OPTICAL IMAGING OF INTRACELLULAR CHLORIDE IN LIVING BRAIN-SLICES

We developed an optical imaging technique to measure changes in intrac ellular levels of Cl- in neurons within the living brain slice. After rat brain slices were incubated with the permeant form of the Cl--sens itive dye, 6-methoxy-N-ethylquinolinium chloride (MEQ), neurons could be imaged within the hippocampus, cerebral cortex and cerebellum using fluorescence microscopy. Both soma and dendrites were clearly visible in pyramidal neurons, interneurons, Purkinje cells and cerebellar gra nule cells. Increased intracellular levels of Cl- were produced by bat h application of the inhibitory neurotransmitter, gamma-aminobutyric a cid (GABA). Within hippocampal pyramidal neurons and interneurons, GAB A produced a concentration-dependent decrease in fluorescence (EC(50) = 200 mu M). The GABA response was mediated via the GABA, receptor sin ce it was blocked by picrotoxin and mimicked by the agonist, muscimol. Muscimol, which is not transported by the GABA re-uptake pump, was ap proximately 20-fold more potent than GABA. The method developed was al so used to image intracellular Cl- levels with UV laser scanning confo cal microscopy. Even greater resolution was obtained and deeper struct ures could be imaged in cerebral cortex and hippocampus. This is the f irst demonstration of optical imaging to measure intracellular Cl- dyn amics in living brain slices using fluorescence microscopy and laser s canning confocal microscopy.