The overall problem with which this project is concerned is how the neuron regulates the type, number and destination of organelles that it sends to the axon and synaptic terminals by axonal transport. All of our experiments will involve the use of single identified giant neurons from the sea hare, Aplysia californica, studied in situ, in short-term organ culture, or in longer-term single cell culture. We want to understand why and how changes in the output of materials from the cell body occur to accommodate transitions between a mature axon with functioning synapses and a growing axon (after axotomy or during development or regeneration). Insofar as the major transported organelle associated with synaptic transmission, the transmitter storage vesicle, is concerned, we will attempt to determine whether its rate of export from the cell body is influenced by the presence of synapses at the ends of the axon and, if so, to determine whether a metabolically functional postsynaptic cell is the key feature of the synapse. These experiments will involve microinjection into an identified cell in culture whose regenerating axon forms chemical synapses. We will also attempt to determine how the cell body changes the rate of export in experiments involving the use of quantitative electron microscopic radioautography and gel electrophoresis. For the growing axon we want to identify the organellar vehicle for growth-associated glycoproteins and to determine whether growth at the sprout tips is the signal for the increased export of glycoproteins during regeneration. We also want to determine whether cAMP-dependent protein phosphorylation is needed for normal axon growth and, if it is, whether the cell body or the axonis its site of action. We will also examine factors underlying the partitioning of transported materials among the branches of the axonal tree. Also, using an identified neuron with a branch axon regenerating in culture, we will attempt to determine whether changes in partitioning are involved in either the maintenance or the initiation of changes in the pattern of growth of the tree during development or regeneration. In addressing basic questions of the cell biology of regeneration, the research is relevant to an understanding of the consequences of spinal cord injury and to the forulation of therapies. It is also relevant to the study of diseases like amyotrophic lateral sclerosis, where compensatory sprouting of healthy axons occurs and may affect the progression of the disease.
