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. 2004 Mar 23;101(12):4280-5.
doi: 10.1073/pnas.0400794101. Epub 2004 Mar 15.

Substance P in the Medial Amygdala: Emotional Stress-Sensitive Release and Modulation of Anxiety-Related Behavior in Rats

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Free PMC article

Substance P in the Medial Amygdala: Emotional Stress-Sensitive Release and Modulation of Anxiety-Related Behavior in Rats

Karl Ebner et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Increasing evidence implicates the substance P (SP)/neurokinin-1 receptor system in anxiety and depression. However, it is not known whether emotional stimulation alters endogenous extracellular SP levels in brain areas important for processing of anxiety and mood, a prerequisite for a contribution of this neuropeptide system in modulating these behaviors. Therefore, we examined in rats whether the release of SP is sensitive to emotional stressors in distinct subregions of the amygdala, a key area in processing of emotions. By using in vivo micropush-pull superfusion and microdialysis techniques, we found a pronounced and long-lasting increase (150%) in SP release in the medial nucleus of the amygdala (MeA), but not in the central nucleus of the amygdala, in response to immobilization stress. SP release in the MeA was transiently enhanced (40%) in response to elevated platform exposure, which is regarded as a mild emotional stressor. Immobilization enhanced the anxiety-related behavior evaluated in the subsequently performed elevated plus-maze test. Bilateral microinjections of the neurokinin-1 receptor antagonist [2-cyclopropoxy-5-(5-(trifluoromethyl)tetrazol-1-yl)benzyl]-(2-phenylpiperidin-3-yl)amine into the MeA blocked the stress-induced anxiogenic-like effect, supporting a functional significance of enhanced SP release. In unstressed rats, the neurokinin-1 receptor antagonist displayed no significant anxiolytic effect but reversed the anxiogenic effect of SP microinjected into the MeA. Our findings identify the MeA as a critical brain area for the involvement of SP transmission in anxiety responses and as a putative site of action for the recently discovered therapeutic effects of SP antagonists in the treatment of stress-related disorders.

Figures

Fig. 1.
Fig. 1.
Schematic drawings showing the reconstructed localizations of the push–pull, microdialysis, and microinjection cannulae. Filled and open triangles represent the tips of the push–pull cannulae and microdialysis membranes, respectively, in the CeA and MeA. Filled circles indicate the microinjection sites in the MeA and outside the amygdala in the internal capsule. The numbers on each section indicate the distance from bregma. The drawings of coronal sections were derived from the atlas of Paxinos and Watson (30).
Fig. 2.
Fig. 2.
Effects of immobilization (IM) stress (A) and superfusion with 100 mM K+ (B) on the content of SP in 20-min push–pull superfusates collected consecutively in either the MeA (Upper) or CeA (Lower) of freely moving rats. Animals were immobilized for 20 min during sampling of the fifth superfusate. After 120 min, the superfusion medium was changed to aCSF containing 100 mM KCl. Animals assigned to the control group (unstressed) remained undisturbed and were not exposed to immobilization. Data (means ± SEM) are expressed as the percentage of the averaged basal values (dotted line). *, P < 0.05; **, P < 0.01 vs. superfusates 1–4; #, P < 0.05; ##, P < 0.01 vs. unstressed controls; +, P < 0.05; ++, P < 0.01 vs. two samples preceding KCl (Newman–Keuls post hoc test).
Fig. 3.
Fig. 3.
Effects of elevated platform (EPF) exposure on the content of SP in 30-min microdialysates collected consecutively in the MeA of freely moving rats. Animals were exposed to the platform for 15 min during sampling of the fourth superfusate. After stress exposure, animals were superfused for a further 90 min. Data (means ± SEM) are expressed as the percentage of the averaged basal values (dotted line). *, P < 0.05 vs. dialysates 1–3 (Fisher's LSD post hoc test).
Fig. 4.
Fig. 4.
Effects of SP and Compound A (ComA) on anxiety-related behavior in unstressed rats. The percentage of the time spent on the open arms (A) and number of entries into the enclosed arms (B) were recorded, starting 5 min after microinjections into the MeA. Data are means ± SEM. Vehicle (Veh; aCSF, n = 9); SP, 0.1 pmol (n = 8), 1 pmol (n = 12), and 10 pmol (n = 4); ComA, 100 pmol (n = 8) and 1 nmol (n = 3); ComA + SP 1.0, ComA (100 pmol, n = 8) preinjected 15 min before SP injection (1 pmol). *, P < 0.05 vs. vehicle treated control group; #, P < 0.05 ComA + SP 1.0 vs. SP 1.0 (Mann–Whitney U test).
Fig. 5.
Fig. 5.
Effect of Compound A (ComA) on anxiety-related behavior in prestressed rats. Rats were prestressed by exposure to 20 min of immobilization 60 min before microinjection of vehicle (Veh; aCSF, n = 9) or ComA (1 nmol, n = 7) into the MeA. Unstressed animals injected with vehicle (n = 6) were used as further controls. The percentage of the time spent on the open arms (A) and number of entries into the enclosed arms (B) were recorded, starting 5 min after microinjections. Data are means ± SEM. *, P < 0.05 vs. unstressed animals; #, P = 0.01 vs. vehicle-treated prestressed animals (Mann–Whitney U test).

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