Calcium Signaling at Single Mossy Fiber Presynaptic Terminals in the Rat Hippocampus

Abstract

We investigated internal Ca(2+) release at mossy fiber synapses on CA3 pyramidal neurons (mossy fiber terminals, MFTs) in the hippocampus. Presynaptic Ca(2+) influx was induced by giving a brief train of 20 stimuli at 100 Hz to the mossy fiber pathway. Using Ca(2+) imaging techniques, we recorded the Ca(2+) response as DeltaF/F, which increased rapidly with stimulation, but was often accompanied by a delayed peak that occurred after the train. The rise in presynaptic [Ca(2+)] could be completely blocked by application of 400 microM Cd(2+). Furthermore, the evoked Ca(2+) signals were reduced by group II mGluR agonists. Under the same experimental conditions, we investigated the effects of several agents on MFTs that disrupt regulation of intracellular Ca(2+) stores resulting in depletion of internal Ca(2+). We found that ryanodine, cyclopiazonic acid, thapsigargin, and ruthenium red all decreased both the early and the delayed increase in the Ca(2+) signals. We applied D,L-2-amino-5-phosphonovaleric acid (D,L-APV; 50 microM) and 6,7-Dinitroquinoxaline-2,3-dione (DNQX; 20 microM) to exclude the action of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Experiments with alternative lower affinity indicators for Ca(2+) (fura-2FF and calcium green-2) and the transient K(+) channel blocker, 4-aminopyridine were performed to control for the possible saturation of fura-2. Taken together, these results strongly support the hypothesis that the recorded terminals were from the mossy fibers of the dentate gyrus and suggest that a portion of the presynaptic Ca(2+) signal in response to brief trains of stimuli is due to release of Ca(2+) from internal stores.