DEVELOPMENT OF ACETYLCHOLINE RECEPTORS ON XENOPUS EMBRYONIC MUSCLE

The following research involves two related but separate problems in synaptogenesis. Xenopus laevis embryos and muscle cultures or nerve-muscle cocultures derived from Xenopus embryos were used in these experiments. Problem 1. Although acetylcholine receptors are known to bind (alpha) bungarotoxin before they are inserted into the plasma membrane, it is not known whether they are physiologically active (bind acetylcholine and control ionic conductance) immediately upon insertion. The following experiments were performed to test the function of newly inserted acetylcholine receptors. Acetylcholine sensitivity was measured either by bath application when recording from embryo somite muscle or iontophoretically by the slope method when recording from cultured muscle cells. Acetylcholine sensitivity was tested slightly before and after the stage in development where sensitivity to acetylcholine first appeared. The same embryos tested with bath application of acetylcholine or the cells which were tested iontophoretically were freeze-fractured and then identified in the platinum replicas. Particle sizes were measured and a distribution of particle sizes for each embryo or cell was produced. Comparison of particle sizes in embryonic muscles and cultured muscle cells without any detectable acetylcholine sensitivity to those with sensitivity indicated in the latter a greater density of dispersed particles in the size range between 10 to 20 nm. As the degree of sensitivity increased in the cultured muscle cells during the first day of culture, dispersed 10 to 20 nm particle densities also increased in a similar manner. (\u27125)I-(alpha)bungarotoxin binding shown by autoradiography, also increased proportionally. Points of high acetylcholine sensitivity, 10 to 20 nm particle aggregates and hot spots in autoradiographs were all first seen after about 16 hours in muscle cultures. On the basis of these data dispersed and aggregated acetylcholine receptors appear to be represented by intramembrane particles 10 to 20 nm in diameter. In addition, it seems that acetylcholine receptors are fully functional with respect to ligand binding and ion gating properties when first inserted into the plasma membrane and before forming aggregates. Problem 2. Filipin, digitonin, and saponin react specifically with membrane cholesterol and produce unique membrane alterations (sterol-specific complexes) which are easily discernible in freeze-fracture replicas. I applied each of these agents to noninnervated and innervated muscle cells in culture. Freeze-fracture of these treated muscle cells revealed that most areas of the muscle plasma membrane contained sterol-specific complexes (19-40 nm proturberances and dimples with filipin; a scalloped appearance with digitonin; or an irregular, rough appearance with saponin). However, these complexes were virtually absent from membrane areas of junctional and nonjunctional aggregates of acetylcholine receptor particles. These results suggest that the membrane matrix of acetylcholine receptor aggregates is low in cholesterol and that this membrane lipid heterogeneity may be linked to the mechanisms involved in the formation and stabilization of acetylcholine receptor aggregates on cultured muscle cells