The effects of the recurrent social isolation stress on fear extinction and dopamine D2 receptors in the amygdala and the hippocampus

Abstract

Background: The present study assessed the influence of recurrent social isolation stress on the aversive memory extinction and dopamine D₂ receptors (D₂R) expression in the amygdala and the hippocampus subnuclei. We also analyzed the expression of epigenetic factors potentially associated with fear extinction: miRNA-128 and miRNA-142 in the amygdala.

Methods: Male adult fear-conditioned rats had three episodes of 48 h social isolation stress before each fear extinction session in weeks intervals. Ninety minutes after the last extinction session, the D₂R expression in the nuclei of the amygdala and the hippocampus (immunocytochemical technique), and mRNA levels for D₂R in the amygdala were assessed (PCR). Moreover, we evaluated the levels of miRNA-128 and miRNA-142 in the amygdala.

Results: It was found that recurrent social isolation stress decreased the fear extinction rate. The extinguished isolated rats were characterized by higher expression of D₂R in the CA1 area of the hippocampus compared to the extinguished and the control rats. In turn, the isolated group presented higher D₂R immunoreactivity in the CA1 area compared to the extinguished, the control, and the extinguished isolated animals. Moreover, the extinguished animals had higher expression of D₂R in the central amygdala than the control and the extinguished isolated rats. These changes were accompanied by the increase in miRNA-128 level in the amygdala in the extinguished isolated rats compared to the control, the extinguished, and the isolated rats. Moreover, the extinguished rats had lower expression of miRNA-128 compared to the control and the isolated animals.

Conclusions: Our results suggest that social isolation stress impairs aversive memory extinction and coexists with changes in the D₂R expression in the amygdala and hippocampus and increased expression of miRNA-128 in the amygdala.

Keywords: Amygdala; Dopamine D2 receptors; Fear extinction; Hippocampus; Social isolation.

Fig. 1

The experimental scheme. The influence of intermittent social isolation on fear extinction. CFT conditioned fear test; isolation—48 h of social isolation; extinction—fear extinction

Fig. 2

The effect of social isolation stress on fear extinction in rats (freezing time during 10 min exposition to aversive context). Extinction sessions were performed in 1 week intervals. The first one was performed 1 week after the test session of fear conditioning. Control—the control rats (n = 10), extinguished—the extinguished rats (n = 10), isolated—the isolated rats (n = 10); isolated extinguished—the isolated extinguished rats (n = 10), and CFT—conditioned fear test. **, *- differs from the control rats, p < 0.01; p < 0.05; ^&&—differs from the isolated rats, p < 0.01; ^##,#—differs from CFT (conditioned fear test) session the extinguished group, p < 0.01, p < 0.05, ^$$—differs from the extinguished rats, p < 0.01. Repeated-measures ANOVA, followed by Newman–Keuls post hoc. The data are shown as the means + SEM

Fig. 3

Expression of dopamine receptors (D₂R) in the hippocampus and the amygdala in rats after the third extinction session was assessed by immunocytochemical staining. Left panel: diagram presenting density of D₂R immunoreactive neurons (number of cells/mm2). Right panel: photomicrographs showing representative expression of D2R in the dentate gyrus (DG), CA1 and CA3 area of the hippocampus, basal nucleus (BA), lateral nucleus (LA), and central nucleus (CeA) of the amygdala with an objective lens at 20 × magnification (total magnification of 200×). Scale bar indicates 120 µm. Control (n = 10), extinguished (n = 10), isolated (n = 10); isolated extinguished (n = 10). Statistics: two-way ANOVA followed by Newman–Keuls post hoc. Data are presented as the mean number of cells per 1 mm2 ± SEM

Fig. 4

A The D₂R–dopamine D₂ receptor mRNA levels. Control (n = 7); extinguished (n = 8); isolated (n = 7); isolated extinguished (n = 8); and miRNA levels: B the miRNA-128 levels, control (n = 7); extinguished (n = 8); isolated (n = 7); isolated extinguished (n = 8); C the miRNA-142 levels, control (n = 7); extinguished (n = 8); isolated (n = 7); isolated extinguished (n = 8) in the amygdala. The mRNA and miRNA levels were measured via PCR and expressed as ΔΔCT. Statistics: two-way ANOVA followed by Newman–Keuls post hoc. Data are presented as the means ± SEM