Extended Data Fig. 5. Extended results of mRNA high throughput sequencing (QuantSeq), and oligodendrocyte analysis by immunofluorescence implicating oligodendrocyte differences in 4EP+ mice.

a, Principal component analysis of all samples analyzed by QuantSeq, with tight clustering by brain region. Each brain region is colored according to the legend. b, Differential gene analysis summary showing number of significantly different genes in 6 tested brain regions. Top pie chart refers to total number of differential genes by one-way group ANOVA with all the 4EP- and 4EP+, at baseline and post-EPM. The bottom pie chart was generated according to the specific significant (p<0.05) contrasts between 4EP+ and 4EP- conditions of post-EPM conditions as calculated with the post-hoc Tukey HSD test. c-d, Gene ontology terms enriched in genes that are upregulated (c) or downregulated (d) in the PVT in 4EP+ relative to 4EP- mice in the baseline, home cage condition. X-axis represents -log10(P) of enrichment analysis. e, p-value histogram of PVT sequencing at baseline, graphed as p-values binned by 0.05 along the x-axis and number of genes along the y-axis. f, Adjusted p-values for 4EP+/4EP- comparison of expression of oligodendrocyte specific genes in all 6 brain regions analyzed by QuantSeq, corresponding to data presented in Main Figure 3c for the PVT. p<0.05 in bold. g, PVT seed analysis correlating 2DG uptake in the PVT to the rest of the brain. Images show 3D rendered, average whole brain correlation data (n=11). The center of the PVT is indicated at the cross section of the red lines, and the number of negatively correlated voxels is shown below in each. Significance is illustrated according to the legend. A minimum threshold of 200 contiguous voxels with p<0.05 was used. h, Schematic of immature OPC and mature, myelinating oligodendrocyte markers used for immunostaining, flow cytometry, or western blots in this study. i, Raw counts of mature oligodendrocytes, measured by CC1+ and OLIG2+ staining in 4EP+ and 4EP- mice in the PVT, used to calculate maturity quotient in Figure 3f (n=8). p=0.04. j, Raw counts of immature oligodendrocytes, measured by NG2+ and OLIG2+ staining in 4EP+ and 4EP- mice in the PVT, used to calculate maturity quotient in Figure 3f (n=8 each group). p=0.055. k, Representative images (corresponding to main Fig. 3e-f) of the PVT in brain sections from 4EP- and 4EP+ mice, stained for OLIG2, using total cumulative counts from 3–5 images per replicate (n=8). Scale bar 100µm. l, Raw counts of OLIG2+ cells in the PVT of 4EP+/− mice. Each data point represents an individual mouse, with total cumulative counts from the PVT (n=8) p=0.8. m, Raw counts of NeuN+ staining in the PVT (4EP- n=10, 4EP+ n=9), using average counts from 3–5 images per replicate. n, Representative images of pan-neuronal NeuN staining in the PVT, with 3–5 images per replicate (4EP- n=10, 4EP+ n=9). Scale bar 100µm. o, NG2+ staining in extended regions of the brain (n=3 each group) p=0.009. Abbreviations: PVT, the paraventricular nucleus of the thalamus; BNST, bed nucleus of the stria terminalis; BLA, basolateral amygdala; mPFC, medial prefrontal cortex; HY, hypothalamus; vHPC, ventral hippocampus; OLIG2, oligodendrocyte transcription factor 2; NG2, neural/glial antigen 2; CC1, antibody (anti-adenomatous polyposis coli (APC) clone) that binds mature oligodendrocyte marker; MOG, myelin oligodendrocyte glycoprotein; MBP, myelin basic protein; ACA, anterior commissure; CC, corpus callosum; LHB, lateral habenula; LS, lateral septum; MHB, medial habenula; MS, medial septum; ME, median eminence; PFC, prefrontal cortex; PVN, paraventricular nucleus of the hypothalamus; SM, stria medullaris of thalamus. Two cohorts of mice from multiple litters were used for experiments in panels g-o in this figure. Data represent mean ± SEM. Analysis performed using two-tailed Welch’s t-tests (i-m) or one-way ANOVA with Dunnett multiple comparison between 4EP+/− pairs (o). * p ≤ 0.05.