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突触系统中Hebbian活动依赖性突触消除的机制。 当双神经支配肌管的一个输入被刺激时，对相同肌管的另一个非活动输入会选择性地丢失。 我们之前已经通过使用适当的抑制剂表明，这一过程取决于蛋白激酶C（PKC）的作用。胆碱能刺激肌肉增加PKC。 我们现在表明，激酶作用于突触后，并对肌肉乙酰胆碱受体（AChR）有重大影响。 用细胞内记录和利用确定突触功效变化的位点的方差方法进行的生理分析表明，PKC激活（通过佛波酯处理）的效果是突触后反应性的降低，在突触前递质释放机制中没有可检测到的变化。 单位突触量子事件的大小也减少了PKC激活。 这些变化是伴随着相应的减少在肌肉表面膜中的AChR的浓度没有检测到突触前的解剖学变化。 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H89以及另一种PKA阻断剂Rp-cAMPS阻断。 我们现在表明，激活的保护作用可以表现为终板电位（EPP）的增加和量子振幅的保留，并且可以通过应用环磷酸腺苷（cAMP）来模拟。 CAMP还可以防止PKC激活产生的AChR从肌膜上丢失 这表明赫布模型的积极方面需要PKA的作用。 大量文献表明，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。 我们假设非刺激输入的丢失和刺激输入的保留的特异性是由于A和C激酶的差异定位。 PKA锚定蛋白发生在神经肌肉接头和未来的工作将集中在显示，适当的本地化的激酶的存在和空间模式的激活可以产生的活动特异性突触调制的特征是广泛调用的Hebbian突触可塑性。

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.