Extended Data Figure 2 |. MnPO^nNOS neurons are necessary for the induction of drinking by SFO^nNOS photostimulation.

a, Casp3-TEVp efficiently eliminates SFO^nNOS neurons (right) without affecting MnPO^nNOS neurons (left). c-Fos expression pattern is shown after water-restriction (red). b, Rastor plots representing licking events during the 5-s session with photostimulation. c, Ablation of MnPO^nNOS (MnPOx) but not SFO^nNOS (SFOx) neurons attenuated the drinking response to OVLT^nNos photostimulation (left, 10 min, blue box). Quantification of the number of licks during the 10-min light-on period (right, n = 9 mice for controls and MnPOx and n = 7 mice for SFOx). d, 5-s brief-access assays to examine the necessity of MnPO^nNOS neurons. Acute inhibition of MnPO^nNOS neurons by CNO injection severely reduced SFO^nNOS-stimulated (left, n = 5 mice for CNO, n = 3 mice for vehicle, and n = 6 mice for no i.p.) and dehydration-induced water intake (middle, n = 7 mice for CNO, n = 5 mice for vehicle, and n = 3 mice for no i.p.). However, the same treatment did not suppress sucrose consumption (300 mM, right, n = 6 mice for CNO, n = 5 mice for vehicle, and n = 3 mice for no i.p.). Control mice transduced by AAV-DIO-mCherry in the MnPO showed no reduction after water or food-restriction (n = 3 mice). e, mCherry control for Fig. 1g. Cumulative water intake in nNOS-cre mice transduced with AAV-DIO-mCherry in the MnPO, AAV-DIO-ChR2-eYFP in the SFO under photostimulated (left, n = 5 mice) or water-restricted conditions (middle, n = 6 mice), and sucrose (300 mM) intake under food-restricted conditions (right, n = 5 mice). f, Intraperitoneal injection of mannitol robustly activated SFO^nNOS neurons with (red trace) or without (black trace) CNO injection (left). CNO injection drastically suppressed drinking behaviour without changing the activity of SFO^nNOS neurons (middle, n = 4 mice). Plasma osmolality was increased by the injection of mannitol (right, n = 5 mice). *P < 0.05, **P < 0.01, by paired two-tailed t-test or Kruskal–Wallis one-way ANOVA test with Dunn’s correction for multiple comparisons. All error bars and shaded areas show mean ± s.e.m. Scale bar, 50 μm.