NEUROBIOLOGIC STUDIES OF NEURONS AND GLIA IN CELL CULTURE

工作总结：1）我们继续研究了体外神经肌肉系统中Hebbian活动依赖性突触消除的机制。我们已经表明，这一过程是依赖于蛋白激酶C（PKC）的行动。我们现在表明，在突触消除过程中激酶作用的一个主要目标是在神经肌肉连接突触的肌肉细胞表面上的乙酰胆碱受体（AChR）。用TPA（一种激活PKC的佛波醇酯）治疗神经-肌肉突触，会导致突触有效性降低，相应的丢失率增加，肌肉膜中AChR浓度降低。长时间高剂量的TPA会导致PKC下调，同时药物对突触有效性和突触AChR水平的影响也会相应降低。在突触器的突触前神经成分中没有可检测到的变化。AChR合成和降解的生化研究表明，TPA处理加速了肌肉AChR的损失，降低了合成速率，后者的作用可能比前者更明显。活动依赖性突触稳定或增强是任何突触可塑性的赫布模型的重要组成部分。我们发现，当突触损失是由TPA引起的，这可以被阻止（突触保存）的突触的低频刺激。这种激活的保护作用被PKA抑制剂H-89阻断。这表明赫布模型的积极方面需要PKA的作用。大量的文献表明PKC和PKA都能磷酸化AChR，这两种激酶产生的差异磷酸化对AChR的稳定性产生不同的影响。PKC使受体不稳定，而PKA使受体稳定。2)体外突触网络的理论和实验工作表明，神经元生理学决定网络性质的关键方面之一是网络内产生自发尖峰活动的神经元的发生率。在缺乏这种神经元的情况下，网络要么表现出非常高的放电率，这是由网络内的兴奋性突触相互作用产生的，要么是沉默的。事实上，大多数网络都显示出持续的低频放电。一些网络表现出突发行为，理论和实验分析都表明，这可以由非常低但有限的自发活动神经元发生率产生。3)生长的轴突已经被证明能够从生长锥向后发展一些实质性的距离（数百微米）的侧分支。这通常发生在对导致轴突生长锥崩溃的刺激的反应中。它可以发生在未受刺激的轴突与受刺激的轴突接触时，塌陷。这一过程可能在轴突向其正确的中央目标的有效向内生长中发挥作用。- 突触消除，蛋白激酶C，蛋白激酶A，活性依赖性，乙酰胆碱受体，神经肌肉接头，体外

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