In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep. However, the sleep-active neurons are spatially intermingled with wake-active neurons, making it difficult to target the sleep neurons specifically for circuit analysis.
Here researchers from the University of California, Berkeley identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, they show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate 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 preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.
Identification of molecular markers for POA sleep neurons
a, Schematics of TRAP (top) and single-cell RNA-seq (bottom) for gene profiling. b, Overlap between haemagglutinin (HA) labelling of GABAPOA→TMN neurons and CCK expression. Shown is a coronal section at the POA stained with haemagglutinin antibody (red) and Hoechst (blue). Region within the square is magnified (inset; scale bar, 50 μm). Arrowheads, haemagglutinin-labelled neurons stained with CCK antibody; 49.1 ± 7.5% of haemagglutinin+ neurons are CCK+ (n = 3 mice). c, Schematic of optogenetic activation of POA CCK neurons (top) and a fluorescence image of POA in a CCK-Cre mouse injected with AAV-DIO-ChR2–eYFP. d, Percentage of time in NREM, REM, or wake state before, during, and after optogenetic stimulation (blue shading, 10 Hz, 120 s) of CCK neurons (P = 0.0007 for REM, P < 0.0001 for NREM and wake, bootstrap; n = 4 mice). e, Overlap between haemagglutinin labelling and CRH expression. 17.1 ± 1.9% of haemagglutinin-labelled neurons are CRH+ (arrowheads; n = 3 mice). f, Fluorescence image of POA in a CRH-Cre mouse injected with AAV-DIO-ChR2–eYFP. g, Percentage of time in NREM, REM, or wake state, averaged from five CRH-Cre mice (P = 0.0014 for NREM, P < 0.0001 for REM and wake). h, Overlap between eYFP labelling of GABAPOA→TMN neurons and TAC1 expression. Arrowheads, eYFP+ neurons expressing TAC1 (FISH, n = 2 mice). i, Fluorescence image of POA in a TAC1-Cre mouse injected with AAV-DIO-ChR2–eYFP. j, Percentage of time in NREM, REM, or wake state, averaged from seven TAC1-Cre mice (P < 0.0001 for NREM and wake). Shading, 95% confidence interval.