Long-term behavioral programming induced by peripuberty stress in rats is accompanied by GABAergic-related alterations in the Amygdala

Abstract

Stress during childhood and adolescence is a risk factor for psychopathology. Alterations in γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain, have been found following stress exposure and fear experiences and are often implicated in anxiety and mood disorders. Abnormal amygdala functioning has also been detected following stress exposure and is also implicated in anxiety and social disorders. However, the amygdala is not a unitary structure; it includes several nuclei with different functions and little is known on the potential differences the impact of early life stress may have on this system within different amygdaloid nuclei. We aimed here to evaluate potential regional differences in the expression of GABAergic-related markers across several amygdaloid nuclei in adult rats subjected to a peripuberty stress protocol that leads to enhanced basal amygdala activity and psychopathological behaviors. More specifically, we investigated the protein expression levels of glutamic acid decarboxylase (GAD; the principal synthesizing enzyme of GABA) and of GABA-A receptor subunits α2 and α3. We found reduced GAD and GABA-A α3, but not α2, subunit protein levels throughout all the amygdala nuclei examined (lateral, basolateral, basomedial, medial and central) and increased anxiety-like behaviors and reduced sociability in peripubertally stressed animals. Our results identify an enduring inhibition of the GABAergic system across the amygdala following exposure to early adversity. They also highlight the suitability of the peripuberty stress model to investigate the link between treatments targeting the dysfunctional GABAergic system in specific amygdala nuclei and recovery of specific stress-induced behavioral dysfunctions.

Figure 1. Experimental design and weight changes following peripuberty stress.

Experimental design (A). All animals were weaned at P21 and at P28 they were assigned randomly to either CTRL or PPS groups. The peripuberty stress protocol consisted of exposure to an open field (OF) on the first day and then to elevated platform (EP) and predator odor (TMT), stressors that were presented in a variable manner until P42. The behavioral testing started at P90. Weight measurements were taken during the early-life period and at adulthood. IHC: Immunohistochemistry. Weight changes immediately after exposure to peripuberty stress and at adulthood (B). A reduction of body weight in PPS animals is evident one day after the end of the stress protocol, at P43, while no difference was observed at P90. ** p<0.01 versus CTRL; results are the mean ± SEM.

Figure 2. Anxiety-like behavior in the elevated plus maze.

PPS animals spent more time in the closed arms of the maze, and less time in the center and open arms as compared to CTRL (A). In addition, they spent less time head-dipping and stretching and more time self-grooming than CTRL (B). * p<0.05, ** p<0.01 versus CTRL; results are the mean ± SEM.

Figure 3. Sociability behavior in the social preference test.

PPS animals spent comparable time exploring the object, but less time sniffing the juvenile rat as compared to CTRL (A). PPS animals exhibited more time self-grooming but comparable rearing behavior to the CTRL rats (B). * p<0.05, ** p<0.01 versus CTRL; results are the mean ± SEM.

Figure 4. Effects of PPS on the expression of glutamic acid decarboxylase 65/67 (GAD6) in the amygdala of rats.

(A) Schematic representation of the different amygdalar nuclei analyzed (Bregma −2.56 mm, Interaural 6.44 mm), modified from the Atlas of Paxinos and Watson (2006). (B & C) Photomicrographs showing the GAD6 neuropil inmunoreactivity in the amygdala of CTRL (B) and PPS (C) rats. Scale bar: 200 µm. Optical densitometry of neuropil immunoreactivity revealed a significant decrease of GAD protein levels in the amygdala of PPS animals as compared to CTRL (D). ** p<0.01 versus CTRL, results are the mean ± SEM.

Figure 5. Effects of PPS on the expression of GABA-A receptor α2 subunit (GABA-A α2) and α3 subunit (GABA-A α3) in the amygdala of rats.

(A & B) Photomicrographs showing the GABA-A α2 inmunoreactivity in the amygdala of CTRL (A) and PPS (B) rats. (D & E) Photomicrographs showing the GABA-A α3 inmunoreactivity in the amygdala of CTRL (D) and PPS (E) rats. Scale bar: 200 µm. Optical densitometry of neuropil immunoreactivity revealed no differences in the expression of GABA-A α2 protein levels between PPS and CTRL groups (C). However, a significant decrease of GABA-A α3 protein levels was observed in the amygdala of PPS as compared to CTRL rats (F). ** p<0.01 versus CTRL, results are the mean ± SEM.