title: Decreased benzodiazepine binding with little effect on gamma-aminobutyric acid binding in rat brain after treatment with antisense oligodeoxynucleotide to the gamma-aminobutyric acidA receptor gamma-2 subunit.
abstract: Benzodiazepine potentiation of gamma-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 [3H]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, [3H]muscimol binding was not changed. [3H]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 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate binding; most brain areas showed small decreases in 4'-ethynyl-4-n-[2, 3-3H2]propylbicycloorthobenzoate binding. However, hippocampal regions showed much larger decreases. Binding of the adenosine A1 receptor antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine was used to examine possible secondary effects of the ASO. There was a decrease in [3H]8-cyclopentyl-1,3-dipropylxanthine binding, but this was much smaller than the change in [3H]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.
authors.affiliation: Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo, Ohio, USA.
authors.forename: T J
authors.initials: TJ
authors.lastname: Zhao
authors.forename: M
authors.initials: M
authors.lastname: Li
authors.forename: T H
authors.initials: TH
authors.lastname: Chiu
authors.forename: H C
authors.initials: HC
authors.lastname: Rosenberg
mesh_terms: D000818:Animals; D001483:Base Sequence; D001569:Benzodiazepines / Q000378:metabolism*; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002531:Cerebellum / Q000187:drug effects* / Q000378:metabolism; D005445:Flunitrazepam / Q000378:metabolism; D008297:Male; D009118:Muscimol / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000235:genetics*; D014316:Tritium; D014970:Xanthines / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D016376:Oligonucleotides, Antisense; D011963:Receptors, GABA-A; D014970:Xanthines; D014316:Tritium; C091518:1-(4-ethynylphenyl)-4-propyl-2,6,7-trioxabicyclo(2.2.2)octane; D001569:Benzodiazepines; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; C051360:1,3-dipropyl-8-cyclopentylxanthine