Communal nesting shapes the sex-dependent glutamatergic response to early life stress in the rat prefrontal cortex

Abstract

Introduction: Early social environment, either positive or negative, shapes the adult brain. Communal nesting (CN), a naturalistic setting in which 2-3 females keep their pups in a single nest sharing care-giving behavior, provides high level of peer interaction for pups. Early social isolation (ESI) from dam and siblings represents, instead, an adverse condition providing no peer interaction.

Methods: We investigated whether CN (enrichment setting) might influence the response to ESI (impoverishment setting) in terms of social behavior and glutamate system in the medial prefrontal cortex (mPFC) of adult and adolescent male and female rats.

Results: Pinning (a rewarding component of social play behavior) was significantly more pronounced in males than in females exposed to the combination of CN and ESI. CN sensitized the glutamate synapse in the mPFC of ESI-exposed male, but not female, rats. Accordingly, we observed (i) a potentiation of the glutamatergic neurotransmission in the mPFC of both adolescent and adult males, as shown by the recruitment of NMDA receptor subunits together with increased expression/activation of PSD95, SynCAM 1, Synapsin I and αCaMKII; (ii) a de-recruiting of NMDA receptors from active synaptic zones of same-age females, together with reduced expression/activation of the above-mentioned proteins, which might reduce the glutamate transmission. Whether similar sex-dependent glutamate homeostasis modulation occurs in other brain areas remains to be elucidated.

Discussion: CN and ESI interact to shape social behavior and mPFC glutamate synapse homeostasis in an age- and sex-dependent fashion, suggesting that early-life social environment may play a crucial role in regulating the risk to develop psychopathology.

Keywords: NMDA receptors; communal nesting; early-life stress; prefrontal cortex; sex difference; social isolation.

Figure 1

Effects of SH, CN and ESI protocol on social play behavior in PND35 male (top) and female (bottom) rats. Panel (A) represents a graphical timeline of the experimental paradigm in both female and male rats at PND35 and PND75. Social play behavior is expressed as number of pinning (B in males and E in females) and number of pouncing (C in males and F in females). The time spent by male (D) and female (G) rats in general social exploration is also shown. ^$p<0.05 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls - no ESI; ESI, Early Social Isolation.

Figure 2

Effect of SH, CN and ESI protocol on synaptic structural markers expression in the crude membrane fraction of the mPFC of PND35 male (top) and female (middle) rats. Protein levels of PSD95 (A), SynCAM 1 (B) in male rats and PSD95 (C), SynCAM 1 (D) in female rats are expressed as percentages of SH-CTRL and represent the mean ± S.E.M. of six rats per group. Representative immunoblots (bottom) are shown for each protein in the respective panels (E, F). ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. SH-CTRL, ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls no ESI; ESI, Early Social Isolation.

Figure 3

Effect of SH, CN and ESI protocol on NMDA receptor subunits in the crude membrane fraction of the mPFC of PND35 male (top) and female (middle) rats. Protein levels of GluN1 (A), GluN2A (B), GluN2B (C), GluN2A/GluN2B ratio (D) in male rats, and GluN1 (E), GluN2A (F), GluN2B (G), GluN2A/GluN2B ratio (H) in female rats are expressed as percentages of SH-CTRL and represent the mean±S.E.M. of six rats per group (GluN2A and GluN2A/GluN2B ratio: 1 outlier in SH-ESI males). Representative immunoblots (bottom) are shown for each protein in the respective panels (I, J). ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. SH-CTRL, ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls no ESI; ESI, Early Social Isolation.

Figure 4

Effect of SH, CN and ESI protocol on proteins regulating neurotransmitter release in the crude membrane fraction of the mPFC of PND35 male (top) and female (middle) rats. Protein levels of pSynapsin I S603/Synapsin I (A), pαCaMKII T286/αCaMKII (B) in male rats and pSynapsin I S603/Synapsin I (C), pαCaMKII T286/αCaMKII (D) in female rats are expressed as percentages of SH-CTRL and represent the mean±S.E.M. of six rats per group. Representative immunoblots (middle) are shown for each protein in the respective panels (E, F). ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. SH-CTRL, ^$p<0.05, ^$$p<0.01 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls no ESI; ESI, Early Social Isolation.

Figure 5

Effect of SH, CN and ESI protocol on synaptic structural markers expression in the crude membrane fraction of the mPFC of PND-75 male (top) and female (bottom) rats. Protein levels of PSD95 (A), SynCAM 1 (B) in male rats and PSD95 (C), SynCAM 1 (D) in female rats are expressed as percentages of SH-CTRL and represent the mean ± S.E.M. of six rats per group (SynCAM: 1 outlier in CN-CTRL males). Representative immunoblots (bottom) are shown for each protein in the respective panels (E, F). ^*p<0.05 vs. SH-CTRL, ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls no ESI; ESI, Early Social Isolation.

Figure 6

Effect of SH, CN and ESI protocol on NMDA receptor subunits in the crude membrane fraction of the mPFC of PND75 male (top) and female (middle) rats. Protein levels of GluN1 (A), GluN2A (B), GluN2B (C), GluN2A/GluN2B ratio (D) in male rats and GluN1 (E), GluN2A (F), GluN2B (G), GluN2A/GluN2B ratio (H) in female rats are expressed as percentages of SH-CTRL and represent the mean ± S.E.M. of six rats per group (I, J). Representative immunoblots (bottom) are shown for each protein in the respective panels. ^*p<0.05, ^**p<0.01, ^***p<0.001 vs. SH-CTRL, ^$$p<0.01, ^$$$p<0.001 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls no ESI; ESI, Early Social Isolation.

Figure 7

Effect of SH, CN and ESI protocol on proteins regulating neurotransmitter release in the crude membrane fraction of the mPFC of PND75 male (top) and female (middle) rats. Protein levels of pSynapsin I S603/Synapsin I (A), pαCaMKII T286/αCaMKII (B) in male rats and pSynapsin I S603/Synapsin I (C), pαCaMKII T286/αCaMKII (D) in female rats are expressed as percentages of SH-CTRL and represent the mean±S.E.M. of six rats per group. Representative immunoblots (bottom) are shown for each protein in the respective panels (E, F). ^*p<0.05 vs. SH-CTRL, ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls no ESI; ESI, Early Social Isolation.