Mode of enlargement of young mouse neuromuscular junctions observed repeatedlyin vivo with visualization of pre- and postsynaptic borders

Abstract

The dynamics of structural remodelling during growth of synapses was studied in identified living neuromuscular junctions observed three times at four-day intervals in three- to five-week-old mice. Nerve terminals and acetylcholine receptors in pectineus muscle were stained and visualized with fluorescent ligands of tetanus toxin C-fragment and alpha-bungarotoxin, respectively. In most observations, about 2.5% of nerve terminal area was observed without underlying acetylcholine receptor (termed ‘preprojection’), and about 0.4% of acetylcholine receptor without overlying nerve terminal (‘postprojection’). Neither overall synaptic growth nor prevalence of pre- and postprojections was affected by repeated observation. In sequential observations, about 80% of the preprojection area at one observation had acquired underlying acetylcholine receptor four days later, while 20% retracted or showed no change. Although approximately one-half of postprojections were also precursors of synaptic regions, their absolute contribution to synaptic growth was small, and some had originated from nerve terminal retraction. Eighty per cent of the disparities between pre- and postsynaptic components in second or third observations were the result of nerve terminal outgrowth or retraction. In the four-day intervals, there was about 7.5% lengthening but also about 3% shortening of synaptic area. Preprojectional induction of acetylcholine receptor accounted for at least 25% of lengthening, and apparent concurrent growth at ends or sides of branches accounted for most of the rest (although level of resolution limits this conclusion). Only about 10% of lengthening was attributable to central intercalary growth. In summary, the pectineus neuromuscular junction grows mainly by nerve terminal outgrowth giving rise four days later to underlying acetylcholine receptor and by conjoint lengthening of synaptic complexes but with relatively little contribution by initial acetylcholine receptor extension or intercalary growth. Growth of the neuromuscular junction is not monotonic: nerve terminals retract and synaptic branches shorten as net lengthening proceeds. Compared with non-growing adult neuromuscular junctions, nerve terminal preprojections in growing neuromuscular functions are more prevalent and more likely to give rise to new synaptic regions.