PRESYNAPTIC CALCIUM DYNAMICS AT THE FROG RETINOTECTAL SYNAPSE

1. We characterized the kinetics of presynaptic Ca2+ ion concentration in optic nerve fibers and terminals of the optic tectum in Rana pipie ns with the use of microfluorimetry. Isolated frog brains were incubat ed with the membrane-permeant tetraacetoxymethyl ester (AM) of the Ca2 + indicator fura-2. An optic nerve shock caused a transient decrease i n the 380-nm excited fluorescence in the optic tectum with a rise time of <15 ms and a recovery to prestimulus levels on a time scale of sec onds. 2. In normal saline, the amplitude of the fluorescence transient s was dependent on stimulus intensity and at all levels it was directl y correlated with the amplitude of postsynaptic field potentials produ ced by activation of unmyelinated optic nerve fibers. In the presence of the non-N-methyl-D-aspartate glutamate receptor antagonist 6-cyano- 7-nitroquinoxaline-2,3-dione, the amplitude and time course of fluores cence transients remained essentially unchanged while postsynaptic fie ld potential amplitude was greatly reduced. Replacing extracellular Ca 2+ with Ba2+ blocked unfacilitated postsynaptic field potentials while fluorescence transients remained significant. In reduced-Ca2+ salines (<1 mM), the amplitude of fluorescence transients increased approxima tely linearly with extracellular [Ca2+], whereas the amplitude the cor responding field potential was nonlinearly related to the fluorescent transient amplitude (similar to 2.5 power). In thin sections of labele d tecta, fluorescence labeling was localized to 1-mu m puncta in the t ermination zone of optic nerve fibers in the superficial layers. Taken together, these results provide strong evidence that the fluorescence transients correspond to an increase in Ca2+ in presynaptic terminals of unmyelinated optic nerve fibers. 3. During trains of optic nerve s timulation, the amplitude of fluorescence transients to succeeding act ion potentials became smaller. The decrement of the amplitudes was not observed in mag-fura-5-labeled tecta, when the intracellular Ca2+ buf fering capacity of fura-2-labeled terminals was increased by incubatio n with bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM or e thylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid ( EGTA)-AM, or in low-Ca2+ saline. We conclude that the Ca2+ influx per action potential is constant during the train and that the reduced res ponse was produced by saturation of the fura-2. We provide a mathemati cal analysis of this saturation effect and use it to estimate the Ca2 change per action potential. 4. Both BAPTA-AM. and EGTA-AM reduced th e overall amplitude of fura-2-measured Ca2+ transients and reduced the saturation effect in action potential trains. However, there was a qu alitative difference in their effects on the shape of the transient. I ncubation with the fast buffer BAPTA prolonged the decay to baseline. In contrast, the slow buffer EGTA (or EDTA) produced an initial decay faster than the control condition while also producing the slower subs equent phase observed with BAPTA. We demonstrate that these results ar e consistent with numerical simulations of Ca2+ dynamics in a single-c ompartment model where the fast initial decay is produced by the forwa rd rate of Ca2+ binding to EGTA.5. Ca2+ influx into tectal presynaptic structures, and also into unmyelinated axons in the isolated optic ne rve, was diminished (60-70%) in the presence of the voltage-activated Ca2+ channel blocker omega-conotoxin GVIA, but was only weakly affecte d (approximate to 10%) by omega-agatoxin IVA. 6. After 10- to 50-Hz st imulus trains, synaptic enhancement of unmyelinated fibers decayed wit h a characteristic time similar to fura-2 fluorescence decays. Incubat ion with EDTA-AM or EGTA-AM produced Little effect on evoked release b ut reduced both the amplitude of the fura-2-measured Ca2+ transient an d the amplitude of short-term synaptic enhancement. Our data are consi stent with the hypothesis that at least part of activity-dependent sho rt-term synaptic enhancement that decays on the time scale of seconds at the retinotectal synapse is produced through the actions of presyna ptic residual Ca2+ ions.