NEUROTRANSMITTER RECEPTOR DYNAMICS IN VIVO

这个项目的主要目标是更好地了解神经递质受体聚集是如何在突触中调节和维持的。将使用新开发的技术来监测活着的小鼠单个神经肌肉接头上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已识别突触的受体增加和丢失的速率。神经肌肉突触活性的修饰将被用来分析突触后活动在调节受体数量方面的作用。使用高分辨率共聚焦显微镜在光漂白后的荧光恢复，以及两种颜色受体的标记，将有助于确定新合成的受体是直接添加到连接处，还是插入到交界区，然后迁移到它们被捕获的连接处。最后，这些实验将检验长期阻断神经肌肉传递对神经肌肉接头后续功能的影响。这个问题在临床上很重要，因为接受神经肌肉阻滞剂治疗数天的患者在移除阻滞剂后偶尔仍会瘫痪数周。这种毁灭性综合征的病因尚不清楚，但似乎类似于在支持这一应用的初步工作中观察到的神经肌肉阻滞的效果。由于活动和不活动引起的受体数量的变化被认为有助于中枢神经系统的长期增强和抑制，因此这里提出的研究可能提供关于活动对较难到达的突触的突触强度的影响的基本信息。

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.