Temporal expression of brainstem neurotrophic proteins following mild traumatic brain injury.

BDNF, a neurotrophic factor, and its receptors have been implicated in the pathophysiology of mild traumatic brain injury (mTBI). The brainstem houses many vital functions, that are also associated with signs and symptoms of mTBI, but has been understudied in mTBI animal models. We determined the extent to which neurotrophic protein and associated receptor expression is affected within the brainstem of adult rats following mTBI. Their behavioral function was assessed and temporal expression of the 'negative' regulators of neuronal function (p75, t-TrkB, and pro-BDNF) and 'positive' neuroprotective (FL-TrkB and m-BDNF) protein isoforms were determined via western blot and immunohistochemistry at 1, 3, 7, and 14 post-injury days (PID) following mTBI or sham (control) procedure. Within the brainstem, p75 expression increased at PID 1 vs. sham animals. t-TrkB and pro-BDNF expression increased at PID 7 and 14. The 'positive' protein isoforms of FL-TrkB and m-BDNF expression were increased only at PID 7. The ratio of t-TrkB:FL-TrkB (negative:positive) was substantial across groups and time points, suggesting a negative impact of neurotrophic signaling on neuronal function. Additional NeuN experiments revealed cell death occurring within a subset of neurons within the medulla. While behavioral measures improved by PID 7-14, negative neurotrophic biochemical responses persisted. Despite the assertion that mTBI produces "mild" injury, evidence of cell death was observed in the medulla. Ratios of TrkB and BDNF isoforms with conflicting functions suggest that future work should specifically measure each subtype since they induce opposing downstream effects on neuronal function.

Skeletal muscle reprogramming enhances reinnervation after peripheral nerve injury.

Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. The onset of PNI is characterized by nerve degeneration distal to the nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of "accepting" innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generated a mouse model in which NANOG, a pluripotency-associated transcription factor can be expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulated the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression led to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation, and downregulation of key muscle atrophy genes. Further, NANOG mice demonstrated extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice showed greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming the muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI.

Blockage of neuromuscular glutamate receptors impairs reinnervation following nerve crush in adult mice.

Motor axons in peripheral nerves are capable of regeneration following injury. However, complete recovery of motor function is rare, particularly when reinnervation is delayed. We have previously found that glutamate receptors play a crucial role in the successful innervation of muscle during mouse development. In particular, blocking N-methyl-D-aspartate (NMDA) receptor activity delays the normal elimination of excess innervation of each neuromuscular junction. Here, we use behavioral, immunohistochemical, electrophysiological, and calcium imaging methods to test whether glutamate receptors play a similar role in the transition from polyneuronal to mono-innervation and in recovery of function following peripheral nerve injury in mature muscle.

Participants with mildly-disabling chronic neck pain perform differently during explicit compared to implicit motor learning of a reaching task.

Chronic musculoskeletal (CMSK) pain associated with musculoskeletal disorders like low back pain or neck pain are the leading causes of disability. While CMSK pain has the potential to negatively influence motor learning, there is limited research to understand the impact of CMSK on motor learning. In order to examine differences in motor learning between individuals with and without CMSK we modified a serial reaction time task to assess motor learning of a repetitive reaching task. The paradigm was used to assess both explicit and implicit motor learning. In a cross-sectional study design, seventeen participants with chronic neck pain (CNP) (5 males) and 21 controls (8 males) were recruited. In addition, physical, cognitive, sensorimotor, disability and pain assessments were used to examine differences between individuals with and without CNP. All participants with CNP were categorized as having mild disability. There was no difference in cognitive assessments and minimal differences in physical measures between groups. Examining motor learning, groups with and without CNP demonstrated similar outcomes in both explicit and implicit motor learning. There was one notable performance difference between groups in the reaching task, the group with CNP demonstrated slower reaching movements outward and inward during blocks without explicit information. This may suggest a cautious approach to movement with reduced explicit information. Findings from this study provide insight on motor learning in individuals with mildly-disabling CNP, further research is necessary to examine how instruction can impact peak performance in people with CMSK pain.

Neuromuscular NMDA Receptors Modulate Developmental Synapse Elimination.

UNLABELLED: At birth, each mammalian skeletal muscle fiber is innervated by multiple motor neurons, but in a few weeks, all but one of those axons retracts (Redfern, 1970) and differential activity between inputs controls this phenomenon (Personius and Balice-Gordon, 2001; Sanes and Lichtman, 2001; Personius et al., 2007; Favero et al., 2012). Acetylcholine, the primary neuromuscular transmitter, has long been presumed to mediate this activity-dependent process (O'Brien et al., 1978), but glutamatergic transmission also occurs at the neuromuscular junction (Berger et al., 1995; Grozdanovic and Gossrau, 1998; Mays et al., 2009). To test the role of neuromuscular NMDA receptors, we assessed their contribution to muscle calcium fluxes in mice and tested whether they influence removal of excess innervation at the end plate. Developmental synapse pruning was slowed by reduction of NMDA receptor activation or expression and by reduction of glutamate production. Conversely, pruning is accelerated by application of exogenous NMDA. We also found that NMDA induced increased muscle calcium only during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and synaptic pruning during development. SIGNIFICANCE STATEMENT: In normal adult muscle, each muscle fiber is innervated by a single axon, but at birth, fibers are multiply innervated. Elimination of excess connections requires neural activity; because the neuromuscular junction (NMJ) is a cholinergic synapse, acetylcholine has been assumed to be the critical mediator of activity. However, glutamate receptors are also expressed at the NMJ. We found that axon removal in mice is slowed by pharmacological and molecular manipulations that decrease signaling through neuromuscular NMDA receptors, whereas application of exogenous NMDA at the NMJ accelerates synapse elimination and increases muscle calcium levels during the first 2 postnatal weeks. Therefore, neuromuscular NMDA receptors play previously unsuspected roles in neuromuscular activity and elimination of excess synaptic input during development.

Innervation of dystrophic muscle after muscle stem cell therapy.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor-derived muscle fibers are structurally innervated. METHODS: Green fluorescent protein (GFP)-expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry. RESULTS: The percent overlap between pre- and postsynaptic immunolabeling was greater in donor-derived GFP(+) myofibers, and fewer GFP(+) myofibers were identified as denervated compared with control GFP(-) fibers (P = 0.001 and 0.03). GFP(+) fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033). CONCLUSION: It is unclear whether GFP(+) fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor-derived fibers demonstrate clear histological innervation. Muscle Nerve 54: 763-768, 2016.

Vitamin D3 intake modulates diaphragm but not peripheral muscle force in young mice.

Recent data support an important role for vitamin D in respiratory health. We tested the hypothesis that dietary vitamin D3 (VD3) intake modulates diaphragm (DIA) strength. Four-week-old female A/J mice (n = 10/group) were randomized to receive diets containing 100 IU VD3/kg (low), 1,000 IU VD3/kg (reference), or 10,000 IU VD3/kg (pharmacologic). After 6 wk of dietary intervention, plasma 25-hydroxyvitamin D3 (25D3) levels, DIA and extensor digitorum longus (EDL) in vitro contractile properties, and fiber cross-sectional area (CSA) were measured. Myosin heavy chain (MHC) composition and Akt/Foxo3A growth signaling were studied in the DIA and tibialis anterior. Mice fed the low, reference, and pharmacologic diets had average 25D3 levels of 7, 21, and 59 ng/ml, respectively. Maximal DIA force, twitch force, and fiber CSA were reduced 26%, 28%, and 10% (P < 0.01), respectively, in mice receiving the low-VD3 diet compared with the reference and pharmacologic diets. EDL force parameters were unaltered by diet. Effects of VD3 intake on DIA force were not observed in mice that began dietary intervention at 12 wk of age. VD3 intake did not alter the MHC composition of the DIA, indicating that decreases in force and CSA in young mice were not due to a switch in fiber type. Paradoxically, low VD3 intake was associated with activation of anabolic signaling in muscle (hyperphosphorylation of Akt and Foxo3A and decreased expression of autophagy marker LC3). These studies identify a potential role of dietary VD3 in regulating DIA development and insulin sensitivity.

Muscleblind-Like 1 and Muscleblind-Like 3 Depletion Synergistically Enhances Myotonia by Altering Clc-1 RNA Translation.

Loss of Muscleblind-like 1 (Mbnl1) is known to alter Clc-1 splicing to result in myotonia. Mbnl1(ΔE3/ΔE3)/Mbnl3(ΔE2) mice, depleted of Mbnl1 and Mbnl3, demonstrate a profound enhancement of myotonia and an increase in the number of muscle fibers with very low Clc-1 currents, where gClmax values approach ~ 1 mS/cm(2), with the absence of a further enhancement in Clc-1 splice errors, alterations in polyA site selection or Clc-1 localization. Significantly, Mbnl1(ΔE3/ΔE3)/Mbnl3(ΔE2) muscles demonstrate an aberrant accumulation of Clc-1 RNA on monosomes and on the first polysomes. Mbnl1 and Mbnl3 bind Clc-1 RNA and both proteins bind Hsp70 and eEF1A, with these associations being reduced in the presence of RNA. Thus binding of Mbnl1 and Mbnl3 to Clc-1 mRNA engaged with ribosomes can facilitate an increase in the local concentration of Hsp70 and eEF1A to assist Clc-1 translation. Dual depletion of Mbnl1 and Mbnl3 therefore initiates both Clc-1 splice errors and translation defects to synergistically enhance myotonia. As the HSA(LR) model for myotonic dystrophy (DM1) shows similar Clc-1 defects, this study demonstrates that both splice errors and translation defects are required for DM1 pathology to manifest. RESEARCH IN CONTEXT: Research in context: Myotonic Dystrophy type 1 (DM1) is a dominant disorder resulting from the expression of expanded CUG repeat RNA, which aberrantly sequesters and inactivates the muscleblind-like (MBNL) family of proteins. In mice, inactivation of Mbnl1 is known to alter Clc-1 splicing to result in myotonia. We demonstrate that concurrent depletion of Mbnl1 and Mbnl3 results in a synergistic enhancement of myotonia, with an increase in muscle fibers showing low chloride currents. The observed synergism results from the aberrant accumulation of Clc-1 mRNA on monosomes and the first polysomes. This translation error reflects the ability of Mbnl1 and Mbnl3 to act as adaptors that recruit Hsp70 and eEF1A to the Clc-1 mRNA engaged with ribosomes, to facilitate translation. Thus our study demonstrates that Clc-1 RNA translation defects work coordinately with Clc-1 splice errors to synergistically enhance myotonia in mice lacking Mbnl1 and Mbnl3.

TrkB expression at the neuromuscular junction is reduced during aging.

INTRODUCTION: Full-length tyrosine kinase B (TrkB.FL) and truncated TrkB (TrkB.t1) receptors are colocalized with acetylcholine receptors (AChRs) at the neuromuscular junction. We have recently shown that reduced TrkB expression leads to age-related alterations in AChR structure, neurotransmission failure, and muscle weakness. METHODS: We investigated whether TrkB expression is reduced in the soleus muscle during aging. RESULTS: TrkB protein expression was decreased in senescent (24-month-old) compared with 3-12-month-old mice. Loss of TrkB expression was concurrent with age-related changes in AChR morphology. Changes in mRNA levels did not correlate with protein expression, because TrkB.FL copy number was increased in the senescent soleus. No change was seen in TrkB.t1 levels. CONCLUSIONS: The results suggest that reduced TrkB expression during aging may result from reduced TrkB.FL mRNA translation or increased TrkB protein turnover. Thus, maintaining adequate TrkB signaling is a potential therapeutic tool to improve muscle function during senescence.

Reduced TrkB expression results in precocious age-like changes in neuromuscular structure, neurotransmission, and muscle function.

Acute blockade of signaling through the tyrosine kinase receptor B (TrkB) attenuates neuromuscular transmission and fragments postsynaptic acetylcholine receptors (AChRs) in adult mice, suggesting that TrkB signaling is a key regulator of neuromuscular function. Using immunohistochemical, histological, and in vitro muscle contractile techniques, we tested the hypothesis that constitutively reduced TrkB expression would disrupt neuromuscular pre- and postsynaptic structure, neurotransmission, muscle fiber size, and muscle function in the soleus muscle of 6- to 8-mo-old TrkB⁺/⁻ mice compared with age-matched littermates. Age-like expansion of postsynaptic AChR area, AChR fragmentation, and denervation was observed in TrkB⁺/⁻ mice similar to that found in 24-mo-old wild-type mice. Neurotransmission failure was increased in TrkB⁺/⁻ mice, suggesting that these morphologic changes were sufficient to alter synaptic function. Reduced TrkB expression resulted in decreased muscle strength and fiber cross-sectional area. Immunohistochemical and muscle retrograde labeling experiments show that motor neuron number and size are unaffected in TrkB⁺/⁻ mice. These results suggest that TrkB- signaling at the neuromuscular junction plays a role in synaptic stabilization, neurotransmission, and muscle function and may impact the aging process of sarcopenia.

Grip force, EDL contractile properties, and voluntary wheel running after postdevelopmental myostatin depletion in mice.

There is no consensus about whether making muscles abnormally large by reducing myostatin activity affects force-generating capacity or the ability to perform activities requiring muscular endurance. We therefore examined grip force, contractile properties of extensor digitorum longus (EDL) muscles, and voluntary wheel running in mice in which myostatin was depleted after normal muscle development. Cre recombinase activity was induced to knock out exon 3 of the myostatin gene in 4-mo-old mice in which this exon was flanked by loxP sequences (Mstn[f/f]). Control mice with normal myostatin genes (Mstn[w/w]) received the same Cre-activating treatment. Myostatin depletion increased the mass of all muscles that were examined (gastrocnemius, quadriceps, tibialis anterior, EDL, soleus, triceps) by approximately 20-40%. Grip force, measured multiple times 2-22 wk after myostatin knockout, was not consistently greater in the myostatin-deficient mice. EDL contractile properties were determined 7-13 mo after myostatin knockout. Twitch force tended to be greater in myostatin-deficient muscles (+24%; P=0.09), whereas tetanic force was not consistently elevated (mean +11%; P=0.36), even though EDL mass was greater than normal in all myostatin-deficient mice (mean +36%; P<0.001). The force deficit induced by eccentric contractions was approximately twofold greater in myostatin-deficient than in normal EDL muscles (31% vs. 16% after five eccentric contractions; P=0.02). Myostatin-deficient mice ran 19% less distance (P<0.01) than control mice during the 12 wk following myostatin depletion, primarily because of fewer running bouts per night rather than diminished running speed or bout duration. Reduced specific tension (ratio of force to mass) and reduced running have been observed after muscle hypertrophy was induced by other means, suggesting that they are characteristics generally associated with abnormally large muscles rather than unique effects of myostatin deficiency.

NMDA receptor blockade maintains correlated motor neuron firing and delays synapse competition at developing neuromuscular junctions.

Mammalian neuromuscular synapses undergo an activity-dependent competitive transition from multiple to single innervation during postnatal life. The presence of temporally correlated motor neuron activity, which, in part, is controlled by gap junctional coupling within the spinal cord, appears to modulate synapse elimination. Postnatal injection of dizocilpine maleate (MK801), a specific NMDA antagonist, has been shown to maintain gap junctional coupling among motor neurons. Thus, we tested the hypothesis that MK801 would maintain correlated motor neuron activity and delay postnatal synapse elimination. Temporally correlated motor neuron activity, which is normally lost during the second postnatal week, was maintained and synaptic competition was delayed by several days in 2-week-old mice injected daily with MK801. MK801 appears to modulate motor neuron activity patterns through enhancing mRNA expression of multiple connexins within the spinal cord and delaying motor neuron growth. Our results suggest that MK801 injection preserves correlated neural activity via both synaptic mechanisms and maintenance of gap junctional coupling among neurons within the spinal cord, ultimately delaying synapse elimination.

Reduced gap junctional coupling leads to uncorrelated motor neuron firing and precocious neuromuscular synapse elimination.

During late embryonic and early postnatal life, neuromuscular junctions undergo synapse elimination that is modulated by patterns of motor neuron activity. Here, we test the hypothesis that reduced spinal neuron gap junctional coupling decreases temporally correlated motor neuron activity that, in turn, modulates neuromuscular synapse elimination, by using mutant mice lacking connexin 40 (Cx40), a developmentally regulated gap junction protein expressed in motor and other spinal neurons. In Cx40-/- mice, electrical coupling among lumbar motor neurons, measured by whole-cell recordings, was reduced, and single motor unit recordings in awake, behaving neonates showed that temporally correlated motor neuron activity was also reduced. Immunostaining and intracellular recording showed that the neuromuscular synapse elimination was accelerated in muscles from Cx40-/- mice compared with WT littermates. Our work shows that gap junctional coupling modulates neuronal activity patterns that, in turn, mediate synaptic competition, a process that shapes synaptic circuitry in the developing brain.

Variability and failure of neurotransmission in the diaphragm of mdx mice.

Loss of specific muscle force and evidence of myopathy are present in the diaphragm of mdx mice by 4 weeks of age. The neuromuscular junction of dystrophic muscle also shows structural abnormalities at this age. Whether these structural alterations result in neural transmission abnormalities is currently unclear, particularly at physiological firing frequencies. Thus, we investigated the extent of neurotransmission variability and failure during 35 and 100 Hz stimulation in the diaphragm of 6 to 8-month-old mdx mice in comparison to age-matched controls. Neurotransmission failure was similar across groups at both stimulation frequencies, despite the presence of disrupted post-synaptic acetylcholine receptors (AChRs). Neural transmission variability, however, measured by comparing variation in force production during direct muscle stimulation compared to variation in force production during phrenic nerve stimulation was significantly greater in dystrophic muscle. Together, these results suggest that neurotransmission is maintained at physiologic firing frequencies in dystrophic muscle, but the precision of neurotransmission is attenuated. A reduced density of functional AChRs likely underlies the increase in neurotransmission variability.

Terminal Schwann cell structure is altered in diaphragm of mdx mice.

The diaphragm muscle of the mdx mouse is a model system of Duchenne muscular dystrophy, since it completely lacks dystrophin and shows severe fiber necrosis and loss of specific muscle force by 4-6 weeks of age. Changes in neuromuscular junction structure also become apparent around 4 weeks including postsynaptic acetylcholine receptor declustering, loss of postsynaptic junctional folds, abnormally complex presynaptic nerve terminals, and muscle fiber denervation. Normally, terminal Schwann cells (TSCs) cap both nerve terminals and acetylcholine receptors at the neuromuscular junction, and play a crucial role in regeneration of motor axons following muscle denervation by guiding axons to grow from innervated junctions to nearby denervated junctions. However, their role in restoring innervation in dystrophic muscle is unknown. We now show that TSCs fail to cap fully the neuromuscular junction in dystrophic muscle; TSCs extend processes, but the organization of these extensions is abnormal. TSC processes of dystrophic muscle do not form bridges from denervated fibers to nearby innervated endplates, but appear to be directed away from these endplates. Adequate signaling for TSC reactivity is present, since significant muscle fiber denervation and acetylcholine receptor declustering are present. Thus, significant structural denervation is present in the diaphragm of mdx mice and the ability of TSCs to form bridges between adjacent endplates to guide reinnervation of muscle fibers is impaired, possibly attenuating the ability of dystrophic muscle to recover from denervation and ultimately leading to muscle weakness.

Myotonia and muscle contractile properties in mice with SIX5 deficiency.

Myotonic dystrophy (DM1) is an autosomal-dominant multisystem disease characterized by progressive skeletal muscle weakness, myotonia, cataracts, cardiac arrhythmias, mild mental retardation, and endocrinopathies. Heterozygous loss of SIX5 in mice causes cataracts and cardiac conduction disease, and homozygous loss also leads to sterility and decreased testicular mass, reminiscent of DM1 in humans. The effect of SIX5 deficiency in muscle is unknown. In this study, we found that muscle contractile properties, electromyographic insertional activity, and muscle histology were normal in SIX5 deficient mice. The implications of these findings for the pathogenesis of DM1 are discussed.

Activity-dependent synaptic plasticity: insights from neuromuscular junctions.

Experience-dependent editing shapes synaptic connections throughout the developing nervous system, but the underlying cellular mechanisms remain poorly understood. A useful model synapse for addressing these mechanisms is the neuromuscular junction, the connection between spinal motor neurons and skeletal muscle fibers. Here the authors review current ideas about the role of activity in editing neuromuscular synaptic connections. A variety of new tools are being used to address some unanswered questions in vivo and in vitro. Understanding activity-dependent plasticity at developing neuromuscular synapses may reveal how neural circuits in the central nervous system are altered by experience throughout life.