Supplementary Figure 3: Chemogenetic activation of Oxtr^PBN neurons decreases fluid intake acutely

(a) Overnight NaCl was not significantly different, but overnight water intake remained slightly decreased following CNO injection in hM₃Dq-injected vs control mCherry-injected mice after 24-h dehydration (n = 7/group; unpaired 2-tailed Student’s t-test; NaCl: t(12) = 0.5656; p = 0.5821; water: t(12) = 2.311; p = 0.0394). (b) Control mCherry-injected mice consumed more NaCl and less water when food was absent; but there was no significant difference in fluid intake after CNO injection in hM₃Dq-injected mice (n = 7/group in food present; 6/group in food absent; 2-way ANOVA; NaCl: interaction F(1,22) = 0.7401; p = 0.3989; main effect of viral genotype F(1,22) = 32.44; p < 0.0001; main effect of food availability F(1,22) = 11.77; p = 0.0024; water: interaction F(1,22) = 4.946; p = 0.0367). (c) Overnight baseline fluid intake was not significantly different following vehicle or CNO injection (n = 7/group; 2-way RM ANOVA; NaCl: interaction F(1,12) = 1.806; p = 0.2038; water: interaction F(1,12) = 0.1977; p = 0.6645). d,e, Acute Oxtr^PBN activation demonstrated no significant difference in Ensure or water intake when Ensure was limited to (d) ~1.6 mL (n = 6/group; 2-way RM ANOVA; Ensure: interaction F(8,80) = 1.168; p = 0.3285; water: interaction F(8,80) = 1.027; p = 0.4228) or (e) ~0.7 mL (n = 6/group; 2-way RM ANOVA; Ensure: interaction F(8,80) = 1.182; p = 0.3202; water: interaction F(8,80) = 1.313; p = 0.2489). (f) Overnight fluid intake was not significantly different after hypertonic saline ip injection in hM₃Dq- or vs mCherry-injected mice (n = 7/group; unpaired 2-tailed Student’s t-test; NaCl: t(12) = 0.2868; p = 0.7791; water: t(12) = 1.354; p = 0.2008). (g) Daily intake of NaCl and water was not significantly different between the start and end of experimentation (n = 7/group; 2-way RM ANOVA; NaCl: interaction F(1,12) = 0.1273; p = 0.7275; water: interaction: F(1,12) = 4.243; p = 0.0618). (h) Percentage Fos in Oxtr^PBN neurons in hM₃Dq-injected vs mCherry-injected Oxtr ^Cre/+ mice (n = 7/group; unpaired 2-tailed Student’s t-test; t(12) = 19.28; p < 0.0001). Data expressed as mean ± s.e.m. ****p < 0.0001; **p < 0.01; *p < 0.05. See Supplementary Information for statistical analyses