The use of in vivo antisense oligonucleotide technology for the investigation of brain GABA receptors.

Antisense oligodeoxynucleotides (ODN) can be used as selective inhibitors of in vivo gene expression in the central nervous system (CNS) of experimental animals. The gamma-aminobutyric acid type A (GABAA) receptor is a member of the ligand-gated ion channel superfamily of neurotransmitter receptors. GABAA receptor function is allosterically modulated by several clinically important compounds, e.g. 1,4-benzodiazepines, barbiturates and certain neurosteroids, which recognize binding sites within the receptor complex. GABAA receptor chloride channel complexes are probably pentamers of different polypeptide subunits. The number of known subunit families and isoforms (six alpha s, four beta s, three gamma s, one delta and two rho s) indicates an extensive heterogeneity of GABAA receptors. The gamma 2 subunit is a functionally integral part of the GABAA receptor, necessary for the high affinity binding of benzodiazepines. The infusion of phosphorothioate ODN antisense to the gamma 2 subunit mRNA, but not control sense or mismatch ODN, into the lateral cerebral ventricle or into the hippocampus of rats leads to significant decreases in benzodiazepine receptor radioligand binding. In the hippocampus this is accompanied by a decrease in the number of GABAA receptors and by a loss of neurones, the latter possibly being due to reduced GABAergic inhibitory neurotransmission. Autoradiographic analysis following continuous intrahippocampal infusion of antisense ODN shows the regional extent of the effect on [3H]flunitrazepam binding. The continuous infusion of antisense ODN, but not of mismatch control ODN, into the right lateral cerebral ventricle induced a significant decrease in benzodiazepine binding and [3H]muscimol binding to membranes of the right cortex. Antisense ODN infused into the striatum decreased benzodiazepine binding and binding to the GABA binding site of the GABAA receptor to an extent similar to that found in the hippocampus. It is concluded that the preferred route of administration of antisense ODN for in vivo studies of the GABAA receptor may be by infusion into defined rat brain regions. The reported data support the idea that antisense ODN can be used as a valuable tool for the investigation of the contribution of individual GABAA receptor subunits to the properties of the receptor complex and of mechanisms of receptor subunit assembly.