Target-dependent regulation of presynaptic calcium influx in an identified neuromuscular synapse in Helisoma trivolvis
Is Version Of
The focus of these studies has been to determine whether contact with an appropriate muscle target can regulate presynaptic calcium handling. An identified neuromuscular synapse from the freshwater pond snail Helisoma trivolvis was reconstructed in cell culture, and used to investigate the effect of target contact on presynaptic calcium handling. Cholinergic buccal moto-neuron 19 (B19) forms chemical synapses with muscle fibers from its in vivo synaptic target, the supralateral radular tensor (SLT) muscle. The reconstruction of this synapse in culture has allowed for experimental control of the extracellular environment, as well as experimental control over the timing of neuron-muscle contact. This control has permitted high-resolution studies of neuromuscular synaptogenesis;Contact with muscle fibers from the SLT causes a rapid enhancement (within 30 minutes) of action potential-evoked calcium accumulation as well as elevate resting calcium levels in neurons B19 at sites of muscle contact. Application of the cAMP analog pCPTcAMP mimics the effect of muscle contact on both presynaptic calcium accumulation, and resting calcium level. Inhibition of the activity of presynaptic cAMP-dependent protein kinase by the synthetic peptide inhibitor IP[subscript]20 blocks both muscle-induced and pCPTcAMP-induced enhancement of calcium levels. Taken together, these data indicate that muscle contact activates presynaptic cAMP-dependent protein kinase to locally-enhance resting calcium levels and action potential-evoked calcium accumulation;Tyrosine kinase activity was also shown to be necessary for the enhancement of presynaptic calcium accumulation. Inhibition of tyrosine kinase activity was shown to block the induction of, and reverse expressed muscle-induced enhancement of calcium accumulation. Inhibition of tyrosine kinase activity had only a small effect on pCPTcAMP-induced enhancement of action potential-evoked calcium accumulation, as did the intrasomatic injection of the broad-based protein tyrosine phosphatase rrbPTP-1. This suggests that the action of tyrosine kinase is upstream of the action of cAMP-dependent protein kinase.