Formation of the active zone at developing neuromuscular junctions in larval and adult bullfrogs

Development of the presynaptic active zone was studied at neuromuscular junctions with freeze-fracture electron microscopy in larval and adult bullfrogs. In rudimentary larval neuromuscular junctions, clusters of active zone particles were scattered over the P-face of the presynaptic membrane. Vesicle openings were observed at these terminals even though active zone particles lacked the mature pattern of two double rows. Gradually, active zone particles became organized into rows, but they were still randomly located and oriented. Once junctional folds were observed in replicas, developing active zones were located opposite to the folds, as in mature terminals. Multiple terminals occupying the same junctional gutters were also observed. At the end of metamorphosis, most active zones were still immature in appearance and had only grown to one third of their mature length. After metamorphosis, the number of active zone segments aligned at the same junctional fold increased. These discontinuous short active zones then elongated, joined together, and finally formed the mature active zones. Signs suggesting synapse elimination such as disorganization of active zones, absence of intramembrane particles in varicosities, and exposed muscle membranes with patches of acetylcholine receptor aggregates were observed. In some multiply innervated junctions, one terminal had mature active zones with vesicle openings, the other in the same gutter displayed disorganized active zones without vesicle openings, although both terminals showed similar sizes and distributions of background particles. This study suggests that developing active zones, as is the case for regenerating active zones in the adult, are functional before the mature organization is formed. The sequence of development of active zones is also similar to that of regeneration except for the random location and orientation of early active zones in tadpoles. The comparison between regeneration and development further indicates that the process of active zone formation is related to junctional folds and/or associated structures. It is also suggested that synapse elimination may involve degenerative changes in presynaptic membranes, although direct evidence remains to be provided.