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. 2023 Feb 28;12:e80517. doi: 10.7554/eLife.80517

Figure 2. Pair bonding leads to persistent and consistent changes in NAc transcription.

(A) Opposite- and same-sex pairs were paired for 2 weeks prior to a baseline partner preference test. Pairs then remain paired for either 48 hours (short-term; ~2 weeks total pairing) or 4 weeks (long-term; ~6 weeks total pairing) prior to collecting fresh nucleus accumbens tissue (dissection sites in red boxes) for RNA sequencing. (B) Baseline partner preference scores of males included in RNA sequencing for the opposite- and same-sex groups (one-tailed t-test relative to 50%: opposite-sex T14=11.76, p=1.21 X 10–8; same-sex T10=7.78, p=1.50 X 10–5). Black dotted line indicates a 50% partner preference score and the grey dotted line indicates 66%. There were no differences in partner preference score between opposite- and same-sex paired animals used for RNAseq (two-tailed t-test: T21.016 = 1.374, p=0.184). (C) Transcriptional analysis workflow. (D) Gene list from both timepoints ordered from the smallest to largest log2FoldChange after short-term pairing with color indicating up- or down-regulation in opposite- vs same-sex pairs. Expression patterns are strongly correlated across timepoints (Rho = 0.38, p=2.2 X 10–16). (E) Schematic of RRHO analysis. The heatmap is arranged into quadrants of genes upregulated in both lists (up-up: quadrant UU), downregulated in both lists (down-down: quadrant DD), or genes that have opposite regulation (up in list 1-down in list 2: quadrant UD; down in list 1-up in list 2: quadrant DU). Genes that are found in both lists at a similar ranked position result in higher p-values and are represented by a yellow color. (F) RRHO comparing short-term and long-term pair bonding (from 2D) indicates a stable pair bond gene signature over time as evidenced by concordant up- or downregulated genes at the two timepoints. (G) The short-term and long-term time points were pooled for opposite- and same-sex pairs to define the combined pair bond gene signature. (H) We compared GO analysis Mus musculus ontology terms between the combined pair bond DEGs and the RRHO quadrants (from 2 F) with strong correspondence between the two analyses.

Figure 2.

Figure 2—figure supplement 1. Cohort shuffled controls for remain paired cohorts.

Figure 2—figure supplement 1.

(A) Spearman’s Ranked Correlation of gene expression (log2FoldChange) at the short-term pair bond and the long-term pair bond. Black line is the regression line with shading indicating a 95% confidence interval. (B) To ensure that our correlations are not driven by genes with consistent but low differential expression values, we only retained the first and fourth quartile of genes in the short-term pair bond and found a stronger Spearman’s Ranked Correlation between pair bond time points. (C) To test if the correlation in gene expression between remain paired timepoints was likely to be due to chance, we shuffled the Cohort identity for each animal and performed differential expression analysis. Heatmaps show every gene from short-term and long-term pairing ordered from the smallest to largest log2FoldChange after short-term pairing in the observed comparison (top) and a representative Cohort shuffle (bottom). Scale represents the scaled log2FoldChange. (D) Histogram of Rho values for 1000 iterations of (C, shuffled data). Only correlations that did not have a –log10(adjusted pvalue) of infinite were retained (n=636). Vertical dashed line indicates the observed Rho value with the percent of shuffled iteration in which Rho was greater than the observed value to the right (0.0%). (E) Unscaled RRHO plot of the representative Cohort shuffle used in (C) indicates elimination of the concordant signal seen with the observed data.
Figure 2—figure supplement 2. Ingenuity Pathway Analysis (IPA) corresponding to GO term analyses.

Figure 2—figure supplement 2.

Figure is also supplemental data for Figure 3. IPA analyses indicating enriched pathways and upstream regulators for applicable comparisons. Representative pathways and regulators were chosen based on significance, activation pattern, and relationship to GO terms. Full lists can be found in Supplementary file 3. Yellow to blue scales indicate the z-scores of enrichment where yellow is activated and blue is repressed. White to blue scale represents the –log10(p-adjusted) where any non-significant terms are white. (A) IPA analysis for the remain-paired cohorts in Figure 2. The strong similarity of the activation/repression of pathways and regulators from short-term to long-term pairing are well represented by the combined pair bond transcriptional signature. Pathways or regulators of interest are bolded. (B) IPA analysis of the combined pair bond, short-term separation, and long-term separation in Figure 3. Grey boxes indicate that the term is not found in the results for that time point. Terms and regulators of interest are bolded while asterisks denote terms or regulators found in A. (C) IPA analysis of the eroded ST to LT genes for the UU and DD quadrants of Figure 3. Results are denoted as adjusted p-values instead of z-scores because there is no log2FoldChange value associated with the RRHO heatmaps. Pathways or regulators of interest are bolded. (D) IPA analysis of the neuronal enriched clusters of interest from Figure 4. Cluster #1 contained few genes and no significant pathways or regulators. Cluster #2 did not have any significant regulators.