Neurobiologic Studies Of Neurons & Glia In Cell Culture

神经元的神经生物学研究

• 批准号: 6508725
• 负责人: PHILLIP G NELSON
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6508725
• 关键词: acetylcholine; bioperiodicity; chickens; developmental neurobiology; enzyme activity; gene targeting; genetically modified animals; glia; laboratory mouse; laboratory rat; neural information processing; neural plasticity; neuromuscular junction; neuronal guidance; neurons; neuropharmacology; neurotrophic factors; protein kinase A; protein kinase C; receptor; staurosporine; superior colliculus; synapses; tissue /cell culture; voltage /patch clamp

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.

工作总结：我们继续在体外神经肌肉突触系统中涉及活性依赖的Hebbian突触消除的细胞生物学机制方面的工作。以往的实验表明，蛋白激酶C(PKC)和蛋白激酶A(PKA)的联合但相互对立的作用是刺激特异性丢失非刺激性输入到由其他输入激活的靶细胞所必需的。今年的发现包括：1)在大鼠或小鼠体内的协同实验证实了PKC在完好动物出生后突触消除过程中的重要性。突触后受体丢失和轴突回缩之间的一些分离在体内是明显的，尽管这两个过程之间的总体相关性是明显的。在PKC的theta亚型被敲除的小鼠中，神经肌肉连接处发生的突触消除出现了延迟，但最终确实发生了多重神经支配的丧失。2)当PKC theta k.O.动物的神经和肌肉在体外形成突触时，刺激PKC不再产生突触丢失。3)当PKC激活佛波酯(如PMA)被放置在我们的三室系统的突触中心时，如果PKC作用于肌肉，我们会看到突触的丢失。同样，PKC阻滞剂如果只在突触中央隔间使用，也是有效的。将PKC缺陷的肌肉与正常神经结合在侧神经元腔中的实验结果也与PKC的肌肉作用轨迹一致。这些制剂显示，由PKC激活产生的突触消除明显减少。更令人惊讶的是正常肌肉和PKC theta基因敲除神经的结果。这些制剂在PKC诱导的突触丢失方面也显示出明显的缺陷，与肌肉K.O.，正常神经组合所显示的完全相同。提示突触前PKC功能必须与突触后PKC结合才能产生突触丢失。4)PKA可能同时具有突触前和突触后的作用。PKA介导了刺激输入的稳定和加强，我们已经证明，突触后注射PKA的抑制剂PKI，结合突触的电激活，会导致突触连接的严重丧失。当PKA阻断剂H-仅应用于侧室时，我们也看到主要的活性依赖性突触丢失。这种损失是由于神经递质释放的概率降低所致。这种对刺激的敏感性需要大约20-30分钟的时间才能形成，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，在那里它是维持递质输出所需的。5)研究了胶质源性神经营养因子(GDNF)对突触稳定作用的可能性。已有研究表明，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对肌肉的治疗，即使在没有神经的情况下，也可以在有神经的纤维中进行，增加AChR插入受体簇的速度。GDNF处理不影响簇上受体的丢失率。GDNF与受体结合的一些细胞生物学机制已经被研究过。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的可塑性，并将在未来更多地关注可能的突触前机制。