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. 2008 Sep 25;150(2):672–678. doi: 10.1210/en.2008-0559

Gastrin-Releasing Peptide Messenger Ribonucleic Acid Expression in the Hypothalamic Paraventricular Nucleus Is Altered by Melanocortin Receptor Stimulation and Food Deprivation

Ellen E Ladenheim 1, Robert R Behles 1, Sheng Bi 1, Timothy H Moran 1
PMCID: PMC2646528  PMID: 18818295

Abstract

Gastrin-releasing peptide (GRP) is a bombesin-like peptide widely distributed in the gastrointestinal tract and central nervous system. In the brain, GRP mRNA is located in the hypothalamic paraventricular nucleus (PVN), a region that receives neural input from the arcuate nucleus and plays a critical role in food intake and energy balance. Because GRP neurons are localized in the vicinity of projection sites in the PVN for peptides that participate in energy homeostasis, we investigated whether GRP mRNA expression in the PVN may be sensitive to challenges imposed by either 38 h food deprivation or stimulation of the melanocortin system by the melanocortin 3/4 receptor agonist, melanotan II (MTII). We found that food deprivation significantly decreased GRP mRNA expression, whereas lateral ventricular MTII administration increased GRP mRNA expression in ad libitum-fed rats 4 h after administration. Furthermore, administration of MTII at a dose that reduces 24 h food intake and body weight prevented the decrease in GRP mRNA expression observed in animals that were pair fed to the amount of food consumed by those injected with MTII. These results demonstrate that food deprivation and stimulation of the melanocortin system produce opposing changes in GRP gene expression in the PVN, suggesting that GRP-containing neurons in the PVN may be part of the hypothalamic signaling pathway controlling food intake and energy balance.

Gastrin-releasing peptide gene expression in the hypothalamic paraventricular nucleus is responsive to signals related to energy balance.

Gastrin-releasing peptide (GRP) is a bombesin (BN)-like peptide with a wide distribution in the mammalian gastrointestinal tract and central nervous system (1,2,3,4,5). Central administration of BN-like peptides elicits a number of behavioral and endocrine responses including a reduction in food intake, increased grooming and locomotion, gastric secretion, changes in body temperature, and elevations in plasma glucose and epinephrine (6).

BN-like immunoreactivity and GRP mRNA are found within discrete regions of the central nervous system (2,7). In the forebrain, GRP mRNA is localized in several hypothalamic nuclei including the medial parvocellular subdivision of the hypothalamic paraventricular nucleus (PVN) (7,8,9,10). This brain region receives a rich projection from neurons within the arcuate nucleus (ARC) that contain neuropeptide Y (NPY) and α-MSH, two peptides that have been implicated in energy homeostasis and are responsive to circulating levels of the adiposity hormone, leptin (11,12,13,14,15,16,17). During fasting, plasma leptin levels are decreased resulting in a reduction in α-MSH and an increase in NPY synthesis in the ARC. The distribution of GRP-containing neurons in the PVN has considerable anatomical overlap with neurons that express NPY receptors and melanocortin-4 receptors (MC4-R), the receptor subtype involved with the feeding inhibitory effects of α-MSH (18,19,20).

Although there have been several reports on the acute effects of intracerebroventricular (icv) administration of BN/GRP-related peptides on short-term food intake (21,22,23,24), little is known about the interaction of brain GRP with other peptides that control feeding and body weight. Previous studies examining in vivo release of BN-like peptides in relation to meal-related food intake demonstrated a robust increase in BN-like immunoreactivity in the PVN after the first spontaneous meal of the nocturnal cycle (25). Furthermore, BN-like immunoreactivity measured by RIA in whole hypothalamus was decreased after an overnight fast compared with levels after refeeding (26,27). These data, combined with the anatomical proximity of GRP-containing neurons to projection sites for peptides involved in energy homeostasis, suggest that PVN GRP may be part of a signaling pathway that is involved in food intake and energy balance.

To address this hypothesis, we evaluated whether GRP mRNA expression in the PVN would be altered in response to energy regulatory challenges that represent negative energy balance (food deprivation) and positive energy balance [stimulation of the melanocortin system by the melanocortin 3/4 receptor agonist, melanotan II (MTII)]. This agonist was chosen to engage a system that would be activated under conditions of positive energy balance and has been demonstrated to be a downstream mediator of leptin’s effects on food intake (16,28). In addition, we compared GRP mRNA levels in animals that received icv administration of MTII at a dose that decreases 24 h food intake and body weight with those that were pair fed to the amount of food consumed by the MTII injected group.

Materials and Methods

Animals and surgery

Male Sprague Dawley rats (Charles River, Kingston, NY) weighing between 250 and 350 g at the onset of the experiments were used as the experimental subjects. They were individually housed in hanging wire-mesh cages maintained in a temperature-controlled room on a 12-h light, 12-h dark schedule and weighed daily. Animals received ad libitum access to standard rodent chow (Prolab RMH 1000, PMI Nutrition, LLC, Brentwood, MO) and water unless otherwise noted. All procedures were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University.

For experiments involving central drug administration, animals were implanted with cannulas directed toward the right lateral ventricle. Rats were anesthetized with a mixture of ketamine (85.5 mg/kg)-xylazine (12.9 mg/kg) given im (1.0 ml/kg) and then mounted in a stereotaxic apparatus with the head level. A guide cannula constructed of 23-g stainless steel tubing was lowered into the ventricle using the coordinates 1.0 mm posterior to bregma, 1.2 mm lateral to midline, and 4.0 mm ventral to dura. The cannulas were permanently affixed to the skull with three stainless steel screws and dental acrylic cement. Cannulas were occluded with a removable 30-g stainless steel obturator at all times except during injections. Immediately after surgery, rats received 1 mg/kg flunixin meglumine im and 60,000 U penicillin im and were allowed to recover for at least 1 wk before experimentation. All injections were delivered via a Gilmont microliter syringe attached to PE-10 tubing and a 30-g beveled stainless steel needle that extended 1.5 mm below the cannula tip.

At least 1 wk after surgery, cannula placement was assessed by evaluating 30 min water intake in response to a 3-μl infusion of 50 ng angiotensin II (Sigma, St. Louis, MO) dissolved in 0.9% saline and compared with water intake after a 3-μl injection of 0.9% saline.

At the conclusion of all experiments, animals were anesthetized with ether and killed by decapitation. Brains were immediately removed, quickly frozen on chilled isopentane on dry ice, and stored at −80 C until ready for determination of GRP gene expression by in situ hybridization.

In situ hybridization

Brains were sectioned on a cryostat in the coronal plane at a thickness of 14 μm and mounted onto electrostatically charged microscope slides (SuperFrost Plus; Fisher Scientific, Pittsburgh, PA). After briefly thawing at room temperature, sections were fixed by immersion in 4% paraformaldehyde, rinsed two times in PBS, and dehydrated through an ascending series of ethanol concentrations. Fixed tissue sections were stored at −80 C until ready for use.

Riboprobes were prepared from a plasmid containing the cDNA template for GRP mRNA (a generous gift from Dr. James Battey, National Institute on Deafness and Other Communication Disorders, The National Institutes of Health, Bethesda, MD) as previously described (7,29) and linearized with the appropriate restriction enzymes. For in situ hybridization, antisense riboprobes for GRP were labeled with 35S-uridine 5-triphosphate (Amersham Pharmacia Biotech, Piscataway. NJ) using a commercially available in vitro transcription kit (Promega, Madison, WI) according to the manufacturer’s specifications. Unincorporated label was separated using Quick Spin RNA columns (Roche Diagnostics Corp., Indianapolis, IN) to yield a specific activity of 5 × 108 cpm/μg.

For the in situ hybridization procedure for GRP mRNA, fixed frozen tissue sections were brought to room temperature, treated with acetic anhydride and ethanol, and incubated overnight at 55 C in hybridization buffer containing 50% formamide, 0.3 m NaCl, 10 mm Tris-HCl (pH 8.0), 1 mm EDTA (pH 8.0), 1× Denhardt’s solution (Eppendorf, Netheler, Germany), 10% dextran sulfate, 10 mm dithiothrietol, 500 μg/ml yeast tRNA, and 107 cpm/ml 35S-uridine 5-triphosphate. The sections were then rinsed three times in 2× standard sodium citrate (SSC), treated at 37 C for 30 min with 20 μg/ml RNase A (Sigma), rinsed twice in 2× SSC at 55 C followed by two rinses in 0.1× SSC at 55 C for 15 min. Slides were then dehydrated in ethanol, air dried, and exposed to BMR-2 film (Kodak, Rochester, NY) for 1–3 d.

The hybridization signal in the PVN was quantified from digitized autoradiographic images that were scanned with an Epson professional scanner (Long Beach, CA) and saved on a computer for subsequent analysis. National Institutes of Health Scion imaging software (Bethesda, MD) was used for quantification by comparing signal density against autoradiographic 14C-microscales (Amersham). Quantification was performed on two to three sections from each brain that were anatomically matched between animals for each hybridization assay. The value for each animal was obtained by determining the mean product of hybridization area times density with the background subtracted. Data were normalized to ad libitum-fed controls as 100%.

Statistical analysis

For statistical analyses, multiple group comparisons were performed by one-way ANOVA followed by Tukey’s test to determine differences between individual means or by Student’s t test where appropriate.

Experiment 1: effect of food deprivation on GRP mRNA expression in the PVN

To assess the effects of food deprivation on GRP mRNA levels in the PVN, rats were either fed ad libitum (n = 7) or fasted for 38 h (n = 7). Animals in the fasted group had food removed at 1800 h. After the period of deprivation, animals in both groups were killed at 0800 h (2 h after lights on) and the brain tissue was prepared for in situ hybridization detection of GRP mRNA as described above.

Experiment 2: effect of melanocortin receptor stimulation on GRP mRNA expression in the PVN

In the first experiment, we evaluated whether GRP mRNA levels in the PVN would be altered in response to activation of anabolic signaling pathways produced by food deprivation. To evaluate whether GRP gene expression in the PVN would also respond to energy regulatory signals that are stimulated under conditions of positive energy balance, we examined the effect of melanocortin receptor stimulation by lateral ventricular injection of the melanocortin 3/4 receptor agonist, MTII (Peninsula Laboratories Inc., Belmont, CA) on GRP mRNA expression under ad libitum-fed conditions. As mentioned previously, the melanocortin system has been demonstrated to be part of a hypothalamic catabolic signaling pathway and is involved in leptin’s effects on food intake (14,15,28).

Rats were stereotaxically implanted with cannulas into the right lateral ventricle and evaluated for their drinking response to angiotensin II as described above. Only rats that drank 5 ml or more above their intake after saline infusion were included in this experiment. Animals were then divided into two groups (n = 5/group) to receive either 0.9% saline (group 1) or MTII (group 2).

Food was removed at 1000 h and 1 h later rats in group 1 were given a lateral ventricular injection of 3 μl 0.9% saline, whereas those in group 2 were administered 0.1 nmol MTII. The MTII dose was chosen because it has previously been shown to elicit a robust effect on food intake and body weight (30). Four hours after the injection (2 h before dark onset), the rats were killed and the brains collected for in situ hybridization in the manner described above.

Experiment 3: effect of MTII on 24 food intake and GRP mRNA expression in the PVN

In experiments 1 and 2, we hypothesized that fasting and administration of MTII would produce reciprocal changes in GRP gene expression levels in the PVN. For this study, we determined whether MTII at a dose that significantly reduces 24 h food intake and body weight would prevent the changes in GRP mRNA expression levels induced by decreased food intake. GRP mRNA levels in the PVN were compared in rats that received MTII with those that were calorically matched (pair fed) to the amount of food consumed in 24 h by the MTII injected group.

Rats were cannulated in the lateral ventricle and tested for cannula placement as previously described. Based on these results, rats were then divided into three groups (n = 6/group) with the highest responders assigned to the group to receive MTII (group 1), medium responders assigned to the group to receive saline (group 2), and the lowest responders assigned to a saline-injected, pair-fed group (group 3) (30 min water intake = 14.7 ± 0.9, 9.8 ± 0.3, and 4.8 ± 1.5 ml, respectively).

On d 1 of the experiment, animals were weighed 2 h before lights out and the food was removed. One hour before lights out, rats in group 1 were injected into the lateral ventricle with 3 μl of 1.0 nmol of MTII, and animals in group 2 received the control injection of 3 μl 0.9% saline. Access to a preweighed portion of rodent chow was given at the time of lights out. On d 2 of the experiment, food intake and body weight were measured 2 h before lights out and the animals were then killed.

The identical protocol was followed for the saline-injected, pair-fed group (group 3) except that the amount of food returned at lights out was calculated based on the mean 24-h intake of the MTII group plus an additional 10% to allow for spillage. As with the other groups, 2 h before lights out on the following day, food intake and body weight were measured and the animals were then killed to evaluate GRP gene expression.

Results

Experiment 1: effect of food deprivation on GRP mRNA expression in the PVN

Figure 1 shows the effects of a 38-h fast on GRP mRNA expression in the PVN. GRP mRNA expression was located primarily in the medial parvocellular portion of the PVN with a higher density in the caudal aspect of this nucleus (Fig. 1C). Results from in situ hybridization demonstrated that 38 h food deprivation produced a significant decrease in the hybridization signal over GRP-expressing neurons in the PVN. The mean density of GRP mRNA in the fasted animals was approximately 51% relative to ad libitum-fed controls (Fig. 1, B and D, P < 0.01).

Figure 1.

Figure 1

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Effect of 38 h food deprivation on GRP mRNA expression in the PVN. A, Stained tissue section at the level of the PVN corresponding to in situ hybridization section shown in C. mPVN, Magnocellular PVN; pPVN, medial portion of the parvocellular PVN. B, Quantification of in situ hybridization. Thirty-eight hours of food deprivation significantly decreased GRP mRNA expression in the PVN compared with ad libitum (Ad lib)-fed controls by approximately 51% (P < 0.05, n = 7/group). C, In situ hybridization for GRP mRNA in the pPVN of the hypothalamus in an ad libitum-fed rat. D, In situ hybridization for GRP mRNA in the pPVN after 38 h food deprivation. *, Significantly different from ad libitum.

Experiment 2: effect of melanocortin receptor stimulation on GRP mRNA expression in the PVN

The effect of lateral ventricular MTII administration on GRP mRNA expression in ad libitum-fed animals is shown in Fig. 2. Lateral ventricular administration of 1 nmol MTII produced a significant increase (∼50%) in GRP gene expression in the PVN 4 h after injection compared with the saline injected group (Fig. 2, P < 0.05).

Figure 2.

Figure 2

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Levels of GRP mRNA expression in the PVN 4 h after lateral ventricular administration of 1 nmol MTII. GRP mRNA levels in the PVN were significantly increased by MTII administration compared with saline-injected controls (P < 0.05, n = 5/group). Data are expressed as percent mean value of control group ± se. *, Significantly different from saline.

Experiment 3: effect of MTII on 24 food intake and GRP mRNA expression in the PVN

At the 24-h time point, MTII significantly decreased food intake by about 40% (P < 0.05) compared with saline-injected rats (Fig. 3A). Consistent with our experimental design, food intake of pair-fed animals did not differ from that of the group that received MTII (P > 0.05). Both MTII and pair feeding significantly reduced 24-h body weight compared with saline injected ad libitum-fed rats (Fig. 3B, P < 0.001), and there was no significant difference in body weight between these treatment groups (P > 0.05).

Figure 3.

Figure 3

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Effect of MTII on food intake and body weight 24 h after lateral ventricular administration. A, MTII significantly reduced 24 h food intake compared with saline injected controls (P < 0.05, n = 6/group). Food intake of pair-fed animals did not differ from those injected with MTII (P > 0.05, n = 6). B, MTII significantly decreased 24 h body weight by 9.5 ± 1.5 g compared with saline-injected controls that exhibited a 6.7 ± 1.8 g increase in body weight. There was no significant difference in body weight change between the MTII-injected and pair-fed groups (P > 0.05). *, Significantly different from saline.

Figure 4 demonstrates GRP gene expression in the PVN 24 h after lateral ventricular saline or 1.0 nmol MTII injection and in rats calorically matched (pair fed) to the intake of the MTII-injected group. In animals pair fed to the amount consumed by the MTII-injected group, GRP mRNA levels in the PVN were significantly reduced compared with the saline-injected control group (P < 0.001). Despite equivalent food intake between the pair-fed and MTII-injected groups, MTII prevented the reduction in GRP gene expression seen in the pair-fed group, resulting in GRP mRNA levels that were not significantly different from those observed in the saline injected control group (P > 0.05).

Figure 4.

Figure 4

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Levels of GRP mRNA in the PVN in ad libitum-fed, saline-injected (saline), ad libitum-fed MTII-injected (1.0 nmol) (MTII), and saline-injected, pair-fed (pair-fed) rats. GRP mRNA expression was significantly reduced in the pair-fed group compared with the saline and MTII groups (P < 0.001). Although food intake was not different between the MTII and pair-fed groups, lateral ventricular administration of MTII prevented the decrease in GRP mRNA levels seen in the pair-fed group (MTII vs. saline, P > 0.05). *, P < 0.001 compared with saline; #, P < 0.001 compared with MTII.

Discussion

The objective of these experiments was to evaluate the effects of opposing energy regulatory challenges on GRP gene expression in the PVN. We chose to use acute food deprivation as a signal of negative energy balance and lateral ventricular administration of the melanocortin 3/4 receptor agonist, MTII, as a signal of positive energy balance. Our results demonstrate that fasting significantly decreased GRP mRNA expression in the PVN, whereas activation of the melanocortin system increased levels of GRP mRNA in ad libitum-fed animals and prevented the reduction in GRP mRNA levels induced by pair feeding.

The mechanism(s) that regulates GRP mRNA levels in the PVN in response to changes in metabolic status is currently unknown. Although GRP is a gastrointestinal peptide that is involved in a number of endocrine and paracrine responses related to food consumption (31,32,33,34,35), it is unlikely that circulating GRP would be a source of direct negative feedback on central GRP neurons, given that levels of GRP in the gastrointestinal tract are significantly lowered after food deprivation (27). A more likely possibility, and one that is consistent with the present data, is that GRP gene expression in the PVN is indirectly responsive to signals, such as circulating levels of leptin, insulin, or glucose, that reflect changes in nutritional state.

As mentioned previously, the ARC contains two populations of neurons that are responsive to circulating levels of leptin. Both populations are involved in food intake and send direct projections to the PVN to regions that anatomically overlap with the distribution of GRP mRNA. One of these populations coexpresses the orexigenic peptides, NPY and agouti gene-related peptide (AgRP), which are increased by fasting and inhibited by leptin (13,15,36). The other coexpresses the anorexigenic peptides proopiomelanocortin (POMC), the precursor to α-MSH, and cocaine and amphetamine-regulated transcript, which are reduced by fasting and stimulated by leptin (16).

Several lines of evidence support an important role for melanocortins in energy balance. Activation of the melanocortin system by icv administration of α-MSH or MTII reduces food intake, whereas administration of endogenous MC4-R antagonist AgRP increases food intake (37). Moreover, mice with either a targeted deletion of MC4-R or overexpression of AgRP exhibit hyperphagia, hyperglycemia, hyperinsulinemia, and obesity (38,39).

As indicated above, plasma leptin levels influence the activity of POMC neurons in the ARC and these neurons express leptin receptor mRNA (40). Previous studies reported that the effects of icv infusion of leptin on food intake and fos activation in the PVN were blocked by the melanocortin 3/4 receptor antagonist, SHU9119, providing evidence that MC4-R is a downstream target of leptin signaling in the ARC (28). In the present study, we demonstrated that icv infusion of MTII stimulated PVN GRP gene expression 4 h after administration. Furthermore, despite no differences in food intake between the pair-fed and MTII injected groups, pretreatment with MTII prevented the decrease in GRP mRNA expression produced by reduced food intake. Although the temporal pattern of food intake in the pair-fed and MTII-injected groups was not matched between groups, it is unlikely that this contributed to our results because both groups also exhibited the same loss in 24 h body weight. The ability of MTII to prevent the reduction in GRP mRNA expression elicited by decreased food intake supports a functional relationship between the central melanocortin and GRP systems and suggests that the reduction in PVN GRP gene expression after fasting or food restriction may be mediated by an inhibition of POMC neurons and decreased synthesis of α-MSH. Whether the effects of melanocortin stimulation on food intake are directly dependent on GRP release remains to be determined. Nonetheless, our data demonstrating that activation of the hypothalamic circuitry by opposing energy regulatory challenges (i.e. fasting or stimulation of the melanocortin system) produced opposite changes in GRP mRNA levels in the PVN provides circumstantial evidence that this neuronal population may be responsive to alterations in leptin signaling. However, we cannot rule out the possibility that the melanocortin system is acting through a system that is independent of leptin to influence GRP expression levels.

The overall contribution of GRP-containing neurons in the PVN to food intake and energy balance has not been established. Previous studies have shown that GRP levels in the hypothalamus, specifically in the PVN, change rapidly during consumption of a meal (25). The relationship between meal-related fluctuations in GRP levels and alterations in GRP mRNA expression resulting from fasting or melanocortin stimulation is not clear. It is possible that changes associated with meal-related food intake occur against a specific background imposed by the metabolic state of the animal. Alternatively, the neuronal population responsible for meal-related changes may be distinct from the population that responds to signals related to long-term energy stores.

A number of studies have demonstrated that central administration of BN, the amphibian homolog of GRP, produces a variety of effects on homeostatic functions. Early work had demonstrated that icv administration of BN produced hypothermia and hyperglycemia (41,42,43). Subsequently it was reported that direct bilateral PVN infusion of BN results in profound increases in blood glucose, free fatty acids, and corticosterone, suggesting that BN binding sites in the PVN may participate in regulating circulating metabolic fuels and the hypothalamus-pituitary-adrenal axis (44).

Although these studies support a local effect of BN-like peptides in the PVN to influence homeostatic controls, an alternative possibility is that changes in PVN GRP synthesis may directly reflect peptide release in projection sites involved in food intake. A study by Kyrkouli et al. (45) demonstrated that direct infusions of BN into various hypothalamic regions including the dorsomedial, ventromedial, and lateral hypothalamic nuclei produced a significant suppression of food intake. These regions receive projections from the PVN (46) and could potentially be influenced by changes in GRP levels within the PVN; however, it is currently unknown whether these projections are GRPergic.

Unlike the hypothalamic nuclei, it is known that the nucleus of the solitary tract (NTS) in the caudal hindbrain is both the recipient of PVN GRP innervation and important for central GRP’s effects on food intake. Anatomical studies by Costello et al. (8) demonstrated that a majority of GRP neurons in the PVN project to the dorsal vagal complex, including the NTS (8). This brain region receives chemical and mechanical sensory input from the gastrointestinal tract and exhibits high affinity binding for GRP and expresses GRP receptor mRNA (47,48). Several studies have demonstrated that injections of GRP or GRP analogs into the fourth cerebral ventricle, or directly into the NTS, reduce food intake, whereas GRP antagonists administered into the fourth cerebral ventricle increase food intake and block peripherally induced satiety signals (21,22,23,49,50,51).

There is accumulating evidence to support the view that signals related to energy stores work in concert with short-term, meal-related controls of food intake to maintain energy homeostasis. A number of studies have demonstrated that leptin enhances the sensitivity of meal-generated satiety signals, such as cholecystokinin, BN, and gastric distension, and increases c-fos activation in the NTS (52,53,54). Conversely, NPY has been shown to reduce the sensitivity of meal-related satiety signals and decrease c-fos activation in the NTS (55).

Previous studies by Blevins et al. (56) provided evidence to support a role for a descending PVN to NTS oxytocinergic pathway in the interaction of leptin and cholecystokinin’s effects on food intake; however, other neural pathways that mediate this integration have not been identified. The results from the present study, combined with anatomical data, raise the possibility that GRP-containing neurons in the PVN that are responsive to energy regulatory signals may provide an alternative neural pathway to integrate adiposity and meal-related satiety signaling.

In summary, our data demonstrate that GRP mRNA expression in the PVN is responsive to food deprivation and melanocortin receptor stimulation. These findings suggest that GRP-containing neurons in the PVN may be part of the hypothalamic neuropeptide circuitry that participates in energy homeostasis.

Footnotes

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46448 and DK-19302.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 25, 2008

Abbreviations: AgRP, Agouti gene-related peptide; ARC, arcuate nucleus; BN, bombesin; GRP, gastrin-releasing peptide; icv, intracerebroventricular; MC4-R, melanocortin-4 receptor; MTII, melanotan II; NPY, neuropeptide Y; NTS, nucleus of the solitary tract; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SSC, standard sodium citrate.

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