Imaging synaptic zinc release in living nervous tissue.
Zinc enriched neurons have a pool of synaptic vesicles which contain free or loosely-bound zinc ions. The movement of the vesicular zinc ions into the synaptic clefts has been previously studied by microdialysis, fluorescence postmortem staining for zinc and radioactive zinc isotope. In this study the zinc fluorescence probe N-6-metoxy-p-toluensulfonamide quinoline (TSQ) has been applied as a tracer of synaptic release of zinc ions. This fluorochrome permeates cell membranes and when exposed to living brain slices gives rise to a staining pattern similar to that seen with autometallography. In the living brain slices, fluorescence emission persists after exposure to calcium saturated ethylen diamino-tetra-acetic acid (Ca-EDTA) because this chelator does not penetrate cell membranes, while sodium dethyldithiocarbamate (DEDTC), that does penetrate membranes, partially suppressed the fluorescence emission. Stimulation of slices bathed in the non-permeant chelator Ca-EDTA with 50 mM potassium chloride leads to a rapid and complete disappearance of fluorescence. In the absence of Ca-EDTA, however, potassium stimulation induces a sudden transitory increase in fluorescence. This increase is caused by a translocation of the fluorochrome (TSQ) zinc molecules from the weakly acid interior of the synaptic vesicles to the neutral extracellular space, whereby the fluorescence emission of the molecules is enhanced sufficiently to be recorded by a high sensitivity TV camera.
Varea
E
E
Neurobiology, Cell Biology Department, University of Valencia, Avda. Dr. Moliner 50, 46100 Burjassot, Spain.
Ponsoda
X
X
Molowny
A
A
Danscher
G
G
Lopez-Garcia
C
C
D000634:Aminoquinolines / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004492:Edetic Acid / Q000494:pharmacology; D001330:Electronic Data Processing / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D008116:Lizards; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D018715:Microscopy, Video / Q000295:instrumentation / Q000379:methods; D009482:Neurophysiology / Q000295:instrumentation / Q000379:methods*; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D013687:Telencephalon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014105:Tosyl Compounds / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D014105:Tosyl Compounds; D011189:Potassium Chloride; C061730:N-(6-methoxy-8-quinolyl)-4-toluenesulfonamide; D004492:Edetic Acid; D015032:Zinc