Neurobiologic Studies Of Neurons & Glia In Cell Culture

工作总结：我们继续在体外神经肌肉突触系统中参与活动依赖性，Hebbian突触消除的细胞生物学机制方面的工作。先前的实验表明，蛋白激酶C （PKC）和蛋白激酶A （PKA）的联合但相反的作用对于未受刺激的输入被其他输入激活的靶细胞的刺激特异性损失是必不可少的。今年的研究成果包括：1)在大鼠或小鼠体内的合作实验证实了PKC在完整动物出生后发生的突触消除过程中的重要性。突触后受体丧失和神经突缩回之间的一些分离在体内是明显的，尽管这两个过程之间的一般相关性是明显的。PKC的θ亚型被敲除的小鼠表现出神经肌肉连接处突触消除的延迟，但最终确实发生了多神经支配的丧失。2)当体外培养的PKC theta k.O.动物神经与肌肉之间形成突触时，刺激PKC不再产生突触丢失。3)当PKC激活phbol酯（如PMA）被放置在我们的3室系统的突触室中心时，如果PKC在肌肉中起作用，我们会看到突触的损失。同样，PKC阻滞剂如果仅应用于突触中心隔室是有效的。PKC缺陷肌与侧神经元室正常神经结合的实验结果也与PKC作用的肌肉位点一致。这些制剂显示出PKC激活产生的突触消除的显著减少。更令人惊讶的是正常肌肉和PKC敲除神经的结果。这些制剂还显示了PKC诱导的突触丢失的明显缺陷，完全与肌肉K.O，正常神经组合所显示的相当。这表明突触前PKC功能必须与突触后PKC结合才能产生突触损失。4) PKA可能同时具有突触前和突触后的作用。PKA介导刺激输入的稳定和加强，我们已经证明突触后注射PKI （PKA的抑制剂）与突触的电激活一起导致突触连通性的主要丧失。当PKA阻滞剂H-89仅应用于侧室时，我们也看到突触的主要活动依赖性损失。这种损失是由于神经递质释放的可能性减少。这种对刺激的敏感性大约需要20-30分钟才能形成，我们认为这是一些依赖PKA的物质从细胞体运输到突触所需的时间，在突触中，它需要维持递质输出。5)我们研究了神经胶质源性神经营养因子（GDNF）可能对突触稳定有影响的可能性。其他人已经证明，从肌肉中释放的GDNF可以影响突触前功能。我们已经测试了GDNF是否对肌肉功能有影响，特别是对乙酰胆碱受体（AChR）的影响。我们发现GDNF治疗肌肉，即使在没有神经的情况下，也在神经支配的纤维中，增加了AChR插入受体簇的速度。GDNF处理不影响细胞簇中受体的丢失率。一些细胞生物学机制，其中GDNF是偶联受体处置已被检查。我们认为我们的结果确定了一些关键的突触后事件介导Hebbian可塑性，并将在未来把更多的注意力放在可能的突触前机制上。

Summary of work: We have continued our work on the cell biological mechanisms involved in activity-dependent, Hebbian synapse elimination in an in vitro neuromuscular synaptic system. Previous experiments showed that conjoint but opposing actions of protein kinase C (PKC) and protein kinase A (PKA) were essential for the stimulus specific loss of unstimulated inputs to target cells activated by other inputs. This years findings include: 1) Collaborative experiments in the rat or mouse in vivo confirm the importance of PKC to the process of synapse elimination that occurs post-natally in the intact animal. Some dissociation between postsynaptic receptor loss and neurite retraction was evident in vivo, although a general correlation between these two processes was evident. Mice in which the theta isoform of PKC was knocked out show a delay in the synapse elimination that occurs at the neuromuscular junction, but eventually loss of multiple innervation does occur. 2) When synapses form in vitro between nerve and muscle from PKC theta k.O. animals, stimulation of PKC no longer produces synapse loss. 3) When a PKC activating phorbol ester such as PMA is placed in the center, synaptic chamber of our 3 compartment system, we see loss of synapses as expected if PKC acted in the muscle. Similarly, a PKC blocker is effective if applied only in the center, synaptic compartment. Also consistent with a muscle locus of PKC action are the results of experiments in which PKC deficient muscle were combined with normal nerve in the side neuronal chambers. These preparation showed a marked decrement in the synapse elimination produced by PKC activation. More surprising were results with normal muscle and PKC theta knockout nerve. These preparations also showed a marked deficit in PKC induced synapsed loss, entirely comparable to that shown with the muscle K.O., normal nerve combination. This suggests that presynaptic PKC function must be combined with postsynaptic PKC to produce synapse loss. 4) PKA also probably has both pre- and post-synaptic action. PKA mediates the stabilization and strengthening of stimulated inputs and we have shown that post-synaptic injection of PKI, an inhibitor of PKA, in conjunction with electrical activation of the synapse results in a major loss of synaptic connectivity. When a PKA blocker, H-89, is applied to the side chamber only, we also see a major activity-dependent loss of synapses. This loss is due to a decrease in the probability of release of neurotransmitter. This sensitivity to stimulation takes some 20-30 minutes to develop, which we interpret as being the time taken for some PKA dependent material to be transported from the cell body to the synapse where it is needed for maintaining transmitter output. 5) We have examined the possibility that the Glia Derived Neurotrophic Factor (GDNF) may have an effect on synapse stabilization. It has been shown by others that GDNF released from muscle can affect presynaptic function. We have tested whether there may be some effect of GDNF on muscle function, specifically on the acetylcholine receptor (AChR). We find that GDNF treatment of muscle, even in the absence of nerve but also in innervated fibers, increases the rate at which AChR are inserted into receptor clusters. The rate of loss of receptors from the clusters is not affected by GDNF treatment. Some of the cell biological mechanisms by which GDNF is coupled to receptor disposition have been examined. We feel that our results identify some of the critical post-synaptic events mediating Hebbian plasticity, and will be putting some increased focus on possible presynaptic mechanisms in the future.