Fig. 8: Inhibition of NAc-ensemble-innervated POA or VTA neurons.

a Experimental scheme. b Schematic showing the injection of pAAV1-PTRE-tight-NLS-Cre into NAc and the injection of AAV8-Ef1a-DIO-eNpHR-mCherry into POA, respectively. NAc ensemble→POA projecting Cre-dependent enhanced natronomonas pharaonic halorhodopsin (eNpHR) validation 4 weeks after injection. c Raster plot (top) and PSTH (bottom) of a NAc-ensemble-innervated POA neuron showing the firing activity before, during, and after 8 s optogenetic silencing. d, e Optogenetic inactivation of NAc-ensemble-innervated POA neurons had no effect on the thermal PWL (n = 14 mice) and mechanical PWT (n = 14 mice). f Optogenetic inactivations of NAc-ensemble-innervated POA neurons (589 nm, yellow laser, 8 s on/2 s off, 120 s) significantly decrease in NREM sleep (n = 11 mice, p < 0.0001, bootstrap), increase in wakefulness (p < 0.0001), and do not affect REM sleep (p = 0.363). Shading represents ± SEM. g Similar to (Fig. 4f), but for inactivation of NAc ensemble-innervated POA neurons. h Similar to (b), but with injection AAV into VTA. Brain figures in (b and h) were adapted from Allen Mouse Brain Atlas (2011 Allen Institute for Cell Science, Allen Mouse Brain Atlas, available at http://atlas.brain-map.org/). i Similar to (c), but for a NAc ensemble-innervated VTA neurons. j, k Optogenetic inactivations of NAc ensemble-innervated VTA neurons significantly decrease the thermal PWL (n = 13 mice) and mechanical PWT (n = 13 mice). l Optogenetic inactivations of NAc ensemble-innervated VTA neurons (589 nm, yellow laser, 8 s on/2 s off, 120 s) do not affect NREM sleep (n = 13 mice, p = 0.490, bootstrap), wakefulness (p = 0.132), and REM sleep (p = 0.140). Shading represents ± SEM. m Similar to (Fig. 4f), but for inactivation of NAc ensemble-innervated VTA neurons. Repeated measures one-way ANOVA test with Bonferroni’s multiple comparisons test for (d, e, j, k). ***p < 0.001, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ns not significant.