CONFOCAL IMAGING OF INTRACELLULAR CHLORIDE IN LIVING BRAIN-SLICES - MEASUREMENT OF GABA(A) RECEPTOR ACTIVITY

We have developed a method using UV laser-scanning confocal microscopy and the fluorescent chloride ion indicator, 6-methoxy-N-ethylquinolin ium chloride (MEQ), to image GABA-mediated changes in intracellular ch loride (Cl-i(-)) in individual neurons of the rat acute brain slice. A fter bath-loading slices with the cell-permeant form (reduced) of MEQ, there was intense fluorescence within neurons of diverse morphologies in the hippocampus, neocortex and cerebellum. MEQ fluorescence locali zed to the cytosolic compartment of both the somata and proximal dendr ites. MEQ fluorescence was calibrated using the ionophores nigericin a nd tributyltin in the presence of varying extracellular Cl- concentrat ions. Neuronal MEQ fluorescence was inversely related to intracellular Cl-, with a Stern-Volmer constant of 16 M-1 (50% quench by 61 mM Cl-) . Application of GABA in the perfusate produced a concentration-depend ent decrease in MEQ fluorescence (EC50 = 40 mu M) that was blocked in the presence of the Cl- channel antagonist, picrotoxin. Bath perfusion of hippocampal slices with modulators of the GABA(A) receptor, pentob arbital and diazepam, potentiated the GABA-mediated response by 85 and 44%, respectively. A regional comparison identified larger GABA. resp onses for both cerebellar Purkinje and granule cells relative to pyram idal neurons of the hippocampus and neocortex and to hippocampal inter neurons. Pressure ejection of the GABA(A) agonist, muscimol (40 mu M), from a micropipet onto individual hippocampal neurons allowed the mea surement of rapid responses (1-5 s), compared to those obtained with b ath application. Thus, optical imaging of [Cl-](i) using MEQ and UV-la ser-scanning confocal microscopy provides investigators with a new met hod to study GABA(A) pharmacology in neighboring neurons and perhaps e ven in the soma versus dendrites, simultaneously, within living brain slices. (C) 1997 Elsevier Science Ireland Ltd.