Unraveling the identity of benzodiazepine binding sites in rat hipppocampus and olfactory bulb
Introduction
Benzodiazepines are therapeutic agents that have hyperphagic, anxiolytic, sedative, hyperthermic, anticonvulsive and cognitive effects Braestrup et al., 1982, Burt and Kamatchi, 1991. These drugs exert their effect by binding to allosteric sites on the hetero-pentameric γ-aminobutyric acid (GABAA) chloride ion channel receptors and modulating the ability of GABAA to open the chloride channel (Olsen and Tobin, 1990). Thus, to further elucidate the molecular basis for their diverse pharmacological effects, it is important to identify the nature and extent of GABAA/benzodiazepine receptor heterogeneity in the brain. Both the number and the uncertainty of subunit composition of the GABAA receptors in the central nervous system contribute to the difficulty of identifying those that are associated with a specific behavioral activity.
Cloning and sequencing have demonstrated the existence of 21 different isoforms of the five GABAA receptor subunits, including α1–α6, β1–β4, γ1–γ4, δ, ρ1–ρ3, π, ε, and θ Bonnert et al., 1999, Burt and Kamatchi, 1991, Olsen and DeLorey, 1999. In principle, these subunits can combine to form a vast number of functional GABAA receptors and the study of cloned receptor subunits in transfected cells cannot by itself lead to the identification of the particular combinations present in the central nervous system receptors. Other experimental approaches can aid in this process. Specifically, immunohistochemistry and in situ hybridization studies have identified different subunits that are expressed in various regions of the brain Thompson et al., 1992, Wisden et al., 1988, Zimprich et al., 1991. Furthermore, co-immunoprecipitation studies have identified the subunits that possibly form the functional receptors Khan et al., 1994, McKernan and Whiting, 1996, Quirk et al., 1994.
These diverse studies provide evidence for at least 13 distinct functional combinations in vivo. In addition, several studies indicate that functional GABAA receptors include α, β and γ subunits Prichett et al., 1989a, Siegel et al., 1990, Smith and Olsen, 1995. It is also now clear that there is more than one type of functional GABAA receptor in virtually every region of the brain Laurie et al., 1992, McKernan and Whiting, 1996, Wisden and Seeburg, 1992.
The nature of the α subunit expressed in each specific pentameric receptor is emerging as the main determinant of receptor affinity for GABAA/benzodiazepine receptor Ebert et al., 1997, Prichett et al., 1989b, Wong and Skolnick, 1992. Thus, one useful indicator of the different possible GABAA/benzodiazepine receptor types in a given brain region is the number of different α subunits expressed in that region. For example in the cerebellum the major GABAA α subunits expressed are α1 and α6 Laurie et al., 1992, Thompson et al., 1992. Thus, in binding studies two major receptor sites are expected to be present, as observed previously Lameh et al., 2000, Uusi-Oukari, 1992, Wisden et al., 1996. In both hippocampus and olfactory bulb, all the major GABAA α subunits are expressed with the exception of α6 subunit Fritschy and Möhler, 1995, Laurie et al., 1992, Sperk et al., 1997, Sur et al., 1999, Thompson et al., 1992. Thus, several receptor types can potentially be expressed in these tissues.
The goals of the present study were two fold: first to identify the number of distinct GABAA/benzodiazepine receptors in rat hippocampus and olfactory bulb, and second to measure the affinities of several benzodiazepine ligands for each of the identified receptor type. Hippocampus and olfactory bulb were chosen because GABAA receptors are expressed at very high levels and have important functions in these tissues. In the olfactory bulb, the GABAA receptors, together with the glutamate receptors are involved in olfactory coding (Wellis and Kauer, 1993) which in turn, at least in rodents, are involved in learning and conditioning Okutani et al., 1999, Trombley and Shepherd, 1992. In the hippocampus, the GABAA receptors are involved in memory processing Frankland et al., 1998, Milanovic et al., 1998, Phillips and LeDeux, 1992.
Studies of structurally diverse benzodiazepine receptor ligands in transfected cells have revealed little or no selective binding to reconstituted GABAA receptors with varying α subunits in combination with β2, γ2 subunits. A major impediment to binding studies as a tool for characterization of native receptors has been the lack of radioligands selective for each of the different types of receptors. The commercial software programs currently available for analysis of receptor binding data require the use of a radioligand capable of distinguishing between different binding sites in order to detect multiple binding affinities at these sites for a non-radioactive ligand. This obstacle was overcome in the present study by use of affinity analysis system developed in our laboratory, which is comprised of two independent analysis programs. The first program, single ligand, performs analysis of saturation and self-competition data. The other program, compete, allows the determination of the affinities and densities of competing cold ligands for multiple binding sites using a non-selective radioligand that binds to all the sites with similar affinity.
Eight different chemical families were represented in the ligands chosen for the present study. These were 1,4-benzodiazepines (flunitrazepam), imidazobenzodiazepines (RO15-1788, RO15-4513, RO16-6028, RO23-0364, RO41-7812 and RO42-8773), imidazopyridines (Zolpidem and AHR 14749), β-carbolines (Abecarnil), pyrazoloquinolines (CGS 8216, CGS 9895 and CGS 9896), pyrroloquinazolinone (AHR 11797), quinoxalinone (U78875) and quinoline (RO23-1590). Systematic binding studies were carried out with these ligands to elucidate the heterogeneity of the GABAA/benzodiazepine receptors in the olfactory bulb and hippocampus and to determine their affinities at each site identified.
Section snippets
Materials
The following compounds were received as generous gifts: zolpidem ([N,N,6-trimethy-1-2-(4-methylphenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate]) (Synthelabo Recherche, Bagneux, France), AHR-11797 (5,6-dihydro-6-methyl-1-phenyl-3H-pyrrolo[3,2,1-ij]quinazolin-3-one), AHR 14749 (1-N,N-dimethyl-amido-imidazo[2,3a]-2-chloropyridine) (A.H. Robins, Richmond, VA, USA), RO15-1788 (8-fluoro-3-carboxy-5,6-dihydro-5-methyl-6-oxo-414-imidazo[1,5-a]1,4 benzodiazepine), RO15-4513 (ethyl
Results
Saturation and competition binding assays were carried out to determine the binding characteristics in the hippocampus and olfactory bulb of 16 benzodiazepine ligands with diverse chemical structures. The binding data were analyzed with new saturation (self-competition) and competition software programs (single ligand and compete, respectively). The competition software (compete) allows characterization of the binding profile of an unlabelled ligand with different binding affinities to sites to
Discussion
Heterogeneity of benzodiazepine receptors in the hippocampus and olfactory bulb were characterized by a combination of saturation and competitive binding studies and the use of affinity analysis system (single ligand and compete) for data analysis. The competition software (compete) allows the determination of the affinities and densities of competing cold ligands for multiple binding sites using non-selective radioactive ligands that bind to all the sites with similar affinities. In these
Acknowledgements
The authors would like to thank Ajna Rivera for carrying out the preliminary work leading to this publication. They also wish to gratefully acknowledge support for this work by a grant from the National Institute of Health, DA06304.
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