Nicotinic acetylcholine receptors (AChRs) undergo functional changes in gating and conductance during the development of skeletal muscle. Using developing muscles of Xenopus laevis, we will address the following questions about the mechanisms of these changes: (1) What is the role of the motor neuron in controlling the development of AChR function? (2) What is the role of muscle activity? (3) In what way does AChR development depend on the identity of the muscle? (4) What is the molecular basis for the developmental change in AChR properties? Our aim is to understand the processes which take place during normal development, and our experiments will therefore be done primarily in vivo. Furthermore, because there is evidence that muscles differ in their patterns of AChR development, we will take a comparative approach in this study. We will study selected muscles (myotomal, extraocular and interhyoideus) which differ in contraction speed, synaptic structure, and in their development of endplate currents. It is important, for the questions we wish to examine, to have a detailed description of the development of AChR function in each of these muscles. With that information as a baseline, we can then manipulate the conditions of development in order to examine the roles of nerve and muscle activity. We will describe the normal development of AChR function by single channel recording. We will compare AChR function at junctional and nonjunctional areas to determine if the motor neurons exert a local influence on receptor properties. Neural regulation of AChR function will be further studied by raising animals in beta-bungarotoxin, which eliminates motor innervation in developing muscle, but appears to leave all other inductive influences in place. The role of motor activity will be studied by raising embryos in tetrodotoxin, which selectively eliminates motor activity while innervation remains intact. To examine the molecular basis for changes in receptor function, we will assess the expression of genes for AChR subunits by in situ hybridization using radiolabeled complementary RNA probes to the subunit mRNAs. We will compare the timing and type of subunit message expressed with the predicted message based on single channel recordings. We will examine subunit mRNA synthesis in normally developing muscle and also in muscle which is aneural and/or immobilized during development.
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