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 ...

Fibrosis is a pathological feature observed in patients with Duchenne muscular dystrophy (DMD) and in mdx mice, the experimental model of DMD. We evaluated the effect of suramin, a transforming growth factor-beta 1 (TGF-β1) blocker, on fibrosis in mdx mice. mdx mice (6 months old) received suramin for 7 weeks. Suramin- and saline-treated (control) mdx mice performed exercise on a treadmill to worsen disease progression. Immunoblotting showed an increase of TGF-β1 in mdx diaphragm, limb, and cardiac muscles. Suramin decreased creatine kinase in mdx mice and attenuated fibrosis in all muscles studied, except for cardiac muscle. Suramin protected limb muscles against damage and reduced the exercise-induced loss of strength over time. These findings support a role for TGF-β1 in fibrinogenesis and myonecrosis during the later stages of disease in mdx mice. Suramin might be a useful therapeutic alternative for the treatment of dystrophinopathies.

Intrinsic laryngeal muscles share many anatomical and physiological properties with extraocular muscles, which are unaffected in both Duchenne muscular dystrophy and mdx mice. We hypothesized that intrinsic laryngeal muscles are spared from myonecrosis in mdx mice and may serve as an additional tool to understand the mechanisms of muscle sparing in dystrophinopathy. Intrinsic laryngeal muscles and tibialis anterior (TA) muscle of adult and aged mdx and control C57Bl/10 mice were investigated. The percentage of central nucleated fibers, as a sign of muscle fibers that had undergone injury and regeneration, and myofiber labeling with Evans blue dye, as a marker of myofiber damage, were studied. Except for the cricothyroid muscle, none of the intrinsic laryngeal muscles from adult and old mdx mice showed signs of myofiber damage or Evans blue dye labeling, and all appeared to be normal. Central nucleation was readily visible in the TA of the same mdx mice. A significant increase in the percentage of central nucleated fibers was observed in adult cricothyroid muscle compared to the other intrinsic laryngeal muscles, which worsened with age. Thus, we have shown that the intrinsic laryngeal muscles are spared from the lack of dystrophin and may serve as a useful model to study the mechanisms of muscle sparing in dystrophinopathy.

Duchenne muscular dystrophy is one of the most devastating myopathies. Muscle fibers undergo necrosis and lose their ability to regenerate, and this may be related to increased interstitial fibrosis or the exhaustion of satellite cells. In this study, we used mdx mice, an animal model of Duchenne muscular dystrophy, to assess whether muscle fibers lose their ability to regenerate after repeated cycles of degeneration-regeneration and to establish the role of interstitial fibrosis or exhaustion of satellite cells in this process. Repeated degenerativeregenerative cycles were induced by the injection of bupivacaine (33 mg/kg), a myotoxic agent. Bupivacaine was injected weekly into the right tibialis anterior muscle of male, 8-week-old mdx (N = 20) and C57Bl/10 (control, N = 10) mice for 20 and 50 weeks. Three weeks after the last injection, the mice were killed and the proportion of regenerated fibers was counted and reported as a fibrosis index. Twenty weekly bupivacaine injections did not change the ability of mdx muscle to regenerate. However, after 50 weekly bupivacaine injections, there was a significant decrease in the regenerative response. There was no correlation between the inability to regenerate and the increase in interstitial fibrosis. These results show that after prolonged repeated cycles of degeneration-regeneration, mdx muscle loses its ability to regenerate because of the exhaustion of satellite cells, rather than because of an increase in interstitial fibrosis. This finding may be relevant to cell and gene therapy in the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common childhood myopathy, characterized by muscle loss and cardiorespiratory failure. While the genetic basis of DMD is well established, secondary mechanisms associated with dystrophic pathophysiology are not fully clarified yet. In order to obtain new insights into the molecular mechanisms of muscle dystrophy during earlier stages of the disease, we performed a comparative proteomic profile of the spared extraocular muscles (EOM) vs. affected diaphragm from the mdx mice, using a label based shotgun proteomic approach. Out of the 857 identified proteins, 42 to 62 proteins had differential abundance of peptide ions. The calcium-handling proteins sarcalumenin and calsequestrin-1 were increased in control EOM compared with control DIA, reinforcing the view that constitutional properties of EOM are important for their protection against myonecrosis. The finding that galectin-1 (muscle regeneration), annexin A1 (anti-inflammatory) and HSP 47 (fibrosis) were increased in dystrophic diaphragm provides novel insights into the mechanisms through which mdx affected muscles are able to counteract dystrophy, during the early stage of the disease. Overall, the shotgun technique proved to be suitable to perform quantitative comparisons between distinct dystrophic muscles and allowed the suggestion of new potential biomarkers and drug targets for dystrophinopaties.