Figure 1 |. Thirst-driving neurons are organized hierarchically in the lamina terminalis.

a, Schematic of monosynaptic rabies tracing (left). Representative images of the MnPO (top right, one of seven mice) and OVLT (bottom right, one of five mice) of an nNOS-cre mouse transduced with AAV-CA-flex-RG and AAV-EF1a-flex-TVA-mCherry (red) followed by RV-SAD-ΔG-eGFP (green). 3V, third ventricle. b, Quantification of eGFP⁺ neurons in the SFO (n = 7 and 5 mice for MnPO and OVLT, respectively). c, Neural epistasis analysis of the circuits of the lamina terminalis by loss-of-function manipulation. Caspase expression is induced in the MnPO, OVLT (left) or SFO (right) of nNOS-cre mice. d, Casp3-TEVp efficiently eliminates nNOS-expressing neurons (green) in the MnPO (93.2 ± 2.5%, n = 4 mice) and OVLT (90.6 ± 1.4%, n = 6 mice). c-Fos expression (red) upon the stimulation of SFO^nNOS neurons is shown. e, Number of licks during the 5-s session (n = 9 mice for controls and OVLTx, n = 7 mice for MnPOx, n = 6 mice for SFOx and SFOx/OVLTx). f, Chemogenetic inhibition of MnPO^nNOS neurons by CNO (left, six out of six neurons), and a diagram of photostimulation of SFO^nNOS and chemogenetic inhibition of MnPO^nNOS neurons (right). g, Cumulative water intake in SFO^nNOS-stimulated mice (left, n = 5 mice) or water-restricted mice (middle, n = 10 mice for CNO and n = 9 mice for vehicle), and sucrose (300 mM) intake in food-restricted mice (right, n = 10 mice for CNO and n = 9 mice for vehicle). h, Fibre photometry of SFO^nNOS neurons while MnPO^nNOS neurons are inhibited by hM4Di-mCherry. i, Intraperitoneal NaCl injection robustly activates SFO^nNOS neurons with (red trace) or without (black trace) CNO injection (left and middle left). By contrast, CNO injection drastically suppressed drinking behaviour (middle right, n = 6 mice). Plasma osmolality (top right) and Na⁺ concentration (bottom right) were measured after NaCl injection (n = 5 mice). **P < 0.01, ***P < 0.001, ****P < 0.0001, by Mann–Whitney U test, paired two-tailed t-test or Kruskal–Wallis one-way analysis of variance (ANOVA) test. All error bars and shaded areas show mean ± s.e.m. Scale bars, 50 μm.