Supplementary Figure 7: Downstream and upstream projections of Oxtr^PBN neurons

(a) Injection of AAV-DIO-synaptophysin:mCherry in Oxtr^PBN neurons demonstrates less prominent downstream projections to (b) the nucleus of the solitary tract (NTS), lateral posterior thalamus, periventricular hypothalamus, tuberal nucleus, paraventricular nucleus of the hypothalamus (PVH) and supraoptic nucleus (SON); (n = 2) scale bar, 200 µm. (c) Percentage Fos in caudal, mid and rostral Oxtr^PBN neurons in Oxt ^Cre/+ ::Oxtr ^Cre/+ mice injected with hM₃Dq or mCherry in PVH (n = 3/group; unpaired 2-tailed Student’s t-test; caudal: t(4) = 4.385; p = 0.0118; mid: t(4) = 1.807; p = 0.1451; rostral: t(4) = 2.278; p = 0.0850). (d) Injection of AAV1-DIO-synaptophysin:mCherry in PVH of Oxtr ^Cre/+ mice demonstrates no visible projections in PBN (n = 3); 3V, third ventricle; thal, thalamic nuclei; scp, superior cerebellar peduncle; dl, dorsolateral PBN; el, external lateral PBN; scale bar, 100 µm. (e) Injection of AAV-DIO-hM₃Dq into the PVH and AAV-DIO-YFP into the PBN of Oxtr ^Cre/+ mice. (f) Following CNO, Fos is robustly expressed in the PVH and thalamus, but (g) there was minimal expression in the PBN (n = 3). (h) Representative live image of the lateral PBN prior to electrophysiological recordings demonstrating Oxt^PVH fibers and Oxtr^PBN neurons; scale bar, 200 μm. (i) Acute Oxt^PVH stimulation decreases water intake following 1 M mannitol ip (n = 7 hM₃Dq, 8 mCherry; 2-way RM ANOVA; NaCl: interaction F(4,52) = 0.6187; p = 0.6512; water: interaction F(4,52) = 4.120; p = 0.0057), but (j) does not significantly alter fluid intake following 48-h salt appetite (n = 7 hM₃Dq, 8 mCherry; 2-way RM ANOVA; NaCl: interaction F(8,104) = 1.056; p = 0.3999; water: interaction F(8,104) = 0.6962; p = 0.6941) or (k) 30% PEG sc (n = 7 hM₃Dq, 8 mCherry; 2-way RM ANOVA; NaCl: interaction F(4,52) = 0.3265; p = 0.8589; water: interaction F(4,52) = 0.5231; p = 0.7191), or (l) food intake at baseline or after a 24-h fast (n = 7 hM₃Dq, 8 mCherry; 2-way RM ANOVA; baseline food: interaction F(8,104) = 0.3734; p = 0.9325; post 24-h fast: interaction F(8,104) = 0.2928; p = 0.9670). (m) Percentage Fos in Oxt^PVH neurons in hM₃Dq-injected vs mCherry-injected Oxt ^Cre/+ mice (n = 7 hM₃Dq, 8 mCherry; unpaired 2-tailed Student’s t-test; t(13) = 12.50; p < 0.0001). Data are expressed as mean ± s.e.m. ****p < 0.0001; *p < 0.05. See Supplementary Information for statistical analyses