Supplementary Figure 9: Characterization of upstream projections to Oxtr^PBN neurons

(a) Injection of AAV-DIO-hM₃Dq:mCherry in Oxt^PVH neurons of Oxt ^Cre/+ mice and bilateral cannula into PBN. (b) Acute Oxt^PVH activation by CNO ip decreases 2-h water consumption after 24-h dehydration during the light cycle (n = 6/group; unpaired 2-tailed Student’s t-test; NaCl: interaction t(10) = 0.3017; p = 0.7690; water: interaction t(10) = 2.484; p = 0.0323), but (c) demonstrates no significant change in 2-h NaCl or water consumption at baseline (n = 6/group; 2-way RM ANOVA; NaCl: interaction F(1,10) = 0.9098; p = 0.3627; water: interaction F(1,10) = 2.118; p = 0.1763). (d) Representative histological image demonstrating cannula placement in PBN (n = 6/group). (e) Injection of AAV-DIO-hM₃Dq:mCherry in Oxt^PVH neurons and AAV-DIO-YFP in Oxtr^PBN neurons of Oxt ^Cre/+ ::Oxtr ^Cre/+ mice and bilateral cannula into PBN. (f) Following intra-PBN CNO, we saw some Fos expression in Oxt^PVH neurons, (g) in Oxtr^PBN neurons, and (h) some scattered Fos in the NTS (n = 3). (i) Injection of AAV-hM₃Dq:mCherry in NTS and AAV-DIO-YFP in Oxtr^PBN neurons of Oxtr ^Cre/+ mice. (j) Following CNO ip, we observed robust Fos expression in hM₃Dq-expressing NTS neurons (n = 6) and (k) minimal Fos expression in control DsRed-expressing NTS neurons (n = 5). (l) Injection of AAV-DIO-hM₃Dq:mCherry in CCK^NTS neurons of Cck ^Cre/+ mice. m,n, Following CNO ip, we observed Fos in the (m) NTS and (n) PBN of hM₃Dq-injected mice (n = 5 hM₃Dq, 4 mCherry). Data are expressed as mean ± s.e.m. dl, dorsolateral PBN; el, external lateral PBN; scp, superior cerebellar peduncle. *p < 0.05. Scale bars, 100 μm. See Supplementary Information for statistical analyses