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The Journal of Neuroscience, September 15, 1999, 19(18):7846-7859
Measurement of Action Potential-Induced Presynaptic Calcium
Domains at a Cultured Neuromuscular Junction
David A.
DiGregorio,
Arthur
Peskoff, and
Julio L.
Vergara
Department of Physiology, University of California at Los Angeles
School of Medicine, Los Angeles, California 90095
Spatially localized Ca2+ 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 Ca2+ indicator Oregon
Green 488 BAPTA-5N to study the spatiotemporal profile of
presynaptic AP-induced Ca2+ domains. Families of
scanned AP-induced fluorescence transients were detected from spot
locations separated by 200-300 nm, within the vicinity of
Ca2+ entry sites. Typically, the largest transient
in a particular scan peaked within ~1 msec and decayed with rapid
( 1 of 1.7 msec) and slow components
(2 of 16 msec, 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 ~7 msec. The size of fluorescence domains were
estimated from the full-width at half-maximum of gaussian fits to
isochronal  F/F plots and ranged
from 0.6 to 3.0 µm, with a mean ± SD of 1.6 ± 0.6 µm. Model simulations of a localized Ca2+ entry site
predicted the major features of the fluorescence transients and
suggested that, within ~1 msec of the initiation of the
Ca2+ current, both the fluorescence domain and the
underlying Ca2+ 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.
Key words:
neuromuscular junction; presynaptic calcium; calcium
transients; calcium indicators; calcium microdomains; synaptic
transmission
Copyright © 1999 Society for Neuroscience 0270-6474/99/19187846-14$05.00/0
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