Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. To help determine how these neurons promote wakefulness, we generated transgenic mice in which orexin neurons expressed halorhodopsin (orexin/Halo mice), an orange light-activated neuronal silencer. Slice patch-clamp recordings of orexin neurons that expressed halorhodopsin demonstrated that orange light photic illumination immediately hyperpolarized membrane potential and inhibited orexin neuron discharge in proportion to illumination intensity. Acute silencing of orexin neurons in vivo during the day (the inactive period) induced synchronization of the electroencephalogram and a reduction in amplitude of the electromyogram that is characteristic of slow-wave sleep (SWS). In contrast, orexin neuron photoinhibition was ineffective during the night (active period). Acute photoinhibition of orexin neurons during the day in orexin/Halo mice also reduced discharge of neurons in an orexin terminal field, the dorsal raphe (DR) nucleus. However, serotonergic DR neurons exhibited normal discharge rates in mice lacking orexin neurons. Thus, although usually highly dependent on orexin neuronal activity, serotonergic DR neuronal activity can be regulated appropriately in the chronic absence of orexin input. Together, these results demonstrate that acute inhibition of orexin neurons results in time-of-day-dependent induction of SWS and in reduced firing rate of neurons in an efferent projection site thought to be involved in arousal state regulation. The results presented here advance our understanding of the role of orexin neurons in the regulation of sleep/wakefulness and may be relevant to the mechanisms that underlie symptom progression in narcolepsy.

The sleep disorder narcolepsy results from loss of hypothalamic orexin/hypocretin neurons. Although narcolepsy onset is usually postpubertal, current mouse models involve loss of either orexin peptides or orexin neurons from birth. To create a model of orexin/hypocretin deficiency with closer fidelity to human narcolepsy, diphtheria toxin A (DTA) was expressed in orexin neurons under control of the Tet-off system. Upon doxycycline removal from the diet of postpubertalorexin-tTA;TetO DTAmice, orexin neurodegeneration was rapid, with 80% cell loss within 7 d, and resulted in disrupted sleep architecture. Cataplexy, the pathognomic symptom of narcolepsy, occurred by 14 d when ∼5% of the orexin neurons remained. Cataplexy frequency increased for at least 11 weeks after doxycycline. Temporary doxycycline removal followed by reintroduction after several days enabled partial lesion of orexin neurons. DTA-induced orexin neurodegeneration caused a body weight increase without a change in food consumption, mimicking metabolic aspects of human narcolepsy. Because the orexin/hypocretin system has been implicated in the control of metabolism and addiction as well as sleep/wake regulation, orexin-tTA; TetO DTAmice are a novel model in which to study these functions, for pharmacological studies of cataplexy, and to study network reorganization as orexin input is lost.

Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neuronsin vivowere generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep.

Controlling neural circuits is a powerful approach to uncover a causal link between neural activity and behaviour. Optogenetics has been widely adopted by the neuroscience community as it offers cell-type-specific perturbation with millisecond precision. However, these studies require light delivery in complex patterns with cellular-scale resolution, while covering a large volume of tissue at depth in vivo. Here we describe a novel high-density silicon-based microscale light-emitting diode (μLED) array, consisting of up to ninety-six 25 μm-diameter μLEDs emitting at a wavelength of 450 nm with a peak irradiance of 400 mW/mm². A width of 100 μm, tapering to a 1 μm point, and a 40 μm thickness help minimise tissue damage during insertion. Thermal properties permit a set of optogenetic operating regimes, with ~0.5 °C average temperature increase. We demonstrate depth-dependent activation of mouse neocortical neurons in vivo, offering an inexpensive novel tool for the precise manipulation of neural activity.

Orexin neurons in the hypothalamus regulate energy homeostasis by coordinating various physiological responses. Past studies have shown the role of the orexin peptide itself; however, orexin neurons contain not only orexin but also other neurotransmitters such as glutamate and dynorphin. In this study, we examined the physiological role of orexin neurons in feeding behavior and metabolism by pharmacogenetic activation and chronic ablation. We generated novel orexin-Cre mice and utilized Cre-dependent adeno-associated virus vectors to express Gq-coupled modified GPCR, hM3Dq or diphtheria toxin fragment A in orexin neurons. By intraperitoneal injection of clozapine-N oxide in orexin-Cre mice expressing hM3Dq in orexin neurons, we could selectively manipulate the activity of orexin neurons. Pharmacogenetic stimulation of orexin neurons simultaneously increased locomotive activity, food intake, water intake and the respiratory exchange ratio (RER). Elevation of blood glucose levels and RER persisted even after locomotion and feeding behaviors returned to basal levels. Accordantly, 83% ablation of orexin neurons resulted in decreased food and water intake, while 70% ablation had almost no effect on these parameters. Our results indicate that orexin neurons play an integral role in regulation of both feeding behavior and metabolism. This regulation is so robust that greater than 80% of orexin neurons were ablated before significant changes in feeding behavior emerged.

Whether increased serotonin (5-HT) release in the forebrain attenuates or enhances anxiety has been controversial for over 25 yr. Although there is co