Supplementary Figure 4: Chronic Oxtr^PBN neuron inactivation does not affect fluid intake at baseline or following various homeostatic challenges

(a) Injection of AAV-DIO-GFP:TetTox in Oxtr^PBN neurons. (b) Representative images of GFP expression in Oxtr^PBN neurons in GFP:TetTox- vs control YFP-injected mice; scp, superior cerebellar peduncle; dl, dorsolateral; el, external lateral; scale bar 100 µm. (c) Chronic Oxtr^PBN inactivation does not significantly alter baseline saline preference at different concentrations (0.075, 0.3, 0.5 M) (n = 6 TetTox, 7 YFP; unpaired 2-tailed Student’s t-test; 0.075 M NaCl: t(11) = 0.4967; p  = 0.6292; 0.3 M NaCl: t(11) = 1.005; p = 0.3364; 0.5 M NaCl: t(11) = 0.09961; p = 0.9224), and does not significantly change fluid intake following normal saline ip, either (d) acutely (n = 6 TetTox, 7 YFP; 2-way RM ANOVA; NaCl: interaction F(8,88) = 0.7729; p = 0.6275; water: interaction F(8,88) = 0.4721; p = 0.8728) or (e) overnight (n = 6 TetTox, 7 YFP; unpaired 2-tailed Student’s t-test; NaCl: t(11) = 0.8014; p = 0.4398; water: t(11) = 0.6858; p = 0.5070). (f) Overnight NaCl intake remained significantly increased in TetTox-injected mice (n = 6 TetTox, 7 YFP; unpaired 2-tailed Student’s t-test; NaCl: t(11) = 2.760; p = 0.0186; water: t(11) = 0.8881; p = 0.3935). Chronic Oxtr^PBN inactivation does not significantly change fluid intake following (g) 1 M mannitol ip (n = 6 TetTox; 7 YFP; 2-way RM ANOVA; NaCl: interaction F(4,44) = 0.4638; p = 0.7619; water: interaction F(4,44) = 1.524; p = 0.2118) or (h) 30% PEG sc (n = 6 TetTox; 7 YFP; 2-way RM ANOVA; NaCl: interaction F(4,44) = 1.494; p = 0.2203; water: interaction F(4,44) = 0.3461; p = 0.8453). (i) There was no significant correlation between the level of GFP:TetTox expression and the amount of fluid intake after 24-h dehydration (n = 6; Pearson product-moment correlation; NaCl: r = 0.3167; p = 0.5408; water: r = 0.4335; p = 0.3905). Data are expressed as mean ± s.e.m. *p < 0.05. See Supplementary Information for statistical analyses