Altered Development of Prefrontal GABAergic Functions and Anxiety-like Behavior in Adolescent Offspring Induced by Prenatal Stress

Abstract

Maternal stress can afflict fetal brain development, putting the offspring at risk of cognitive deficits, including anxiety. The prefrontal cortex (PFC), a protracted maturing region, is notably affected by prenatal stress (PS). However, it remains unclear how PS interferes with the maturation of the GABAergic system, considering its functional adjustment in the PFC during adolescence. The present study thus investigated the long-lasting consequences of PS on the prefrontal GABAergic functions of adolescent offspring. Pregnant Sprague-Dawley rats were divided into controls and the PS group, which underwent restraint stress during the last week of gestation. Male pups from postnatal days (PND) 40-42 were submitted to the elevated plus maze (EPM) test. Proteins essentially involved in GABAergic signaling were then examined in PFC tissues, including the K⁺-Cl^- cotransporter (KCC2), Na⁺-K⁺-Cl^- cotransporter (NKCC1), α1 and α5 subunits of GABA type A receptors (GABA_A receptors), and parvalbumin (PV), along with cAMP response element-binding protein phosphorylation (pCREB), which reacts in the plasticity regulation of PV-positive interneurons. The results revealed that the higher anxiety-like behavior of PS adolescent rats concurred with the significant decreases of the KCC2 and α1 subunits, with PV- and pCREB-lowered levels. The findings suggested that PS disrupts the continuance of PFC maturity by reducing the essential elements of GABAergic functions. These changes likely underlie the anxiety emerging in adolescence, possibly progressing to mental disorders.

Keywords: GABAergic functions; adolescence; anxiety; prefrontal cortex; prenatal stress.

Figure 1

Anxiety-like behavior in adolescent rats. (A) Time spent in the open arms. (B) Time spent in the closed arms. Bar graphs display the results from the percentage calculation compared between the control and prenatal stress (PS) groups; the significant differences are at * p < 0.05. (C) Number of entries into the open arms. (D) Number of entries into the closed arms. Bar graphs display the results from counted number calculation compared between the control and PS groups; non-significance (ns) was found. (E) Distance in the open arms. (F) Distance in the closed arms. Bar graphs display the results from the percentage calculation compared between the control and PS groups; non-significance (ns) was found. Data are shown as the mean ± SEM, n = 5.

Figure 2

Developmental shift of cation–chloride cotransporters. (A) Immunohistochemical staining of KCC2. Expression in the medial prefrontal cortex between the control (left) and prenatal stress (PS) (right) groups; white arrows indicate KCC2-positive cells (upper; red) merged with the nuclear stain DAPI (lower; blue), scale bar = 20 µm. (B) Western blotting of KCC2. Upper, band comparing the control (left) and PS (right) groups; lower, bar graph displays the analysis of protein band densities of the KCC2/ß–actin ratio; the significant differences at *** p < 0.001. (C) Western blotting of NKCC1. Upper, band comparing the control (left) and PS (right) groups; lower, bar graph displays the analysis of protein band densities of the NKCC1/ß–actin ratio; non-significance (ns) was found. Data are shown as the mean ± SEM, n = 5.

Figure 2

Developmental shift of cation–chloride cotransporters. (A) Immunohistochemical staining of KCC2. Expression in the medial prefrontal cortex between the control (left) and prenatal stress (PS) (right) groups; white arrows indicate KCC2-positive cells (upper; red) merged with the nuclear stain DAPI (lower; blue), scale bar = 20 µm. (B) Western blotting of KCC2. Upper, band comparing the control (left) and PS (right) groups; lower, bar graph displays the analysis of protein band densities of the KCC2/ß–actin ratio; the significant differences at *** p < 0.001. (C) Western blotting of NKCC1. Upper, band comparing the control (left) and PS (right) groups; lower, bar graph displays the analysis of protein band densities of the NKCC1/ß–actin ratio; non-significance (ns) was found. Data are shown as the mean ± SEM, n = 5.

Figure 3

GABA type A receptors functional development. (A) Western blotting of GABA_A receptor α1 subunits. Upper, band comparing the control (left) and prenatal stress (PS) (right) groups; lower, bar graph displays the analysis of protein band densities of the GABA_A receptor α1/ß–actin ratio; the significant differences at *** p < 0.001. (B) Western blotting of the GABA_A receptor α5 subunits. Upper, band comparing the control (left) and PS (right) groups; lower, bar graph displays the analysis of protein band densities of the GABA_A receptor α5/ß–actin ratio; non-significance (ns) was found. Data are shown as the mean ± SEM, n = 5.

Figure 4

The plasticity regulation of GABAergic interneurons. (A) Western blotting of PV. Upper, band comparing the control (left) and prenatal stress (PS) (right) groups; lower, bar graph displays the analysis of protein band densities of the PV/β–actin ratio; the significant differences at *** p < 0.001. (B) Western blotting of pCREB. Upper, band comparing the control (left) and PS (right) groups; lower, bar graph displays the analysis of protein band densities of the pCREB/Tubulin-β ratio; the significant differences at *** p < 0.001. Data are shown as Mean ± SEM, n = 5.