Confocal imaging of intracellular chloride in living brain slices: measurement of GABAA receptor activity.
We have developed a method using UV laser-scanning confocal microscopy and the fluorescent chloride ion indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ), to image GABA-mediated changes in intracellular chloride (Cli-) in individual neurons of the rat acute brain slice. After 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 localized to the cytosolic compartment of both the somata and proximal dendrites. MEQ fluorescence was calibrated using the ionophores nigericin and tributyltin in the presence of varying extracellular Cl- concentrations. 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-dependent decrease in MEQ fluorescence (EC50 = 40 microM) that was blocked in the presence of the Cl- channel antagonist, picrotoxin. Bath perfusion of hippocampal slices with modulators of the GABAA receptor, pentobarbital and diazepam, potentiated the GABA-mediated response by 85 and 44%, respectively. A regional comparison identified larger GABA responses for both cerebellar Purkinje and granule cells relative to pyramidal neurons of the hippocampus and neocortex and to hippocampal interneurons. Pressure ejection of the GABAA agonist, muscimol (40 microM), from a micropipet onto individual hippocampal neurons allowed the measurement of rapid responses (1-5 s), compared to those obtained with bath application. Thus, optical imaging of [Cl-]i using MEQ and UV-laser-scanning confocal microscopy provides investigators with a new method to study GABAA pharmacology in neighboring neurons and perhaps even in the soma versus dendrites simultaneously, within living brain slices.
Inglefield
J R
JR
Department of Pharmacology, Duke University Medical Center, Durham, NC 27710, USA. ingle004@acpub.duke.edu
Schwartz-Bloom
R D
RD
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D001921:Brain / Q000166:cytology; D001923:Brain Chemistry / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism; D011806:Quinolinium Compounds; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000378:metabolism*
D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D011963:Receptors, GABA-A; C070311:6-methoxy-N-ethylquinolinium