Comparative Biochemistry and Physiology, Part A 148 (2007) 657 – 663 www.elsevier.com/locate/cbpa

Anorexigenic effects of central neuropeptide S involve the hypothalamus in chicks (Gallus gallus) Mark A. Cline ⁎, David C. Godlove, Wint Nandar, Christie N. Bowden, Brian C. Prall Department of Biology P.O. Box 6931, Radford University, Radford, Virginia 24142, USA Received 31 July 2007; received in revised form 14 August 2007; accepted 14 August 2007 Available online 21 August 2007

Abstract Neuropeptide S (NPS) affects appetite-related processes in mammals. However, its role in avian biology is unreported. We hypothesized that intracerebroventricular (ICV) NPS would cause anorexigenic effects in chicks (Gallus gallus). To evaluate this, Cobb-500 chicks were centrally injected with multiple doses (0, 0.313, 0.625 and 1.250 μg) of NPS. NPS-treated chicks responded with decreased feed and water intake. The effect on water intake was secondary to feed intake, because fasted NPS-treated chicks did not reduce water intake. ICV NPS injection also reduced plasma corticosterone concentration. We monitored behavior and found decreased ingestive and exploratory pecking, jumping, locomotion, and increased time spent in deep rest. We hypothesized that the anorexigenic effects were hypothalamic in origin and quantified c-Fos reactivity in the lateral hypothalamus (LH), paraventricular nucleus (PVN) and ventromedial hypothalamus (VMH) after NPS treatment. NPS was associated with decreased c-Fos reactivity in the LH, increased reactivity in the PVN and had no effect in the VMH. When NPS was injected directly into the LH and PVN, chicks responded with decreased feed and water intake, suggesting that effects were directly mediated by these nuclei. We conclude that ICV NPS causes anorexigenic effects in chicks, without directly affecting water intake, and the hypothalamus is involved. © 2007 Elsevier Inc. All rights reserved. Keywords: Chick; Feed intake; Hypothalamus.; Neuropeptide S

1. Introduction Neuropeptide S (NPS), a 20 residue neurotransmitter, affects many physiological processes in mammals. The name “S” comes from its N-terminal serine residue. In humans, NPS is part of a larger precursor molecule that contains several proteolytic processing sites (Reinscheid and Xu, 2005). NPS binds to a previously orphan G protein-coupled receptor (Xu et al., 2004), now named the NPS receptor (Bernier et al., 2006; Roth et al., 2006). Although NPS appears to be absent from fish genomes, sequences coding for NPS are present in the chicken and other later-diverging vertebrate genomes (Reinscheid, 2007). In rats, NPS is primarily expressed in the brainstem with proximity to Abbreviations: ICV, intracerebroventricular, LH; lateral hypothalamus; LIN, linear contrast; NPS, neuropeptide S; PTSE, pooled standard error of the treatment mean; PVN, paraventricular nucleus; QUD, quadratic contrast; TIM, time post injections; TRT, treatment effect; VMH, ventromedial hypothalamus. ⁎ Corresponding author. Tel.: +1 540 831 6431; fax: +1 540 831 5129. E-mail address: [email protected] (M.A. Cline). 1095-6433/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2007.08.016

the locus coeruleus and Barrington's nucleus and NPS precursor mRNA has been isolated from peripheral organs including the thyroid, salivary and mammary glands (Xu et al., 2004). When NPS was administered directly into the paraventricular nucleus (PVN) and lateral ventricle system of rats, a linear dosedependent decrease in feed intake was observed (Smith et al., 2006; Beck et al., 2005). The decrease in feed intake coincided with an increase in plasma corticosterone concentration (Smith et al., 2006). However, Niimi (2006) reported that NPS causes a potent orexigenic effect in rats. In rats, NPS activates the PVN and lateral hypothalamus (LH; Niimi, 2006). Behaviors unrelated to ingestion are also affected by central NPS. Rats which had been habituated to a testing chamber exhibited increased locomotion after central NPS, suggesting stimulated arousal (Xu et al., 2004). This conclusion is further supported by the observation that NPS-increased rearing activity in addition to increasing ambulatory movement in rats. NPS reduced the magnitude of anxiety-like responses of rats caused by stressors or unfamiliar environments (Xu et al., 2004).

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Information on the biological role of NPS in birds is lacking. We hypothesized that NPS would reduce feed intake in broilertype chicks, and designed experiments to further explore this effect. Specifically, we tested multiple NPS doses on feed and water intake and plasma corticosterone concentration after intracerebroventricular (ICV) injection. We also designed an experiment to measure the effects of ICV NPS on behaviors unrelated to appetite, and measured the effects on neuronal activation in the LH, PVN and ventromedial (VMH) hypothalamus. Finally, we measured the effects of direct microinjection of NPS into the LH and PVN on feed and water intake.

2.3. Experiment 1: effect on feed intake

2. Experimental procedures

Chicks, fasted for 180 min, were randomly assigned to receive 0, 0.313 (0.14 nmol), 0.625 (0.28 nmol) or 1.25 μg (0.56 nmol) NPS by ICV injection. Doses were based on a preliminary study. After injection chicks were returned to their individual cages and given ad libitum access to both feed and water, with feed intake monitored (0.01 g) every 30 min for 180 min post injection. Data were analyzed using analysis of variance at each time point. The model included NPS dose, sex and the interaction of sex with NPS dose. NPS dose effects were partitioned into linear and quadratic contrasts to determine dose relationships at each time point. Statistical significance was set at P b 0.05 for all exp.

2.1. Animals

2.4. Experiment 2: effect on water intake of fasted/fed chicks

Morning of hatch Cobb-500 broiler chicks (Gallus gallus) from breeders 30 to 40 weeks of age were obtained from a commercial hatchery. They were caged individually in a room at 30 ± 2 °C and 50 ± 5% relative humidity with ad libitum access to a mash diet (20% crude protein) and tap water. All trials were conducted 4 d post hatch unless otherwise noted. The sequential experiments reported consisted of 7 hatches (5 for exp 1 through 6 and 2 for exp 7). All experimental procedures were performed according to the National Research Council publication, Guide for Care and Use of Laboratory Animals and were approved by the Radford University Institutional Animal Care and Use committee.

The experimental procedures were identical to those in Exp 1 except that water intake (0.01 g) was recorded. Chicks were fasted 180 min before injection and were given ad libitum access to both feed and water post injection. Water mass (g) was converted to volume (1 g = 1 mL).

2.2. Intracerebroventricular (ICV) injection procedure Chicks were injected using a method adapted from Davis et al. (1979). The head of the chick was briefly inserted into a restraining device that left the cranium exposed and allowed for free-hand injection. Injection coordinates were 3 mm anterior to the coronal suture, 1 mm lateral from the sagittal suture, and 2 mm deep targeting the left lateral ventricle. Anatomical landmarks were determined visually and by palpation. Injection depth was controlled by placing a plastic tubing sheath over the needle. The needle remained at injection depth for 10 s post injection to reduce backflow. Chicks were assigned to treatments at random. Rat NPS (SFRNGVGSGVKKTSFRRAKQ; SigmaAldrich Co., St. Louis, MO, USA) was dissolved in artificial cerebrospinal fluid (aCSF, Anderson and Heisey, 1972) for a total injection volume of 5 μL with 0.1% Evans Blue dye to facilitate injection site localization. aCSF was prepared by dissolving 0.75 g sodium chloride, 0.028 g potassium chloride, 0.024 g magnesium chloride hexahydrate, 0.19 g sodium bicarbonate, 0.02 g sodium phosphate dibasic, 0.1 g L-ascorbic acid and 0.018 g calcium chloride dehydrate in 100 mL of filtered deionized water. Control treatments (0 μg NPS) consisted of aCSF only. After data collection, the chick was decapitated and its head sectioned along the frontal plane to determine site of injection. Any chick without dye present in the ventricle system was eliminated from analysis. Numbers of chicks in an experiment are provided in the results section. After decapitation, sex was visually detected by dissection.

2.5. Experiment 3: plasma corticosterone concentration All chicks from Exp 1 and 2 were decapitated 180 min after injection and blood was collected into microcentrifuge tubes that contained 0.06 mg EDTA. Microcentrifuge tubes were immediately centrifuged at 3000 ×g for 10 min and the supernatant was collected. Plasma was stored at − 80 °C until assay. Plasma corticosterone concentrations were determined using a commercially available enzyme immunoassay kit (Correlate-EIA, Assay Designs Inc., Ann Arbor, MI, USA). The within-assay precision was 8.4%. Data were analyzed using analysis of variance. The model included NPS dose, sex and the interaction of sex with NPS dose. NPS dose effects were partitioned into linear and quadratic contrasts to determine dose relationships. 2.6. Experiment 4: effect on water intake in fed/fasted chicks The experimental procedures were identical to those in Exp 2 except that chicks were not fasted prior to injection, and feed was withheld during the observation period. 2.7. Experiment 5: behavior One day post hatch chicks were kept in individual cages with auditory but not visual contact with each other. At 5 days post hatch chicks were randomly assigned to receive either 0 or 0.625 μg NPS ICV. Following injection, chicks were immediately placed in a 290 × 290 mm acrylic recording arena with feed and water containers in diagonal corners. Chicks were simultaneously and automatically recorded from 3 angles for 30 min post injection on DVD and data were analyzed in 5 min intervals using ANY-maze behavioral analysis software (Stoelting, Wood Dale, IL). Feed consumption was quantified at 30 min post injection. Locomotion (m traveled), the amount of time spent standing, sitting, preening, or in deep rest, and the

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number of jumps, feed and exploratory pecks, drinks, vocalizations and escape attempts were quantified. Feed pecks were defined as pecks within the feed container, whereas any other pecks were counted as exploratory. Drinks were defined as the chick dipping its beak in water, then raising and extending its head to swallow. Deep rest was defined as the eyes closed for greater than 3 s, starting 3 s after eye closure. Data were analyzed by the Mann–Whitney U test. 2.8. Experiment 6: immunocytochemistry Chicks, fasted for 180 min, were randomly assigned to receive either 0 or 0.625 μg NPS ICV and then were given ad libitum access to both feed and water post injection. Thirty min after central injection chicks were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg body mass) and then decapitated. The brain was immediately fixed with a 2% paraformaldehyde 0.1% glutaraldehyde solution via a carotid artery. The head was positioned in a stereotaxic instrument and the brain sectioned frontally according to Kuenzel and Masson (1988). The blocked brain was placed in 20% sucrose in phosphate buffer saline for 40 h at 4 °C. Using a cryostat, sections 40 μm thick were cut from areas of the brain that contained the LH, PVN and VMH and mounted on poly-L-lysine coated slides. Sections were incubated with anti-Fos polyclonal antibody (1:400 v/v; Sigma-Aldrich Co., St. Louis, MO, USA; significant homology with chicken c-Fos) for 48 h at 4 °C and then with an alkaline phosphatase-conjugated secondary monoclonal antibody (1:400 v/v; Sigma-Aldrich Co.) at room temperature for 2 h. The secondary antibody was visualized using alkaline phosphatase substrate kit III (Vector Laboratories Ltd., Burlingame, CA, USA). The number of reactive cells was counted from the injected side of the brain (left) in an area 0.2 mm2 located in the center of respective nucleus using light microscopy by a technician blind to treatment, according to co-

Fig. 1. Cumulative feed intake following ICV injection of NPS (Exp 1). LIN, linear contrast; TIM, time post injections in min; PTSE, pooled standard error of the treatment mean; TRT, treatment effect; QUD, quadratic contrast; + P ≤ 0.05.

Fig. 2. Cumulative water intake following ICV injection of NPS in fed chicks (Exp 2). LIN, linear contrast; TIM, time post injections in min; PTSE, pooled standard error of the treatment mean; TRT, treatment effect; QUD, quadratic contrast; + P ≤ 0.05.

ordinates based on Kuenzel and Masson (1988). Two sections were counted and averaged to arrive at the value for each chick. Data were analyzed by two tailed t-test. 2.9. Experiment 7. effect of NPS microinjection into the LH and PVN on feed and water intake Chicks, 14 d post hatch, were anesthetized with 30 mg/kg body mass sodium pentobarbital via the brachial vein. Chicks were then unilaterally implanted with a 22 gauge stainless steel guide cannula stereotaxically. Stereotaxic coordinates were 1.0 mm lateral to midline (L); 7.5 mm below the skull surface (Y); 7.0 mm anterior to interaural line (A) for the LH and L, 0.3; Y, 4.3; A, 7.0 for the PVN based on Kuenzel and Masson (1988). Using these coordinates the guide cannula's bevel point

Fig. 3. Cumulative water intake following ICV injection of NPS in fasted, feedrestricted chicks (Exp 4). TIM, time post injections in min; PTSE, pooled standard error of the treatment mean.

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Table 1 Changes in behaviors of chicks after ICV injection of NPS (Exp 5) Parameter

Feeding pecks Exploratory pecks Drinks Vocalizations Locomotion (m) Jumps Escape attempts Stand time (s) Sit time (s) Deep rest time (s) Preen time (s)

Treatment

NPS Control NPS Control NPS Control NPS Control NPS Control NPS Control NPS Control NPS Control NPS Control NPS Control NPS Control

Time post injection (min) 5

10

15

20

25

30

1.2 ± 1.2 8.6 ± 8.5 5.3 ± 9.8 32.0 ± 15.4 0 0 197.3 ± 56.0 272.0 ± 70.0 0.5 ± 0.2 0.9 ± 0.2 1.2 ± 1.1 2.6 ± 1.5 0.6 ± 0.4 3.9 ± 2.1 244.5 ± 26.1 247.2 ± 29.3 42.1 ± 26.0 36.4 ± 20.8 11.2 ± 8.9 13.9 ± 6.1 0.3 ± 0.3 1.0 ± 1.0

1.2 ± 1.2⁎ 62.3 ± 32.9 11.0 ± 5.4⁎ 49.1 ± 19.9 0 0.1 ± 0.1 337.9 ± 100.0 490.1 ± 122.0 0.8 ± 0.4⁎ 1.5 ± 0.4 2.0 ± 1.7 3.9 ± 1.9 1.2 ± 0.9 6.3 ± 2.7 479.8 ± 26.0 502.1 ± 47.7 65.7 ± 38.2 60.3 ± 24.8 50.9 ± 20.5 35.0 ± 25.9 1.2 ± 0.6 1.0 ± 1.0

4.1 ± 2.9⁎ 68.7 ± 32.7 15.4 ± 6.2⁎ 76.4 ± 30.0 0 0.3 ± 0.2 491.6 ± 153.7 705.0 ± 167.6 1.2 ± 0.4⁎ 2.3 ± 0.4 2.8 ± 1.9 4.6 ± 1.9 2.0 ± 1.2 8.4 ± 3.0 721.7 ± 56.8 751.5 ± 66.8 76.3 ± 42.3 99.9 ± 42.8 98.0 ± 39.1 45.1 ± 26.6 1.7 ± 0.8 1.4 ± 1.4

4.2 ± 2.9⁎ 138.1 ± 51.7 23.8 ± 10.5⁎ 101.9 ± 46.4 0 0.4 ± 0.3 605.9 ± 203.7 871.0 ± 202.5 1.5 ± 0.6⁎ 3.3 ± 0.6 3.3 ± 2.4⁎ 6.0 ± 1.9 3.7 ± 2.3 11.6 ± 4.2 941.0 ± 80.8 1002.8 ± 74.8 107.7 ± 58.0 141.3 ± 55.7 147.0 ± 64.4 49.8 ± 26.1 1.9 ± 0.8 3.0 ± 2.0

26.9 ± 23.4⁎ 162.0 ± 57.8 30.1 ± 13.6⁎ 109.6 ± 48.6 0 0.4 ± 0.3 716.1 ± 246.4 1084.1 ± 248.1 1.9 ± 0.7⁎ 4.0 ± 0.7 3.9 ± 2.9⁎ 7.4 ± 2.3 4.9 ± 3.1 14.7 ± 5.5 1179.1 ± 103.9 1222.4 ± 103.9 116.5 ± 63.6 203.9 ± 85.8 200.0 ± 94.28⁎ 64.7 ± 27.9 1.9 ± 0.8 3.1 ± 2.0

26.9 ± 23.4⁎ 226.6 ± 55.7 36.6 ± 16.7⁎ 127.3 ± 52.3 0 0.4 ± 0.3 776.3 ± 278.0 1241.4 ± 283.1 2.0 ± 0.8⁎ 4.5 ± 0.8 4.1 ± 3.0⁎ 8.9 ± 2.8 6.1 ± 3.6 16.7 ± 6.2 1368.5 ± 135.7 1443.6 ± 127.4 152.1 ± 76.1 265.0 ± 103.6 273.0 ± 119.9⁎ 79.2 ± 32.7 3.4 ± 1.5 4.9 ± 2.0

Values are means ± S.E.M. Significance from control is indicated by ⁎ which implies P b 0.05.

was positioned 1 mm superior to the respective nucleus. The guide cannula was anchored to the skull via three stainless steel screws using cranial cement. A dummy injector remained in the guide cannula when it was not in use. Chicks were allowed at least 3 d recovery from the cannulation procedure. Microinjections were conducted after a 180 min fast. Chicks were randomly assigned to receive either 0 or 0.625 μg NPS in a cross-over design. The 0.5 μl injection was made using a 30 gauge stainless steel injector connected to a 10 μl microsyringe by PE-20 tubing. When in vivo, the injector protruded 1 mm below the guide cannula and was left at injection depth for 15 s post injection. Feed and water intake were monitored concurrently (0.01 g) every 15 min for 60 min post injection for each chick. For this experiment, chicks were kept in a room at 24 ± 2 °C with 50 ± 10% relative humidity. After the observation period, the chick was decapitated and its brain fixed with Heidenhain's solution via a

carotid artery. A 0.5 μl injection of Evans Blue dye was made post fixation. Using a cryostat, the brain was sectioned frontally to verify cannula placement. Data from any chick with a mispositioned cannula were excluded from the analysis. Data were analyzed using analysis of variance at each time point. 3. Results 3.1. Experiment 1: effect on feed intake Chicks responded to central NPS with a linear decrease in feed intake as NPS dose increased (Fig. 1). The NPS effect was significant by 30 min post injection and remained significant through the end of observation. Feed intake was not affected by sex or a sex by NPS dose interaction. Eight to 9 chicks per dose were available for analysis.

Fig. 4. Effect of ICV injection of NPS on the number of reactive cells in the LH, PVN and VMH within the chick hypothalamus (Exp 6). (⁎) = different from control (P ≤ 0.05). Values are means ± S.E.M.

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3.2. Experiment 2: effect on water intake of fasted/fed chicks Chicks responded to central NPS with reduced water intake when both feed and water were available (Fig. 2). This effect was significant by 90 min post injection and remained significant through the end of observation. Water intake was not affected by sex or a sex by dose interaction. Nine to 10 chicks per dose were available for the analysis. 3.3. Experiment 3: plasma corticosterone concentration The range of NPS doses caused a quadratic decrease in plasma corticosterone concentration; 0 μg, 6.7 ± 1.3 ng/mL; 0.313 μg, 3.8 ± 0.9 ng/mL; 0.625 μg, 3.2 ± 0.6 ng/mL; 1.25 μg, 3.8 ± 0.7 ng/mL. The middle dose, 0.625 μg NPS, was associated with the lowest plasma corticosterone concentration. The NPSinduced decrease in plasma corticosterone was not affected by sex or a sex by dose interaction. Blood was collected from chicks after Exp 1 and 2, and no effect of replicate was detected. 3.4. Experiment 4: effect on water intake of fed/fasted chicks When chicks were feed restricted, central NPS did not affect water intake (Fig. 3). For this experiment 9 to 10 chicks per dose were available for the analysis. 3.5. Experiment 5: behavior NPS-treated chicks had reduced feeding pecks that were significant by 10 min post injection (Table 1). Feed intake was decreased by NPS treatment during the behavior observation (1.80 ± 0.67, control; and 0.11 ± 0.08 g, NPS). Additionally, the number of exploratory pecks was significantly reduced by 10 min post injection. The magnitude of suppression for feeding pecks was higher than exploratory pecks (88 vs. 72% suppression respectively). No NPS-treated chicks drank during the observation period. NPS-treated chicks jumped less by 20 min post

Fig. 5. Cumulative feed intake following microinjection of NPS into the LH (Exp 7). PTSE, pooled standard error of the treatment mean; TIM, time post injections in min; TRT, treatment effect; + P ≤ 0.05.

Fig. 6. Cumulative water intake following microinjection of NPS into the LH (Exp 7). PTSE, pooled standard error of the treatment mean; TIM, time post injections in min; TRT, treatment effect; + P ≤ 0.05.

injection and moved less distance by 10 min post injection. NPS also caused chicks to deeply rest more, an effect that was significant by 25 min post injection. Other behaviors including time spent standing, sitting, preening and the number of vocalizations were not affected by NPS. Although escape attempts were not significantly affected by NPS, control chicks tended (P = 0.10) to have more escape attempts. In this experiment there were 8 chicks in the control group and 9 in the NPS group. 3.6. Experiment 6: immunocytochemistry The LH was deactivated by NPS (Fig. 4) and chicks treated with NPS had a pronounced activation of the PVN. The magnitude of LH deactivation was less than the increase in activation associated with the PVN (24.4 vs. 46.6% respectively). NPS did not affect c-Fos immunoreactivity in the VMH. Data from 6 chicks per treatment were used in the analysis.

Fig. 7. Cumulative feed intake following microinjection of NPS into the PVN (Exp 7). PTSE, pooled standard error of the treatment mean; TIM, time post injections in min; TRT, treatment effect; + P ≤ 0.05.

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Fig. 8. Cumulative water intake following microinjection injection of NPS into the PVN (Exp 7). PTSE, pooled standard error of the treatment mean; TIM, time post injections in min; TRT, treatment effect; + P ≤ 0.05.

3.7. Experiment 7: NPS direct microinjection Chicks injected with NPS directly into the LH had reduced feed (Fig. 5) and water (Fig. 6) intake. These effects were significant at all observation times. Similarly, chicks that received NPS directly into the PVN also had reduced feed (Fig. 7) and water (Fig. 8) intake. However, the magnitude of feed intake suppression was less after PVN than LH NPS administration. Data from 6 LH-cannulated and 9 PVNcannulated chicks were used for the analysis of the cross-over design in two replicates; no effect of replicate was detected. 4. Discussion The present study supports the hypothesis that central NPS causes anorexigenic effects in chicks and that NPS is highly conserved across multiple species. The NPS we injected (SFRNGVGSGVKKTSFRRAKQ) is structurally similar to naturally occurring chicken NPS (AFRNGVGSGIKKTSFRRAKS), differing by only three amino acids (Xu et al., 2004). In the chicken, the gene coding for NPS is located on chromosome 6 (Reinscheid, 2007). Central NPS linearly decreased feed intake in a dosedependent manner in chicks, consistent with the anorexigenic effects of NPS reported in rats (Smith et al., 2006; Beck et al., 2005). The linear decrease with doses of 0.14 (0.313 μg) to 0.28 nmol (0.625 μg) suggests that NPS may act via a different neuronal network in chicks than in mammals, since ICV 0.1 and 1.0 nmol did not affect rat feed intake while 10 nmol tended to reduce feed intake (Smith et al., 2006). First order neurons involved in this cascade may be in proximity to the lateral ventricle system in the chick. It is also possible that the chick is hypersensitive to the effects of intracerebroventricular (ICV) NPS as compared to rats (Smith et al., 2006). However, our results are similar to Beck et al. (2005) who reported 1 and 10 μg injected ICV in rats strongly inhibited feed intake. With this comparison, the chick and rat central NPS appetite-related

systems appear similar. However, our results do not support the orexigenic effects of NPS similar to the effect reported by Niimi (2006) in rats. The anorexic effect of NPS appear to be weaker than the effects of other anorexigenic peptides in chicks, including CRH (Furuse et al., 1997), α-melanocyte stimulating hormone (Kawakami et al., 2000) and ghrelin (Furuse et al., 2001). When others reported the appetite-related effects of NPS, only male rats were used (Smith et al., 2006; Beck et al., 2005; Niimi, 2006); we tested both sexes and found that sex did not affect the NPS anorexigenic effect. Central NPS caused activation of the satiety-related PVN (Leibowitz et al., 1981) and deactivation in the LH, a nucleus classically associated with hunger (Brobeck, 1946; Anad and Brobeck, 1951). Thus in chicks, NPS may simultaneously increase the magnitude of satiety while decreasing the magnitude of hunger. Our results are consistent with NPS-activation of the PVN in rats (Niimi, 2006). However, in rats NPS causes LH activation. Comparison of effects at the LH support that hypothalamic NPS modulation is different between chicks and rats. Since in rats feed intake was reduced with PVN administration (Smith et al., 2006), we hypothesized that NPS may be acting directly at the PVN. Chicks that received microinjection of NPS into both the LH and PVN had reduced feed intake, indicating that both the LH and PVN are directly affected by central NPS; the results of Exp 7 demonstrates that the observed anorexigenic effects of NPS may not be secondary to other nuclei. In a preliminary study, 0.0625 μg NPS did not affect feed intake in LH or PVN-cannulated chicks. The effective dose of 0.625 μg may seem high at first considering that usually one tenth of an effective ICV dose is used for interhypothalamic injection. However, the body weights of chicks in Exp 7 are nearly 4 times that of Exp 1 and the brain of a 18 d old chick is nearly twice the size of a 4 d old chick (Kunezel and Masson, 1988). Thus, the dose of NPS used in Exp 7 is near one tenth that 1.25 μg used in Exp 1 for ICV when these factors are considered. Central NPS caused behaviors that may be competitive with appetite and contribute to the anorexigenic effect. NPS effects, including reduced locomotion, the tendency to attempt escape less and decreased plasma corticosterone, were consistent with anxiolytic-like effects observed in rats (Xu et al., 2004). NPS stimulates arousal in rats (Xu et al., 2004); however, NPStreated chicks spent more time in deep rest. Also, NPS increases locomotion in rats (Smith et al., 2006); an effect that differed from our observations in chicks. NPS reduces the magnitude of anxiety-like responses caused by stressors or unfamiliar environments in rats (Xu et al., 2004); since the observation arenas were novel environments for the chicks, their decreased exploratory pecking is consistent with rat findings. When water intake was measured concurrently with feed intake, NPS potently decreased water intake. When chicks were feed restricted, NPS did not significantly affect water intake, supporting the thesis that the effect was secondary. That is, NPS did not reduce water intake directly at doses that reduce feed intake. In conclusion, the results of our study provide evidence that NPS causes behavioral and physiological responses in chicks that are related to appetite. Although central NPS reduced water intake, this effect was secondary to a reduction in feed intake. The

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behavioral and plasma corticosterone results may be interpreted as central NPS causes anxiolytic-like effects in chicks. However, our data provide evidence that NPS does not increase arousal in chicks. The anorexigenic effects of NPS are likely mediated at the hypothalamus, in particular directly at the PVN and LH in chicks. Based on the effects at appetite-related nuclei, we conclude that the anorexigenic signal associated with NPS is primarily central in origin rather than secondary to other behavioral effects. Through divergent evolution from birds to mammals, the anorexigenic effect of NPS may have been conserved while its arousal associated effects were not. Thus, we conclude that central NPS causes anorexigenic effects via the hypothalamus in chicks, and its arousal-related effects differ from rats. Acknowledgement A portion of this research was financed by a Virginia Academy of Sciences grant. References Anderson, D.K., Heisey, S.R., 1972. Clearance of molecules from cerebrospinal fluid in chickens. Am. J. Physiol. 222, 645–648. Anand, B.K., Brobeck, J.R., 1951. Hypothalamic control of food intake in rats and cats. Yale J. Biol. Med. 24, 123–146. Beck, B., Fernette, B., Stricker-Krongrad, A., 2005. Peptide S is a novel potent inhibitor of voluntary a–d fast-induced food intake in rats. Biochem. Biophys. Res. Commun. 332, 859–865. Bernier, V., Stocco, R., Bogusky, M.J., Joyce, J.G., Parachoniak, C., Grenier, K., Arget, M., Mathieu, M.C., O'Neill, G.P., Slipetz, D., Crackower, M.A., Tan, C.M., Therien, A.G., 2006. Structure/function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I. J Biol. Chem. 281, 24704–24712. Brobeck, J.R., 1946. Mechanism of the development of obesity in animals with hypothalamic lesions. Physiol. Rev. 26, 541–559.

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