Michihiro Mieda

Hypothalamic orexin neurons regulate arousal according to energy balance in mice

Mammals respond to reduced food availability by becoming more wakeful and active, yet the central pathways regulating arousal and instinctual motor programs (such as food seeking) according to homeostatic need are not well understood. We demonstrate that hypothalamic orexin neurons monitor indicators of energy balance and mediate adaptive augmentation of arousal in response to fasting. Activity of isolated orexin neurons is inhibited by glucose and leptin and stimulated by ghrelin. Orexin expression of normal and ob/ob mice correlates negatively with changes in blood glucose, leptin, and food intake. Transgenic mice, in which orexin neurons are ablated, fail to respond to fasting with increased wakefulness and activity. These findings indicate that orexin neurons provide a crucial link between energy balance and arousal.

The dorsomedial hypothalamic nucleus as a putative food-entrainable circadian pacemaker

Temporal restriction of feeding can phase-shift behavioral and physiological circadian rhythms in mammals. These changes in biological rhythms are postulated to be brought about by a food-entrainable oscillator (FEO) that is independent of the suprachiasmatic nucleus. However, the neural substrates of FEO have remained elusive. Here, we carried out an unbiased search for mouse brain region(s) that exhibit a rhythmic expression of the Period genes in a feeding-entrainable manner. We found that the compact part of the dorsomedial hypothalamic nucleus (DMH) demonstrates a robust oscillation of mPer expression only under restricted feeding. The oscillation persisted for at least 2 days even when mice were given no food during the expected feeding period after the establishment of food-entrained behavioral rhythms. Moreover, refeeding after fasting rapidly induced a transient mPer expression in the same area of DMH. Taken in conjunction with recent findings (i) that behavioral expression of food-entrainable circadian rhythms is blocked by cell-specific lesions of DMH in rats and (ii) that DMH neurons directly project to orexin neurons in the lateral hypothalamus, which are essential for proper expression of food-entrained behavioral rhythms, the present study suggests that DMH plays a key role as a central FEO in the feeding-mediated regulation of circadian behaviors.

Orexin Neurons Function in an Efferent Pathway of a Food-Entrainable Circadian Oscillator in Eliciting Food-Anticipatory Activity and Wakefulness

Temporal restriction of feeding can entrain circadian behavioral and physiological rhythms in mammals. Considering the critical functions of the hypothalamic orexin (hypocretin) neuropeptides in promoting wakefulness and locomotor activity, we examined the role of orexin neurons in the adaptation to restricted feeding. In orexin neuron-ablated transgenic mice, the food-entrained rhythmicity of mPer2 expression in the brain and liver, the reversal of the sleep-wake cycle, and the recovery of daily food intake were unaltered compared with wild-type littermates. In contrast, orexin neuron-ablated mice had a severe deficit in displaying the normal food-anticipatory increases in wakefulness and locomotor activity under restricted feeding. Moreover, activity of orexin neurons markedly increased during the food-anticipatory period under restricted feeding in wild-type mice. Orexin neurons thus convey an efferent signal from putative food-entrainable oscillator or oscillators to increase wakefulness and locomotor activity.

Pharmacogenetic Modulation of Orexin Neurons Alters Sleep/Wakefulness States in Mice

Hypothalamic neurons expressing neuropeptide orexins are critically involved in the control of sleep and wakefulness. Although the activity of orexin neurons is thought to be influenced by various neuronal input as well as humoral factors, the direct consequences of changes in the activity of these neurons in an intact animal are largely unknown. We therefore examined the effects of orexin neuron-specific pharmacogenetic modulation in vivo by a new method called the Designer Receptors Exclusively Activated by Designer Drugs approach (DREADD). Using this system, we successfully activated and suppressed orexin neurons as measured by Fos staining. EEG and EMG recordings suggested that excitation of orexin neurons significantly increased the amount of time spent in wakefulness and decreased both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep times. Inhibition of orexin neurons decreased wakefulness time and increased NREM sleep time. These findings clearly show that changes in the activity of orexin neurons can alter the behavioral state of animals and also validate this novel approach for manipulating neuronal activity in awake, freely-moving animals.

Central control of bone remodeling by neuromedin U

Bone remodeling, the function affected in osteoporosis, the most common of bone diseases, comprises two phases: bone formation by matrix-producing osteoblasts and bone resorption by osteoclasts. The demonstration that the anorexigenic hormone leptin inhibits bone formation through a hypothalamic relay suggests that other molecules that affect energy metabolism in the hypothalamus could also modulate bone mass. Neuromedin U (NMU) is an anorexigenic neuropeptide that acts independently of leptin through poorly defined mechanisms. Here we show that Nmu-deficient (Nmu−/−) mice have high bone mass owing to an increase in bone formation; this is more prominent in male mice than female mice. Physiological and cell-based assays indicate that NMU acts in the central nervous system, rather than directly on bone cells, to regulate bone remodeling. Notably, leptin- or sympathetic nervous system–mediated inhibition of bone formation was abolished in Nmu−/− mice, which show an altered bone expression of molecular clock genes (mediators of the inhibition of bone formation by leptin). Moreover, treatment of wild-type mice with a natural agonist for the NMU receptor decreased bone mass. Collectively, these results suggest that NMU may be the first central mediator of leptin-dependent regulation of bone mass identified to date. Given the existence of inhibitors and activators of NMU action, our results may influence the treatment of diseases involving low bone mass, such as osteoporosis.

Connectomics of orexin-producing neurons: interface of systems of emotion, energy homeostasis and arousal.

Avoiding danger and finding food, which are life-sustaining activities that are regulated by emotion, reward and energy balance, require proper wakefulness. The orexin system controls sleep and wakefulness through interactions with systems that regulate emotion, reward and energy homeostasis. Recent findings have brought about the possibility of novel therapies targeting the orexin system for sleep disorders, including insomnia and narcolepsy-cataplexy, as well as other pathological conditions such as obesity and drug addiction [1]. In this review, we will discuss the current understanding of the integrative physiology and clinical perspectives of the orexin system. We will briefly review signaling through orexin A and B receptors and discuss the role of orexins in the pathophysiology of narcolepsy. We will also examine connections between orexin neurons and other brain areas involved in feeding behavior, reward and emotion. Finally, we will consider the therapeutic potential of drugs that target orexin receptors.

Differential Roles of Orexin Receptor-1 and -2 in the Regulation of Non-REM and REM Sleep

Orexin-A and orexin-B are hypothalamic neuropeptides that play critical roles in the maintenance of wakefulness. Intracerebroventricular (ICV) administration of orexin-A has been shown to promote wakefulness and suppress both rapid eye movement (REM) sleep and non-REM (NREM) sleep through the orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). Here, we elucidated the differential roles of orexin receptors in the regulation of sleep and wakefulness by comparing the effects of ICV orexin-A administration in wild-type, OX1R−/−, and OX2R−/− mice. The effects of orexin-A on wakefulness and NREM sleep were significantly attenuated in both knock-out mice as compared with wild-type mice, with substantially larger attenuation in OX2R−/− mice than in OX1R−/− mice. These results suggest that although the OX2R-mediated pathway has a pivotal role in the promotion of wakefulness, OX1R also plays additional roles in promoting arousal. In contrast, suppression of REM sleep by orexin-A administration was slightly and similarly attenuated in both OX1R−/− and OX2R−/− mice, suggesting a comparable contribution of the two receptors to REM sleep suppression. Histological studies demonstrated differential distributions of each receptor subtype in distinct neuronal populations with specific neurotransmitter identities in brainstem cholinergic/monoaminergic neurons. In the laterodorsal tegmental and pedunculopontine tegmental nuclei especially, cholinergic neurons exclusively expressed OX1R mRNA, but OX2R mRNA was expressed mainly in GABAergic putative interneurons. Thus, each orexin receptor subtype plays differential roles in gating NREM and REM sleep through distinct neuronal pathways.

The orexin system: roles in sleep/wake regulation

The neuropeptides orexin A and orexin B, produced in hypothalamic neurons, are critical regulators of sleep/wake states. Deficiency of orexin signaling results in narcoleptic phenotype in humans, dogs, and rodents. Recently, accumulating evidence has indicated that the orexin system regulates sleep and wakefulness through interactions with neuronal systems that are closely related with emotion, reward, and energy homeostasis. In this review, we will discuss the current understanding of the physiology of the orexin system especially focusing on its roles in the regulation of sleep/wakefulness states.

Sleep, feeding, and neuropeptides: roles of orexins and orexin receptors

Recent studies using molecular genetics in mice and dogs, as well as histopathological analyses of human disease, have come to the same conclusion: the human sleep disorder narcolepsy is caused by failure of signaling mediated by orexin (hypocretin) neuropeptides. These and other findings strongly suggest that the orexin system plays a critical role in sleep/wake regulation. In addition, the orexin system may link energy homeostasis to the regulation of sleep/wake cycles.

Indispensability of the glutamate transporters GLAST and GLT1 to brain development

Previous in vitro studies have shown that the neurotransmitter glutamate is important in brain development. Paradoxically, loss-of-function mouse models of glutamatergic signaling that are generated by genetic deletion of glutamate receptors or glutamate release show normal brain assembly. We examined the direct consequences on brain development of extracellular glutamate buildup due to the depletion of the glutamate transporters GLAST and GLT1. GLAST/GLT1 double knockout mice show multiple brain defects, including cortical, hippocampal, and olfactory bulb disorganization with perinatal mortality. Here, we report abnormal formation of the neocortex in GLAST/GLT1 mutants. Several essential aspects of neuronal development, such as stem cell proliferation, radial migration, neuronal differentiation, and survival of SP neurons, were impaired. These results provide direct in vivo evidence that GLAST and GLT1 are necessary for brain development through regulation of extracellular glutamate concentration and show that an important mechanism is likely to be maintenance of glutamate-mediated synaptic transmission.