Comparison of the activation of somatostatin- and neuropeptide Y-containing neuronal populations of the rat amygdala following two different anxiogenic stressors

Abstract

Rats exposed to the odor of a predator or to the elevated plus maze express fear behaviors without a prior exposure to either stimulus. The expression of innate fear provides for an ideal model of anxiety which can aid in the elucidation of brain circuits involved in anxiety-related behaviors. The current experiments compared activation of neuropeptide-containing neuronal populations in the amygdala of rats exposed to either the elevated plus maze (EPM; 5 min) versus home cage controls, or predator ferret odor versus butyric acid, or no odor (30 min). Sections of the brains were prepared for dual-labeled immunohistochemistry and counts of c-Fos co-localized with somatostatin (SOM) or neuropeptide Y (NPY) were made in the basolateral (BLA), central (CEA), medial (MEA) nuclei of the amygdala. Ferret odor and butyric acid exposure significantly decreased the percentage of SOM-positive neurons also immunoreactive for c-Fos in the anterior BLA compared to controls, whereas EPM exposure yielded a significant increase in the activation of SOM-positive neurons versus home cage controls. In the CEA, ferret odor and butyric exposure significantly decreased the percentage of SOM-positive neurons also immunoreactive for c-Fos compared to no-odor controls whereas EPM exposure yielded no change versus controls. In the MEA, both ferret odor exposure and EPM exposure resulted in increased SOM co-localized with c-Fos compared to control groups whereas NPY co-localized with c-Fos occurred following ferret odor exposure, but not EPM exposure. These results indicate that phenotypically distinct neuronal populations of the amygdala are differentially activated following exposure to different anxiogenic stimuli. These studies further elucidate the fundamental neurocircuitry of anxiety and could possibly explain the differential behavioral effects of predator versus novelty-induced stress.

Figure 1

Depiction of the anatomical levels of the amygdala analyzed in this study. Separate counts were made in the anterior (−1.80 to −2.30 mm from bregma) and posterior (−2.56 to −3.30 mm from bregma) portions of the basolateral/lateral (BLA), central (CEA), and medial (MEA) nuclei of the amygdala (Paxinos G. and Watson C, 1998).

Figure 2

Elevated plus maze exposure-induced changes in c-Fos expression in limbic brain regions. The data represent counts from single sections in the representative region. **P<0.01, *P<0.05 (N=7–8).

Figure 3

Odor exposure-induced changes in c-Fos expression of A) SOM+ and B) NPY+ neurons of the basolateral amygdala (BLA). (Top) Representative photomicrograph with SOM (brown) and c-Fos (blue/black) labeling in control (left) and ferret odor-exposed (right) rats. (Middle) Representative photomicrograph with NPY (brown) and c-Fos (blue/black) labeling in control (left) and ferret odor-exposed (right) rats. Exposure to butyric acid or ferret odor decreased the percentage of SOM-positive neurons with c-Fos in the BLA (Bottom, A) compared to control odor. **P<0.01, (N=5–7). The data represent counts from the anterior sections (A) and cumulative counts from anterior and posterior sections (B). Percent SOM or NPY with c-Fos neurons represent: (the total number of double-labeled neurons / total NPY or SOM-labeled neurons) * 100. Pictures are at 40× magnification. Scale bar represents approximately 50 microns. The solid arrow points to a single-labeled neuron (blue/black = c-Fos and brown = SOM or NPY), and the dotted arrow points to a double-labeled SOM/c-Fos neuron.

Figure 4

Elevated plus maze (EPM) exposure-induced changes in c-Fos expression of A) SOM+ and B) NPY+ neurons of the basolateral amygdala (BLA). (Top) Representative photomicrograph with SOM (brown) and c-Fos (blue/black) labeling in control (left) and EPM-exposed (right) rats. (Middle) Representative photomicrograph with NPY (brown) and c-Fos (blue/black) labeling in control (left) and EPM-exposed (right) rats. Exposure to the EPM increased the percentage of SOM-positive neurons with c-Fos in the BLA (Bottom, A) compared to controls. ***P<0.001 (N=4–5). The data represent cumulative counts from anterior and posterior sections. Percent SOM or NPY with c-Fos neurons represent: (the total number of double-labeled neurons / total NPY or SOM-labeled neurons) * 100. Pictures are at 40× magnification. Scale bar represents approximately 50 microns. The solid arrow points to a single-labeled neuron (blue/black = c-Fos and brown = SOM or NPY), and the dotted arrow points to a double-labeled SOM/c-Fos neuron.

Figure 5

Odor exposure-induced changes in c-Fos expression of A) SOM+ and B) NPY+ neurons of the central amygdala (CEA). (Top) Representative photomicrograph with SOM (brown) and c-Fos (blue/black) labeling in control (left) and ferret odor-exposed (right) rats. (Middle) Representative photomicrograph with NPY (brown) and c-Fos (blue/black) labeling in control (left) and ferret odor-exposed (right) rats. Exposure to butyric acid or ferret odor decreased the percentage of SOM-positive neurons with c-Fos in the CEA (Bottom, A) compared to control odor. **P<0.01, *P<0.05 (N=5–7). The data represent counts from the anterior sections (A) and cumulative counts from anterior and posterior sections (B). Percent SOM or NPY with c-Fos neurons represent: (the total number of double-labeled neurons / total NPY or SOM-labeled neurons) * 100. Pictures are at 40× magnification. Scale bar represents approximately 50 microns. The solid arrow points to a single-labeled neuron (blue/black = c-Fos and brown = SOM or NPY), and the dotted arrow points to a double-labeled SOM/c-Fos neuron.

Figure 6

Elevated plus maze (EPM) exposure-induced changes in c-Fos expression of A) SOM+ and B) NPY+ neurons of the basolateral amygdala (BLA). (Top) Representative photomicrograph with SOM (brown) and c-Fos (blue/black) labeling in control (left) and EPM-exposed (right) rats. (Middle) Representative photomicrograph with NPY (brown) and c-Fos (blue/black) labeling in control (left) and EPM-exposed (right) rats. Exposure to the EPM did not significantly alter percentage of SOM- or NPY-positive neurons with c-Fos in the CEA (Bottom, A and B) compared to controls. ***P<0.001 (N=4–5). The data represent cumulative counts from anterior and posterior sections (A and B). Percent SOM or NPY with c-Fos neurons represent: (the total number of double-labeled neurons / total NPY or SOM-labeled neurons) * 100. Pictures are at 40× magnification. Scale bar represents approximately 50 microns. The solid arrow points to a single-labeled neuron (blue/black = c-Fos and brown = SOM or NPY).

Figure 7

Odor exposure-induced changes in c-Fos expression of A) SOM+ and B) NPY+ neurons of the medial amygdala (MEA). (Top) Representative photomicrograph with SOM (brown) and c-Fos (blue/black) labeling in control (left) and ferret odor-exposed (right) rats. (Middle) Representative photomicrograph with NPY (brown) and c-Fos (blue/black) labeling in control (left) and ferret odor-exposed (right) rats. Exposure to ferret odor increased the percentage of SOM- and NPY-positive neurons with c-Fos in the MEA (Bottom, A and B) compared to control odor and butyric acid. **P<0.01, *P<0.05 (N=5–7). The data represent cumulative counts from anterior and posterior sections (A and B). Percent SOM or NPY with c-Fos neurons represent: (the total number of double-labeled neurons / total NPY or SOM-labeled neurons) * 100. Pictures are at 40× magnification. Scale bar represents approximately 50 microns. The solid arrow points to a single-labeled neuron (blue/black = c-Fos and brown = SOM or NPY), and the dotted arrow points to a double-labeled SOM/c-Fos neuron.

Figure 8

Elevated plus maze (EPM) exposure-induced changes in c-Fos expression of A) SOM+ and B) NPY+ neurons of the medial amygdala (MEA). (Top) Representative photomicrograph with SOM (brown) and c-Fos (blue/black) labeling in control (left) and EPM-exposed (right) rats. (Middle) Representative photomicrograph with NPY (brown) and c-Fos (blue/black) labeling in control (left) and EPM-exposed (right) rats. Exposure to the EPM increased the percentage of SOM-positive neurons with c-Fos in the MEA (Bottom, A) compared to controls. **P<0.01 (N=4–5). The data represent cumulative counts from anterior and posterior sections. Percent SOM or NPY with c-Fos neurons represent: (the total number of double-labeled neurons / total NPY or SOM-labeled neurons) * 100. Pictures are at 40× magnification. Scale bar represents approximately 50 microns. The solid arrow points to a single-labeled neuron (blue/black = c-Fos and brown = SOM or NPY), and the dotted arrow points to a double-labeled SOM/c-Fos neuron.