Dario I. Carrasco

Neurotrophin-4/5 is required for the early growth of regenerating axons in peripheral nerves.

The requirement of the trkB ligand, neurotrophin‐4/5 (NT‐4/5), for the growth of regenerating axons in the peripheral nervous system (PNS) is not well established. We studied regenerating axon growth in transected peripheral nerves of thy‐1‐YFP‐H mice that had been repaired using allografts obtained from brain‐derived neurotrophic factor (BDNF) or NT‐4/5 knockout mice. Lengths of profiles of YFP+ axons measured in these grafts were compared with those measured in grafts obtained from wild‐type donors. When compared with axon profiles measured in grafts from wild‐type donors, axon profile lengths measured in grafts from homozygous (NT‐4/5−/−) or heterozygous (NT‐4/5+/−) mice were significantly shorter. In contrast, the lengths of axon profiles measured in grafts from BDNF+/− mice were not significantly different from those measured in grafts from wild‐type mice. A reduced amount of BDNF, but not NT‐4/5, is sufficient to promote the elongation of regenerating axons in the PNS. When grafts from wild‐type or NT‐4/5−/− mice were treated acutely at the time of surgical repair either with exogenous BDNF or NT‐4/5, the lengths of axon profiles measured in the grafts were significantly longer than those measured in grafts from untreated wild‐type mice. These findings are consistent with a requirement for NT‐4/5 from within the pathway used by regenerating axons for the successful growth of those axons in peripheral nerves.

Sexual Dimorphism in the Rabbit Masseter Muscle: Myosin Heavy Chain Composition of Neuromuscular Compartments

The myosin heavy chain (MyHC) isoform composition of six adult (>7 months old) male and female rabbit masseter muscles was studied using seven monoclonal antibodies. In matched serial tissue sections, muscle fibers in 10 different neuromuscular compartments were analyzed. Nearly all fibers were found to express one of five phenotypes. They either contained one of four different slow/beta MyHC phenotypes (I1–I4), nearly all of which co-express cardiac alpha MyHC, or they contained type IIa MyHC. Very few fibers contained slow/beta or cardiac alpha MyHC only or both the alpha/slow/beta and IIa isoforms. Most, but not all, of the compartments studied contained similar proportions of fibers of the five major phenotypes, at least within sex. For 7 of the 10 compartments studied, significant sex differences in the proportion of I1 and IIa fibers were found. Males contained more IIa fibers and fewer I1 fibers than females. Fibers of the IIa phenotype were significantly larger than fibers of all of the other phenotypes and larger in males than females.

Differential activation of neuromuscular compartments in the rabbit masseter muscle during different oral behaviors

The rabbit masseter muscle is composed of multiple anatomical partitions that produce different mechanical actions. The purpose of this study was to test the hypothesis that these compartments are differentially activated during the performance of different oral behaviors. Rhythmic activation of the masticatory muscles was elicited by stimulating the cortical masticatory area (CMA) while recording forces generated at the incisors in three dimensions with the mandible immobilized. Torques about the right temporomandibular joint (TMJ) were calculated using these forces recorded during isometric function. A set of 1–15 unique rhythmic behaviors was identified for each rabbit using torque phase plot patterns. Electromyographic recordings were made at nine different compartments in the right masseter, two compartments in the left masseter, two regions in the right digastric, and single locations in the left digastric and right and left medial pterygoid muscles. In activation cycles producing similar mechanical actions, activity patterns at the 16 recording sites were clustered into three to six groups using principal component analysis (PCA). To test for similarities in the activation of masseter compartments, pair-wise comparisons of the PCA assignment for the nine masseter compartments were conducted and frequencies of common assignment were compiled for each unique rhythmic behavior for each rabbit. Masseter muscle compartments were found to vary significantly in their PCA from the expected distribution of 100% common principal component (PC) assignment (i.e., similar activation pattern). This finding is consistent with the independent activation of masseter compartments.

Neurotrophin 4/5 is required for the normal development of the slow muscle fiber phenotype in the rat soleus

SUMMARY During normal postnatal development, rat soleus (SOL) muscle fibers undergo a dramatic fast-to-slow myosin heavy chain (MyHC) isoform transformation. We exploited this phenomenon to evaluate the role of neurotrophin 4/5 (NT-4/5) in the regulation of muscle fiber phenotype. Intramuscular injections of recombinant NT-4/5 into the SOL muscle of rat neonates significantly accelerated the normal fast-to-slow MyHC isoform transformation. Sequestration of endogenous NT-4/5 with TrkB–IgG prevented this transformation from occurring. Administration of the other TrkB ligand, brain-derived neurotrophic factor (BDNF), did not affect the normal course of the MyHC isoform transformation in this muscle, indicating that the observed effect is NT-4/5 specific. Botulinum toxin blockade of synaptic transmission significantly disrupted the normal fast-to-slow MyHC isoform switch. Because administration of NT-4/5 to paralyzed muscles failed to restore the normal course of this MyHC transformation, we believe that the effect of NT-4/5 is not directly on the muscle fibers but that it probably activates or forms a type of retrograde signal to motoneurons. The developmental upregulation of NT-4/5 mRNA in rat SOL muscle fibers occurred earlier than the upregulation of MyHC I/b mRNA associated with muscle fiber transformation. This timing is consistent with the idea that NT-4/5 is involved in early events that lead to the upregulation of the slow MyHC isoform in this muscle.

Regulation of Motoneuron Excitability via Motor Endplate Acetylcholine Receptor Activation

Motoneuron populations possess a range of intrinsic excitability that plays an important role in establishing how motor units are recruited. The fact that this range collapses after axotomy and does not recover completely until after reinnervation occurs suggests that muscle innervation is needed to maintain or regulate adult motoneuron excitability, but the nature and identity of underlying mechanisms remain poorly understood. Here, we report the results of experiments in which we studied the effects on rat motoneuron excitability produced by manipulations of neuromuscular transmission and compared these with the effects of peripheral nerve axotomy. Inhibition of acetylcholine release from motor terminals for 5-6 d with botulinum toxin produced relatively minor changes in motoneuron excitability compared with the effect of axotomy. In contrast, the blockade of acetylcholine receptors with α-bungarotoxin over the same time interval produced changes in motoneuron excitability that were statistically equivalent to axotomy. Muscle fiber recordings showed that low levels of acetylcholine release persisted at motor terminals after botulinum toxin, but endplate currents were completely blocked for at least several hours after daily intramuscular injections of α-bungarotoxin. We conclude that the complete but transient blockade of endplate currents underlies the robust axotomy-like effects of α-bungarotoxin on motoneuron excitability, and the low level of acetylcholine release that remains after injections of botulinum toxin inhibits axotomy-like changes in motoneurons. The results suggest the existence of a retrograde signaling mechanism located at the motor endplate that enables expression of adult motoneuron excitability and depends on acetylcholine receptor activation for its normal operation.

Nerve Terminal Degeneration Is Independent of Muscle Fiber Genotype in SOD1G93A Mice

Background Motor neuron degeneration in SOD1G93A transgenic mice begins at the nerve terminal. Here we examine whether this degeneration depends on expression of mutant SOD1 in muscle fibers. Methodology/Principal Findings Hindlimb muscles were transplanted between wild-type and SOD1G93A transgenic mice and the innervation status of neuromuscular junctions (NMJs) was examined after 2 months. The results showed that muscles from SOD1G93A mice did not induce motor terminal degeneration in wildtype mice and that muscles from wildtype mice did not prevent degeneration in SOD1G93A transgenic mice. Control studies demonstrated that muscles transplanted from SOD1G93A mice continued to express mutant SOD1 protein. Experiments on wildtype mice established that the host supplied terminal Schwann cells (TSCs) at the NMJs of transplanted muscles. Conclusions/Significance These results indicate that expression of the mutant protein in muscle is not needed to cause motor terminal degeneration in SOD1G93A transgenic mice and that a combination of motor terminals, motor axons and Schwann cells, all of which express mutant protein may be sufficient.

Rat motoneuron properties recover following reinnervation in the absence of muscle activity and evoked acetylcholine release

Available evidence supports the idea that muscle fibres provide retrograde signals that enable the expression of adult motoneuron electrical properties but the mechanisms remain unknown. We showed recently that when acetylcholine receptors are blocked at motor endplates, the electrical properties of rat motoneurons change in a way that resembles changes observed after axotomy. This observation suggests that receptor blockade and axotomy interrupt the same signalling mechanisms but leaves open the possibility that the loss of muscle fibre activity underlies the observed effects. To address this issue, we examined the electrical properties of axotomized motoneurons following reinnervation. Ordinarily, these properties return to normal following reinnervation and re‐activation of muscle, but in this study muscle fibre activity and evoked acetylcholine release were prevented during reinnervation by blocking axonal conduction. Under these conditions, the properties of motoneurons that successfully reinnervated muscle fibres recovered to normal despite the absence of muscle fibre activity and evoked release. We conclude that the expression of motoneuron electrical properties is not regulated by muscle fibre activity but rather by a retrograde signalling system coupled to activation of endplate acetylcholine receptors. Our results indicate that spontaneous release of acetylcholine from regenerated motor terminals is sufficient to operate the system.

Altered terminal Schwann cell morphology precedes denervation in SOD1 mice

In mice that express SOD1 mutations found in human motor neuron disease, degeneration begins in the periphery for reasons that remain unknown. At the neuromuscular junction (NMJ), terminal Schwann cells (TSCs) have an intimate relationship with motor terminals and are believed to help maintain the integrity of the motor terminal. Recent evidence indicates that TSCs in some SOD1 mice exhibit abnormal functional properties, but other aspects of possible TSC involvement remain unknown. In this study, an analysis of TSC morphology and number was performed in relation to NMJ innervation status in mice which express the G93A SOD1 mutation. At P30, all NMJs of the fast medial gastrocnemius (MG) muscle were fully innervated by a single motor axon but 50% of NMJs lacked TSC cell bodies and were instead covered by the processes of Schwann cells with cell bodies located on the preterminal axons. NMJs in P30 slow soleus muscles were also fully innervated by single motor axons and only 5% of NMJs lacked a TSC cell body. At P60, about 25% of MG NMJs were denervated and lacked labeling for TSCs while about 60% of innervated NMJs lacked TSC cell bodies. In contrast, 96% of P60 soleus NMJs were innervated while 9% of innervated NMJs lacked TSC cell bodies. The pattern of TSC abnormalities found at P30 thus correlates with the pattern of denervation found at P60. Evidence from mice that express the G85R SOD1 mutation indicate that TSC abnormalities are not unique for mice that express G93A SOD1 mutations. These results add to an emerging understanding that TSCs may play a role in motor terminal degeneration and denervation in animal models of motor neuron disease.

Motor terminal degeneration unaffected by activity changes in SOD1G93A mice; a possible role for glycolysis

This study examined whether activity is a contributing factor to motor terminal degeneration in mice that overexpress the G93A mutation of the SOD1 enzyme found in humans with inherited motor neuron disease. Previously, we showed that overload of muscles accomplished by synergist denervation accelerated motor terminal degeneration in dogs with hereditary canine spinal muscular atrophy (HCSMA). In the present study, we found that SOD1 plantaris muscles overloaded for 2months showed no differences of neuromuscular junction innervation status when compared with normally loaded, contralateral plantaris muscles. Complete elimination of motor terminal activity using blockade of sciatic nerve conduction with tetrodotoxin cuffs for 1month also produced no change of plantaris innervation status. To assess possible effects of activity on motor terminal function, we examined the synaptic properties of SOD1 soleus neuromuscular junctions at a time when significant denervation of close synergists had occurred as a result of natural disease progression. When examined in glucose media, SOD1 soleus synaptic properties were similar to wildtype. When glycolysis was inhibited and ATP production limited to mitochondria, however, blocking of evoked synaptic transmission occurred and a large increase in the frequency of spontaneous mEPCs was observed. Similar effects were observed at neuromuscular junctions in muscle from dogs with inherited motor neuron disease (HCSMA), although significant defects of synaptic transmission exist at these neuromuscular junctions when examined in glucose media, as reported previously. These results suggest that glycolysis compensates for mitochondrial dysfunction at motor terminals of SOD1 mice and HCSMA dogs. This compensatory mechanism may help to support resting and activity-related metabolism in the presence of dysfunctional mitochondria and prolong the survival of SOD1 motor terminals.

Abnormal response of distal Schwann cells to denervation in a mouse model of motor neuron disease

In several animal models of motor neuron disease, degeneration begins in the periphery. Clarifying the possible role of Schwann cells remains a priority. We recently showed that terminal Schwann cells (TSCs) exhibit abnormalities in postnatal mice that express mutations of the SOD1 enzyme found in inherited human motor neuron disease. TSC abnormalities appeared before disease-related denervation commenced and the extent of TSC abnormality at P30 correlated with the extent of subsequent denervation. Denervated neuromuscular junctions (NMJs) were also observed that lacked any labeling for TSCs. This suggested that SOD1 TSCs may respond differently than wildtype TSCs to denervation which remain at denervated NMJs for several months. In the present study, the response of SOD1 TSCs to experimental denervation was examined. At P30 and P60, SC-specific S100 labeling was quickly lost from SOD1 NMJs and from preterminal regions. Evidence indicates that this loss eventually becomes complete at most SOD1 NMJs before reinnervation occurs. The loss of labeling was not specific for S100 and did not depend on loss of activity. Although some post-denervation labeling loss occurred at wildtype NMJs, this loss was never complete. Soon after denervation, large cells appeared near SOD1 NMJ bands which colabeled for SC markers as well as for activated caspase-3 suggesting that distal SOD1 SCs may experience cell death following denervation. Denervated SOD1 NMJs viewed 7 days after denervation with the electron microscope confirmed the absence of TSCs overlying endplates. These observations demonstrate that SOD1 TSCs and distal SCs respond abnormally to denervation. This behavior can be expected to hinder reinnervation and raises further questions concerning the ability of SOD1 TSCs to support normal functioning of motor terminals.