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. 2023 Nov 10:28:100587.
doi: 10.1016/j.ynstr.2023.100587. eCollection 2024 Jan.

Genomic glucocorticoid receptor effects guide acute stress-induced delayed anxiety and basolateral amygdala spine plasticity in rats

Leonardo S Novaes  1 , Leticia M Bueno-de-Camargo  1 , Amadeu Shigeo-de-Almeida  1 , Vitor A L Juliano  1 , Ki Goosens  2 , Carolina D Munhoz  1
Affiliations

Genomic glucocorticoid receptor effects guide acute stress-induced delayed anxiety and basolateral amygdala spine plasticity in rats

Leonardo S Novaes et al. Neurobiol Stress. .

Abstract

Anxiety, a state related to anticipatory fear, can be adaptive in the face of environmental threats or stressors. However, anxiety can also become persistent and manifest as anxiety- and stress-related disorders, such as generalized anxiety or post-traumatic stress disorder (PTSD). In rodents, systemic administration of glucocorticoids (GCs) or short-term restraint stress induces anxiety-like behaviors and dendritic branching within the basolateral complex of the amygdala (BLA) ten days later. Additionally, increased arousal-related memory retention mediated by elevated GCs requires concomitant noradrenaline (NE) signaling, both acting in the BLA. It is unknown whether GCs and NE play a role in the delayed acute stress-induced effects on behavior and BLA dendritic plasticity. Here, inhibiting corticosterone (CORT) elevation during 2 h of restraint stress prevents stress-induced increases in delayed anxiety-like behavior and BLA dendritic spine density in rats. Also, we show that the delayed acute stress-induced effects on behavior and morphological alterations are critically dependent on genomic glucocorticoid receptor (GR) actions in the BLA. Unlike CORT, the pharmacological enhancement of NE signaling in the BLA was insufficient to drive delayed anxiety-related behavior. Nonetheless, the delayed anxiety-like behavior ten days after acute stress requires NE signaling in the BLA during stress exposure. Therefore, we define the essential roles of two stress-related hormones for the late stress consequences, acting at two separate times: CORT, via GR, immediately during stress, and NE, via beta-adrenoceptors, during the expression of delayed anxiety.

Keywords: Amygdala; Anxiety; Behavior; Glucocorticoid signaling; Neuroplasticity; Restraint.

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Conflict of interest statement

None.

Figures

Fig. 1
Fig. 1
CORT synthesis inhibition during stress prevented the delayed emergence of anxiety-like behavior. (A) Schematic representation of the experimental design showing the timeline of vehicle or metyrapone (Met) injections, the stress submission, and the EPM behavioral test. One cohort of animals was euthanized immediately after the end of the stress session. (B) Previous administration of Met prevented the stress-induced increase in CORT plasma levels measured immediately after stress (n = 6–7 animals for each group, two-way ANOVA: stress F1, 21 = 40.34, P < 0.0001; Met F1, 21 = 22.81, P = 0.0001; stress-Met interaction F1, 21 = 44.35, P < 0.0001). (C and D) Stressed animals treated with vehicle exhibited reduced open arm entries and time spent in the EPM test compared to vehicle-treated non-stressed animals and stressed and non-stressed Met-treated animals (C; n = 10–12 animals for each group, two-way ANOVA: stress F1, 40 = 4.692, P = 0.0363; Met F1, 40 = 3.23, P = 0.0799; stress-Met interaction F1, 40 = 8.9, P = 0.0048; D; n = 10–12 animals for each group, two-way ANOVA: stress F1, 39 = 8.712, P = 0.0053; Met F1, 39 = 5.783, P = 0.0210; stress-Met interaction F1, 39 = 2.352, P = 0.1332). (E) Stress increased the anxiety index in vehicle-treated but not in Met-treated rats (n = 10–12 animals for each group, two-way ANOVA: stress F1, 39 = 6.85, P = 0.0126; Met F1, 39 = 3.786, P = 0.0589; stress-Met interaction F1, 39 = 9.282, P = 0.0041). (F) Neither stress nor Met administration influenced the animal's locomotor activity in the EPM (n = 9–12 animals for each group, two-way ANOVA: stress F1, 34 = 2.388, P = 0.1315; Met F1, 34 = 3.78, P = 0.0602; stress-Met interaction F1, 34 = 0.1839, P = 0.6707). (G) Representative tracking plots for the average motion of each experimental group in the EPM. Results are represented as the mean ± SEM. Tukey's multiple comparisons post-hoc test. Significance differences between groups are indicated as * P < 0.05; **P < 0.01; and ***P < 0.001.
Fig. 2
Fig. 2
Pre-stress Met administration prevented the stress-induced increase in BLA dendritic spine density. (A–D) Stressed vehicle-treated animals showed an increase in spine density, an effect prevented by pre-stress Met administration in the primary (A; n = 15–20 neurons for each group, four animals per group, two-way ANOVA: stress F1, 69 = 9.351, P = 0.0032; Met F1, 69 = 7.786, P = 0.0079; stress-Met interaction F1, 69 = 7.72, P = 0.0070), secondary (B; n = 15–20 neurons for each group, two-way ANOVA: stress F1, 69 = 9.871, P = 0.0025; Met F1, 69 = 20.65, P < 0.0001; stress-Met interaction F1, 69 = 8.895, P = 0.0038), tertiary (C; n = 15–20 neurons for each group, two-way ANOVA: stress F1, 69 = 42.17, P < 0.0001; Met F1, 69 = 39.75, P < 0.0001; stress-Met interaction F1, 69 = 9.809, P = 0.0025), and quaternary (D; n = 14–20 neurons for each group, two-way ANOVA: stress F1, 65 = 18.62, P < 0.0001; Met F1, 65 = 15.92, P = 0.0002; stress-Met interaction F1, 65 = 6.267, P = 0.0148) dendrite segments compared to non-stressed animals. (Top, left) Representative photomicrography of a BLA's secondary dendrite segment. (Scale bar, 10 μm). Results are represented as the mean ± SEM. Tukey's multiple comparisons post-hoc test). Significant differences between groups are indicated as **P < 0.01 and ***P < 0.001.
Fig. 3
Fig. 3
dnGR overexpression in the BLA prevented the delayed emergence of stress-induced anxiety-like behavior. (A, left) Schematic representation of the experimental design shows the virus injection timeline into BLA (GFP or dnGR), the stress submission, and the EPM behavioral test. A cohort of animals was euthanized immediately after the end of the stress session. (Right) Representative figure showing the viral injection into BLA and photomicrography of an infected BLA (GFP, green). (Scale bar, 100 μm). CeA: central nucleus of the amygdala. (B) GFP-stressed animals exhibited reduced open arm entries in the EPM test compared to GFP-non-stressed and stressed and non-stressed animals overexpressing dnGR in the BLA (n = 10–12 animals for each group, two-way ANOVA: stress F1, 39 = 7.527, P = 0.0091; dnGR F1, 39 = 2.612, P = 0.1114; stress-dnGR interaction F1, 39 = 6.724, P = 0.0133). (C) GFP-stressed animals spent less time in the EPM open arms than GFP-non-stressed animals. dnGR animals (stressed and non-stressed) exhibited no differences in time spent in open arms compared to GFP non-stressed animals (n = 10–12 animals for each group, two-way ANOVA: stress F1, 39 = 6.595, P = 0.0142; dnGR F1, 39 = 0.6368, P = 0.4297; stress-dnGR interaction F1, 39 = 8.864, P = 0.0050). (D) Stress increased the anxiety index in GFP-expressing but not in dnGR-expressing rats (n = 10–12 animals for each group, two-way ANOVA: stress F1, 39 = 7.943, P = 0.0075; dnGR F1, 39 = 2.06, P = 0.1592; stress-dnGR interaction F1, 39 = 8.047, P = 0.0072). (E) Neither stress nor BLA virus infection influenced the animal's locomotor activity in the EPM (n = 10–12 animals for each group, two-way ANOVA: stress F1, 42 = 0.212, P = 0.6476; dnGR F1, 42 = 2.257, P = 0.1405; stress-dnGR interaction F1, 42 = 2.283, P = 0.1383). (F) Representative tracking plots for the average motion of each experimental group in the EPM. (G) Acute restraint stress increases CORT plasma levels in GFP- and dnGR-overexpressing animals immediately after the stressor stimulus (n = 6–9 animals for each group, two-way ANOVA: stress F1, 25 = 36.25, P < 0.0001; dnGR F1, 25 = 0.007222, P = 0.9330; stress-dnGR interaction F1, 25 = 0,07801, P = 0.7823). Results are represented as the mean ± SEM. Tukey's multiple comparisons post-hoc test. Significant differences between groups are indicated as * P < 0.05, **P < 0.01, and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
dnGR expression in the BLA prevented the stress-related increase in BLA's dendritic spine density. (A–C) GFP-stressed animals showed an increase in spine density, an effect prevented by the dnGR overexpressing in the BLA in the primary (A; n = 19–27 neurons for each group, four animals per group, two-way ANOVA: stress F1, 83 = 17.32, P < 0.0001; dnGR F1, 83 = 0.188, P = 0.6657; stress-dnGR interaction F1, 83 = 0.02408, P = 0.8771), secondary (B; n = 19–27 neurons for each group, two-way ANOVA: stress F1, 85 = 10.49, P = 0.0017; dnGR F1, 85 = 10.34, P = 0.0018; stress-dnGR interaction F1, 85 = 0.6021, P = 0.4399), and tertiary (C; n = 15–25 neurons for each group, two-way ANOVA: stress F1, 69 = 16.8, P = 0.0001; dnGR F1, 69 = 8.421, P = 0.0050; stress-dnGR interaction F1, 69 = 7.85, P = 0.0066) dendrite segments compared to non-stressed animals. (Top, left) Representative photomicrography of a BLA's secondary dendrite segment. (Scale bar, 10 μm). Results are represented as the mean ± SEM. Tukey's multiple comparisons post-hoc test. Significant differences between groups are indicated as * P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 5
Fig. 5
Intra-BLA dexamethasone administration promotes an anxiety-like behavior ten days after infusion. (A, left) Schematic representation of the experimental design showing the timeline of dexamethasone (Dex) or vehicle injection into BLA, the stress submission, and the behavioral tests. (Right) Representative figure and photomicrography showing a representative location of cannulae and injection needle tips into BLA. (Scale bar, 100 μm); (B–D) Intra-BLA dexamethasone administered animals exhibited reduced open arms entries (B; n = 8 animals per group, two-tailed student's t-test: P = 0.0482) and time spent (C; n = 7–8 animals per group, two-tailed student's t-test: P = 0.0285), as well as increased anxiety index (D; n = 7–8 animals per group, two-tailed student's t-test: P = 0.0127) in the EPM test comparing to vehicle administered animals. (E) Intra-BLA dexamethasone promoted a delayed reduced motor activity in the EPM test (n = 8 animals per group, two-tailed student's t-test: P = 0.0048). (F) Intra-BLA dexamethasone administration promoted a delayed decrease in time spent in the lit compartment comparing to vehicle administered animals in the light/dark (L/D) test (n = 7 animals per group, two-tailed student's t-test: P = 0.0369). (G) Representative tracking plots for the average motion of each experimental group in the EPM. Results are represented as mean ± SEM. Significance differences between groups are indicated as * P < 0.05.
Fig. 6
Fig. 6
Intra-BLA yohimbine administration promotes an acute, but not delayed, anxiety-like behavior. (A, left) Schematic representation of the experimental design showing the timeline of yohimbine or saline injection into BLA and the behavioral tests. (Right) Representative figure and photomicrography showing a representative location of cannulae and injection needle tips in the BLA. (B–C) Animals that received intra-BLA yohimbine exhibited reduced time spent (B; n = 7 animals per group, two-tailed student's t-test: P = 0.0225) and entries (C; n = 7 animals per group, two-tailed student's t-test: P = 0.0234) in the center area of the OF. (D) Intra-BLA yohimbine promoted an acute decrease in motor activity in the OF (n = 7 animals per group, two-tailed student's t-test: P = 0.00462). (E) Representative tracking plots for the average motion of each experimental group in the OF. (F–G) There were no differences in the open arms entries (F; n = 7–8 animals per group, two-tailed student's t-test: P = 0.9058) and time spent (G; n = 7–8 animals per group, two-tailed student's t-test: P = 0.8491) in the EPM test of BLA yohimbine administered animals comparing to saline administered animals. (H) Intra-BLA yohimbine did not influence the animal's locomotor activity in the EPM (n = 7–8 animals per group, two-tailed student's t-test: P = 0.1617). (I) Representative tracking plots for the average motion of each experimental group in the EPM. Results are represented as mean ± SEM. Significance differences between groups are indicated as * P < 0.05.
Fig. 7
Fig. 7
Pre-EPM test propranolol administration in the BLA prevented the delayed manifestation of the stress-induced anxiety-like behavior. (A, left) Schematic representation of the experimental design shows the propranolol or saline injection timeline into BLA, the stress submission, and the EPM test. (Right) Representative figure and photomicrograph showing the representative location of cannulae and injection needle tips into the BLA. (B and C) Stressed animals treated with saline exhibited reduced open arm entries and time spent in the EPM test compared to saline-treated non-stressed animals and stressed and non-stressed propanolol-treated animals (B; n = 8–11 animals for each group, two-way ANOVA: stress F1, 34 = 9.403, P = 0.0042; propranolol F1, 34 = 2.663, P = 0.1119; stress-propranolol interaction F1, 34 = 8.800, P = 0.0055; C; n = 8–11 animals for each group, two-way ANOVA: stress F1, 33 = 2.791, P = 0.1042; propranolol F1, 33 = 4.866, P = 0.0345; stress-propranolol interaction F1, 33 = 4.157, P = 0.0495). (D) Stress increased the anxiety index in saline-treated but not in propranolol-treated rats (n = 8–12 animals for each group, two-way ANOVA: stress F1, 32 = 7.739, P = 0.0090; propranolol F1, 32 = 2.723, P = 0.1087; stress-propranolol interaction F1, 32 = 8.960, P = 0.0053). Results are represented as the mean ± SEM. Tukey's multiple comparisons post-hoc test. Significance differences between groups are indicated as * P < 0.05 and ***P < 0.001.
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References

    1. Abdullahi P.R., Raeis-Abdollahi E., Sameni H., Vafaei A.A., Ghanbari A., Rashidy-Pour A. Protective effects of morphine in a rat model of post-traumatic stress disorder: role of hypothalamic-pituitary-adrenal axis and beta- adrenergic system. Behav. Brain Res. 2020;395 - PubMed
    1. Arango-Lievano M., Borie A.M., Dromard Y., Murat M., Desarmenien M.G., Garabedian M.J., Jeanneteau F. Persistence of learning-induced synapses depends on neurotrophic priming of glucocorticoid receptors. Proc. Natl. Acad. Sci. U.S.A. 2019;116:13097–13106. - PMC - PubMed
    1. Arango-Lievano M., Lambert W.M., Bath K.G., Garabedian M.J., Chao M.V., Jeanneteau F. Neurotrophic-priming of glucocorticoid receptor signaling is essential for neuronal plasticity to stress and antidepressant treatment. Proc. Natl. Acad. Sci. U.S.A. 2015;112:15737–15742. - PMC - PubMed
    1. Aston-Jones G., Rajkowski J., Cohen J. Role of locus coeruleus in attention and behavioral flexibility. Biol. Psychiatr. 1999;46:1309–1320. - PubMed
    1. Belda X., Fuentes S., Labad J., Nadal R., Armario A. Acute exposure of rats to a severe stressor alters the circadian pattern of corticosterone and sensitizes to a novel stressor: relationship to pre-stress individual differences in resting corticosterone levels. Horm. Behav. 2020;126 - PubMed

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