NEUROBIOLOGIC STUDIES OF NEURONS AND GLIA IN CELL CULTURE

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Summary of work: 1) We have continued our work on the mechanisms underlying Hebbian, activity dependent synapse elimination in an in vitro neuromuscular system. We have shown that this process is dependent on the action of protein kinase C (PKC). We now show that a major target for kinase action during the synapse elimination is the acetyl choline receptor (AChR) on the muscle cell surface at neuromuscular junctional synapses. Treatment of nerve-muscle synapses with TPA, a phorbol ester that activates PKC, produces a decrement in synapse effectiveness and a corresponding increased rate of loss and decrease in the concentration of AChR in the muscle membrane. Prolonged, high doses of TPA produce a down regulation of PKC with a parallel decrease in the effect of the drug on synapse effectiveness and on the levels of AChR at the synapse. There is not a detectable change in the pre- synaptic, neural component of the synaptic apparatus. Biochemical studies of AChR synthesis and degradation indicate that loss of AChR from the muscle is accelerated and the synthesis rate is diminished by TPA treatment and the latter effect may be more pronounced than the former. An activity dependent synapse stabilization or augmentation is a crucial component of any Hebbian model of synaptic plasticity. We find that when synapse loss is induced by TPA this can be blocked (synapses preserved) by low frequency stimulation of the synapses. This preserving effect of activation is blocked by a PKA inhibitor, H-89. This suggests that the positive aspect of the Hebbian model requires the action of PKA. Considerable evidence from the literature indicates that both PKC and PKA phosphorylate the AChR and the differential phosphorylation produced by these two kinases results in different effects on AchR stability. PKC destabilizes while PKA stabilizes the receptor. 2) Theoretical and experimental work on synaptic networks in vitro have shown that one of the key aspects of neuronal physiology determining the properties of networks is the incidence of the neurons within the network that generate spontaneous spiking activity. In the absence of such neurons a network will either exhibit very high rates of firing generated by sustaining excitatory synaptic interactions within the network, or be silent. Most networks in fact show sustained low frequency firing. Some networks show bursty behavior and both theory and experimental analysis indicate that this can be produced by a very low but finite incidence of spontaneously active neurons. 3) Growing axons have been shown to be capable of developing side branches some substantial distance (hundreds of microns) back from the growth cone. This usually happens in response to a stimulus that causes a collapse of the axons growth cone. It can happen to an unstimulated axon that is in contact with a stimulated, collapsing axon. This process may play a role in the efficient ingrowth of axons to their correct central targets. - Synapse elimination, Protein kinase C, Protein kinase A, activity dependent, acetylcholine receptor, neuromuscular junction, in vitro