NEUROBIOLOGIC STUDIES OF NEURONS AND GLIA IN CELL CULTURE

工作总结：1)在体外神经肌肉突触系统中，我们继续研究Hebbian，活性依赖的突触消除的机制。当刺激双神经支配的肌管的一个输入时，同一肌管的另一个不活跃的输入被选择性地丢失。我们先前已经证明，通过使用适当的抑制剂，这一过程依赖于蛋白激酶C(PKC)的作用。对肌肉的胆碱能刺激增加了PKC。我们现在证明，该激酶在突触后起作用，并对肌乙酰胆碱受体(AChR)有重要影响。细胞内记录的生理分析和利用确定突触效能变化位置的方差方法表明，PKC激活(通过佛波酯处理)的效果是突触后反应性的降低，而突触前递质释放机制没有可检测到的变化。PKC的激活也减小了单一突触量子事件的大小。伴随着这些变化的是肌肉表面膜上AChR浓度的相应降低，而没有检测到突触前解剖结构的变化。依赖活动的突触稳定或增强是任何突触可塑性的Hebbian模型的关键组成部分。我们发现，当TPA诱导突触丢失时，这可以被突触的低频刺激所阻断(突触被保留)。这种激活的保护作用可被PKA抑制剂H89和另一种PKA阻滞剂RP-cAMP阻断。我们现在证明，激活的保存效应可以表现为终板电位(EPP)的增加和量子幅度的保存，并且可以被环腺苷一磷酸(CAMP)的应用所模拟。CAMP还可以阻止PKC激活所产生的肌膜AChR的丢失，这表明Hebbian模型的积极方面需要PKA的作用。大量的文献证据表明，PKC和PKA都能使AChR磷酸化，并且这两种酶产生的不同磷酸化对AChR稳定性的影响是不同的。PKC使受体不稳定，而PKA使受体稳定。非刺激输入丢失和刺激输入保留的特异性，我们假设是由于A和C激酶的不同定位所致。PKA锚定蛋白存在于神经肌肉接头处，未来的工作将集中于证明存在适当的激酶定位及其激活的空间模式可以产生活性特异性的突触调节，这是被广泛激活的Hebbian突触可塑性的特征。

Summary of work: 1) We have continued our work on the mechanisms underlying Hebbian, activity dependent synapse elimination in an in vitro neuromuscular synaptic system. When one input to doubly innervated myotubes is stimulated there is a selective loss of the other, inactive input to the same myotubes. We have previously shown, by the use of appropriate inhibitors, that this process is dependent on the action of protein kinase C (PKC). Cholinergic stimulation of muscle increases PKC. We now show that the kinase acts postsynaptically and has a major effect on the muscle acetyl choline receptor(AChR). Physiological analysis with intracellular recordings and utilizing a variance method of determining the locus for changes in synapse efficacy shows that the effect of PKC activation (by phorbol ester treatment) is a decrease in post-synaptic responsiveness, with no detectable change in the presynpatic transmitter release mechanism. The size of the unitary synaptic quantal event is also reduced by PKC activation. These changes are accompanied by a corresponding decrease in the concentration of AChR in the muscle surface membrane without detectable presynaptic anatomical change. 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, H89, as well as another PKA blocker, Rp-cAMPS. We now show that the preserving effect of activation can be demonstrated as an increase in the end plate potential (EPP) and a preservation of quantal amplitude, and can be mimicked by the application of cyclic adenosine monophosphate (cAMP). CAMP also prevents the loss of AChR from the muscle membrane produced by PKC activation 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. The specificity of loss of non-stimulated inputs and retention of stimulated inputs we hypothesize to be due to differential localization of the A and C kinases. PKA anchoring proteins occur at the neuromuscular junction and future work will focus on showing that the appropriate localization of the kinases exists and spatial patterns of their activation can generate the activity specific synaptic modulation that is characteristic of the widely invoked Hebbian synaptic plasticity.