Natsuko Tsujino

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.

Input of Orexin/Hypocretin Neurons Revealed by a Genetically Encoded Tracer in Mice

The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.

Orexin/Hypocretin: A Neuropeptide at the Interface of Sleep, Energy Homeostasis, and Reward System

Recent studies have implicated the orexin system as a critical regulator of sleep/wake states as well as feeding behavior and reward processes. Orexin deficiency results in narcolepsy in humans, dogs, and rodents, suggesting that the orexin system is particularly important for maintenance of wakefulness. In addition, orexin deficiency also cause abnormalities in energy homeostasis and reward systems. Orexin activates waking active monoaminergic and cholinergic neurons in the hypothalamus and brainstem regions to maintain a long, consolidated waking period. Orexin neurons receive abundant input from the limbic system. Orexin neurons also have reciprocal links with the hypothalamic arcuate nucleus, which regulates feeding. Moreover, the responsiveness of orexin neurons to peripheral metabolic cues, such as leptin and glucose, suggest that these neurons have important role as a link between the energy homeostasis and vigilance states. Orexin neurons also have a link with the dopaminergic reward system in the ventral tegmental nucleus. These findings suggest that the orexin system interacts with systems that regulate emotion, reward, and energy homeostasis to maintain proper vigilance states. Therefore, this system may be a potentially important therapeutic target for treatment of sleep disorder, obesity, emotional stress, and addiction.

Orexin-induced food intake involves neuropeptide Y pathway

Orexins (orexin-A and -B) are recently identified neuropeptides, which are thought to be implicated in the regulation of feeding behavior. We used a NPY-Y1 receptor specific antagonist, BIBO3304, to examine whether NPY is involved in orexin-induced feeding behavior. Intracerebroventricular administration of orexin-A (10 nmol) induced food intake in rats (food intake for 3 h; vehicle 0.3+/-0.2 g vs. orexin-A 10 nmol, 4.0+/-0.5 g, n=4). Orexin-induced feeding behavior was partially inhibited by prior administration of BIBO3304 (3 h food intake: orexin-A 10 nmol, 4.0+/-0.5 g vs. BIBO3304 (60 microgram) + orexin-A 10 nmol, 2.2+/-0.2 g, n=4). A low dose of BIBO3304 (30 microgram) did not show a significant inhibitory effect. BIBO3457, an inactive enantiomer, used as a negative control, did not show any inhibitory effect on orexin-A-induced feeding behavior. Fos expression was observed in NPY-containing neurons in the arcuate nucleus 1 h after orexin-A (10 nmol) was administered intracerebroventricularly (control 0.3+/-0.08%, orexin-A 10.2+/-0.8%, n=5 rats/group). These observations suggest that NPY is involved in orexin-induced feeding behavior. However, BIBO3304 did not completely abolish the effect of orexin-A. These results suggest that orexin-A elicits feeding behavior partially via the NPY pathway. The NPY system could be the one of downstream pathways by which orexin-A induces feeding behavior. Another pathway may also be involved in orexin-A-induced feeding behavior, because BIBO3304 did not completely abolish orexin-A-induced feeding behavior.

Regulation of orexin neurons by the monoaminergic and cholinergic systems

Orexins are a pair of neuropeptides implicated in energy homeostasis and arousal. Here we characterize the electrophysiological properties of orexin neurons using slice preparations from transgenic mice in which orexin neurons specifically express green fluorescent protein. Orexin neurons showed high frequency firing with little adaptation by injecting a positive current. The hyperpolarization-activated current was observed in orexin neurons by a negative current injection. The neurotransmitters, which were implicated in sleep/wake regulation, affected the activity of orexin neurons; noradrenaline and serotonin hyperpolarized, while carbachol depolarized orexin neurons in either the presence or absence of tetrodotoxin. It has been reported that orexins directly or indirectly activate the nuclei that are the origin of the neurons containing these neurotransmitters. Our data suggest that orexin neurons have reciprocal neural circuitries between these nuclei for either a positive or negative feedback loop and orchestrate the activity of these neurons to regulate the vigilance states.

Serotonergic Regulation of the Orexin/Hypocretin Neurons through the 5-HT1A Receptor

Both orexin and serotonin (5-HT) have important roles in the regulation of sleep-wakefulness, as well as in feeding behavior. We examined the effects of 5-HT on orexin/hypocretin neurons, using hypothalamic slices prepared from orexin/enhanced green fluorescent protein (EGFP) transgenic mice in which EGFP is expressed exclusively in orexin neurons. Patch-clamp recording from EGFP-expressing cells showed that 5-HT hyperpolarized all orexin neurons in a concentration-dependent manner. The response was inhibited by the 5-HT1A receptor antagonist WAY100635. A 5-HT1A receptor agonist, 8-hydroxy-2-(dl-N-propyl-amino)tetralin, also evoked hyperpolarization on orexin neurons with potency comparable with 5-HT. A low concentration of Ba2+ (30 μm) inhibited 5-HT-induced hyperpolarization. Single-channel recording revealed that the conductance of 5-HT-induced channel activity was 33.8 pS, which is in good agreement with that of the G-protein-coupled inward rectifier potassium channel (GIRK). Moreover, 5-HT1A receptor-like immunoreactivity was observed on orexin neurons, and 5-HT transporter immunoreactive nerve endings are in close apposition to orexin neurons. Intracerebroventricular injection of the 5-HT1A receptor-selective antagonist WAY100635 (100 ng) increased locomotor activity during the latter half of dark phase in wild-type mice but not in orexin/ataxin-3 mice in which orexin neurons are specifically ablated, suggesting that activation of orexin neurons is necessary for the WAY100635-induced increase in locomotor activity. These results indicate that 5-HT hyperpolarizes orexin neurons through the 5-HT1A receptor and subsequent activation of the GIRK and that this inhibitory serotonergic input to the orexin neurons is likely to be important for the physiological regulation of this neuropeptide system.

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.

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC50) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC50 values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 and/or GPR8.

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.

Role of orexin in modulating arousal, feeding, and motivation

Orexin deficiency results in narcolepsy in humans, dogs, and rodents, suggesting that the orexin system is particularly important for maintenance of wakefulness. However, orexin neurons are “multi-tasking” neurons that regulate sleep/wake states as well as feeding behavior, emotion, and reward processes. Orexin deficiency causes abnormalities in energy homeostasis, stress-related behavior, and reward systems. Orexin excites waking-active monoaminergic and cholinergic neurons in the hypothalamus and brain stem regions to maintain a long, consolidated waking period. Orexin neurons also have reciprocal links with the hypothalamic nuclei, which regulates feeding. Moreover, the responsiveness of orexin neurons to peripheral metabolic cues suggests that these neurons have an important role as a link between energy homeostasis and vigilance states. The link between orexin and the ventral tegmental nucleus serves to motivate an animal to engage in goal-directed behavior. This review focuses on the interaction of orexin neurons with emotion, reward, and energy homeostasis systems. These connectivities are likely to be highly important to maintain proper vigilance states.