Optical imaging of intracellular chloride in living brain slices.
We developed an optical imaging technique to measure changes in intracellular levels of Cl- in neurons within the living brain slice. After rat brain slices were incubated with the permeant form of the Cl(-)-sensitive 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 granule cells. Increased intracellular levels of Cl- were produced by bath application of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). Within hippocampal pyramidal neurons and interneurons, GABA produced a concentration-dependent decrease in fluorescence (EC50 = 200 microM). The GABA response was mediated via the GABA receptor since it was blocked by picrotoxin and mimicked by the agonist, muscimol. Muscimol, which is not transported by the GABA re-uptake pump, was approximately 20-fold more potent than GABA. The method developed was also used to image intracellular Cl- levels with UV laser scanning confocal microscopy. Even greater resolution was obtained and deeper structures could be imaged in cerebral cortex and hippocampus. This is the first demonstration of optical imaging to measure intracellular Cl- dynamics in living brain slices using fluorescence microscopy and laser scanning confocal microscopy.
Schwartz
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RD
Department of Pharmacology, Duke University Medical Center, Durham, NC 27710, USA.
Yu
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X
D000818:Animals; D000831:Animals, Newborn; D002463:Cell Membrane Permeability; D002531:Cerebellum / Q000737:chemistry* / Q000166:cytology; D002540:Cerebral Cortex / Q000737:chemistry* / Q000166:cytology; D002712:Chlorides / Q000032:analysis*; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D018755:GABA Agonists / Q000494:pharmacology; D006624:Hippocampus / Q000737:chemistry* / Q000166:cytology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009118:Muscimol / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009924:Organ Culture Techniques; D017966:Pyramidal Cells / Q000166:cytology / Q000378:metabolism; D011806:Quinolinium Compounds / Q000493:pharmacokinetics; D051381:Rats; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002712:Chlorides; D005456:Fluorescent Dyes; D018755:GABA Agonists; D011806:Quinolinium Compounds; C070311:6-methoxy-N-ethylquinolinium; D009118:Muscimol; D005680:gamma-Aminobutyric Acid