Structure of cortical network activity across natural wake and sleep states in mice.

Cortical neurons fire intermittently and synchronously during non-rapid eye movement sleep (NREMS), in which active and silent periods are referred to as ON and OFF periods, respectively. Neuronal firing rates during ON periods (NREMS-ON-activity) are similar to those of wakefulness (W-activity), raising the possibility that NREMS-ON neuronal-activity is fragmented W-activity. To test this, we investigated the patterning and organization of cortical spike trains and of spike ensembles in neuronal networks using extracellular recordings in mice. Firing rates of neurons during NREMS-ON and W were similar, but showed enhanced bursting in NREMS with no apparent preference in occurrence, relative to the beginning or end of the on-state. Additionally, there was an overall increase in the randomness of occurrence of sequences comprised of multi-neuron ensembles in NREMS recorded from tetrodes. In association with increased burst firing, somatic calcium transients were increased in NREMS. The increased calcium transients associated with bursting during NREM may activate calcium-dependent, cell-signaling pathways for sleep related cellular processes.

Dynamics of Cortical Local Connectivity during Sleep-Wake States and the Homeostatic Process.

Sleep exerts modulatory effects on the cerebral cortex. Whether sleep modulates local connectivity in the cortex or only individual neural activity, however, is poorly understood. Here we investigated functional connectivity, that is, covarying activity between neurons, during spontaneous sleep-wake states and during and after sleep deprivation using calcium imaging of identified excitatory/inhibitory neurons in the motor cortex. Functional connectivity was estimated with a statistical learning approach glasso and quantified by "the probability of establishing connectivity (sparse/dense)" and "the strength of the established connectivity (weak/strong)." Local cortical connectivity was sparse in non-rapid eye movement (NREM) sleep and dense in REM sleep, which was similar in both excitatory and inhibitory neurons. The overall mean strength of the connectivity did not differ largely across spontaneous sleep-wake states. Sleep deprivation induced strong excitatory/inhibitory and dense inhibitory, but not excitatory, connectivity. Subsequent NREM sleep after sleep deprivation exhibited weak excitatory/inhibitory, sparse excitatory, and dense inhibitory connectivity. These findings indicate that sleep-wake states modulate local cortical connectivity, and the modulation is large and compensatory for stability of local circuits during the homeostatic control of sleep, which contributes to plastic changes in neural information flow.

Serotonergic Input to Orexin Neurons Plays a Role in Maintaining Wakefulness and REM Sleep Architecture.

Neurons expressing neuropeptide orexins (hypocretins) in the lateral hypothalamus (LH) and serotonergic neurons in the dorsal raphe nucleus (DR) both play important roles in the regulation of sleep/wakefulness states, and show similar firing patterns across sleep/wakefulness states. Orexin neurons send excitatory projections to serotonergic neurons in the DR, which express both subtypes of orexin receptors (Mieda et al., 2011), while serotonin (5-HT) potently inhibits orexin neurons through activation of 5HT1A receptors (5HT1ARs). In this study, we examined the physiological importance of serotonergic inhibitory regulation of orexin neurons by studying the phenotypes of mice lacking the 5HT1A receptor gene (Htr1a) specifically in orexin neurons (ox5HT1ARKO mice). ox5HT1ARKO mice exhibited longer NREM sleep time along with decreased wakefulness time in the later phase of the dark period. We also found that restraint stress induced a larger impact on REM sleep architecture in ox5HT1ARKO mice than in controls, with a larger delayed increase in REM sleep amount as compared with that in controls, indicating abnormality of REM sleep homeostasis in the mutants. These results suggest that 5HT1ARs in orexin neurons are essential in the regulation of sleep/wakefulness states, and that serotonergic regulation of orexin neurons plays a crucial role in the appropriate control of orexinergic tone to maintain normal sleep/wake architecture.

Sleep as a biological problem: an overview of frontiers in sleep research.

Sleep is a physiological process not only for the rest of the body but also for several brain functions such as mood, memory, and consciousness. Nevertheless, the nature and functions of sleep remain largely unknown due to its extremely complicated nature and lack of optimized technology for the experiments. Here we review the recent progress in the biology of the mammalian sleep, which covers a wide range of research areas: the basic knowledge about sleep, the physiology of cerebral cortex in sleeping animals, the detailed morphological features of thalamocortical networks, the mechanisms underlying fluctuating activity of autonomic nervous systems during rapid eye movement sleep, the cutting-edge technology of tissue clearing for visualization of the whole brain, the ketogenesis-mediated homeostatic regulation of sleep, and the forward genetic approach for identification of novel genes involved in sleep. We hope this multifaceted review will be helpful for researchers who are interested in the biology of sleep.

Effects of a newly developed potent orexin-2 receptor-selective antagonist, compound 1 m, on sleep/wakefulness states in mice.

Orexins (also known as hypocretins) play critical roles in the regulation of sleep/wakefulness states by activating two G-protein coupled receptors (GPCRs), orexin 1 (OX1R) and orexin 2 receptors (OX2R). In order to understand the differential contribution of both receptors in regulating sleep/wakefulness states we compared the pharmacological effects of a newly developed OX2R antagonist (2-SORA), Compound 1 m (C1 m), with those of a dual orexin receptor antagonist (DORA), suvorexant, in C57BL/6J mice. After oral administration in the dark period, both C1m and suvorexant decreased wakefulness time with similar efficacy in a dose-dependent manner. While C1m primarily increased total non-rapid eye movement (NREM) sleep time without affecting episode durations and with minimal effects on REM sleep, suvorexant increased both total NREM and REM sleep time and episode durations with predominant effects on REM sleep. Fos-immunostaining showed that both compounds affected the activities of arousal-related neurons with different patterns. The number of Fos-IR noradrenergic neurons in the locus coeruleus was lower in the suvorexant group as compared with the control and C1m-treated groups. In contrast, the numbers of Fos-IR neurons in histaminergic neurons in the tuberomamillary nucleus and serotonergic neurons in the dorsal raphe were reduced to a similar extent in the suvorexant and C1m groups as compared with the vehicle-treated group. Together, these results suggest that an orexin-mediated suppression of REM sleep via potential activation of OX1Rs in the locus coeruleus may possibly contribute to the differential effects on sleep/wakefulness exerted by a DORA as compared to a 2-SORA.

GABAergic neurons in the preoptic area send direct inhibitory projections to orexin neurons.

Populations of neurons in the hypothalamic preoptic area (POA) fire rapidly during sleep, exhibiting sleep/waking state-dependent firing patterns that are the reciprocal of those observed in the arousal system. The majority of these preoptic "sleep-active" neurons contain the inhibitory neurotransmitter GABA. On the other hand, a population of neurons in the lateral hypothalamic area (LHA) contains orexins, which play an important role in the maintenance of wakefulness, and exhibit an excitatory influence on arousal-related neurons. It is important to know the anatomical and functional interactions between the POA sleep-active neurons and orexin neurons, both of which play important, but opposite roles in regulation of sleep/wakefulness states. In this study, we confirmed that specific pharmacogenetic stimulation of GABAergic neurons in the POA leads to an increase in the amount of non-rapid eye movement (NREM) sleep. We next examined direct connectivity between POA GABAergic neurons and orexin neurons using channelrhodopsin 2 (ChR2) as an anterograde tracer as well as an optogenetic tool. We expressed ChR2-eYFP selectively in GABAergic neurons in the POA by AAV-mediated gene transfer, and examined the projection sites of ChR2-eYFP-expressing axons, and the effect of optogenetic stimulation of ChR2-eYFP on the activity of orexin neurons. We found that these neurons send widespread projections to wakefulness-related areas in the hypothalamus and brain stem, including the LHA where these fibers make close appositions to orexin neurons. Optogenetic stimulation of these fibers resulted in rapid inhibition of orexin neurons. These observations suggest direct connectivity between POA GABAergic neurons and orexin neurons.

Chronic alterations in monoaminergic cells in the locus coeruleus in orexin neuron-ablated narcoleptic mice.

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.

Differential roles of orexin receptors in the regulation of sleep/wakefulness.

Orexin A and orexin B are hypothalamic neuropeptides that play critical roles in the regulation of sleep/wakefulness, as well as in a variety of physiological functions such as emotion, reward, and energy homeostasis. The actions of orexins are mediated by two receptors, orexin 1 (OX1R) and orexin 2 (OX2R) receptors. OX1R and OX2R show partly overlapping but distinct distributions throughout the central nervous system, suggesting their differential roles. This review presents and discusses the current knowledge concerning the physiological roles of each orexin receptor subtype, focusing on the regulation of sleep/wakefulness.

Neurotensin co-expressed in orexin-producing neurons in the lateral hypothalamus plays an important role in regulation of sleep/wakefulness states.

Both orexin and neurotensin are expressed in the lateral hypothalamic area (LHA) and have been implicated in the regulation of feeding, motor activity and the reward system. A double label immunofluorescence and in situ hybridization studies showed that neurotensin colocalizes with orexin in neurons of the LHA. Pharmacological studies suggested that neurotensin excites orexin-producing neurons (orexin neurons) through activation of neurotensin receptor-2 (NTSR-2) and non-selective cation channels. In situ hybridization study showed that most orexin neurons express neurotensin receptor-2 mRNA but not neurotensin receptor-1 (Ntsr-1) mRNA. Immunohistochemical studies showed that neurotensin-immunoreactive fibers make appositions to orexin neurons. A neurotensin receptor antagonist decreased Fos expression in orexin neurons and wakefulness time in wild type mice when administered intraperitoneally. However, the antagonist did not evoke any effect on these parameters in orexin neuron-ablated mice. These observations suggest the importance of neurotensin in maintaining activity of orexin neurons. The evidence presented here expands our understanding of the regulatory mechanism of orexin neurons.

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.

[Circadian rhythm of leptin, orexin and ghrelin].

Both metabolic syndrome and sleep disorder are major health problems. Leptin, orexin, ghrelin are implicated in regulation of energy homeostasis and sleep/wakefulness. The levels of these substances exhibit circadian fluctuations, and abnormalities in these rhythms were observed in those disorders. Serum leptin levels were increased during night. Cerebrospinal fluid orexin A levels were increased during active phase. Plasma ghrelin concentrations were increased before meals and during night. High concentrations of leptin during sleep might help keeping sleep by inhibition of feeding behavior and arousal through inhibition of neuropeptide Y and orexin neurons. On the contrary, high concentrations of ghrelin before meal might enhance wakefulness through activation of orexin neurons. These results suggest that the circadian rhythms of these substances are important for maintenance of normal energy homeostasis and sleep/wakefulness.

A single nucleotide polymorphism of the neuropeptide B/W receptor-1 gene influences the evaluation of facial expressions.

Neuropeptide B/W receptor-1 (NPBWR1) is expressed in discrete brain regions in rodents and humans, with particularly strong expression in the limbic system, including the central nucleus of the amygdala. Recently, Nagata-Kuroiwa et al. reported that Npbwr1(-/-) mice showed changes in social behavior, suggesting that NPBWR1 plays important roles in the emotional responses of social interactions.The human NPBWR1 gene has a single nucleotide polymorphism at nucleotide 404 (404A>T; SNP rs33977775). This polymorphism results in an amino acid change, Y135F. The results of an in vitro experiment demonstrated that this change alters receptor function. We investigated the effect of this variation on emotional responses to stimuli of showing human faces with four categories of emotional expressions (anger, fear, happiness, and neutral). Subjects' emotional levels on seeing these faces were rated on scales of hedonic valence, emotional arousal, and dominance (V-A-D). A significant genotype difference was observed in valence evaluation; the 404AT group perceived facial expressions more pleasantly than did the 404AA group, regardless of the category of facial expression. Statistical analysis of each combination of [V-A-D and facial expression] also showed that the 404AT group tended to feel less submissive to an angry face than did the 404AA group. Thus, a single nucleotide polymorphism of NPBWR1 seems to affect human behavior in a social context.

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.

Critical role of neuropeptides B/W receptor 1 signaling in social behavior and fear memory.

Neuropeptide B/W receptor 1 (NPBWR1) is a G-protein coupled receptor, which was initially reported as an orphan receptor, and whose ligands were identified by this and other groups in 2002 and 2003. To examine the physiological roles of NPBWR1, we examined phenotype of Npbwr1⁻/⁻ mice. When presented with an intruder mouse, Npbwr1⁻/⁻ mice showed impulsive contact with the strange mice, produced more intense approaches toward them, and had longer contact and chasing time along with greater and sustained elevation of heart rate and blood pressure compared to wild type mice. Npbwr1⁻/⁻ mice also showed increased autonomic and neuroendocrine responses to physical stress, suggesting that impairment of NPBWR1 leads to stress vulnerability. We also observed that these mice show abnormality in the contextual fear conditioning test. These data suggest that NPBWR1 plays a critical role in limbic system function and stress responses. Histological and electrophysiological studies showed that NPBWR1 acts as an inhibitory regulator on a subpopulation of GABAergic neurons in the lateral division of the CeA and terminates stress responses. These findings suggest important roles of NPBWR1 in regulating amygdala function during physical and social stress.

Nicorandil ameliorates impulse conduction disturbances during ischemia in isolated arterially perfused canine atria.

BACKGROUND: Nicorandil has protective effects on the ischemic atrial myocardium. However, effects of nicorandil on ischemia-induced impulse conduction disturbances are still uncertain. METHODS: Optical action potentials were recorded from 256 sites of the left atrium in isolated arterially perfused canine atria during the left atrial ischemia. Constant pacing (BCL = 350 ms) from the left superior pulmonary vein (LSPV) and the posterior left atrium (PLA) was performed, and local conduction velocity (CV) was calculated at the LSPV-left atrial (LA) junction and the right inferior PV (RIPV)-LA junction. Impulse conduction failure was elucidated within the optical mapping field during sinus rhythm. RESULTS: In the control, ischemia slowed the local CV at both regions regardless of the pacing site, and impulse conduction failure occurred within the mapping field during sinus rhythm. Nicorandil suppressed the ischemic conduction slowing at both regions and prevented the conduction failure. Nicorandil also reduced the dispersion of local CV during ischemia. HMR1098, a blocker of cardiac sarcolemmal K(ATP) channels abolished suppression of the ischemic conduction slowing by nicorandil at the RIPV-LA junction but not at the LSPV-LA junction and induced the conduction failure. 5-HD, a blocker of mitochondrial K(ATP) channels also abolished it at both regions and induced the conduction failure. 5-HD abolished the decreased dispersion of local CV by nicorandil, and HMR1098 further increased the dispersion of local CV compared with the control. CONCLUSIONS: These results indicate that nicorandil suppresses ischemia-induced impulse conduction disturbances by its action on both the mitochondrial and sarcolemmal K(ATP) channels.

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.

Activation of bombesin receptor subtype-3 influences activity of orexin neurons by both direct and indirect pathways.

The neuropeptides orexin A and orexin B (also known as hypocretin 1 and hypocretin 2), produced in lateral hypothalamic neurons, are critical regulators of feeding behavior, the reward system, and sleep/wake states. Orexin-producing neurons (orexin neurons) are regulated by various factors involved in regulation of energy homeostasis and sleep/wakefulness states. Bombesin receptor subtype 3 (BRS3) is an orphan receptor that might be implicated in energy homeostasis and is highly expressed in the hypothalamus. However, the neural pathway by which BRS3 regulates energy homeostasis is largely unknown. We examined whether BRS3 is involved in the regulation of orexin neurons. Using a calcium imaging method, we found that a selective BRS3 agonist [Ac-Phe-Trp-Ala-His-(tauBzl)-Nip-Gly-Arg-NH2] increased the intracellular calcium concentration of orexin neurons. However, intracellular recordings from slice preparations revealed that the BRS3 agonist hyperpolarized orexin neurons. The BRS3 agonist depolarized orexin neuron in the presence of tetrodotoxin. Moreover, in the presence of GABA receptor blockers, picrotoxin and CGP55845, the BRS3 agonist induced depolarization and increased firing frequency. Additionally, double-label in situ hybridization study revealed that Brs3 mRNA was expressed in almost all orexin neurons and many cells around these neurons. These findings suggest that the BRS3 agonist indirectly inhibited orexin neurons through GABAergic input and directly activated orexin neurons. Inhibition of activity of orexin neurons through BRS3 might be an important pathway for regulation of feeding and sleep/wake states. This pathway might serve as a novel target for the treatment of obesity.

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.

Contribution of L-type Ca2+ channels to early afterdepolarizations induced by I Kr and I Ks channel suppression in guinea pig ventricular myocytes.

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels.