Specificity and Competition During Maturation of Neuromuscular Synapses

Fast and slow contracting fibers in neonatal mammalian skeletal muscle are each innervated in a highly specific manner by motor neurons of the corresponding type, even at an age when polyinnervation is widespread. Chemospecific recognition is a possible mechanism by which this pattern of innervation could be established. I have investigated this possibility by studying the degree of specificity during reinnervation of neonatal rabbit soleus muscle. Fiber type composition was assayed by measuring the twitch rise times of motor units within two days of the onset of functional reinnervation. In contrast to the broad, bimodal distribution of single motor unit twitch rise times seen in normal muscles, motor units in reinnervated muscles yielded a narrower, unimodal distribution of rise times. Rise times of reinnervated units were intermediate to those of normal fast and slow units, suggesting that reinnervated units were composed of a mixture of fast and slow contracting muscle fibers. An alternative possibility, that specific reinnervation was masked by contractile de-differentiation of muscle fibers, was examined by maintaining a transmission blockade induced by botulinum toxin poisoning for an equivalent interval. Twitch rise times of treated motor units exhibited the distinctly bimodal distribution characteristic of normal muscles, suggesting that muscle fibers can retain contractile diversity during a transient period of denervation. Computer simulations were employed to estimate the amount of rise time diversity induced by varying degrees of specificity during reinnervation. Based on this analysis, I conclude that there is little if any selective reinnervation of muscle fiber types at the ages studied. In a second experiment, I compared the development of fast and slow motor innervation in the neonatal rabbit soleus, a muscle which contains two distinct motor unit types during the early period of polyneuronal innervation. The innervation state of individual muscle fibers was ascertained using an intracellular electrode; a fluorescent dye was then injected into particular fibers to permit subsequent identification of histochemical type. No significant difference in the time course of synapse elimination was observed for fast and slow motor units as judged by the percentage of fibers remaining polyneuronally innervated at two ages: 7-8 days, when most fibers are multiply innervated, and 10-11 days, when the level of polyinnervation is low. In a third experiment, I examined a phenomenon in which compound endplate potentials were occasionally seen in muscle fibers at an age (17-23 days) well past the major episode of synapse elimination. Several lines of evidence indicate that this apparent polyinnervation in fact derives from an electrode-induced electrical coupling artifact, and that genuinely polyinnervated fibers are very rare at this stage, if present at all. A computer model of neuromuscular synapse elimination was developed to serve as an analytical tool in exploring the potential roles of candidate mechanisms in regulating the normal process and in shaping its response to experimental perturbations. Synapse elimination is a complex process likely to involve the dynamic interaction of several specific mechanisms. This situation limits the reliability of a strictly inductive theoretical investigation into how these mechanisms might act. Three mechanisms which have been previously proposed and discussed in the literature are simulated, including a synaptic stabilization molecule, a muscle derived trophic factor, and a hypothesized intrinsic tendency of motor neurons to limit their arbor. The model is stochastic rather than deterministic in character, and is also dynamic, tracing the growth and retraction of individual presynaptic terminals at each iteration as they compete for limited synaptic space. Nine experimental observations were selected to guide development of the model and evaluate its performance. All but one of the experimental observations canbe simulated by at least one of the mechanisms studied. No single mechanism, however, is adequate to duplicate the entire body of experimental evidence. A relative advantage for larger terminals appears critical for convergence in both the scaffolding and trophic factor mechanisms. Several alternative roles for activity are compared.