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.
