To understand how sleep-wakefulness cycles are regulated, it is essential to disentangle structural and functional relationships between the preoptic area (POA) and lateral hypothalamic area (LHA), since these regions play important yet opposing roles in the sleep-wakefulness regulation. GABA- and galanin (GAL)-producing neurons in the ventrolateral preoptic nucleus (VLPO) of the POA (VLPOGABA and VLPOGAL neurons) are responsible for the maintenance of sleep, while the LHA contains orexin-producing neurons (orexin neurons) that are crucial for maintenance of wakefulness. Through the use of rabies virus-mediated neural tracing combined with in situ hybridization in male and female orexin-iCre mice, we revealed that the vesicular GABA transporter (Vgat, Slc32a1)- and galanin (Gal)-expressing neurons in the VLPO directly synapse with orexin neurons in the LHA. A majority (56.3卤8%) of all VLPO input neurons connecting to orexin neurons were double-positive for Vgat and Gal. Using projection-specific rabies virus-mediated tracing in male and female Vgat-ires-Cre and Gal-Cre mice, we discovered that VLPOGABA and VLPOGAL neurons that send projections to the LHA received innervations from similarly distributed input neurons in many brain regions, with the POA and LHA being among the main upstream areas. Additionally, we found that acute optogenetic excitation of axons of VLPOGABA neurons, but not VLPOGAL neurons, in the LHA of male Vgat-ires-Cre mice induced wakefulness. This study deciphers the connectivity between the VLPO and LHA, provides a large-scale map of upstream neuronal populations of VLPO鈫扡HA neurons, and reveals a previously uncovered function of the VLPOGABA鈫扡HA pathway in the regulation of sleep and wakefulness.

Significance statement:

We identified that neurons in the VLPO, that are positive for vesicular GABA transporter (Vgat)- and/or galanin (Gal), serve as presynaptic partners of orexin-producing neurons in the LHA. We depicted monosynaptic input neurons of GABA- and galanin-producing neurons in the VLPO that send projections to the LHA throughout the entire brain. Their input neurons largely overlap, suggesting that they comprise a common neuronal population. However, acute excitatory optogenetic manipulation of VLPOGABA鈫扡HA pathway, but not VLPOGAL鈫扡HA pathway, evoked wakefulness. This study shows the connectivity of major components of the sleep/wake circuitry in the hypothalamus and unveils a previously unrecognized function of the VLPOGABA鈫扡HA pathway in sleep-wakefulness regulation. Furthermore, we suggest the existence of subpopulations of VLPOGABA neurons that innervate LHA.

The authors declare no competing financial interests.

This study was supported by a JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (JP 18H02595) (TS); AMED Grant Number JP21zf0127005 (TS); and JST CREST Grant Number JPMJCR1655 Japan (TS); TMFC Japan Foundation for applied Enzymology (AH). We thank Dr. P. Vergara for conducting multidimensional scaling and Spearman correlation analyses; Dr. Y. Cherasse for preparing virus vectors; and M. Fukatsu and Y. Katsuyama for technical assistance.

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