Two-photon chloride imaging in neurons of brain slices.
Two-photon laser scanning microscopy has been used successfully for imaging activity-dependent changes of intracellular calcium and sodium levels. Here we introduce a simple technique for two-photon chloride imaging in intact neurons. It involves the use of the membrane-permeable Cl(-) indicator dye MQAE [N-(6-methoxyquinolyl) acetoethyl ester]. Brief incubation with MQAE produced a robust loading of cells in slices from various brain regions including hippocampus, cortex and cerebellum. In contrast to conventional fluorescence measurements using MQAE, two-photon imaging was not affected in a major way by dye bleaching and phototoxic damage. Instead, it allowed prolonged recordings of time-resolved changes in intracellular chloride concentration in somata and dendrites. As an example of an application we imaged GABA-mediated Cl(-) transients in pyramidal cells of cortical and hippocampal slices as well as in cerebellar Purkinje neurons. By combining Cl(-) imaging with the gramicidin-based perforated-patch-clamp technique we showed that changes in MQAE fluorescence are proportional to the magnitudes of GABA-evoked transmembrane Cl(-) fluxes. Thus, MQAE-based two-photon microscopy promises to be a valuable technique for many applications requiring chloride imaging in single cells.
Marandi
Nima
N
Institut für Physiologie, Ludwig-Maximilians Universität München, Pettenkoferstrasse 12, 80336 Munich, Germany.
Konnerth
Arthur
A
Garaschuk
Olga
O
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008956:Models, Chemical; D009474:Neurons / Q000378:metabolism*; D017785:Photons; D011804:Quinolines; D051381:Rats; D017207:Rats, Sprague-Dawley
D002712:Chlorides; D005456:Fluorescent Dyes; D011804:Quinolines; C078229:N-(6-methoxyquinolyl)acetoethyl ester