Calcium signaling at single mossy fiber presynaptic terminals in the rat hippocampus

We investigated internal Ca2+ release at mossy fiber synapses on CA3 pyramidal neurons (mossy fiber terminals, MFTs) in the hippocampus. Presynaptic Ca2+ influx was induced by giving a brief train of 20 stimuli at 100 Hz to the mossy fiber pathway. Using Ca2+ imaging techniques, we recorded the Ca2+ 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 [Ca2+] could be completely blocked by application of 400 muM Cd2+. Furthermore, the evoked Ca2+ 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 Ca2+ stores resulting in depletion of internal Ca2+. We found that ryanodine, cyclopiazonic acid, thapsigargin, and ruthenium red all decreased both the early and the delayed increase in the Ca2+ signals. We applied D, L-2-amino-5-phosphonovaleric acid (D, L-APV; 50 muM) and 6,7-Dinitroquinoxaline-2,3-dione (DNQX; 20 muM) to exclude the action of N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Experiments with alternative lower affinity indicators for Ca2+ (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 Ca2+ signal in response to brief trains of stimuli is due to release of Ca2+ from internal stores.