From Plaque to Pretzel: Fold Formation and Acetylcholine Receptor Loss at the Developing Neuromuscular Junction

Abstract

Although there has been progress in understanding the initial steps in the formation of synapses, less is known about their subsequent maturation (Sanes and Lichtman, 1999). Two alterations on the postsynaptic side of the mammalian neuromuscular junction occur during early postnatal life: acetylcholine receptors (AChRs) disappear from parts of the developing junction as all but one axonal inputs are removed, and the topography of the postsynaptic membrane becomes more complicated as gutters and folds are established. We have studied the maturation of the AChR distribution and postsynaptic topography simultaneously by imaging labeled AChRs at the mouse neuromuscular junction in a new way, using reflected light confocal microscopy. At birth postsynaptic receptors were localized in irregular patches within a spoon-shaped plaque. Beginning several days later, receptor regions within a single endplate were divided into differentiated and less organized compartments. Folds generally oriented orthogonal to the long axis of the muscle fiber were seen in developing gutters, although the orientation of the gutters seemed to be imposed by the branching pattern of the nerve. Eventually, superficial regions lacking AChR labeling were apparent in all junctions. In junctions denervated in the neonatal period both gutter formation and the disappearance of superficial receptors regions were prevented. We suggest that tension between growing muscle fibers and the relatively inelastic synaptic terminals that adhere to them causes the topographic features of the postsynaptic membrane. This view provides a mechanical explanation for gutters, folds, and the location of folds at sites of neurotransmitter release. Key words: reflected light confocal microscopy; postsynaptic membrane topography; acetylcholine receptor distribution; neuromuscular junction development; synapse elimination; active zonesThe use of fluorescently tagged ␣-bungarotoxin to label acetylcholine receptors (AChRs) at the neuromuscular junction has shown that there is a dramatic maturational alteration in receptor distribution in early postnatal life. At birth, AChRs are arranged in an oval plaque in what appears to be a relatively uniform density. Over several weeks this plaque perforates (Nyström, 1968;Steinbach, 1981;Slater, 1982; such that the regions that maintain AChRs become the pretzel-shaped branches of the mature singly innervated neuromuscular junction, whereas regions that lose membraneassociated AChRs also lose nerve terminal innervation during the process of synapse elimination (Balice-Gordon and Lichtman, 1993; Gan and Lichtman, 1998). Although fluorescence labeling of AChRs allows for the study of the overall distribution of receptors during development, this technique has not been useful in studying alterations in the three-dimensional topography of the muscle fiber membrane as neuromuscular junctions mature. Hence, the formation of gutters (primary synaptic clefts) and the elaboration of folds within the gutters could not be related to the changes ...

Acute and long-term effects of botulinum neurotoxin on the function and structure of developing extraocular muscles

“…6A) that appears to be a progression of what has been observed during embryonic development. As previously described for the embryonic period of development (between embryonic day 14 and P2 (Croes and von Bartheld, 2005), the AChRs that were initially clustered in the shape of oval plaques with a relatively uniform density eventually became perforated and displayed a "pretzel-like" morphology similar to that described for the NMJs of skeletal muscles (Balice-Gordon et al, 1990;Balice-Gordon and Lichtman, 1993;Sanes and Lichtman, 1999;Marques et al, 2000). This "pretzel-like" appearance continued throughout the experimental time period of P2 through P112.…”

Regulation of the Intermediate Filament Protein Nestin at Rodent Neuromuscular Junctions by Innervation and Activity

“…One would be maintenance of the junctional folds themselves. In this regard, it is interesting that nestin becomes localized to the synaptic site during the postnatal period when junctional folds are reported to be first forming (Marques et al, 2000). Arguing against this possibility is the observation that nestin essentially disappears from rat neuromuscular junctions after denervation, whereas the folds persist (Miledi and Slater, 1968), although some observations suggest that these folds begin to change the size of their openings to the synaptic space, their frequency, and the depth of their penetration into the sarcoplasm with longer periods of denervation (Matsuda et al, 1988).…”

“…1 B). These AChR bands represent a diffraction-limited view of the openings of the secondary (junctional) folds that are present in the sacrolemma underneath the nerve terminal (Marques et al, 2000). The puncta of nestin staining are commonly situated between the bands of AChR labeling (Fig.…”

Myosin Va cooperates with PKA RIα to mediate maintenance of the endplate in vivo

“…Down-regulation of myosin Va led to a reduced t 1/2 of AChRs (4) similar to pathological conditions, such as denervation (6,(10)(11)(12)(13) or dystrophy (14). A shortened t 1/2 of AChRs accompanies morphological changes of the NMJ: while NMJs in mouse muscles are normally elliptic and pretzel-shaped (15), they elongate and fragment under pathological conditions (16,17).…”