Inhibition of the central extended amygdala by loud noise and restraint stress

Abstract

It is well established that the central nucleus of the amygdala (CEA) is involved in responses to stress, fear and anxiety. Many studies have used c-fos expression to map the brain's response to processive stress, but curiously the CEA generally is not highly activated. We have previously shown that exposure to a novel vs. home environment reduces amphetamine-induced activation of the lateral CEA (CEAl) and the oval nucleus of the bed nucleus of the stria terminalis (BSTov). This is consistent with the idea that processive stress inhibits neurons in these nuclei. We have tested this hypothesis by exposing rats to noise, at a range of intensities from non-stressful to stressful, or to restraint conditions, immediately after a remote injection of amphetamine, 2 mg/kg i.p., or interleukin-1beta (IL-1beta) 0.5 microg/kg i.p. (used to obtain a level of c-fos mRNA against which to measure inhibition). In keeping with our hypothesis, amphetamine- or IL-1beta-induced c-fos and zif-268 mRNA were significantly decreased in the CEAl and BSTov under conditions of loud noise or restraint stress compared with control conditions. This inhibition does not require a stress-induced rise in corticosterone because data were similar in animals that had been adrenalectomized with a low-dose corticosterone replacement. As both the CEAl and BSTov are highly gamma-aminobutyric acid (GABA) -ergic and project to the medial CEA (CEAm), their inhibition potentially causes an increased input to the CEAm. As the CEAm is a major output nucleus of the amygdala, this could have important consequences within the neural circuitry controlling responses to processive stress.

Fig. 1

Digital images of X-ray films to show c-fos mRNA in the BSTov (A, D, G, J, M and P), CEA (B, E, H, K, N and Q) and PVN (C, F, I, L, O and R) after exposure to background noise conditions (60 dBA; A–C), stressful noise conditions (30 min at 100 dBA; D–F), unhandled cage control (naïve; G–I), restraint stress for 30 min (J–L), a remote injection of saline (M–O) or a remote injection of amphetamine, 2 mg/kg i.p. (P–R). Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus. Arrows indicate these respective regions. Note the very low level of c-fos mRNA in the BSTov and CEA of noise- and restraint-stressed animals.

Fig. 2

Graphs to show the relative levels of (A) c-fos and (B) zif-268 mRNA in the BSTov, CEA and PVN, 50 min after a remote injection of amphetamine (2 mg/kg i.p.) under conditions of background (60 dBA) or stressful (100 dBA) white noise. Values represent means + SEM. *P < 0.05; **P < 0.01; ***P < 0.001 with respect to the relevant 60 dBA group. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus.

Fig. 3

Graphs to show the relative levels of c-fos mRNA in the (A) BSTov, (B) CEA and (C) PVN at 30, 60 or 90 min after a remote injection of amphetamine (2 mg/kg i.p.) under conditions of background (60 dBA) or stressful (100 dBA) white noise. The level of c-fos mRNA 30 min after a saline injection under background noise conditions is shown to illustrate baseline c-fos mRNA levels in this experimental set-up. Values represent means + SEM. ***P < 0.001 with respect to the time-equivalent 60 dBA group. †P < 0.05, †††P < 0.001 with respect to the 30 min saline/60 dBA group. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus.

Fig. 4

Graphs to show the relative levels of c-fos mRNA in the (A) BSTov, (B) CEA and (C) PVN 30 min after a remote injection of amphetamine (2 mg/kg i.p.) under conditions of background (60 dBA) or increasing intensities of white noise. Values represent means + SEM. *P < 0.05; **P < 0.01; ***P < 0.001 with respect to the relevant 60 dBA group. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus.

Fig. 5

Digital images of X-ray films to show c-fos mRNA in the BSTov (A and D), CEA (B and E) and PVN (C and F) 30 min after a remote injection of amphetamine (2 mg/kg i.p.) under conditions of background (60 dBA; A–C) or stressful (100 dBA; D–F) levels of white noise. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus.

Fig. 6

Photomicrographs to illustrate the distribution of c-fos mRNA within the BSTov (A and B) and CEA (C and D) 30 min after an amphetamine injection, 2 mg/kg i.p. under conditions of background (60 dBA; A and C) or stressful (100 dBA; B and D) white noise. Note that within the CEA, amphetamine-induced c-fos mRNA expression is largely restricted to the lateral division. Abbreviations: ac, anterior commissure; BLA, basolateral nucleus of the amygdala; BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; CP, caudate putamen; int, internal capsule; opt, optic tract; PVN, paraventricular nucleus of the hypothalamus; st, stria terminalis. Scale bar, 300 μm.

Fig. 7

Graph to show the relative levels of c-fos mRNA in the BSTov, CEA and PVN 30 min after a remote injection of amphetamine (2 mg/kg i.p.) under home-cage or restraint stress conditions. Values represent means + SEM. ***P < 0.001 with respect to control values. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus.

Fig. 8

Graph to show the relative levels of c-fos mRNA in the BSTov, CEA and PVN 30 min after a remote injection of IL-1β (0.5 μg/kg i.p.) under 60 dBA (control) or 100 dBA (stressful) noise conditions. Values represent means + SEM. *P < 0.05; **P < 0.01 with respect to control values. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus.

Fig. 9

Graphs to show the effect of adrenalectomy and low-dose corticosterone replacement (ADX) vs. sham surgery on the relative levels of c-fos mRNA in the (A) BSTov, (B) CEA and (C) PVN 30 min after a remote injection of amphetamine (2 mg/kg i.p.) under conditions of background (60 dBA) or stressful (100 dBA) levels of white noise. Values represent means + SEM. *P < 0.05; **P < 0.01; ***P < 0.001 with respect to the 60 dBA sham group; †P < 0.05; ††P < 0.01 with respect to the 60 dBA ADX group; ##P < 0.01 with respect to the 100 dBA sham group. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEA, central nucleus of the amygdala; PVN, paraventricular nucleus of the hypothalamus.

Fig. 10

Schematic diagram to show possible mechanisms for processive stress inhibition of c-fos mRNA expression (−) in the BSTov and CEAl by an activating stimulus (+). Model A: activating stimuli use a single pathway to stimulate the CEAl and BSTov, and processive stress inhibits neurons along this activational pathway. Model B: activating stimuli use different pathways to stimulate the CEAl and BSTov, and processive stress inhibits neurons along these different activational pathways. Model C, on which our working hypothesis is based: activating stimuli use different pathways to stimulate the CEAl and BSTov, but stress directly inhibits the CEAl and BSTov through a separate pathway, thus setting up a competition between activation and inhibition. Abbreviations: BSTov, bed nucleus of the stria terminalis, oval subdivision; CEAl/CEAm, central nucleus of the amygdala, lateral/medial division; PVN, paraventricular nucleus of the hypothalamus.