Anxiolytic effects of 5-HT₁A receptors and anxiogenic effects of 5-HT₂C receptors in the amygdala of mice

Abstract

The aim of the present study is to test a hypothesis that 5-HT(1A) and 5-HT(2C) receptors in the amygdala play an important role in the regulation of anxiety behaviors. We examined alterations in anxiety-like behaviors after manipulation of the expression of 5-HT(1A) and 5-HT(2C) receptors in the amygdala using recombinant adenovirus approaches. Recombinant adenoviruses containing a 5-HT(1A) promoter-controlled 5-HT(1A) antisense sequence or a 5-HT(2C) promoter-controlled 5-HT(2C) sense sequence were injected into the amygdala. Elevated plus-maze (EPM) and open field tests were conducted to determine anxiety-like behavior and locomotor activity. Reductions in the expression of 5-HT(1A) receptors in the amygdala significantly attenuated the time spent in the open arms of EPM and time spent in the center of an open field. Reduction in the percent of time spent in the open arms of EPM is negatively correlated with the density of 5-HT(1A) receptors in the central amygdala. On the other hand, increased expression of 5-HT(2C) receptors reduced the time spent in the open arms of EPM and time spent in the center of an open field. The reductions in the time spent and distance traveled in the open arms of EPM were correlated to the density of 5-HT(2C) receptors in the basolateral nucleus of amygdala. These data suggest that amygdaloid 5-HT(1A) receptors produce anxiolytic and 5-HT(2C) receptors produce anxiogenic effects. Together, the present results demonstrate the important role of the amygdaloid 5-HT(1A) and 5-HT(2C) receptors in the regulation of anxiety-like behaviors. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Figure 1

Generation and evaluation of recombinant adenovirus containing 5-HT_2C receptor sense sequence (P_2C-5HT_2C-S-Ad). A: Scheme for the 5-HT_2C sense construct in P_2C-5HT_2C-S-Ad. The construct includes a proximal 5-HT_2C promoter region (1247 bp upstream of the start site of 5-HT_2C mRNA (= 1) and a 2200 bp 5-HT_2C cDNA sequence, including 5′ UTR (1–690) and coding region (691–2070, Access No. NM_008312). The arrow heads indicate the direction of gene expression. The arrow lines indicate the primers used to amplify 5-HT_2C promoter and mRNA regions. The lines represent the PCR products generated from the primers. The promoter fragment was inserted into Kpn I and Sal I sites and then 5-HT_2C receptor sequence was ligated into Sal I and Xho I sites. B: Test for 5-HT_2C promoter-controlled expression of 5-HT_2C receptors. Autoradiography of ¹²⁵I-DOI binding shows 5-HT_2C receptor expression after injection of P_2C-5HT_2C-S-Ad into the amygdala and cerebellum. The viral infection was indicated by the expression of green fluorescent protein (GFP) in an adjacent section, as indicated by the box. C: Time course of P_2C-5HT_2C-S-Ad-induced expression of 5-HT_2C receptors. Autoradiography of ¹²⁵I-DOI binding shows the expression of 5-HT_2C receptor 3, 7 and 10 days after injection of P_2C-5HT_2C-S-Ad (indicated by arrows).

Figure 2

The amygdaloid injection of P_1A-5-HT_1A-AS-Ad reduced the density of 5-HT_1A receptors in the nuclei of the amygdala. A: An example of autoradiography of ¹²⁵I-MPPI binding for the density of 5-HT_1A receptors. Sections 1 & 3 are from a mouse injected with Ad-track and sections 2 & 4 are from a P_1A-5HT_1A-AS-Ad injected mouse. (1), (2), (3) and (4) show the inserts in the section 1, 2, 3 and 4, respectively, as indicated by rectangular boxes. Dashed circles indicate outlines of the area measured for each nucleus. The outline in sections 2 and 4 indicate the reduced density of 5-HT_1A receptors induced by P_1A-5HT_1A-AS-Ad (also see white outline in the inserts). B. The density of 5-HT_1A receptors was reduced in the central (CeA) and basolateral nucleus (BLA), but not in the basomedial nucleus (BMA). The data were represented as mean ± SEM (n = 8–10 mice). * Significantly different from AD-track injected mice, P<0.05.

Figure 3

Amygdaloid injection with P_1A-5-HT_1A-AS-Ad significantly increases anxiety-like behavior as measured by EPM (A) and open field (B). The data are presented as the mean ± SEM (n = 13–14 mice), *: Significantly different from AD-track injected mice, P<0.05.

Figure 4

Correlation between the density of 5-HT_1A receptors in the amygdaloidal nuclei and the % of time spent or distance traveled in the open arms of the EPM. Non-linear regression analysis was conducted to compare the ¹²⁵I-MPPI binding sites in the CeA, BLA and BMA to the % of time spent or % of distance traveled in the open arms of the EPM (n = 10). R: Correlation coefficient; R²: R squared.

Figure 5

Amygdaloidal injection of P_2C-5HT_2C-S-Ad produces an over-expression of 5-HT_2C receptors in the BLA. The density of 5-HT_2C receptors was measured by autoradiography of ¹²⁵ antagonist, spiperone I-DOI binding in the presence of a 5-HT_2A (see methods for details). A: an example of autoradiography of ¹²⁵I-DOI binding. Sections 1 & 3 are from a mouse injected with Ad-track and sections 2 & 4 are from a P_2C-5HT_2C-S-Ad injected mouse. (1), (2), (3) and (4) show the inserts in the sections 1, 2, 3 and 4, respectively, as indicated by rectangular boxes. Dashed circles represent the outlines of the area measured for each nucleus (also see white outline in the inserts). Top sections present the total binding, while bottom sections are non-specific binding (in the presence of RS 102221, a 5-HT_2C antagonist). The abbreviations are same as those in Figure 2. CP: Caudoputamen. B: The effect of P_2C-5HT_2C-S-Ad on the density of 5-HT_2C receptors in the amygdaloidal nuclei. Data are presented as the mean ± SEM (n=10–12).

Figure 6

Amygdaloidal injections with P_2C-5-HT_2C-S-Ad significantly increase anxiety-like behavior as measured by EPM (A) and open field (B). The data are presented as the mean ± SEM (n = 7–8 mice). * Significantly different from AD-track injected mice, P<0.05.

Figure 7

Correlation between the density of 5-HT_2C receptors in the amygdaloidal nuclei and the % of time spent or distance traveled in the open arms of the EPM. Nonlinear regression analysis was conducted to compare the ¹²⁵I-DOI binding sites in the BLA, CeA, BMA and CP to the % of time spent or % of distance traveled in the open arms of the EPM (n = 8). R: Correlation coefficient; R²: R squared.