During development, mammalian neuromuscular junctions (NMJs) transit from multiple-innervation to single-innervation through axonal competition via unknown molecular mechanisms. Previously, using an in vitro model system, we demonstrated that the postsynaptic secretion of pro-brain-derived neurotrophic factor (proBDNF) stabilizes or eliminates presynaptic axon terminals, depending on its proteolytic conversion at synapses. Here, using developing mouse NMJs, we obtained in vivo evidence that proBDNF and mature BDNF (mBDNF) play roles in synapse elimination. We observed that exogenous proBDNF promoted synapse elimination, whereas mBDNF infusion substantially delayed synapse elimination. In addition, pharmacological inhibition of the proteolytic conversion of proBDNF to mBDNF accelerated synapse elimination via activation of p75 neurotrophin receptor (p75 NTR ). Furthermore, the inhibition of both p75 NTR and sortilin signaling attenuated synapse elimination. We propose a model in which proBDNF and mBDNF serve as potential "punishment" and "reward" signals for inactive and active terminals, respectively, in vivo.

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Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M 2 mAChRs were present solely in motor neurons, whereas M 1 , M 3 , and M 5 mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M 2/4 -preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M 2Ϫ/Ϫ but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M 2 Ϫ/Ϫ NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M 2 or other mAChR subtypes. We also found that many NMJs of M 5 Ϫ/Ϫ mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M 2 and that muscle fiber growth is defective without M 5 . Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.

Neurotrophins and their receptors, the Trks, are differentially expressed among the cell types that make up neuromuscular and other synapses, but the function and directionality of neurotrophin signaling at synapses are poorly understood. Here we demonstrate, via immunostaining, Western blotting, and RT-PCR analyses, that TrkC, the receptor for neurotrophin-3 (NT3), is expressed by mouse perisynaptic and myelinating Schwann cells from birth through adulthood and is unaltered after denervation. Analyses of transgenic mice in which the NT3 coding sequence is replaced by lacZ showed that NT3 is expressed in motor neurons and Schwann cells during perinatal development, but not in adult mice. In muscle, NT3 is expressed by intrafusal muscle fibers within spindles, as has been previously reported. Surprisingly, NT3 is also expressed in extrafusal muscle fibers during perinatal life and in adults. Genetic approaches were used to explore the roles of NT3 and TrkC signaling at neuromuscular synapses. Overexpression of NT3 in muscle fibers during development resulted in an increased number of perisynaptic Schwann cells at neuromuscular synapses, without altering synaptic size, suggesting that muscle-derived NT3 might act as a mitogen or trophic factor for Schwann cells. Conditional deletion of NT3 from motor neurons did not alter the number of Schwann cells or other aspects of neuromuscular synaptic structure, suggesting that motor-neuron-derived NT3 is not required for normal development of perisynaptic Schwann cells or synapses. Together, these results demonstrate that NT3 expression is developmentally regulated in skeletal muscle and may modulate the number of Schwann cells at neuromuscular synapses.