Measurement of action potential-induced presynaptic calcium domains at a cultured neuromuscular junction.
Spatially localized Ca(2+) domains are thought to play a key role in action potential (AP)-evoked neurotransmitter release at fast synapses. We used a stage-scan confocal spot-detection method and the low-affinity Ca(2+) indicator Oregon Green 488 BAPTA-5N to study the spatiotemporal profile of presynaptic AP-induced Ca(2+) domains. Families of scanned AP-induced fluorescence transients were detected from spot locations separated by 200-300 nm, within the vicinity of Ca(2+) entry sites. Typically, the largest transient in a particular scan peaked within approximately 1 msec and decayed with rapid (tau(1) of 1.7 msec) and slow components (tau(2) of 16 msec, tau(3) of 78 msec). As the spot was incrementally displaced, transients progressively exhibited a slowing in their time-to-peak and a loss of the fast decay component. Three-dimensional graphs of fluorescence versus time and spot displacement revealed the presence of AP-induced fluorescence domains that dissipated within approximately 7 msec. The size of fluorescence domains were estimated from the full-width at half-maximum of gaussian fits to isochronal DeltaF/F plots and ranged from 0.6 to 3.0 micrometer, with a mean +/- SD of 1.6 +/- 0.6 micrometer. Model simulations of a localized Ca(2+) entry site predicted the major features of the fluorescence transients and suggested that, within approximately 1 msec of the initiation of the Ca(2+) current, both the fluorescence domain and the underlying Ca(2+) domain do not extend significantly beyond the site of entry. Consistent with this prediction, the intracellular addition of EGTA (up to 2 mM) accelerated the decay of the measured transients but did not affect the domain size.
DiGregorio
D A
DA
Department of Physiology, University of California at Los Angeles School of Medicine, Los Angeles, California 90095, USA.
Peskoff
A
A
Vergara
J L
JL
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology*; D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004533:Egtazic Acid / Q000494:pharmacology; D004625:Embryo, Nonmammalian; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D008959:Models, Neurological; D008962:Models, Theoretical; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D017729:Presynaptic Terminals / Q000502:physiology*; D014981:Xenopus
D005456:Fluorescent Dyes; D004533:Egtazic Acid; D002118:Calcium