Functional GABA(A) receptor heterogeneity of acutely dissociated hippocampal CA1 pyramidal cells

CA1 pyramidal cells were voltage clamped, and GABA was applied to individua l cells with a modified U-tube, rapid drug application system. With V-h = - 50 mV; inward currents elicited by 10 mu M GABA were inhibited by GABA, rec eptor (GABAR) antagonists and were baclofen insensitive, suggesting that GA BA actions on isolated CA1 pyramidal cells were GABAR mediated. GABA concen tration-response curves averaged from all cells were fitted best with a two -site equation, indicating the presence of at least two GABA binding sites, a higher-affinity site (EC50-1 = 11.0 mu M) and a lower-affinity site (EC5 0-2 = 334.2 mu M), On two or more populations of cells. The effects of GABA R allosteric modulators on peak concentration-dependent GABAR currents were complex and included monophasic (loreclezole) or multiphasic (diazepam) en hancement, mixed enhancement/inhibition (DMCM, zolpidem) or multiphasic inh ibition (zinc). Monophasic (70% of cells) or biphasic (30% of cells) enhanc ement of GABAR currents by diazepam suggested three different sites on GABA Rs (EC50-1 = 1.8 nM; EC50-2 = 75.8 nhl; EC50-3 275.9 nM) revealing GABAR he terogeneity. The imidazopyridine zolpidem enhanced GABAR currents in 70% of cells with an EC50 = 222.5 nM, suggesting a predominance of moderate affin ity alpha 2 (or alpha 3-) subtype containing BZ Type IIA receptors. a small fraction of cells (10%) had a high affinity for zolpidem, something that i s suggestive of alpha 1 subtype-containing BZ Type I receptors. The remaini ng 30% of cells were insensitive to or inhibited by zolpidem, suggesting th e presence of alpha 5 subtype-containing BZ Type IIB receptors. Whether BZ Type I and Type II receptors coexist could not be determined. The beta-carb oline methyl 6,7-dimethoxy-3-ethyl-beta-carboline-3-carboxylate (DMCM) inhi bited GABAR currents in all cells at midnanomolar concentrations, but in ad dition, potentiated GABAR currents in some cells at low nanomolar concentra tions, characterizing two groups of cells, the latter likely due to functio nal assembly of alpha 5 beta x gamma GABARs. In all cells, GABAR currents w ere moderately sensitive (EC50 = 9 mu M) to loreclezole, consistent with a relatively greater beta 3 subtype, than pi subtype, subunit mRNA expression . Two populations of cells were identified based on their sensitivities to zinc (IC50 = 28 and 182 mu M), suggesting the presence of at least two GABA R isoforms including alpha 5 beta 3 gamma 2 GABARs. Consistent with the het erogeneity of expression of GABAR subunit mRNA and protein in the hippocamp us and based on their differential responses to GABA and to allosteric modu lators, distinct populations of CAI pyramidal cells likely express multiple , functional GABAR isoforms.