Multiple innervation of tonic endplates revealed by activity-dependent uptake of fluorescent probes

During development of the vertebrate nervous system, there is a widespread reduction in the number of axons innervating target cells. This phenomenon, often called synapse elimination, has been particularly well studied at the neuromuscular junction of developing twitch muscle fibres: following a period of polyneuronal innervation, axonal branches are retracted, usually leaving each twitch fibre endplate innervated by only one axon. Here we describe a new technique for the study of synapse elimination--activity-mediated uptake of fluorescent probes. These probes selectively and supravitally label all the terminals of individual axons. The technique is used here in adult and embryonic snakes to study the innervation pattern of a thin muscle containing two fibre types: twitch fibres, which are fast-contracting and have propagated action potentials, and tonic fibres, which are slow-contracting and lack action potentials. We find that twitch muscle fibres, as expected, eliminate all polyneuronal innervation during development; in contrast, tonic fibre endplates remain polyneuronally innervated into adulthood. The persistence of multiple innervation at tonic endplates may be related to the lack of action potential activity in tonic muscle fibres.