Experiments will be conducted to investigate the role of synaptic glia (terminal Schwann cells) in the formation and maintenance of neuromuscular junctions (nmjs). Recent evidence suggests that these glia explain many of the responses to loss of muscle innervation: these cells apparently extend processes that induce and guide growth of nerve sprouts. Here it is proposed to rigorously test the importance of these cells by vitally imaging the cellular components of the nmj during recovery from nerve injuries. Muscle acetylcholine receptors will be labeled with rhodamine-alpha-bungarotoxin. Doubly transgenic mice expressing fluorescent proteins (CFP and GFP) of different colors in their axons and Schwann cells will allow visualization of these components as well. By repeated imaging of the same nmjs in living animals it will be possible to determine whether Schwann cell processes lead or follow several forms of axon growth and regression, whether Schwann cell processes grow in a direct manner to synaptic sites in muscle, and whether the growth induced in response to nerve injuries actually disrupts existing synaptic connections. In addition to these vital observations, Schwann cells will be manipulated in the living animal. First, these cells will be ablated and the consequences for synaptic maintenance and repair evaluated. Second, additional transgenic animals will be prepared in which it will be possible to induce selectively the expression of specific genes in Schwann cells and examine the consequences for muscle innervation. Initially these experiments will employ a dominant negative receptor for a trophic factor implicated in the previous application period as important for the maintenance of Schwann cells. Taken together these experiments should add to the recent explosion of information about synaptic glia. They also will help us understand the growth of nerves in muscles by sprouting, the major means by which muscle strength is maintained following loss of motor neurons in neurodegenerative disease or peripheral nerve lesions.
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