Visual identification of nerve terminals in living isolated skeletal muscle

Abstract

1. The unmyelinated terminal branches of motor nerve fibres were clearly resolved in live, unstained skeletal muscles of the frog and of the mudpuppy (Necturus maculosus), using Nomarski optics. The observations were supplemented by several histological procedures, including electron microscopy, and by extracellular recordings from the nerve terminals. 2. In live motor nerve terminals of the mudpuppy one can see a series of varicosities, which in the electron microscope are shown to contain accumulations of synaptic vesicles. Junctional folds in the muscle fibres are confined to the areas opposite the varicosities. Terminal branches of the frog's motor axon are also varicose, but the swellings are so closely spaced that they can be seen only after staining or by electron microscopy. 3. Nuclei of Schwann cells are recognized along living nerve terminals. Electrophoretic injection of a fluorescent dye, Procion yellow, into the cell bodies of Schwann cells enables one to see the distribution of their processes with the light microscope. 4. Visibility of terminal arborizations was improved by bathing nerve-muscle preparations in solutions of collagenase for 15 to 30 min, thereby removing much of the connective tissue. After longer collagenase treatment nerve terminals could be lifted off muscle fibres with a micropipette, thus exposing the postsynaptic membrane.