Calcium in motor nerve terminals associated with posttetanic potentiation

We have used fura-2 fluorescence to study the effects of repetitive stimulation producing posttetanic potentiation (PTP) at crayfish neuromuscular junctions on presynaptic calcium concentration. Fura-2 was injected into the preterminal axon of the excitor motor neuron to the claw opener muscle of a walking leg. Pictures of presynaptic terminals on the muscle surface were obtained with a charge-coupled device camera, ratioed, and converted to spatial images of intracellular calcium concentration. Stimulation of the motor nerve for 7-10 min at 20-33 Hz produced potentiation during the tetanus and PTP following the tetanus. Presynaptic calcium levels in terminal boutons and varicosities rose to about 2 microM during the tetanus and decayed at first rapidly and then slowly back to levels near the initial concentration of about 200 nM. The decay rate of potentiated synaptic transmission was the same as the decay rate of the elevated calcium concentration during the posttetanic period dominated by PTP, when facilitation and augmentation had dissipated. A 13-fold potentiation corresponded to a 500 nM elevation of calcium to about 700 nM. The linear dependence we observed is not consistent with the power law formulation of a residual calcium hypothesis for PTP. During the tetanus, the enhancement of synaptic transmission due to facilitation, augmentation, and potentiation exceeded that expected from the correspondence between PTP and posttetanic calcium levels. This may occur because during the tetanus there is insufficient time for calcium to equilibrate spatially between action potentials, and the submembrane calcium will be higher than the volume-average calcium levels that we detect. Following low-frequency trains (typically 8 Hz for about 35 sec), enhanced synaptic transmission and elevated presynaptic calcium decayed rapidly, within a few seconds. Short high-frequency trains (50-100 Hz for 1-2 min) elicited an additional hours-long elevation of presynaptic calcium, corresponding to, and perhaps responsible for, part of the long-term potentiation of transmission that such stimulation produces at this synapse.