Optogenetic stimulation of astrocytes in the posterior hypothalamus increases sleep at night in C57BL/6J mice.

A distributed network of neurons regulates wake, non-rapid eye movement (NREM) sleep, and REM sleep. However, there are also glia in the brain, and there is growing evidence that neurons and astroglia communicate intimately to regulate behaviour. To identify the effect of optogenetic stimulation of astrocytes on sleep, the promoter for the astrocyte-specific cytoskeletal protein, glial fibrillary acidic protein (GFAP) was used to direct the expression of channelrhodopsin-2 (ChR2) and the linked reporter gene, enhanced yellow fluorescent protein (EYFP), in astrocytes. rAAV-GFAP-ChR2 (H134R)-EYFP or rAAV-GFAP-EYFP was microinjected (750 nL) into the posterior hypothalamus (bilateral) of mice. Three weeks later baseline sleep was recorded (0 Hz) and 24 h later optogenetic stimulation applied during the first 6 h of the lights-off period. Mice with ChR2 were given 5, 10 or 30 Hz stimulation for 6 h (10-ms pulses; 1 mW; 1 min on 4 min off). At least 36 h elapsed between the stimulation periods (5, 10, 30 Hz) and although 0 Hz was always first, the order of the other three stimulation rates was randomised. In mice with ChR2 (n = 7), 10 Hz, but not 5 or 30 Hz stimulation increased both NREM and REM sleep during the 6-h period of stimulation. Delta power did not increase. In control mice (no ChR2; n = 5), 10 Hz stimulation had no effect. This study demonstrates that direct stimulation of astrocytes powerfully induces sleep during the active phase of the sleep-wake cycle and underlines the inclusion of astrocytes in network models of sleep-wake regulation.

Orexin gene transfer into the amygdala suppresses both spontaneous and emotion-induced cataplexy in orexin-knockout mice.

Narcolepsy is a chronic sleep disorder linked to the loss of orexin-producing neurons in the hypothalamus. Cataplexy, a sudden loss of muscle tone during waking, is an important distinguishing symptom of narcolepsy and it is often triggered by strong emotions. The neural circuit underlying cataplexy attacks is not known, but is likely to involve the amygdala, a region implicated in regulating emotions. In mice models of narcolepsy, transfer of the orexin gene into surrogate neurons has been successful in ameliorating narcoleptic symptoms. However, it is not known whether this method also blocks cataplexy triggered by strong emotions. To examine this possibility, the gene encoding mouse prepro-orexin was transferred into amygdala neurons of orexin-knockout (KO) mice (rAAV-orexin; n = 8). Orexin-KO mice that did not receive gene transfer (no-rAAV; n = 7) or received only the reporter gene (rAAV-GFP; n = 7) served as controls. Three weeks later, the animal's sleep and behaviour were recorded at night (no-odour control night), followed by another recording at night in the presence of predator odour (odour night). Orexin-KO mice given the orexin gene transfer into surrogate amygdala neurons had significantly less spontaneous bouts of cataplexy, and predator odour did not induce cataplexy compared with control mice. Moreover, the mice with orexin gene transfer were awake more during the odour night. These results demonstrate that orexin gene transfer into amygdala neurons can suppress both spontaneous and emotion-induced cataplexy attacks in narcoleptic mice. It suggests that manipulating amygdala pathways is a potential strategy for treating cataplexy in narcolepsy.

Optogenetic stimulation of MCH neurons increases sleep.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

Neurons containing orexin or melanin concentrating hormone reciprocally regulate wake and sleep.

Neurons containing orexin (hypocretin), or melanin concentrating hormone (MCH) are intermingled with each other in the perifornical and lateral hypothalamus. Each is a separate and distinct neuronal population, but they project to similar target areas in the brain. Orexin has been implicated in regulating arousal since loss of orexin neurons is associated with the sleep disorder narcolepsy. Microinjections of orexin into the brain or optogenetic stimulation of orexin neurons increase waking. Orexin neurons are active in waking and quiescent in sleep, which is consistent with their role in promoting waking. On the other hand, the MCH neurons are quiet in waking but active in sleep, suggesting that they could initiate sleep. Recently, for the first time the MCH neurons were stimulated optogenetically and it increased sleep. Indeed, optogenetic activation of MCH neurons induced sleep in both mice and rats at a circadian time when they should be awake, indicating the powerful effect that MCH neurons have in suppressing the wake-promoting effect of not only orexin but also of all of the other arousal neurotransmitters. Gamma-Aminobutyric acid (GABA) is coexpressed with MCH in the MCH neurons, although MCH is also inhibitory. The inhibitory tone of the MCH neurons is opposite to the excitatory tone of the orexin neurons. We hypothesize that strength in activity of each determines wake vs. sleep.