NEUROBIOLOGIC STUDIES OF NEURONS AND GLIA IN CELL CULTURE

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We have demonstrated activity-dependent synapse reduction in a 3- compartment tissue culture system in which two populations of cholinergic neurons converge to innervate a common population of muscle fibers. This synapse reduction is blocked by a specific thrombin inhibitor, hirudin; this implicates the action of thrombin in the process of synapse reduction. We now show that the source of the thrombin is the muscle or nerve cells (and not the serum usually present in the culture medium) by demonstrating activity-dependent synapse reduction in serum free medium. Exogenous thrombin produces synapse reduction even in the absence of neural activity and the thrombin induced reduction is blocked by hirudin. Thrombin takes some three days to produce synapse loss, while stimulation produces similar loss in only one day, so some synergy between thrombin action and nerve activity may occur. We have measured thrombin activity (hirudin sensitive proteolytic activity) in the supernatant media from inactive (tetrodotoxin silenced) and active (acetyl choline stimulated) muscle fibers. Activated muscle cells produce about twice as much thrombin as do inactive muscle. We have used semi-quantitative polymerase chain reaction methods to examine prothrombin mRNA levels in activated and inactive muscle cell cultures. After 48 hours of stimulation, prothrombin mRNA levels are about two times higher in activated than in inactive muscle cells. Thus in active muscle cells, and in correlation with the synapse loss that occurs with activation, thrombin secretion and the capacity for thrombin synthesis increases by some two fold. We have examined quantitatively the voltage-dependent Ca++ currents in dorsal root ganglion neurons, as a function of the activation of these cells. We find that the Ca++ currents are reduced substantially and significantly by 24 hours or more of electrical stimulation. This may be the basis for the adaptation of the growth cones of these cells to prolonged electrical stimulation that we had described earlier. mRNA for a cell adhesion molecule, L1, is strikingly down regulated by electrical stimulation of dorsal root ganglion neurons at only 0.1 Hz.