Identification of a stress-responsive subregion of the basolateral amygdala in male rats

Abstract

The basolateral amygdala (BLA) is reliably activated by psychological stress and hyperactive in conditions of pathological stress or trauma; however, subsets of BLA neurons are also readily activated by rewarding stimuli and can suppress fear and avoidance behaviours. The BLA is highly heterogeneous anatomically, exhibiting continuous molecular and connectivity gradients throughout the entire structure. A critical gap remains in understanding the anatomical specificity of amygdala subregions, circuits, and cell types explicitly activated by acute stress and how they are dynamically activated throughout stimulus exposure. Using a combination of topographical mapping for the activity-responsive protein FOS and fiber photometry to measure calcium transients in real-time, we sought to characterize the spatial and temporal patterns of BLA activation in response to a range of novel stressors (shock, swim, restraint, predator odour) and non-aversive, but novel stimuli (crackers, citral odour). We report four main findings: (1) the BLA exhibits clear spatial activation gradients in response to novel stimuli throughout the medial-lateral and dorsal-ventral axes, with aversive stimuli strongly biasing activation towards medial aspects of the BLA; (2) novel stimuli elicit distinct temporal activation patterns, with stressful stimuli exhibiting particularly enhanced or prolonged temporal activation patterns; (3) changes in BLA activity are associated with changes in behavioural state; and (4) norepinephrine enhances stress-induced activation of BLA neurons via the ß-noradrenergic receptor. Moving forward, it will be imperative to combine our understanding of activation gradients with molecular and circuit-specificity.

Fig. 1. Novel psychological stressors induce a graded neuroendocrine response.

A Overview of experimental procedures. B Plasma CORT 30min following stimulus onset (n = 8-10 per group; H = 45.26, p < 0.0001). C Plasma CORT 90min following stimulus onset (n = 6–9 per group; H = 36.68, p < 0.0001). D Area-under-the-curve (AUC) of plasma CORT from t30 to t90 (n = 6–10 per group; H = 40.77, p < 0.0001). All comparisons were performed using the Kruskal-Wallis test. Post-hoc comparisons were performed using Dunn’s multiple comparisons test to the naive condition in (B) and (C), and between all conditions in (D). Error bands represent mean +/- SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2. Spatial gradients of activation in response to novel aversive and non-aversive stimuli.

A Representative coronal image of FOS+ neurons in naïve condition (left) or following exposure to restraint (right); red = FOS, blue = DAPI; scalebar = 500 um. Brightness was adjusted equally to enhance contrast. B Average density of FOS+ neurons in the BLA following exposure to each novel stimulus (n = 5–10 per group; H = 22.68, p = 0.0009). C BLA subdivisions at AP -2.80: lateral amygdala (LA), lateral basal amygdala (LBA), and medial basal amygdala (mBA). D Heatmaps representing density of normalized FOS+ expression in 25 um × 25 um bins at AP −2.80. E Heat matrix representing average normalized density of FOS+ cells in each subregion (rows) in response to each stimulus (columns). Significance is to the average normalized density of the naive condition for each respective subregion (n = 5–10 per group, as shown in Fig. 2F-H). F Average FOS+ density in the LA following exposure to each novel stimulus (n = 5–10 per group; H = 21.40, p = 0.0016). G Average FOS+ density in the mBA following exposure to each novel stimulus (n = 5–10 per group; H = 24.92, p = 0.0004). H Average FOS+ density in the LA following exposure to each novel stimulus (n = 5–10 per group; H = 19.23, p = 0.0038). FOS+ neurons were quantified across the entire BLA (AP -2.12 to AP -3.60) and normalized to average BLA dimensions at each AP position and for number of slices. Error bands represent mean +/- SEM. Data were analyzed using Kruskal-Wallis test followed by Dunn’s post-hoc comparison to the naive condition; ^p = 0.0598; & p = 0.0708; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Compass represents orientation of slice (D = dorsal; L = lateral). Scalebar = 250 um.

Fig. 3. Distinct novel stimuli elicit distinct temporal patterns of activation in the BLA.

A Overview of experimental procedures. Blue represents exposure to stimulus; gray epoch indicates 120sec baseline epoch; green epochs indicate 5min epochs following stimulus onset analyzed in (D)–(I); dashed vertical lines indicate stimulus initiation or termination. B Representative image of GCaMP6s expression and fiber optic tip placement. Scalebar = 250 um; dashed lines delineate BLA. Compass represents orientation of slice (D = dorsal; M = medial). Brightness was adjusted to enhance contrast. C Representative images of GCaMP6s expression (green) and locations of fiber optic tips (-) for each group. Some animals were exposed to multiple conditions. D–I GCaMP6s fluorescence in BLA CaMKII+ neurons following exposure to each stimulus (n = 4–7 per group). Values were normalized to the mean z-score of the 120sec baseline epoch immediately preceding disruption of the animal. Data were plotted after averaging z-score into consecutive 10sec bins. Dashed vertical line indicates moment animals were exposed to the discrete stimulus. Dark green line represents mean z-score of all animals in that condition; light green bands represent SEM. J Mean change from baseline of z-score in the first minute following stimulus onset. Data were analyzed using ordinary one-way ANOVA (n = 4–7 per group; F(5,27) = 3.238, p = 0.0203) followed by Fisher’s LSD to the citral condition. K Average z-score of baseline vs. 3x5min epochs following stimulus onset (n = 4–7 per group). Data were analyzed using RM one-way ANOVA followed by Fisher’s LSD to the baseline epoch. Citral: F(1.304, 6.519) = 1.972, p = 0.2108; L Crackers: F(2.405, 12.03) = 3.083, p = 0.0762; M Bobcat: F(1.219, 7.313) = 10.23, p = 0.0120; N Swim: F(1.870, 7.479) = 4.237, p = 0.0606; O Restraint: F(1.165, 4.662) = 11.80, p = 0.0193; P Shock: F(1.402, 4.206) = 36.04, p = 0.0027. Error bars represent mean +/- SEM. #p = 0.0762; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4. Change in BLA activity is associated with transition in behavioural state.

A Average GCaMP6s fluorescence in BLA CaMKII+ neurons during swim stress in immobile vs mobile state. Data were analyzed using Wilcoxon matched-pairs signed rank test (n = 5 animals; p = 0.1250). B Left: GCaMP6s fluorescence in BLA CaMKII+ neurons surrounding the transition from immobile to mobile state during swim stress (n = 40 transitions from 5 animals); Right: Average GCaMP6s fluorescence in BLA CaMKII+ neurons during swim stress in 4sec epochs surrounding transition from immobile to mobile state (n = 40 trials from 5 animals). Data were averaged across trials and analyzed using a paired t-test (t(39) = 2.680, p = 0.0107). C Left: GCaMP6s fluorescence in BLA CaMKII+ neurons surrounding the transition from mobile to immobile state during swim stress (n = 49 transitions from 5 animals). Right: Average GCaMP6s fluorescence in BLA CaMKII+ neurons during swim stress in 4sec epochs surrounding transition from mobile to immobile state (n = 49 trials from 5 animals). Data were averaged across trials and analyzed using Wilcoxon matched-pairs signed rank test (p = 0.0113). D Average GCaMP6s fluorescence in BLA CaMKII+ neurons during shock stress when displaying each behaviour. Data were averaged for each animal and analyzed using a mixed-effects model (F(1.202, 3.205) = 15.13, p = 0.0257). E Left: GCaMP6s fluorescence in BLA CaMKII+ neurons surrounding the transition from immobile (freezing) to mobile state during shock stress (n = 37 transitions from 4 animals). Right: Average GCaMP6s fluorescence in BLA CaMKII+ neurons during shock stress in 4sec epochs surrounding transition from immobile to mobile state (n = 37 trials from 4 animals). Data were averaged across trials and analyzed using a paired t-test (t(36) = 4.883, p < 0.0001). F Left: GCaMP6s fluorescence in BLA CaMKII+ neurons surrounding the transition from mobile to immobile (freezing) state during shock stress (n = 44 transitions from 4 animals). Right: Average GCaMP6s fluorescence in BLA CaMKII+ neurons during swim stress in 4sec epochs surrounding transition from mobile to immobile state (n = 44 trials from 4 animals). Data were averaged across trials and analyzed using a paired t-test (t(43) = 5.818, p < 0.0001). Values were normalized to the mean z-score of the 120sec baseline epoch (see Fig. 3). Dashed vertical line indicates transitions between states. Dark line represents mean z-score of all animals in that condition; light bands represent SEM. Each transition was comprised of a minimum of 4 consecutive seconds of the first state followed by 4 consecutive seconds of the subsequent state. Error bars represent mean +/- SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.

Fig. 5. Propranolol reduces stress-induced activation of the basolateral amygdala.

A GRAB:NE fluorescence in BLA neurons during exposure to restraint stress (n = 7). Blue block indicates stress exposure; solid green represents mean; light green represents SEM. Traces were aligned to time animal was picked up. B Average GRAB:NE z-score of baseline vs. first 10 min of stress. Data were analyzed using a two-tailed Wilcoxon matched-pairs signed rank test (p = 0.0156). C Average GRAB:NE z-score of baseline vs. first min of stress. Data were analyzed using a two-tailed Wilcoxon matched-pairs signed rank test (p = 0.0156). D GCaMP6s fluorescence in BLA CaMKII+ neurons following exposure to restraint stress, with either saline or propranolol (10mg/kg i.p.) administered 30min prior to stress exposure (n = 6 per group). Traces were aligned to the moment the cage was first disturbed. For representation, data were averaged every 10sec epoch for each animal and plotted as a series of points. Dashed vertical lines indicate stress initiation and termination; solid dark lines represent mean; shaded boundaries represent SEM. E Average GCaMP6s z-score during entire 15min stress (n = 6 per group; t(10) = 2.251, p = 0.0481); Data were analyzed using an unpaired t-test; F Average GCaMP6s z-score during first min of stress; data were analyzed using the Mann Whitney test (n = 6 per group; p = 0.0043). G Average GCaMP6s z-score for the 20min epoch following stress (n = 5 per group; t(8) = 1.506, p = 0.1705); data were analyzed using an unpaired t-test. Error bars represent mean +/- SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.