Neuroscience 144 (2007) 412– 423

SHORT-TERM EXPOSURE TO AN ENRICHED ENVIRONMENT ENHANCES DENDRITIC BRANCHING BUT NOT BRAIN-DERIVED NEUROTROPHIC FACTOR EXPRESSION IN THE HIPPOCAMPUS OF RATS WITH VENTRAL SUBICULAR LESIONS B. BINDU, P. A. ALLADI, B. M. MANSOORALIKHAN, B. N. SRIKUMAR, T. R. RAJU AND B. M. KUTTY*

of spatial memory functions in aged mice when exposed continuously for a long period to enriched environment (EE) than providing daily enrichment. The neuroanatomical changes are attributed to alterations in gene expression linked to neuronal structure, synaptic plasticity and transmission (Rampon and Tsien, 2000). Changes in neurotrophins’ mRNA levels in the cortex and hippocampus may also play an important role in experience induced shaping of neuronal connection (Torasdotter et al., 1996). The neurotrophins like brain-derived neurotrophic factor (BDNF) and nerve growth factor play a major role in synaptic plasticity in the hippocampus and other brain regions such as somatosensory and visual cortex (McAllister et al., 1995, 1996). Experience-associated changes in brain morphology leading to functional recovery have been reported after brain injury (Kolb and Gibb, 1991; Kolb et al., 1998; Passineau et al., 2001). Rats subjected to environmental enrichment following transient global cerebral ischemia exhibited enhanced morphological and behavioral plasticity (Briones et al., 2000). Enriched rehabilitation following middle cerebral artery occlusion improved motor performances in staircase reaching and beam traversing tasks in ischemic rats. Enriched housing has in fact produced enhanced dendritic complexity in the layer V pyramidal cells within undamaged motor cortex. Task specific rehabilitation augments neuronal plasticity in the affected region of the brain and thereby promotes functional outcome (Biernaskie and Corbett, 2001). Similarly, studies have reported increased dendritic complexity in the undamaged sensorimotor cortex following exposure to EE in rats with cortical lesion and cerebral infarction (Jones and Schallert, 1994; Johansson, 1996). Young et al. (1999) have also reported the various plasticity evoking properties of environmental enrichment following brain insults. They have suggested that enriched housing helps to optimize the structural morphology in the intact brain. Gobbo and O’Mara (2005) found that EE enhances functional recovery after ischemia and they attributed the recovery to enhanced BDNF expression following environmental enrichment. BNDF/tyrosine kinase B signaling has been shown to mediate morphological plasticity in the hippocampal neurons (Tyler and Pozzo-Miller, 2001). Additionally this may act as an environmental stimuli to preserve some aspects of molecular machinery responsible for neuronal plasticity (Restivo et al., 2005).

Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, P.O. Box 2900, Hosur Road, Bangalore 560 029, India

Abstract—Environmental enrichment promotes structural and behavioral plasticity in the adult brain. We have evaluated the efficacy of enriched environment on the dendritic morphology and brain-derived neurotrophic factor (BDNF) expression in the hippocampus of ventral subicular–lesioned rats. Bilateral ventral subicular lesion has significantly reduced the dendritic architecture and spine density of hippocampal pyramidal neurons. The lesioned rats exposed to enriched housing for 10 days showed a significant degree of morphological plasticity in terms of enhanced dendritic branching and spine density. However, the BDNF expression in the hippocampus remained unchanged following subicular lesion and following environmental enrichment. We suggest the participation of other neurotrophic factors in mediating the synaptic plasticity events following exposure to environmental enrichment in ventral subicular–lesioned rats. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: brain lesion, dendritic atrophy, neuronal plasticity.

Environmental enrichment has been extensively used to demonstrate the behavioral and brain plasticity in response to experience (Rosenzweig et al., 1962; Greenough et al., 1973). Various studies have reported that adult rats exposed to a complex environment consisting of a combination of social stimulation and physical activity induce chemical and anatomical changes and enhanced behavioral performance (Connor and Diamond, 1982; Faherty et al., 2003; Fiala et al., 1978; Knafo et al., 2001; Rampon et al., 2000; Bennett et al., 1969; Diamond et al., 1964, 1966, 1975; Altman and Das, 1965). Additionally different types of enrichment may have different effects as reported by Bennett et al. (2006). They have observed an overall enhancement *Corresponding author. Tel: ⫹91-80-26995170; fax: ⫹91-80-26562121 or ⫹91-80-2656-4830. E-mail address: [email protected] (B. M. Kutty). Abbreviations: BDNF, brain-derived neurotrophic factor; CA1, cornu ammonis 1; CA3, cornu ammonis 3; EE, enriched environment; FITC, fluorescein thiocyanate; NC, normal control; PBS, phosphate-buffered saline; PBSTx, phosphate-buffered saline with Triton X-100; TBS– Tween, 250 nM Tris–HCl, 0.9% NaCl and 0.1% Tween 20; VC, vehicle control; VSL, ventral subicular–lesioned; VSL⫹EE, ventral subicular– lesioned rats reared in enriched housing condition; VSL⫹SH, ventral subicular–lesioned rats reared in standard housing condition.

0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.09.057

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The hippocampus is an area of increasing interest notably due to its vital role in learning and memory (Scoville and Milner, 1957; Amaral and Witter, 1989, 1995). Numerous studies have demonstrated the role of hippocampal formation in spatial information processing (O’Keefe and Nadel, 1978; Morris et al., 1982; Rawlins and Tsaltas, 1983). The hippocampal formation is not a unitary structure; consists of many sub divisions; dentate gyrus, hippocampus proper, entorhinal cortex and subiculum which together constitute to the ‘hippocampal learning system’ (Jarrard et al., 1984; Naber et al., 2000; O’Mara et al., 2001; Witter and Amaral, 1991). The structures of hippocampal formation are connected via the trisynaptic pathway (Amaral and Witter, 1995). These structures have distinct anatomical and physiological characteristics and are likely to have functional contributions to the hippocampal learning system. Previous studies from our laboratory showed that selective lesioning of ventral subiculum reduces the dendritic branching pattern of cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) hippocampal neurons (Nutan and Meti, 1998; Shankaranarayana Rao et al., 2001) neurodegeneration of hippocampal and entorhinal cortical cells (Devi et al., 2003; Govindaiah et al., 1997) and spatial learning impairment in T maze (Laxmi et al., 1999), eight-arm radial maze (Devi et al., 2003) and water maze tasks (Bindu et al., 2005). The behavioral impairment observed has been attributed to the dendritic atrophy or neurodegeneration of the CA1 and CA3 neurons following subicular lesion (Nutan and Meti, 1998; Shankaranarayana Rao et al., 2001; Devi et al., 2003; Govindaiah et al., 1997). In addition, short-term environmental enrichment following subicular lesion has brought behavioral recovery in eight arm radial maze tasks in rats (Bindu et al., 2005). In the present study, we aimed to determine the extent of morphological plasticity of hippocampal pyramidal neurons in ventral subicular–lesioned (VSL) rats following short-term enriched housing. Additionally the effect of enriched housing on BDNF expression in the hippocampus has been investigated.

EXPERIMENTAL PROCEDURES

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neurons that met the inclusion criteria, were included in the study. Accordingly, all 33 animals met the selection criterion and were included for dendritic analysis of CA1 subsector whereas only 26 rats have met the criteria for analysis of dendritic morphology from the CA3 area. Similarly for spine analysis, only 27 animals were used and six were excluded. For immunohistochemical assessment (n⫽25) and Western blot analysis (n⫽25) of BDNF expression in hippocampus, a total of 50 rats were used. Rats were randomly divided into normal control (NC) group (reared in their home cages), vehicle control (VC) group [subjected to bilateral injections of 0.5 ␮l of phosphate buffered saline (PBS, pH⫽7.4) into ventral subiculum stereotaxically] and ventral subicular–lesioned (VSL) group [subjected to chemical lesioning of ventral subiculum bilaterally using ibotenic acid]. The VSL groups were again divided into VSL⫹SH and VSL⫹EE groups. The VSL⫹EE group was exposed to enriched environment and the VSL⫹SH group was reared in standard housing conditions. The fifth group was the NC rats exposed to enriched environment (EE). Environmental enrichment was given for duration of 6 h/day for a period of 10 days. Each day, following 6 h of enrichment, the rats (VSL⫹EE and EE groups) were kept in the standard polypropylene cages (dimensions⫽22.5⫻35.5⫻ 15 cm) with the same littermates.

Housing conditions Standard housing condition. Rats from NC, VC, VSL⫹SH were housed in polypropylene cages of dimensions (22.5⫻35.5⫻15 cm). Two to three rats were housed in each cage containing paddy husk as bedding material that was changed on alternate days. Food and water were provided ad libitum. They were kept in a well-aerated room and 12-h light/dark cycle was maintained. The room temperature was maintained at 26⫾2 °C.

Enriched housing conditions Both the VSL⫹EE and EE groups were exposed to enriched housing conditions for 6 h daily (from 10:00 AM to 4:00 PM) for 10 days. Enriched housing was provided by exposing the rats to a specially designed cage with a dimension of 81.5⫻61⫻45 cm, the walls made of metal wire mesh and the bottom with wooden platform. A sliding door was provided for replacing the paddy husk and placing the rats. Eight to 10 rats were housed in this cage to provide social stimulation. The cage was equipped with various exploratory materials like plastic tunnels (30 cm long and 12 cm diameter), metal platforms (40 cm long⫻25 cm width and 25 cm long⫻15 cm width), balls, rattle, ladders, and toys of various sizes and textures (wood, metal and plastics). The rats were exposed to novelty stimulation by changing the exploratory objects daily. Food and water were provided ad libitum.

Subjects Two months old male Wistar rats were obtained from the Central Animal Research Facility of the National Institute of Mental Health and NeuroSciences, Bangalore, India. A total of 83 rats were used for the study. Rats were housed in polypropylene cages (dimensions 22.5⫻35.5⫻15 cm) at a room temperature 26⫾2 °C and were maintained on a 12-h light/dark schedule. Food and water were provided ad libitum. The experiments were carried out in accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996) and the institutional animal ethics committee. All efforts were made to minimize both the number and suffering of the animals used. Thirty-three rats were used for histological assessment of dendritic morphology by Golgi staining. All 33 rats were considered for analysis for CA1 morphology whereas, only 26 rats were considered for analysis for CA3 morphology and seven rats were excluded since they did not meet the selection criterion. Those rats, which had 10

Lesioning of the ventral subiculum The animals from VSL groups (VSL⫹SH and VSL⫹EE) were anesthetized with combination of ketamine (75 mg/kg body weight, i.p.) and xylazine (10 mg/kg body weight, i.p.) and subsequently with lignocaine (1%) as local anesthesia. The animal was fixed on a stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). The flat skull stereotaxic coordinates were adapted from (Paxinos and Watson, 1982) rat atlas. Ibotenic acid (0.5 ␮g/0.5 ␮l/site) was injected into the ventral subiculum with the pre-determined stereotaxic coordinates: AP⫽⫺7.0 mm, ML⫽5.0 mm and DV⫽5.3 mm with the help of a micro injector at the rate of 0.5 ␮l/min. The VC underwent the same surgical procedures, with the exception that they received the same volume of PBS instead of ibotenic acid; 24 h after surgery, the VSL rats were exposed to either standard housing or enriched housing conditions. Ten days following surgery, the rats were subjected to various experimental protocols.

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Fig. 1. Schematic representation of Sholl’s (1956) method for quantification of dendritic branching in the hippocampal pyramidal neurons. The dendritic branching points and intersection of the apical dendrites were counted in successive radial segments of 50 ␮m taking the center of soma as the center of the circle. The point at which dendrites cross the concentric circles was considered the intersection point. Scale bar⫽50 ␮m.

Morphological analysis of hippocampal pyramidal neurons At the end of the experimental procedures, the rats from all five groups were killed by an overdose of pentobarbitone. The brains

were exposed rapidly and the hippocampus was immediately dissected and processed for rapid Golgi staining as described in earlier studies (Shankaranarayana Rao et al., 2001). Sections 120 ␮m thick were taken in horizontal plane using sledge microtome (Meopta, Czechoslovakia). Sections were collected serially, dehydrated in absolute alcohol, cleared in xylene and mounted onto glass slides containing distyrene phosphate xylene and coverslipped. All the slides were coded to overcome the experimenter’s bias. The CA1 and CA3 neurons were viewed randomly using Leitz microscope (Leica, Wetzlar, Germany) and the neurons, which fulfilled the following criteria, were selected for the study; (i) the cell type must be identifiable (ii) dark and consistent silver impregnation throughout the extent of all the dendrites (iii) the presence of untruncated dendrites (iv) relative isolation from the neighboring impregnated neurons. In the CA3 subsector, we have chosen the short-shafted neurons for camera lucida tracing as they have greater number of apical dendritic branches compared with long-shafted neurons and it is considered that they receive more number of mossy fiber input. The short-shaft pyramidal neurons are characterized by short apical shafts, a large number of thorny excrescences, and densely branched apical and basilar trees. Short-shaft neurons have more total dendritic length than long-shaft neurons indicating that each type receives a different number of synapses per neuron (Fitch et al., 1989). Camera lucida tracings were obtained from CA1 and short shaft CA3 pyramidal neurons at a magnification 625⫻ using a Leitz microscope. The quantification and analysis of dendritic branching points and dendritic intersections were carried out using Sholl’s (1956) analysis. Concentric circles of 50-␮m distances apart were drawn on a tracing paper using a stage micrometer scale at the same magnification at which the neurons were drawn (Fig. 1). Keeping the center of the circle on the center of the cell body as the reference point, dendritic intersections and branching points were measured from the soma by calculating values in each successive concentric segment. The dendritic branching and intersections were studied up to a length of 250-␮m distance from the center of the soma, to include the stratum lucidum and radiatum of CA1 and CA3 (short shaft) pyramidal neurons wherein the apical dendrites receive major afferent inputs (Paxinos, 1995).

Spine density Spines from the primary dendrites of CA1 neurons were visualized under oil immersion (cedar wood oil, Merck Ltd., Mumbai, India) objective using Olympus BX51 microscope (Olympus, Melville, NY, USA) and analyzed using Neurolucida image analysis 6.21.2

Fig. 2. Representative camera lucida tracings of hippocampal CA1 pyramidal neurons from different groups; NC, EE, VC, VSL⫹SH and VSL⫹EE drawn under 625⫻ magnification. Note the reduced dendritic arborization in the VSL⫹SH rat and the restoration of dendritic branching in VSL⫹EE rat following EE. Scale bar⫽50 ␮m.

B. Bindu et al. / Neuroscience 144 (2007) 412– 423 (Microbrightfield, Williston, VT, USA) system. Any branch arising from the main shaft between 50 and 250 ␮m was considered as primary branch. All protrusions, which had direct contact with the primary dendrite, were counted as spines. Five primary apical dendrites were selected randomly from the main shaft of CA1 pyramidal neurons. Spine density was counted up to 80 ␮m length along each primary dendrite. The 80 ␮m length was further divided into eight segments of 10 ␮m length was estimated along five primary apical dendrites in the CA1 region. A total of 27 rats were used (NC⫽5 rats, VC⫽6 rats, EE⫽5 rats, VSL⫹SH⫽6, and VSL⫹EE⫽5 rats).

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white (highly fluorescent/bright) pixels. Thereafter, using the ‘detect’ command, the CA3 sub-region of hippocampus falling in the ‘image frame of 500⫻500 ␮m2’ was ‘selected.’ The image was manually ‘thresholded’ between the values of 0 –255, to select only the stained area for intensity measurement and thereby obviating the interference by ‘non-specifically stained area/background’ if any. The intensity values were generated by the software and tabulated using the Excel program. Similar protocol was followed for CA1 sub-region. The images of the hippocampus were captured from left and right side of the coronal brain section and the values of each of the slides were pooled to obtain mean values of each animal (Alladi et al., 2002).

Immunohistochemical assessment of BDNF in CA1 and CA3 regions of hippocampus

SDS PAGE and Western blotting

For immunohistochemical studies a total of 25 rats were used. Each group comprised five rats. The groups were NC, VC, EE, VSL⫹SH and VSL⫹EE. Rats were deeply anesthetized and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde.

For Western blotting, five rats from each group (NC, VC, VSL⫹SH, VSL⫹EE and EE) were used. The rats were decapitated under halothane anesthesia and brains were removed. The hippocampus from both sides was dissected out and homogenized in ice cold buffer containing 10% sucrose, 1 mM EDTA in 20 mM Tris–HCl (pH 7.4) and centrifuged at 12,000 r.p.m. for 12 min

BDNF immunohistochemistry The brains were postfixed in the same fixative (4% PFA) for 48 h, followed by three washes in chilled 0.1 M phosphate buffer (pH 7.4). Each wash lasted for an hour. The brains were then cryoprotected in 15%, 20% and 40% sucrose and finally embedded in tissue freezing medium for cryosectioning. Coronal sections of 30 ␮m were obtained at the level of dorsal hippocampus using cryostat (Leica) and collected in a deep well plate containing 0.1 M-phosphate buffer (pH 7.4). Every sixth section was used for the study. The sections were washed with PBSTx (3⫻5 min, phosphate-buffered saline with 0.9% NaCl and 0.05% Triton X-100) followed by incubation in 5% fat free skimmed milk solution for 4 h at room temperature to block nonspecific staining. The sections were then incubated in primary antibody (antirabbit BDNF IgG Fraction, 1:200, Chemicon International, Temecula, CA, USA) for 72 h at 4 °C. Primary antibody was removed and sections washed with PBSTx thrice for 5 min each and then incubated in secondary antibody (1:200, antirabbit IgG FITC (fluorescein thiocyanate) conjugated; Sigma, St. Louis, MO, USA) overnight at 4 °C. The sections were then washed thrice with PBSTx, mounted with 65% glycerol to avoid dehydration of the sections. The sections were coverslipped and sealed using nail enamel.

Microscopy and quantification Confocal imaging. The sections were then viewed under confocal microscope for BDNF expression using blue filter at an excitation of 488 nm under 4⫻ magnification using an Olympus FV1000 confocal microscope of our central facility. For fluorescence intensity measurement, the sections were viewed and images captured using a Leica confocal microscope (Leica TCS SL). The FITC fluorochrome bound to the primary antibody against BDNF was excited using an argon laser (488 nm) and detected using standard FITC filters. All the images were captured under 20⫻ magnification at a constant PMT (photo multiplier tube) voltage of 557 V and each image was averaged four times. The image format was kept constant at 1024⫻1024 pixel resolution. Other features such as pinhole diameter, scan speed etc. were also maintained uniformly. For each brain, three sections were stained along the rostrocaudal axis and subjected to intensity measurement. The eight bit images captured on the confocal microscope were analyzed ‘off-line’ using Q-Win Plus software for image analysis (Leica). Briefly, the captured images were converted into monochrome i.e. black and white images for estimation of the staining intensity. The fluorescence intensity was measured on a scale of 0 –255, where ‘0’ refers to black (dark) and ‘255’ refers to

Fig. 3. (A, B) Apical dendritic branching points and intersections of the hippocampal CA1 pyramidal neurons in NC (n⫽6 rats), NC exposed to EE (n⫽9 rats), VC (n⫽6 rats), the VSL⫹SH (n⫽6 rats) or VSL⫹EE (n⫽6 rats). Each value represents the mean⫾S.E.M. VSL⫹SH vs. NC, VC, EE (** P⬍0.01,*** P⬍0.001) and VSL⫹EE vs. VSL⫹SH (␺␺ P⬍0.01, ␺␺␺ P⬍0.001); two way repeated measures ANOVA followed by Tukey’s post hoc test. Note the significant decrease in the branching points and intersections of CA1 apical dendrites in the VSL⫹SH rats and the restoration of dendritic branching points and intersections in VSL⫹EE rats.

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at 4 °C using refrigerated centrifuge (Mikro22R, Hettich, Zentrifugen, Germany). Protein concentration was determined (Lowry’s method; Bangalore Genei Pvt. Ltd. India). Sixty microgram samples were loaded onto 10% bis-acrylamide gel and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylene difluoride membrane (BioTraceTM PVDF, Muncie, IN, USA). The membrane was washed with TBS–Tween (250 nM Tris–HCl, 0.9% NaCl and 0.1% Tween 20) blocked with 10% non-fat milk in TBS–Tween for 2 h. The membrane was incubated with rabbit polyclonal antibody against BDNF (1:500, Chemicon International) overnight. Subsequently the membranes were washed in TBS–Tween and incubated with biotinylated antirabbit secondary antibody (1:500, Vector Laboratories, Burlingame, CA, USA) for 90 min. The blots were further developed with avidin biotin complex (ABC, Vector laboratories, USA) for 90 min and the color developed using 3=3= diaminobenzidine hydrochloride. ␤-Actin (Sigma) was used as loading control.

Quantification of Western blotting The blots were quantified using Quality 1 Gel documentation system (Gel Doc 2000, Bio-Rad Inc., Hercules, CA, USA). The peak optical intensity of bands was detected using Image analysis software.

Histological verification of subicular lesion site The rats from VSL⫹SH and VSL⫹EE (n⫽5/group) were used for histological verification of subicular lesion site. The rats were deeply anesthetized and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. The brains were subsequently removed and kept in 4% paraformaldehyde for 48 h. Coronal sections 30 ␮m thick were taken using vibratome (Vibratome Company, St. Louis, MO, USA) at the level of the ventral subiculum to examine the site of lesion and the extent of subicular damage. Sections were stained with Cresyl Violet. The data presented here are with lesion confined exclusively to bilateral ventral subiculum and the lesion had not spread to other areas with a lesion size of 1.5–2 mm3.

Statistical analysis The dendritic branching points, intersections and spines were statistically analyzed using two way repeated measures analysis of variance followed by Tukey’s post hoc test. For Golgi analysis 10 neurons were averaged to give one value for each rat. The

intensity for BDNF immunostaining was statistically analyzed by one-way ANOVA followed by Tukey’s post hoc test. Peak optical density of the BDNF bands (Western blots) was statistically analyzed by one-way ANOVA.

RESULTS Dendritic morphology of hippocampal pyramidal neurons Bilateral ventral subicular lesion produced dendritic atrophy in the CA1 (main effect of group: F(4,112)⫽39.94 P⬍0.001) and CA3 pyramidal neurons (main effect of group: F(4,84)⫽57.25 P⬍0.001) of hippocampus. The dendritic branches were reduced by 52 and 55% in the CA1 and CA3 areas, respectively. Two-way ANOVA with repeated measures revealed a significant interaction between the groups and segments in both CA1 branching points (F(16,112)⫽4.96 P⬍0.001) and CA3 branching points (F(16,84)⫽4.82 P⬍0.001). Further, there was a significant interaction in terms of number of dendritic intersections in the CA1 (F(16,112)⫽2.73 P⬍0.01) and CA3 (F(16,84)⫽5.5 P⬍0.001) subsectors. Representative camera lucida drawings of CA1 and CA3 pyramidal neurons were depicted in the Figs. 2 and 4 to highlight the dendritic morphology in each group. The decrease was prominent along the 50 –250 ␮m and 100 –250 ␮m distance from the soma in the CA1 (Fig. 3A, B) and CA3 neurons (Fig. 5 A, B), respectively. Short-term exposure to EE produced considerable amount of neuronal plasticity in the subicular-lesioned rats, as they showed more dendritic branching in the hippocampal pyramidal neurons. Accordingly, the VSL⫹EE group showed significant increase in the dendritic branching points and intersections in both CA1 (Figs. 2, 3A, B) and CA3 pyramidal neurons when compared with the VSL⫹SH group (Figs. 4, 5A, B). However, such shortterm enrichment produced only partial recovery in the lesioned rats (VSL⫹EE) and was not comparable with that of the NC and VC groups.

Fig. 4. Representative tracings (camera lucida) of hippocampal CA3 pyramidal (short shaft) neurons of NC, EE, VC, VSL⫹SH and VSL⫹EE drawn at 625⫻ magnification. Note the lesion induced dendritic atrophy in the VSL⫹SH rat and the restoration of dendritic branching in the VSL⫹EE rat following exposure to EE. Scale bar⫽50 ␮m.

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NC/VC groups (Figs. 7, 8A, B). BDNF expression remains unchanged in the dorsal hippocampus following selective lesioning of ventral subiculum. No significant difference was observed between the VSL⫹SH and VSL⫹EE groups (Figs. 7, 8A, B). Western blot analysis (Fig. 9A) and the relative optical density (Fig. 9B) of BDNF in the hippocampus showed a specific band at 30 kDa. No significant change in BDNF levels was observed between the groups; NC, VC, VSL⫹SH, VSL⫹EE and EE groups (F(4,20)⫽0.0155, P⬎0.05). No enhancement in BDNF expression was observed in the EE group when the whole hippocampus was analyzed. Histological studies Histological examination of brain sections revealed that the ibotenic acid injections had damaged completely the ven-

Fig. 5. (A, B) Apical dendritic branching points and intersections of the hippocampal CA3 pyramidal neurons of NC (n⫽5 rats), VC (n⫽5 rats), NC exposed to EE (n⫽5 rats), the VSL⫹SH (n⫽6 rats) or VSL⫹EE (n⫽5 rats). Each value represents the mean⫾S.E.M. VSL⫹SH vs. NC/VC/EE (*** P⬍0.001) and VSL⫹EE vs. VSL⫹SH (␺␺ P⬍0.01, ␺␺␺ P⬍0.001); two way repeated measures ANOVA followed by Tukey’s post hoc test. Note the significant reduction in the number of apical dendritic branching points and intersections in VSL⫹SH and an increase in branching points and intersections in the VSL⫹EE group following exposure to EE.

The spine density along the primary branches (20 – 80 ␮m length) of CA1 neurons was also reduced significantly (main effect of group: F(4,154)⫽26.89 P⬍0.001; group⫻segment: F(28,154)⫽2.48 P⬍0.001) following ventral subicular lesion (Fig. 6A, B). In addition to enhanced dendritic branching, the VSL⫹EE rats showed a complete reversal of spine density and were comparable to that of NC and VC and EE groups (Fig. 6A and B). We did not find any enhancement of dendritic branching and spine density in the EE group following exposure to EE and both were comparable with that of the NC and VC groups. Assessment of BDNF expression in the hippocampus EE rats (NC rats exposed to enriched housing) showed a significant increase in intensity of BDNF expression in the CA1 F(4,20)⫽8.28 P⬍0.001 and CA3 F(4,20)⫽11.13 P⬍0.001) regions of the dorsal hippocampus when compared with

Fig. 6. (A) Representative images of the primary branch of hippocampal CA1 pyramidal neurons under 100⫻ magnification to show the spine density in NC, EE, VC, VSL⫹SH and VSL⫹EE. The spine density is relatively reduced in the VSL⫹SH. The spine density in the VSL⫹EE group is comparable to that of NC, VC and EE. Scale bar⫽5 ␮m. (B) Spine density/80 ␮m distance in the primary apical dendrites of hippocampal CA1 pyramidal neurons along 50–250 ␮m distance from the soma. The groups include the NC (n⫽5 rats), VC (n⫽6 rats), NC exposed to EE (n⫽5 rats), the VSL⫹SH (n⫽6 rats) or VSL⫹EE (n⫽5 rats). Each value represents the mean⫾S.E.M. VSL⫹SH vs. NC/VC/EE (* P⬍0.05, *** P⬍0.001) and VSL⫹EE vs. VSL⫹SH (␺ P⬍0.05, ␺␺ P⬍0.01, ␺␺␺ P⬍0.001); two way repeated measures ANOVA followed by Tukey’s post hoc test. The spine density in the VSL⫹SH group is reduced significantly where as the VSL⫹EE group shows a significant degree of reversal.

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Fig. 7. Confocal immunofluorescent images of dorsal hippocampus showing BDNF expression in the CA1 and CA3 areas in the NC, EE, VC, VSL⫹SH or VSL⫹EE and negative control observed under 4⫻ magnification of confocal laser scanning microscope. Note the enhanced intensity of BDNF expression in the EE group. Scale bar⫽300 ␮m.

tral subiculum and the lesion has not spread to other areas (Fig. 10A, B). Ibotenic acid has produced a lesion size of 1.5–2 mm3 in the VSL rats. No differences in the lesion size was observed between the VSL⫹SH and VSL⫹EE groups.

DISCUSSION Bilateral ventral subicular lesion resulted in considerable atrophy of dendritic arborization in both the CA1 and CA3 pyramidal neurons of hippocampus. The spine density in the stratum radiatum of CA1 pyramidal neurons was also significantly reduced following subicular lesion. Earlier studies have also demonstrated the dendritic atrophy of hippocampal neurons following selective subicular lesion (Nutan and Meti, 1998; Shankaranarayana Rao et al., 2001). Subiculum is an area of hippocampal formation known to be a major output structure of CA1 hippocampus and has got both afferent and efferent connections with the hippocampus and entorhinal cortex (Witter et al., 1989; Sharp et al., 1990). The anatomical connections between hippocampal structures suggest the possibility of dendritic atrophy of hippocampal pyramidal neurons following subicular lesion. The dendritic atrophy was studied mainly in the stratum lucidum and radiatum wherein the apical dendrites receive major afferent inputs; CA1 receives most of the Schaffer collaterals from CA3. The apical dendrites of CA3 (short shaft neurons) pyramidal neurons receive mossy fiber inputs, at the proximal segment and entorhinal fibers at the molecular layer, septohippocampal fibers at the middle and terminal dendritic regions and nor adrenergic fibers as well as associational, commissural and collateral inputs at the stratum radiatum. We have observed the dendritic atrophy mainly in the stratum radiatum following ventral subicular lesion. Perhaps the ventral subicular lesion would have affected the afferent inputs to CA1 and CA3 neurons leading to dendritic atrophy. Subicular lesion-induced dendritic atrophy of hippocampal structures

has been correlated with impaired learning performances (Bindu et al., 2005; Devi et al., 2003; Jarrard, 1978; Jarrard et al., 1984; Laxmi et al., 1999) and we have observed that subicular lesion impaired the behavioral performances in radial arm maze and water maze tasks (Bindu et al., 2005). Hence, it may be plausible to correlate the dendritic atrophy and decreased spine density of hippocampal neurons with that of impaired behavioral performances. Other studies have also reported such correlation between hippocampal neuronal atrophy and learning (Volpe et al., 1989; Sunanda et al., 1995). Chronic exposure to stress has been shown to cause hippocampal neuronal cell loss and dendritic atrophy of hippocampal neurons (Sunanda et al., 1995). However, they have reported a significant increase in the spine density and excrescences following chronic restraint stress. They have suggested this phenomenon of enhanced spine density as a result of increased activity of the glutamatergic system since restraint stress is reported to enhance the activity of glutamatergic neurons (Gilad et al., 1990; Moghaddam, 1993; Moghaddam et al., 1994). Volpe et al. (1989) have also correlated the selective loss of hippocampal CA1 neurons with memory impairment. Human studies have also shown the correlation between structural plasticity and learning. The decline of cognitive and other behavioral performances associated with Downs syndrome, Alzheimer’s disease, senile dementia and schizophrenia has in fact been correlated with the altered neuronal plasticity (Catala et al., 1988; Garey et al., 1998; Glantz and Lewis, 2000; Mehraein et al., 1975). Short-term exposure to EE reversed the lesion-induced dendritic atrophy and spine density in the subicularlesioned rats. Environmental enrichment has been extensively used to demonstrate the behavioral and brain plasticity in response to experience (Rosenzweig et al., 1962; Greenough et al., 1973). Adult rats exposed to a complex environment consisting of a combination of social interac-

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control rats reared in standard housing conditions. Other studies have also reported the positive effect of EE following lesion and ischemia (Gobbo and O’Mara, 2005; Johansson, 2003). It is assumed that the enrichment would have caused an augmentation of intrinsic plasticity process of the surrounding non-injured regions and thereby compensate for the lost function (Biernaskie and Corbett, 2001; Briones et al., 2000; Johansson, 1996). Perhaps to produce a complete recovery of hippocampal neurons following subicular lesion, appropriate environmental enrichment may be warranted for a prolonged period of time. The dendritic morphology of CA3 neurons has been reported to be modulated by early environmental conditions in guinea pigs (Bartesaghi and Serrai, 2001). They have observed that the dendritic morphology of long shaft neurons of CA3 area is significantly reduced following isolation. We have chosen the short-shafted CA3 neurons for dendritic analysis as they have greater number of apical dendritic branches compared with long-shafted neurons and studies have reported the changes of dendritic morphology of short shaft CA3 pyramidal neurons following exposure to EE (Juraska et al., 1989). In the present study, short-term enrichment however has brought a total reversal of spine density in the VSL rats. Increased spine density and associated enhancement in learning and memory functions have been reported in the ischemic rats following enrichment (Moser et al., 1994, 1997; Rampon et al., 2000). Exposure to enriched housing has been shown to

Fig. 8. (A, B) Intensity of BDNF expression in the CA1 and CA3 region of the hippocampus (n⫽5/group) in NC, VC, NC exposed to EE, in the VSL⫹SH or VSL⫹EE. Each value represents the mean⫾S.E.M. *** NC vs. EE (P⬍0.001); one way ANOVA followed by Tukey’s post hoc test. Note a significant increase in BDNF expression in the CA1 and CA3 areas of dorsal hippocampus in the EE group when compared with NC/VC/VSL⫹SH/VSL⫹EE.

tion and learning show many positive effects on behavioral performance along with associated changes in brain morphology, increase in hippocampal thickness, dendritic arborization, increased synapses per neuron, neurogenesis, glial proliferation and expression of various growth promoting factors such and nerve growth factor, BDNF (Falkenberg et al., 1992; Fiala et al., 1978; Torasdotter et al., 1996, 1998; Turner and Greenough, 1985; van Praag et al., 2002; Walsh et al., 1969; Williams et al., 2001). However in the present study, with such short-term enrichment (6 h per day for 10 days), no obvious changes in dendritic morphology was seen in NC rats (EE group). Perhaps, long-term exposure may be required for observing any obvious changes in dendritic morphology in the normal rats as reported by Greenough et al., 1973; Rosenzweig et al., 1962. However, the VSL⫹EE rats showed enhanced dendritic morphology following such short exposure to EE though it could not match with the dendritic arbors of

Fig. 9. (A, B) Representative immunoblot and relative optical density for BDNF in the hippocampus in different groups; NC, EE, VC, VSL⫹SH and VSL⫹EE). Data are expressed as mean⫾S.E.M. Oneway ANOVA showed no significant changes across groups.

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Fig. 10. (A, B) Schematic representation to show the extent of lesion of ventral subiculum by ibotenic acid in the VSL⫹SH and VSL⫹EE rats. Shaded areas indicate the extent of lesion at different planes from bregma (⫺4.8 to ⫺7.8) (Paxinos and Watson, 1982). Ibotenic acid injections had damaged completely the ventral subiculum and the lesion has not spread to other areas. No differences in the lesion size were observed between the VSL⫹SH and VSL⫹EE groups.

enhance the spine density in the N-methyl D-aspartate knockout mice (Rampon et al., 2000) and hence the learning performances. It may be hypothesized that the presence of spines may enhance the synaptic efficacy and thereby enhance the excitability of the network involved in learning processes. Probably the restoration of dendritic architecture in the CA1 and CA3 pyramidal neurons together with increased spine density may contribute for better learning performance of VSL rats exposed to EE. This kind of short-term enrichment showed the behavioral recovery only in the radial maze task and not in the water maze task (Bindu et al., 2005). Differences in the extent to which differing brain systems benefit from enrichment may account for such differential effects on specific tasks. The BDNF levels remain unchanged in the lesioned rats housed in either standard or enriched conditions. On the other hand, enriched housing has promoted both structural and functional plasticity in the lesioned rats (VSL⫹EE) when compared with those exposed to standard housing (VSL⫹SH). Lapchak et al. (1993) have also reported the hippocampal synaptic plasticity–related changes following entorhinal cortical lesion. Their study

also failed to support the role of BDNF in hippocampal remodeling following lesion. They suggested the involvement of other neurotrophins such as NT3 or NT4/5 in inducing the hippocampal plasticity. Gobbo and O’Mara (2005) found that EE enhances functional recovery after ischemia and they attributed the recovery to enhanced BDNF expression following environmental enrichment. Enhanced BDNF levels were seen in ischemic rats reared in both housing conditions: enriched as well as standard housing conditions though the behavioral recovery was seen only in the groups exposed to EE and not in the rats kept in standard conditions. They suggest that ischemic insults perhaps induce enhanced BDNF expression as a compensatory reaction of the brain (Gobbo and O’Mara, 2005; Korte et al., 1995). In the present study however no such enhancement in BDNF expression was observed following lesion. This may be due to the differences in the methodology adopted. They have exposed the rats to enriched housing conditions for a long duration of 10 weeks; 6 weeks before ischemic insults and 4 weeks following ischemia was induced in the rats. Additionally, BDNF expression was determined after studying the behavioral

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performances in the rats, whereas in the present study, the rats (VSL⫹EE) were exposed to enriched housing for a short duration; 6 h per day for a period of 10 days following ventral subicular lesion. They were not given any prior exposure to EE before lesioning of ventral subiculum. Moreover these rats were not subjected for behavioral performances prior to the assessment of BDNF. In the EE group, immunocytochemical studies showed an enhanced expression of BDNF in the CA1 and CA3 areas separately in the dorsal hippocampus. However with Western blotting studies the collective effect on the hippocampus per se has been studied using the whole hippocampus (dorsal and ventral) for analysis of BDNF expression. This could be the reason that we could not replicate the increase in BDNF with environmental enrichment. On the whole, short-term exposure to enriched housing has brought a significant degree of plasticity in terms of enhanced dendritic architecture, and spine density of hippocampal neurons in the subicular-lesioned rats. The BDNF levels remain unchanged following lesion and following exposure to environmental enrichment. Exposure to different environmental stimulations such as continuous exposure (Bennett et al., 2006) and physical exercise using a voluntary running wheel (Pietropaolo et al., 2006) has been shown to exert multiple effects on the brain. However in our study, we have adopted a short-term daily enrichment without physical exercise in a running wheel. This may account for the unchanged BDNF levels in the hippocampus. We also suggest the participation of other neurotrophic factors in mediating the synaptic plasticity events following environmental enrichment as reported earlier (Moser et al., 1994; Puurunen et al., 2001). Acknowledgment—The authors greatly acknowledge the Life Science Research Board (LSRB, DRDO), New Delhi for providing the financial support to carry out the research study (Project No. LSRB-48/2003/EPB). We wish to acknowledge with thanks the timely help rendered by Prof. M. N. Subhash and Mr. Devaraj from the Department of Neurochemistry for providing the Gel Documentation facility, Prof. Preeti Joshi, Department of Biophysics for taking the confocal images of hippocampus and Dr. K. Thennarasu, Associate Professor, Department of Biostatistics for statistical help.

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(Accepted 20 September 2006) (Available online 9 November 2006)