The proposed research examines roles for neurotransmitters and their cellular signal transduction mechanisms (electrical activity, intracellular calcium and other second messengers in the regulation of dendritic and axonal outgrowth in isolated individual and mitotic sister hippocampal pyramidal neurons. This model allows us to address several key problems not previously approachable including; 1) How are axons and dendrites different with respect to their outgrowth and its control by neurotransmitters, electrical activity and intracellular calcium? 2) Which of the neurotransmitters that provide input to pyramidal neurons in situ affect outgrowth in isolated neurons and where do they act on the neuron (growth cones, neurite shaft,soma)? 3) Based upon the fact that different classes of afferents provide different neurotransmitter influences in a sequential order during development, we will ask whether this is mirrored in the chronology of expression of outgrowth responses to the different neurotransmitters? 4) What are the cellular mechanisms by which neurotransmitters affect outgrowth? Are they the same mechanisms used in synaptic transmission? 5) What influence does cell lineage have on the expression of neuronal form and its modification by neurotransmitters? This question will be addressed using mitotically- related pyramidal neurons. The techniques used to address these questions include: culture of isolated pyramidal neurons: analyses of axonal and dendritic outgrowth rates, physical isolation of axons and dendrites: electrophysiological recording and stimulation: measurement of intracellular calcium levels with fura-2. The results obtained from these studies will provide important insights into how neurotransmitters and electrical activity might be involved in the generation of functional neuronal circuitry, its modification during adult life, and its degeneration in neurodegenerative disorders.
