Corticosterone after acute stress prevents the delayed effects on the amygdala

Abstract

Even a single 2-hour episode of immobilization stress is known to trigger anxiety-like behavior and increase spine-density in the basolateral amygdala (BLA) of rats 10 days later. This delayed build-up of morphological and behavioral effects offers a stress-free time window of intervention after acute stress, which we used to test a protective role for glucocorticoids against stress. We observed that post-stress corticosterone, given 1 day after acute stress in drinking water, reversed enhanced anxiety-like behavior 10 days later. Quantification of spine-density on Golgi-stained BLA principal neurons showed that the same intervention also prevented the increase in spine numbers in the amygdala, at the same delayed time-point. Further, stress elevated serum corticosterone levels in rats that received vehicle in the drinking water. However, when stress was followed 24 h later by corticosterone in the drinking water, the surge in corticosterone was prevented. Together, these observations suggest that corticosterone, delivered through drinking water even 24 h after acute stress, is capable of reversing the delayed enhancing effects on BLA synaptic connectivity and anxiety-like behavior. Strikingly, although the immobilization-induced surge in corticosterone by itself has delayed detrimental effects on amygdalar structure and function, there exists a window of opportunity even after stress to mitigate its impact with a second surge of exogenously administered corticosterone. This provides a framework in the amygdala for analyzing how the initial physiological and endocrine processes triggered by traumatic stress eventually give rise to debilitating emotional symptoms, as well as the protective effects of glucocorticoids against their development.

Fig. 1. Corticosterone in drinking water after stress prevents increase in anxiety-like behavior 10 days later.

a Experimental design. Following 2 h of immobilization on day 0, rats were subjected to elevated plus-maze 10 days later. b Stressed rats show reduced open arm time on the elevated plus-maze, 10 days after stress. c Stressed rats also show decreased open-arm entries. d Stressed rats show enhanced Anxiety Index on the elevated plus-maze, 10 days after stress. (Control, N = 11 rats, Stress¸ N = 15 rats). ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 in Unpaired t-test. e Experimental design. Rats received either corticosterone or vehicle in their drinking water bottle for 12 h (noon to midnight), 24 h after stress. Anxiety-like behavior was quantified 10 days later. f Post-stress corticosterone treatment, but not vehicle, prevented the decrease in open-arm time as well as (g) open-arm entries. h Corticosterone also prevents increased Anxiety Index, 10 days after stress. (Control + Vehicle, N = 25 rats; Control + Corticosterone, N = 14 rats; Stress + Vehicle, N = 30 rats; Stress + Corticosterone, N = 14 rats). ^Ψp < 0.05 in ‘interaction’ between factors stress and corticosterone, in Two-way ANOVA. **p < 0.01 in post-hoc Sidak’s test.

Fig. 2. Corticosterone in drinking water after stress prevents impairment in social interaction with a juvenile rat 10 days later.

a Experimental design. Following acute immobilization stress on day 0, rats underwent social interaction with a male, unfamiliar juvenile rat, 10 days later. b Stressed rats spend significantly less time interacting with a juvenile conspecific. c Stress leads to a significant impairment in anogenital exploration of a juvenile. (Control, N = 19 rats, Stress, N = 24 rats). ^##p < 0.01 and ^###p < 0.001 in Unpaired t-test. d Experimental design. Rats received either corticosterone or vehicle in their drinking water bottle for 12 h (noon to midnight), 24 h after stress. Social interaction with a male, unfamiliar juvenile rat was quantified 10 days later. e Vehicle-treated stressed rats show a significant reduction in total social interaction, which is rescued by post-stress corticosterone. f Post-stress corticosterone significantly prevents decreased anogenital exploration of a juvenile conspecific. (Control + Vehicle, N = 16 rats; Control + Corticosterone, N = 15 rats; Stress + Vehicle, N = 18 rats; Stress + Corticosterone, N = 16 rats). ^{Ψ Ψ Ψ}p < 0.001 in ‘interaction’ between factors stress and corticosterone, in Two-way ANOVA. **p < 0.01 in post-hoc Sidak’s test.

Fig. 3. Post-stress corticosterone prevents the delayed increase in spine-density in the basolateral amygdala (BLA).

a Experimental design. Rats received either corticosterone or vehicle in their drinking water bottle for 12 h (noon to midnight), 1 day after stress. Spine-density in the BLA were quantified 10 days later. b Representative image showing a Golgi-stained coronal section of a rat BLA. Scale bar 200 µm. c Representative images of dendritic spines from the experimental groups. Scale bar 10 µm. d Post-stress corticosterone, but not vehicle, also prevented elevated spine-density in the BLA. (Control + Vehicle, N = 6 rats; Control + Corticosterone, N = 6 rats; Stress + Vehicle, N = 7 rats; Stress + Corticosterone, N = 7 rats). ^{Ψ Ψ} p < 0.01 in ‘interaction’ between factors stress and corticosterone, in Two-way ANOVA. e Percentage change in BLA spine-density, normalized to respective controls, has been plotted along the x-axis and change in Anxiety Index of the same rats along the y-axis. While vehicle-treated stressed rats showed an increase in both spine-density and anxiety (diagonal solid arrow), these large changes in both cellular and behavioral measures was not seen with post-stress corticosterone treatment (diagonal dashed arrow). (Control + Vehicle, N = 6 rats, Stress + Vehicle, N = 7 rats, Control + Corticosterone, N = 6 rats, Stress + Corticosterone, N = 7 rats).

Fig. 4. Post-stress corticosterone treatment leads to a reduction in stress-induced increase in serum corticosterone levels.

a Following 2 h of immobilization stress (day 0), rats received either corticosterone or vehicle in their drinking water bottle 24 h later (day 1). Treatment was carried out for a period of 12 h (noon to midnight). 12 h after the end of corticosterone treatment, rats were sacrificed on day 2 and serum levels of corticosterone were quantified with an ELISA. b Day 2: Vehicle-treated stressed rats showed significantly elevated levels of serum corticosterone. In comparison, corticosterone treatment causes a significant reduction in serum corticosterone on day 1, although levels are higher than corticosterone-treated control rats. (Control + Vehicle, N = 7 rats; Control + Corticosterone, N = 8 rats; Stress + Vehicle, N = 4 rats; Stress + Corticosterone, N = 5 rats). ^{Ψ Ψ Ψ Ψ}p < 0.0001 for ‘interaction’ between factors stress and corticosterone, in Two-way ANOVA. ****p < 0.0001 and *p < 0.05 in post-hoc Sidak’s test. c Day 1: Consumption of drinking water containing vehicle or corticosterone was measured per cage every 4 h between noon and midnight. Regular drinking water was replaced at the end of the treatment period. d Cumulative plot of consumption over time show that stressed and control rats consumed vehicle and e corticosterone to the same extent over the treatment period (Control + Vehicle, N = 8 cages; Control + Corticosterone, N = 6 cages; Stress + Vehicle, N = 9 cages; Stress + Corticosterone, N = 9 cages). f To quantify elevation in systemic levels of corticosterone, control rats were sacrificed at midnight after consumption of either vehicle or corticosterone during the treatment period. g Corticosterone treatment causes a significant elevation of serum levels of the glucocorticoid, in contrast to vehicle-treated individuals. ^####p < 0.0001 in Unpaired t-test (Control + Vehicle, N = 8 rats; Control + Corticosterone, N = 5 rats). h Summary of findings. (Top) Stressed rats treated with vehicle 24 h after stress show elevated levels of corticosterone, which is followed by enhanced anxiety-like behavior and elevated spine-density in the BLA 10 days later. (Bottom) By contrast, when corticosterone is administered 24 h after stress, this prevents the increase in corticosterone, and also the delayed increase in anxiety-like behavior and BLA spine-density 10 days later. CORT Corticosterone.