Extended Data Fig. 1: Thirst-state-dependent drinking behaviour and genetic labelling of active neurons.

a, FOS expression in the SFO (left) and the OVLT (right) under the four thirst states (SFO: n = 6 sections from 4 mice for control, n = 6 sections from 6 mice for osmotic thirst, n = 5 sections from 4 mice for hypovolaemic thirst, and n = 6 sections from 6 mice for water deprivation; OVLT: n = 3 sections from 3 mice for control, n = 8 sections from 7 mice for osmotic thirst, n = 5 sections from 4 mice for hypovolaemic thirst, n = 7 sections from 7 mice for water deprivation). b, Water and 0.3M NaCl consumption in sated control animals. The number of total licks for water (grey) and 0.3M saline (red) were quantified during a one-hour session (n = 9 mice for each group). c, Water (grey) and 0.3M KCl intake (orange) under osmotic and hypovolaemic thirst states. The number of total licks was quantified during a one-hour session (n = 6 mice). d, Experimental diagram for TRAP2 activity-dependent genetic labelling. TRAP2/Ai14 double transgenic animals were challenged with osmotic stress by i.p. injection of NaCl solution in the presence of 4-OHT. Osmolality sensitive cells (upper) express Cre-ER under the promoter of Fos gene, which turns on tdTomato expression (red). In osmolality insensitive cells, the same stimulus does not induce tdTomato expression (bottom). e, Genetic labelling of thirst-sensitive neurons in the OVLT of TRAP2/Ai14 mice. Experimental design to label activated neurons under osmotic thirst and hypovolaemic thirst (top). Osmolality sensitive neurons (Osm-TRAP, red) in the OVLT (bottom) overlapped with NaCl-induced acute FOS expression (green). Individual labelling and merged images are shown. By contrast, a significantly smaller fraction of Osm-TRAP neurons was co-labelled with hypovolaemia-induced FOS. Scale bars, 50 μm. f, Quantification of OVLT TRAP2 experiments (n = 6 sections from 4 mice for Osm-Osm, n = 5 sections from 5 mice for Osm-Hvol). g, TRAP labelling in the SFO and the OVLT of sated control animals (n = 6 sections from 3 mice for SFO, n = 3 sections from 2 mice for OVLT). *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Wilcoxon matched-pairs signed rank test or Mann–Whitney test. Data are shown as mean ± s.e.m.