Neuropeptide Distribution and Release from Aplysia californica Neurons

One challenge of contemporary neurobiology is to understand the cellular mechanisms responsible for neurotransmitter targeting and release. Essential to these studies is knowledge of the amounts and locations of neuropeptides present in the neuron. Although multiple neurotransmitters are found in many neurons, it is assumed that a given neuron releases the same set of neurotransmitters from all of its terminals. The situation appears to be more complex in the giant marine mollusk Aplysia californica. Multiple neuroactive peptides found within the bag cell neurons are differentially packed into individual vesicles and the vesicles (containing different neuropeptides) may be differentially distributed among the process of the neuron. These results prompt additional questions: does the neuron target different neuropeptides to specific release sites, and can the neuron release different neuropeptides at specific terminals? The goals of our research are twofold. The first is to develop the analytical instrumentation and methodology capable of identifying and quantifying neuronal releasates from a single nerve terminal and the contents of individual varicosities along a single nerve process. The second research area is in applying these techniques to study the distribution and release of neurotransmitters from individual cultured neurons of Aplysia and Lymnaea.

We have developed new analytical instrumentation and methodology to allow the distribution and release of neuroactive peptides to be measured. Our approach is both matrix assisted laser-desoprtion mass spectrometry and capillary electrophoresis separations followed by multichannel laser-induced fluorescence and radiochemical detection. The fluorescence method can separate and detect zeptomole (10-21 mole) amounts of these analytes in nanoliter volumes. These methods allow the assay of the peptides present in a neuron as well as the release of peptides from an intact cluster. We are using these methods to assay the peptides from individual cells and have started to follow peptide release from clusters of peptidergic neurons. In leading to a description of the subcellular dynamics of neuronal signaling, this work contributes to our basic understanding of the nervous system.