Serotonin receptor 1A knockout: an animal model of anxiety-related disorder

Abstract

To investigate the contribution of individual serotonin (5-hydroxytryptamine; 5-HT) receptors to mood control, we have used homologous recombination to generate mice lacking specific serotonergic receptor subtypes. In the present report, we demonstrate that mice without 5-HT1A receptors display decreased exploratory activity and increased fear of aversive environments (open or elevated spaces). 5-HT1A knockout mice also exhibited a decreased immobility in the forced swim test, an effect commonly associated with antidepressant treatment. Although 5-HT1A receptors are involved in controlling the activity of serotonergic neurons, 5-HT1A knockout mice had normal levels of 5-HT and 5-hydroxyindoleacetic acid, possibly because of an up-regulation of 5-HT1B autoreceptors. Heterozygote 5-HT1A mutants expressed approximately one-half of wild-type receptor density and displayed intermediate phenotypes in most behavioral tests. These results demonstrate that 5-HT1A receptors are involved in the modulation of exploratory and fear-related behaviors and suggest that reductions in 5-HT1A receptor density due to genetic defects or environmental stressors might result in heightened anxiety.

Figure 1

Autoradiography studies. (a) Autoradiography of brain sections from wild-type (+/+), heterozygote (+/−), and knockout (−/−) mice using p-[¹²⁵I]MPPI, antagonist of the 5-HT1A receptor (Upper) and 8-OH-[³H]DPAT, agonist of the 5-HT1A receptor (Lower), demonstrating the intermediate level and the absence of binding sites in the heterozygote and knockout mice, respectively. (b) Level of p-[¹²⁵I]MPPI binding sites (mean ± SEM, n = four adult male mice) in different structures. Amy, amygdala; Cx, layers IV-VI of cerebral cortex; DR, dorsal raphe, Hip, layer CA1 of the hippocampus; L, lateral septum. (c) 5-HT1A receptor density obtained with p-[¹²⁵I]MPPI and 8-OH-[³H]DPAT in coronal brain section of wild-type (+/+) and heterozygote mice (+/−) expressed as percentage ± SEM of wild-type level (n = four adult male mice).

Figure 2

Superfusion experiments. (a) Effect of the 5-HT1A agonist 8-OH-DPAT and the 5-HT1A antagonist WAY 100635 on the inhibition of electrically evoked release of 5-[³H]HT from preloaded mesencephalic brain slices obtained from wild-type and knockout mice (mean ± SEM). The number of experiments per group is indicated at the bottom of each column. For each group, slices were obtained from five mice. Drug values were compared with control values unless otherwise indicated by a bracket, by using Student’s t test. (∗, P ≤ 0.05; ∗∗, P ≤ 0.01; ∗∗∗, P ≤ 0.001). (b) Effect of the 5-HT1B agonist CP93129 on the inhibition of electrically evoked release of 5-[³H]HT from preloaded hippocampus slices (mean ± SEM). The number of experiments per group is indicated in parentheses; P = 0.026 by using a two-tailed Student’s t test.; S1, electrically evoked release of 5-[³H]HT was not significantly different between wild-type and knockout mice.

Figure 3

Open field: n = 20 mice per genotype and per sex. Planned contrasts were performed between knockouts and wild types unless indicated by a bracket. ∗: P < 0.05, ∗∗: P < 0.01. Anxiety-related panels: (b). Time in the center showed a marginally significant effect of genotype [F(2,115) = 2.974; P < 0.055). Mutant males spent significantly less time in the center than wild-type males [planned contrast: F(1,115) = 7.52; P < 0.01]. In addition, wild-type males spent significantly more time in the center than wild-type females [F(1,115) > 10.412; P < 0.01]. (d) The ratio between locomotion in the center and total locomotion showed a significant effect of genotype [F(2,115) = 3.088; P < 0.05] and a marginally significant sex × genotype interaction [F(2,115) = 3.037; P < 0.052]. Mutant males were less active in the center than wild-type males [planned contrast: F(1,115) = 9.265; P = 0.003] and wild-type males were significantly more active than wild-type females [planned contrast: F(1,115) = 7.347; P < 0.01]. Exploratory related panels: (a) Path length of the three genotypes for a total of 1 hr shows a significant effect of genotype [F(2,115) = 5.502; P < 0.01]. (c) Knockout males tended to rear less than wild-type males [planned contrast: F(1,113) = 3.634; P = 0.06], which reared more than wild-type females [planned contrast: F(1,113) = 4.384; P < 0.01]. (e) There was a significant effect of genotype on nose pokes [F(2,115) = 4.372; P < 0.05]. Planned contrasts revealed no significant pairwise comparisons.

Figure 4

Elevated Plus Maze. Anxiety-related panels: a–e. A significant effect of genotype was found for time in open-arms and closed-arms, number of entries in the open-arms, percentage of open-arm entries versus total entries, and number of head-dips. This reflected increased anxiety in the 5-HT1A knockout mice [Fs(2,76) > 3.2; ps < 0.05]. Exploratory-related panels: f–h. The number of closed-arm entries, rearings, and total entries showed no difference between the three genotypes [Fs(2,75) < 1.4; ps > 0.26]. n = 25–26 mice per genotype. As no significant effect of sex was found in any category, the two sexes were pooled. ∗, P < 0.05, ∗∗, P < 0.01, ∗∗∗, P < 0.001.

Figure 5

Forced swim test. A significant effect of genotype was found in the percentage of immobility [F(2,78) = 3.457; P < 0.05]. Independent ANOVAs showed that in wild types, immobility increased throughout the session [F(2,52) = 8.9, P < 0.001], while it did not change in the knockouts [F(2,52) = 2.3, P > 0.1]. As no significant effect of sex was found in any category, the two sexes were pooled. n = 27 mice per genotype. ∗∗, P < 0.01.

Figure 6

Effect of WAY100635 in the open field. (a) Mean path length ± SEM during 1 hr. There was no significant effect of genotype or treatment. (b) Mean time spent in the center of the open field (± SEM). There was a main effect of treatment [F(2,68) = 4.110; P < 0.05], a main effect of genotype [F(1,68) = 4.248; P < 0.05], and a treatment by genotype interaction [F(2,68) = 4.101; P < 0.05]. n = 12–14 mice per group. Fisher post-hoc comparisons: ∗, P < 0.05.