Decreased Benzodiazepine Binding with Little Effect on g-Aminobutyric Acid Binding in Rat Brain After Treatment with Antisense Oligodeoxynucleotide to the g-Aminobutyric AcidA Receptor Gamma-2 Subunit

Benzodiazepine potentiation of g-aminobutyric acid (GABA) neurotransmission is associated with the presence of a gamma-2 subunit in the GABAA receptor. A method was developed to modify the gamma-2 subunit expression in adult rat brain. Unilateral intracerebroventricular (i.c.v.) infusion of a 17-base phosphorothioate-modified antisense oligodeoxynucleotide (ASO) was performed every 12 hr for 3 days. Controls were treated with a sense oligodeoxynucleotide. Parasagittal brain sections were used for quantitative autoradiographic analysis of radioligand binding. ASO treatment caused a 15% to 25% decrease of specific [H]flunitrazepam binding in most brain areas, with statistically significant decreases in frontal cortex, cerebellar molecular layer, zona reticulata of substantia nigra and CA3 of hippocampus. In contrast, [H]muscimol binding was not changed. [H]GABA binding was also unchanged, except for a 10% decrease in cerebellar granule cell layer. The effect on the chloride channel of the GABAA receptor complex was examined by 49ethynyl-4-n-[2,3-H2]propylbicycloorthobenzoate binding; most brain areas showed small decreases in 49-ethynyl-4-n-[2,3H2]propylbicycloorthobenzoate binding. However, hippocampal regions showed much larger decreases. Binding of the adenosine A1 receptor antagonist [ H]8-cyclopentyl-1,3-dipropylxanthine was used to examine possible secondary effects of the ASO. There was a decrease in [H]8-cyclopentyl-1,3-dipropylxanthine binding, but this was much smaller than the change in [H]flunitrazepam binding, and no area showed a significant effect. Quantitative immunoblotting with a monoclonal antibody that recognizes GABAA receptor beta-2 and beta-3 subunits showed no change in immunoreactivity in cerebellar tissue after ASO treatment. The results indicate a selective effect on benzodiazepine binding to GABAA receptors and a possible change in receptor subunit composition. The GABAA receptor, a ligand-gated anion channel, carries sites of action for several clinically important compounds, such as benzodiazepines and barbiturates. The receptors consist of several glycoprotein subunits, most of which exist in several isoforms, encoded by different genes (e.g., alpha-1–6, beta-1–3, gamma-1–3) (Macdonald and Olsen, 1994). The distribution of these subunits in the brain shows regional and cellular differences (Wisden et al., 1992). It is thought that there are several GABAA receptor subtypes in brain due to the differential assembly of subunits. GABAA receptors can be investigated using radioligands recognizing different binding sites, including the benzodiazepine binding site, GABA binding site and TBPS site (associated with the ion channel domain of the receptor). Benzodiazepines act via the benzodiazepine binding site, which is a modulatory site located on the GABAA receptor. The presence of a gamma subunit in the GABAA receptor is necessary for formation of a benzodiazepine recognition site and for potentiation of the GABA response by benzodiazepines (Pritchett et al., 1989b; Günther et al., 1995). This binding site may be located at the interface between alpha and gamma subunits (Sigel and Buhr, 1997; Stephenson, 1995). In contrast, the GABA recognition site may be at the interface of alpha and beta subunits (Sigel and Buhr, 1997). In the rat brain, the gamma-2 isoform is the predominant gamma isoform, is widely expressed throughout most regions of the rat brain and most often is associated with alpha-1 and beta-2 subunits (Benke et al., 1994). Received for publication April 6, 1998. 1 This work constitutes a portion of the doctoral dissertation work of T.-J. Z. and was supported by National Institutes of Health Grant DA02194 to H.C.R. and a predoctoral fellowship to T.-J. Z. from the Medical College of Ohio. 2 Preliminary data were reported at the 26th meeting of the Society for Neuroscience, November 16–21, Washington, DC. 3 Present address: Department of Pharmacology, Tzu Chi College of Medicine, 701, Section 3, Chung Yang Road, Hualien 970, Taiwan. ABBREVIATIONS: ASO, antisense oligodeoxynucleotide; b-CCM, methyl-b-carboline-3-carboxylate; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; GABA, g-aminobutyric acid; [H]EBOB, 49-ethynyl-4-n-[2,3-H2]propylbicycloorthobenzoate; TBOB, t-butylbicycloorthobenzoate; TBPS, t-buytlbicyclophosphorothionate; i.c.v., intracerebroventricular. 0022-3565/98/2872-0752$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 287, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 287:752–759, 1998