Acute social isolation alters neurogenomic state in songbird forebrain

Abstract

Prolonged social isolation has negative effects on brain and behavior in humans and other social organisms, but neural mechanisms leading to these effects are not understood. Here we tested the hypothesis that even brief periods of social isolation can alter gene expression and DNA methylation in higher cognitive centers of the brain, focusing on the auditory/associative forebrain of the highly social zebra finch. Using RNA sequencing, we first identified genes that individually increase or decrease expression after isolation and observed general repression of gene sets annotated for neurotrophin pathways and axonal guidance functions. We then pursued 4 genes of large effect size: EGR1 and BDNF (decreased by isolation) and FKBP5 and UTS2B (increased). By in situ hybridization, each gene responded in different cell subsets, arguing against a single cellular mechanism. To test whether effects were specific to the social component of the isolation experience, we compared gene expression in birds isolated either alone or with a single familiar partner. Partner inclusion ameliorated the effect of solo isolation on EGR1 and BDNF, but not on FKBP5 and UTS2B nor on circulating corticosterone. By bisulfite sequencing analysis of auditory forebrain DNA, isolation caused changes in methylation of a subset of differentially expressed genes, including BDNF. Thus, social isolation has rapid consequences on gene activity in a higher integrative center of the brain, triggering epigenetic mechanisms that may influence processing of ongoing experience.

Keywords: DNA methylation; RNA-seq; forebrain; isolation; zebra finch.

Fig. 1.

Top genes from differential gene expression analysis (RNA-seq) in female auditory lobule after overnight isolation. Normalized counts for 33 genes with most significant changes in expression between aviary and solo conditions (FDR < 0.001) and the largest fold-changes in expression [abs(log-twofold change) > 0.4]. Data are plotted for 23 individual female zebra finches (11 aviary and 12 solo, red and blue symbols, respectively). Genes are sorted left to right by fold change.

Fig. 2.

Competitive gene set enrichment analysis. Gene sets representing 186 KEGG pathways were tested for sensitivity to solo isolation, using cameraPR and the Wald test statistic from DESeq2 to rank the gene sets by relative expression. Two gene sets were down-regulated significantly relative to other gene sets, and the barcode plots show the ranking statistics for the individual genes (vertical bars) in each set. Ranking statistics are displayed with genes down-regulated in the solo condition toward the left and up-regulated toward the right. The wavy line at the top shows enrichment of vertical bars in each part of the plot. Pink and blue shading: P < 0.05 threshold for differential up- or down-regulation of individual genes.

Fig. 3.

Gene expression changes persist after 2 d in isolation. Changes in expression of EGR1, BDNF, FKBP5, and UTS2B were independently replicated with qRT-PCR for the solo (“1 d” solo isolation, n = 9 females) versus aviary (n = 10 females) comparison, with the addition of a group housed alone for an additional day (“2 d” solo isolation, n = 9 females), and modeled with MCMC.qpcr. Fixed effects of solo isolation (relative to aviary) are plotted for 1 d (black) and 2 d (blue); points represent posterior means with 95% credible intervals. The relative stability of HPRT, PGK1, and YWHAZ is reflected in the model (which is naive to the prediction that these are “control” genes).

Fig. 4.

Anatomical expression patterns of mRNAs within the auditory lobule. Shown are images from in situ hybridizations with riboprobes (4 genes labeled on the Left), from adult females (2015 experiment). The AVIARY and SOLO columns show sections in the sagittal plane (0.2 to 0.5 mm from midline). (Scale bar: 1 mm.) Column 3 shows high-magnification images (40×) illustrating the cellular labeling pattern in NCM for condition of highest expression (BDNF and EGR1: AVIARY; FKBP5 and UTS2B: SOLO). (Scale bar: 50 µm.)

Fig. 5.

Including a partner mitigates some, but not all, effects of solo isolation. Female (red) and male (blue) zebra finches were housed overnight in sound attenuation chambers either alone (solo) or with a partner of the same sex (duo). qRT-PCR measured expression of EGR1, BDNF, FKBP5, and UTS2B, and changes were modeled with MCMC.qpcr. Data for HPRT and PGK1 were also incorporated in the model to improve estimation of random effects (Materials and Methods). Points represent inferred transcript abundances, and whiskers represent 95% credible intervals.

Fig. 6.

Solo isolation leads to altered methylation of the BDNF gene. Schematic gene model shows the 3 currently annotated transcripts (Top). Each circle is the value in 1 animal for the fraction of DNA methylated at that site from RRBS of auditory lobule DNA (solo isolation for 2 d, blue; group aviary, red). Two sites are marked with an asterisk (*): chr5:1328465 (methylation difference +26%, q = 3.03E-05) is localized to a region (“a”) immediately upstream and adjacent to the start site for XM_012573738.1; chr5:1308195 (methylation difference −41%, q = 1.70E-72) is within the BDNF protein coding exon (“b”). Note that both changes are consistent with decreased transcription of the BDNF gene, based on precedents in mammals (54, 55): increased methylation at BDNF exonic start sites (as in A) has been linked to transcriptional suppression (56), whereas increased methylation in coding exons (A) is associated with more active transcription and/or regulation of alternative splicing (57).