Sex-Dependent Modulation of AMPA and GABAA Receptors in Response to Perinatal Stress: Implications for Cognitive and Emotion-related Behaviors

Abstract

Background: Early-life stress can severely impact brain health and neuronal plasticity, potentially leading to psychiatric disorders, with excitatory and inhibitory neurotransmission changes being key to understanding and mitigating these effects.

Objective: We investigated the effects of Perinatal Stress (PRS) on the balance of excitatory and inhibitory neurotransmission, particularly focusing on AMPA and GABA_A receptor protein levels and their relationship with cognition and risk-taking behavior in male and female Sprague-Dawley rats.

Methods: Adult PRS (3-4 months old) offspring of dams exposed to 10 days of gestational restraint stress, which led to reduced maternal care, were evaluated at 3-4 months for behavioral responses to novelty, adverse environments, and recognition memory, with biochemical analyses conducted in the prefrontal cortex and the ventral and dorsal hippocampus.

Results: PRS and sex notably affected behavior and AMPA/GABA_A receptor subunit expression. PRS males showed reduced risk-taking behavior when exposed to novel and adverse environments and impaired recognition memory, while PRS females demonstrated better behavioral performance compared to both PRS males and control females. In the dorsal hippocampus, PRS increased the GluA2:GluA1 ratio and GABA_A-α1 subunit in females but reduced them in males, modulating the AMPA/GABA_A balance to enhance synaptic GABAergic inhibition and behavioral resilience in PRS females and control males.

Conclusion: Our findings indicate that increased synaptic inhibition and reduced excitatory noise may underlie enhanced recognition memory and risk-taking behavior. The sex differences in PRS rats suggest that targeting AMPA or GABA_A receptors could help treat early-life stress-related disorders and underscore the need for developing gender-specific therapies.

Keywords: Perinatal Stress (PRS) animal model; adverse environment.; behavioral response to novelty; cognition and risk-taking behaviors; dorsal and ventral hippocampus; glutamate/GABA; prefrontal cortex.

Fig. (1)

Experimental design. Male and female CONT and PRS rats, aged 3-4 months, were used for behavioral and biochemical analysis. Separate sets of animals were used for the two behavioral tests as well as for the biochemical analysis. Specifically, animals designated for biochemical analysis were behaviorally naïve. Biochemical analysis was performed in the hippocampus (ventral and dorsal) and the prefrontal cortex.

Fig. (2)

Sex-specific effect of PRS on spatial recognition memory in the two-chamber apparatus and risk-taking behavior and exploration in the Elevated-Plus Maze (EPM) of adult rats. Adult PRS and control rats of both sexes were assessed for spatial recognition memory using the 2-chamber apparatus (A, B) and for risk-taking behavior in the elevated plus maze (C-G). Values are expressed as means ± S.E.M. (n = 5-7 rats/group); main sex effect: #= p <0.05; PRS x sex interaction: $$= p <0.01; $$$= p <0.001.

Fig. (3)

AMPAR, GABA_A expression, and GABA_A receptor cationic transporters KCC2 and NKCC1 expression in the prefrontal cortex of adult rats. Immunoblot analysis of AMPAR GluA1, GluA2, and GluA3 subunits and the resulting GluA₂:GluA₁ ratio in crude synaptosomal fractions in PRS and CONT unstressed rats of both sexes. (A-D). Immunoblot analysis of GABA_A receptor α1, α2, α4, δ, and γ2 subunits (E-I) and NKCC1, KCC2, and their resulting NKCC1: KCC2 ratio (J-L) in the same synaptosomal fraction of groups above reported. Values are expressed as means ± S.E.M. (n = 9-12 PFC tissue/group, adults); PRS x sex interaction: $= p <0.05. O.D.=optical density.

Fig. (4)

AMPAR, GABA-_A receptor subunits, and GABA_A receptor cationic transporters KCC2 and NKCC1 expression in the dorsal hippocampus of adult rats. Immunoblot analysis of AMPAR GluA1, GluA2, and GluA3 subunits and the resulting GluA2:GluA1 ratio in crude synaptosomal fractions in PRS and CONT unstressed rats of both sexes. (A-D). Immunoblot analysis of GABA_A receptor α1, α2, α4, γ2 and δ subunits (E-I) and NKCC1, KCC2, and their resulting NKCC1:KCC2 ratio (J-L) in the same synaptosomal fraction of groups above reported. Values are expressed as means ± S.E.M. (n = 5-14 HPC-D/group); main sex effect: #= p <0.05; ##= p< 0.01; PRS x sex interaction: $= p <0.05, $$= p <0.01, $$$= p <0.001; O.D.=optical density.

Fig. (5)

AMPAR and GABA_A receptor subunits and GABA_A receptor cationic transporters KCC2 and NKCC1 expression in the ventral hippocampus of adult rats. Immunoblot analysis of AMPAR GluA1, GluA2, and GluA3 subunits and the resulting GluA2:GluA1 ratio in crude synaptosomal fractions in PRS and CONT unstressed rats of both sexes. (A-D). Immunoblot analysis of GABA_A receptor α1, α2, α4, γ2 and δ subunits (E-I) and NKCC1, KCC2, and their resulting NKCC1: KCC2 ratio (J-L) in the same synaptosomal fraction of groups above reported. Values are expressed as means ± S.E.M. (n = 5-12 HPC-V/group); main PRS effect: * = p <0.05; main sex effect: # = p <0.05; PRS x sex interaction: $= p <0.05, $$$= p <0.001. O.D.=optical density.

Fig. (6)

AMPA and GABA_A Receptors Ratio in the dorsal and ventral hippocampus of adult rats. AMPAR:GABA_AR ratios in PRS and CONT unstressed rats of both sexes, considering both the presence and absence of the GluA2 subunit and the synaptic (α_1, α_2, γ2) or extra-synaptic (α_4, δ) localization of GABA_A receptors subunits in the dorsal hippocampus (A-D) and ventral hippocampus (E-H). Values are expressed as means ± S.E.M. (n = 8-13 HPC-D or HPC-V/group); main PRS effect: * = p <0.05; main sex effect: ### = p <0.001; PRS x sex interaction: $$= p <0.01, $$$= p <0.001. O.D.=optical density.

Fig. (7)

Multidimensional analysis. PCA-Principal component analysis in PRS and CONT unstressed rats of both sexes of the protein data set (AMPAR GluA1, GluA2, GluA3 subunits and the resulting GluA2:GluA1 ratio; GABA_A receptor α1, α2, α4, γ2 and δ subunits; NKCC1, KCC2 and their resulting NKCC1: KCC2 ratio). (A) Dorsal hippocampus, (B) ventral hippocampus, and (C) prefrontal cortex. PERMANOVA analysis was used to test group differences and the effects of variables in a multivariate manner.