Charlotte E. Lee

Narcolepsy in orexin Knockout Mice: Molecular Genetics of Sleep Regulation

Neurons containing the neuropeptide orexin (hypocretin) are located exclusively in the lateral hypothalamus and send axons to numerous regions throughout the central nervous system, including the major nuclei implicated in sleep regulation. Here, we report that, by behavioral and electroencephalographic criteria, orexin knockout mice exhibit a phenotype strikingly similar to human narcolepsy patients, as well as canarc-1 mutant dogs, the only known monogenic model of narcolepsy. Moreover, modafinil, an anti-narcoleptic drug with ill-defined mechanisms of action, activates orexin-containing neurons. We propose that orexin regulates sleep/wakefulness states, and that orexin knockout mice are a model of human narcolepsy, a disorder characterized primarily by rapid eye movement (REM) sleep dysregulation.

DIFFERENTIAL EXPRESSION OF OREXIN RECEPTORS 1 AND 2 IN THE RAT BRAIN

Orexins (hypocretins) are neuropeptides synthesized in the central nervous system exclusively by neurons of the lateral hypothalamus. Orexin‐containing neurons have widespread projections and have been implicated in complex physiological functions including feeding behavior, sleep states, neuroendocrine function, and autonomic control. Two orexin receptors (OX1R and OX2R) have been identified, with distinct expression patterns throughout the brain, but a systematic examination of orexin receptor expression in the brain has not appeared. We used in situ hybridization histochemistry to examine the patterns of expression of mRNA for both orexin receptors throughout the brain. OX1R mRNA was observed in many brain regions including the prefrontal and infralimbic cortex, hippocampus, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, dorsal raphe nucleus, and locus coeruleus. OX2R mRNA was prominent in a complementary distribution including the cerebral cortex, septal nuclei, hippocampus, medial thalamic groups, raphe nuclei, and many hypothalamic nuclei including the tuberomammillary nucleus, dorsomedial nucleus, paraventricular nucleus, and ventral premammillary nucleus. The differential distribution of orexin receptors is consistent with the proposed multifaceted roles of orexin in regulating homeostasis and may explain the unique role of the OX2R receptor in regulating sleep state stability. J. Comp. Neurol. 435:6–25, 2001.

Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area

Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin‐concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), α‐melanin‐stimulating hormone (α‐MSH), and agouti‐related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual‐label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP‐ and NPY‐immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas α‐MSH‐immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY‐, AgRP‐, and α‐MSH‐immunoreactive fibers. Projections from populations of leptin‐responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight. J. Comp. Neurol. 402:442–459, 1998.

Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area

Recent studies have reinforced the view that the lateral hypothalamic area (LHA) regulates food intake and body weight. We identified leptin-sensitive neurons in the arcuate nucleus of the hypothalamus (Arc) that innervate the LHA using retrograde tracing with leptin administration. We found that retrogradely labeled cells in the Arc contained neuropeptide Y (NPY) mRNA or proopiomelanocortin (POMC) mRNA. Following leptin administration, NPY cells in the Arc did not express Fos but expressed suppressor of cytokine signaling-3 (SOCS-3) mRNA. In contrast, leptin induced both Fos and SOCS-3 expression in POMC neurons, many of which also innervated the LHA. These findings suggest that leptin directly and differentially engages NPY and POMC neurons that project to the LHA, linking circulating leptin and neurons that regulate feeding behavior and body weight homeostasis.

Leptin Activates Hypothalamic CART Neurons Projecting to the Spinal Cord

The adipocyte-derived hormone leptin decreases body weight in part by activating the sympathetic nervous system, resulting in increased thermogenesis and energy expenditure. We investigated hypothalamic pathways underlying leptins effects on stimulating the sympathetic nervous system. We found that leptin activates neurons in the retrochiasmatic area (RCA) and lateral arcuate nucleus (Arc) that innervate the thoracic spinal cord and also contain cocaine- and amphetamine-regulated transcript (CART). We also found that most CART-containing neurons in the RCA and Arc of the hypothalamus also contain proopiomelanocortin (POMC) mRNA. The finding that leptin activates CART/POMC neurons innervating sympathetic preganglionic neurons in the thoracic spinal cord suggests that this pathway may contribute to the increased thermogenesis and energy expenditure and decreased body weight observed following leptin administration.

Divergence of Melanocortin Pathways in the Control of Food Intake and Energy Expenditure

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.

Expression of ghrelin receptor mRNA in the rat and the mouse brain

Ghrelin is a hormone that stimulates growth hormone secretion and signals energy insufficiency via interaction with its receptor, the growth hormone secretagogue receptor (GHSR). The GHSR is located in both the central nervous system and the periphery. Its distribution in the CNS, as assessed by in situ hybridization histochemistry (ISHH), has been described previously in a few mammalian species, although these studies were limited by either the detail provided or the extent of the regions examined. In the present study, we systematically examined the distribution of GHSR mRNA in the adult rat and mouse brains and cervical spinal cords by using ISHH with novel cRNA probes specific for the mRNA encoding functional GHSR (the type 1a variant). We confirmed GHSR mRNA expression in several hypothalamic nuclei, many of which have long been recognized as playing roles in body weight and food intake. GHSR also was found in several other regions previously unknown to express GHSR mRNA, including many parasympathetic preganglionic neurons. Additionally, we found GHSR mRNA within all three components of the dorsal vagal complex, including the area postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus. Finally, we examined the coexpression of GHSR with tyrosine hydroxylase and cholecystokinin and demonstrate a high degree of GHSR mRNA expression within dopaminergic, cholecystokinin‐containing neurons of the substantia nigra and ventral tegmental area. J. Comp. Neurol. 494:528–548, 2006.

Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis

Neuroanatomical and electrophysiological studies have shown that hypothalamic POMC neurons are targets of the adipostatic hormone leptin. However, the physiological relevance of leptin signaling in these neurons has not yet been directly tested. Here, using the Cre/loxP system, we critically test the functional importance of leptin action on POMC neurons by deleting leptin receptors specifically from these cells in mice. Mice lacking leptin signaling in POMC neurons are mildly obese, hyperleptinemic, and have altered expression of hypothalamic neuropeptides. In summary, leptin receptors on POMC neurons are required but not solely responsible for leptins regulation of body weight homeostasis.

Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity

A subset of neurons in the brain, known as ‘glucose-excited’ neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic β-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (KATP) channels. Although β-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of KATP channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.

Mice lacking ghrelin receptors resist the development of diet-induced obesity

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Since its discovery, accumulating evidence has suggested that ghrelin may play a role in signaling and reversing states of energy insufficiency. For example, ghrelin levels rise following food deprivation, and ghrelin administration stimulates feeding and increases body weight and adiposity. However, recent loss-of-function studies have raised questions regarding the physiological significance of ghrelin in regulating these processes. Here, we present results of a study using a novel GHSR-null mouse model, in which ghrelin administration fails to acutely stimulate food intake or activate arcuate nucleus neurons. We show that when fed a high-fat diet, both female and male GHSR-null mice eat less food, store less of their consumed calories, preferentially utilize fat as an energy substrate, and accumulate less body weight and adiposity than control mice. Similar effects on body weight and adiposity were also observed in female, but not male, GHSR-null mice fed standard chow. GHSR deletion also affected locomotor activity and levels of glycemia. These findings support the hypothesis that ghrelin-responsive pathways are an important component of coordinated body weight control. Moreover, our data suggest that ghrelin signaling is required for development of the full phenotype of diet-induced obesity.