Investigations into the control of nerve fiber growth and survival by local variations in extracellular potassium are proposed. A compartmentalized culture preparation will be employed in which axons from cell bodies of rat neurons located in one compartment grow across silicone grease barriers to enter separate compartments. In this way the fluid environment of the distal axons can be controlled independently of the fluid environment of the cell bodies and proximal axons. The distal axons can be removed at any time and promptly regenerate. This allows axonal regeneration of the same population of neurons to be observed sequentially under different experimental conditions and as the neurons develop and age in vitro. Micrometric measurements of neurite extension and photomicrographic comparisons of neurite density will be used to evaluate growth. Also, neurons in compartmentalized cultures can be chronically electrically stimulated, permitting the performance of experiments concerned with the role of electrical activity in growth and development. In addition to work with compartmentalized cultures, it is proposed to measure changes in [K+]e that occur in vivo by means of K+-selective microelectrodes. The questions addressed in this proposal arise because of observations made in this laboratory employing compartmentalized cultures. Neurites of sympathetic neurons, dorsal root ganglion neurons and spinal cord neurons can be excluded from entering a region of locally increased extracellular potassium ([K+]e). Also, a proximo-distal increase in [K+]e along the neurites has a variety of regressive effects on neurite growth, regeneration, and survival: elongation can be slowed, an extreme retraction of neurites can ensue, and complete degeneration can occur. These observations have implications for the mechanism of nerve fiber elongation, and they also raise the far-reaching possibility that natural variations in [K+]e in vivo during normal development may strongly influence the establishment and/or maintenance of connections between neurons. For example, K+ released by nerve fibers during activity may mediate competitive interactions for postsynaptic sites. It is also possible that regressive changes induced by variations in [K+]e may have implications for the problems of regeneration, aging, and seizure activity in the nervous system. Experiments are proposed to: (1) characterize the effects of locally elevated K+ on peripheral and central neurons with particular emphasis on changes that occur with development and aging in vivo and in vitro, (2) investigate the mechanisms of these effects, (3) begin to collect information about what magnitude of changes in [K+]e can occur during development in vivo, and (4) establish an in vitro system to study the long-term effects of activity on synapse formation and maintenance.
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