Progress in Neuro-Psychopharmacology and Biological Psychiatry
Treatment with haloperidol and diazepam alters GABAA receptor density in the rat brain
Introduction
There is a significant body of data implicating deficiencies in the γ-aminobutyric acid (GABA) system in the pathology of schizophrenia (Lewis et al., 2004). Strongly underpinning this hypothesis are the many postmortem studies that have reported increased levels of GABA type A receptors (GABAAR) (Benes et al., 1992, Benes et al., 1996a, Benes et al., 1996b, Benes et al., 1997, Dean et al., 1999, Hanada et al., 1987, Owen et al., 1981, Kiuchi et al., 1989); with the upregulation of the GABAAR being attributable to an underactive GABAergic system (Benes et al., 1996a).
The GABAAR is the primary mediator of fast inhibitory neurotransmission in the central nervous system (CNS) and modulates levels of anxiety, vigilance, memory and epileptogenesis (Mohler et al., 1995a, Mohler et al., 1995b). The receptor is a 275 kDa heteropentameric glycoprotein formed by distinct classes of polypeptide subunits (α1–6, β1–4, γ1–4, δ, ρ1–3, ɛ, θ, π) (Mohler et al., 1995a, Mohler et al., 1995b) that form membrane spanning regions that associate to form a ligand-gated Cl− ion channel pore (Barnard et al., 1988). The assembled receptor contains multiple binding sites which includes one that binds GABA and another that is the target for benzodiazepines (BZs); a receptor must contain the BZ binding site to be a target for the BZs (Mohler et al., 1995a, Mohler et al., 1995b). Because the BZ binding site on the GABAAR is allosterically coupled to the GABA binding site, BZs increase the affinity of the GABA binding site for GABA to increase receptor occupancy at low GABA concentrations by increasing the frequency of channel openings (Pritchett et al., 1989).
Given the data on GABAARs in the CNS of subjects with schizophrenia it is significant that treatment with both typical and atypical antipsychotic drugs has been shown to affect the density of GABAARs in the rat CNS. For example, chronic haloperidol treatment has generally been shown to cause CNS regionally specific increases in the density of GABAARs (Frey et al., 1987, Frey et al., 1989, Gale, 1980, Huffman and Ticku, 1983, See et al., 1989, See et al., 1990). However, the effects of haloperidol appear complex in that it has been shown that the drug increases GABAARs in the striatum, anterior cingulate cortex, infralimbic cortex and the core of the nucleus accumbens but decreases levels of the receptor in the temporal and parietal cortex in the CNS of the same animals (Zink et al., 2004). Notably, as the levels of the GABAAR have been shown to be unaltered in the orbitofrontal cortex (Brodmann's area (BA) 10) of neuroleptic-naïve schizophrenic subjects (Benes et al., 1996b), it has been suggested that the increase in the receptor in the CNS of antipsychotic drug-treated subjects with schizophrenia might be a drug effect. To add complexity to the study of GABAARs in schizophrenia, BZs are sometimes used in conjunction with antipsychotic drugs when treating the disorder (Alexander et al., 2004, Greenblatt et al., 1990) and, predictably, BZ drug treatment alone significantly affects GABAAR density in the rat brain (Hutchinson et al., 1996, Toki et al., 1996a, Toki et al., 1996b, Wu et al., 1994).
Given the concurrent use of BZs and antipsychotic drugs in the treatment of schizophrenia it is noteworthy that no reported studies have examined the effects of such combined treatment on levels of the GABAAR. To address this deficiency, we investigated the effects of BZ and antipsychotic drug treatment, alone or in concert, on GABAAR density in rat CNS. The GABAAR density was measured using [3H]muscimol (GABA binding site) and [3H]flumazenil (BZ binding site) (McLeod et al., 2002). In addition, we previously determined that [3H]flumazenil binding in rodent and human brain comprises zolpidem-sensitive and -insensitive sites, with the latter corresponding to GABAARs that contain the α5 subunit (McLeod et al., 2002). Therefore, in order to investigate possible alterations in radioligand binding following drug treatment specific to GABAARs containing the α5 subunit, zolpidem-insensitive and -sensitive [3H]flumazenil binding was also measured.
Section snippets
Materials
[3H]Flumazenil ([N-methyl-3H]-Ro 15-1788, 87 Ci/mmol, NEN) and [3H]muscimol ([methylene-3H-(N)]-3-hydroxy-5-aminomethylisoxazole 20 Ci/mmol, NEN) were obtained from Amrad Biotech, Melbourne, Australia. Clonazepam and paraformaldehyde were obtained from Sigma Aldrich Pty. Ltd., Castle Hill, New South Wales, Australia. SR95531 was obtained from Research Biochemical International, USA. Zolpidem was obtained from Tocris Pty. Ltd., Sydney, Australia. [3H]Microscales® were obtained from Amersham
The distribution of [3H]muscimol and [3H]flumazenil binding in the rat brain
Both radioligands were effectively displaced by the unlabelled drugs used in our study. Importantly, it is known that [3H]muscimol binding is not specific to the GABAA receptor (DeFeudis et al., 1979, Wang et al., 1979). Therefore our use of a SR 95531, a highly selective and high affinity antagonist of the rat GABAA receptor (Heaulme et al., 1987), as a displacing agent is critical to ensure that the specific binding we measure is made up only of [3H]muscimol bound to the GABAA receptor. The
Discussion
The distribution and density of GABAAR sites in vehicle-treated rats, as described in this study, are in general agreement with the regional pattern observed previously using [3H]muscimol and the BZ binding site ligands [3H]flunitrazepam and [3H]flumazenil (McLeod et al., 2002, Nabeshima et al., 1994, Niddam et al., 1987, Olsen et al., 1990, Palacios et al., 1981, Young and Kuhar, 1980). Similarly, the distribution of zolpidem-sensitive and -insensitive binding sites was as we would have
Conclusions
This study shows that treatment with diazepam, haloperidol or a combination of the two drugs has differential outcomes in levels of GABAAR in different regions of the rat CNS. These are the first data to suggest that the outcome of such combined treatment, when used in subjects with schizophrenia, could bring about therapeutic effects by differentially changing levels of GABAARs in the CNS from subjects with the disorder. This conclusion must be tempered by the clear differences in treatment
Acknowledgements
These studies were funded in part by the State Government of Victoria and the Rebecca L. Cooper Medical Research Foundation. BD is an NHMRC Senior Research Fellow (Level B: (# 400016)). MM was the recipient of a University of Melbourne, Australia Postgraduate Award. The authors wish to thank Geoffrey Pavey for technical assistance. The study was also supported by NIMH Grant # MH069691-01A1 and The Rebecca L. Cooper Medical Research Foundation.
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