NEUROTRANSMITTER RECEPTOR DYNAMICS IN VIVO

该项目的主要目的是更好地了解神经递质受体聚集是如何在突触中被调节和维持的。将使用新开发的技术来监测活小鼠个体神经肌肉连接处乙酰胆碱受体数量的变化。这种方法可以在几分钟到几个月的时间内量化一个确定的突触的受体增加和损失的速率。神经肌肉突触活动的改变将用于分析突触后活动在调节受体数量中的作用。利用高分辨率共聚焦显微镜，光漂白后荧光恢复，以及两种颜色受体标记，将有助于确定新合成的受体是直接添加到结上，还是插入到结周区域，然后迁移到被捕获的结上。最后，这些实验将检查长期阻断神经肌肉传递对神经肌肉连接的后续功能的影响。这个问题在临床上很重要，因为用神经肌肉阻滞剂治疗几天的患者，有时在移除阻滞剂后仍会瘫痪数周。这种破坏性综合征的病因尚不清楚，但似乎类似于支持该应用的初步工作中观察到的神经肌肉阻断的效果。由于活动和不活动诱导的受体数量的变化被认为是中枢神经系统长期增强和抑制的工具，因此本文提出的研究可能为活动对不易接近的突触的突触强度的影响提供基本信息。

The main objective of this project is to better understand how neurotransmitter receptor aggregations are regulated and maintained at a synapse. Newly developed techniques for monitoring changes in the number of acetylcholine receptors at individual neuromuscular junctions in living mice will be used. This approach permits quantification of the rates of receptor addition and loss from an identified synapse over periods of minutes to months. Modifications of the activity of the neuromuscular synapse will be used to analyze the role of postsynaptic activity in modulating receptor numbers. The use of high resolution confocal microscopy fluoresce recovery after photobleaching, and two color receptor labeling, will help resolve whether newly synthesized receptors are added directly to the junction or are inserted into the perijunctional region and then migrate to the junction where they are trapped. Lastly, these experiments will examine the consequences of long-term blockade of neuromuscular transmission on the subsequent function of the neuromuscular junction. This question is clinically important because patients who have been treated with neuromuscular blocking agents for several days occasionally remain paralyzed for weeks after removing the blocking agents. The etiology of this devastating syndrome is not known but seems analogous to the effects of neuromuscular blockade observed in the preliminary work that supports this application. Because activity and inactivity induced changes in receptor numbers are thought to be instrumental in long-term potentiation and depression in the central nervous system, the studies proposed here may provide fundamental information concerning the effects of activity on synaptic strength at less accessible synapses.