NEUROBIOLOGIC STUDIES OF NEURONS AND GLIA IN CELL CULTURE

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We have previously shown that chronic (>24 hr.) stimulation of sensory neurons in vitro produces an alteration in growth cone sensitivity to stimulation and a down regulation of voltage-sensitive Ca++ channels (VSCC). We now show specificity in this effect both with regard to the type of VSCC affected and for different patterns of stimulation. The low voltage activated VSCC (T current) is extremely sensitive to stimulation; single stimuli at a steady rate of 0.5 Hz completely abolish the T current while not affecting the high voltage activated (N and L) VSCC. The same number of impulses organized into different patterns of tetanic ('bursty') or steady stimulations have differential effects on the N and L currents. Binding of a radio-labelled VSCC ligand, PN200-110 is also down regulated by chronic stimulation confirming that the number of Ca++ channels is affected by the stimulation. Activation of cultured skeletal muscle cells with acetylcholine produces an increase in prothrombin mRNA, which is consistent with the role we have proposed for thrombin in the process of activity dependent synapse elimination. We fail to demonstrate a modulation of the serine protease inhibitor Protease Nexin 1 in muscle, however. If this endogenous inhibitor does play a role in synapse elimination it may be because of its presence (and modulation) in some other cell type, either nerve or glia. Evidence for PN 1's presence and modulation in glia cells has indeed been obtained by Brenneman's group in the LDN. To investigate the cellular localization of activity-independent guidance cues in the developing visual system, we have established retinal and tectal cultures in modified 3-compartment chambers. Using embryonic chick we have localized a previously identified aversive component to tectal neurons. We have discovered a possibly novel, positive guidance component on radial glia. Both of these guidance mechanisms are restricted to one portion of the tectum during development.