To eat or to sleep: That is a lateral hypothalamic question.

The lateral hypothalamus (LH) is a functionally and anatomically complex brain region that is involved in the regulation of many behavioral and physiological processes including feeding, arousal, energy balance, stress, reward and motivated behaviors, pain perception, body temperature regulation, digestive functions and blood pressure. Despite noteworthy experimental efforts over the past decades, the circuit, cellular and synaptic bases by which these different processes are regulated by the LH remains incompletely understood. This knowledge gap links in large part to the high cellular heterogeneity of the LH. Fortunately, the rapid evolution of newer genetic and electrophysiological tools is now permitting the selective manipulation, typically genetically-driven, of discrete LH cell populations. This, in turn, permits not only assignment of function to discrete cell groups, but also reveals that considerable synergistic and antagonistic interactions exist between key LH cell populations that regulate feeding and arousal. For example, we now know that while LH melanin-concentrating hormone (MCH) and orexin/hypocretin neurons both function as sensors of the internal metabolic environment, their roles regulating sleep and arousal are actually opposing. Additional studies have uncovered similarly important roles for subpopulations of LH GABAergic cells in the regulation of both feeding and arousal. Herein we review the role of LH MCH, orexin/hypocretin and GABAergic cell populations in the regulation of energy homeostasis (including feeding) and sleep-wake and discuss how these three cell populations, and their subpopulations, may interact to optimize and coordinate metabolism, sleep and arousal. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

Defense of Elevated Body Weight Setpoint in Diet-Induced Obese Rats on Low Energy Diet Is Mediated by Loss of Melanocortin Sensitivity in the Paraventricular Hypothalamic Nucleus.

Some animals and humans fed a high-energy diet (HED) are diet-resistant (DR), remaining as lean as individuals who were naïve to HED. Other individuals become obese during HED exposure and subsequently defend the obese weight (Diet-Induced Obesity- Defenders, DIO-D) even when subsequently maintained on a low-energy diet. We hypothesized that the body weight setpoint of the DIO-D phenotype resides in the hypothalamic paraventricular nucleus (PVN), where anorexigenic melanocortins, including melanotan II (MTII), increase presynaptic GABA release, and the orexigenic neuropeptide Y (NPY) inhibits it. After prolonged return to low-energy diet, GABA inputs to PVN neurons from DIO-D rats exhibited highly attenuated responses to MTII compared with those from DR and HED-naïve rats. In DIO-D rats, melanocortin-4 receptor expression was significantly reduced in dorsomedial hypothalamus, a major source of GABA input to PVN. Unlike melanocortin responses, NPY actions in PVN of DIO-D rats were unchanged, but were reduced in neurons of the ventromedial hypothalamic nucleus; in PVN of DR rats, NPY responses were paradoxically increased. MTII-sensitivity was restored in DIO-D rats by several weeks' refeeding with HED. The loss of melanocortin sensitivity restricted to PVN of DIO-D animals, and its restoration upon prolonged refeeding with HED suggest that their melanocortin systems retain the ability to up- and downregulate around their elevated body weight setpoint in response to longer-term changes in dietary energy density. These properties are consistent with a mechanism of body weight setpoint.

Melanin-concentrating hormone is necessary for olanzapine-inhibited locomotor activity in male mice.

Olanzapine (OLZ), an atypical antipsychotic, can be effective in treating patients with restricting type anorexia nervosa who exercise excessively. Clinical improvements include weight gain and reduced pathological hyperactivity. However the neuronal populations and mechanisms underlying OLZ actions are not known. We studied the effects of OLZ on hyperactivity using male mice lacking the hypothalamic neuropeptide melanin-concentrating hormone (MCHKO) that are lean and hyperactive. We compared the in vivo effects of systemic or intra-accumbens nucleus (Acb) OLZ administration on locomotor activity in WT and MCHKO littermates. Acute systemic OLZ treatment in WT mice significantly reduced locomotor activity, an effect that is substantially attenuated in MCHKO mice. Furthermore, OLZ infusion directly into the Acb of WT mice reduced locomotor activity, but not in MCHKO mice. To identify contributing neuronal mechanisms, we assessed the effect of OLZ treatment on Acb synaptic transmission ex vivo and in vitro. Intraperitoneal OLZ treatment reduced Acb GABAergic activity in WT but not MCHKO neurons. This effect was also seen in vitro by applying OLZ to acute brain slices. OLZ reduced the frequency and amplitude of GABAergic activity that was more robust in WT than MCHKO Acb. These findings indicate that OLZ reduced Acb GABAergic transmission and that MCH is necessary for the hypolocomotor effects of OLZ.

Melanin-concentrating hormone neurons release glutamate for feedforward inhibition of the lateral septum.

Melanin-concentrating hormone (MCH) regulates vital physiological functions, including energy balance and sleep. MCH cells are thought to be GABAergic, releasing GABA to inhibit downstream targets. However, there is little experimental support for this paradigm. To better understand the synaptic mechanisms of mouse MCH neurons, we performed neuroanatomical mapping and characterization followed by optogenetics to test their functional connectivity at downstream targets. Synaptophysin-mediated projection mapping showed that the lateral septal nucleus (LS) contained the densest accumulation of MCH nerve terminals. We then expressed channel rhodopsin-2 in MCH neurons and photostimulated MCH projections to determine their effect on LS activity. Photostimulation of MCH projections evoked a monosynaptic glutamate release in the LS. Interestingly, this led to a feedforward inhibition that depressed LS firing by a robust secondary GABA release. This study presents a circuit analysis between MCH and LS neurons and confirms their functional connection via monosynaptic and polysynaptic pathways. Our findings indicate that MCH neurons are not exclusively GABAergic and reveal a glutamate-mediated, feedforward mechanism that inhibits LS cells.

Optogenetic-mediated release of histamine reveals distal and autoregulatory mechanisms for controlling arousal.

Histaminergic neurons in the tuberomammillary nucleus (TMN) are an important component of the ascending arousal system and may form part of a "flip-flop switch" hypothesized to regulate sleep and wakefulness. Anatomical studies have shown that the wake-active TMN and sleep-active ventrolateral preoptic nucleus (VLPO) are reciprocally connected, suggesting that each region can inhibit its counterpart when active. In this study, we determined how histamine affects the two branches of this circuit. We selectively expressed channelrhodopsin-2 (ChR2) in TMN neurons and used patch-clamp recordings in mouse brain slices to examine the effects of photo-evoked histamine release in the ventrolateral TMN and VLPO. Photostimulation decreased inhibitory GABAergic inputs to the ventrolateral TMN neurons but produced a membrane hyperpolarization and increased inhibitory synaptic input to the VLPO neurons. We found that in VLPO the response to histamine was indirect, most likely via a GABAergic interneuron. Our experiments demonstrate that release of histamine from TMN neurons can disinhibit the TMN and suppresses the activity of sleep-active VLPO neurons to promote TMN neuronal firing. This further supports the sleep-wake "flip-flop switch" hypothesis and a role for histamine in stabilizing the switch to favor wake states.

Expression of melanin-concentrating hormone receptor 2 protects against diet-induced obesity in male mice.

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide that is a ligand for two subtypes of MCH receptors, MCHR1 and MCHR2. MCHR1 is universally expressed in mammals ranging from rodents to humans, but the expression of MCHR2 is substantially restricted. In mammals, MCHR2 has been defined in primates as well as other species such as cats and dogs but is not seen in rodents. Although the role of MCHR1 in mediating the actions of MCH on energy balance is clearly defined using mouse models, the role of MCHR2 is harder to characterize because of its limited expression. To determine any potential role of MCHR2 in energy balance, we generated a transgenic MCHR1R2 mouse model, where human MCHR2 is coexpressed in MCHR1-expressing neurons. As shown previously, control wild-type mice expressing only native MCHR1 developed diet-induced obesity when fed a high-fat diet. In contrast, MCHR1R2 mice had lower food intake, leading to their resistance to diet-induced obesity. Furthermore, we showed that MCH action is altered in MCHR1R2 mice. MCH treatment in wild-type mice inhibited the activation of the immediate-early gene c-fos, and coexpression of MCHR2 reduced the inhibitory actions of MCHR1 on this pathway. In conclusion, we developed an experimental animal model that can provide insight into the action of MCHR2 in the central nervous system and suggest that some actions of MCHR2 oppose the endogenous actions of MCHR1.

Neurochemical characterization of neurons expressing melanin-concentrating hormone receptor 1 in the mouse hypothalamus.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that acts via MCH receptor 1 (MCHR1) in the mouse. It promotes positive energy balance; thus, mice lacking MCH or MCHR1 are lean, hyperactive, and resistant to diet-induced obesity. Identifying the cellular targets of MCH is an important step to understanding the mechanisms underlying MCH actions. We generated the Mchr1-cre mouse that expresses cre recombinase driven by the MCHR1 promoter and crossed it with a tdTomato reporter mouse. The resulting Mchr1-cre/tdTomato progeny expressed easily detectable tdTomato fluorescence in MCHR1 neurons, which were found throughout the olfactory system, striatum, and hypothalamus. To chemically identify MCH-targeted cell populations that play a role in energy balance, MCHR1 hypothalamic neurons were characterized by colabeling select hypothalamic neuropeptides with tdTomato fluorescence. TdTomato fluorescence colocalized with dynorphin, oxytocin, vasopressin, enkephalin, thyrothropin-releasing hormone, and corticotropin-releasing factor immunoreactive cells in the paraventricular nucleus. In the lateral hypothalamus, neurotensin, but neither orexin nor MCH neurons, expressed tdTomato. In the arcuate nucleus, both Neuropeptide Y and proopiomelanocortin cells expressed tdTomato. We further demonstrated that some of these arcuate neurons were also targets of leptin action. Interestingly, MCHR1 was expressed in the vast majority of leptin-sensitive proopiomelanocortin neurons, highlighting their importance for the orexigenic actions of MCH. Taken together, this study supports the use of the Mchr1-cre mouse for outlining the neuroanatomical distribution and neurochemical phenotype of MCHR1 neurons.

Magel2 is required for leptin-mediated depolarization of POMC neurons in the hypothalamic arcuate nucleus in mice.

Prader-Willi Syndrome is the most common syndromic form of human obesity and is caused by the loss of function of several genes, including MAGEL2. Mice lacking Magel2 display increased weight gain with excess adiposity and other defects suggestive of hypothalamic deficiency. We demonstrate Magel2-null mice are insensitive to the anorexic effect of peripherally administered leptin. Although their excessive adiposity and hyperleptinemia likely contribute to this physiological leptin resistance, we hypothesized that Magel2 may also have an essential role in intracellular leptin responses in hypothalamic neurons. We therefore measured neuronal activation by immunohistochemistry on brain sections from leptin-injected mice and found a reduced number of arcuate nucleus neurons activated after leptin injection in the Magel2-null animals, suggesting that most but not all leptin receptor-expressing neurons retain leptin sensitivity despite hyperleptinemia. Electrophysiological measurements of arcuate nucleus neurons expressing the leptin receptor demonstrated that although neurons exhibiting hyperpolarizing responses to leptin are present in normal numbers, there were no neurons exhibiting depolarizing responses to leptin in the mutant mice. Additional studies demonstrate that arcuate nucleus pro-opiomelanocortin (POMC) expressing neurons are unresponsive to leptin. Interestingly, Magel2-null mice are hypersensitive to the anorexigenic effects of the melanocortin receptor agonist MT-II. In Prader-Willi Syndrome, loss of MAGEL2 may likewise abolish leptin responses in POMC hypothalamic neurons. This neural defect, together with increased fat mass, blunted circadian rhythm, and growth hormone response pathway defects that are also linked to loss of MAGEL2, could contribute to the hyperphagia and obesity that are hallmarks of this disorder.

The role of NPY in hypothalamic mediated food intake.

Neuropeptide Y (NPY) is a highly conserved neuropeptide with orexigenic actions in discrete hypothalamic nuclei that plays a role in regulating energy homeostasis. NPY signals via a family of high affinity receptors that mediate the widespread actions of NPY in all hypothalamic nuclei. These actions are also subject to tight, intricate regulation by numerous peripheral and central energy balance signals. The NPY system is embedded within a densely-redundant network designed to ensure stable energy homeostasis. This redundancy may underlie compensation for the loss of NPY or its receptors in germline knockouts, explaining why conventional knockouts of NPY or its receptors rarely yield a marked phenotypic change. We discuss insights into the hypothalamic role of NPY from studies of its physiological actions, responses to genetic manipulations and interactions with other energy balance signals. We conclude that numerous approaches must be employed to effectively study different aspects of NPY action.

Neuropeptide Y suppresses anorexigenic output from the ventromedial nucleus of the hypothalamus.

Output from the hypothalamic ventromedial nucleus (VMN) is anorexigenic and is supported by the excitatory actions of leptin. The VMN is also highly sensitive to the orexigenic actions of Neuropeptide Y (NPY). We report that NPY robustly inhibits VMN neurons by hyperpolarizing them and decreasing their ability to fire action potentials. This action was mediated by Y(1) receptors coupled to the activation of GIRKs (G-protein-coupled inwardly rectifying potassium channels). Approximately 80% of VMN neurons expressing leptin receptors were sensitive to the actions of NPY, whereas 75% of NPY-sensitive neurons in VMN also responded to glucose by being uniformly inhibited by elevations in glucose. Interestingly, only approximately 36% of NPY-sensitive, leptin receptor b-expressing neurons were also glucosensitive. We suggest that NPY inhibits VMN neurons that are excited by leptin, thereby arresting the anorexigenic tone exerted by VMN neurons. The results further suggest a dynamic interplay between anorexigenic and orexigenic neuromodulators within the VMN to directly affect energy balance.

Neuropeptide Y and gamma-melanocyte stimulating hormone (gamma-MSH) share a common pressor mechanism of action.

Central circuits known to regulate food intake and energy expenditure also affect central cardiovascular regulation. For example, both the melanocortin and neuropeptide Y (NPY) peptide families, known to regulate food intake, also produce central hypertensive effects. Members of both families share a similar C-terminal amino acid residue sequence, RF(Y) amide, a sequence distinct from that required for melanocortin receptor binding. A recently delineated family of RFamide receptors recognizes both of these C-terminal motifs. We now present evidence that an antagonist with Y1 and RFamide receptor activity, BIBO3304, will attenuate the central cardiovascular effects of both gamma-melanocyte stimulating hormone (gamma-MSH) and NPY. The use of synthetic melanocortin and NPY peptide analogs excluded an interaction with melanocortin or Y family receptors. We suggest that the anatomical convergence of NPY and melanocortin neurons on cardiovascular control centers may have pathophysiological implications through a common or similar RFamide receptor(s), much as they converge on other nuclei to coordinately control energy homeostasis.

The third intracellular loop stabilizes the inactive state of the neuropeptide Y1 receptor.

Constitutively active G-protein-coupled receptors (GPCRs) can signal even in the absence of ligand binding. Most Class I GPCRs are stabilized in the resting conformation by intramolecular interactions involving transmembrane domain (TM) 3 and TM6, particularly at loci 6.30 and 6.34 of TM6. Signaling by Gi/Go-coupled receptors such as the Neuropeptide Y1 receptor decreases already low basal metabolite levels. Thus, we examined constitutive activity using a biochemical assay mediated by a Gi/Gq chimeric protein and a more direct electrophysiological assay. Wild-type (WT-Y1) receptors express no measurable, agonist-independent activation, while mu-opioid receptors (MOR) and P2Y12 purinoceptors showed clear evidence of constitutive activation, especially in the electrophysiological assay. Neither point mutations at TM6 (T6.30A or N6.34A) nor substitution of the entire TM3 and TM6 regions from the MOR into the Y1 receptor increased basal WT-Y1 activation. By contrast, chimeric substitution of the third intracellular loop (ICL3) generated a constitutively active, Y1-ICL3-MOR chimera. Furthermore, the loss of stabilizing interactions from the native ICL3 enhanced the role of surrounding residues to permit basal receptor activation; because constitutive activity of the Y1-ICL3-MOR chimera was further increased by point mutation at locus 6.34, which did not alter WT-Y1 receptor activity. Our results indicate that the ICL3 stabilizes the Y1 receptor in the inactive state and confers structural properties critical for regulating Y receptor activation and signal transduction. These studies reveal the active participation of the ICL3 in the stabilization and activation of Class I GPCRs.

Y eat?

Feeding behavior is tightly regulated by peptidergic transmission within the hypothalamus. Neuropeptide Y (NPY) is one of the most potent known stimulators of food intake and has robust effects on the hypothalamic feeding neuronal networks. A vast body of literature has documented the substantial effects of NPY on feeding behavior. However, the cellular mechanisms underlying the actions of NPY have only recently begun to be explored. The NPYergic signal, including its expression in hypothalamic neurons, its release into the synaptic space, and its direct or indirect receptor-mediated actions, is highly responsive to decreases in the metabolic state. The orexigenic NPY signal can suppress the anorexigenic drive to restore energy balance homeostasis when energy levels are low, such as after food deprivation. The NPY signal interacts with glucose- and fat-sensitive signals arriving in the hypothalamus and effects changes in anorexigenic pathways, such as those mediated by the melanocortins. Recent applications of electrophysiological methods to examine the neuronal activity and pathways engaged by NPY-mediated signaling have advanced our understanding of this orexigenic system. Furthermore, crucial roles for NPY pathways in the development of hypothalamic feeding circuitry have been identified by these means. Orexigenic NPY signaling is critical during development and its absence is lethal in adults, thus reflecting the essential role of NPY for the regulation of energy homeostasis.