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1.  Developmental and Functional Considerations of Masseter Muscle Partitioning 
Archives of oral biology  2006;52(4):305-308.
The masseter muscle participates in a wide variety of activities including mastication, swallowing and speech. The functional demands for accurate mandibular positioning and generation of forces during incising or a power stroke require a diverse set of forces that are determined by the innate muscle form. The complex internal tendon architecture subdivides the masseter into multiple partitions that can be further subdivided into neuromuscular compartments representing small motor unit territories. Individual masseter compartments have unique biomechanical properties that, when activated individually or in groups, can generate a wide range of sagittal and off-sagittal torques about the temporomandibular joint. The myosin heavy chain (MyHC) fiber-type distribution in the adult masseter is sexually dimorphic and is influenced by hormones such as testosterone. These testosterone-dependent changes cause a phenotype switch from slower to faster fiber-types in the male. The development of the complex organization of the masseter muscle, the MyHC fiber-type message and protein expression, and the formation of endplates appear to be pre-programmed and not under control of the muscle nerve. However, secondary myotube generation and endplate maturation are nerve dependent. The delayed development of the masseter muscle compared to the facial, tongue and jaw opening muscles may be related to the delayed functional requirements for chewing. In summary, masseter muscle form is pre-programmed prior to birth while muscle fiber contractile characteristics are refined postnatally in response to functional requirements. The motor control mechanisms that are required to coordinate the activation of discrete functional elements of this muscle remain to be determined.
doi:10.1016/j.archoralbio.2006.09.015
PMCID: PMC1861846  PMID: 17109812
masseter; masticatory muscle; tongue; compartments; development; myosin; endplate
2.  Development of Postural Muscles and Their Innervation 
Neural Plasticity  2005;12(2-3):141-151.
Control of posture is a prerequisite for efficient motor performance. Posture depends on muscles capable of enduring contractions, whereas movements often require quick, forceful muscle actions. To serve these different goals, muscles contain fibers that meet these different tasks. Muscles with strong postural functions mainly consist of slow muscle fibers with a great resistance against fatigue. Flexor muscles in the leg and arm muscles are mainly composed of fast muscle fibers producing relatively large forces that are rapidly fatigable. Development of the neuromuscular system continues after birth. We discuss in the human baby and in animal experiments changes in muscle fiber properties, regression from polyneural into mononeural innervation, and developmental changes in the motoneurons of postural muscles during that period. The regression of poly-neural innervation in postural muscles and the development of dendrite bundles of their motoneurons seem to be linked to the transition from the immature into the adult-like patterns of moving and postural control.
doi:10.1155/NP.2005.141
PMCID: PMC2565456  PMID: 16097482
3.  Functional Characterization of a Novel Family of Acetylcholine-Gated Chloride Channels in Schistosoma mansoni 
PLoS Pathogens  2014;10(6):e1004181.
Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the iodide flux assay used to characterize SmACC-1 represents a new high-throughput tool for drug screening against these unique parasite ion channels.
Author Summary
Schistosomiasis is a widespread, chronic disease affecting over 200 million people in developing countries. Currently, there is no vaccine available and treatment depends on the use of a single drug, praziquantel. Reports of reduced praziquantel efficacy, as well as its ineffectiveness against larval schistosomula highlight the need to develop new therapeutics. Interference with schistosome motor function provides a promising therapeutic target due to its importance in a variety of essential biological processes. The cholinergic system has been shown previously to be a major modulator of parasite motility. In this study, we have described a novel clade of schistosome acetylcholine-gated chloride channels (SmACCs) that act as inhibitory modulators of this pathway. Our results suggest that these receptors are absent in the human host and indirectly modulate inhibitory neuromuscular responses, making them an attractive drug-target. We have also validated a new functional assay to characterize these receptors, which may be modified for future use as a high-throughput drug screening method for parasite chloride channels.
doi:10.1371/journal.ppat.1004181
PMCID: PMC4055736  PMID: 24945827
4.  Dendritic Targeting in the Leg Neuropil of Drosophila: The Role of Midline Signalling Molecules in Generating a Myotopic Map 
PLoS Biology  2009;7(9):e1000199.
During development of the Drosophila motor system, global guidance cues control and coordinate the targeting of both input and output elements of the neural system.
Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. Within the embryonic nervous system of Drosophila motoneuron dendrites are organized topographically as a myotopic map that reflects their pattern of innervation in the muscle field. Here we reveal that this fundamental organizational principle exists in adult Drosophila, where the dendrites of leg motoneurons also generate a myotopic map. A single postembryonic neuroblast sequentially generates different leg motoneuron subtypes, starting with those innervating proximal targets and medial neuropil regions and producing progeny that innervate distal muscle targets and lateral neuropil later in the lineage. Thus the cellular distinctions in peripheral targets and central dendritic domains, which make up the myotopic map, are linked to the birth-order of these motoneurons. Our developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. We demonstrate that the medio-lateral positioning of motoneuron dendrites in the leg neuropil is controlled by the midline signalling systems Slit-Robo and Netrin-Fra. These results reveal that dendritic targeting plays a major role in the formation of myotopic maps and suggests that the coordinate spatial control of both pre- and postsynaptic elements by global neuropilar signals may be an important mechanism for establishing the specificity of synaptic connections.
Author Summary
During development the axons of sensory neurons generate highly ordered ”sensory maps„ within the nervous system that represent specific qualities of the environment. Much less is known about the anatomical organization and development of motor systems. Here, we show that the leg motoneurons of Drosophila organize their dendrites within the central nervous system in a way that reflects the position of the muscles they innervate. These motoneurons generate a ‘myotopic map’ by targeting the growth of their dendrites (sites of synaptic input) into discrete territories during development. The precise targeting of dendrites along the mediolateral axis is controlled by the signaling molecules Slit and Netrin, which are secreted by midline cells. These proteins act as global guidance cues and exert their effects via distinct signaling pathways using receptors called Roundabout and Frazzled, respectively. Previous studies have shown that Slit also helps to position the termini of axons (sites of synaptic output), independent of their synaptic partners. We suggest that the coordinated targeting of both input and output elements of a neural system into a common space using shared global guidance cues could be a simple way of establishing the specificity of synaptic connections within neural networks.
doi:10.1371/journal.pbio.1000199
PMCID: PMC2737123  PMID: 19771147
5.  A Conditioning Lesion Provides Selective Protection in a Rat Model of Amyotrophic Lateral Sclerosis 
PLoS ONE  2009;4(10):e7357.
Background
Amyotrophic Lateral Sclerosis (ALS) is neurodegenerative disease characterized by muscle weakness and atrophy due to progressive motoneuron loss. The death of motoneuron is preceded by the failure of neuromuscular junctions (NMJs) and axonal retraction. Thus, to develop an effective ALS therapy you must simultaneously preserve motoneuron somas, motor axons and NMJs. A conditioning lesion has the potential to accomplish this since it has been shown to enhance neuronal survival and recovery from trauma in a variety of contexts.
Methodology/Principal Findings
To test the effects of a conditioning lesion in a model of familial ALS we administered a tibial nerve crush injury to presymptomatic fALSG93A rats. We examined its effects on motor function, motoneuron somas, motor axons, and NMJs. Our experiments revealed a novel paradigm for the conditioning lesion effect. Specifically we found that the motor functional decline in fALSG93A rats that received a conditioning lesion was delayed and less severe. These improvements in motor function corresponded to greater motoneuron survival, reduced motor axonopathy, and enhanced NMJ maintenance at disease end-stage. Furthermore, the increased NMJ maintenance was selective for muscle compartments innervated by the most resilient (slow) motoneuron subtypes, but was absent in muscle compartments innervated by the most vulnerable (fast fatigable) motoneuron subtypes.
Conclusions/Significance
These findings support the development of strategies aimed at mimicking the conditioning lesion effect to treat ALS as well as underlined the importance of considering the heterogeneity of motoneuron sub-types when evaluating prospective ALS therapeutics.
doi:10.1371/journal.pone.0007357
PMCID: PMC2752158  PMID: 19806196
6.  Independent Passive Mechanical Behavior of Bovine Extraocular Muscle Compartments 
Purpose.
Intramuscular innervation of horizontal rectus extraocular muscles (EOMs) is segregated into superior and inferior (transverse) compartments, while all EOMs are also divided into global (GL) and orbital (OL) layers with scleral and pulley insertions, respectively. We sought evidence of potential independent action by examining passive mechanical coupling between EOM compartments.
Methods.
Putative compartments of each of the six whole bovine anatomical EOMs were separately clamped to a physiologically controlled, dual channel microtensile load cell (5-mN force resolution) driven by independent, high-speed, linear motors having 20-nm position resolution. One channel at a time was extended or retracted by 3 to 5 mm, with the other channel stationary. Fiducials distributed on the EOM global surface enabled optical tracking of local deformation. Loading rates of 5 to 100 mm/sec were applied to explore speeds from slow vergence to saccades. Control loadings employed transversely loaded EOM and isotropic latex.
Results.
All EOM bellies and tendons exhibited substantial compartmental independence when loaded in the physiologic direction, both between OL and GL, and for arbitrary transverse parsings of EOM width ranging from 60%:40% to 80%:20%. Intercompartmental force coupling in the physiologic direction was less than or equal to 10% in all six EOMS even for saccadic loading rates. Coupling was much higher for nonphysiologic transverse EOM loading and isotropic latex. Optical tracking demonstrated independent strain distribution between EOM compartments.
Conclusions.
Substantial mechanical independence exists among physiologically loaded fiber bundles in bovine EOMs and tendons, providing biomechanical support for the proposal that differential compartmental function in horizontal rectus EOMs contributes to novel torsional and vertical actions.
Dual-channel tensile loading demonstrates that adjacent extraocular muscle (EOMs) regions have marked mechanical independence. This finding supports the active pulley hypothesis and the proposal that topographic innervation within horizontal rectus EOMs could command torsional and vertical actions.
doi:10.1167/iovs.12-10318
PMCID: PMC4113332  PMID: 23188730
7.  Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle 
The Journal of Cell Biology  1995;130(4):959-968.
In innervated adult skeletal muscles, the Golgi apparatus (GA) displays a set of remarkable features in comparison with embryonic myotubes. We have previously shown by immunocytochemical techniques, that in adult innervated fibers, the GA is no longer associated with all the nuclei, but appears to be concentrated mostly in the subneural domain under the nerve endings in chick (Jasmin, B. J., J. Cartaud, M. Bornens, and J.- P. Changeux. 1989. Proc. Natl. Acad. Sci. USA. 86:7218-7222) and rat (Jasmin, B. J., C. Antony, J.-P. Changeux, and J. Cartaud. 1995. Eur. J. Neurosci. 7:470-479). In addition to such compartmentalization, biochemical modifications take place that suggest a functional specialization of the subsynaptic GA. Here, we focused on the developmental regulation of the membrane traffic organization during the early steps of synaptogenesis in mouse diaphragm muscle. We investigated by immunofluorescence microscopy on cryosections, the distribution of selected subcompartments of the exocytic pathway, and also of a representative endocytic subcompartment with respect to the junctional or extrajunctional domains of developing myofibers. We show that throughout development the RER, the intermediate compartment, and the prelysosomal compartment (mannose 6-phosphate receptor-rich compartment) are homogeneously distributed along the fibers, irrespective of the subneural or extrajunctional domains. In contrast, at embryonic day E17, thus 2-3 d after the onset of innervation, most GA markers become restricted to the subneural domain. Interestingly, some Golgi markers (e.g., alpha-mannosidase II, TGN 38, present in the embryonic myotubes) are no longer detected in the innervated fiber even in the subsynaptic GA. These data show that in innervated muscle fibers, the distal part of the biosynthetic pathway, i.e., the GA, is remodeled selectively shortly after the onset of innervation. As a consequence, in the innervated fiber, the GA exists both as an evenly distributed organelle with basic functions, and as a highly differentiated subsynaptic organelle ensuring maturation and targeting of synaptic proteins. Finally, in the adult, denervation of a hemidiaphragm causes a burst of reexpression of all Golgi markers in extrasynaptic domains of the fibers, hence showing that the particular organization of the secretory pathway is placed under nerve control.
PMCID: PMC2199963  PMID: 7642711
8.  Intramuscular Innervation of Primate Extraocular Muscles: Unique Compartmentalization in Horizontal Recti 
Segregation of intramuscular motor nerves indicates distinct superior and inferior zones within the horizontal but not vertical rectus extraocular muscles in humans and monkeys, supporting a potential functional role for differential innervation that might mediate oculorotary actions.
Purpose.
It has been proposed that the lateral rectus (LR), like many skeletal and craniofacial muscles, comprises multiple neuromuscular compartments subserving different physiological functions. To explore the anatomic potential of compartmentalization in all four rectus extraocular muscles (EOMs), evidence was sought of possible regional selectivity in intramuscular innervation of all rectus EOMs.
Methods.
Whole orbits of two humans and one macaque monkey were serially sectioned at 10 μm thickness and stained with Masson's trichrome. Three-dimensional reconstruction was performed of the intramuscular courses of motor nerves from the deep orbit to the anterior extents of their arborizations within all four rectus EOMs in each orbit.
Results.
Findings concorded in monkey and human orbits. Externally to the global surface of the lateral (LR) and medial rectus (MR) EOMs, motor nerve trunks bifurcated into approximately equal-sized branches before entering the global layer and observing a segregation of subsequent arborization into superior zones that exhibited minimal overlap along the length of the LR and only modest overlap for MR. In contrast, intramuscular branches of the superior and the nasal portion of the inferior rectus were highly mixed.
Conclusions.
Consistent segregation of intramuscular motor nerve arborization suggests functionally distinct superior and inferior zones within the horizontal rectus EOMs in both humans and monkeys. Reduced or absent compartmentalization in vertical rectus EOMs supports a potential functional role for differential innervation in horizontal rectus zones that could mediate previously unrecognized vertical oculorotary actions.
doi:10.1167/iovs.10-6651
PMCID: PMC3088565  PMID: 21220556
9.  Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae 
In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP’s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP’s and minature excitatory junction potentials (mEJP’s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP’s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.
doi:10.3791/1109
PMCID: PMC2762897  PMID: 19229189
10.  Striking Denervation of Neuromuscular Junctions without Lumbar Motoneuron Loss in Geriatric Mouse Muscle 
PLoS ONE  2011;6(12):e28090.
Reasons for the progressive age-related loss of skeletal muscle mass and function, namely sarcopenia, are complex. Few studies describe sarcopenia in mice, although this species is the mammalian model of choice for genetic intervention and development of pharmaceutical interventions for muscle degeneration. One factor, important to sarcopenia-associated neuromuscular change, is myofibre denervation. Here we describe the morphology of the neuromuscular compartment in young (3 month) compared to geriatric (29 month) old female C57Bl/6J mice. There was no significant difference in the size or number of motoneuron cell bodies at the lumbar level (L1–L5) of the spinal cord at 3 and 29 months. However, in geriatric mice, there was a striking increase (by ∼2.5 fold) in the percentage of fully denervated neuromuscular junctions (NMJs) and associated deterioration of Schwann cells in fast extensor digitorum longus (EDL), but not in slow soleus muscles. There were also distinct changes in myofibre composition of lower limb muscles (tibialis anterior (TA) and soleus) with a shift at 29 months to a faster phenotype in fast TA muscle and to a slower phenotype in slow soleus muscle. Overall, we demonstrate complex changes at the NMJ and muscle levels in geriatric mice that occur despite the maintenance of motoneuron cell bodies in the spinal cord. The challenge is to identify which components of the neuromuscular system are primarily responsible for the marked changes within the NMJ and muscle, in order to selectively target future interventions to reduce sarcopenia.
doi:10.1371/journal.pone.0028090
PMCID: PMC3229526  PMID: 22164231
11.  Correlated Electrophysiological and Ultrastructural Studies of a Crustacean Motor Unit 
The Journal of General Physiology  1972;59(5):586-615.
Structural and functional interrelationships between the pre- and postsynaptic elements of a singly motor innervated crab muscle (stretcher of Hyas araneus L.) were examined using electrophysiological and electron microscopic techniques. Excitatory postsynaptic potential (EPSP) amplitude at 1 Hz was found to be inversely related to the extent of facilitation, and directly related both to the amount of transmitter released at 1 Hz and the muscle fiber input resistance (Rin). The extent of facilitation (Fe), taken as the ratio of the EPSP amplitude at 10 Hz to that 1 Hz, was inversely related to muscle fiber Rin, τm, and sarcomere length. Sarcomere length was directly related to Rin and τm. The excitatory nerve terminals of low Fe muscle fibers had larger neuromuscular synapses than did those of high Fe fibers. Inhibitory axo-axonal synapses were more often found in low Fe muscle fibers. These structural features may account for the greater release of transmitter at low frequencies from the low Fe nerve terminals as well as provide for a greater amount of presynaptic inhibition of low Fe muscle fibers. The implications of these findings for the development and physiological performance of the crustacean motor unit are discussed. It is proposed that both nerve and muscle fiber properties may be determined by the developmental pattern of nerve growth.
PMCID: PMC2203193  PMID: 5027760
12.  A synaptic nidogen: Developmental regulation and role of nidogen-2 at the neuromuscular junction 
Neural Development  2008;3:24.
Background
The skeletal neuromuscular junction is a useful model for elucidating mechanisms that regulate synaptogenesis. Developmentally important intercellular interactions at the neuromuscular junction are mediated by the synaptic portion of a basal lamina that completely ensheaths each muscle fiber. Basal laminas in general are composed of four main types of glycosylated proteins: laminins, collagens IV, heparan sulfate proteoglycans and nidogens (entactins). The portion of the muscle fiber basal lamina that passes between the motor nerve terminal and postsynaptic membrane has been shown to bear distinct isoforms of the first three of these. For laminins and collagens IV, the proteins are deposited by the muscle; a synaptic proteoglycan, z-agrin, is deposited by the nerve. In each case, the synaptic isoform plays key roles in organizing the neuromuscular junction. Here, we analyze the fourth family, composed of nidogen-1 and -2.
Results
In adult muscle, nidogen-1 is present throughout muscle fiber basal lamina, while nidogen-2 is concentrated at synapses. Nidogen-2 is initially present throughout muscle basal lamina, but is lost from extrasynaptic regions during the first three postnatal weeks. Neuromuscular junctions in mutant mice lacking nidogen-2 appear normal at birth, but become topologically abnormal as they mature. Synaptic laminins, collagens IV and heparan sulfate proteoglycans persist in the absence of nidogen-2, suggesting the phenotype is not secondary to a general defect in the integrity of synaptic basal lamina. Further genetic studies suggest that synaptic localization of each of the four families of synaptic basal lamina components is independent of the other three.
Conclusion
All four core components of the basal lamina have synaptically enriched isoforms. Together, they form a highly specialized synaptic cleft material. Individually, they play distinct roles in the formation, maturation and maintenance of the neuromuscular junction.
doi:10.1186/1749-8104-3-24
PMCID: PMC2567315  PMID: 18817539
13.  Severe neuromuscular denervation of clinically relevant muscles in a mouse model of spinal muscular atrophy 
Human Molecular Genetics  2011;21(1):185-195.
Spinal muscular atrophy (SMA), a motoneuron disease caused by a deficiency of the survival of motor neuron (SMN) protein, is characterized by motoneuron loss and muscle weakness. It remains unclear whether widespread loss of neuromuscular junctions (NMJs) is involved in SMA pathogenesis. We undertook a systematic examination of NMJ innervation patterns in >20 muscles in the SMNΔ7 SMA mouse model. We found that severe denervation (<50% fully innervated endplates) occurs selectively in many vulnerable axial muscles and several appendicular muscles at the disease end stage. Since these vulnerable muscles were located throughout the body and were comprised of varying muscle fiber types, it is unlikely that muscle location or fiber type determines susceptibility to denervation. Furthermore, we found a similar extent of neurofilament accumulation at NMJs in both vulnerable and resistant muscles before the onset of denervation, suggesting that neurofilament accumulation does not predict subsequent NMJ denervation. Since vulnerable muscles were initially innervated, but later denervated, loss of innervation in SMA may be attributed to defects in synapse maintenance. Finally, we found that denervation was amendable by trichostatin A (TSA) treatment, which increased innervation in clinically relevant muscles in TSA-treated SMNΔ7 mice. Our findings suggest that neuromuscular denervation in vulnerable muscles is a widespread pathology in SMA, and can serve as a preparation for elucidating the biological basis of synapse loss, and for evaluating therapeutic efficacy.
doi:10.1093/hmg/ddr453
PMCID: PMC3235013  PMID: 21968514
14.  Compartmentalized Innervation of Primate Lateral Rectus Muscle 
Innervation to monkey and human lateral rectus muscles is segregated into well-defined superior and inferior zones, so that the lateral rectus may function as two parallel muscles under separate control. Differential activation of the two lateral rectus zones could impart previously unrecognized torsional and vertical actions to this nominally “horizontal” rectus muscle, potentially resolving an important paradox in ocular kinematics.
Purpose.
Skeletal and craniofacial muscles are frequently composed of multiple neuromuscular compartments that serve different physiological functions. Evidence of possible regional selectivity in LR intramuscular innervation was sought in a study of the anatomic potential of lateral rectus (LR) muscle compartmentalization.
Methods.
Whole orbits of two humans and five macaque monkeys were serially sectioned at 10-μm thickness and stained with Masson trichrome. The abducens nerve (CN6) was traced anteriorly from the deep orbit as it branched to enter the LR and arborized among extraocular muscle (EOM) fibers. Three-dimensional reconstruction was performed in human and monkey orbits.
Results.
Findings were in concordance in the monkey and human orbits. External to the LR global surface, CN6 bifurcated into approximately equal-sized trunks before entering the global layer. Subsequent arborization showed a systematic topography, entering a well-defined inferior zone 0.4 to 2.5 mm more posteriorly than branches entering the largely nonoverlapping superior zone. Zonal innervation remained segregated anteriorly and laterally within the LR.
Conclusions.
Consistent segregation of intramuscular CN6 arborization in humans and monkeys suggests functionally distinct superior and inferior zones for the LR. Since the LR is shaped as a broad vertical strap, segregated control of the two zones could activate them separately, potentially mediating previously unappreciated but substantial torsional and vertical oculorotary LR actions.
doi:10.1167/iovs.10-5330
PMCID: PMC2941164  PMID: 20435590
15.  NEUROMUSCULAR DETERMINANTS OF FORCE COORDINATION DURING MULTIDIGIT GRASPING 
The biomechanical structure of the hand and its underlying neurophysiology contribute to the coordination of the kinematics and kinetics necessary for multidigit grasping. We recently examined the neural organization of inputs to different extrinsic finger flexors during multi‐digit object hold and found moderate to strong motor unit short‐term synchrony. This suggests a common neural input to the motoneurons innervating these different hand muscles/muscle compartments, which may in turn influence the coordination of grip forces.
To further characterize this common input to the hand muscles during multidigit grasping, we used the frequency‐based measure of coherence. Motor unit coherence provides information with regards to the oscillatory frequency of a common input, as well as the coupling of the discharges of a motor unit pair at both short and long latencies.
Preliminary results indicate that a large proportion of trials are characterized by significant coherence in the 1–12 Hz frequency range, which is more pronounced in the within‐ than between‐muscle/muscle compartment analysis. This indicates a differential organization of common oscillatory inputs to pairs of motoneurons innervating the same vs. different muscles/ muscle compartments. The functional role of the 1–12 Hz oscillatory modulation of motor unit behavior is currently being investigated.
doi:10.1109/IEMBS.2004.1404287
PMCID: PMC2040330  PMID: 17271343
EMG; grasping; hand
16.  Abnormal development of the neuromuscular junction in Nedd4-deficient mice 
Developmental biology  2009;330(1):153-166.
Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5–E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.
doi:10.1016/j.ydbio.2009.03.023
PMCID: PMC2810636  PMID: 19345204
Neuromuscular junction; Mouse genetics; Motoneuron; Synaptogenesis; Mammalian development
17.  A connectivity model for the locomotor network of Caenorhabditis elegans 
Worm  2012;1(2):125-128.
Recently, we described a new method for representing and analyzing the connectivity of a motoneuronal network. We used it to deduce a connectivity model for the neuromuscular network that generates locomotion in the nematode Caenorhabditis elegans. The network regulates muscle contraction and for this reason we used the location or function of body wall muscles to map every element (neuron or muscle cell) in a new framework, namely the peri-motor space. The previously published connectivity data for C. elegans locomotion network are incomplete; in particular, the connectivity of motoneurons in the posterior half of the animal is missing or partial. When we analyzed the connectivity data for motoneurons in the anterior half, we detected repeating patterns which we named iterativity. We analyzed the iterativity of each class of motoneuron and statistically validated that it is higher than expected by chance. We could then extrapolate the iteration into the posterior half. Here we will explain the new terms and elaborate on the process of analysis and the features of the new connectivity model.
doi:10.4161/worm.19392
PMCID: PMC3670228  PMID: 24058836
Caenorhabditis elegans; locomotion; motoneurons; network connectivity; peri-motor
18.  Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular development even in the absence of failure in neuromuscular transmission or loss of motor neurons 
Developmental biology  2011;356(2):432-444.
A mouse model of the devastating human disease "spinal muscular atrophy" (SMA) was used to investigate the severe muscle weakness and spasticity that precedes the death of these animals near the end of the 2nd postnatal week. Counts of motor units to the soleus muscle as well as of axons in the soleus muscle nerve showed no loss of motor neurons. Similarly, neither immunostaining of neuromuscular junctions nor the measurement of the tension generated by nerve stimulation gave evidence of any significant impairment in neuromuscular transmission, even when animals were maintained up to 5 days longer via a supplementary diet. However, the muscles were clearly weaker, generating less than half their normal tension. Weakness in 3 muscles examined in the study appears due to a severe but uniform reduction in muscle fiber size. The size reduction results from a failure of muscle fibers to grow during early postnatal development and, in soleus, to a reduction in number of fibers generated. Neuromuscular development is severely delayed in these mutant animals: expression of myosin heavy chain isoforms, the elimination of polyneuronal innervation, the maturation in the shape of the AChR plaque, the arrival of SCs at the junctions and their coverage of the nerve terminal, the development of junctional folds. Thus, if SMA in this particular mouse is a disease of motor neurons, it can act in a manner that does not result in their death or disconnection from their targets but nonetheless alters many aspects of neuromuscular development.
doi:10.1016/j.ydbio.2011.05.667
PMCID: PMC3143211  PMID: 21658376
Spinal muscular atrophy; neuromuscular junction; Schwann cells; skeletal muscle development; mouse model
19.  Expanding Repertoire In The Oculomotor Periphery: Selective Compartmental Function In Rectus Extraocular Muscles 
Since connective tissue pulleys implement Listing's law by systematically changing rectus extraocular muscle (EOM) pulling directions, non-Listing's law gaze-dependence of the vestibulo-ocular reflex is currently inexplicable. Differential activation of compartments within rectus EOMs may endow the ocular motor system with more behavioral diversity than previously supposed. Innervation to horizontal, but not vertical, rectus EOMs of mammals is segregated into superior and inferior compartments. Magnetic resonance imaging in normal subjects demonstrates contractile changes in the lateral rectus (LR) inferior, but not superior, compartment during ocular counter-rolling (OCR) induced by head tilt. In human orbits ipsilesional to unilateral superior oblique palsy, neither LR compartment exhibits contractile change during head tilt, although the inferior compartment contracts normally in contralesional orbits. This suggests that differential compartmental LR contraction assists normal OCR. Computational simulation suggests that differential compartmental action in horizontal rectus EOMs could achieve more force than required by vertical fusional vergence.
doi:10.1111/j.1749-6632.2011.06112.x
PMCID: PMC3286355  PMID: 21950970
extraocular muscles; magnetic resonance imaging; motor nerve; pulleys; vestibulo-ocular reflex
20.  Alterations in muscle mass and contractile phenotype in response to unloading models: role of transcriptional/pretranslational mechanisms 
Skeletal muscle is the largest organ system in mammalian organisms providing postural control and movement patterns of varying intensity. Through evolution, skeletal muscle fibers have evolved into three phenotype clusters defined as a motor unit which consists of all muscle fibers innervated by a single motoneuron linking varying numbers of fibers of similar phenotype. This fundamental organization of the motor unit reflects the fact that there is a remarkable interdependence of gene regulation between the motoneurons and the muscle mainly via activity-dependent mechanisms. These fiber types can be classified via the primary type of myosin heavy chain (MHC) gene expressed in the motor unit. Four MHC gene encoded proteins have been identified in striated muscle: slow type I MHC and three fast MHC types, IIa, IIx, and IIb. These MHCs dictate the intrinsic contraction speed of the myofiber with the type I generating the slowest and IIb the fastest contractile speed. Over the last ~35 years, a large body of knowledge suggests that altered loading state cause both fiber atrophy/wasting and a slow to fast shift in the contractile phenotype in the target muscle(s). Hence, this review will examine findings from three different animal models of unloading: (1) space flight (SF), i.e., microgravity; (2) hindlimb suspension (HS), a procedure that chronically eliminates weight bearing of the lower limbs; and (3) spinal cord isolation (SI), a surgical procedure that eliminates neural activation of the motoneurons and associated muscles while maintaining neurotrophic motoneuron-muscle connectivity. The collective findings demonstrate: (1) all three models show a similar pattern of fiber atrophy with differences mainly in the magnitude and kinetics of alteration; (2) transcriptional/pretranslational processes play a major role in both the atrophy process and phenotype shifts; and (3) signaling pathways impacting these alterations appear to be similar in each of the models investigated.
doi:10.3389/fphys.2013.00284
PMCID: PMC3795307  PMID: 24130531
spaceflight; hindlimb suspension (unloading); spinal cord isolation; myosin isoforms; non-coding RNAs
21.  THE SPINAL NUCLEUS OF THE BULBOCAVERNOSUS: FIRSTS IN ANDROGEN-DEPENDENT NEURAL SEX DIFFERENCES 
Hormones and behavior  2007;53(5):596-612.
Cell number in the spinal nucleus of the bulbocavernosus (SNB) of rats was the first neural sex difference shown to differentiate under the control of androgens, acting via classical intracellular androgen receptors. SNB motoneurons reside in the lumbar spinal cord and innervate striated muscles involved in copulation, including the bulbocavernosus (BC) and levator ani (LA). SNB cells are much larger and more numerous in males than in females, and the BC/LA target muscles are reduced or absent in females. The relative simplicity of this neuromuscular system has allowed for considerable progress in pinpointing sites of hormone action, and identifying the cellular bases for androgenic effects. It is now clear that androgens act at virtually every level of the SNB system, in development and throughout adult life. In this review we focus on effects of androgens on developmental cell death of SNB motoneurons and BC/LA muscles; the establishment and maintenance of SNB motoneuron soma size and dendritic length; BC/LA muscle morphology and physiology; and behaviors controlled by the SNB system. We also describe new data on neurotherapeutic effects of androgens on SNB motoneurons after injury in adulthood.
doi:10.1016/j.yhbeh.2007.11.008
PMCID: PMC2423220  PMID: 18191128
Motoneuron; Bulbocavernosus; Levator ani; Androgen; Neuromuscular; Dendrite; Sexual differentiation; Cell death; Testosterone; Dihydrotestosterone
22.  A functional system for high-content screening of neuromuscular junctions in vitro 
Technology  2013;1(1):37-48.
High-content phenotypic screening systems are the logical extension of the current efficient, yet low information content, pre-clinical screens for drug discovery. A physiologically accurate in vitro neuromuscular junction (NMJ) screening system would therefore be of tremendous benefit to the study of peripheral neuropathies as well as for basic and applied neuromuscular research. To date, no fully-defined, selective assay system has been developed which would allow investigators to determine the functional output of cultured muscle fibers (myotubes) when stimulated via the NMJ in real time for both acute and chronic applications. Here we present the development of such a phenotypic screening model, along with evidence of NMJ formation and motoneuron initiated neuromuscular transmission in an automated system. Myotubes assembled on silicon cantilevers allowed for measurement of substrate deflection in response to contraction and provided the basis for monitoring the effect of controlled motoneuron stimulation on the contractile behavior. The effect was blocked by treatment with D-tubocurarine, confirming NMJ functionality in this highly multiplexed assay system.
doi:10.1142/S2339547813500015
PMCID: PMC4092002  PMID: 25019094
23.  Extraocular Muscles in Patients With Infantile Nystagmus 
Archives of ophthalmology  2012;130(3):343-349.
Objective
To test the hypothesis that the extraocular muscles (EOMs) of patients with infantile nystagmus have muscular and innervational adaptations that may have a role in the involuntary oscillations of the eyes.
Methods
Specimens of EOMs from 10 patients with infantile nystagmus and postmortem specimens from 10 control subjects were prepared for histologic examination. The following variables were quantified: mean myofiber cross-sectional area, myofiber central nucleation, myelinated nerve density, nerve fiber density, and neuromuscular junction density.
Results
In contrast to control EOMs, infantile nystagmus EOMs had significantly more centrally nucleated myofibers, consistent with cycles of degeneration and regeneration. The EOMs of patients with nystagmus also had a greater degree of heterogeneity in myofiber size than did those of controls, with no difference in mean myofiber cross-sectional area. Mean myelinated nerve density, nerve fiber density, and neuromuscular junction density were also significantly decreased in infantile nystagmus EOMs.
Conclusions
The EOMs of patients with infantile nystagmus displayed a distinct hypoinnervated phenotype. This represents the first quantification of changes in central nucleation and myofiber size heterogeneity, as well as decreased myelinated nerve, nerve fiber, and neuromuscular junction density. These results suggest that deficits in motor innervation are a potential basis for the primary loss of motor control.
Clinical Relevance
Improved understanding of the etiology of nystagmus may direct future diagnostic and treatment strategies.
doi:10.1001/archophthalmol.2011.381
PMCID: PMC3759680  PMID: 22411664
24.  Kv1.1 knock-in ataxic mice exhibit spontaneous myokymic activity exacerbated by fatigue, ischemia and low temperature 
Neurobiology of Disease  2012;47(3):310-321.
Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K+ channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1V408A/+). Here, we investigated the neuromuscular transmission of Kv1.1V408A/+ ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve–muscle from Kv1.1+/+ and Kv1.1V408A/+ mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca2 + signals that occurred abnormally only in preparations dissected from Kv1.1V408A/+ mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca2 + homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K+ channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult.
Highlights
► Kv1.1V408A/+ mice recapitulate the spontaneous and stress-induced neuromuscular hyper-excitability of EA1. ► The motor nerve is an important generator of myokymic activity. ► Dysfunction of Kv1.1 channels alters Ca2+ homeostasis in motor axons that likely contributes to myokymic activity ► Muscle fatigue contributes more than PNS fatigue to exacerbate myokymia/neuromyotonia. ► Juxtaparanodal K+ channels composed of Kv1.1 subunits tone down nerve excitability during fatigue or ischemic insult.
doi:10.1016/j.nbd.2012.05.002
PMCID: PMC3402927  PMID: 22609489
EA1, episodic ataxia type 1; Kv, voltage-gated potassium channel; LG, lateral gastrocnemius; EMG, electromyography; HFS, high frequency stimulation; CMAP, compound muscle action potential; GSL I, griffonia simplicifolia lectin I; 2P-LSM, two-photon laser scanning microscopy; Ataxia; Voltage-gated potassium channel; Kv1.1; KCNA1; Myokymia; Sciatic nerve; Stress; Fatigue; Ischemia; Ca2 + signals
25.  GDNF Secreting Human Neural Progenitor Cells Protect Dying Motor Neurons, but Not Their Projection to Muscle, in a Rat Model of Familial ALS 
PLoS ONE  2007;2(8):e689.
Background
Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by rapid loss of muscle control and eventual paralysis due to the death of large motor neurons in the brain and spinal cord. Growth factors such as glial cell line derived neurotrophic factor (GDNF) are known to protect motor neurons from damage in a range of models. However, penetrance through the blood brain barrier and delivery to the spinal cord remains a serious challenge. Although there may be a primary dysfunction in the motor neuron itself, there is also increasing evidence that excitotoxicity due to glial dysfunction plays a crucial role in disease progression. Clearly it would be of great interest if wild type glial cells could ameliorate motor neuron loss in these models, perhaps in combination with the release of growth factors such as GDNF.
Methodology/Principal Findings
Human neural progenitor cells can be expanded in culture for long periods and survive transplantation into the adult rodent central nervous system, in some cases making large numbers of GFAP positive astrocytes. They can also be genetically modified to release GDNF (hNPCGDNF) and thus act as long-term ‘mini pumps’ in specific regions of the rodent and primate brain. In the current study we genetically modified human neural stem cells to release GDNF and transplanted them into the spinal cord of rats over-expressing mutant SOD1 (SOD1G93A). Following unilateral transplantation into the spinal cord of SOD1G93A rats there was robust cellular migration into degenerating areas, efficient delivery of GDNF and remarkable preservation of motor neurons at early and end stages of the disease within chimeric regions. The progenitors retained immature markers, and those not secreting GDNF had no effect on motor neuron survival. Interestingly, this robust motor neuron survival was not accompanied by continued innervation of muscle end plates and thus resulted in no improvement in ipsilateral limb use.
Conclusions/Significance
The potential to maintain dying motor neurons by delivering GDNF using neural progenitor cells represents a novel and powerful treatment strategy for ALS. While this approach represents a unique way to prevent motor neuron loss, our data also suggest that additional strategies may also be required for maintenance of neuromuscular connections and full functional recovery. However, simply maintaining motor neurons in patients would be the first step of a therapeutic advance for this devastating and incurable disease, while future strategies focus on the maintenance of the neuromuscular junction.
doi:10.1371/journal.pone.0000689
PMCID: PMC1925150  PMID: 17668067

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