Development of Myotomal Neuromuscular Junction

Kullberg, Richard

Abstract

The embryonic neuromuscular junction of Xenopus laevis will be used to study the development of synaptic currents and their dependence upon changes in acetylcholine receptor (AChR) channel kinetics, junctional densities of AChRs and acetylcholine esterase (AChE), and muscle activity. The mean channel open time of embryonic AChRs present on myotomal muscle at the time of first nerve-muscle contact will be estimated by spectral analysis of ACh-induced current fluctuations. Subsequent developmental changes in channel kinetics will be examined to determine whether the population of receptors is homogeneous at each stage, or whether two populations of channels (embryonic and mature) are present, as in developing rat neuromuscular junctions. The contribution of AChE development to changes in duration of synaptic currents will be examined by blocking the enzyme at the mature synapse with methanesulfonyl fluoride and comparing the resultant synaptic currents with those recorded from early developmental stages where AChE is absent. The hypothesis that the developmental changes in shape and duration of synaptic currents are due solely to an increase in AChE and a reduction in AChR channel open time will be tested by simultaneously blocking AChE and lengthening channel open time at the mature synapse. The dependence of developmental changes in AChR kinetics on muscle activity will be investigated by measuring AChR channel open time in embryos which have developed in the presence of immobilizing anesthetic. In parallel with these physiological studies, the developmental changes in densities of AChR and AChE sites on myotomal muscle membrane will be studied quantitatively by electron microscope autoradiography. AChR sites will be selectively labeled by 125I-alpha-BTX; AChE sites will be labeled with 3H-DFP following 2-PAM reactivation of cold DFP-blocked esterase. The degradation rate of embryonic AChRs, as estimated by EM autoradiography, will be compared with the rate of developmental change in channel kinetics to determine whether the change in kinetics is due to replacement or modification of receptors.