Implication of nNOS in the enlargement of AChR aggregates but not the initial aggregate formation in a novel coculture model.

The aggregation of nicotinic acetylcholine receptors (AChRs) is an early hallmark of the formation of neuromuscular junction (NMJ), and nitric oxide is recently known to play an important role. In many NMJ studies, nerve-muscle coculture model was used, and NG108-15 cells, a neuroblastoma x glioma hybrid cell line, were the most frequently used nerve cells. However, possible contributions from glial cells could not be excluded. In this study, Neuro-2a neuroblastoma cells were used instead of [corrected] coculture with myotubes, and the relationship between AChR aggregation and spatiotemporal expression and activation of nNOS (neuronal nitric oxide synthase) was examined. Upon coculture, AChR aggregates were observed by FITC-conjugated alpha-bungarotoxin, and double labeling of AChRs and neurofilament showed that the neurites of a Neuro-2a cell innervated several myotubes. After treating the cocultures with single dose of L-NAME at the end of 1-day [corrected] coculturing, only slight effect on AChR aggregation could be found indicating that nNOS is not related to the initial formation of AChR aggregates. In contrast, when L-NAME treatment was given at the end of a 3-day coculturing, the day just before reaching the maximum extent of AChR aggregation, new AChR aggregates were hardly formed and the preformed AChR aggregates were even dispersed indicating that the enlargement of AChR aggregates is highly dependent on the nNOS activity. Double-labeling study of nNOS and AChR further showed that the coupling of membranous nNOS to regions nearby the AChR aggregates was essential for the enlargement of AChR aggregates. These results not only revealed the spatiotemporal relationship between AChR aggregation and nNOS activity but also verified the feasibility and usefulness of using Neuro-2a cells in a coculture model.