Calcium Dynamics of the Ventrolateral Preoptic GABAergic Neurons during Spontaneous Sleep-Waking and in Response to Homeostatic Sleep Demands

Abstract

The ventrolateral preoptic area (VLPO) contains GABAergic sleep-active neurons. However, the extent to which these neurons are involved in expressing spontaneous sleep and homeostatic sleep regulatory demands is not fully understood. We used calcium (Ca2+) imaging to characterize the activity dynamics of VLPO neurons, especially those expressing the vesicular GABA transporter (VGAT) across spontaneous sleep-waking and in response to homeostatic sleep demands. The VLPOs of wild-type and VGAT-Cre mice were transfected with GCaMP6, and the Ca2+ fluorescence of unidentified (UNID) and VGAT cells was recorded during spontaneous sleep-waking and 3 h of sleep deprivation (SD) followed by 1 h of recovery sleep. Although both VGAT and UNID neurons exhibited heterogeneous Ca2+ fluorescence across sleep-waking, the majority of VLPO neurons displayed increased activity during nonREM/REM (VGAT, 120/303; UNID, 39/106) and REM sleep (VGAT, 32/303; UNID, 19/106). Compared to the baseline waking, VLPO sleep-active neurons (n = 91) exhibited higher activity with increasing SD that remained elevated during the recovery period. These neurons also exhibited increased Ca2+ fluorescence during nonREM sleep, marked by increased slow-wave activity and REM sleep during recovery after SD. These findings support the notion that VLPO sleep-active neurons, including GABAergic neurons, are components of neuronal circuitry that mediate spontaneous sleep and homeostatic responses to sustained wakefulness.

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“…This characteristic enables high detection rates for single spikes. However, due to its slower decay time, GCaMP6 may not accurately capture high-frequency neuronal spikes and might not precisely reflect the actual firing rate of neurons compared to electrophysiology methods [48][49][50]. The NAc consists of two distinct subregions, the shell (NAcSh, a part of extended amygdala) and the core (NAcC, a part of dorsal striatum) [51,52].…”

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“…VLPO lesions cause sleep fragmentation and insomnia [ 25 ], whereas selective activation of VLPO neurons projecting to the hypocretin field promotes sleep [ 26 ]. VLPO neurons exhibit sleep-associated c-fos expression (Fos-IR) and increased Ca 2+ activity, markers of neuronal activation during nonREM and or REM sleep [ 27 , 28 , 29 ]. The VLPO sleep-active neurons typically exhibit increasing discharge from waking → nonREM sleep transition → stable nonREM/REM sleep [ 30 , 31 ].…”

“…The VLPO sleep-active neurons typically exhibit increasing discharge from waking → nonREM sleep transition → stable nonREM/REM sleep [ 30 , 31 ]. Their activity profiles further suggest that VLPO sleep-active neurons are involved in responding to homeostatic sleep need [ 29 , 31 ]. While VLPO contains a high concentration of sleep-active neurons, these neurons are diffusely distributed in the VLPO core and its adjoining POA areas and are mixed with wake-active neurons [ 32 , 33 , 34 ].…”