Probes for INS

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.

Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.

Single-cell RNA sequencing has generated catalogs of transcriptionally defined neuronal subtypes of the brain. However, the cellular processes that contribute to neuronal subtype specification and transcriptional heterogeneity remain unclear. By comparing the gene expression profiles of single layer 6 corticothalamic neurons in somatosensory cortex, we show that transcriptional subtypes primarily reflect axonal projection pattern, laminar position within the cortex, and neuronal activity state. Pseudotemporal ordering of 1,023 cellular responses to sensory manipulation demonstrates that changes in expression of activity-induced genes both reinforced cell-type identity and contributed to increased transcriptional heterogeneity within each cell type. This is due to cell-type biased choices of transcriptional states following manipulation of neuronal activity. These results reveal that axonal projection pattern, laminar position, and activity state define significant axes of variation that contribute both to the transcriptional identity of individual neurons and to the transcriptional heterogeneity within each neuronal subtype.

The perioculomotor (pIII) region of the midbrain was postulated as a sleep-regulating center in the 1890s but largely neglected in subsequent studies. Using activity-dependent labeling and gene expression profiling, we identified pIII neurons that promote non-rapid eye movement (NREM) sleep. Optrode recording showed that pIII glutamatergic neurons expressing calcitonin gene-related peptide alpha (CALCA) are NREM-sleep active; optogenetic and chemogenetic activation/inactivation showed that they strongly promote NREM sleep. Within the pIII region, CALCA neurons form reciprocal connections with another population of glutamatergic neurons that express the peptide cholecystokinin (CCK). Activation of CCK neurons also promoted NREM sleep. Both CALCA and CCK neurons project rostrally to the preoptic hypothalamus, whereas CALCA neurons also project caudally to the posterior ventromedial medulla. Activation of each projection increased NREM sleep. Together, these findings point to the pIII region as an excitatory sleep center where different subsets of glutamatergic neurons promote NREM sleep through both local reciprocal connections and long-range projections.

Thermoregulation is one of the most vital functions of the brain, but how temperature information is converted into homeostatic responses remains unknown. Here, we use an unbiased approach for activity-dependent RNA sequencing to identify warm-sensitive neurons (WSNs) within the preoptic hypothalamus that orchestrate the homeostatic response to heat. We show that these WSNs are molecularly defined by co-expression of the neuropeptides BDNF and PACAP. Optical recordings in awake, behaving mice reveal that these neurons are selectively activated by environmental warmth. Optogenetic excitation of WSNs triggers rapid hypothermia, mediated by reciprocal changes in heat production and loss, as well as dramatic cold-seeking behavior. Projection-specific manipulations demonstrate that these distinct effectors are controlled by anatomically segregated pathways. These findings reveal a molecularly defined cell type that coordinates the diverse behavioral and autonomic responses to heat. Identification of these warm-sensitive cells provides genetic access to the core neural circuit regulating the body temperature of mammals.