Early Social Isolation Stress and Perinatal NMDA Receptor Antagonist Treatment Induce Changes in the Structure and Neurochemistry of Inhibitory Neurons of the Adult Amygdala and Prefrontal Cortex

Abstract

The exposure to aversive experiences during early life influences brain development and leads to altered behavior. Moreover, the combination of these experiences with subtle alterations in neurodevelopment may contribute to the emergence of psychiatric disorders, such as schizophrenia. Recent hypotheses suggest that imbalances between excitatory and inhibitory (E/I) neurotransmission, especially in the prefrontal cortex and the amygdala, may underlie their etiopathology. In order to understand better the neurobiological bases of these alterations, we studied the impact of altered neurodevelopment and chronic early-life stress on these two brain regions. Transgenic mice displaying fluorescent excitatory and inhibitory neurons, received a single injection of MK801 (NMDAR antagonist) or vehicle solution at postnatal day 7 and/or were socially isolated from the age of weaning until adulthood (3 months old). We found that anxiety-related behavior, brain volume, neuronal structure, and the expression of molecules related to plasticity and E/I neurotransmission in adult mice were importantly affected by early-life stress. Interestingly, many of these effects were potentiated when the stress paradigm was applied to mice perinatally injected with MK801 ("double-hit" model). These results clearly show the impact of early-life stress on the adult brain, especially on the structure and plasticity of inhibitory networks, and highlight the double-hit model as a valuable tool to study the contribution of early-life stress in the emergence of neurodevelopmental psychiatric disorders, such as schizophrenia.

Keywords: PSA-NCAM; interneuron; neuronal plasticity; schizophrenia; social isolation; stress.

Figure 1.

Experimental procedure. Seven days after birth (P7), male pups from two different transgenic strains: the GIN mice (Oliva et al. 2000), which express EGFP in interneurons, and the THY1 mice (Feng et al. 2000), which express the YFP in pyramidal neurons) were intraperitoneally injected with MK801 or NaCl (vehicle solution). After weaning (P21), mice were randomly selected and housed alone (isolation) or in groups of four mice (social rearing) for 10 weeks. At P90, all mice were tested in the hole-board apparatus. Brains from GIN molecular mice were destined to protein and gene expression studies, whereas brains from GIN structure mice were used for the structural and neurochemical analysis of interneurons. The THY structure set of mice was used to study structural alterations of pyramidal neurons. For further details, see Material and Methods.

Figure 2.

Behavioral analysis of GAD-EGFP expressing mice. A, Representative track-plot reports recorded during the hole-board test session (ANY-maze). Observe the increased distance traveled (purple line) in the two groups subjected to post-weaning isolation, especially in the border of the apparatus. Social isolation rearing increased locomotor activity (B) and anxiety-related behavior (D) but did not change exploratory behavior (number of head dips into novel holes/total number of head dips; C). Gray lines in graphs (B–D) represent statistically significant effects of rearing in a two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Behavioral analysis of Thy1-YFP expressing mice. A, Representative track-plot reports showing the increased distance traveled (purple line) of double-hit mice (MK801-Isolation group) in the hole-board test compared with the other groups. Double-hit mice showed increased locomotor activity (B) and anxiety-related behavior (D) but no changes in exploratory behavior (number of head dips into novel holes/total number of head dips; C). B–D, Horizontal lines in graphs represent statistically significant effects of MK801 treatment (black), rearing (gray), or interaction (gray, dashed) in a two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Colored symbols in bars represent trends and statistically significant differences among groups after post hoc analysis: # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Volumetric analysis. Treatment, rearing, and the interaction of both treatments influenced the volume of the amygdala and the PFC. Although the total volume of both regions was not significantly affected (A), the volume of some nuclei of the amygdala (B, C) and some regions of the PFC (D, E) was affected by treatment (BMA, Cg1), rearing (BLA, IL), or their interaction (Ce, Cg1). Horizontal lines in graphs represent trends and statistically significant effects of MK801 treatment (black), rearing (gray), or interaction (gray, dashed) in a two-way ANOVA. # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Colored symbols in bars represent trends and statistically significant differences among groups after post hoc analysis: # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. B, D, Schematic representations of the regions that were measured in our volumetric study (amygdala: all brain slices from bregma −0.58 to −2.30 mm; PFC: all brain slices from bregma +2.96 to −0.22 mm).

Figure 5.

Structural analysis of pyramidal neurons. A, Panoramic view of the PFC of THY1-YFP expressing mice. Arrowheads point to fluorescent pyramidal neurons. B, Representative confocal image of a pyramidal neuron from these animals. C1 (NaCl-Group) and C2 (MK801-Isolation) represent sections of the spiny apical dendrites of these pyramidal neurons. D, E, Graphs showing the results of the analysis of dendritic spine density. D, The segmented analysis of the spine densities only showed a significant interaction effect in the first segment (0–50 µm of distance from the soma; Table 3) but no statistically significant differences among groups where observed after post hoc comparison. Despite the significant effects of treatment or rearing in some of the other segments (Table 3), the interaction (two-way ANOVA) was not significant in any of them and, therefore, post hoc comparisons were not applicable (n/a). E, No statistical significant effects of treatment, rearing, or their interaction (two-way ANOVA) were observed when analyzing the spines density of the total length of the dendrite (200 µm). Scale bars: A, 100 µm; B, 20 µm; C1, C2, 2.5 µm.

Figure 6.

Structural analysis of interneurons. Panoramic view of the amygdala (A) and PFC (H) of GIN mice. B1, B2, I1, I2, Representative 3D reconstructions of the dendritic arbor of GAD-EGFP expressing interneurons. Double-hit mice showed increased dendritic arborization (number of intersections in the Sholl analysis) in interneurons form the amygdala (D, E) but not from the PFC (K, L). C1, C2, J1, J2, Representative images of spiny dendrites from GAD-EGFP expressing interneurons. Insets are magnified views of the squared sections of their respective images. Arrowheads point to dendritic spines. Analysis of the dendritic spines showed increased density in the PFC (M, N) but not in the amygdala (F, G) of double-hit mice. Horizontal lines in graphs (E, N) represent statistically significant effects of MK801 treatment (black), rearing (gray), or interaction (gray, dashed) in a two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Colored symbols (D, E, M, N) represent statistically significant differences among groups after post hoc analysis: *p < 0.05, **p < 0.01, ***p < 0.001. If the interaction (two-way ANOVA) was not significant, post hoc comparisons were not applicable (n/a). Scale bars: A, H, 800 µm; B, I, 40 µm; C, J, 14 µm; C, J (insets), 2 µm.

Figure 7.

Excitatory and inhibitory neurotransmission. Schematic view of all the analyzed regions (A1, amygdala; B1, PFC). Right, Representative confocal images of excitatory (blue, VGLUT1) and inhibitory (red, VGAT) puncta in the neuropil of NaCl-Social and MK801-Isolated animals. Scale bars: images, 16 µm; insets, 6 µm. Significant differences in VGLUT1 puncta density (A2, B2), VGAT puncta density (A3, B3), and E/I ratio (A4, B4) among groups were observed. Horizontal lines in graphs represent trends and statistically significant effects of MK801 treatment (black), rearing (gray), or interaction (gray, dashed) in a two-way ANOVA. # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Colored symbols in bars represent trends and statistically significant differences among groups after post hoc analysis: # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8.

Protein and gene expression studies. Bar graphs showing the presence or lack of effect of the treatment, rearing, or their interaction in GAD67, SYN, and PSA-NCAM protein expression (A–C) and in GAD67, BDNF, ErbB4, CB1-R, ST8SiaII, and ST8SiaIV mRNA levels (D–I). Horizontal lines in graphs represent trends and statistically significant effects of MK801 treatment (black), rearing (gray) or interaction (gray, dashed) in a two-way ANOVA. # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. J & K: Representative bands from immunoblots for GAD67 (J) and SYN (K) in the amygdala and the PFC.

Figure 9.

Expression of PNNs in PV-immunoreactive neurons. A, Representative confocal images showing the distribution of PV (red) and PNNs (blue) in the different nuclei of the amygdala (left) and regions of the PFC (right) of GIN mice (GAD-GFP, green). The squared section in the panoramic images was 2× magnified to show the different nuclei and areas that were analyzed. In a detailed view (below), two PV-immunoreactive neurons expressing PNN can be observed. GAD-GFP expressing interneurons do not express PNN. Scale bars: panoramic views, 875 µm; detailed view, 25 µm. B, C, Treatment, rearing, or their interaction affected the total number of PV neurons (B1, C1), PNNs (B2, C2), or their colocalization (B3, C3) in some nuclei of the amygdala and regions of the PFC. Horizontal lines in graphs represent trends and statistically significant effects of MK801 treatment (black), rearing (gray), or interaction (gray, dashed) in a two-way ANOVA. # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Colored symbols in bars represent trends and statistically significant differences among groups after post hoc analysis: # 0.1 > p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.