Interventions after acute stress prevent its delayed effects on the amygdala

Abstract

Stress is known to elicit contrasting patterns of plasticity in the amygdala and hippocampus. While chronic stress leads to neuronal atrophy in the rodent hippocampus, it has the opposite effect in the basolateral amygdala (BLA). Further, even a single episode of acute stress is known to elicit delayed effects in the amygdala. For example, 2 h of immobilisation stress has been shown to cause a delayed increase in dendritic spine density on BLA principal neurons 10 days later in young rats. This is paralleled by higher anxiety-like behaviour at the same delayed time point. This temporal build-up of morphological and behavioural effects 10 days later, in turn, provides a stress-free time window of intervention after exposure to acute stress. Here, we explore this possibility by specifically testing the efficacy of an anxiolytic drug in reversing the delayed effects of acute immobilisation stress. Oral gavage of diazepam 1 h after immobilisation stress prevented the increase in anxiety-like behaviour on the elevated plus-maze 10 days later. The same post-stress intervention also prevented delayed spinogenesis in the BLA 10 days after acute stress. Surprisingly, gavage of only the vehicle also had a protective effect on both the behavioural and synaptic effects of stress 10 days later. Vehicle gavage was found to trigger a significant rise in corticosterone levels that was comparable to that elicited by acute stress. This suggests that a surge in corticosterone levels, caused by the vehicle gavage 1 h after acute stress, was capable of reversing the delayed enhancing effects of stress on anxiety-like behaviour and BLA synaptic connectivity. These findings are consistent with clinical reports on the protective effects of glucocorticoids against the development of symptoms of post-traumatic stress disorder. Taken together, these results reveal strategies, targeted 1 h after stress, which can prevent the delayed effects of a brief exposure to a severe physical stressor.

Keywords: Anxiety; Basolateral amygdala; Corticosterone; Dendritic spines; Diazepam; Stress.

Graphical abstract

Fig. 1

Stress causes a delayed increase in anxiety-like behaviour. (a) Experimental design. Rats underwent a single 2-h session of acute immobilisation stress, and anxiety-like behaviour was measured on the elevated plus-maze 10 days later. Control animals remained unstressed. (b) Stressed animals spend significantly less time in the open-arm. (c) Stressed animals show a trend towards decreased open-arm entries that did not reach statistical significance. (d) Stress leads to a delayed increase in the Anxiety Index. (e) Stress did not affect total entries on the maze. (Control, N = 12 animals; Stress, N = 11 animals). ** indicates p < 0.01 and * indicates p < 0.05 in Student's unpaired t-test.

Fig. 2

Stress triggers a delayed increase in dendritic spine density in the basolateral amygdala. (a) Experimental design. Animals underwent a single 2-h session of acute immobilisation stress, and they were sacrificed 10 days later for Golgi-Cox staining to visualise dendritic spines on the principal neurons of the basolateral amygdala (BLA). Control animals remained unstressed. (b) Stress leads to a delayed increase in dendritic spine density in the apical dendrites of the BLA pyramidal neurons. (Control, n = 22 dendrites, N = 6 animals; Stress, n = 28 dendrites, N = 6 animals). (c) Representative images of dendritic segments analysed in the control (left) and stressed (right) animals. Scale bar 10 μm ** indicates p < 0.01 in Student's unpaired t-test.

Fig. 3

Diazepam and vehicle oral gavage, 1 h after stress, both prevent anxiety-like behaviour 10 days later. (a) Experimental design. Animals underwent a single 2 h episode of immobilisation stress (Stress) or remained unstressed (Control), and received either a single dose of diazepam or vehicle gavage 1 h after stress. Anxiety on the elevated plus-maze was quantified 10 days later. (b) Open-arm time, (c) Open-arm entries and (d) Anxiety Index are comparable across all four groups. (e) All groups show comparable locomotion on the maze as well (Control + Vehicle: N = 20 animals, Control + Diazepam: N = 25 animals, Acute Stress + Vehicle: N = 18 animals, Acute Stress + Diazepam: N = 22 animals). Dotted lines indicate mean values of each parameter in control (no gavage) (black) and stressed (no gavage) (red) groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Diazepam and vehicle oral gavage, 1 h after stress, both prevent the delayed increase in BLA spinogenesis. (a) Experimental design. Animals underwent a single 2 h episode of immobilisation stress (Stress) or remained unstressed (Control), and received either a single dose of diazepam or vehicle gavage 1 h after stress. Dendritic spine density in the BLA was quantified 10 days later. (b) Diazepam and vehicle both prevent spinogenesis in the BLA. (Control + Vehicle: n = 32 dendrites, N = 4 animals, Control + Diazepam: n = 28 dendrites, N = 4 animals, Acute Stress + Vehicle: n = 27 dendrites, N = 4 animals, Acute Stress + Diazepam: n = 29 dendrites, N = 4 animals). (c) Representative apical dendritic segments from BLA pyramidal neurons from all four groups. Dotted lines indicate mean spine density of control (no gavage) (black) and stressed (no gavage) (red) groups. ** indicates p < 0.01 and * indicates p < 0.05 in post-hoc Tukey's test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Oral gavage of vehicle is stressful to rats. (a) Experimental design. Different cohorts of rats were sacrificed after handling (baseline), after vehicle gavage and after acute immobilisation stress, and corticosterone levels were quantified subsequently. (b) Oral gavage of vehicle as well as stress significantly increases corticosterone levels. Increase in corticosterone due to gavage and stress were not significantly different from each other (Baseline, N = 7, After Gavage, N = 8, After Stress, N = 5). ^^^^ indicates p < 0.0001 in One-way ANOVA. **** indicate p < 0.0001 in post-hoc Tukey's test.