Prenatal stress programs neuroendocrine stress responses and affective behaviors in second generation rats in a sex-dependent manner

Abstract

An adverse environment in early life is often associated with dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis and higher rates of mood disorders in adulthood. In rats, exposure to social stress during pregnancy results in hyperactive HPA axis responses to stress in the adult offspring and heightened anxiety behavior in the males, but not the females. Here we tested whether, without further intervention, the effects of prenatal stress (PNS) in the first filial generation (F1) are transmitted to the F2 generation via the maternal line. F1 control and PNS female rats were mated with control males and housed under non-stress conditions throughout pregnancy. HPA axis responses to acute stress, anxiety- and depressive-like behavior were assessed in the adult F2 offspring. ACTH and corticosterone responses to an acute stressor were markedly enhanced in F2 PNS females compared with controls. This was associated with greater corticotropin releasing hormone (Crh) mRNA expression in the paraventricular nucleus and reduced hippocampal glucocorticoid (Gr) and mineralocorticoid receptor (Mr) mRNA expression. Conversely, in the F2 PNS males, HPA axis responses to acute stress were attenuated and hippocampal Gr mRNA expression was greater compared with controls. F2 PNS males exhibited heightened anxiety-like behavior (light-dark box and elevated plus maze) compared with F2 control males. Anxiety-like behavior did not differ between F2 control and PNS females during metestrus/diestrus, however at proestrus/estrus, F2 control females displayed a reduction in anxiety-like behavior, but this effect was not observed in the F2 PNS females. Heightened anxiety in the F2 PNS males was associated with greater Crh mRNA expression in the central nucleus of the amygdala compared with controls. Moreover, Crh receptor-1 (Crhr1) mRNA expression was significantly increased, whereas Crhr2 mRNA was significantly decreased in discrete regions of the amygdala in F2 PNS males compared with controls, with no differences in the F2 females. No differences in depressive-like behavior (sucrose preference or forced swim test) were observed in either sex. In conclusion, the effects of maternal stress during pregnancy on HPA axis regulation and anxiety-like behavior can be transmitted to future generations in a sex-dependent manner. These data have implications for human neuropsychiatric disorders with developmental origins.

Keywords: Anxiety; Glucocorticoids; HPA axis; Prenatal stress; Sex differences; Trans-generational.

Fig. 1

Effect of acute stress exposure on ACTH and corticosterone secretion in second generation control and PNS rats. Age-matched second generation control (CON; filled circles) and PNS (open circles) male and female rats were blood sampled under non-stress (ns) conditions or before and after administration of intravenous interleukin-1β (IL-1β; 500ng/kg) or exposure to 30 min restraint (grey bars). A basal (B) blood sample was collected (at t = −1 min). At t = 0 min rats remained undisturbed in their home cage (non-stress), were placed in a rodent restraint tube for 30 min or were administered IL-1β. Further blood samples were collected at t = 15, 30, 60, 90 and 120 min. Plasma ACTH concentrations in female rats in the (a) non-stress; (b) IL-1β-treated; and (c) restraint groups. *p < 0.05 vs B values in the same group; #p < 0.04 vs the other group at the same time point (two-way RM ANOVA with SNK). The increase in plasma corticosterone concentrations from basal levels in female rats in the (d) non-stress; (e) IL-1β-treated; and (f) restraint groups. *p < 0.01 vs B values in the same group; #p < 0.02 vs the other group at the same time point (two-way RM ANOVA with SNK). Plasma ACTH concentrations in male rats in the (g) non-stress; (h) IL-1β-treated; and (i) restraint groups. *p < 0.002 vs B values in the same group; #p < 0.001 vs the other group at the same time point (two-way RM ANOVA with SNK). The increase in plasma corticosterone concentrations from basal levels in female rats in the (j) non-stress; (k) IL-1β-treated; and (l) restraint groups. *p < 0.01 vs B values in the same group; #p < 0.005 vs the other group at the same time point (two-way RM ANOVA with SNK). Rat numbers/group are n = 5–7 for females and n = 6–7 for males. Data are group means ± SEM.

Fig. 2

Effect of acute stress exposure on Crh and Avp mRNA expression in the mpPVN in second generation control and PNS rats. Age-matched F2 control (CON, filled bars) and F2 PNS (PNS, open bars) rats were killed 4 h after the onset of acute stress and brains were processed by in situ hybridization. Quantification of Crh mRNA expression in the medial parvocellular paraventricular nucleus (mpPVN) in (a) females and (e) males. #p < 0.02 versus respective non-stress group; *p < 0.02 versus respective F2 control group (two-way ANOVA with SNK). Quantification of Avp mRNA expression in the mpPVN in the (b) females and (f) males. Rat numbers/group are n = 5–7 for females and n = 5–11 for males. Representative dark-field images of (c) Crh mRNA and (d) Avp mRNA expression in the PVN of (i) F2 control female and (ii) F2 PNS female 4 h after restraint. Scale bar = 100 μm; 3 V, 3rd ventricle; ROI, region of interest. Data are group means ± SEM.

Fig. 3

Hippocampal Gr and Mr mRNA expression under basal conditions in second generation control and PNS rats. Brains from age-matched F2 control (CON, filled bars) and F2 PNS (PNS, open bars) were processed by in situ hybridization. Quantification of Gr mRNA expression in the hippocampal subfields CA1, CA2, CA3 and dentate gyrus (DG) in (a) females and (e) males. Statistics: *p ≤ 0.03 vs F2 control group (Student’s t-test). Quantification of Mr mRNA expression in the hippocampus in (b) females and (f) males. Statistics: *p < 0.04 vs F2 control group (Student’s t-test). Rat numbers/group are n = 6 for females and n = 7 for males. Representative dark-field autoradiographs of basal expression of (c) Gr and (d) Mr mRNA in the hippocampus of a (i) F2 control female and (ii) F2 PNS female. Scale bar = 500 μm; ROI, region of interest.

Fig. 4

Anxiety-like behavior in the light-dark box and on the elevated plus maze. Age-matched F2 control (CON, filled bars) and F2 PNS (PNS, open bars) male and female rats were tested in the light-dark box (LDB) and 2 days later on the elevated plus maze (EPM). Females are subdivided based on estrus cycle stage. Time spent (seconds) in (a) the light compartment of the LDB and (b) the open arms of the EPM in females during metestrus/diestrus (M/D) and proestrus/estrus (P/E). Statistics: #p < 0.02 control M/D group; *p < 0.03 vs PNS P/E group (two-way ANOVA with SNK). (c) Time spent in the light compartment of the LDB and on the open arms of the EPM in males. Statistics: *p ≤ 0.05 vs F2 control males (Student’s t-test). In each case data are group means ± SEM and n = 10 rats/group. Representative tracking of (d) M/D female rats; (e) P/E female rats and (f) male rats in the LDB. Light compartment delineated with dashed line and dark compartment with a solid line. Representative tracking of (g) M/D female rats; (h) P/E female rats and (i) male rats on the EPM. O, open arms; C, closed arms.

Fig. 5

Crhr1 and Crhr2 mRNA expression in the amygdala under basal conditions in second generation control and PNS rats. Brains from age-matched F2 control (CON, filled bars) and F2 PNS (PNS, open bars) were processed by in situ hybridization. Quantification of Crhr1 mRNA in (a) females and (c) males and Crhr2 mRNA expression in (b) females and (d) males. Statistics: *p < 0.02 vs F2 control group (Student’s t-test). In each case data are group means ± SEM. Rat numbers/group are n = 5–6 for females and n = 8–9 for males. Abbreviations: BLA, basolateral amygdala; BMA, basomedial amygdala; CeA, central amygdala; MeA, medial amygdala; ROI, region of interest.