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.     

Hypocretin/orexin, sleep and narcolepsy

The discovery that hypocretins are involved in narcolepsy, a disorder associated with excessive daytime sleepiness, cataplexy and unusually rapid transitions to rapid-eye-movement sleep, opens a new field of investigation in the area of sleep control physiology. Hypocretin-1 and -2 (also called orexin-A and -B) are newly discovered neuropeptides processed from a common precursor, preprohypocretin. Hypocretin-containing cells are located exclusively in the lateral hypothalamus, with widespread projections to the entire neuroaxis. Two known receptors, Hcrtr1 and Hcrtr2, have been reported. The functional significance of the hypocretin system is rapidly emerging in both animals and humans. Hypocretin abnormalities cause narcolepsy in dogs, human and mice. The role of the hypocretin system in normal sleep regulation is more uncertain. We believe hypocretin cells drive cholinergic and monoaminergic activity across the sleep cycle. Input from the suprachiasmatic nucleus to hypocretin-containing neurons may explain the occurrence of clock-dependent alertness. Other functions are suggested by pharmacological and neurochemical experiments. These include regulation of food intake, neuroendocrine function, autonomic nervous system activity and energy balance.     

Lack of association between orexin receptor gene polymorphisms and obstructive sleep apnea syndrome in Japanese

Sleep and Biological Rhythms - The pathogenesis of obstructive sleep apnea (OSA) is not fully understood, particularly at the genetic level. The orexins have pleiotropic effects on several aspects...     

Wakefulness: an eye-opening perspective on orexin neurons

Orexin-containing neurons regulate wakefulness, and loss of orexin produces narcolepsy. Recent studies of mice lacking orexin neurons have shown that these cells also play essential roles in the control of feeding and energy balance.     

Elevated body temperature during sleep in orexin knockout mice

Core body temperature (Tb) is influenced by many physiological factors, including behavioral state, locomotor activity, and biological rhythms. To determine the relative roles of these factors, we examined Tb in orexin knockout (KO) mice, which have a narcolepsy-like phenotype with severe sleep-wake fragmentation. Because orexin is released during wakefulness and is thought to promote heat production, we hypothesized that orexin KO mice would have lower Tb while awake. Surprisingly, Tb was the same in orexin KO mice and wild-type (WT) littermates during sustained wakefulness. Orexin KO mice had normal diurnal variations in Tb, but the ultradian rhythms of Tb, locomotor activity, and wakefulness were markedly reduced. During the first 15 min of spontaneous sleep, the Tb of WT mice decreased by 1.0 degrees C, but Tb in orexin KO mice decreased only 0.4 degrees C. Even during intense recovery sleep after 8 h of sleep deprivation, the Tb of orexin KO mice remained 0.7 degrees C higher than in WT mice. This blunted fall in Tb during sleep may be due to inadequate activation of heat loss mechanisms or sustained activity in heat-generating systems. These observations reveal an unexpected role for orexin in thermoregulation. In addition, because heat loss is an essential aspect of sleep, the blunted fall in Tb of orexin KO mice may provide an explanation for the fragmented sleep of narcolepsy.     

Regulation of orexin neurons by the monoaminergic and cholinergic systems

Sleep and Biological Rhythms -     

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.     

Associations between daytime and nighttime plasma orexin A levels and cognitive function in patients with obstructive sleep apnea

Sleep and Biological Rhythms - The relationship between plasma orexin A (OXA) levels and cognitive function in patients with obstructive sleep apnea (OSA) remains unclear. This study aimed to...     

Serotoninergic mechanisms and sleep

Serotoninergic neurons play a critical role in the sleep mechanism. This is supported by a lot of converging experiments and has provided the basis for a great deal of research. A critical analysis is first developed, supported by more recent data which are not in complete agreement with the theory that raphe nuclei are actively implied in slow wave sleep. On the other hand, numerous experimental evidences were collected during the sixties on the EEG synchronizing influence of the lower brain stem and preoptic area. Recent data showed that serotonin could also play here a crucial role in the induction of sleep. Nevertheless, at the moment, it is difficult to make a critical examination of the interaction and regulation of these putative 5-HT-related areas of the brain, but we can postulate that the occurrence of true physiological sleep-waking continuum necessitates their successive or conjoint activation.     

Interaction mechanisms between Gracilaria chilensis (Rhodophyta) and epiphytes

Epiphytes can have a considerable effect on Gracilaria production, and Ulva is one of the commonest algal species identified as an epiphyte, reaching loads of 60% (g of epiphytes per g of Gracilaria) in the intertidal cultures of southern Chile. This study evaluates the relative importance of light reduction, addition of weight to the host thalli and nutrient depletion, as mechanisms determining the interaction effects of Ulva epiphytes on Gracilaria cultivation. Using field experiments undertaken during the main Gracilaria growth season (spring), we evaluate the mechanisms of epiphyte-host algae interaction by manipulating artificial epiphytes. The results indicate that Ulva can significantly depress Gracilaria biomass production and that the addition of weight to the host algae and the consequent dislodgement increase, appear to be the main mechanisms involved in the Ulva-Gracilaria interaction. However, the light reduction caused by the epiphytes can also partially explain the reduction in Gracilaria production. Nutrients depletion would not appear to fully account for the detrimental effects of Ulva over Gracilaria in intertidal farming areas of southern Chile.     

Metabolic state signalling through central hypocretin/orexin neurons

Hypothalamic neurons that produce the peptide transmitters hypocretins/orexins have attracted much recent attention. They provide direct and predominantly excitatory inputs to all major brain areas except the cerebellum, with the net effect of stimulating wakefulness and arousal. These inputs are essential for generating sustained wakefulness in mammals, and defects in hypocretin signalling result in narcolepsy. In addition, new roles for hypocretins/orexins are emerging in reward-seeking, learning, and memory. Recent studies also indicate that hypocretin/orexin neurons can alter their intrinsic electrical activity according to ambient fluctuations in the levels of nutrients and appetite-regulating hormones. These intriguing electrical responses are perhaps the strongest candidates to date for the elusive neural correlates of after-meal sleepiness and hunger-induced wakefulness. Hypocretin/orexin neurons may thus directly translate rises and falls in body energy levels into different states of consciousness.     

Peripheral ghrelin interacts with orexin neurons in glucostatic signalling

Ghrelin interactions with glycemia in appetite control as well as the potential mechanisms involving the orexin and melanin-concentrating hormone (MCH) neurons in the orexigenic ghrelin signals were investigated by using a specific anti-ghrelin antibody (AGA). Our results confirm that peripheral ghrelin is an important signal in meal initiation and appetite. Employing immunohistochemistry techniques, we found that c-fos positive neurons in the lateral hypothalamus (LH) and perifornical area (PFA) increased after insulin or 2-deoxyglucose administration. Moreover, we have also demonstrated that peripheral ghrelin blockade by the AGA, reduces the orexigenic signal induced by insulin and 2-DG administration probably partly producing a decrease of c-fos immunoreactivity in the LH and PFA as well as a lower activation of orexin neurons. In contrast, the c-fos positive MCH neurons were not apparently affected. In summary, our findings suggest that peripheral ghrelin plays an important role in regulatory "glucostatic" feeding mechanisms by means of its role as a "hunger" signal affecting the LH and PFA areas, which may contribute to energy homeostasis through orexin neurons.     

Interaction between central and peripheral temperature signals on temperature-sensitive hypothalamic neurons

Changes in the mean firing rate of posterior hypothalamic neurons were studied in experiments on unanesthetized cats in response to elevation of the brain temperature by 0.7–1.5°C and the skin temperature by 3–5°C separately or simultaneously. Altogether 85 neurons were studied in 14 animals: 11 responded to only one form of temperature stimulation, whereas in 16 neurons changes in the firing pattern (in most cases in the same direction) were observed in response to both forms of temperature stimulation. Different types of responses of these neurons were established. Sensitivity to the central temperature stimulus was increased in some neurons of this group when skin temperature stimulation was intensified.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 8, No. 6, pp. 613–619, November–December, 1976.     

Interaction between pentobarbital and GABA-activated ionic channels in rat cerebellar neurons

Using techniques of voltage clamping at the membrane, intracellular perfusion, and concentration clamping, GABA- and barbiturate-activated currents were investigated in single neurons isolated from the rat cerebellum. The dissociation constant for interaction between GABA and GABA receptors was measured at 3±0.8 × 10^–5 M. The presence of pentobarbital in the bathing solution exerts a potentiating effect on GABA-induced conductance in isolated neurons, shifting the dose-response curve for GABA towards lower concentration values without increasing peak chloride conductance. The concentrations at which GABA effects are potentiated range between 10^–6–10^–4 M. High concentrations of pentobarbital inhibit GABA-activated conductance; at concentrations in excess of 5 × 10^–4 M, it also brings about activation of chloride conductance, depressed by bicuculline and picrotoxin, in the absence of GABA. A short-term increase in membrane conductance is produced by rapid pentobarbital washout.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 93–98, January–February, 1990.     

Local network regulation of orexin neurons in the lateral hypothalamus

Obesity and inadequate sleep are among the most common causes of health problems in modern society. Thus, the discovery that orexin (hypocretin) neurons play a pivotal role in sleep/wake regulation, energy balance, and consummatory behaviors has sparked immense interest in understanding the regulatory mechanisms of these neurons. The local network consisting of neurons and astrocytes within the lateral hypothalamus and perifornical area (LH/PFA), where orexin neurons reside, shapes the output of orexin neurons and the LH/PFA. Orexin neurons not only send projections to remote brain areas but also contribute to the local network where they release multiple neurotransmitters to modulate its activity. These neurotransmitters have opposing actions, whose balance is determined by the amount released and postsynaptic receptor desensitization. Modulation and negative feedback regulation of excitatory glutamatergic inputs as well as release of astrocyte-derived factors, such as lactate and ATP, can also affect the excitability of orexin neurons. Furthermore, distinct populations of LH/PFA neurons express neurotransmitters with known electrophysiological actions on orexin neurons, such as melanin-concentrating hormone, corticotropin-releasing factor, thyrotropin-releasing hormone, neurotensin, and GABA. These LH/PFA-specific mechanisms may be important for fine tuning the firing activity of orexin neurons to maintain optimal levels of prolonged output to sustain wakefulness and stimulate consummatory behaviors. Building on these exciting findings should shed further light onto the cellular mechanisms of energy balance and sleep-wake regulation.