Differential effects of long-term treatment with clozapine or haloperidol on GABA_A receptor binding and GAD₆₇ expression

Abstract

One of the most consistent findings in postmortem studies of schizophrenia is increased GABA_A receptor binding and reduced glutamic acid decarboxylase (GAD₆₇) expression. Due to long-term antipsychotic treatment before death, these findings may reflect not only the consequences of schizophrenia but also medication effects. To differentiate between these options, we used an animal model and evaluated long-term effects of typical (haloperidol) and atypical (clozapine) antipsychotic drugs on the GABAergic system.

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 (45 mg/kg/day), another one received haloperidol (1.5 mg/kg/day) and a third one received pH-adapted minimal concentrations of HCl in the drinking water. Receptor autoradiography of the GABA_A receptor ([³H]-muscimol binding) and in situ hybridization in adjacent sections with ³⁵S-labeled cRNA probes of the γ-aminobutyric acid (GABA)-producing enzyme, GAD₆₇, was performed.

While haloperidol increased GABA_A receptor binding in striatum and nucleus accumbens (NA), it suppressed GABA_A receptor binding in temporal (TEMPC) and parietal (PARC) cortex. Clozapine induced GABA_A receptor binding in infralimbic cortex (ILC) and similar like haloperidol in anterior cingulate cortex (ACC), two regions of the limbic cortex. In addition, either drug increased gene expression of GAD₆₇.

It is concluded that antipsychotic drugs differentially alter the GABAergic system, strongly suggesting that drug effects are partially responsible for the up-regulation of GABA_A receptor binding in certain brain regions as observed in postmortem brains of schizophrenic patients. However, the reduced GAD₆₇ expression seen in postmortem brains does not appear to reflect drug effects, since our animal model demonstrated increased gene 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 GABA_A 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 GABA_A 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 GABA_A 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 GABA_A 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.