Automated Patch Clamp Recordings of GPCR-Gated Ion Channels: Targeting the MC4-R/Kir7.1 Potassium Channel Complex.

Automated patch clamp recording is a valuable technique in drug discovery and the study of ion channels. It allows for the precise measurement and manipulation of channel currents, providing insights into their function and modulation by drugs or other compounds. The melanocortin 4 receptor (MC4-R) is a G protein-coupled receptor (GPCR) crucial to appetite regulation, energy balance, and body weight. MC4-R signaling is complex and involves interactions with other receptors and neuropeptides in the appetite-regulating circuitry. MC4-Rs, like other GPCRs, are known to modulate ion channels such as Kir7.1, an inward rectifier potassium channel, in response to ligand binding. This modulation is critical for controlling ion flow across the cell membrane, which can influence membrane potential, excitability, and neurotransmission. The MC4-R is the target for the anti-obesity drug Imcivree. However, this drug is known to lack optimal potency and also has side effects. Using high-throughput techniques for studying the MC4-R/Kir7.1 complex allows researchers to rapidly screen many compounds or conditions, aiding the development of drugs that target this system. Additionally, automated patch clamp recording of this receptor-channel complex and its ligands can provide valuable functional and pharmacological insights supporting the development of novel therapeutic strategies. This approach can be generalized to other GPCR-gated ion channel functional complexes, potentially accelerating the pace of research in different fields with the promise to uncover previously unknown aspects of receptor-ion channel interactions.

Structure of the Ion Channel Kir7.1 and Implications for its Function in Normal and Pathophysiologic States.

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

Functional coupling between MC4R and Kir7.1 contributes to clozapine-induced hyperphagia.

Most antipsychotic drugs (APDs) induce hyperphagia and weight gain. However, the neural mechanisms are poorly understood, partly due to challenges replicating their metabolic effects in rodents. Here, we report a new mouse model that recapitulates overeating induced by clozapine, a widely prescribed APD. Our study shows that clozapine boosts food intake by inhibiting melanocortin 4 receptor (MC4R) expressing neurons in the paraventricular nucleus of the hypothalamus. Interestingly, neither clozapine nor risperidone, another commonly used APD, affects receptor-ligand binding or the canonical Gαs signaling of MC4R. Instead, they inhibit neuronal activity by enhancing the coupling between MC4R and Kir7.1, leading to the open state of the inwardly rectifying potassium channel. Deletion of Kir7.1 in Mc4r-Cre neurons prevents clozapine-induced weight gain, while treatment with a selective Kir7.1 blocker mitigates overeating in clozapine-fed mice. Our findings unveil a molecular pathway underlying the effect of APDs on feeding behavior and suggest its potential as a therapeutic target.

Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design.

Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.

Inhibition of the melanocortin-3 receptor (MC3R) causes generalized sensitization to anorectic agents.

The melanocortin-3 receptor (MC3R) acts presynaptically to regulate GABA release from agouti-related protein (AgRP) nerve terminals and thus may be a negative regulator of multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of MC3R in regulating the response to various anorexigenic agents. Our findings reveal that genetic deletion or pharmacological inhibition of MC3R improves the dose responsiveness to Glucagon-like peptide 1 (GLP1) agonists, as assayed by inhibition of food intake and weight loss. An enhanced anorectic response to other agents, including the acute satiety factors peptide YY (PYY3-36) and cholecystokinin (CCK) and the long-term adipostatic factor, leptin, demonstrated that increased sensitivity to anorectic agents is a generalized result of MC3R antagonism. Enhanced neuronal activation in multiple nuclei, including ARH, VMH, and DMH, was observed using Fos immunohistochemistry following low-dose liraglutide in MC3R knockout mice (Mc3r-/-), supporting the hypothesis that the MC3R is a negative regulator of circuits regulating multiple aspects of feeding behavior. The enhanced anorectic response in Mc3r -/- mice after administration of GLP1 analogs was also independent of the incretin effects and malaise induced by GLP1R analogs, suggesting that MC3R antagonists may have value in enhancing the dose-response range of obesity therapeutics.

Melanocortin-3 receptor expression in AgRP neurons is required for normal activation of the neurons in response to energy deficiency.

The melanocortin-3 receptor (MC3R) is a negative regulator of the central melanocortin circuitry via presynaptic expression on agouti-related protein (AgRP) nerve terminals, from where it regulates GABA release onto secondary MC4R-expressing neurons. However, MC3R knockout (KO) mice also exhibit defective behavioral and neuroendocrine responses to fasting. Here, we demonstrate that MC3R KO mice exhibit defective activation of AgRP neurons in response to fasting, cold exposure, or ghrelin while exhibiting normal inhibition of AgRP neurons by sensory detection of food in the ad libitum-fed state. Using a conditional MC3R KO model, we show that the control of AgRP neuron activation by fasting and ghrelin requires the specific presence of MC3R within AgRP neurons. Thus, MC3R is a crucial player in the responsiveness of the AgRP soma to both hormonal and neuronal signals of energy need.

Aqueous remote loading of setmelanotide in poly(lactic-co-glycolic acid) microspheres for long-term obesity treatment.

Setmelanotide (Imcivree™) was developed as a daily injectable therapeutic peptide for the treatment of rare forms of syndromic obesity, such as POMC deficiency and leptin receptor deficiency. The important option of poly(lactic-co-glycolic acid) (PLGA) controlled release microspheres has become more attractive for this class of drugs upon the discovery that net positively charged peptides can be remote-loaded rapidly from aqueous peptide solution into blank microspheres at high loading and encapsulation efficiency. Here we sought to remote-load setmelanotide in PLGA microspheres and examine its potential for long-term controlled release and body weight control. The influence of PLGA microsphere porosity was investigated with respect to morphology, drug loading, and in vitro release profiles. Increased density of the microspheres inhibited the progress of encapsulation of the dicationic peptide. A diet-induced obese murine model was then used to determine the pharmacokinetic profile and to evaluate long-term efficacy of an optimal formulation. Remote loaded PLGA formulations encapsulated setmelanotide as high as ∼63% (∼6.3% w/w loading) and exhibited slow and continuous peptide release over ∼6 weeks in vitro largely independent of microsphere porosity. The obtained in vivo release pattern from deconvolution of the pharmacokinetics after subcutaneous microsphere injection was consistent with the in vitro release profile but with a lower initial burst release and overall slightly faster release rate. After a single injection of remote-loaded setmelanotide, continuous long-term inhibition of food intake and body weight control was observed over 17 and 30 days, respectively. The improvement in body weight control over drug-free microsphere vehicle-treated control groups matched the observed PK profile. This study provides the first report of long-acting release formulation for 1-month controlled release of setmelanotide and body weight control in a diet induced obese murine model, and supports the further development of long-acting treatment options for obese patients.

Cell autonomous regulation of the activation of AgRP neurons by the melanocortin-3 receptor.

The melanocortin-3 receptor (MC3R) is a negative regulator of the central melanocortin circuitry via presynaptic expression on AgRP nerve terminals, from where it regulates GABA release onto secondary MC4R-expressing neurons. Hence, animals lacking MC3R (MC3R KO) exhibit hypersensitivity to MC4R agonists. However, MC3R KO mice also exhibit defective behavioral and neuroendocrine responses to fasting. Here, we demonstrate that MC3R KO mice exhibit defective activation of AgRP neurons in response to fasting and cold exposure, while exhibiting normal inhibition of AgRP neurons by sensory detection of food. Further, using an AgRP-specific MC3R knockout model, we show that the control of AgRP neuron activation by MC3R is cell-autonomous. One mechanism underlying this involves the response to ghrelin, which is also blunted in mice with AgRP-specific deletion of the MC3R. Thus, MC3R is a crucial player in the control of energy homeostasis by the central melanocortin system, not only acting presynaptically on AgRP neurons, but via AgRP cell-autonomous regulation of fasting- and cold-induced neuronal activation as well.

CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccßA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, ß-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%. Fish containing both the ccßA-msElovl2 transgene knock-in and mc4r knockout (Elovl2) were 41.8% larger than controls at 6 months post-hatch (p = 0.005). Mean eicosapentaenoic acid (EPA, C20:5n-3) levels in Elov2 mutants and mc4r knockout mutants (MC4R) were 121.6% and 94.1% higher than in controls, respectively (p = 0.045; p = 0.025). Observed mean docosahexaenoic acid (DHA, C22:6n-3) and total EPA + DHA content was 32.8% and 45.1% higher, respectively, in Elovl2 transgenic channel catfish than controls (p = 0.368; p = 0.025). To our knowledge this is the first example of genome engineering to simultaneously target transgenesis and knock-out a gene in a commercially important aquaculture species for multiple improved performance traits. With a high transgene integration rate, improved growth, and higher omega-3 fatty acid content, the use of Elovl2 transgenic channel catfish appears beneficial for application on commercial farms.

Targeting the central melanocortin system for the treatment of metabolic disorders.

A large body of preclinical and clinical data shows that the central melanocortin system is a promising therapeutic target for treating various metabolic disorders such as obesity and cachexia, as well as anorexia nervosa. Setmelanotide, which functions by engaging the central melanocortin circuitry, was approved by the FDA in 2020 for use in certain forms of syndromic obesity. Furthermore, the FDA approvals in 2019 of two peptide drugs targeting melanocortin receptors for the treatment of generalized hypoactive sexual desire disorder (bremelanotide) and erythropoietic protoporphyria-associated phototoxicity (afamelanotide) demonstrate the safety of this class of peptides. These approvals have also renewed excitement in the development of therapeutics targeting the melanocortin system. Here, we review the anatomy and function of the melanocortin system, discuss progress and challenges in developing melanocortin receptor-based therapeutics, and outline potential metabolic and behavioural disorders that could be addressed using pharmacological agents targeting these receptors.

Loss of Agrp1 in zebrafish: Effects on the growth and reproductive axis.

Loss of agouti related neuropeptide (AgRP) does not lead to overt phenotypes in mammals unless AgRP neurons are ablated. In contrast, in zebrafish it has been shown that Agrp1 loss of function (LOF) leads to reduced growth in Agrp1 morphant as well as Agrp1 mutant larvae. Further, it has been shown that multiple endocrine axes are dysregulated upon Agrp1 LOF in Agrp1 morphant larvae. Here we show that adult Agrp1 LOF zebrafish show normal growth and reproductive behavior in spite of a significant reduction in multiple related endocrine axes namely reduced expression in pituitary growth hormone (gh) follicle stimulating hormone (fshb) as well as luteinizing hormone (lhb). We looked for compensatory changes in candidate gene expression but found no changes in growth hormone and gonadotropin hormone receptors that would explain the lack of phenotype. We further looked at expression in the hepatic and muscular insulin-like growth factor (Igf) axis which appears to be normal. Fecundity as well as ovarian histology also appear largely normal while we do see an increase in mating efficiency specifically in fed but not fasted AgRP1 LOF animals. This data shows that zebrafish can grow and reproduce normally in spite of significant central hormone changes and suggests a peripheral compensatory mechanism additional to previously reported central compensatory mechanisms in other zebrafish neuropeptide LOF lines.

The unique structural characteristics of the Kir 7.1 inward rectifier potassium channel: a novel player in energy homeostasis control.

The inward rectifier potassium channel Kir7.1, encoded by the KCNJ13 gene, is a tetramer composed of two-transmembrane domain-spanning monomers, closer in homology to Kir channels associated with potassium transport such as Kir1.1, 1.2, and 1.3. Compared with other channels, Kir7.1 exhibits small unitary conductance and low dependence on external potassium. Kir7.1 channels also show a phosphatidylinositol 4,5-bisphosphate (PIP2) dependence for opening. Accordingly, retinopathy-associated Kir7.1 mutations mapped at the binding site for PIP2 resulted in channel gating defects leading to channelopathies such as snowflake vitreoretinal degeneration and Leber congenital amaurosis in blind patients. Lately, this channel's role in energy homeostasis was reported due to the direct interaction with the melanocortin type 4 receptor (MC4R) in the hypothalamus. As this channel seems to play a multipronged role in potassium homeostasis and neuronal excitability, we will discuss what is predicted from a structural viewpoint and its possible implications for hunger control.

Common Genetic Variation and Age of Onset of Anorexia Nervosa.

Background: Genetics and biology may influence the age of onset of anorexia nervosa (AN). The aims of this study were to determine whether common genetic variation contributes to age of onset of AN and to investigate the genetic associations between age of onset of AN and age at menarche. Methods: A secondary analysis of the Psychiatric Genomics Consortium genome-wide association study (GWAS) of AN was performed, which included 9335 cases and 31,981 screened controls, all from European ancestries. We conducted GWASs of age of onset, early-onset AN (<13 years), and typical-onset AN, and genetic correlation, genetic risk score, and Mendelian randomization analyses. Results: Two loci were genome-wide significant in the typical-onset AN GWAS. Heritability estimates (single nucleotide polymorphism-h 2) were 0.01-0.04 for age of onset, 0.16-0.25 for early-onset AN, and 0.17-0.25 for typical-onset AN. Early- and typical-onset AN showed distinct genetic correlation patterns with putative risk factors for AN. Specifically, early-onset AN was significantly genetically correlated with younger age at menarche, and typical-onset AN was significantly negatively genetically correlated with anthropometric traits. Genetic risk scores for age of onset and early-onset AN estimated from independent GWASs significantly predicted age of onset. Mendelian randomization analysis suggested a causal link between younger age at menarche and early-onset AN. Conclusions: Our results provide evidence consistent with a common variant genetic basis for age of onset and implicate biological pathways regulating menarche and reproduction.

Improved Growth and High Inheritance of Melanocortin-4 Receptor (mc4r) Mutation in CRISPR/Cas-9 Gene-Edited Channel Catfish, Ictalurus punctatus.

Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing. An overall mutation rate of 33% and 33% homozygosity/bi-allelism was achieved in 2017. Approximately 71% of progeny inherited the mutation. Growth was generally higher in MC4R mutants than controls (CNTRL) at all life stages and in both pond and tank environments. There was a positive relationship between zygosity and growth, with F1 homozygous/bi-allelic mutants reaching market size 30% faster than F1 heterozygotes in earthen ponds (p = 0.022). At the stocker stage (~ 50 g), MC4R × MC4R mutants generated in 2019 were 40% larger than the mean of combined CNTRL × CNTRL families (p = 0.005) and 54% larger than F1 MC4R × CNTRL mutants (p = 0.001) indicating mutation may be recessive. With a high mutation rate and inheritance of the mutation as well as improved growth, the use of gene-edited MC4R channel catfish appears to be beneficial for application on commercial farms.

Organization of neural systems expressing melanocortin-3 receptors in the mouse brain: Evidence for sexual dimorphism.

The central melanocortin system is fundamentally important for controlling food intake and energy homeostasis. Melanocortin-3 receptor (MC3R) is one of two major receptors of the melanocortin system found in the brain. In contrast to the well-characterized melanocortin-4 receptor (MC4R), little is known regarding the organization of MC3R-expressing neural circuits. To increase our understanding of the intrinsic organization of MC3R neural circuits, identify specific differences between males and females, and gain a neural systems level perspective of this circuitry, we conducted a brain-wide mapping of neurons labeled for MC3R and characterized the distribution of their projections. Analysis revealed MC3R neuronal and terminal labeling in multiple brain regions that control a diverse range of physiological functions and behavioral processes. Notably, dense labeling was observed in the hypothalamus, as well as areas that share considerable connections with the hypothalamus, including the cortex, amygdala, thalamus, and brainstem. Additionally, MC3R neuronal labeling was sexually dimorphic in several areas, including the anteroventral periventricular area, arcuate nucleus, principal nucleus of the bed nucleus of the stria terminalis, and ventral premammillary region. Altogether, anatomical evidence reported here suggests that MC3R has the potential to influence several different classes of motivated behavior that are essential for survival, including ingestive, reproductive, defensive, and arousal behaviors, and is likely to modulate these behaviors differently in males and females.

Demonstration of a Common DPhe7 to DNal(2')7 Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Histone deacetylase 6 inhibition restores leptin sensitivity and reduces obesity.

The adipose tissue-derived hormone leptin can drive decreases in food intake while increasing energy expenditure. In diet-induced obesity, circulating leptin levels rise proportionally to adiposity. Despite this hyperleptinemia, rodents and humans with obesity maintain increased adiposity and are resistant to leptin's actions. Here we show that inhibitors of the cytosolic enzyme histone deacetylase 6 (HDAC6) act as potent leptin sensitizers and anti-obesity agents in diet-induced obese mice. Specifically, HDAC6 inhibitors, such as tubastatin A, reduce food intake, fat mass, hepatic steatosis and improve systemic glucose homeostasis in an HDAC6-dependent manner. Mechanistically, peripheral, but not central, inhibition of HDAC6 confers central leptin sensitivity. Additionally, the anti-obesity effect of tubastatin A is attenuated in animals with a defective central leptin-melanocortin circuitry, including db/db and MC4R knockout mice. Our results suggest the existence of an HDAC6-regulated adipokine that serves as a leptin-sensitizing agent and reveals HDAC6 as a potential target for the treatment of obesity.

Deletion of the Feeding-Induced Hepatokine TSK Ameliorates the Melanocortin Obesity Syndrome.

Work in recent decades has established that metabolic hormones released by endocrine cells and diverse other cell types serve to regulate nutrient intake and energy homeostasis. Tsukushi (TSK) is a leucine-rich repeat-containing protein secreted primarily by the liver that exerts an inhibitory effect on brown fat sympathetic innervation and thermogenesis. Despite this, physiological regulation of TSK and the mechanisms underlying its effects on energy balance remain poorly understood. Here we show that hepatic expression and plasma concentrations of TSK are induced by feeding and regulated by melanocortin-4 receptor (MC4R) signaling. We generated TSK and MC4R-double-knockout mice to elucidate the nature of cross talk between TSK and the central regulatory circuit of energy balance. Remarkably, TSK inactivation restores energy balance, ameliorates hyperphagia, and improves metabolic health in MC4R-deficient mice. TSK ablation enhances thermogenic gene expression in brown fat, dampens obesity-association inflammation in the liver and adipose tissue, and protects MC4R-null mice from diet-induced nonalcoholic steatohepatitis. At the cellular level, TSK deficiency augments feeding-induced c-Fos expression in the paraventricular nucleus of the hypothalamus. These results illustrate physiological cross talk between TSK and the central regulatory circuit in maintaining energy balance and metabolic homeostasis.

Cardiac Phenotype and Tissue Sodium Content in Adolescents With Defects in the Melanocortin System.

CONTEXT: Pro-opiomelanocortin (POMC) and the melanocortin-4 receptor (MC4R) play a pivotal role in the leptin-melanocortin pathway. Mutations in these genes lead to monogenic types of obesity due to severe hyperphagia. In addition to dietary-induced obesity, a cardiac phenotype without hypertrophy has been identified in MC4R knockout mice. OBJECTIVE: We aimed to characterize cardiac morphology and function as well as tissue Na+ content in humans with mutations in POMC and MC4R genes. METHODS: A cohort of 42 patients (5 patients with bi-allelic POMC mutations, 6 heterozygous MC4R mutation carriers, 19 obese controls without known monogenic cause, and 12 normal weight controls) underwent cardiac magnetic resonance (CMR) imaging and 23Na-MRI. RESULTS: Monogenic obese patients with POMC or MC4R mutation respectively had a significantly lower left ventricular mass/body surface area (BSA) than nonmonogenic obese patients. Left ventricular end-diastolic volume/BSA was significantly lower in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. Subcutaneous fat and skin Na+ content was significantly higher in POMC- and MC4R-deficient patients than in nonmonogenic obese patients. In these compartments, the water content was significantly higher in patients with POMC and MC4R mutation than in control groups. CONCLUSION: Patients with POMC or MC4R mutations carriers had a lack of transition to hypertrophy, significantly lower cardiac muscle mass/BSA, and stored more Na+ within the subcutaneous fat tissue than nonmonogenic obese patients. The results point towards the role of the melanocortin pathway for cardiac function and tissue Na+ storage and the importance of including cardiologic assessments into the diagnostic work-up of these patients.

Network dynamics of hypothalamic feeding neurons.

Mutations in the melanocortin 4 receptor (MC4R) result in hyperphagia and obesity and are the most common cause of monogenic obesity in humans. Preclinical rodent studies have determined that the critical role of the MC4R in controlling feeding can be mapped in part to its expression in the paraventricular nucleus of the hypothalamus (paraventricular nucleus [PVN]), where it regulates the activity of anorexic neural circuits. Despite the critical role of PVN MC4R neurons in regulating feeding, the in vivo neuronal activity of these cells remains largely unstudied, and the network activity of PVN MC4R neurons has not been determined. Here, we utilize in vivo single-cell endomicroscopic and mathematical approaches to determine the activity and network dynamics of PVN MC4R neurons in response to changes in energy state and pharmacological manipulation of central melanocortin receptors. We determine that PVN MC4R neurons exhibit both quantitative and qualitative changes in response to fasting and refeeding. Pharmacological stimulation of MC4R with the therapeutic MC4R agonist setmelanotide rapidly increases basal PVN MC4R activity, while stimulation of melanocortin 3 receptor (MC3R) inhibits PVN MC4R activity. Finally, we find that distinct PVN MC4R neuronal ensembles encode energy deficit and energy surfeit and that energy surfeit is associated with enhanced network connections within PVN MC4R neurons. These findings provide valuable insight into the neural dynamics underlying hunger and energy surfeit.