Figure 4. Valence-specific organization of the lateral parabrachial area.

A. Intraperitoneal doses of CCK and LiCl, were titrated to induce ~50% reduction in food intake. Main effect of treatment F[2,12]=38.65 p<0.001. N=5; Paired t-test saline vs. CCK Bonferroni p<0.001; saline vs. LiCl Bonferroni p<0.001; CCK vs. LiCl p=0.8. B. The mouse parabrachial area. PBNdl = parabrachial nucleus, dorsolateral region; PBNel = externolateral parabrachial nucleus; PBNm = medial parabrachial nucleus; scp = superior cerebellar peduncle (brachium conjunctivum). C-D. Compounded image of parabrachial sections showing Fos expression patterns in response to CCK (C) and LiCl (D) across 5 mice. E. Fos+ cells in response to CCK or LiCl. PBNdl specifically responded to CCK, whereas PBNel specifically responded to LiCl. No responses for saline injections. N=5 in each group. Main effect of treatment in PBNdl: F[2,12]=21.9, p<0.0001; Post-hoc two-sample t-tests saline vs. LiCl p=0.249; saline vs. CCK Bonferroni *p<0.001; LiCl vs. CCK Bonferroni **p=0.002. Main effect of treatment in PBNel: F[2,12]=59.5, p<0.0001; Post-hoc two-sample t-tests saline vs. LiCl Bonferroni *p<0.001; saline vs. CCK p=0.9; LiCl vs. CCK Bonferroni **p<0.001. Main effect of treatment in medial PBN (PBNm): F[2,12]=0.8, p=0.471; Post-hoc two-sample t-tests all p>0.85. F. No Fos expression in response to CCK was observed in PBNel (left), in particular no CGRP+ neurons (center) were found to express Fos in response to CCK (right). G. Robust Fos expression in response to LiCl was observed in PBNel (left). The region containing CGRP+ neurons (center) was found to include most of the LiCl-responding Fos-positive neurons (right). H. The anterograde tracer PHA-L was iontophoretically injected into either PBNdl or PBNel; in the same PBNdl case the retrograde tracer FluoroGold was iontophoretically injected into the dorsal striatum. I. Darkfield photomicrographs of coronal sections revealing a substantial anterograde labeling in the SNc after an injection in PBNdl. Note that these projection patterns were highly specific, with the Substantia nigra, pars reticulata (SNr) devoid of parabrachial terminals. Scale bar 100μm. J. Brightfield photomicrograph showing PHA-L anterograde labeling from PBNdl in register with FluoroGold retrograde labeling from the dorsal striatum in the SNc, suggesting the existence of a parabrachio-nigro-striatal pathway. Scale bar 20μm. K. Fibers of passage running dorsal to the SNc, itself unlabeled, after an injection in the PBNel. Scale bar 100μm. L. To assess the cell-type specificity of the SNc targets of parabrachial projections, we transfected the SNc of both DAT-ires-Cre and VGat-ires-Cre mice with the construct AAV5-EF1a-FLEX-TVAmCherry, and two weeks after with the Cre-inducible retrogradely transported pseudotyped rabies virus SAD G-GFP. M-N. In both DAT-ires-Cre (M) and VGat-ires-Cre (N) mice, robust expression of rabies-infected cells were observed in PBNdl and to a lesser extent in PBNm on 7days post rabies infection, but virtually no rabies-infected cells were observed in PBNel, including regions containing CGRP+ cells. Bars=100μM. O. Open-field heatmaps after intraperitoneal injections of saline (upper), CCK (center), or LiCl (lower). P-Q. LiCl injections significantly reduced distance traveled (P) and velocities (Q) compared to both saline and CCK. CCK produced no significant effects on either. ANOVA F[2,12]=15.9, p=0.001 (distance), F[2,12]=16.5, p<0.001 (velocity). Post-hoc t-tests Bonferroni *p<0.05. R. CCK injections significantly enhanced dopamine release in dorsal striatum N=5; Two-way RM-ANOVA injection × sampling time interaction effect F[13,52]=5.8, *p<0.0001. S. LiCl injections significantly inhibited dopamine levels in dorsal striatum N=5; F[13,52]=29.7 *p<0.0001. Data reported as mean±SEM.