Dysregulation of 5HT2A and mGlu2 receptors in rat cortex after chronic treatment with antipsychotics

A García-Bea1, P Miranda-Azpiazu1, C Muguruza1, AM Gabilondo1, R Díez-Alarcia1, LF Callado1, J González-Maeso2, JJ Meana1. 1University of the Basque Country (UPV/EHU), Department of Pharmacology and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM). Leioa, Bizkaia 48940, Spain, 2Mount Sinai School of Medicine, Departments of Psychiatry, Neurology, and Friedman Brain Institute. New York, NY 10029, USA

While atypical antipsychotic drugs all have in common a high affinity for the serotonin 5-HT2A receptor (5HT2AR), the metabotropic glutamate 2 receptor (mGlu2R) has become a promising new target for the treatment of schizophrenia (Patil et al., 2007). These two receptors form a heterocomplex that may be responsible for some psychotic symptoms in schizophrenia (González-Maeso et al., 2008).

The aim of the present study was to evaluate the modulation of 5HT2AR and mGlu2R (mRNA and protein) expression by chronic treatment with typical (haloperidol) or atypical (clozapine and risperidone) antipsychotic drugs in rat cortex.

Male Sprague-Dawley rats (8 weeks old) were treated during 21 days with saline (2 ml/kg/day, i.p.; n=10), clozapine (10 mg/kg/day, i.p.; n=10), risperidone (1 mg/kg/day, i.p.; n=10) or haloperidol (1 mg/kg/day, i.p.; n=10). Rats were sacrificed 48-72 hours after the last injection, being their brain removed and the cortex dissected and stored at -70ºC until assays. 5HT2AR and mGlu2R mRNA was measured by qRT-PCR with Taq-Man® probes, using GAPDH, beta-actin and RPS29 as housekeeping genes. The receptor protein expression was measured by both, Western Blot and radioligand binding assays. Immunodetection of 5HT2AR and mGlu2R was made following standard protocols, and using commercially available antibodies. Beta-actin signal was used as loading control. Binding saturation curves of [3H]ketanserin (5HT2A/CR ligand) and [3H]LY341495 (mGlu2/3R ligand) were performed in order to determine the 5HT2AR and mGlu2/3R density. The statistical comparisons were carried out by one-way ANOVA (qRT-PCR), Student´s t-test (Western Blot) and extra-sum-of-squares (F test) (binding), using GraphPad Prism® software.

A statistically significant decrease of 5HT2AR mRNA expression was observed in the rats chronically treated with both clozapine (-30±5%, p<0.05) and risperidone (-29±8%, p<0.05) compared to saline, but not in those treated with haloperidol. However, mGlu2R mRNA expression was decreased only after chronic treatment with clozapine (-25±3%, p<0.05). Immunoreactive density of 5HT2AR was decreased in brain cortex membrane preparations (P2 fraction) of rats chronically treated with either clozapine (-36±6%, p<0.01) or risperidone (-26±9%, p<0.05), while no changes were observed when measured in total homogenates. Immunodetection of mGlu2R protein was also decreased in total homogenates of rat cortex after chronic treatment with clozapine (-20±7%, p<0.05), with no differences in either risperidone or haloperidol groups compared to saline. A significant decrease in the maximum number of binding sites (Bmax) of [3H]ketanserin was observed in clozapine treated rats (431±29 fmol/mg prot., p<0.0001) compared to those treated with saline (670±24 fmol/mg prot.), without changes in the apparent dissociation constant (KD). However, there were no differences in the parameters of [3H]LY341495 saturation curves in any of the groups.

These results suggest that chronic treatment with atypical, but not with typical, antipsychotic drugs modulates the expression of 5HT2A and mGlu2 receptors in rat cortex. Our data also reveal that the primary methods (western blot and radioligand binding) and protein preparation protocols (total protein and membrane fractionation) are factors to be taken into account in receptor protein expression studies.

González-Maeso J et al., (2008) Nature, 452(7183): 93-97.

Patil S.T. et al., (2007) Nature Medicine, 13(9): 1102-1107.