Melanocortin 3 receptor-expressing neurons in the ventromedial hypothalamus promote glucose disposal.

The ventromedial hypothalamus (VMH) is a critical neural node that senses blood glucose and promotes glucose utilization or mobilization during hypoglycemia. The VMH neurons that control these distinct physiologic processes are largely unknown. Here, we show that melanocortin 3 receptor (Mc3R)-expressing VMH neurons (VMHMC3R) sense glucose changes both directly and indirectly via altered excitatory input. We identify presynaptic nodes that potentially regulate VMHMC3R neuronal activity, including inputs from proopiomelanocortin (POMC)-producing neurons in the arcuate nucleus. We find that VMHMC3R neuron activation blunts, and their silencing enhances glucose excursion following a glucose load. Overall, these findings demonstrate that VMHMC3R neurons are a glucose-responsive hypothalamic subpopulation that promotes glucose disposal upon activation; this highlights a potential site for targeting dysregulated glycemia.

An open-source device for measuring food intake and operant behavior in rodent home-cages.

Feeding is critical for survival, and disruption in the mechanisms that govern food intake underlies disorders such as obesity and anorexia nervosa. It is important to understand both food intake and food motivation to reveal mechanisms underlying feeding disorders. Operant behavioral testing can be used to measure the motivational component to feeding, but most food intake monitoring systems do not measure operant behavior. Here, we present a new solution for monitoring both food intake and motivation in rodent home-cages: the Feeding Experimentation Device version 3 (FED3). FED3 measures food intake and operant behavior in rodent home-cages, enabling longitudinal studies of feeding behavior with minimal experimenter intervention. It has a programmable output for synchronizing behavior with optogenetic stimulation or neural recordings. Finally, FED3 design files are open-source and freely available, allowing researchers to modify FED3 to suit their needs.

Obesity and anorexia nervosa are two health conditions related to food intake. Researchers studying these disorders in animal models need to both measure food intake and assess behavioural factors: that is, why animals seek and consume food. Measuring an animal's food intake is usually done by weighing food containers. However, this can be inaccurate due to the small amount of food that rodents eat. As for studying feeding motivation, this can involve calculating the number of times an animal presses a lever to receive a food pellet. These tests are typically conducted in hour-long sessions in temporary testing cages, called operant boxes. Yet, these tests only measure a brief period of a rodent's life. In addition, it takes rodents time to adjust to these foreign environments, which can introduce stress and may alter their feeding behaviour. To address this, Matikainen-Ankney, Earnest, Ali et al. developed a device for monitoring food intake and feeding behaviours around the clock in rodent home cages with minimal experimenter intervention. This 'Feeding Experimentation Device' (FED3) features a pellet dispenser and two 'nose-poke' sensors to measure total food intake, as well as motivation for and learning about food rewards. The battery-powered, wire-free device fits in standard home cages, enabling long-term studies of feeding behaviour with minimal intervention from investigators and less stress on the animals. This means researchers can relate data to circadian rhythms and meal patterns, as Matikainen-Ankney did here. Moreover, the device software is open-source so researchers can customise it to suit their experimental needs. It can also be programmed to synchronise with other instruments used in animal experiments, or across labs running the same behavioural tasks for multi-site studies. Used in this way, it could help improve reproducibility and reliability of results from such studies. In summary, Matikainen-Ankney et al. have presented a new practical solution for studying food-related behaviours in mice and rats. Not only could the device be useful to researchers, it may also be suitable to use in educational settings such as teaching labs and classrooms.

Integrating Hunger with Rival Motivations.

Motivated behaviors have fascinated neuroscientists and ethologists for decades due to their necessity for organism survival. Motivations guide behavioral choice through an intricate synthesis of internal state detection, external stimulus exposure, and learned associations. One critical motivation, hunger, provides an accessible example for understanding purposeful behavior. Neuroscientists commonly focus research efforts on neural circuits underlying individual motivations, sacrificing ethological relevance for tight experimental control. This restrictive focus deprives the field of a more nuanced understanding of the unified nervous system in weighing multiple motivations simultaneously and choosing, moment-to-moment, optimal behaviors for survival. Here, we explore the reciprocal interplay between hunger, encoded via hypothalamic neurons marked by the expression of Agouti-related peptide, and alternative need-based motivational systems.

Paraventricular, subparaventricular and periventricular hypothalamic IRS4-expressing neurons are required for normal energy balance.

Understanding the neural components modulating feeding-related behavior and energy expenditure is crucial to combating obesity and its comorbidities. Neurons within the paraventricular nucleus of the hypothalamus (PVH) are a key component of the satiety response; activation of the PVH decreases feeding and increases energy expenditure, thereby promoting negative energy balance. In contrast, PVH ablation or silencing in both rodents and humans leads to substantial obesity. Recent studies have identified genetically-defined PVH subpopulations that control discrete aspects of energy balance (e.g. oxytocin (OXT), neuronal nitric oxide synthase 1 (NOS1), melanocortin 4-receptor (MC4R), prodynorphin (PDYN)). We previously demonstrated that non-OXT NOS1PVH neurons contribute to PVH-mediated feeding suppression. Here, we identify and characterize a non-OXT, non-NOS1 subpopulation of PVH and peri-PVH neurons expressing insulin-receptor substrate 4 (IRS4PVH) involved in energy balance control. Using Cre-dependent viral tools to activate, trace and silence these neurons, we highlight the sufficiency and necessity of IRS4PVH neurons in normal feeding and energy expenditure regulation. Furthermore, we demonstrate that IRS4PVH neurons lie within a complex hypothalamic circuitry that engages distinct hindbrain regions and is innervated by discrete upstream hypothalamic sites. Overall, we reveal a requisite role for IRS4PVH neurons in PVH-mediated energy balance which raises the possibility of developing novel approaches targeting IRS4PVH neurons for anti-obesity therapies.

Lateral Hypothalamic Mc3R-Expressing Neurons Modulate Locomotor Activity, Energy Expenditure, and Adiposity in Male Mice.

The central melanocortin system plays a crucial role in the control of energy balance. Although the decreased energy expenditure and increased adiposity of melanocortin-3 receptor (Mc3R)-null mice suggest the importance of Mc3R-regulated neurons in energy homeostasis, the roles for specific subsets of Mc3R neurons in energy balance have yet to be determined. Because the lateral hypothalamic area (LHA) contributes to the control of energy expenditure and feeding, we generated Mc3rcre mice to determine the roles of LHA Mc3R (Mc3RLHA) neurons in energy homeostasis. We found that Mc3RLHA neurons overlap extensively with LHA neuron markers that contribute to the control of energy balance (neurotensin, galanin, and leptin receptor) and project to brain areas involved in the control of feeding, locomotion, and energy expenditure, consistent with potential roles for Mc3RLHA neurons in these processes. Indeed, selective chemogenetic activation of Mc3RLHA neurons increased locomotor activity and augmented refeeding after a fast. Although the ablation of Mc3RLHA neurons did not alter food intake, mice lacking Mc3RLHA neurons displayed decreased energy expenditure and locomotor activity, along with increased body mass and adiposity. Thus, Mc3R neurons lie within LHA neurocircuitry that modulates locomotor activity and energy expenditure and contribute to energy balance control.

The Role of PVH Circuits in Leptin Action and Energy Balance.

Although it has been known for more than a century that the brain controls overall energy balance and adiposity by regulating feeding behavior and energy expenditure, the roles for individual brain regions and neuronal subtypes were not fully understood until recently. This area of research is active, and as such our understanding of the central regulation of energy balance is continually being refined as new details emerge. Much of what we now know stems from the discoveries of leptin and the hypothalamic melanocortin system. Hypothalamic circuits play a crucial role in the control of feeding and energy expenditure, and within the hypothalamus, the arcuate nucleus (ARC) functions as a gateway for hormonal signals of energy balance, such as leptin. It is also well established that the ARC is a primary residence for hypothalamic melanocortinergic neurons. The paraventricular hypothalamic nucleus (PVH) receives direct melanocortin input, along with other integrated signals that affect energy balance, and mediates the majority of hypothalamic output to control both feeding and energy expenditure. Herein, we review in detail the structure and function of the ARC-PVH circuit in mediating leptin signaling and in regulating energy balance.

Control of food intake and energy expenditure by Nos1 neurons of the paraventricular hypothalamus.

The paraventricular nucleus of the hypothalamus (PVH) contains a heterogeneous cluster of Sim1-expressing cell types that comprise a major autonomic output nucleus and play critical roles in the control of food intake and energy homeostasis. The roles of specific PVH neuronal subtypes in energy balance have yet to be defined, however. The PVH contains nitric oxide synthase-1 (Nos1)-expressing (Nos1(PVH)) neurons of unknown function; these represent a subset of the larger population of Sim1-expressing PVH (Sim1(PVH)) neurons. To determine the role of Nos1(PVH) neurons in energy balance, we used Cre-dependent viral vectors to both map their efferent projections and test their functional output in mice. Here we show that Nos1(PVH) neurons project to hindbrain and spinal cord regions important for food intake and energy expenditure control. Moreover, pharmacogenetic activation of Nos1(PVH) neurons suppresses feeding to a similar extent as Sim1(PVH) neurons, and increases energy expenditure and activity. Furthermore, we found that oxytocin-expressing PVH neurons (OXT(PVH)) are a subset of Nos1(PVH) neurons. OXT(PVH) cells project to preganglionic, sympathetic neurons in the thoracic spinal cord and increase energy expenditure upon activation, though not to the same extent as Nos1(PVH) neurons; their activation fails to alter feeding, however. Thus, Nos1(PVH) neurons promote negative energy balance through changes in feeding and energy expenditure, whereas OXT(PVH) neurons regulate energy expenditure alone, suggesting a crucial role for non-OXT Nos1(PVH) neurons in feeding regulation.

Leptin-inhibited PBN neurons enhance responses to hypoglycemia in negative energy balance.

Hypoglycemia initiates the counter-regulatory response (CRR), in which the sympathetic nervous system, glucagon and glucocorticoids restore glucose to appropriate concentrations. During starvation, low leptin levels restrain energy utilization, enhancing long-term survival. To ensure short-term survival during hypoglycemia in fasted animals, the CRR must overcome this energy-sparing program and nutrient depletion. Here we identify in mice a previously unrecognized role for leptin and a population of leptin-regulated neurons that modulate the CRR to meet these challenges. Hypoglycemia activates neurons of the parabrachial nucleus (PBN) that coexpress leptin receptor (LepRb) and cholecystokinin (CCK) (PBN LepRb(CCK) neurons), which project to the ventromedial hypothalamic nucleus. Leptin inhibits these cells, and Cck(cre)-mediated ablation of LepRb enhances the CRR. Inhibition of PBN LepRb cells blunts the CRR, whereas their activation mimics the CRR in a CCK-dependent manner. PBN LepRb(CCK) neurons are a crucial component of the CRR system and may be a therapeutic target in hypoglycemia.

AVP neurons in the paraventricular nucleus of the hypothalamus regulate feeding.

Melanocortins and their receptors are critical components of energy homeostasis and the paraventricular nucleus of the hypothalamus (PVH) is an important site of melanocortin action. Although best known for its role in osmoregulation, arginine vasopressin (AVP) has been implicated in feeding and is robustly expressed in the PVH. Since the anorectic melanocortin agonist MTII activates PVH-AVP neurons, we hypothesized that PVH-AVP neurons contribute to PVH-mediated anorexia. To test this, we used an AVP-specific Cre-driver mouse in combination with viral vectors to acutely manipulate PVH-AVP neuron function. Using designer receptors exclusively activated by designer drugs (DREADDs) to control PVH-AVP neuron activity, we show that activation of PVH-AVP neurons acutely inhibits food intake, whereas their inhibition partially reverses melanocortin-induced anorexia. We further show that MTII fails to fully suppress feeding in mice with virally-induced PVH-AVP neuron ablation. Thus PVH-AVP neurons contribute to feeding behaviors, including the acute anorectic response to MTII.

An obligate role of oxytocin neurons in diet induced energy expenditure.

Oxytocin neurons represent one of the major subsets of neurons in the paraventricular hypothalamus (PVH), a critical brain region for energy homeostasis. Despite substantial evidence supporting a role of oxytocin in body weight regulation, it remains controversial whether oxytocin neurons directly regulate body weight homeostasis, feeding or energy expenditure. Pharmacologic doses of oxytocin suppress feeding through a proposed melanocortin responsive projection from the PVH to the hindbrain. In contrast, deficiency in oxytocin or its receptor leads to reduced energy expenditure without feeding abnormalities. To test the physiological function of oxytocin neurons, we specifically ablated oxytocin neurons in adult mice. Our results show that oxytocin neuron ablation in adult animals has no effect on body weight, food intake or energy expenditure on a regular diet. Interestingly, male mice lacking oxytocin neurons are more sensitive to high fat diet-induced obesity due solely to reduced energy expenditure. In addition, despite a normal food intake, these mice exhibit a blunted food intake response to leptin administration. Thus, our study suggests that oxytocin neurons are required to resist the obesity associated with a high fat diet; but their role in feeding is permissive and can be compensated for by redundant pathways.