Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions.

The distribution of acetylcholine receptors (AChRs) in neuromuscular junctions of embryonic and newborn mice (strain 129/ReJ) was examined on the fine structural level using [125I]alpha-bungarotoxin labeling and quantitative electron microscope autoradiography. Changes in junctional receptor site density were related to changes in the structure of the postjunctional membrane, in particular the differentiation of morphologically distinct regions of thickened membrane and the formation of junctional folds. The following sequence of development is described: (1) At the earliest age examined, embryonic day 16 of gestation (i.e., at approximately the time when junctional receptor accumulations are first detected), subneural receptor aggregates have poorly defined boundaries and seem to extend beyond the region of direct axonal or Schwann cell contact. At that time the subneural AChR site density is somewhat variable but averages approximately 2000 to 3000 sites/micron 2, and the subneural muscle membrane shows discontinuous membrane specialization (membrane thickening plus a cytoplasmic amorphous layer). There seems to be no preferential labeling of contact regions rich in such specialization compared with those where membrane thickening was less obvious or absent. (2) By birth, junctional AChRs are strongly correlated with morphologically specialized membrane, at a constant density of approximately 9000/micron 2 (comparable to that in adult animals of this strain of mouse). During the first postnatal week, a large fraction of the primary cleft is covered by Schwann cell rather than axon terminal. Receptor-rich dense membrane and incipient junctional folds are found under axon terminals and, to a lesser extent, under Schwann cell extensions, but they are not seen beyond the edge of the "junctional complex." Coated vesicles are a prominent feature of the subjunctional sarcoplasm in neonatal junctions, and their number declines sharply during the first week. (3) Junctional folds differentiate mainly after the first postnatal week. Development of folds is associated with a 1.5- to 1.8-fold increase in length of specialized thickened membrane and a parallel increase in number of receptor sites per endplate. Since this is also a period of rapid overall endplate growth (Steinbach, J.H. (1981) Dev. Biol. 84: 267-276) the total number of endplate receptors increases greater than 30-fold during the maturation period following the first week while the concentration of receptors/micron 2 of thickened membrane remains constant.