The regulation of synaptic efficacy at regenerated and cultured neuromuscular junctions

Abstract

The neuromuscular junction (NMJ) is a synapse formed between a motoneuron and a muscle fiber which transmits the signals required to initiate muscle contraction. The functional state of the NMJ is intimately tied to the structure and function of the motoneuron, such that reductions in postsynaptic activity retrogradely stimulate sustained reorganization of presynaptic motor terminals in an attempt to maintain normal contractile output. In the adult, these plastic changes occur most notably as regenerative responses following traumatic injury and during the progression of motoneuron diseases (MNDs), and can contribute to a considerable amount of functional repair. However, limitations to the regeneration capacity of motoneurons place an upper limit on the effectiveness of endogenous repair mechanisms and can restrict the extent of functional recovery. Using a combination of immunofluorescence, sharp electrode electrophysiology and live labeling of synaptic vesicle recycling during various forms of synaptic growth and regeneration in vivo and in vitro, I have identified that the neural cell adhesion molecule (NCAM) is a key regulator of the regenerative capacity of motoneurons. In vivo experiments revealed that NCAM influences the maturation and stabilization of regenerated synapses via the recruitment and recycling of synaptic vesicles necessary for effective synaptic transmission. The presence of both pre- and post-synaptic NCAM were necessary to maintain the abundance of recycling synaptic vesicles at regenerated synapses, demonstrating a coordinated influence of these molecules in regenerative synaptic plasticity in vivo. To accurately assess the regenerative potential of motoneurons in vitro, it was necessary to develop a system which could reliably and consistently generate mature NMJs amenable to experimental investigation. Motoneurons differentiated from embryonic stem cells were grown for 3-5 weeks in co-culture with muscle fibers and generated mature NMJs which possessed morphological and functional criteria consistent with NMJs formed in vivo. NMJs formed by NCAM-/- motoneurons did not mature and were found to exhibit deficits consistent with their in vivo counterparts. These studies have revealed that NCAM is a key mediator of regenerative plasticity at the NMJ and may be a target for efforts to enhance endogenous repair following traumatic injury or during the progression of neurodegenerative disease.