In humans and other mammalian species, lesions in the preoptic area (POA) of the hypothalamus cause profound sleep impairment1–5, indicating a crucial role of the POA in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry showed the existence of sleep-active neurons in the POA, especially in the ventrolateral preoptic area (VLPO) and median preoptic nucleus (MnPO)6–9. Pharmacogenetic activation of c-Fos-labeled sleep-active neurons has been shown to induce sleep10. However, the sleep-active neurons are spatially intermingled with wake-active neurons6,7, making it difficult to target the sleep neurons specifically for circuit analysis. Here, we have identified a population of POA sleep neurons based on their projection target and discovered their molecular markers. Using a lentivirus expressing channelrhodopsin-2 (ChR2) or a light-activated chloride channel (iC++) for retrograde labeling, bidirectional optogenetic manipulation, and optrode recording, we showed that the POA GABAergic neurons projecting to the tuberomammillary nucleus (TMN) are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification (TRAP) and single-cell RNA-seq identified candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrated that several peptide markers (cholecystokinin, corticotropin releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting POA neurons and a valuable entry point for dissecting the sleep control circuit.

Sleep is a fundamental biological process observed widely in the animal kingdom, but the neural circuits generating sleep remain poorly understood. Understanding the brain mechanisms controlling sleep requires the identification of key neurons in the control circuits and mapping of their synaptic connections. Technical innovations over the past decade have greatly facilitated dissection of the sleep circuits. This has set the stage for understanding how a variety of environmental and physiological factors influence sleep. The ability to initiate and terminate sleep on command will also help us to elucidate its functions within and beyond the brain.

Rapid eye movement (REM) sleep is a distinct brain state characterized by activated electroencephalogram (EEG) and complete skeletal muscle paralysis, and it is associated with vivid dreams1-3. Transection studies by Jouvet first demonstrated that the brainstem is both necessary and sufficient for REM sleep generation2, and the neural circuits in the pons have since been studied extensively4-8. The medulla also contains neurons that are active during REM sleep9-13, but whether they play a causal role in REM sleep generation remains unclear. Here we show that a GABAergic pathway originating from the ventral medulla (vM) powerfully promotes REM sleep. Optogenetic activation of vM GABAergic neurons rapidly and reliably initiated REM sleep episodes and prolonged their durations, whereas inactivating these neurons had the opposite effects. Optrode recordings from channelrhodopsin 2 (ChR2)-tagged vM GABAergic neurons showed that they were most active during REM sleep (REM-max), and during wakefulness they were preferentially active during eating and grooming. Furthermore, dual retrograde tracing showed that the rostral projections to the pons and midbrain and caudal projections to the spinal cord originate from separate vM neuron populations. Activating the rostral GABAergic projections was sufficient for both the induction and maintenance of REM sleep, which are likely mediated in part by inhibition of REM-suppressing GABAergic neurons in the ventrolateral periaqueductal gray (vlPAG). These results identify a key component of the pontomedullary network controlling REM sleep. The capability to induce REM sleep on command may offer a powerful tool for investigating its functions.

Mammalian sleep consists of distinct rapid eye movement (REM) and non-REM (NREM) states. The midbrain region ventrolateral periaqueductal gray (vlPAG) is known to be important for gating REM sleep, but the underlying neuronal mechanism is not well understood. Here, we show that activating vlPAG GABAergic neurons in mice suppresses the initiation and maintenance of REM sleep while consolidating NREM sleep, partly through their projection to the dorsolateral pons. Cell-type-specific recording and calcium imaging reveal that most vlPAG GABAergic neurons are strongly suppressed at REM sleep onset and activated at its termination. In addition to the rapid changes at brain state transitions, their activity decreases gradually between REM sleep and is reset by each REM episode in a duration-dependent manner, mirroring the accumulation and dissipation of REM sleep pressure. Thus, vlPAG GABAergic neurons powerfully gate REM sleep, and their firing rate modulation may contribute to the ultradian rhythm of REM/NREM alternation.