Probes for GAL

The preoptic area (POA) is necessary for sleep, but the fundamental POA circuits have remained elusive. Previous studies showed that galanin (GAL)- and GABA-producing neurons in the ventrolateral preoptic nucleus (VLPO) express cFos after periods of increased sleep and innervate key wake-promoting regions. Although lesions in this region can produce insomnia, high frequency photostimulation of the POAGAL neurons was shown to paradoxically cause waking, not sleep. Here we report that photostimulation of VLPOGAL neurons in mice promotes sleep with low frequency stimulation (1-4 Hz), but causes conduction block and waking at frequencies above 8 Hz. Further, optogenetic inhibition reduces sleep. Chemogenetic activation of VLPOGAL neurons confirms the increase in sleep, and also reduces body temperature. In addition, chemogenetic activation of VLPOGAL neurons induces short-latency sleep in an animal model of insomnia. Collectively, these findings establish a causal role of VLPOGAL neurons in both sleep induction and heat loss.

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.