Early life adversity promotes resilience to opioid addiction-related phenotypes in male rats and sex-specific transcriptional changes

Abstract

Experiencing some early life adversity can have an "inoculating" effect that promotes resilience in adulthood. However, the mechanisms underlying stress inoculation are unknown, and animal models are lacking. Here we used the limited bedding and nesting (LBN) model of adversity to evaluate stress inoculation of addiction-related phenotypes. In LBN, pups from postnatal days 2 to 9 and their dams were exposed to a low-resource environment. In adulthood, they were tested for addiction-like phenotypes and compared to rats raised in standard housing conditions. High levels of impulsivity are associated with substance abuse, but in males, LBN reduced impulsive choice compared to controls. LBN males also self-administered less morphine and had a lower breakpoint on a progressive ratio reinforcement schedule than controls. These effects of LBN on addiction-related behaviors were not found in females. Because the nucleus accumbens (NAc) mediates these behaviors, we tested whether LBN altered NAc physiology in drug-naïve and morphine-exposed rats. LBN reduced the frequency of spontaneous excitatory postsynaptic currents in males, but a similar effect was not observed in females. Only in males did LBN prevent a morphine-induced increase in the AMPA/NMDA ratio. RNA sequencing was performed to delineate the molecular signature in the NAc associated with LBN-derived phenotypes. LBN produced sex-specific changes in transcription, including in genes related to glutamate transmission. Collectively, these studies reveal that LBN causes a male-specific stress inoculation effect against addiction-related phenotypes. Identifying factors that promote resilience to addiction may reveal novel treatment options for patients.

Keywords: nucleus accumbens; sex difference; stress; substance use disorder; transcription.

Fig. 1.

LBN reduces impulsive choice in male rats. (A) LBN exposure reduced impulsive choice in male rats, as evidenced by the increase in preference of the larger/delayed option at the 15 s, 30 s, and 45 s delays. *P < 0.05. (B) Discounting did not differ significantly between LBN and control females. LBN exposure did not affect male (C) or female (D) latency to respond.

Fig. 2.

LBN reduces self-administration of a low dose of morphine in male rats. LBN exposure reduced self-administration on FR1 (A) and PR (B) of a low dose of morphine (0.25 mg/kg/infusion) in male rats. *P < 0.05. LBN did not significantly alter FR1 (C) or PR (D) for this low dose in females. FR1 and PR for the high dose of morphine (0.75 mg/kg/infusion) was not affected by LBN in male rats (E and F) or female rats (G and H).

Fig. 3.

LBN alters both baseline and morphine-induced glutamate transmission in male rats. (A) LBN exposure reduced sEPSC frequency in male rats regardless of morphine history. *P < 0.05. (B) sEPSC amplitude was lower following morphine exposure, and this effect was driven primarily by the LBN group. *P < 0.05, drug-naïve LBN males vs. morphine LBN males. (C) While no differences were seen in drug-naïve rats, control males exposed to morphine exhibited an increase in the AMPA/NMDA ratio that was not present in the LBN males. ***P < 0.001. (D) Example sEPSC traces from male rats. LBN exposure did not significantly affect sESPC frequency (E), sEPSC amplitude (F), or AMPA/NMDA ratio (G) in females. (H) Example sEPSC traces from female rats.

Fig. 4.

LBN produces sex-specific changes in gene expression in the NAc. (A) Threshold-free comparison of DEGs by rank-rank hypergeometric overlap. Hotspots represent the overlap between the impact of LBN on gene expression in females and males. Hotter colors indicate more overlap. The upper right quadrant includes co–up-regulated genes, while the lower left quadrant represents co–down-regulated genes. The top left and bottom right quadrants represent genes that are changed by LBN in opposite directions in males compared to females. (B) LBN induces 170 DEGs in males and 335 DEGs in females, with 11 overlapping DEGs between the sexes. (C) Heatmap sorted by fold change of DEGs in males (Top) compared to the expression of those genes in females (Bottom). LBN reduced the expression of 55 genes and increased the expression of 115 genes in males. (D) Heatmap sorted by fold change of DEGs in females (Top) compared to the expression of those genes in males (Bottom). LBN increased the expression of 162 genes and decreased the expression of 173 genes. (E) KEGG and Panther enrichment terms identified in males following LBN. The number of DEGs within each term is in parentheses to the right of the term. (F) KEGG and Panther enrichment terms identified in females following LBN. The number of DEGs within each term is in parentheses to the right of the term. (G) KEGG enrichment terms identified in both males and females following LBN. The number of DEGs within each term is in parentheses to the right of the term, with males in blue and females in red.