As part of the training program of the applicant, this research project's aim is to elucidate further the function of calcium in neuromodulation of ion currents and in secretion, and to study the effects of neuroactive peptides on the calcium currents in Aplysia bag cell neurons. By using whole cell voltage clamping and single channel recording techniques, and by looking at the calcium concentration using fura-2 and image processing techniques, the changes in calcium current and intracellular calcium concentration in response to bag cell peptides can be studied in the intact nervous system. A set of experiments will attempt to determine if the peptides work by activating protein kinase C. The effects of the peptides on the calcium currents will be examined in the presence of protein kinase inhibiting agents to see if observed changes in calcium current can be prevented in the presence of these substances. The effect of the peptide on calcium current will be examined during the neurons' refractory period to determine if calcium and neuroactive peptides play a role in bringing on or maintaining the refractory period. Bag cell neurons will be examined in primary culture to study specific properties of the ion currents and the influence of the peptides on the currents. The influence of culturing the neurons on the calcium current will be studied. In order to see if ion channels can move during the life of a cell, cultured bag cell neurons will be examined using electrophysiological techniques and fluorescence imaging techniques to look at changes in the concentration of calcium in the soma as compared to the neurites during the process of neurite-sprouting. Cytoskeleton inhibiting agents will used to try to inhibit changes in calcium current observed. Neurons will be studied in the absence of external calcium to see if autoactive peptides can influence the release of intracellular calcium. The ultimate goal of this research will be to determine how neurons communicate in order to provide insight into prevalent neuropathological processes.
