Fig. 6. VTA^Vgat neurons inhibit wakefulness. Lesioning of VTA^Vgat neurons produces extended wakefulness, but VTA^Vgat neurons are selectively wake- and REM-active.

(a) Lesioning of VTA^Vgat neurons. Injection of AAV-DIO-GFP (control) or AAV-DIO-taCASP3-TEV into the VTA area of Vgat-ires-Cre mice. Pictures show GFP control expression in the VTA area of VTA^Vgat-GFP mice and that this GFP expression has been greatly diminished in the caspase treated mice. The experiment was repeated independently 5 times. IF, interfasicular nucleus; PBP, parabrachial pigmented nucleus; PN, paranigral nucleus; PBP, parabrachial pigmented nucleus; RLi, rostral linear nucleus

(b) Lesioning of VTA^Vgat neurons increases wakefulness. Percentage of wake, NREM and REM sleep in control VTA^Vgat-GFP mice (n=7 mice) and VTA^Vgat-CASP3 mice (n=5 mice), and the total vigilance times in the “lights on” and “lights off” periods.

(c, d) Lesioning of VTA^Vgat neurons reduces the transitions between vigilance states and stabilizes wakefulness. Episode number and duration for wake, NREM and REM sleep, and vigilance state transitions during the “lights off” periods in VTA^Vgat-GFP control mice (n=7 mice) and VTA^Vgat-CASP3 mice (n=5 mice).

(e) Fiber photometry for Ca²⁺ levels in VTA^Vgat neurons. Injection of AAV-DIO-GCaMP6 into the VTA of Vgat-ires-Cre mice. GCaMP6 expression can be detected in the VTA area and was co-stained with GABA. The trace of where the optical fiber was placed is illustrated. The experiment was repeated independently 7 times.

(f) Fiber photometry for VTA^Vgat neurons. Neurons are more active in wake and REM sleep. Ca²⁺ photometry spectra (bottom trace) recorded in the VTA of VTA^Vgat-GCaMP6 mice aligned with the EEG spectra (middle trace) and EMG (top trace) during wakefulness, NREM and REM sleep. “Epoch” indicates vigilance state: blue: wake; green: NREM sleep; magenta, REM sleep.

(g) Fiber photometry for VTA^Vgat neurons. ΔF/F ratio of the Ca²⁺ photometry signal in VTA^Vgat-GCaMP6f mice during wakefulness, NREM sleep and REM sleep (n=7 mice; 41 trials).

(h) Fiber photometry for VTA^Vgat neurons. Detail of how the Ca²⁺ signal in Vgat neurons of VTA^Vgat-GCaMP6 mice changes at the boundaries of the vigilance states (n=7 mice). “Epoch” indicates vigilance state: blue, wake; green, NREM sleep; magenta: REM sleep. Grey shaded regions represent SEM.

*p<0.05, **p<0.01, ****p<0.0001; For b-d, two-sided unpaired t-test, for g, one-way ANOVA. All error bars represent the SEM. For detailed statistics information, see Supplementary Table1.