Transient and permanent effects of androgen during synapse elimination in the levator ani muscle of the rat

Young male rats were castrated at 7 days of age, and treated with testosterone propionate daily from 7 to 34 days of age. At 13 months of age, motor axons and terminals innervating the levator ani (LA) muscle were stained with tetranitroblue tetrazolium (TNBT). The number of separate axons innervating individual muscle fibers was counted, and muscle fiber diameter was measured. Previous studies have shown that this androgen treatment increases muscle fiber diameter and delays synapse elimination, measured as (1) a greater percentage of muscle fibers innervated by multiple axons and (2) larger motor units. The present results indicate that the androgenic effect on synapse elimination is permanent, in that high levels of multiple innervation persisted for 12 months after the end of androgen treatment. In contrast, the effect on muscle fiber diameter was not maintained for this period. This dissociation of androgenic effects on the pattern of innervation from androgenic effects on muscle fiber diameter offers further evidence that the androgenic maintenance of multiple innervation is not dependent on muscle fiber size. In addition, circulating testosterone levels were measured at 50 and 60 days of age in animals similarly treated with androgen or oil from 7 to 34 days of age. By 60 days of age, testosterone levels in hormone-treated animals had dropped below detectability, comparable to levels in oil-treated controls. This provides additional evidence that androgen treatment during juvenile development can have permanent effects on the adult pattern of innervation in the LA muscle.     

A novel pathway for MuSK to induce key genes in neuromuscular synapse formation

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.     

Nerve-independent formation of a topologically complex postsynaptic apparatus

As the mammalian neuromuscular junction matures, its acetylcholine receptor (AChR)-rich postsynaptic apparatus is transformed from an oval plaque into a pretzel-shaped array of branches that precisely mirrors the branching pattern of the motor nerve terminal. Although the nerve has been believed to direct postsynaptic maturation, we report here that myotubes cultured aneurally on matrix-coated substrates form elaborately branched AChR-rich domains remarkably similar to those seen in vivo. These domains share several characteristics with the mature postsynaptic apparatus, including colocalization of multiple postsynaptic markers, clustering of subjacent myonuclei, and dependence on the muscle-specific kinase and rapsyn for their formation. Time-lapse imaging showed that branched structures arise from plaques by formation and fusion of AChR-poor perforations through a series of steps mirroring that seen in vivo. Multiple fluorophore imaging showed that growth occurs by circumferential, asymmetric addition of AChRs. Analysis in vivo revealed similar patterns of AChR addition during normal development. These results reveal the sequence of steps by which a topologically complex domain forms on a cell and suggest an unexpected nerve-independent role for the postsynaptic cell in generating this topological complexity.     

豚鼠眶外肌中快肌与慢肌纤维的小终板电流

豚鼠眶外肌中有两类不同电活动模式的肌纤维。一类纤维有动作电位,小终板电位时程较短并且较一致;另一类纤维不能产生动作电位,小终板电位时程较长而且多变。用电泳注射普胺天蓝分别标记这两类纤维各6条,并结合胆碱酯酶染色,结果表明,前一类肌纤维都是只有一个神经肌肉接头的快纤维;而另一类肌纤维都是有多个接头的慢纤维。根据双微电极电庄箱位实验结果,计算了上述决、慢肌纤维小终板电流下降相时间常数τ及其电压系数A。实验表明:(1)在静息电位附近,慢肌纤维小终板电流的τ值大于快肌纤维的;(2)慢肌纤维的A值比快肌纤维的小的多,即τ值较少随膜电位变化。本文对上述不同曾加以讨论。     

Exercise-dependent regulation of glial cell line-derived neurotrophic factor (GDNF) expression in skeletal muscle and its importance for the neuromuscular system

The focus of this review is to highlight the importance of glial cell line-derived neurotrophic factor (GDNF) for the motor nervous system. GDNF is the most potent survival factor for motor neurons, where it enhances maintenance and survival of both developing and mature motor neurons in vivo and in vitro. GDNF aids in neuromuscular junction formation, maintenance, and plasticity, where skeletal muscle-derived GDNF may be responsible for this phenomenon. Increased levels of physical activity can increase GDNF protein levels in skeletal muscle, where alterations in acetylcholine and acetylcholine receptor activation may be involved in regulation of these changes observed. With inactivity and disuse, GDNF expression shows different patterns of regulation in the central and peripheral nervous systems. Due to its potent effects for motor neurons, GDNF is being extensively studied in neuromuscular diseases.     

Distinct domains of MuSK mediate its abilities to induce and to associate with postsynaptic specializations.

Agrin released from motor nerve terminals activates a muscle-specific receptor tyrosine kinase (MuSK) in muscle cells to trigger formation of the skeletal neuromuscular junction. A key step in synaptogenesis is the aggregation of acetylcholine receptors (AChRs) in the postsynaptic membrane, a process that requires the AChR-associated protein, rapsyn. Here, we mapped domains on MuSK necessary for its interactions with agrin and rapsyn. Myotubes from MuSK(-/)- mutant mice form no AChR clusters in response to agrin, but agrin-responsiveness is restored by the introduction of rat MuSK or a Torpedo orthologue. Thus, MuSK(-/)- myotubes provide an assay system for the structure-function analysis of MuSK. Using this system, we found that sequences in or near the first of four extracellular immunoglobulin-like domains in MuSK are required for agrin responsiveness, whereas sequences in or near the fourth immunoglobulin-like domain are required for interaction with rapsyn. Analysis of the cytoplasmic domain revealed that a recognition site for the phosphotyrosine binding domain-containing proteins is essential for MuSK activity, whereas consensus binding sites for the PSD-95/Dlg/ZO-1-like domain-containing proteins and phosphatidylinositol-3-kinase are dispensable. Together, our results indicate that the ectodomain of MuSK mediates both agrin- dependent activation of a complex signal transduction pathway and agrin-independent association of the kinase with other postsynaptic components. These interactions allow MuSK not only to induce a multimolecular AChR-containing complex, but also to localize that complex to a primary scaffold in the postsynaptic membrane.     

Synapse elimination from the mouse neuromuscular junction in vitro: A non-hebbian activity-dependent process

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2–7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p< 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i. e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p< 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development. © 1993 John Wiley & Sons, Inc.