Fig 2: Brainstem nuclei identification for LCM. Immunohistological localization of brainstem nuclei confirmed brain tissue to be included for proteomics analyses on standard slides in a parallel section to the section on LCM slides that were subjected to the same immunohistology. (A) The midbrain DR were evaluated at the level of the inferior colliculus. The presence of the DR were confirmed by TPH2(+) neurons, depicted in a representative case. Scale bar represents 200 µm. (B) The region microdissected, 4.5 mm2, is depicted in the dashed box on an overview image on the right. Scale represents 5 mm. (C) The MR were evaluated in available brain tissue from one to four sections per case, from ∼6 to 10 mm above obex. The presence of the MR were confirmed by TPH2(+) neurons located medially, depicted in a representative case. Scale bar represents 200 µm. (D) The region microdissected, 4.5 mm2, is depicted in the dashed box on an overview image on the right. Scale represents 4 mm. (E) The VLM was evaluated in available brain tissue from 1 to 4 sections per case, from ∼6 to 10 mm above obex. The presence of the VLM was confirmed by NK1R(+) cells bilaterally with lateral localization, and TPH2(-) neurons, depicted in a representative case. Scale bar represents 200 µm. (F) The region microdissected, 12 mm2, is depicted in the dashed box on an overview image on the right. Scale bar represents 4 mm.

Fig 3: Optogenetic bidirectional modulation of 5-HT neuron in the DR in the DR-ACC neural circuit.(A) Schematic of optogenetic manipulation. (B) Timeline of experiments. (C) Immunohistological image showing virus expression in the DR (c1) and amplified images in the left box showing the mCherry, TPH2, and the colocalization of the two (‘c2-c4’). (D) Electrophysiological recording model. (E and F) Representative traces from electrophysiological recordings showing photostimulation (E) and photoinhibition of a 5-HT neuron (F). (G) Quantification of allogrooming time in the consolation test of the CHR2 and control animals (n = 8 in each group; CHR2 vs CTR, independent samples t-test and Bayesian independent samples t-test; male: t(14) = 0.340, p=0.739, BF+0 = 0.445 with median posterior δ = 0.104, 95% CI = [−0.673 to 0.930]; female: t(14) = −0.279, p = 0.785, BF+0 = 0.439 with median posterior δ = −0.085, 95% CI = [−0.906 to 0.694]). (H) Quantification of allogrooming time in the consolation test of the eNPHR3.0 and control animals (n = 8 in each group; CHR2 vs eNPHR3.0, independent samples t-test and Bayesian independent samples t-test; male: t(14) = 7.293, p < 0.001, BF+0 = 6000.583; female: t(14) = 6.327, p < 0.001, BF+0 = 1562.921). (I) Quantification of social preference ratio in the three-chamber test of the CHR2 and control animals (n = 7 in CHR2 groups, one male and one female were excluded from analysis due to immobility; n = 8 in CTR groups; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 13) = 3.042, p = 0.105, BF(incl) = 1.184; light: F(1, 13) = 0.531, p = 0.479, BF(incl) = 0.425; group × light: F(1, 13) = 0.246, p = 0.628, BF(incl) = 0.479; female: group: F(1, 13) = 2.088, p = 0.172, BF(incl) = 0.790; light: F(1, 13) = 0.180, p = 0.678, BF(incl) = 0.426; group × light: F(1, 13) = 0.233, p = 0.638, BF(incl) = 0.358). (J) Quantification of social preference ratio in the three-chamber test of the eNPHR3.0 and control animals (n = 8 in each group; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 14) = 4.084, p = 0.063, BF(incl) = 1.236; light: F(1, 14) = 28.361, p < 0.001, BF(incl) = 25.390; group × light: F(1, 14) = 22.959, p < 0.001, BF(incl) = 87.850; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p = 0.981; eNPHR_Off vs eNPHR_On, p<0.001; female: group: F(1, 14) = 11.892, p = 0.004, BF(incl) = 4.965; light: F(1, 14) = 22.678, p < 0.001, BF(incl) = 7.067; group × light: F(1, 14) = 33.771, p < 0.001, BF(incl) = 623.339; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p = 0.879; eNPHR_Off vs eNPHR_On, p < 0.001). (K) Quantification of time spent in the central area in the open-field test of the CHR2 and control animals (n = 8 in each group; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 14) = 0.009, p = 0.465, BF(incl) = 1.184; light: F(1, 14) = 0.808, p = 0.384, BF(incl) = 0.442; group × light: F(1, 14) = 2.266, p = 0.155, BF(incl) = 0.964; female: group: F(1, 14) = 0.240, p = 0.632, BF(incl) = 0.602; light: F(1, 14) = 0.341, p = 0.568, BF(incl) = 0.371; group × light: F(1, 14) = 2.192, p = 0.161, BF(incl) = 0.910). (L) Quantification of time spent in the central area in the open-field test of the eNPHR3.0 and control animals (male_mCherry, n = 7 (one was excluded from analysis due to immobility); male_eNPHR3.0, n = 8; female_mCherry, n = 8; male_eNPHR3.0, n = 8; two-way repeated measures ANOVA along with two-way Bayesian repeated measures ANOVA; male: group: F(1, 13) = 6.326, p = 0.026, BF(incl)=1.935; light: F(1, 13) = 1.176, p = 0.298, BF(incl)=0.605; group × light: F(1, 13) = 0.039, p = 0.846, BF(incl) = 0.578; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p = 0.928; eNPHR_Off vs eNPHR_On, p = 0.785; female: group: F(1, 14) = 0.794, p = 0.388, BF(incl) = 0.660; light: F(1, 14) = 0.632, p = 0.440, BF(incl) = 0.402; group × light: F(1, 14) = 3.390, p = 0.087, BF(incl) = 1.352; post-hoc comparisons (Tukey): mCherry_Off vs mCherry_On, p = 0.928; eNPHR_Off vs eNPHR_On, p = 0.785). Scale bars, 100 μm. Error bars are ± SEM. **p < 0.01. For raw data in this figure, please refer to Figure 1—source data 1. ACC: anterior cingulate cortex; Aq: aqueduct; ANOVA: analysis of variance; CTR: control; DR: dorsal raphe nucleus; TPH2: tryptophan hydroxylase 2; 5-HT: serotonin.Figure 1—source data 1.Source data indicating behavioral performances of bidirectional optogenetic modulation of 5-HT neurons in the DR in the DR-ACC neural circuit.

Fig 4: Neuropathology findings in the medulla (examined at obex 6 mm) A. p62 at low power demonstrates the distribution of neuronal inclusions in specific regions of the ventrolateral medulla (VLM, arrow) and in the dorsomedial medulla (arrowhead) in the vicinity of the XIIth cranial nerve nucleus, mainly in axons and process here. In addition, numerous inclusions were present in the external cuneate nucleus (ECu) and solitary tract (ST) nuclei. There were not identified in the median raphe (MR). B. Tyrosine hydroxylase (TH) labelling confirmed preservation and normal distribution of neurones in the VLM, dorsomedial medulla and around the solitary tract. C. Tryptophan hydroxylase (TPH) confirms neurones of normal morphology and distribution mainly in the MR and VLM. D-J. All represent high magnification images of the VLM region. D. H&E revealed numerous basophilic, irregular cytoplasmic inclusions in neuronal perikarya and dendrites. E. These irregular, globular inclusions, distending process were PAS positive in keeping with polyglucosan bodies. A high-resolution version of this slide for use with the Virtual Microscope is available as eSlide: VM06202 F. p62 labelling at lower magnification showed numerous positive structures in the VLM neurones and G. at higher magnification with structures of similar morphology to the PAS stain. H. Inclusions of similar morphology were also evident in many TH labelled neurones but not in TPH2 neurones or in pre-Botzinger neurones, characterised by I. NK1R and J. SST peripheral labelling. Bar approximately equivalent in A, B and C to 2 mm in F to 25 microns and D-J to 50 microns.

Fig 5: Fiber photometry recording DR 5-HT neural dynamics during the consolation test.(A) Schematic diagrams depicting the virus injection and recording sites. (B) Histology showing the expression of GCaMP6 (left) and GFP control (right) in the DR. (C) Experimental timeline for photometry experiments. (D) Representative fluorescence changes of GCaMP6 (red line) and GFP (blue line) during photometry recordings. (E1–I1) Representative peri-event plot of GCaMP6 fluorescence signals aligned to onsets of various behaviors (for all peri-event plots, the red line denotes the mean signals of four to six bouts of behaviors, whereas the red shaded region denotes the SEM). (E2) Quantification of change in GCaMP6 fluorescence signals before and after allogrooming (n = 6 in each group; male: t(5) = −5.967, p = 0.002, BF+0 = 24.488 with median posterior δ = −1.904, 95% CI = [−3.749,–0.424]; female: t(5) = −7.420, p<0.01, BF+0 = 52.689 with median posterior δ = −2.397, 95% CI = [−4.610,–0.625]). (F2) Quantification of change in GCaMP6 fluorescence signals before and after approaching (n = 6 in each group; male: t(5) = −19.871, p<0.001, BF+0 = 2233.691; female: t(5) = −8.448, p < 0.001, BF+0 = 84.470 with median posterior δ = −2.747, 95% CI = [−5.225,–0.767]). (G2) Quantification of change in GCaMP6 fluorescence signals before and after sniffing (n = 6 in each group; male: t(5) = −3.689, p = 0.011, BF+0 = 6.449 with median posterior δ = −1.221, 95% CI = [−2.576,–0.138]; female: t(5) = −3.689, p = 0.014, BF+0 = 5.312 with median posterior δ = −1.137, 95% CI = [−2.434,–0.101]). (H2) Quantification of change in GCaMP6 fluorescence signals before and after selfgrooming (n = 6 in each group; male: t(5) = −1.032, p = 0.350, BF+0 = 0.559 with median posterior δ = −0.300, 95% CI = [−1.076, 0.383]; female: t(5) = −0.707, p = 0.511, BF+0 = 0.456 with median posterior δ = −0.205, 95% CI = [−0.904, 0.466]). (I2) Quantification of change in GCaMP6 fluorescence signals before and after running (n = 6 in each group; male: t(5) = −1.032, p = 0.350, BF+0 = 0.559 with median posterior δ = −0.300, 95% CI = [−1.076, 0.383]; female: t(5) = −0.707, p = 0.511, BF+0 = 0.456 with median posterior δ = −0.205, 95% CI = [−0.904, 0.466]). (E3–I3) Representative peri-event plot of GFP signals aligned to onsets of various behavioral events (for all peri-event plots, the blue line denotes the mean signals of four to six bouts of behaviors, whereas the blue shaded region denotes the SE). (E4) Quantification of change in GFP fluorescence signals before and after allogrooming (n = 5 in each group; male: t(4) = −0.145, p = 0.885, BF+0 = 0.401 with median posterior δ = −0.047, 95% CI = [−0.3787, 0.675]; female: t(4) = −1.085, p = 0.339, BF+0 = 0.610 with median posterior δ = −0.32, 95% CI = [−1.198,–0.406]). (F4) Quantification of change in GFP fluorescence signals before and after approaching (n = 5 in each group; male: t(4) = −1.211, p = 0.293, BF+0 = 0.667 with median posterior δ = −0.371, 95% CI = [−1.260, 0.377]; female: t(4) = −0.723, p = 0.510, BF+0 = 0.488 with median posterior δ = −0.220, 95% CI = [−1.026, 0.499]). (G4) Quantification of change in GFP fluorescence signals before and after sniffing (n = 5 in each group; male: t(4) = 1.001, p = 0.373, BF+0 = 0.577 with median posterior δ = 0.306, 95% CI = [−0.427, 1.156]; female: t(4) = −0.687, p = 0.530, BF+0 = 0.479 with median posterior δ = −0.209, 95% CI = [−1.009, 0.510]). (H4) Quantification of change in GFP fluorescence signals before and after selfgrooming (n = 5 in each group; male: t(4) = 0.237, p = 0.825, BF+0 = 0.407 with median posterior δ = 0.072, 95% CI = [−0.647, 0.819]; female: t(4) = −0.350, p = 0.744, BF+0 = 0.418 with median posterior δ = −0.106, 95% CI = [−0.865, 0.610]). (I4) Quantification of change in GFP fluorescence signals before and after running (n = 5 in each group; paired t-test and Bayesian paired samples t-test, two-tailed; male: t(4) = −0.202, p = 0.850, BF+0 = 0.404 with median posterior δ = −0.061, 95% CI = [−0.806, 0.659]; female: t(4) = −1.813, p = 0.144, BF+0 = 52.689 with median posterior δ = 0.560, 95% CI = [−1.580,–0.251]). Error bars are ± SEM. Scale bars, 100 μm. *p<0.05, **p<0.01. Paired samples t-test along with Bayesian paired samples t-test. For raw data in this figure, please refer to Figure 4—source data 1. ACC: anterior cingulate cortex; GFP: green fluorescent protein; TPH2: tryptophan hydroxylase 2; Aq: aqueduct; Allo: allogrooming; Sni: sniffing; App: approaching; Gro: selfgrooming; Run: running; 5-HT: serotonin.Figure 4—source data 1.Source data indicating GCaMP6 and GFP fluorescent signals align to some behaviors.