The goal of this project is to understand how mRNAs, proteins, and subcellular organelles are distributed and organized into functional domains in excitable cells. We continue to use the model system of hybrid myotubes, based on the mouse muscle cell line C2, described previously. In addition, new tools have been developed in the past year. First, a tissue culture system of low-density rat hippocampal neurons has been established in our laboratory. It will allow us to extend our work to nerve cells. The advantage of this specific system is that individual neurons can be observed in the process of differentiating and acquiring polarity. Second, we have obtained expression of the betagalactosidase cDNA in these neurons. Several approaches were attempted: transfection of plasmid DNA with a commercial reagent (Transfectam), direct injection of DNA, and electroporation. So far, transfection has given the best results in terms of cell survival and optimization of expression. In the near future we will start transfections with a series of plasmids that will help us determine the effect of mRNA stability on protein and mRNA distribution in muscle and nerve cells. Expression of these plasmids has already been tested on C2 muscle cells. For this purpose, a new protocol using transient rather than permanent transfections to form hybrid myotubes, containing a single transfected nucleus in a background of unmodified cells, has been established. The next step will be to determine the distribution of the mRNAs of transfected plasmids by in situ hybridization. Thus, we have several new tools available to study protein and mRNA distribution in muscle and nerve cells. Studies on the distribution of subcellular organelles, especially the Golgi complex, have also progressed. A developmental study of the Golgi complex distribution in muscle cells in vitro and muscle fibers in vivo was completed by an electron microscopy (EM) study carried out in the NINDS EM Facility. It confirmed our light microscopy results that suggested that the Golgi complex was distributed throughout the length of a muscle fiber, both near and away from the neuromuscular junction. This was an important point to establish. Finally, a new collaborative study of the dynamics and changes of distribution of the Golgi complex and associated membranes in muscle in vivo has been started, using the muscle-specific glucose transporter GLUT4 as a marker and combining both light and electron microscopy.