1. In this study we investigated whether electrical stimulation accelerates the upregulation of Talpha1-tubulin and GAP-43 (regeneration-associated genes; RAGs) and the downregulation of the medium-molecular-weight neurofilament (NFM), in concert with stimulation-induced acceleration of BDNF and trkB gene expression and axonal regeneration. 2. Two weeks prior to unilateral femoral nerve transection and suture, fluorogold (Fluorochrome Inc., Denver) or fluororuby (Dextran tetramethylrhodamine, Mol. Probes, D-1817, Eugene, OR) was injected into quadriceps muscles of the left and right hindlimbs to label the femoral motoneuron pools as previously described. Over a period of 7 days, fresh spinal cords were processed for semiquantitation of mRNA by using in situ hybridization. 3. There was an increase in Talpha1-tubulin and GAP-43 mRNA and a decline in the NFM mRNA at 7 days after nerve suture and sham stimulation but not in intact nerves. In contrast, 1-h stimulation of sutured but not intact nerves dramatically accelerated the changes in gene expression: mRNA levels of Talpha1-tubulin and GAP-43 were significantly elevated above control levels by 2 days while NFM mRNA was significantly reduced by 2 days in the sutured nerves. Thereby, the neurofilament/tubulin expression ratio was reduced at 2 days after suture and stimulation, possibly allowing more tubulin to be transported faster into the growing axons to accelerate the elongation rate following stimulation. Importantly, the changes in RAGs and NFM gene expression were delayed relative to the accelerated upregulation of BDNF and trkB mRNA by electrical stimulation. 4. The temporal sequence of upregulation of BDNF and trkB, altered gene expression of RAGs and NFM, and accelerated axonal outgrowth from the proximal nerve stump are consistent with a key role of BDNF and trkB in mediating the altered expression of RAGs and, in turn, the promotion of axonal outgrowth after electrical stimulation.

Motor unit (MU) enlargement by sprouting is an important compensatory mechanism for loss of functional MUs during normal aging and neuromuscular disease. Perisynaptic Schwann cells at neuromuscular junctions extend processes that bridge between denervated and reinnervated endplates, and guide axonal sprouts to reinnervate the denervated endplates. In a rat model of partial denervation, high levels of daily neuromuscular activity have been shown to inhibit the outgrowth of sprouts by preventing Schwann cell bridging. In this review, we consider (1) the relative roles of increasing levels of oxidative stress and neuromuscular activity to the destabilization of neuromuscular junctions with age and disease, and (2) how a progressive increase in the neuromuscular activity of declining numbers of functional MUs contributes to the progressive failure of adaptive sprouting and, in turn, to the progressive muscle weakness in the motoneuron diseases of post-polio syndrome and amyotrophic lateral sclerosis. We conclude that there is a time-related progression of MU loss, adaptive sprouting followed by maladaptive sprouting, and continuing recession of terminals during normal aging. The progression is accelerated in motoneuron disease, progressing more rapidly in the post-polio syndrome after prolonged denervation and extremely rapidly in ALS.

The effects of increasing neural activity on sprouting remain unclear and controversial. In a rat model of partial denervation of skeletal muscles, we investigated the effect of neuromuscular activity on sprouting. Rat hindlimb muscles were partially denervated by avulsion of either L4 or L5 spinal root. Immediately after partial denervation, the rats were divided into three groups: (1) normal caged activity, (2) running exercise on wheels, 8 hr daily, and (3) functional electrical stimulation (FES) of sciatic nerves, 20 Hz for 8 hr daily. At 1 month, muscle unit (MU) enlargement was quantitated electrophysiologically and histochemically. MU twitch force was increased by four-to fivefold by partial denervation in extensively denervated tibialis anterior (TA) and medial gastrocnemius (MG) and by approximately twofold in moderately denervated plantaris (PL) and soleus (SOL). For the extensively denervated TA and MG muscles, MU enlargement, measured electrophysiologically, declined significantly after an average of 1757 Ϯ 310 m/d running exercise and daily FES for 1 month. The detrimental effects on MU enlargement were much less but significant in the moderately denervated PL and did not reach statistical significance in the moderately denervated SOL muscle. Histochemical evaluation of sprouting showed a reduction in the number of sprouts in the extensively denervated TA muscle, but not the moderately denervated PL and SOL muscles, by increased neuromuscular activity. Thus, increased neuromuscular activity is detrimental primarily in muscles that are extensively denervated, and the MUs are smaller than under conditions in which the muscles experience normal physiological levels of activation.Key words: sprouting; motor unit; motoneuron disease; neuromuscular activity; partial denervation; poliomyelitis Poliomyelitis, the early stages of amyotrophic lateral sclerosis (ALS), spinal cord trauma, and motoneuron destruction associated with cancer are only some of the neuromuscular conditions resulting in compensatory axonal sprouting and, in turn, MU enlargement (Brown et al., 1981;Halstead and Wiechers, 1987). MU enlargement is unfortunately restricted to a limit of five-to eightfold such that sprouting compensates for up to 85% loss of muscle units (MUs) (Thompson and Jansen, 1977;Brown and Ironton, 1978;Yang et al., 1990;Rafuse et al., 1992). Thus when Ͻ20% of intact MUs remain and sprouting cannot reinnervate all denervated muscle fibers, muscle weakness becomes evident (Luff et al., 1988;Rafuse et al., 1992; Rafuse and Gordon, 1996a,b).The strong association of exercise with muscle strength and endurance has led naturally to attempts to optimize muscle function with exercise. However, the effects of neuromuscular activity on sprouting are both unclear and controversial because of the conflicting findings of previous studies of these effects. Some studies have shown that activity can promote sprouting or reinnervation (Ribchester, 1988;Einsiedel and Luff, 1994) or that it has no effect at all (Gardiner and Faltus, ...

This review considers the relative roles of sprouting stimuli, perisynaptic Schwann cells and neuromuscular activity in axonal sprouting at the neuromuscular junction in partially denervated muscles. A number of sprouting stimuli, including insulin-like growth factor II, which are generated from inactive muscle fibers in partially denervated and paralyzed skeletal muscles, has been considered. There is also evidence that perisynaptic Schwann cells induce and guide axonal sprouting in adult partially denervated muscles. Excessive neuromuscular activity significantly reduces bridging of perisynaptic Schwann cell processes between innervated and denervated endplates and thereby inhibits axonal sprouting in partially denervated adult muscles. Elimination of neuromuscular activity is also detrimental to sprouting in these muscles, suggesting that calcium influx into the nerve is crucial for axonal sprouting. The role of neuromuscular activity in axonal sprouting will be considered critically in the context of the roles of sprouting stimuli and perisynaptic Schwann cells in the process of axonal sprouting.

Following partial denervation of rat hindlimb muscle, terminal Schwann cells extend processes from denervated endplates to induce and guide sprouting from the remaining intact axons. Increased neuromuscular activity significantly reduces motor unit enlargement and sprouting during the acute phase of sprouting. These findings led to the hypothesis that increased neuromuscular activity perturbs formation of Schwann cell bridges and thereby reduces sprouting. Adult rat tibialis anterior (TA) muscles were extensively denervated by avulsion of L4 spinal root and were immediately subjected to normal caged activity or running exercise (8 h daily) for 3, 7, 14, 21, and 28 days. Combined silver/cholinesterase histochemical staining revealed that the progressive reinnervation of denervated endplates by sprouts over a 1 month period in the extensively partially denervated TA muscles was completely abolished by increased neuromuscular activity. Immunohistochemical staining and triple immunofluorescence revealed that the increased neuromuscular activity did not perturb the production of Schwann cell processes, but prevented bridging between Schwann cell processes at innervated and denervated endplates. Our findings suggest that failure of Schwann cell processes to bridge between endplates accounts, at least in part, for the inhibitory effect of increased neuromuscular activity on sprouting.