we have also investigated the occurrence of modifications in the hypothalamic aromatase-IR, possibly related to xenoestrogen treatment. Anyway our preliminary observations do not indicate

257

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

variations in number and distribution of aromatase-containing neurons. Accordingly, the aromatase mRNA expression do not seem to be modified by 4NP treatment. Since it is known that sex steroid receptors are widely distributed in the brain of Vertebrates, amphibians included [1, 2], the possibility that 4-NP treatment might interact with central estrogen receptor expression and distribution was also investigated. Financial support: MIUR ex-60% to MFF

Reference List [1] G.A. Davis, F.L. Moore. Neuroanatomical distribution of androgen and estrogen receptorimmunoreactive cells in the brain of the male roughskin newt, J Comp Neurol. 372 (1996) 294-308. [2] G. Guerriero, C.E. Roselli, M. Paolucci, V.Botte, G. Ciarcia. Estrogen receptors and aromatase activity in the hypothalamus of the female frog, Rana esculenta. Fluctuations throughout thereproductive cycle, Brain Res. 880 (2000) 92-101. [3] B.S. McEwen. Neural gonadal steroid actions, Science. 211 (1981) 1303-11. [4] G. Mosconi, O. Carnevali, M.F. Franzoni, E. Cottone, I. Lutz, W. Kloas, K. Yamamoto, S. Kikuyama, A.M. Polzonetti-Magni. Environmental estrogens and reproductive biology in amphibians, Gen Comp Endocrinol. 126 (2002) 125-9. [5] C.E. Roselli, L.E. Horton, J.A. Resko. Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system, Endocrinology. 117 (1985) 2471-7.

258

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EFFECTS OF EXPOSURE TO BISPHENOL A ON MATERNAL BEHAVIOR, IN RATS Della Seta D.1, Minder I. 2, Dessì-Fulgheri F.2 and Farabollini F.1 1

Dipartimento di Fisiologia, Sezione di Neuroscienze e Fisiologia Applicata, Università degli Studi di Siena, Nuovi Istituti Biologici, via Aldo Moro, 53100, Siena, fax: 0577/234037, e-mail: [email protected]; 2 Dipartimento di Biologia Animale e Genetica, Università di Firenze. In mammals, endogenous estrogens are crucial for sexual differentiation during the perinatal period and, in adulthood, for modulating many neuroendocrine and behavioral functions involved in reproduction. During pregnancy and lactation, the estrogenic environment directly affects maternal behavior. In rats, gestation and the first phases of lactation are directed from estradiol, afterwards suckling by pups becomes more important. Bisphenol-A (BPA), is an environmental estrogenic-like compound which proved to affect fertility and the reproductive success. Its estrogenic activity, depends on its capacity to interact with estrogen receptors in brain areas involved in the activation of reproductive behavior, including maternal behavior. Our previous studies have shown a decrease in estrogen receptor-alfa cells in the arcuate nucleus of lactating female rats exposed to BPA. The aim of the present study was to test the hypothesis that exposure to BPA (0.040 mg/kg/die, orally) of adult female rats from mating to weaning of the pups, could alter the processes affecting maternal behavior. An appropriate methodology was applied, to reveal differences in the behavior of dams directed to male and female pups. Before testing, each litter (consisting in 4 males and 4 females) was removed from the mother’s cage for 30’, thereafter four pups of the same sex were reintroduced for the duration of the test (30 min.); the day after, the same procedure was applied, but the dam was exposed to pups of the other sex. Tests took place on post-natal days (PND) 3,4 and were repeated on PND 8-9. Statistical analysis of control mothers (treated with the vehicle peanuts oil) confirmed previous studies on maternal behavior, revealing differences in ano-genital licking, directed more to male than female pups. Moreover, our study revealed a different pattern of maternal behavior in the two periods of observation. Exposure of mothers to BPA modified their behavior towards pups, reducing specific components of maternal behavior, both active (directed to pups) and passive (postures on nest), irrespective of the sex of pups and the period of observation. On the whole, this experiment shows that maternal behavior is affected by a prolonged exposure to BPA during pregnancy and lactation. It is not unlike that these modifications in maternal care could be the basis of alterations in socio-sexual behavior of the adult offspring of treated mothers, observed in previous experiments.

259

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

NEUROBIOLOGICAL ACTIVITIES OF THE ENVIRONMENTAL ESTROGEN BISPHENOL A IN LIMBIC REGIONS OF THE RAT OCCUR VIA ITS INTERACTION WITH THE SRIF RECEPTOR SUBTYPE SST3 Facciolo R.M.1, Madeo M.1, Alò R.1, Canonaco M.1 and Dessì-Fulgheri F.2 1

Comparative Neuroanatomy Laboratory of the Ecology Department., University of Calabria, Ponte Pietro Bucci, 87030 Arcavacata di Rende, Cosenza-ITALY-E-mail: [email protected], Fax number: +39 984 492986; 2Animal Biology Department, University of Florence, Florence-ITALY. Recently, scientific interests dealing with estrogenic functions have mostly been aroused by both the toxic and beneficial effects of some environmental chemicals, known as xenoestrogens. One of these environmental estrogens is the industrial phenolic chemical bisphenol A (BPA) which is widely used in the manifacture of polycarbonate plastics [2]. Earlier studies demonstrated its potentially toxic steroidal actions as displayed by some reproductive malformations of offsprings after maternal exposure to this xenoestrogen [6]. Consequently, it appears that not only estrogens but also xenoestrogens derivatives (despite their 1000-fold less potent activity with respect to that of estradiol) have important and various pleiotropic actions in both reproductive and non reproductive tissues. On the basis of the above considerations plus on the key role played by the somatostatin (SRIF) receptor system in neurosecretory function at the brain level, it is the aim of the present investigation to evaluate the neurobiological activities of the environmental estrogen BPA on one of the biologically more active SRIF receptor subtype (sst3) in the limbic system of the rat. At the moment, five different SRIF receptor subtypes have been cloned and all possess a heptahelical architecture suggesting that they belong to the G-protein coupled receptor family [3]. From recent studies, it has been shown that sst3 is widely distributed in numerous brain regions including those of the limbic system and, interestingly enough, it appears relatively early as well as being relatively stable throughout development [7]. In addition, studies have begun to show an interaction of the SRIF receptor complex with specific subunits of other neurotransmitter systems, such as the GABA type A [8] that may be considered an important factor operating at the brain level after exposure to BPA. With this intention, after treating pregnat female Sprague-Dawley rats with two doses (400 µg/kg/day, BPA400; 40 µg/kg/day, BPA40) of BPA, the transcriptional activity of sst 3 mRNA was evaluated in some limbic regions of the offspring, at the age of postnatal days 7 (PND 7) and of adults (55 days). These two doses were chosen on the basis of their capability to promote evident morphometrical changes in the offsprings as well as to represent the concentrations that may be released when resins for lining food cans and dental sealants come into contact with food products. Brains of both treatment groups (BPA400 and BPA40) anf of controls (OIL) were quickly removed for in situ hybridization and competition autoradiography. In a preliminary phase the binding changes of [125I]Tyr1-SRIF14 (25pM), in the presence of different concentrations (100nM-100pM) of the highly specific nonpeptide agonists (L-779,976 and L-796,778), selective for sst2 and sst3, respectively, permitted us to demonstrate high levels of the sst 3 in a similar fashion to those of sst2. In view of these results, comparison of the effects of the two BPA doses consented us to identify a

260

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

heterogeneous expression pattern of the mRNA encoding for sst3, especially in the case of the higher dose which has also been shown to be the most effective one for the sst2 receptor subtype [5]. The variations of the sst 3 expression levels after treatment with the two BPA doses proved to be primarily of a mixed nature in hypothalamic and extra-hypothalamic regions especially in adults with respect to PND 7 rats. Indeed, some adult limbic regions such as the frontoparietal cortex lamina V and the stratum lacunosum CA1 layer of the hippocampus, displayed significantly (p < 0.001) diminished transcriptional levels in offsprings which received the higher BPA dose. On the contrary, enhanced (p < 0.01) and highly enhanced mRNA densities were reported for the arcuate nucleus of the hypothalamus and the ventromedial hypothalamic nucleus (VMN), respectively, of adult BPA 400 treatment groups. Subsequently, different concentrations (5 nM-500µM) of some selective agonists such as the imidazopyridine zolpidem and the imidazobenzodiazepine RY 080 were used to evaluate the role of α GABA type A receptor isoforms on the trascriptional activities of sst3 . From this part, it was possible to observe an even greater potentiating effect of BPA on sst 3 expression activities, especially in some limbic regions of PND 7 rats. On the other hand, the fact that zolpidem is not able to modify sst3 mRNA levels in the VMN, during such a biological age, could be due to the GABA type A receptor not being assembled with α 1 isoform in this hypothalamic station [4]. Taken together, these results provide, for the first time, direct evidence of BPA being capable of alterating the neurobiological activities, at an early developmental phase, via the regulation of mRNA levels of the sst 3 receptor subtype. It is obvious that we are still at the beginning but knowledge of the estrogenic-like activities might bring us closer to understand molecular and neural pathways involved not only in sociosexual behaviors [1] but also in cognitive and emotional processes.

References List [1] A.M. Aloisi, D. Della Seta, C. Rendo, I. Ceccarelli, A. Scaramuzzino, F. Farabollini, Exposure to estrogenic pollutant bisphenol A affects pain behavior induced by subcutaneous formalin injection in male and female rats, Bran Res. 937 (2002) 1-7. [2] J.E.. Biles, K.D. White, T.P.. McNeal, T.H. Begley, Determination of Diglycidyl Ether of Bisphenol A and its derivatives in canned foods, J. Agric. Food Chem. 47 (1999) 1965-1969. [3] Z. Csaba, P. Dournaud, Cellular biology of somatostatin receptors, Neuropeptides 35 (2001) 1-23. [4] A.M. Davies, M.M. McCarthy, Development increas in 3H-muscimol binding to the g-aminobutyric acidA receptor in hypothalamic and limbic areas of the rat: why is the ventromedial nucleus of the hypothalamus an exception? Neurosci. Lett. 288 (2000) 223-227. [5] R.M. Facciolo R. Alò, M. Madeo, M. Canonaco, F. Dessì-Fulgheri Early cerebral activities of the environmental estrogen Bisphenol A appear to act via the somatostatin receptor subtype sst2, 110 (2002) 397-402. [6] C. Gupta, Reproductive malformation of the male offspring following maternal exposure to estrogenic chemicals, Proc. Soc. Exp. Biol. 224 (2000) 61-68. [7] V.S. Thoss, J. Perez, D. Duc, D. Hoyer, Embryonic and postnatal mRNA distribution of five somatostatin receptor subtypes in the rat brain, Neuropharmacol. 34 (1995) 1673-1688. [8] M. Vincens, F. Mauvais-Jarvis, S. Behar, A novel recognition site for somatostatin-14 on the GABAA receptor complex, Eur. J. Pharmacol. 344 (1998) R1-R2.

261

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

EFFECT OF PERINATAL EXPOSURE TO ENVIRONMENTAL ESTROGENS ON LEARNING ABILITY AND WORKING MEMORY IN ADULT MICE Gioiosa L., Fissore E. and Palanza P. Dipartimento di Biologia Evolutiva e Funzionale, Universita’ degli Studi di Parma viale delle Scienze 11/a, 43100 – Parma, Italia. [email protected], fax 0521-905657 Early ontogeny is considered a markedly plastic and crucial stage in the organization of CNS structures. Early events, such as small perturbations of sensory experiences or hormonal milieu, as well as exposure to psychoactive agents, have been found to have a major role in modulating developmental trajectories. In this context, particular attention should be addressed to possible effects of perinatal exposure to a large class of man-made compounds present in the environment named “endocrine disrupters”, which show the characteristic to interfere with the endocrine system. At this time, the best characterized endocrine-disrupting chemicals are those able to bind to estrogen receptors in cells, such as the naturally occurring phytoestrogens (e.g., soybeans), herbicides and pesticides (e.g., o,p'-DDT, methoxychlor and chlordane), products associated with plastics (bisphenol A and nonylphenol), pharmaceuticals (ethinyl estradiol, diethylstilbestrol (DES)), or industrial chemicals ( PCBs) [12]. While previous studies on endocrine disrupters have focused on effects on male fertility and gross reproductive disturbances, several reports have recently shown wider behavioral effects. In particular, from epidemiological data and from studies on laboratory animals, common effects such as attention deficits, hyperactivity, altered behavioral response to novelty and learning and memory deficits, have been reported after prenatal exposure to low doses of estrogenic endocrine disruptors (such as PCBs, methoxychlor, bisphenol A) [1,2,3,4,5,6,7,9,10,11]. The present study aimed to investigate the timing and effects of controlled maternal exposure, during critical periods of development (fetal and/or neonatal) to a low, environmentally relevant dose of the estrogenic chemicals, bisphenol A (BPA) and methoxychlor (MXC), on cognitive responses in the House mouse as animal model. Bisphenol A is an essential component used in the manufacture of resins and polycarbonate and it is reported to be released in food cans during autoclaving; it is also a component of plastic used in dental fillings. Methoxychlor is a pesticide characterized by both weak androgenic and estrogenic actions. Pregnant females were fed daily doses of corn oil with or without 10 microg/Kg body weight BPA or 20 microg/Kg bw MXC from gestation day 11 post-partum day 8 (16 days of treatment). Pregnant females were trained to drink corn oil from a modified syringe (syringe without needle). This procedure allows not to stress the animals during gestation. In order to discriminate prenatal (via placenta) and postnatal (via lactation) BPA exposure effects, at PND 1 we cross-fostered part of the control and BPA-treated mothers. We thus obtained 3 experimental categories in relation to the timing of exposure of the offspring: prenatal, postnatal and perinatal (both pre- and post-natal) exposure to BPA. As adults (70-80 days old) the male and female offspring were tested in a spatial learning task to assess learning ability and working memory. Mice were subjected to six testing sessions in a small open-field. During session 1, each mouse was placed in a empty open field to became familiar with the apparatus, and to record a baseline level of locomotor activity. During session 2-3, three objects were placed in the

262

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

open field. In the following sessions changes in the environment were introduced by either changing the distribution of some objects or by replacing a familiar object with a totally new one. Behavior was recorded before and during the test for measuring lomotor activity, attention and memory skills. Results shows that control mice showed sexual differences in the basal amount of locomotion in the OPF, with females being more active than males, and in the habituation process, with males spending more time in exploring the objects than females in both the familiarization sessions. Pre- and perinatal exposure to BPA decreased such differences between males and females and affected the response to non-spatial novelty (change of object). BPA-exposed females spent more time exploring the replaced object than the familiar ones. Perinatal exposure to MXC affected the response to the spatial change, with exposed females being more reactive to the spatial change than males, whereas controls did not show sexual differences. These results suggest that perinatal exposure to BPA and MXC affected sexual dimorphic responses to spatial novelty and spatial change. However, the timing of exposure (pre- or/and post-natal) and the type of chemical appear to differentially affect these responses, though the cross-fostering procedures can be a confounding factor.

Reference List 1. Farabollini F, Porrini S, Dessi-Fulgheri F. Perinatal exposure to the estrogenic pollutant bisphenol A affects behavior in male and female rats. Pharmacol Biochem Behav. 64 (1999) 687-94. 2. Jacobson JL, Jacobson SW. Intellectual impairment in children exposed to polychlorinated biphenyls in utero. N Engl J Med. 335 (1996) 783-9. 3. Palanza P, Morellini F, Parmigiani S, vom Saal FS. Prenatal exposure to endocrine disrupting chemicals: effects on behavioral development. Neurosci Biobehav Rev. 23 (1999) 1011-27. 4. Palanza P, Parmigiani S, Liu H, vom Saal FS. Prenatal exposure to low doses of the estrogenic chemicals diethylstilbestrol and o,p'-DDT alters aggressive behavior of male and female house mice. Pharmacol Biochem Behav. 64 (1999) 665-72. 5. Palanza P, Parmigiani S, vom Saal FS. Effects of prenatal exposure to low doses of diethylstilbestrol, o,p'DDT, and methoxychlor on postnatal growth and neurobehavioral development in male and female mice. Horm Behav. 40 (2001) 252-65. 6. Palanza P, Morellini F, Parmigiani S, vom Saal FS. Ethological methods to study the effects of maternal exposure to estrogenic endocrine disrupters: a study with methoxychlor. Neurotoxicol Teratol. 24 (2002) 55-69. 7. Palanza PL, Howdeshell KL, Parmigiani S, vom Saal FS. Exposure to a low dose of bisphenol A during fetal life or in adulthood alters maternal behavior in mice. Environ Health Perspect. 110 (2002) 415-22. 8. Ricceri L, Usiello A, Valanzano A, Calamandrei G, Frick K, Berger-Sweeney J. Neonatal 192 IgGsaporin lesions of basal forebrain cholinergic neurons selectively impair response to spatial novelty in adult rats. Behav Neurosci. 113(1999) 1204-15. 9. Rice DC, Hayward S. Effects of postnatal exposure of monkeys to a PCB mixture on concurrent random interval-random interval and progressive ratio performance. Neurotoxicol Teratol. 21 (1999) 47-58. 10. Rice DC, Hayward S. Comparison of visual function at adulthood and during aging in monkeys exposed to lead or methylmercury. Neurotoxicology. 20 (1999) 767-84. 11. Rice DC, Hayward S. Effects of exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126) throughout gestation and lactation on behavior (concurrent random interval-random interval and progressive ratio performance) in rats. Neurotoxicol Teratol. 21 (1999) 679-87. 12. Sonnenschein C, Soto AM. An updated review of environmental estrogen and androgen mimics and antagonists. J Steroid Biochem Mol Biol. 65 (1998) 143-50.

263

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

D-AMPHETAMINE-RELATED REINFORCING EFFECTS ARE REDUCED IN MICE EXPOSED PRENATALLY TO ESTROGENIC ENDOCRINE DISRUPTORS Laviola G.*, Gioiosa L.+, Adriani W.* and Palanza P.+ +Dept. of Biologia Evolutiva e Funzionale, Universita’di Parma, Parma, Italy; [email protected] *Sect. Behavioural Pathophysiology, Lab. F.O.S., Istituto Superiore di Sanità, Roma, Italy Estrogenic Endocrine Disruptors (EECS) are hormonally active compounds that can bind to the intracellular receptors for estradiol. At level of the central nervous system, the mesolimbic and nigrostriatal dopamine pathways represent major structures underlying fundamental behavioural regulations, which have been reported to be affected by early developmental exposure to endocrine disruptors. With the aim of assessing the impact of prenatal EECs, separate groups of pregnant female CD-1 mice were allowed to drink spontaneously during gestation days 11 to 17, oil or low, environmentally relevant doses of two estrogenic compounds, methoxychlor (MXC, 20 microg/kg) or bisphenol-A (BPA, 10 microg/kg). Their adult offspring were assessed for response to conditioned place preference (CPP) produced by d-amphetamine (AMPH, 0, 1 or 2mg/kg doses). Results revealed a marked reduction in sensitivity to conditioned reinforcing efficacy of AMPH. A clear profile of AMPH-CPP was evidenced in fact only in oil-exposed females, as compared to BPA- and MXC-exposed subjects. Interestingly, some of these effects were sex-dependent and apparently no changes emerged in the male offspring. A detailed analysis of acute d-amphetamine effects also evidenced a dose-dependent increase in locomotor activity, which was more pronounced in females than in males. Such a sex difference also appeared to be significantly reduced in the group exposed to BPA or MXC during prenatal life. As a whole, these findings represent a clear evidence that the prenatal exposure to EECs interacts with some steps in the organization of the brain dopaminergic systems in the female offspring thus leading to long-term alterations in neurobehavioral function. This should be a cause of concern for public health, confirming that exposure to weak environmental estrogens in the period of sexual differentiation of the brain can influence adult behaviour.

264

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

EFFECTS OF p,p’-DDE ON SEXUAL MATURATION AND COPULATORY BEHAVIOR IN THE JAPANESE QUAIL (COTURNIX JAPONICA) Quinn M.J.Jr., Summitt C.L. and Ottinger M.A. University of Maryland, College Park, Maryland, 20742, USA; [email protected] There is growing concern about reproductive effects of endocrine active chemicals (EACs) in wildlife. Most of the research in this area has focused on the effects of estrogenic environmental chemicals, with little attention being paid to environmental chemicals that disrupt androgen mediated actions. Testosterone has strong affects on the development of the sexually dimorphic nucleus of the preoptic area (SDN-POA), the area of the brain that controls copulatory behavior, in the Japanese quail (Coturnix japonica). Testosterone also plays a key role in the limiting step of aromatase driven copulatory behavior in quail. Chemicals that block the action of testosterone could cause disruption of embryonic development of the SDN-POA, which could have potentially serious impacts on adult copulatory behavior. The objectives of our study, therefore, were to determine if in ovo exposure to p,p’-DDE [ethylene, 1, 1-dichloro-2,2-bis(p-chlorophenyl)], an antiandrogenic metabolite of the pesticide DDT, at 2 and 4 ppm on day one of incubation would affect sexual maturation and reproductive behavior in Japanese quail. Developmental endpoints included onset of puberty in females, ovarian follicle counts, male proctodeal gland size, testicular weight, and sperm motility. Behavioral endpoints measured included latency to mount, latency to successful copulation, and number of successful cloacal contacts. Onset of puberty was significantly accelerated in DDE-treated females by approximately 8 days (p < 0.05). Male copulatory behavior was significantly depressed in birds exposed to DDE, as measured by the number of attempts to mount (p < .01) and latency to successfully copulate (p < 0.05). No significant effects were seen with other measurements. These data add to the existing information concerning the harmful effects of DDE exposure on reproduction, which has mostly focused on eggshell thickness. This study shows that this anti-androgenic chemical can also have significant consequences on sexual development and behavior at very low levels of exposure.

265

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

CHRONIC EXPOSURE TO XENOESTROGENS INTERFERE WITH PAIRBONDING AND MATERNAL BEHAVIOR IN FEMALE MONGOLIAN GERBILS Razzoli M., Massardi B., Valsecchi P. and Palanza P. Dipartimento di Biologia Evolutiva e Funzionale, Università degli Studi di Parma, Parco Area delle Scienze 11/A, 43100 Parma, Italy. email: [email protected]. fax: 0039-0521905657 Sex steroid hormones concentrations show wide fluctuations in different species. Such variations may account for species-specific sensitivity to hormones, which, in turn, may relate to the number and/or distribution of hormone receptors in the central nervous system. This can explain the reported species differences in sensitivity to endocrine disrupters (EDCs), man-made compounds present in the environment and capable of interfering with the vertebrate endocrine system. It is therefore of great interest to widen the range of different species included in the research of EDCs effects on the development of the neuro-endocrine and reproductive systems. The neuro-endocrine mechanisms that regulate reproduction in all groups of vertebrates share a basic similarity. There is now considerable evidence that environmental contaminants that mimic steroid hormones can interfere with the functioning of the vertebrate HPG axis. Particularly, EDCs mimicking the actions of endogenous hormones can interfere with the neuro-endocrine substrates of behavior, exerting either organizational or activational effects. Several behavioral systems can potentially be a target of such interference: aggression, group cohesion, courtship, pair bonding, mating, and parental care can be considered. The expression of socio-sexual behaviors, as well as their neuroendocrine control, is associated with social organization or the mating system of a species. In many mammalian species social interactions between males and females are restricted to reproductive periods, whereas in socially monogamous mammals prolonged periods of social contact occur independently from the reproductive season. However, the majority of the (few) studies examining the effects of EDCs on socio-sexual behavior have been conducted in rats and mice, i.e., polygynous species. Mongolian gerbils (Meriones unguiculatus) are socially monogamous rodents, organized in stable reproductive pairs with their offspring, who inhabit a complex burrow system. Their territory is defended by both pair members against conspecific intruders. Both sexes display parental care. Philopatric offspring is reproductively suppressed, so that the dominant female is the only reproductively active female of the group. Females are regular ovulators, thus they are expected to rely at a great extent on gonadal hormones fluctuations. The species is widely used in biomedical research, constituting a model for studies on cerebral ischemia, auditory physiological research, aging, epilepsy, etc. A series of studies was aimed at presenting the Mongolian gerbil female as a new animal model for the study of the behavioral effects of endocrine disruptors, namely xenoestrogens. Pair bond formation represents one of the sensitive periods for highlighting the role of estrogenic compounds in shaping behavioral responses. Furthermore it can be considered one of the critical time-points in the orchestration of sexual behavior, influencing the reproductive success in monogamous species. Estrogen and progesterone (P) have been

266

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

employed to test the fineness of their action in modulating female gerbil social behavior. Adult females were observed during 48hr periods of housing with a sexually experienced male and as a function of hormonal manipulations. Females were either gonadally intact (INT) and tested during vaginal estrus, or ovariectomized (OVXed) and subsequently received s.c. injections according to one of the following treatments: vehicle (days 1-5) and estradiol benzoate (EB: 6.6microg, days 6-7) – short-EB; vehicle (days 1-7) – OVXed controls; EB (6.6microg, days 1-7) – long-EB; EB (6.6microg, days 1-2) and P (100microg, day 3) - EBP. INT females displayed extremely low levels of aggression and high levels of affiliation, contrary to short-eb and OVXed control females. long-eb and ebp females were less aggressive and more affiliative than OVXed controls, resembling more closely the behavioral performance of INT females. These results suggest a major role for estrogens in modulating aggression and affiliation in gerbils, revealing on one hand a great reliance on ovarian products and on the other an exquisite sensitivity of this species to gonadal steroids actions. A second study aimed to assess the effects of chronic exposure of females to low, environmentally relevant doses of the estrogenic chemical, bisphenol A (BPA), as well as to a physiological dose of the estrogen 17alfa-estradiol (17aE), on both pair-bonding and maternal behavior. Paired females were daily administered with one of the following treatments: a low BPA dose (2microg/kg body weight BPA), a medium BPA dose (10microg/kg body weight BPA), and an high BPA dose (20microg/kg body weight BPA); 17aE (0.04microg/kg body weight); corn oil (vehicle). Females received the treatment from the day of pairing to the day of birth of pups. Gerbils were trained to drink corn oil from a modified syringe (syringe without needle) starting a week prior pairing. This procedure allows not to stress the animals during chronic treatment. Due to the gestation length of this species, the treatment was administered for 26-28 days. Starting the day of pairing, the behavior of the couples was observed daily for 1 hour (instantaneous sampling) recording social behaviors such as agonistic interactions, social investigation, affiliation and nest related activities. The maternal performance of the dams was evaluated through repeated retrieving tests run on day of parturition and at postnatal day 5. Starting the day of parturition through postnatal day 30, the maternal behavior was daily recorded for 1 hour following an instantaneous sampling method, assessing either the pup-related behaviors or the general activities of the females. due to the aforementioned sensitivity of this species to both estrogens and estrogen-like compounds, a change in female behavior in the critical periods of pair-bonding and/or the expression of maternal care is predicted.

267

Posters’ Exhibition: The Role of Neuroactive Steroids in Healthy Ageing: Therapeutical Perspectives • Ibanez C., Guennoun R., Liere P., Eychenne B., Pianos A., El-Etr M., Baulieu E.E and Schumacher M. (Bicêtre, France, EU) Developmental expression of genes involved in neurosteroidogenesis: 3ß-hydroxysteroid-dehydrogenase/∆5-∆4 isomerase in the rat brain.

• Lacreuse A. and Herndon J.G. (Atlanta, GA, USA) Estrogen and cognition in young and aged female rhesus monkeys

• Leonelli E., Azcoitia I., Ballabio M., Cavarretta I., Garcia-Segura L.M., Gonzalez L.C., Magnaghi V., Veiga S.and Melcangi R.C. (Milano, Italy, EU) progesterone and its derivatives dihydroprogesterone and tetrahydroprogesterone stimulate remyelination of sciatic nerve in aged male rat

• Matsumoto A. (Hongo, Japan) Androgen regulates expression of androgen receptor protein in the perineal motoneurons of aged male rats

• Sujkovic E., Fry J.P., Mileusnic R. and Rose S.P.R. (Milton, UK, EU) Dehydroepiandrosterone sulphate potentiates memory in the day-old chick

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

DEVELOPMENTAL EXPRESSION OF GENES INVOLVED IN 5 4 ∆ ∆ -∆ NEUROSTEROIDOGENESIS: 3ß-HYDROXYSTEROID-DEHYDROGENASE/∆ ISOMERASE IN THE RAT BRAIN Ibanez C., Guennoun R., Liere P., Eychenne B., Pianos A., El-Etr M., Baulieu E.E. and Schumacher M. INSERM U488, Stéroïdes et Système Nerveux, 80 rue du Général Leclerc, 94276 Bicêtre, France. Email: [email protected]. Fax: 00 33 (0)1 45 21 19 40. In the central nervous system (CNS), neurosteroids, in particular progesterone, have neurotrophic and neuroprotective effects. We thus decided to study the developmental expression of the 3ß-hydroxysteroid-dehydrogenase/∆5-∆4 isomerase (3ßHSD), enzyme which converts pregnenolone to progesterone, in the male rat brain at 0, 7, 14, 70 days after birth. 3ßHSD mRNA was found to be widely distributed throughout the brain as shown by in situ hybridisation. At all ages, the same cerebral structures were labelled, but the intensity of the hybridisation signal constantly decreased during postnatal development. As the hippocampus is of particular interest because of its neuronal plasticity, we chose to quantify the changes in 3ßHSD mRNA levels, as well as progesterone and pregnenolone concentrations in this structure. Quantitative in situ hybridisation confirmed a decrease in the expression of 3ßHSD mRNA with progressing age as revealed by a significant reduction in the density of silver grains per cell in the CA1 layer. This decrease was confirmed by semi-quantitative RT-PCR on hippocampal samples. Concentrations of hippocampal pregnenolone and progesterone measured by gas chromatography/mass spectrometry (GC/MS) were highest at P0 and lower at the other ages. Plasma concentrations of these steroids were lower than in the hippocampus, suggesting that they may have been mostly synthesized in situ since P0. These results demonstrate variations in the expression of a gene coding for an enzyme critically involved in progesterone synthesis in the hippocampus throughout the postnatal development.

269

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ESTROGEN AND COGNITION IN YOUNG AND AGED FEMALE RHESUS MONKEYS Lacreuse A. and Herndon J.G. Division of Neuroscience, Yerkes Research Center, Emory University, 954 Gatewood Rd, Atlanta GA 30322, USA. Email: [email protected] Fax: 404-727-3278 Because women can expect to live more than one third of their life in a postmenopausal state, it is of critical importance to understand the consequences of estrogen loss and replacement on a variety of systems, including cognitive function. So far, several studies have suggested potential benefits of estrogen replacement therapy (ERT) on verbal memory and risk of Alzheimer’s disease, but results have been rather inconsistent. Because studies in women are limited by a number of confounds, the effects of estrogens on cognition may be best studied in an animal model of human menopause. The female rhesus monkey is an important and relevant model because this species is capable of performing complex cognitive tasks and has a reproductive endocrinology that is very similar to that of the woman. We have investigated the relationships between estrogen and cognitive function in a series of experiments involving young and aged female rhesus monkeys. In a first experiment in intact young females, we examined whether cognitive function fluctuates across the menstrual cycle. Four young females were tested daily during one menstrual cycle on a task of visual recognition memory, the Matching-to-Sample (MTS), and a task of spatial memory, the spatial condition of the Delayed Recognition Span Test (spatial-DRST). MTS performance did not vary as a function of the menstrual cycle, but spatial-DRST performance worsened during the periovulatory phase of the cycle, when estrogen levels were at their highest. These findings support the notion that high estrogen levels are detrimental to spatial memory. Following bilateral ovariectomy, the monkeys (n=6) were tested on the MTS-mixed delays and the spatial, object and face conditions of the DRST. They were tested 5 days a week, 1 task per week, for a total of 8 months, while undergoing monthly treatments with placebo and ethinyl estradiol (EE2, 450 ng/kg/day). EE2 failed to improve performance on any task, but clearly worsened performance on the face-DRST, a task involving the discrimination of rhesus monkey faces. A follow up experiment with new categories of face stimuli (rhesus monkeys, humans and chimpanzees) revealed that the detrimental effect of EE2 was restricted to the processing of conspecifics’ faces. These results suggest that estrogen may enhance the emotional reactivity of the monkeys to socially relevant stimuli. Finally, we examined the effects of EE2 (450 ng/kg/day) and the Selective Estrogen Receptor Modulator (SERM) raloxifene (1 mg/kg/day) on cognitive performance in aged, long-term ovariectomized (OVX), rhesus monkeys. We tested 5 aged females (21-24 years old) that had been OVX for many years (10-16 years) on the Delayed Response (DR), the Delayed Non Matching-to-Sample with a 10 minute delay (DNMS-10 min) and the spatial-DRST. Monkeys were tested 5 days a week on each task, 1 task per week, for 9 consecutive months, while undergoing monthly treatments with placebo, EE2, and raloxifene. We found that EE2 enhanced performance on the spatial-DRST, but did not affect performance on the other tasks of the battery. Further, the improvement was

270

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

restricted to working memory, as opposed to reference memory. Raloxifene had no effect on cognitive performance for any tasks. These findings show that estradiol is able to enhance spatial memory in aged monkeys despite many years of estrogenic deprivation. Because data in rodents and humans suggest that estradiol affects motor performance, we also assessed fine motor ability in the same animals using a ‘lifesaver task’. Monkeys removed a lifesaver candy from three rods of different complexity using both hands alternatively. They were tested twice a week for 8 weeks, while treated with placebo and EE2 in alternating 14-days blocks. EE2 had no detectable effect on fine motor performance. These results suggest that the effects of EE2 on cognition are unlikely to be mediated by changes in motor performance. Overall, these findings in young and aged female rhesus monkeys suggest that (1) during endogenous ovarian cycles, spatial memory is impaired around the time of the monthly peak in estradiol (2) exogenous estradiol, as administered in these studies, does not benefit memory in young females, but does improve spatial working memory in aged females (3) estradiol may enhance emotional reactivity (4) the effects of estradiol on cognitive performance are not mediated by changes in motor performance. Important lines of future research will be to determine the influence of different doses and timing of estrogen administration on cognitive performance, to elucidate the interactions among estrogen replacement, aging, and duration of estrogen deprivation, and to consider the influences of estrogens on non mnemonic factors, such as emotional behavior.

271

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

PROGESTERONE AND ITS DERIVATIVES DIHYDROPROGESTERONE AND TETRAHYDROPROGESTERONE STIMULATE REMYELINATION OF SCIATIC NERVE IN AGED MALE RAT Leonelli E.*, Azcoitia I.°, Ballabio M.*, Cavarretta I.*, Garcia-Segura L.M.#, Gonzalez L.C.*, Magnaghi V.*, Veiga S.* and Melcangi R.C.* *Department of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milano, Italy, Tel. +39-02-50318238, Fax: +39-02-50318204, Email: : [email protected] # Instituto Cajal, C.S.I.C., 28002 Madrid, Spain. °Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, E28040 Madrid, Spain. Our recent observations have indicated that, in adult male rats and in rat Schwann cell cultures, neuroactive steroids [e.g., progesterone (P), dihydroprogesterone (DHP), tetrahydroprogesterone, (THP), testosterone (T), dihydrotestosterone (DHT), and 5alphaandrostan-3alpha,17beta-diol (3alpha-diol)] are able to stimulate the expression of two important myelin proteins of peripheral nervous system, [i.e., glycoprotein Po (Po), and peripheral myelin protein 22 (PMP22)] [1-6]. These two myelin proteins play an important physiological role for the maintenance of the multilamellar structure of PNS myelin [see for review 6], and modification in their synthesis, with a corresponding influence on the myelin structure occurs during the aging process [see for review 5]. Loss of myelinated fibers and alterations in myelin structure and morphology of myelinated fibers are common observations in studies of aged peripheral nerves [see for review 5]. To this purpose, we have now evaluated the effect of neuroactive steroids (i.e., P, DHP, THP, T, DHT and 3alpha-diol) on the mRNA and protein levels of Po and PMP22 present in the sciatic nerve of aged male rats. Moreover, we have also assessed whether these neuroactive steroids may influence the morphology of myelinated fibers in the sciatic nerves of aged male rats. 22-24 month-old male rats were treated for a month with eight subcutaneous injections of 1 mg of neuroactive steroids. Injections were administered every four days and 24 hours after the last treatment mRNA and protein levels of Po and PMP22 were evaluated in sciatic nerve, by Northern and Western blot analysis respectively. Data obtained have indicated that only DHP is able to increase significantly the low messenger levels of Po. On the contrary, P, THP, T, DHT and 3alpha-diol were unable to significantly modify the gene expression of Po. However, when we have analyzed the protein levels of this myelin protein, we have observed that not only DHP but also P is effective. The low mRNA levels of PMP22 found in the sciatic nerve of aged male rats were unaffected by the in vivo treatment with all the neuroactive steroids tested. However, THP is able to significantly increase the protein levels of this myelin protein. Data obtained by morphometric analysis of the myelin compartment of sciatic nerve of aged male rats have indicated that treatments with P, DHP and THP result in a significant increase in the number of myelinated fibers of small caliber, in a significant reduction in the frequency of myelin abnormalities and in a significant increase in the g ratio of small myelinated fibers.

272

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

Moreover, P treatment significantly reduces the frequency of myelinated fibers with irregular shapes. In contrast, treatments with T, DHT or 3alpha-diol did not significantly affect any of the morphological parameters examined [7]. In conclusion, the present data indicating that P, DHP and THP stimulate the low expression of myelin proteins in the sciatic nerve of aged male rats and reduce agingassociated morphological abnormalities of myelin and aging-associated myelin fiber loss in this peripheral nerve suggest that these neuroactive steroids may represent useful therapeutic alternatives to maintain peripheral nerve integrity in aged animals.

(This study has been carried out with financial support from the Commission of the European Communities, specific RTD programme “Quality of Life and Management of Living Resources”, QLK6CT-2000-00179)

Reference List 1. Melcangi, R.C., Magnaghi, V., Cavarretta, I., Martini, L. and Piva, F., Age-induced decrease of glycoprotein Po and Myelin Basic Protein gene expression in the rat sciatic nerve. Repair by steroid derivatives, Neuroscience, 85 (1998) 569-578. 2. Magnaghi V, Cavarretta I, Zucchi I, Susani L, Rupprecht R, Hermann B, Martini L, Melcangi RC. Po gene expression is modulated by androgens in the sciatic nerve of adult male rats. Mol Brain Res 1999; 70:36¯44. 3. Melcangi, R.C., Magnaghi, V., Cavarretta, I., Zucchi, I., Bovolin, P., D'Urso, D. and Martini, L., Progesterone derivatives are able to influence peripheral myelin protein 22 and Po gene expression: possible mechanisms of action, J. Neurosci. Res., 56 (1999) 349-357. 4. Melcangi, R.C., Magnaghi, V., Galbiati, M., Ghelarducci, B, Sebastiani, L. and Martini, L., The action of steroid hormones on peripheral myelin proteins: a possible new tool for the rebuilding of myelin? J. Neurocytol., 29 (2000) 327-339. 5. Melcangi, R.C., Magnaghi, V. and Martini, L., Aging in peripheral nerves: regulation of myelin protein genes by steroid hormones, Prog. Neurobiol., 60 (2000) 291-308. 6. Magnaghi, V., Cavarretta, I., Galbiati, M., Martini, L. and Melcangi, R.C., Neuroactive steroids and peripheral myelin proteins. Brain Res Rev 37 (2001) 360-371. 7. Azcoitia, I., Leonelli, E., Magnaghi, V., Veiga, S., Garcia-Segura, L.M. and Melcangi, R.C., Progesterone and its derivatives dihydroprogesterone and tetrahydroprogesterone reduce myelin fiber morphological abnormalities and myelin fiber loss in the sciatic nerve of aged rats, Neurobiol. Aging (2003), in press.

273

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ANDROGEN REGULATES EXPRESSION OF ANDROGEN RECEPTOR PROTEIN IN THE PERINEAL MOTONEURONS OF AGED MALE RATS Matsumoto A. Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 1130033, Japan; e-mail: [email protected]; fax: 81-3-5800-0245 Sex steroids are known to play a crucial role in reproductive neuroendocrine functions. A number of neurons in the neuroendocrine brain contain sex steroid receptors, and are thought to be a key element of functional neural circuits that are regulated by sex steroids. Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) in adult male rodents are one of the androgen receptor (AR)-containing neurons. These motoneurons and their target muscles, the bulbocavernosus and levator ani that attach to the penis, have an important role in copulatory behavior. During the process of aging, disorders of the reproductive function occur in male rats. Aged male rats show a decrease in plasma levels of testosterone, associated with decline in copulatory behavior. Recent immunohistochemical study revealed that, in aged rats, both the intensity of AR immunoreactivity and number of AR-immunoreactive nuclei of the SNB motoneurons are significantly reduced. With regard to morphology, decreases in the number of neurons and synapses as well as dendritic deterioration have been observed in the preoptic area-hypothalamus containing androgen-sensitive neurons of aged male rats and mice. These age-related changes may be related to the decline in the neural function of that region. Androgen has been reported to play a significant role in maintaining expression of ARs of the SNB motoneurons of young male rats [1]. Nuclear immunoreactivity of SNB motoneurons of the castrated animals was absent 5 days after castration. There was no significant difference in AR immunoreactivity of SNB motoneurons between controls and castrated males treated with testosterone propionate (TP). In the present experiment, it was examined whether expression of ARs was also regulated by androgen in aged ones. Twelve aged male rats were castrated at 26 months of age. Five days following castration, the animals were injected subcutaneously with 500 µg of TP (6 males) or vehicle (6 males), and killed two hours later. Six sham-castrated rats served as controls. AR immunoreactivity was examined in motoneurons of the SNB in these animals by immunohistochemistry using the polyclonal antibody, PG21 (a gift from Dr. G.S. Prins, Univ. Illinois at Chicago College of Medicine). All SNB motoneurons containing AR-immunoreactive nuclei with nucleolus were counted. The density of AR immunoreactivity was judged on a three point scale; 1 for low-, 2 for medium, and 3 for high-density labeling. The immunoreactivity was confined to the nuclei of the SNB cells in aged controls and castrated, aged males treated with TP. In aged male rats, AR immunostaining of the SNB motoneurons was found to be not as intense as that in castrated, aged animals treated with TP. Nuclear AR immunoreactivity of SNB moto- neurons of aged males was absent 5 days following castration. AR immunoreactivity of SNB motoneurons was more intense in castrated, aged rats treated with TP compared with that in controls. Low-density AR-immunoreactive nuclei of SNB motoneurons were dominant in control animals. There were no significant difference in the number of medium-density AR-immunoreactive nuclei of SNB motoneurons between controls and castrated, aged males treated with TP (p<0.05). On the other hand, high-

274

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

density AR-immunoreactive nuclei of SNB motoneurons were dominant in castrated, aged male rats treated with TP. There was no significant difference in the total number of ARimmunoreactive nuclei of the SNB motoneurons between controls and castrated, aged male rats treated with TP (p>0.05). Plasma levels of testosterone of castrated, aged male rats treated with TP were significantly greater than those of control animals (p<0.01). Plasma levels of testosterone were not detected in castrated animals. These results suggest that expression of ARs in the SNB motoneurons of aged male rats is up-regulated in response to androgen, and that androgen may be, at least in part, involved in the process of aging of the SNB system in male rats.

(Supported by grants from the Ministry of Education, Culture, Science, Sports and Technology of Japan).

Reference List [1] A. Matumoto, Y. Arai, G.S. Prins, Androgenic regulation of androgen receptor immunoreactivity in motoneurons of the spinal nucleus of the bulbocavernosus of male rats, J. Neuroendocrinol. 8(1996) 553-559.

275

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

DEHYDROEPIANDROSTERONE SULPHATE POTENTIATES MEMORY IN THE DAY-OLD CHICK Sujkovic E., Fry J.P.*, Mileusnic R. and Rose S.P.R. The Open University, Department of Biological Sciences, Brain and Behaviour Research Group, Walton Hall, Milton Keynes, MK7 6AA, UK E-mail: [email protected], Fax: ++44 1908 654 167 *University College London, London WC1E 6BT, UK The neurosteroid Dehydroepiandrosterone (DHEA) and its sulphate ester (DHEAS) have been shown to enhance memory formation in various learning paradigms. Moreover, DHEAS produced in the brain can be converted to free steroid, a process mediated by the steroid sulphatase (STS) enzyme. Chronic STS inhibition increases levels of DHEAS enhancing learning and memory [3]. Recent findings from our group suggest that DHEA and DHEAS are present in the chick brain and facilitate memory recall when administered intracerebrally [1, 2]. In this study we investigated the effects of peripherally administered DHEAS on early events of memory formation in day old chicks, using the one trial passive avoidance task. This task is quick, reproducible and sharply timed and has two versions: strong and weak training, characterised by different durations of retention. Intraperitoneal administration (i.p.) of DHEAS 20mg/kg at either 30 minutes pretraining or 30 minutes post-training enhanced recall 24 hours post-training, in the weak version (usually recalled for less than 9 hrs) of the passive avoidance learning task. Assays of steroid sulphatase in the day old chick using DHEAS as the substrate showed no detectable activity in homogenates of both liver and brain, suggesting that the effects of DHEAS on memory are not consequent on desulphation to the free steroid. Our results suggest that DHEAS may play a pivotal role in memory consolidation.

References List [1] Johnston AN, Migues PV. Task- and time-dependent memory enhancement by dehydroepiandosterone in day-old chicks. Neural Plasticity 8(2001):255-70 [2] Migues PV, Johnston AN, Rose SP. Dehydroepiandosterone and its sulphate enhance memory retention in day-old chicks. Neuroscience 109(2002):243-51. [3] Rhodes ME, Li PK, Burke AM, Johnson DA. Enhanced plasma DHEAS, brain acetylcholine and memory mediated by steroid sulfatase inhibition. Brain Research 773(1997):28-32

276

Posters’ Exhibition: Estrogen Receptors •

Axelsson, J., Halldin, K. and Brunström, B. (Uppsala, Sweden, EU) Expression of estrogen receptor -alpha and -beta mRNA in embryonic quail brain

•

Halldin K., Axelsson J., Holmgren C. and Brunström B. (Uppsala, Sweden, EU) Localization of estrogen receptor- α and -β mRNA in the brain of embryonic and adult Japanese quail

•

Kalita K., Szymczak S., Markowska A., Konopka W. and Kaczmarek L. (Warsaw, Poland) Estrogen receptor beta isoforms in the function of the rat brain

•

Szymczak S. and Kaczmarek L. (Warsaw, Poland) Estrogen receptor beta in the rat hippocampus following stimulation with kainic acid

•

Vegeto E., Belcredito S., Etteri S., Ciana P. and Maggi A. (Milano, Italy, EU) Estrogen receptors mediate the inhibitory activity of estradiol on brain macrophage reactivity

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

EXPRESSION OF ESTROGEN RECEPTOR -ALPHA AND -BETA mRNA IN EMBRYONIC QUAIL BRAIN Axelsson J., Halldin K. and Brunström B. Dept. of Environmental Toxicology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, SE-75236 Uppsala, Sweden. E-mail: [email protected]. Fax number: +46-18-518843. Sexual differentiation of brain and behavior in birds is dependent on the hormonal environment of the embryos in ovo. During embryonic development, the female Japanese quail brain undergoes organizational changes due to the demasculinizing action of ovarian estrogens [1]. These changes result in loss of ability of adult females to show male copulatory behavior even after testosterone treatment. If male quail embryos are exposed to estrogenic chemicals before day twelve of incubation, they will not display male sexual behavior as adults regardless of the hormonal milieu [4,6]. The neurological changes in the embryonic brain caused by estrogens and the underlying mechanisms for the effects of estrogen on brain differentiation are still to a great extent unknown, although binding to estrogen receptors (ERs) is believed to be necessary for these effects. Two types of ERs, ER alpha and ER beta, are known to be present in the brain of adult birds [3,2] and immunohistochemical studies have shown the presence of ER alpha also in the embryonic quail brain [5]. The expression of ER alpha and ER beta mRNA at various stages of embryonic development in quail has not been described. In this study we used RT-PCR to determine the expression of ER alpha and ER beta in the brain of quail embryos at various developmental stages. We found that ER beta is expressed as early as day six of incubation, while ER alpha seems to appear at a later stage in development. The finding that ER beta mRNA appears in the brain at an earlier developmental stage than ER alpha mRNA was supported by in situ hybridisation studies performed in our lab (see abstract by Halldin et al.). The physiological significance of the differential expression of the two ERs in the embryonic quail brain remains to be elucidated. Furthermore, knowledge about the expression of ER alpha and ER beta during development is important for the understanding of how various xenoestrogens can interfere with the differentiation of the brain and reproductive behavior in quail. Reference List [1] E.K. Adkins, Hormonal Basis of Sexual Differentiation in the Japanese Quail, J. Comp. Physiol. Psychol. 89:1 (1975) 61-71. [2] G. F. Ball, D. J. Bernard, A. Foidart, B. Lakaye, J. Balthazart, Steroid Sensitive Sites in the Avian Brain: Does the Distribution of the Estrogen Receptor α and β Types provide Insight into Their Function?, Brain Behav. Evol. 54 (1999) 28-40. [3] A. Foidart, B. Lakaye, T. Grisar, G. F. Ball, Estrogen Receptor-β in quail: Cloning, Tissue Expression and Neuroanatomical Distribution, J. Neurobiol. 40 (1999) 327-342. [4] K. Halldin, C. Berg, I. Brandt, B. Brunström, Sexual Behavior in Japanese Quail as a Test End Point for Endocrine Disruption: Effects of in Ovo Exposure to Ethinylestradiol and Diethylstilbestrol, Environ. Health Persp. 107:11 (1999) 861-866. [5] M. Gahr, E. Balaban, The development of a species difference in the local distribution of brain estrogen receptive cells, Dev. Brain Res. 92 (1996) 182-189. [6] M. Schumacher, J. C. Hendrick, J. Balthazart, Sexual differentiation in quail: critical period and hormonal specificity, Horm. Behav. 23 (1989) 130-149.

278

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

α AND -β β mRNA IN THE BRAIN LOCALIZATION OF ESTROGEN RECEPTOR -α OF EMBRYONIC AND ADULT JAPANESE QUAIL Halldin K.a, Axelsson J.a, Holmgren C.b and Brunström B.a a

Department of Environmental Toxicology, bDepartment of Developmental Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, SE-752 36 Uppsala, SWEDEN, E-mail; [email protected], Fax; +46 18 518843.

We cloned partial ERα and ERβ cDNA sequences in Japanese quail and made riboprobes for in situ hybridization to study the localization of the mRNAs in brains of adults and embryos of both sexes. The brains of two male and two female adult quail and one brain from each sex at embryonic day 9 and 17 were used. In adult brains, high levels of ERα mRNA were present throughout the medial preoptic nucleus (POM). In the bed nucleus striae terminalis (BST) only a few labeled cells were detected, localized posterior to the anterior commissure (CA). Hybridization signal was also detected in the telencephalic nucleus taeniae (Tn) and in the nucleus supraopticus externus (SOE). The hybridization signal of ERβ was generally more intense than that for ERα and strong signal was found in the POM, BST and Tn. In the sections collected posterior to the CA, the signal of BST merged with the signal of the caudal part of the POM. The expression within the Tn was higher than that for ERα, and appeared t o be stronger in sections collected from males than in sections from females. The distribution of ERα mRNA in 17-day-old embryos closely matched that found in adult brains. Weak labeling was found in the POM and a somewhat more intense signal in the SOE. The Tn also showed expression of ERα mRNA, whereas no signal was found in the BST. As for ERα, the localization of ERβ mRNA in 17-day-old embryos was very similar to that in the adult brain. The POM showed strong hybridization signal and the BST was intensely labeled for ERβ. The ventral part of the Tn showed strong and discrete hybridization signal. Localization of ERα and ERβ mRNA was the same in both female and male brain sections. No ERα hybridization signal was found in sections from 9-day-old embryos. However, brains hybridized for ERβ showed marked signal in areas that presumably are the POM and the BST. This expression was found in both the male and female brain. To conclude, both ERα and ERβ mRNA are expressed in areas of the quail brain known to be important for activation of sexual behavior. The expressions of the mRNAs largely overlap, but there are differences in localization and in density. Furthermore, there seems to be differences in expression of ERβ in the Tn of males and females. We detected ERβ earlier than ERα in the embryonic quail brain. The distribution of ERβ mRNA in adult males as shown by ISH using a specific riboprobe confirms the results reported using oligoprobes [2]. Furthermore, the distribution of ERα mRNA in the brain of adult quail was similar to the distribution of the receptor protein [1]. Consequently, our study supports previous reports on the distribution of ERs in adult quail brain and provides new data on the distribution of ERβ in adult females and of both ERs during differentiation of the embryonic brain. Reference List [1] Balthazart J, Gahr M, Surlemont C. Distribution of estrogen receptors in the brain of the Japanese quail: an immunocytochemical study. Brain Res 501:205-14(1989). [2] Foidart A, Lakaye B, Grisar T, Ball GF, Balthazart J. Estrogen receptor-beta in quail: cloning, tissue expression and neuroanatomical distribution. J Neurobiol 40:327-42.(1999).

279

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ESTROGEN RECEPTOR BETA ISOFORMS IN THE FUNCTION OF THE RAT BRAIN Kalita K. 1, Szymczak S. 1, Markowska A. 2, Konopka W. 1 and Kaczmarek L. 1 1

Departament of Molecular and Cellular Neurobiology, Nencki Institute, Warsaw, Poland Department of Psychology, The Johns Hopkins University, Baltimore USA; [email protected] 2

Estrogens play an important role not only in brain development, but also in promotion of neuronal growth, regeneration, and neuroprotection throughout the whole lifespan. Estrogens are known to act through estrogen receptors (ER), transcription factors, which exist in two subtypes α and β. Recently, five different isoforms of ERβ, which are produced from one RNA as a result of alternative splicing, have been identified in the rat. Protein structures predicted from the mRNAs sequence differ significantly, suggesting their differential function as well. To approach ERβ mRNA isoforms expressed in the rat brain we have developed an RNase protection assay that allows investigating a variety of isoforms in the same RNA sample. The samples were taken from different parts of the brain such as: frontal cortex, striatum, olfactory bulbs, sensor cortex, cerebellum, entorhinal cortex and hippocampus. The results of experiment demonstrate significant differences in the expression of isoforms in the analyzed brain structures. In cerebellum, entorhinal cortex, olfactory bulbs and striatum we have observed the highest level of expression of isoform ERβ1 as well as isoform with 54 bp insert in LBD region and with deletion in DBD. On the other hand in the hippocampus, forebrain and sensory cortex mRNA expression level of these isoforms was significantly lower. Transcript expression level is different among analyzed brain structures, yet the ratio of these three trancripts' levels is stable in the investigated structures. Estrogens are known to affect neuronal plasticity in vivo and in vitro in the hippocampus structure involved learning and memory formation. This is indicated by their ability to influence density of dendritic spines, which represents synaptic contacts between neurons. During estrous cycle, when estrogen level correlates with plastic changes in the hippocampus, we observed an increase in the levels of ERβ mRNA isoforms in estrous phase vs. proestrus. Also ERβ mRNA expression level is increased in OVX animal's hippocampus vs estrogen treatment. The lower levels of estrogens correlated with higher expression of mRNA of ERβ in the hippocampus. Immunohistochemistry methods shown that ERβ protein is found in the hippocampus outside the nucleus, whereas in the medial amygdala is nuclear. We observed a subcellular co-localization of ERβ protein and HSP90 in those structures. To further investigate subcellular localization of estrogen receptors beta we introduced an adenovirus with ERβ into the primary hippocampal and cortical cell cultures as well as into the brain. In both systems ERβ protein was expressed in glial as well as in neuron-like cells. Adenoviraldirect expession of ERβ was observed in the cell nucleus the primary cell cultures and in CA1 and DG fields of hippocampus. We conclude, that ERβ responds to changes in neuronal plasticity. However, its subcellulal localization suggests varied mechanisms of action and requires further studies.

280

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ESTROGEN RECEPTOR BETA IN THE RAT HIPPOCAMPUS FOLLOWING STIMULATION WITH KAINIC ACID Szymczak S. and Kaczmarek L. Department of Molecular Neurobiology, The Nencki Institute of Experimental Biology, Pasteura 3, 02-093, Warsaw, Poland [email protected], fax (48)(22) 822 5481 Introduction: Estrogen receptors (ER), transcription factors and that mediate the mechanism of action of estrogens in the body, are the object of a growing number of studies of brain function as well as dysfunction. Receptors for estrogen exist in two known subtypes, α and β, and multiple isoforms of both. This accounts for high complexity of action, which is cell/ tissue specific and may modulate the response to estradiol. Estrogens are known to act as neuroprotectants, promote neuronal growth and regeneration as well as induce synapse formation. The rat hippocampus, structure associated with learning and memory, has shown particular responsiveness to estrogens [12, 8] while expressing both ERs. In this paper the role of the ER in plasticity events in the hippocampus has been studied using the kainic acid (KA)induced seizures model. When administered to male Wistar rats at a dose of 10 mg/kg kainic acid, a glutamate analog, causes varied effects across space and time within the hippocampus: depolarization leading to convulsions, and eventually leading to neurodegeneration in CA subfields and plasticity in DG. Underlying every process occurring in these cells are changes in gene expression. ER β, aside from being the main ER expressed in the hippocampus, seems t o also be one of the gene products experiencing an increase as a result of the processes activated by the excitatory neurotransmitter analog, kainic acid. We have noticed that, both on the level of mRNA as well as protein, ER β appears to undergo an increase as early as 1h (peaking at 6h) following the initiation of seizures. Results:. Following the i.p. administration of KA to male rats, we have collected either their hippocampi or the entire brains and processed the tissue for total RNA extraction or as 35 µm coronal floating sections, respectively, at 1, 6, and 24h post-seizure. Care was taken that only animals exhibiting full status epilepticus were used in the study. Through the studies of the level of ER β mRNA by semi-quantitative RT-PCR and in situ hybridization we noticed that the expression level of ERβ increases at 6 and 24 h post-seizure. In situ hybrydization confirmed these results, further localizing the change in ER β to CA3 hippocampal region. Protein analysis through immunohistochemistry has confirmed that following stimulation with kainic acid the intensity of ERβ signal indeed increases, however it is localized to the outside of the nucleus, particularly in the CA3 and the hilus of the DG. In double immunostaining procedure, the strong ER β signal after KA stimulation collocalizes with neuronal markers (NeuN and MAP-2) rather than glial cell markers (GFAP). Furthermore, the strong extranuclear immuno-localization of ER β was also noted in primary cultures from hippocampi of newborn rats, where following the addition of either KCl (20 mM) or Glutamate (10 uM), there is a focusing of the ER β signal outside the nucleus. Conclusion: ER β has been found in the normal, unstimulated hippocampus [11] and localized mostly to the nucleus. However, Milner et.al. (2001) have also localized estrogen receptors (particularly ER α) to non-nuclear sites within the principal cells of the hippocampus. In our model ER β was found to collocalize with NeuN and MAP-2. This suggests a role for this protein in events taking place in stimulated neurons. The fact that its location under these conditions is extra-nuclear would point to its function in a process other than the direct activation of gene transcription. Results from various studies suggest that ERs couple to second messenger systems for local signalling within neurons [6, 9]. Cardona-Gomez et.al. (2000) have co-localized ER β with IGF-I receptors, suggesting a cross-coupling of their signalling pathways. Putative membrane ERs appear to mediate rapid activation by E of members of the MAPK and src tyrosine kinase [10,3] and c-AMP [5] signalling pathways, among other effects enhancing NMDA receptor function and LTP and affecting AMPA/kainate receptors [1, 4]. We found that the expression of ER β is affected by KAdriven depolarization of neurons, particularly at 6h post seizure, at which time there is also an

281

Trabajos del Instituto Cajal. Tomo LXXIX, 2003 increased presence of activated astrocytes. These were found to have a significantly increased aroamtase activity [13]. Thus, a change in the expression and location of ER β following KAinduced stimulation may be a response to an increased local production of E, which is known t o be neuroprotective. This suggests that ER β might be active in brain repair processes. Furthermore, the evident increase in ER β signal post-seizure is localized to CA3 subfield, known to be very susceptible to KA-induced damage, where brain repair processes might be activated.

DG

Amount of PCR Product representing amount of mRNA

ER beta 2 0,6

ER beta 1

0,4

*

**

CA3 0,2

0

control

1h

6h

24h

Time point post-KA

Fig. 1 Level of mRNA For ERβ isoforms in rat hippocampus increases after stimulation with kainic acid

Fig. 3 Extranuclear localization of ERβ in CA3 prior to and following 6h of KA treatment visualized by confocal microscopy.

Fig. 2 ERβ expression in hippocampus prior to and following 6h of KA treatment

Fig. 4 Focal accumulation of ERβ in hippocampal primary cells following6h of stimulation with KCl

Reference List 1. Bi, R., Broutman, G., Foy, R., Thompson, R.F., Boudry, M. (2000) PNAS 97, 3602-3607. 2. Cardona-Gomez, G.P., Doncarlos, L., Garcia-Segura, L.M. (2000) Neuroscience 99, 751-760. 3. Kuroki, y., Fukushima, K., Kanda, Y., Mizuno, K., Watanabe, Y. (2000) Eur J Pharmacol 400, 205209. 4. Lason, W. (2000) Polish J Pharmacology 52, 59-62. 5. Lazennec, G., Thomas, J.A., Katzenellenbogen, B.S. (2001) J Steroid Biochem Mol Biol 77, 193203. 6. Levin, E.R. (1999) Trends Endocrinil Metab 10, 374-377. 7. Milner, T.A., McEwen, B.S., Hayashi, S., Li, C.J., Reagen, L., Alves, S.E. (2001) J.Comp. Neurol. 429, 538-541. 8. Sandstrome, N.J. and Williams, C.L. (2001) Behav. Neurosci 115, 384-393. 9. Simoncini, T., Hfezi-Moghadam, A., Brazil, D.P., Ley, K., Chin, W.W., Liao, J.K. (2000) Nature 407, 538-541. 10. Singh, M., Setalo, G., Guam, X., Frail, D.E., Toran-Allerand, C. (2000) J Neuroscience 20, 16941700. 11. Shughrue, P.j., Lane, M.V., Merchenthaler, I. (1999) Endocrinology 140, 2613-2620. 12. Wooley, C.S and McEwen, B.S. (1994) J Neurosci. 14, 7680-7687. 13. Garcia-Segura, L.M., Wozniak, A., Azcitia, I., Rodriguez, J.R., Hutchison, R.E., Hutchison, J.B. (1999) Neuroscience 89, 567-578.

282

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ESTROGEN RECEPTORS MEDIATE THE INHIBITORY ESTRADIOL ON BRAIN MACROPHAGE REACTIVITY

ACTIVITY

OF

Vegeto E., Belcredito S., Etteri S., Ciana P. and Maggi A. Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, University of Milan, 20133-Milan, Italy. [email protected]; FAX 0039-0250318284 Beyond the well-recognized role in the control of reproduction, estrogens act in the adult nervous system to preserve cognitive functions and to protect the brain against neurodegenerative stimuli. The mechanism underlying this effect still needs to be fully elucidated. Previous studies have highlighted the role of 17β-estradiol (E2) on neurons and its activity on metabolic and survival pathways. We have recently described that E2 targets monoblastoid cells and brain macrophages, i.e. microglia. Here we demonstrate that, in rat, E2 regulates brain macrophage reactivity in vivo, showing that systemic administration of the hormone prevents morphological and biochemical activation of microglia and recruitment of peripheral monocytes induced by intraventricular injections of lipopolysaccharide (LPS). Hormone action involves all the brain regions sensible to the LPS effect and occurs at a time and dosage compatible with the activation profile of brain estrogen receptors (ER). The effect of hormone on LPS-induced macrophage reactivity in ER-knock out mice will be discussed Altogether, our data reveal a novel function for E2 in the brain, highlighting the inhibitory role of the hormone-ER complex on local inflammatory cells. Since inflammation is a key element in degenerative processes of the injured brain, our results provide a novel mechanism to explain the beneficial activity of hormone in neurological disorders associated with inflammation.

283

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

α-REDUCTASE ISOZYMES DIFFERENTIAL REGULATION OF STEROID 5α EXPRESSION BY ANDROGENS IN THE ADULT RAT BRAIN Torres J.M.1 and Ortega E.1,2 (1) Department of Biochemistry and Molecular Biology. Faculty of Medicine. University of Granada. 18012 Granada. Spain. (2) Institute of Neurosciences, University of Granada. 18012 Granada. Spain.

The enzyme 5α-reductase (5α-R) is present in many mammalian tissues, including the brain. The physiological importance of 5α-R in the brain derives from its capability to convert testosterone (T) to a more potent androgen, dihydrotestosterone (DHT), and to convert progesterone and deoxycorticosterone (DOC) to their respective 5α-reduced derivatives, precursors of allopregnanolone and tetrahydroDOC, potent allosteric modulators of the γ-aminobutyric acid receptor (GABAA-R). 5α-R occurs as two isoforms, 5α-R type 1 (5α-R1) and 5α-R type 2 (5α-R2). We studied the effects of T and DHT on the mRNA levels of both 5α-R isozymes in the prefrontal cortex of the adult rat, using an accurate and precise method that combines the high specificity of one-step quantitative RT-PCR with the sensitivity of capillary electrophoresis (CE). Our results demonstrate that both isozymes of 5α-R are expressed in the cerebral cortex of adult rats. The gene expression of 5α-R type 2 is under the positive control of T and DHT. The gene that codes for 5α-R type 1 is not constitutive, because its expression is negatively regulated by T and DHT. These results open up a new research line that may lead to a better understanding of the role of 5α-R isozymes in the physiology of the central nervous system (CNS).

284

2nd International Meeting STEROIDS AND NERVOUS SYSTEM Villa Gualino, TORINO, Italy. February 22-26 2003

ASSAY DEVELOPMENT FOR INVESTIGATION OF β -ISOFORM SPECIFIC GLUCOCORTICOID RECEPTOR MODULATORS TO REGULATE THE GLUCOCORTICOID SENSITIVITY Tóth Sz., Likó I. and Pázmány* T. Chemical Works of Gedeon Richter Ltd. Department of Molecular Biology * corresponding author, e-mail: [email protected] Multiple sclerosis (MS) is a neurodegenerative disease, characterized by inflammatory mononuclear cell infiltration in the CNS that is mediated by activated macrophages/glial cells and CD4+ T cells, which recognize myelin protein-derived peptides. Pro-inflammatory cytokines are suspected to be important in the pathogenesis of MS. Numerous studies demonstrate that the relapsing-remitting type of MS can be treated successfully by high-dose methylprednisolone, prednisolone and dexamethasone since glucocorticoids decrease the disability score. Despite the beneficial effects, the employment of glucocorticoids is limited on account of their adverse effects. Increasing body of evidence suggest that glucocorticoid receptor (GR) β plays a critical role in the regulation of target cell sensitivity to glucocorticoids. GRβ isoform was found to act as a dominant negative inhibitor of glucocorticoid action, forming a heterodimer with GRα and trapping it within an inactive complex. Moreover, overexpression of the β isoform has been published in different inflammatory diseases (e.g. asthma bronchiale, ulcerative colitis), leading to GC resistance. Involvement of β-isoform specific glucocorticoid receptor modulators that decrease the amount of GRβ could increase the efficacy of glucocorticoid therapy of multiple sclerosis and improve the outcome of the patients. Here we report the establishment and characterization of a colon carcinoma (Caco-2) derived cell line overexpressing recombinant GRβ and containing glucocorticoid response element (GRE) driven secreted alkaline phosphatase (SEAP) reporter plasmide (pGRESEAP) in which decreased transcriptional activity of reporter gene was detected in presence of dexamethasone compared to control cell containing normal amount beta isoform, suggesting, that elevated expression of GRβ impaires GRα-mediated glucocorticoid action. This result confirms the hypothesis that low GRα/GRβ ratio may play important role in the mechanism of glucocorticoid resistance. This hGRβoverexpressing cell line is suitable for high throughput screening of β-isoform specific glucocorticoid receptor modulators.

285

Trabajos del Instituto Cajal. Tomo LXXIX, 2003

ALCOHOL INFLUENCES RAT PLASMA AND BRAIN STEROID CONTENTS MEASURED BY GAS CHROMATOGRAHY/MASSS SPECTROMETRY Vallée M.1,*, Alomary A.A1,2, Koob G.F.1, Fitzgerald R.L.2 and Purdy R.H.1,2. 1

Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California, USA. 2Veterans Affairs Healthcare Center, Veterans Medical Research Foundation, San Diego, California, USA. * Current address: INSERM U259, Institut F. Magendie, Bordeaux, FRANCE

The stress response to acutely administered ethanol has become a well-documented marker for the predisposition to alcoholism in men. The common substrate, GABAAR, for alcohol and neurosteroids suggests that alcohol effects can be mediated by neurosteroids. Accordingly, alcohol-related change of rat neurosteroid levels in brain, such as allopregnanolone, has been recently proposed as a novel mechanism of action for alcohol. We previously found that an acute stress influences plasma and cortical neurosteroid contents in male rats. The purpose of this work was to investigate the impact of an acute administration of ethanol on the concentrations of the steroids testosterone (T), dehydroepiandrosterone (DHEA), pregnenolone (PREG), allopregnanolone (3α,5α-TH PROG, 3α,5α-tetrahydroprogesterone) and epiallopregnanolone (3β,5α-TH PROG) in plasma and frontal cortex of adult male rats. The steroids were extracted from both plasma and brain tissue by a simple solid-phase extraction method, and the steroid contents were quantified by gas chromatography/negative chemical ionization-mass spectrometry. Neurosteroids were measured 30min, 60min and 6h following an acute ethanol administration (2g/kg, i.p.). The blood alcohol levels were approximately 200 mg/dL 30 and 60 min post-injection of ethanol, while 6h post-injection alcohol was no longer detectable in blood. We found that 30 and 60 min following ethanol levels of T, PREG, 3α,5α-TH PROG and 3β,5α-TH PROG markedly increased in plasma and frontal cortex. Finally, 6h following ethanol administration, the steroid levels significantly decreased in plasma and cortex. These data are consistent with an acute effect of alcohol increasing plasma and brain neurosteroids, possibly via steroidogenesis and secretion of these steroids by the adrenals and gonads.

Supported by National Institutes of Health Grants AA06420 and AA07456 from the National Institute on Alcohol Abuse and Alcoholism.

286

AUTHOR INDEX

A Abdelnabi M ..............................1 0 Ábrahám I.M. ............................9 7 Adachi N...................................159 Adriani W................................264 Agnati L.F................................158 Akwa Y.........................................2 0 Allard P ....................................187 Alleva E.............................12, 253 Allieri F ....................................244 Almeida O.F.X ........................154 Alò R.................................251, 260 Aloisi A.M.......................124, 236 Alomari A.A........................286 Alonso A...................................130 Alonso R......................................4 8 Alt J.J........................................176 Amateau S.K ..............................7 7 Andbjer B.................................152 Andréen L ................................169 Anglade I .................................196 Antoniou K..............................227 Apostoli P .....................................7 Arab-Yarmohammadi M ....160 Arai T........................................159 Arnold A.P ...............................109 Arteaga R.................................219 Augustine R.A.........................208 Avola R.....................................190 Axelsson J ...............13, 278, 279 Azcoitia I ...24, 26, 81, 184, 272 B Bäckström T. 50, 72, 127, 144, 147, 150, 169, 177, 187, 248 Bakker J................120, 227, 244 Balfour D.J.K .........................162 Ball G.F ....................................113 Ballabio M ..............24, 192, 272 Balthazart J. 120, 122, 204, 227, 244 Bartolomucci A......................221 Bass A.H...................................206 Baulieu E.E ........19, 20, 22, 269 Beattie J.E ...............................162 Belcredito S....................189, 283 Belelli D....................30, 133, 138 Belle M.C.D..............................223 Bellido I ...................................152 Belloli S.........................................7 Beyer C ..............................39, 235 Bhatt R.........................................8 7 Biasiotto G...................................7 Biggio G...................................238 Birch L......................................212 Birzniece V .....................127, 248 Bixo M ......................................169 Blaustein J.D ..........................115 Boscaro V ................................170

Bourguignon J.-P...........99, 215 Branchi I...........................12, 253 Brito V ...............................85, 235 Broekhoven F. van...................5 0 Brunström B ...........13, 278, 279 Brussaard A.B...........................4 1 Bunn S.J ...................................208 C Caccamo D...............................190 Callachan H...............................3 0 Cambiasso M.J .........................8 5 Campisi A ................................190 Caniglia S .......................194, 216 Cannavò G ..............................190 Canoine V ................................202 Canonaco M...................251, 260 Capone F ..................................253 Carelli A...................................251 Carrer H.F ........................85, 231 Carrillo B ................................225 Carter C.S ................................214 Cascio C ............................44, 128 Cassone M.C ...........................170 Castelino C.B..........................113 Catania C.................................154 Cavarretta I............24, 192, 272 Ceccarelli I.....................124, 236 Celotti F....................................255 Ceresini G................................221 Chavis J.A ..................................8 7 Chiavegatto S.........................155 Chirieleison A ........................221 Ciana P .......................7, 189, 283 Ciarcia G ........................210, 212 Colciago A...............................255 Collado P ........................199, 225 Conrad H.E ......................82, 198 Constantinescu C.S.* ...........157 Cornil C.A................................204 Cortez M.A...............................180 Costa L.G.................................253 Cottone E .................................257 Currò M....................................190 Cushing B.S.............................214 Cutter W......................................7 0 D Dalla C......................................227 Davey H.W ..............................208 De Acetis G .................................1 2 De Bortoli M ...........................240 de Lacalle S.............................171 De Leo G......................................4 4 De Nicola A.F .............................8 3 DeFazio R.A................................9 5 Del Cerro M.C.R.....................242 Della Seta D.............................259 Dessì-Fulgheri F ...........259, 260 Di Lorenzo D................................7 Di S................................................4 2 Díaz M................................48, 130 Dominguez R...........................171

DonCarlos L.L ..........................2 6 Drew P.D ......................................8 7 Droogleever Fortuyn, H.A.....5 0 Druzin M ..................................132 E Edinger K ................................229 El-Etr M ............................22, 269 Etgen A.M ................................102 Etteri S.............................189, 283 Eva C.........................................238 Evrard H.C..............................122 Eychenne B..............................269 F Facciolo R.M..................251, 260 Farabollini F ..........................259 Fasolo A ......................................8 9 Fiorenzani P...................124, 236 Fissore E ..................................262 Fitzgerald R.L.....................286 Forlano P.M ............................206 Franklin R.J.M..........................2 2 Franzoni M.F ..........................257 Freund-Mercier M.J .............178 Freytes P...................................231 Fry J.P.......................................276 Frye C.A. 46, 139, 141, 181, 185, 229 Fusani L ...................................202 Fuxe K..............................152, 158 G Gago N.........................................2 0 Gahr M ..........................................8 Galbiati M........................89, 104 García-Falgueras A .............225 García-Ovejero D ....................2 6 Garcia-Segura L.M. 24, 26, 81, 184, 272 Gass P. .........................................5 8 Gavish M .................................137 Gennuso F.......................194, 216 Gérard A .....................................9 9 Giachino C .................................8 9 Gioiosa L........................262, 264 Giusi G .....................................251 Glaser M ..................82, 198, 200 Gómez-Luque A .....................152 Gonzalez Deniselle M.C .........8 3 Gonzalez L.C ..........24, 192, 272 Gonzalez S..................................8 3 Gorosito S ..................................8 5 Gottlob I ..................................157 Grattan D.R.............................208 Guarneri M ......................44, 128 Guarneri P........................44, 128 Guarneri R .......................44, 128 Guastalla A.............................257 Guennoun R ..............20, 83, 269 Guerra B .....................................4 8 Guerriero G ...................210, 212

Guillamón A ..................199, 225 Gustafsson J.-Å .....................234 Guyomarc’h C........................243 H Haage D....................................132 Halldin K.................13, 278, 279 Han S.-K......................................9 7 Hansson A.C ..................152, 158 Harada N ..............120, 227, 244 Harney S.A .................................3 0 Hau M .......................................202 Havlíková H...........................135 Hazelton J.L ....................10, 214 Hejazi J ....................................160 Herbison A.E .............................9 7 Herd M.B..................................133 Herndon J.G ...........................270 Hill M........................................135 Hines M ....................................118 Hoffman G.E...........................214 Holmes M.C................................5 5 Holmgren C.............................279 Holsboer F..................................5 9 Honda S.................120, 227, 244 Hurd Y.L .....................................6 5 I Ibanez C .....................20, 22, 269 Ientile R....................................190 Ishunina T.A...........................173 Izquierdo M.A.P.....................242 J Jalali K ....................................171 Joëls M ........................................6 1 Johansson I.-M.............127, 248 Johansson S............................132 K Kaczmarek L .................280, 281 Kah O........................................196 Kalita K ...................................280 Kappes V..................................178 Keller E .......................................6 5 Kelly M.J ....................................3 5 Kikuyama S ............................257 Konopka W.............................280 Koob G.F.............................286 Koshibu K ...............................232 Kotelevtsev Y ............................5 5 Kudwa A.E...............................234 Küppers E.........................39, 235 L L’Episcopo F .................194, 216 Labombarda F ..........................8 3 Lacreuse A...............................270 LaFollette A .....................82, 200 Lambert J.J......................30, 145 Lariviere W.R ........................236 Laviola G ................................264 Le Guevel R.............................196 Le W.W.....................................214 Lea R.W....................................223 Lebrethon M.-C...............99, 215

Leonelli E ................24, 192, 272 Levine K...................................214 Levitt P .....................................232 Li W.-W.......................................2 2 Liere P................................20, 269 Likò I...................................285 Lindblad C ..............................127 Liu L..........................................208 Liu R.............................................6 9 Lundgren P..............................144 M Macaione S..............................190 Madeo M .........................251, 260 Maggi A......................7, 189, 283 Magnaghi V ............24, 192, 272 Malcher-Lopes R......................4 2 Malpaux B...............................106 Maney D.L ...............................113 Marchetti B.....................194, 216 Marin R .......................................4 8 Markowska A .........................280 Martini L ........................104, 192 Massardi B..............................266 Matagne V ........................99, 215 Matsumoto A ..........................274 Mayo W.......................................2 8 McCarthy M.M................77, 176 Melcangi R.C. 24, 89, 104, 192, 272 Mele P........................................238 Mendelowitsch A ...................183 Méndez P ..............................26, 81 Mennerick S ............................147 Mensah-Nyagan A.G............178 Menuet A ..................................196 Merchenthale I..........................7 3 Metsis M...................................158 Micevych P ..............................246 Michaelidis T .........................154 Mielke J.G ...............................148 Mileusnic R .............................276 Milman A .................................137 Minder I. ..................................259 Mitchell E.A ............................138 Mitsuyo T ................................159 Mjörndal T..............................187 Moenter S.M...............................9 5 Moore F.L ...................................5 7 Morale M.C.....................194, 216 Morales A ...................................4 8 Morales M ...............................130 Moreno N .................................242 Mosconi G ...............................257 Motte P......................................204 Mullins J.J .................................5 5 Murphy D.G.M ..........................7 0 Mussi P ..........................................7 N Nadal A.....................................130 Negri-Cesi P ............................255 Nelson R.J................................155 Ng B.K................................82, 198 Nichols N.R.................................9 1 Nobahar M ..............................174 Nunemaker C.S..........................9 5 Nuñez J.L .................................176

Nyberg S..........................169, 177 O Orso F .......................................240 Ortega E......................242, 284 Östlund H ...................................6 5 Ottinger M.A.10, 214, 246, 265 P Pakdel F....................................196 Palanza P. 15, 221, 262, 264, 266 Panerai A.E .............................221 Panzica G.C..........225, 240, 244 Papadopoulou-Daifoti Z ....227 Parízek A..................................135 Parmigiani S...........................221 Patte-Mensah C......................178 Paz L..........................................137 Pazmany T..........................285 Peden D ........................................3 0 Pederzani T .............................221 Pellegrini E .............................196 Peretto P ......................................8 9 Pérez-Laso C...........................242 Pérez-Torrero E.....................225 Persad V ..........................148, 180 Peter Y.......................................137 Petralia S.M ............................139 Pianos A ...................................269 Piccoli F.............................44, 128 Pick C.G....................................137 Pincemy G................................243 Pinos H ............................199, 225 Pisu M.G...................................238 Plumari L.................................244 Polzonetti-Magni A.M .........257 Pravettoni A............................255 Prins G.S ..................................212 Purdy R.H...........................286

Q Qiu J.............................................3 5 Quinn M.J.Jr ...................10, 265 R ρnnekleiv O.K ........................3 5 Rρ Raciti G ....................................190 Raica M ....................................167 Rashidy-Pour A ..160, 163, 165 Raviscioni M ...............................7 Razzoli M.................................266 Reid I.C.....................................162 Rhodes M.E ....................141, 181 Rissman E.F ............................234 Ritz M.-F...................................183 Rivera L ...................................242 Robert F.......................................2 0 Robertson D.A ........................162 Rodriguez M ...........................199 Rodriguez-Waitkus P. M ......82, 200 Ropero A.B ..............................130 Rose S.P.R ................................276 Roselli C.E......................210, 212

Rosellini R.A...........................141 Ruggeri G .....................................7 Ruscio M .....................................1 0 Russo D..............................44, 128 S Sacerdote P..............................221 Santucci D...................................1 2 Sartor J.J.................................113 Schlinger B.A..........................202 Schmidt P .................................183 Schreiber S ..............................137 Schumacher M...20, 22, 83, 269 Seckl J.R......................................5 5 Segovia S .................................242 Serra M.....................................238 Seutin V ....................................204 Sharp P.J..................................223 Shi D ..........................................143 Shields S.A..................................2 2 Sica M ..............................240, 244 Sierra A .......................................8 1 Simpkins J.W ............................6 9 Sisk C ........................................101 Snead III O.C ................148, 180 Sommer W ...............................158 Sotiropoulos I........................154 Span P.N ......................................5 0 Spigset O..................................187 Stoffel-Wagner B .....................6 7 Straume M ..................................9 5 Strömberg I.............................158

Strömberg J ............................144 Suidan G ..................................200 Sujkovic E ...............................276 Summitt C.L ............................265 Sundström-Poromaa I ........169, 177, 187 Svare B .....................................141 Swaab D.F ................................173 Swann J.M ...............................117 Szymczak S.....................280, 281

Vegeto E ..........................189, 283 Veiga S. 24, 26, 81, 184, 192, 272 Verkes R.J...................................5 0 Verkuyl M...................................6 1 Viglietti-Panzica C ...............244 Villa R............................................7 Vlad A.G...................................167

T

Wagner E.J ................................3 5 Wahlström G.................127, 150 Walf A.A..........................185, 229 Wang M.D.......................147, 150 Wang Y.T.................................148 Weil-Engerer S .........................2 0 Weizman R ..............................137 Wihlbäck A.-C........................187 Wong C.G.T ...................148, 180 Wu J .............................................1 0

Taherian A.A .................160, 163 Tasker J.G........................42, 143 Testa N.............................194, 216 Thiéry J.-C ..............................106 Thompson N.....................10, 246 Tirolo C...........................194, 216 Todman M ..................................9 7 Torres J.M..........................284 Toth Sz................................285 Tsutsui K .................................178 Turkmen S ......................127, 248 V Vafaei A.A....160, 163, 165, 174 Vallee M..............................286 Valsecchi P ..............................266 van Riel E ...................................6 1 Vanella A .................................190 Vardy A.W........................30, 145

W

Y Yang S.-H....................................6 9 Yau J.L ........................................5 5 Z Zhu D................................127, 150 Zhu T.S ..............................82, 200 Zorumski C.F..........................147