Differential effects of long-term treatment with clozapine or haloperidol on GABAA receptor binding and GAD67 expression
Introduction
Evidence for disturbances in the metabolism of γ-aminobutyric acid (GABA) in schizophrenic postmortem brain samples has been repeatedly reported Benes and Berretta, 2001, Lewis et al., 1999. GABA is synthesized by glutamic acid decarboxylase (GAD67) (Meltzer and Fonnum, 1987). Three distinct classes of GABAergic neurons are defined due to their immunoreactivity to calretinin, parvalbumin or calbindin (Hendry et al., 1989). GAD67 has been found to be reduced in patients with tardive dyskinesia (Andersson et al., 1989). Reduced expression was also prominent in prefrontal cortex of schizophrenic patients Akbarian et al., 1995b, Guidotti et al., 2000, likely indicating a loss of expression in a subset of GABAergic neurons (Volk et al., 2000).
GABA receptors and transporters have been cloned and functionally characterized in many species including human (Gadea and Lopez-Colome, 2001) and rat (Durkin et al., 1995). The number of GABAA receptors measured by [3H]-muscimol binding in prefrontal cortex, the caudate nucleus Hanada et al., 1987, Benes et al., 1996b, Dean et al., 1999, Mizukami et al., 2000 and in anterior cingulate cortex of schizophrenics (Benes et al., 1992) was increased, whereas gene expression of the receptor subunits seemed to be unchanged (Akbarian et al., 1995a).
These findings gave rise to the hypothesis that a deficit in GABAergic interneurons and secondary up-regulation of GABAA receptors paired with hypoglutamatergic function underlie the pathomechanism in schizophrenia Squires and Saederup, 1991, Deakin and Simpson, 1997, Carlsson et al., 2001 with regard to cortical (Lewis et al., 1999) and hippocampal (Benes, 1999) dysfunction.
Since the abovementioned studies used brain tissue after antipsychotic treatment, attempts were made to control for drug effects by examining treated monkeys (Volk et al., 2000) or antipsychotically treated patients without schizophrenia (Woo et al., 1998). Data from the available literature is not without ambiguity. However, while unchanged mesolimbic GABA content or GAD activity has been reported after chlorpromazine or haloperidol treatment (Perry et al., 1979), others found inhibitions of GAD activity after neuroleptic treatment of monkeys (Gunne et al., 1984) as well as reduced GAD67 expression of globus pallidus in an animal model of tardive dyskinesia (Delfs et al., 1995b). The same group (Delfs et al., 1995b) reported induced GAD67 expression in striatum after haloperidol treatment of rats for either 28 days or 8 months. Treatment of rats lasting for 7 days with haloperidol (Jolkkonen et al., 1994)—in another study with haloperidol or sertindole, but not with olanzapine (Sakai et al., 2001)—showed elevations of GAD67 mRNA in striatum and a reduction in globus pallidus. In contrast, a comparison of haloperidol and clozapine revealed increased GAD67 expression in globus pallidus only after haloperidol treatment (Delfs et al., 1995a) similar to striatal GAD67 induction after treatment with haloperidol or sulpiride (Laprade and Soghomonian, 1995). Short-term application of fluphenazine (Chen and Weiss, 1993) and treatment over a period of 6 months (Johnson et al., 1994) increased the expression of GAD67 in striatum. Clozapine but not haloperidol interacts antagonistically with a subset of GABAA receptors Squires and Saederup, 1997, Michel and Trudeau, 2000. The atypical antipsychotic drugs clozapine and olanzapine but not haloperidol or chlorpromazine decreased the number of GABAA receptors in cortical and limbic brain regions (Farnbach-Pralong et al., 1998).
In summary, the reported changes of GABAergic markers in schizophrenia or in animal models of antipsychotic treatment pose the question whether they are because of disease or medication. We therefore established an animal model that very closely reflects long-term antipsychotic treatment.
Section snippets
Animal trials
Male Sprague–Dawley rats (initial weight 250–280 g, Janvier, France) were grouped and housed three per cage under a 12-h light/dark cycle and permitted food and water ad libitum. A total of 33 adult male rats were treated in three cohorts over a period of 6 months. One cohort of 11 animals received clozapine (Novartis, 45 mg/kg/day), another one received haloperidol (Janssen, 1.5 mg/kg/day) and a third one received minimal concentrations of HCl in the drinking water adjusted to pH 6.5. Water
Results
Three cohorts of 11 adult male rats were treated with the antipsychotic drugs haloperidol and clozapine or pH-adapted water for 6 months. There were no obvious behavioral or morphological differences in either of the groups, but behavioral tests were not performed. GABAA receptor autoradiography and in situ hybridization for GAD67 were performed in the following regions: infralimbic (ILC) and anterior cingulate cortex (ACC), dorsolateral prefrontal cortex (DFC), the core of the nucleus
Discussion
This animal model of long-term antipsychotic treatment explores molecular changes in the GABAergic system. The main results were increased expression of GAD67 in the infralimbic cortex and in anterior cingulate cortex and differential effects of haloperidol and clozapine on [3H]-muscimol binding to GABAA receptors within cortical, limbic and subcortical areas. The traditional antipsychotic haloperidol increased GABAA receptor binding in the basal ganglia along with a reduction in parietal and
Acknowledgments
The Forschungsfond of the Central Institute of Mental Health funded this work. The authors thank Dr. Gaebicke-Härter and W. VanSyckel for text revision.
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