Figure 4. VMPO^LPS neurons regulate body temperature and appetite through direct and indirect synaptic connections.

(a,b) Anterograde tracing from LPS-TRAP neurons in the VMPO(c). (d) Expression of GFP-labeled VMPO^LPS axonal fibers, n=6 mice. (e) Retrograde tracing from warm-sensitive neurons in AVPe/MnPO (warm-TRAP). (f) Percent overlap of warm-TRAP neurons with adcyap1 and sncg, n=3. (g) Experimental timeline for retrograde tracing. (h) Starter cells in the AVPe/MnPO, green=g-deleted rabies, magenta=AAV-TVA-mcherry. (i) Overlap of rabies-infected input cells (green) and LPS-induced Fos (magenta). (i’) magnification of boxed region in (i). (j) Optogenetic activation of projections from VMPO^LPS neurons to AVPe/MnPO. (k) Fiber placement. (l) Body temperature, stimulation (green) or no stimulation (grey) of VMPO^LPS-AVPe/MnPO projections, light lines = individual mice, dark lines = mean, n=5, two-way ANOVA, * = p<0.05, ** = p<0.01, *** =p<0.001 (m) Chow consumed, n=5, ns=not significant, two-way Mann-Whitney test (n) Identification of downstream targets. (o) Mean Fos expression following CNO, n=3/group, two-way ANOVA, p=<0.0001 (p) Fos expression in LPS-TRAP neurons, control (top), VMPO activated (bottom). (q) Mean marker expression in DMH LPS-TRAP neurons, n=3 mice. (r) Fos expression following CNO. (s) Mean marker expression in LPS-TRAP neurons in the Arc, n=3 mice. (t) Retrograde tracing from appetite-controlling neurons in Arc. (u) Overlap of agrp and pomc in hunger-TRAP neurons, n=3/group. (v) Timeline of retrograde tracing. (w) Starter cells in the Arc. (x) Overlap of rabies-infected input neurons (green) and LPS-induced Fos (magenta). (x’) high magnification of (x). (y) Activation of VMPO^LPS projections to Arc. (z) Fiber placement. (aa) Mean chow consumed following stimulation (on) or no stimulation (off) of VMPO^LPS-Arc projections. dot-lines=individuals, n=6, p=0.0065, two-way Mann-Whitney test. (bb) Body temperature after VMPO^LPS-Arc stimulation, (n=6). All scale bars = 200μm. All error bars = SEM.