Neurobiologic Studies Of Neurons & Glia In Cell Culture

工作总结：我们继续我们的工作，在体外神经肌肉突触系统的活动依赖性，赫布突触消除的细胞生物学机制。以前的实验表明，蛋白激酶C（PKC）和蛋白激酶A（PKA）的联合但相反的行动是必不可少的刺激特异性损失的未受刺激的输入到靶细胞激活的其他输入。今年的调查结果包括：1）在大鼠或小鼠体内的合作实验证实了PKC对完整动物出生后发生的突触消除过程的重要性。突触后受体丢失和轴突回缩之间的一些解离在体内是明显的，虽然这两个过程之间的一般相关性是显而易见的。PKC的theta亚型被敲除的小鼠表现出在神经肌肉接头处发生的突触消除的延迟，但最终确实发生了多个神经支配的丧失。2)当神经和肌肉之间的突触在体外由PKC θ k.O.在动物中，PKC的刺激不再产生突触损失。3)当PKC激活佛波酯如PMA被放置在我们的3隔室系统的中心突触室时，我们看到如果PKC在肌肉中起作用，则如预期的那样失去突触。类似地，PKC阻断剂如果仅应用于中心突触隔室是有效的。与PKC作用的肌肉位点一致的还有将PKC缺陷的肌肉与侧神经元腔中的正常神经结合的实验结果。这些制备物显示由PKC激活产生的突触消除显著减少。更令人惊讶的是正常肌肉和PKC theta敲除神经的结果。这些制备物还显示出PKC诱导的突触丢失的显著缺陷，与肌肉K.O.所显示的完全相当，正常的神经组合这表明突触前PKC功能必须与突触后PKC结合才能产生突触丢失。4)PKA也可能具有突触前和突触后作用。PKA介导的稳定和加强刺激的输入，我们已经表明，突触后注射的PKI，PKA的抑制剂，与电激活的突触的结果在一个重大损失的突触连接。当PKA阻断剂H-89仅应用于侧腔时，我们也看到主要的活动依赖性突触丢失。这种损失是由于神经递质释放的可能性降低。这种对刺激的敏感性需要大约20-30分钟来发展，我们将其解释为一些PKA依赖性物质从细胞体运输到突触所需的时间，在突触中需要维持递质输出。5)我们已经研究了胶质源性神经营养因子（GDNF）可能对突触稳定性有影响的可能性。其他人已经表明，从肌肉释放的GDNF可以影响突触前功能。我们已经测试了GDNF是否可能对肌肉功能，特别是对乙酰胆碱受体（AChR）有一些影响。我们发现，GDNF治疗的肌肉，即使在没有神经，但也在神经支配的纤维，增加乙酰胆碱受体插入到受体簇的速率。受体从簇中丢失的速率不受GDNF处理的影响。GDNF与受体结合的一些细胞生物学机制已被研究。我们认为，我们的研究结果确定了一些关键的突触后事件介导的赫布可塑性，并将在未来把一些可能的突触前机制的重点。

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.