Ghrelin-induced Food Intake, but not GH Secretion, Requires the Expression of the GH Receptor in the Brain of Male Mice.

Ghrelin stimulates both GH secretion and food intake. The orexigenic action of ghrelin is mainly mediated by neurons that coexpress agouti-related protein (AgRP) and neuropeptide Y (NPY) in the arcuate nucleus of the hypothalamus (ARH). GH also stimulates food intake and, importantly, ARHAgRP/NPY neurons express GH receptor (GHR). Thus, ghrelin-induced GH secretion may contribute to the orexigenic effect of ghrelin. Here, we investigated the response to ghrelin in male mice carrying GHR ablation specifically in neurons (brain GHR knockout [KO] mice) or exclusively in ARHAgRP/NPY neurons (AgRP GHR KO mice). Although brain GHR KO mice showed normal ghrelin-induced increase in plasma GH levels, these mutants lacked the expected orexigenic response to ghrelin. Additionally, brain GHR KO mice displayed reduced hypothalamic levels of Npy and Ghsr mRNA and did not elicit ghrelin-induced c-Fos expression in the ARH. Furthermore, brain GHR KO mice exhibited a prominent reduction in AgRP fiber density in the ARH and paraventricular nucleus of the hypothalamus (PVH). In contrast, AgRP GHR KO mice showed no changes in the hypothalamic Npy and Ghsr mRNAs and conserved ghrelin-induced food intake and c-Fos expression in the ARH. AgRP GHR KO mice displayed a reduced AgRP fiber density (~16%) in the PVH, but this reduction was less than that observed in brain GHR KO mice (~61%). Our findings indicate that GHR signaling in the brain is required for the orexigenic effect of ghrelin, independently of GH action on ARHAgRP/NPY neurons.

Projections from the dorsomedial division of the bed nucleus of the stria terminalis to hypothalamic nuclei in the mouse.

As stressful environment is a potent modulator of feeding, we seek in the present work to decipher the neuroanatomical basis for an interplay between stress and feeding behaviors. For this, we combined anterograde and retrograde tracing with immunohistochemical approaches to investigate the patterns of projections between the dorsomedial division of the bed nucleus of the stria terminalis (BNST), well connected to the amygdala, and hypothalamic structures such as the paraventricular (PVH) and dorsomedial (DMH), the arcuate (ARH) nuclei and the lateral hypothalamic areas (LHA) known to control feeding and motivated behaviors. We particularly focused our study on afferences to proopiomelanocortin (POMC), agouti-related peptide (AgRP), melanin-concentrating-hormone (MCH) and orexin (ORX) neurons characteristics of the ARH and the LHA, respectively. We found light to intense innervation of all these hypothalamic nuclei. We particularly showed an innervation of POMC, AgRP, MCH and ORX neurons by the dorsomedial and dorsolateral divisions of the BNST. Therefore, these results lay the foundation for a better understanding of the neuroanatomical basis of the stress-related feeding behaviors.

Diurnal Patterns for Cortisol, Cortisone and Agouti-Related Protein in Human Cerebrospinal Fluid and Blood.

CONTEXT: Cortisol in blood has a robust circadian rhythm and exerts potent effects on energy balance that are mediated in part by central mechanisms. These interactions involve orexigenic agouti-related protein (AgRP) neurons that are stimulated by glucocorticoids. However, diurnal changes in brain or cerebrospinal fluid (CSF) cortisol and cortisone, which are interconverted by 11ß-HSD1, have not been characterized in humans. OBJECTIVE: To conduct a secondary analysis of existing samples to characterize diurnal changes in cortisol and cortisone in CSF and examine their relationships to changes in AgRP. METHODS: Stored CSF and plasma samples were obtained from 8 healthy subjects who served as controls for a sleep study. CSF was collected every 2h for 36h via indwelling lumbar catheter; plasma was collected every 2h. RESULTS: There was a diurnal rhythm for cortisol and cortisone in CSF that closely followed the plasma rhythm by 2 h with peak and nadir levels at 0900h and 0100h. The ratio of cortisol (active) to cortisone (inactive) in CSF was 48% higher at the peak versus nadir. There was a diurnal rhythm for AgRP in plasma that was out of phase with the cortisol rhythm. There was a less distinct diurnal rhythm for AgRP in CSF that oscillated with a similar phase as cortisol. CONCLUSIONS: There is a robust diurnal rhythm for cortisol and cortisone in CSF. Diurnal changes were noted for AgRP that are related to the cortisol changes. It remains to be determined if AgRP mediates adverse metabolic effects associated with disruption of the cortisol circadian rhythm.

Unlike calorie restriction, Roux-en-Y gastric bypass surgery does not increase hypothalamic AgRP and NPY in mice on a high-fat diet.

OBJECTIVES: Dieting often fails because weight loss triggers strong counter-regulatory biological responses such as increased hunger and hypometabolism that are thought to be critically dependent on the master fuel sensor in the mediobasal hypothalamus (MBH). Because prolonged starvation has been shown to increase AgRP and NPY, the expression level of these two orexigenic genes has been taken as an experimental readout for the presence or absence of hunger. Roux-en-Y gastric bypass (RYGB) surgery leads to a significant weight loss without inducing the associated hunger, indicating possible changes in hypothalamic neuropeptides and/or signaling. Our goal was to assess key genes in the MBH involved in regulating body weight, appetite, and inflammation/oxidative stress after RYGB surgery in mice. METHODS: Obese mice on a high-fat diet were subjected to either sham or RYGB surgery, or caloric restriction to match the weight of RYGB group. Chow-fed mice without surgery served as an additional control group. After 2 or 12 weeks post-surgery, hypothalamic genes were analyzed by real-time qPCR. RESULTS: During the rapid weight loss phase at 2 weeks after RYGB surgery, hypothalamic AgRP and NPY gene expression was not increased compared to mice with sham surgery, indicating that the mice are not hungry. In contrast, the same weight loss induced by caloric restriction promptly triggered increased AgRP and NPY expression. This differential effect of RYGB and caloric restriction was no longer observed during the weight-maintenance phase at 12 weeks after surgery. A similar differential effect was observed for ObRb, but not for POMC and CART expression. Furthermore, RAGE and IBA-1, two markers for inflammation/oxidative stress, were significantly suppressed after RYGB compared to caloric restriction at 2 weeks post-surgery. CONCLUSIONS: These findings suggest that RYGB prevents the biologically adaptive hunger response triggered by undernutrition and weight loss, and suppresses weight loss-induced hypothalamic inflammation markers.

The physiological and neuroendocrine correlates of hunger in the Red Junglefowl (Gallus gallus).

The ability to regulate food intake is critical to survival. The hypothalamus is central to this regulation, integrating peripheral signals of energy availability. Although our understanding of hunger in rodents is advanced, an equivalent understanding in birds is lacking. In particular, the relationship between peripheral energy indices and hypothalamic 'hunger' peptides, agouti-related protein (AgRP), pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) is poorly understood. Here, we compare AgRP, POMC and NPY RNA levels in the hypothalamus of Red Junglefowl chicks raised under ad libitum, chronic restriction and intermittent feeding regimens. Hypothalamic gene expression differed between chronically and intermittently restricted birds, confirming that different restriction regimens elicit different patterns of hunger. By assessing the relationship between hypothalamic gene expression and carcass traits, we show for the first time in birds that AgRP and POMC are responsive to fat-related measures and therefore represent long-term energy status. Chronically restricted birds, having lower indices of fat, show elevated hunger according to AgRP and POMC. NPY was elevated in intermittently fasted birds during fasting, suggesting a role as a short-term index of hunger. The different physiological and neuroendocrine responses to quantitative versus temporal feed restriction provide novel insights into the divergent roles of avian hunger neuropeptides.

Hypothalamic effects of neonatal diet: reversible and only partially leptin dependent.

Early life diet influences metabolic programming, increasing the risk for long-lasting metabolic ill health. Neonatally overfed rats have an early increase in leptin that is maintained long term and is associated with a corresponding elevation in body weight. However, the immediate and long-term effects of neonatal overfeeding on hypothalamic anorexigenic pro-opiomelanocortin (POMC) and orexigenic agouti-related peptide (AgRP)/neuropeptide Y (NPY) circuitry, and if these are directly mediated by leptin, have not yet been examined. Here, we examined the effects of neonatal overfeeding on leptin-mediated development of hypothalamic POMC and AgRP/NPY neurons and whether these effects can be normalised by neonatal leptin antagonism in male Wistar rats. Neonatal overfeeding led to an acute (neonatal) resistance of hypothalamic neurons to exogenous leptin, but this leptin resistance was resolved by adulthood. While there were no effects of neonatal overfeeding on POMC immunoreactivity in neonates or adults, the neonatal overfeeding-induced early increase in arcuate nucleus (ARC) AgRP/NPY fibres was reversed by adulthood so that neonatally overfed adults had reduced NPY immunoreactivity in the ARC compared with controls, with no further differences in AgRP immunoreactivity. Short-term neonatal leptin antagonism did not reverse the excess body weight or hyperleptinaemia in the neonatally overfed, suggesting factors other than leptin may also contribute to the phenotype. Our findings show that changes in the availability of leptin during early life period influence the development of hypothalamic connectivity short term, but this is partly resolved by adulthood indicating an adaptation to the metabolic mal-programming effects of neonatal overfeeding.

Hunger neurons drive feeding through a sustained, positive reinforcement signal.

The neural mechanisms underlying hunger are poorly understood. AgRP neurons are activated by energy deficit and promote voracious food consumption, suggesting these cells may supply the fundamental hunger drive that motivates feeding. However recent in vivo recording experiments revealed that AgRP neurons are inhibited within seconds by the sensory detection of food, raising the question of how these cells can promote feeding at all. Here we resolve this paradox by showing that brief optogenetic stimulation of AgRP neurons before food availability promotes intense appetitive and consummatory behaviors that persist for tens of minutes in the absence of continued AgRP neuron activation. We show that these sustained behavioral responses are mediated by a long-lasting potentiation of the rewarding properties of food and that AgRP neuron activity is positively reinforcing. These findings reveal that hunger neurons drive feeding by transmitting a positive valence signal that triggers a stable transition between behavioral states.

Excitatory transmission onto AgRP neurons is regulated by cJun NH2-terminal kinase 3 in response to metabolic stress.

The cJun NH2-terminal kinase (JNK) signaling pathway is implicated in the response to metabolic stress. Indeed, it is established that the ubiquitously expressed JNK1 and JNK2 isoforms regulate energy expenditure and insulin resistance. However, the role of the neuron-specific isoform JNK3 is unclear. Here we demonstrate that JNK3 deficiency causes hyperphagia selectively in high fat diet (HFD)-fed mice. JNK3 deficiency in neurons that express the leptin receptor LEPRb was sufficient to cause HFD-dependent hyperphagia. Studies of sub-groups of leptin-responsive neurons demonstrated that JNK3 deficiency in AgRP neurons, but not POMC neurons, was sufficient to cause the hyperphagic response. These effects of JNK3 deficiency were associated with enhanced excitatory signaling by AgRP neurons in HFD-fed mice. JNK3 therefore provides a mechanism that contributes to homeostatic regulation of energy balance in response to metabolic stress.

Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss.

Molecular and cellular processes in neurons are critical for sensing and responding to energy deficit states, such as during weight-loss. Agouti related protein (AGRP)-expressing neurons are a key hypothalamic population that is activated during energy deficit and increases appetite and weight-gain. Cell type-specific transcriptomics can be used to identify pathways that counteract weight-loss, and here we report high-quality gene expression profiles of AGRP neurons from well-fed and food-deprived young adult mice. For comparison, we also analyzed Proopiomelanocortin (POMC)-expressing neurons, an intermingled population that suppresses appetite and body weight. We find that AGRP neurons are considerably more sensitive to energy deficit than POMC neurons. Furthermore, we identify cell type-specific pathways involving endoplasmic reticulum-stress, circadian signaling, ion channels, neuropeptides, and receptors. Combined with methods to validate and manipulate these pathways, this resource greatly expands molecular insight into neuronal regulation of body weight, and may be useful for devising therapeutic strategies for obesity and eating disorders.

Arcuate hypothalamic AgRP and putative POMC neurons show opposite changes in spiking across multiple timescales.

Agouti-related-peptide (AgRP) neurons-interoceptive neurons in the arcuate nucleus of the hypothalamus (ARC)-are both necessary and sufficient for driving feeding behavior. To better understand the functional roles of AgRP neurons, we performed optetrode electrophysiological recordings from AgRP neurons in awake, behaving AgRP-IRES-Cre mice. In free-feeding mice, we observed a fivefold increase in AgRP neuron firing with mounting caloric deficit in afternoon vs morning recordings. In food-restricted mice, as food became available, AgRP neuron firing dropped, yet remained elevated as compared to firing in sated mice. The rapid drop in spiking activity of AgRP neurons at meal onset may reflect a termination of the drive to find food, while residual, persistent spiking may reflect a sustained drive to consume food. Moreover, nearby neurons inhibited by AgRP neuron photostimulation, likely including satiety-promoting pro-opiomelanocortin (POMC) neurons, demonstrated opposite changes in spiking. Finally, firing of ARC neurons was also rapidly modulated within seconds of individual licks for liquid food. These findings suggest novel roles for antagonistic AgRP and POMC neurons in the regulation of feeding behaviors across multiple timescales.

Early weaning is associated with higher neuropeptide Y (NPY) and lower cocaine- and amphetamine-regulated transcript (CART) expressions in the paraventricular nucleus (PVN) in adulthood.

The interruption of lactation for a short period, without the use of pharmacological substances or maternal separation, causes offspring malnutrition and hypoleptinaemia and programmes for metabolic disorders such as higher body weight and adiposity, hyperphagia, hyperleptinaemia and central leptin resistance in adulthood. Here, in order to clarify the mechanisms underlying the phenotype observed in adult early-weaned (EW) rats, we studied the expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in different hypothalamic nuclei by immunohistochemistry and Western blot. In the EW group, the teats of lactating rats were blocked with a bandage to interrupt lactation during the last 3 d, while control pups had free access to milk throughout the entire lactation period. At age 180 d, EW offspring showed higher NPY staining in the paraventricular nucleus (PVN), as well as NPY protein content (+68 %) in total hypothalamus than control ones. AgRP showed no changes in staining or Western blot. POMC content was not affected; however, its distribution pattern was altered. CART-positive cells of EW offspring had lower immunoreactivity associated with reduced cell number in the PVN and lower protein content ( - 38 %) in total hypothalamus. The present data indicate that precocious weaning can imprint the neuronal circuitry, especially in the PVN, and cause a long-term effect on the expression of specific orexigenic and anorexigenic neuropeptides, such as NPY and CART, that can be caused by leptin resistance and are coherent with the hyperphagia observed in these animals.

Cognitive flexibility and Agouti-related protein in adolescent patients with anorexia nervosa.

INTRODUCTION: Cognitive flexibility and the flexible learning and relearning of stimulus-reward-associations are important for decision-making and goal-directed behavior. Studies on patients with anorexia nervosa (AN) have shown difficulties in cognitive functions associated with malnutrition and extreme underweight. However, to date we find a lack of neuropsychological studies on cognitive flexibility among adolescent patients with AN. Furthermore, the underlying biological mechanisms remain unclear. Therefore, we aim to examine cognitive functions, especially reward association learning, as a measure of cognitive flexibility in adolescent patients with AN and investigate the relationship between Agouti-related protein (AGRP) and cognitive functions. METHODS: The study population consists of 30 patients with AN (M(age)=16.2 ± 1.2) and a healthy control group (CG) of 28 female adolescents (M(age)=16.3 ± 1.3). All subjects completed a neuropsychological test battery including the probabilistic Object Reversal Task, the Digit Symbol Test and the Trail Making Test. Patients with AN were explored before and after weight gain, the CG initially and after 3 months. RESULTS: Subtle deficits in cognitive flexibility were found in patients with AN compared to the CG. After weight gain, the AN group improved relative to their baseline values in most of the variables but did not reach CG values. They still showed slight impairments. Moreover the study revealed a clear association between AGRP levels and cognitive flexibility. DISCUSSION: Cognitive flexibility plays an important role in AN and may be modulated by abnormal levels of the appetite-regulating peptide AGRP. Even subtle impairments in cognitive flexibility can be relevant for the ability to fully engage in therapy and therefore may hinder a prosperous treatment.

Carbohydrate-to-fat ratio affects food intake and body weight in Wistar rats.

The aim of the study was to evaluate the impact of carbohydrate-to-fat ratio on body weight and appetite regulation in Wistar rats. Twenty-four Wistar rats were randomized to three dietary groups (n = 8): normal carbohydrate diet (NC), low-carbohydrate diet (LC) and high-carbohydrate diet (HC) for 12 weeks. Body weight and food intake were recorded. Circulating leptin and insulin levels were measured by radioimmunoassay method. The expression levels of leptin receptor, insulin receptor, orexin, neuropeptide Y (NPY), agouti-related protein (AgRP) and melanocortin-4 receptor (MC-4R) in the hypothalamus were also measured by realtime polymerase chain reaction (PCR). In the LC group, food intake reduced while body weight increased significantly compared with the NC and HC groups. Plasma leptin levels increased in the LC (18.5 +/- 8.2 ng/mL) group compared with the NC (8.6 +/- 3.8 ng/mL, P < 0.001) and HC (6.6 +/- 1.9 ng/mL, P < 0.001) groups. Realtime reverse transcription-PCR revealed a decrease in the hypothalamic expression level of only leptin receptor in the LC (0.764, 0.471-4.648 copy/mL) and HC (0.357, 0.129-0.781 copy/mL) groups compared with the NC (1.323, 0.616-2.392 copy/mL; P = 0.01) group, and that there was no significant change in those of insulin receptor, AgRP, Orexin, NPY and MC-4R. Low-carbohydrate, high-fat diet raised body weight, which led to a rising of circulating leptin levels and a reduced expression of leptin receptor in the hypothalamus.

Sleeve gastrectomy induces loss of weight and fat mass in obese rats, but does not affect leptin sensitivity.

BACKGROUND & AIMS: Surgical intervention produces sustainable weight loss and metabolic improvement in obese individuals. Vertical sleeve gastrectomy (VSG) produces dramatic, sustained weight loss; we investigated whether these changes result from improved sensitivity to leptin. METHODS: VSG was performed in Long-Evans rats with diet-induced obesity. Naïve or sham-operated rats, fed either ad libitum or pair-fed with the VSG group, were used as controls. Following surgery, body weights and food intake were monitored. We investigated energy expenditure, meal patterns, leptin sensitivity, and expression of pro-opiomelanocortin/agouti-related peptide/neuropeptide Y in the hypothalamus of the rats. RESULTS: We observed sustained losses in weight and body fat in male and female rats after VSG. Weight loss persisted after the disappearance of a transient, postsurgical food intake reduction. Resting energy expenditure was similar between control and VSG rats. VSG rats maintained their reduced body weights. However, they responded to a chronic food restriction challenge by overeating, which resulted in prerestriction, rather than pre-VSG, body weights. Consistent with lower adiposity, VSG decreased plasma leptin levels. Although VSG slightly improved leptin's anorectic action, the response was comparable to that observed in controls matched for adiposity by caloric restriction. Changes in hypothalamic neuropeptide expression were consistent with the lower body weight and lower leptin levels but cannot account for the sustained weight loss. CONCLUSIONS: VSG causes sustained reduction in body weight, which results from loss of fat mass. The maintenance of weight loss observed did not result from changes in sensitivity to leptin.

Nutritional programming affects hypothalamic organization and early response to leptin.

Nutritional programming, taking place in utero or early after birth, is closely linked with metabolic and appetite disorders in adulthood. Following the hypothesis that nutritional programming impacts hypothalamic neuronal organization, we report on discrepancies of multiple molecular and cellular early events that take place in the hypothalamus of rats submitted to intrauterine growth restriction (IUGR). Expression screening performed on hypothalami from IUGR rats at birth and at postnatal d 12 identified changes in gene expression of neurodevelopmental process (cell differentiation and cytoskeleton organization). Additionally, a slight reduction of agouti-related protein and a strong reduction of alpha-MSH-immunoreactive efferent fibers were demonstrated in the paraventricular nucleus of IUGR rats. Rapid catch-up growth of IUGR rats, 5 d after birth, had a positive effect on neurodevelopmental factors and on neuronal projections emanating from the arcuate nucleus. The molecular and cellular anomalies detected in IUGR rats can be related to the reduced and delayed plasma leptin surge from d 0-16 when compared with control and IUGR rats with catch-up growth. However, the ability of leptin to activate intracellular signaling in arcuate nucleus neurons was not reduced in IUGR rats. Other mechanism such as epigenetic regulation of the major appetite-regulating neuropeptides genes was analyzed in parallel with their mRNA expression during postnatal development. This study reveals the importance of an early catch-up growth that reduces abnormal organization of hypothalamic pathways involved in energy homeostasis, whereas protein restriction, maintained during postnatal development leads to an important immaturity of the hypothalamus.

Established maternal obesity in the rat reprograms hypothalamic appetite regulators and leptin signaling at birth.

OBJECTIVE: Key appetite regulators and their receptors are already present in the fetal hypothalamus, and may respond to hormones such as leptin. Intrauterine food restriction or hyperglycemia can reprogram these circuits, possibly predisposing individuals to adverse health outcomes in adulthood. Given the global obesity epidemic, maternal overweight and obesity is becoming more prevalent. Earlier, we observed rapid growth of pups from obese dams during the suckling period. However, it is unclear whether this is because of alterations in leptin and hypothalamic appetite regulators at birth. DESIGN: Female Sprague-Dawley rats were fed palatable high-fat diet (HFD) or chow for 5 weeks to induce obesity before mating. The same diet continued during gestation. At day 1, after birth, plasma and hypothalamus were collected from male and female pups. MEASUREMENTS: Body weight and organ mass were recorded. Leptin and insulin levels were measured in the plasma by radioimmunoassay. Hypothalamic mRNA expression of neuropeptide-Y (NPY), pro-opiomelanocortin, leptin receptor and its downstream signal, STAT3 (signal transducer and activator of transcription 3), were measured using real-time PCR. RESULTS: Body and organ weights of pups from obese dams were similar to those from lean dams, across both genders. However, plasma leptin levels were significantly lower in offspring from obese dams (male: 0.53+/-0.13 vs 1.05+/-0.21 ng ml(-1); female: 0.33+/-0.09 vs 2.12+/-0.57 ng ml(-1), respectively; both P<0.05). Hypothalamic mRNA expression of NPY, pro-opiomelanocortin, leptin receptor and STAT3 were also significantly lower in pups from obese dams. CONCLUSION: Long-term maternal obesity, together with lower leptin levels in pups from obese dams may contribute to the lower expression of key appetite regulators on day 1 of life, suggesting altered intrauterine neuron development in response to intrauterine overnutrition, which may contribute to eating disorders later in life.

Decreased immunoreactivity of the melanocortin neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) after chronic ethanol exposure in Sprague-Dawley rats.

BACKGROUND: The melanocortin (MC) system is composed of peptides that are cleaved from the polypeptide precursor proopiomelanocortin (POMC). Recent pharmacologic and genetic evidence suggests that MC receptor (MCR) signaling modulates neurobiologic responses to ethanol and ethanol intake. Because ethanol decreases POMC mRNA levels, we determined if exposure to an ethanol-containing diet (ED) would significantly reduce central immunoreactivity of the MC peptide alpha-MSH in rats. We also determined if ethanol exposure would alter the immunoreactivity of agouti-related protein (AgRP), an endogenous MCR antagonist. METHODS: Male Sprague-Dawley rats were given 18 days of access to normal rodent chow or a control diet (CD), or short-term (4 days) or long-term (18 days) access to an ED. At the end of the study, rats were perfused with 4% paraformaldehyde and their brains were sectioned into two sets for processing with alpha-MSH or AgRP immunohistochemistry. RESULTS: Rats exposed to an ED showed significant reductions of central alpha-MSH immunoreactivity relative to rats exposed to a control diet (CD) or normal rodent chow. Ethanol-induced reductions of alpha-MSH immunoreactivity were site-specific and were noted in regions of the hypothalamus and extended amygdala, as well as the paraventricular nucleus of the thalamus. Because there were no differences in body weights or caloric intake between the CD and ED groups, reductions of alpha-MSH immunoreactivity in ED-treated rats are best explained by ethanol exposure rather than altered energy balance. No significant ethanol-induced alterations in hypothalamic AgRP immunoreactivity were detected. CONCLUSIONS: The present study shows that ethanol site specifically reduces alpha-MSH immunoreactivity in rat brain. These observations, in tandem with recent pharmacologic and genetic studies, suggest that the endogenous MC system modulates neurobiologic responses to ethanol. Thus, compounds which target MCRs may prove to have therapeutic value in the treatment of excessive ethanol consumption and/or the symptoms associated with ethanol withdrawal.