Reduced levels of serotonin 2A receptors underlie resistance of Egr3-deficient mice to locomotor suppression by clozapine

Abstract

The immediate-early gene early growth response 3 (Egr3) is associated with schizophrenia and expressed at reduced levels in postmortem patients' brains. We have previously reported that Egr3-deficient (Egr3(-/-)) mice display reduced sensitivity to the sedating effects of clozapine compared with wild-type (WT) littermates, paralleling the heightened tolerance of schizophrenia patients to antipsychotic side effects. In this study, we have used a pharmacological dissection approach to identify a neurotransmitter receptor defect in Egr3(-/-) mice that may mediate their resistance to the locomotor suppressive effects of clozapine. We report that this response is specific to second-generation antipsychotic agents (SGAs), as first-generation medications suppress the locomotor activity of Egr3(-/-) and WT mice to a similar degree. Further, in contrast to the leading theory that sedation by clozapine results from anti-histaminergic effects, we show that H1 histamine receptors are not responsible for this effect in C57BL/6 mice. Instead, selective serotonin 2A receptor (5HT(2A)R) antagonists ketanserin and MDL-11939 replicate the effect of SGAs, repressing the activity in WT mice at a dosage that fails to suppress the activity of Egr3(-/-) mice. Radioligand binding revealed nearly 70% reduction in 5HT(2A)R expression in the prefrontal cortex of Egr3(-/-) mice compared with controls. Egr3(-/-) mice also exhibit a decreased head-twitch response to 5HT(2A)R agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI). These findings provide a mechanism to explain the reduced sensitivity of Egr3(-/-) mice to the locomotor suppressive effects of SGAs, and suggest that 5HT(2A)Rs may also contribute to the sedating properties of these medications in humans. Moreover, as the deficit in cortical 5HT(2A)R in Egr3(-/-) mice aligns with numerous studies reporting decreased 5HT(2A)R levels in the brains of schizophrenia patients, and the gene encoding the 5HT(2A)R is itself a leading schizophrenia candidate gene, these findings suggest a potential mechanism by which putative dysfunction in EGR3 in humans may influence risk for schizophrenia.

Figure 1

Histamine H₁ antagonism is not responsible for resistance of Egr3-deficient (Egr3^−/−) mice to locomotor suppression by clozapine. Locomotor activity was monitored for 60 min in wild-type (WT) mice following administration of highly specific H₁ receptor antagonist pyrilamine. Pyrilamine was not sedating at 10 or 50 mg/kg, the highest dose reported in the literature (Roth, 2008) (n=3 per group).

Figure 2

First-generation antipsychotics (FGAs) reduce activity to a similar degree in Egr3-deficient (Egr3^−/−) and wild-type (WT) mice. The locomotor activity of Egr3^−/− and WT mice was monitored for 60 min following administration of chlorpromazine, a low-potency, highly sedating FGA. As previously reported with haloperidol (Gallitano-Mendel et al., 2008), Egr3^−/− mice demonstrate a similar susceptibility to locomotor suppression by chlorpromazine as WT controls (see also Supplementary Video S2). (a) Vehicle-treated Egr3^−/− mice are hyperactive in comparison to vehicle-treated WT mice. Chlorpromazine reduced locomotor activity in a dose-dependent manner to a similar degree in both Egr3^−/− and WT mice (n=10 per group). (b) The average activity of vehicle-treated mice for each genotype was used to calculate the percent decrease from basal activity for each animal (see Methods). The average percent decrease in activity is presented for both Egr3^−/− and WT mice treated with either 0, 5, or 10 mg/kg chlorpromazine.

Figure 3

Egr3-deficient (Egr3^−/−) mice are resistant to locomotor inhibition by second-generation antipsychotic agents (SGAs). The locomotor activity of Egr3^−/− and WT mice was monitored for 60 min following administration of SGAs. (a) Olanzapine suppressed the activity of WT controls at a dosage of 1 mg/kg, while a dosage of 3 mg/kg reduced the activity of Egr3^−/− mice to normal WT activity levels (n=8 per group) (see also Supplementary Video S3). (b) The average activity of vehicle-treated mice for each genotype was used to calculate the percent decrease from basal activity for each animal (see Materials and Methods). The average percent decrease is presented for both Egr3^−/− and WT mice treated with 0, 1, 2, or 3 mg/kg olanzapine. (c) Quetiapine reduced the activity of Egr3^−/− mice to vehicle-treated WT activity levels, while abolishing almost all locomotor activity in WT mice, at 20 mg/kg (n=7 per group). (d) The average percent decrease in activity from vehicle group is presented for both Egr3^−/− and WT mice treated with 0, 10, or 20 mg/kg quetiapine. (e) Ziprasidone (2.5 mg/kg) suppressed the activity of WT mice, while 5 mg/kg was required to reduce the hyperactivity of Egr3^−/− mice to normal WT levels (n=10 per group). (f) The average percent decrease in activity from vehicle group is presented for both Egr3^−/− and WT mice treated with 0, 2.5, or 5 mg/kg ziprasidone. ^*Significant post hoc comparisons of simple main effects between Egr3^−/− and WT mice at the dose leading to an extreme suppression in activity in WT controls (a, c, and e), or Student's t-test after Bonferroni correction for multiple comparisons (b, d, and f) (^*p<0.05; ^**p<0.001).

Figure 4

Stereotypic behavior does not account for the differential response of Egr3-deficient (Egr3^−/−) mice to first-generation antipsychotics (FGAs) vs second-generation antipsychotics (SGAs). Drowsiness, motor impairment, and stereotypy scores were assessed at 30 and 60 min following administration of the drug (clozapine, 7 mg/kg, a, b, and c; or haloperidol, 3 mg/kg, d, e, and f) or corresponding vehicle. Egr3^−/− and wild-type (WT) mice responded differently to clozapine than to haloperidol in measures of drowsiness (a, d) and motor impairment (b, e), but not stereotypy (c, f) (n=8 per group for clozapine; n=9 per group for haloperidol). ^*Significant comparisons between vehicle and drug treatment groups within genotype (^*p<0.05; ^**p<0.01; ^***p<0.005; by Student's t-test).

Figure 5

Serotonin 2A receptor (5HT_2AR) antagonists suppress the locomotor activity of wild-type (WT), but not Egr3-deficient (Egr3^−/−) mice. Locomotor activity was monitored for 60 min following administration of 5HT_2AR-specific agents or vehicle. (a) Ketanserin suppresses the locomotor activity of WT mice at 5 mg/kg, a dose that decreases the hyperactivity of Egr3^−/− mice, but does not reduce it to normal WT levels (n=7–9 per group). (b) The average activity of vehicle-treated mice for each genotype was used to calculate the percent decrease from basal activity for each animal (see Methods). The average percent decrease in activity from vehicle group is presented for both Egr3^−/− and WT mice treated with 0, 2.5, or 5.0 mg/kg ketanserin. (c) MDL-11939 (10 mg/kg) suppresses the activity in WT mice, but fails to reduce the hyperactivity of Egr3^−/− mice to normal WT activity levels (n=8 per group) (see also Supplementary Video S4). (d) The average percent decrease in activity from vehicle group is presented for both Egr3^−/− and WT mice treated with 0, 2.5, 5, and 10 mg/kg MDL-11939. ^*Significant post hoc comparisons of simple main effects between Egr3^−/− and WT mice at the dose leading to an extreme reduction in activity in WT controls (a and c) or Student's t-test after Bonferroni correction for multiple comparisons (b and d) (^*p<0.005; ^**p<0.001).

Figure 6

Serotonin 2A receptor (5HT_2AR) levels are decreased in the prefrontal cortex (PFC) of Egr3-deficient (Egr3^−/−) mice. (a) [³H]Ketanserin binding saturation curves in the frontal cortex of WT (black) and Egr3^−/− (white) mice (n=8 per group). There was no change in binding affinity. (b) Maximum number of binding sites (B_max) for [³H]ketanserin obtained from individual saturation curves. (c) Egr3^−/− mice display a reduced behavioral response to 5HT_2AR hallucinogenic agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI). Head-twitch responses were recorded for 30 min post-administration of DOI (1 mg/kg) or vehicle (^*p<0.0001, Student's t-test).