Probes for CCK

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.

Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.

The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.