Discussion
Using DREADDs, we showed that chemo-activation of the SCN, the RCA, the PVH and PVH projecting neurons to the PB, all promoted arousal. We hypothesize that SCN-RCA-PVH-PB forming an arousal network promotes arousal.
Lesions of the medial PB and lateral PB result in 40% and 10% increase in sleep, respectively, and lesions of the entire PB produce a coma-like state (Fuller et al. , 2011). PVH projections to the PB are mostly glutamatergic (Chen et al. , 2021) and RCA projections to the PB may also be glutamatergic. Given that both PVH and RCA receive SCN inputs, chemo-stimulation of the SCN induces Fos in RCA and PVH, and chemo-stimulations of these sites promoted arousal, it is speculative that the SCN activating RCA-PVH-PB network promotes arousal.
Single-unit recordings of the SCN in vivo in freely moving rats reveals not only circadian firing pattern (Kawamura & Hashimoto, 1979) ,but also mixed populations of sleep and wake dependent firing in mouse, with predominantly wake-active neurons (Sakai, 2014). A small sample of single-unit recording in the SCN in rats demonstrates wake-REM sleep active firing (Deboer et al. , 2003). These wake-active neurons may mediate retinal inputs on arousal but may not be critical for circadian regulation. It is not clear whether these neurons receive direct retinal inputs. SCN wake-active neurons may particularly be important in diurnal species such as primates (Mistlberger, 2005). Despite of existence of small amounts of sleep-active neurons in the SCN, we never observed sleep promotion effects by stimulation of the SCN, PVH, or RCA.
Although DREADD clozapine-N-oxide (CNO) on sleep have been systematically tested, it intraperitoneal injections of commonly used mouse CNO doses (1, 5, and 10 mg/kg) dose-dependent suppression of rapid eye movement (REM) sleep, alter sleep in male mice (Traut et al., 2023), CNO at much lower dose 0.2 mg/kg did not alter sleep architecture in rats (Wen et al., 2020).
Compared to wake effects of PVH or PVH-PB pathway stimulation as well as other arousal neurons such as orexinergic neurons, unilateral SCN stimulation showed weaker effects and only effective during the daytime, the highest sleep period. On the other hand, we expect that the bilateral SCN stimulation would show stronger arousal effects and may promote arousal during the night time. In the PVH, melanocortin-4 receptor containing glutamatergic neurons that project to the PB regulate appetite (Garfield et al. , 2015). These neurons may regulate arousal and serve as a neural circuit by which hunger and appetite influence on sleep-wake behavior. The PVH-PB pathway may mediate arousal by other neural factors such as stress (Radley et al. , 2009), dehydration (Watts et al. , 1999) and cold exposure (Cano et al. , 2003).