Neuromuscular Systems in Molluscs

Molluscs have become increasingly popular in the study of centralneural mechanisms. More recently, there have been attempts torelate activity in central neurons with behavior in animalsof this phylum. The latter studies necessitate an understandingof the effectors of such behaviors. This requires not only informationabout the neuromuscular junction, but also an awareness of thecapabilities of the muscles themselves. Therefore, we have discussedsome structural and related functional characteristics of molluscanmuscle. We suggest that invertebrate mucles might be comparedon three scales: the amount of myofilament organization, theamount of vesicular specialization and organization, and theamount of paramyosin. We have considered some characteristicsof the widely-studied sustained contraction, known as "catch."Finally, we have discussed the neuromuscular junction—thetypes of junctions, the multiplicity of innervation, and someaspects of pharmacology. The results of such a study indicatedmany areas in which further research is essential before wecan understand behavior in terms of activity in the centralnervous system.     

Excitatory Transmission in Insect Neuromuscular Systems

Many, but not all, visceral muscles in insects are innervatedby neurosecretory axons. The neurosecretory junctions with theheart muscle of the American cockroach, Periplaneta americana,show ultrastructural and electrophysiological evidence of chemicallytransmitting synapses, and cytochemical evidence for the presenceof monoamines. Electron microscopy of nerve terminals showsthat synaptic vesicles may be formed directly from electron-dense"neurosecretory" granules Neurotomy of motor axons to skeletal muscles in insects leadsto aggregation and clumping of synaptic vesicles after 48 hours.Treatment of in vitro nerve-muscle preparations with variousrespiratory poisons caused aggregation similar to that developedin neurotomized animals. This suggested that vesicle aggregationin both cases may have resulted from a decrease in availableadenosine triphosphate in the nerve terminal with subsequentalteration in the normal charge density which supports a repulsiveforce between the vesicles.     

Neuromuscular Systems in Neurogenic Arthropod Hearts

Neuromuscular transmission has been studied in detail by variousauthors in neurogenic hearts of decapod and stomatopod crustaceans,horseshoe crabs, and spiders. In these hearts, bursts of impulsesgenerated in the cardiac ganglion at regular intervals producedepolarizations of the muscle fibers. Each depolarization isassociated with a heart contraction. The depolarization is composedof many excitatory junction potentials (ejp's), each producedby a single nerve impulse. There is no evidence in Homarus,Squilla, or Limulus hearts that single ejp's or composites ofejp's give rise to regenerative membrane responses; in thesehearts, spontaneous depolarizations never overshoot the zeroreference level. Overshooting occurs in certain crab and crayfishhearts, and it is possible that muscle fibers of these heartsproduce regenerative membrane events. The muscle fibers of Limulus, Tachypleus and Homarus heartsare polyneuronally innervated. Pulse stimuli applied to nerve branches evoke ejp's that facilitatein hearts of Squilla and Homarus. In addition to facilitationin Homarus, there is also depression; at certain frequenciesof stimulation both facilitation and depression can be observed.Experiments in tarantula, Limulus, and Homarus hearts show thatL-glutamic acid mimics the natural transmitter substance.     

Remodelling of Neuromuscular Systems During Insect Metamorphosis

During metamorphosis in the hawkmoth, Manduca sexta, the larvalthoracic legs are replaced by a new set of adult legs that includenew sensory neurons and muscles, and participate in new patternsof locomotor activity. Larval leg motoneurons persist to innervatethe new adult leg muscles, but undergo striking changes in dendriticmorphology that are regulated by the insect steroid, 20-hydroxyecdysone.In the periphery, the motor terminals regress as larval musclesdegenerate, and expand as new adult muscles form from myoblasts.Evidence obtained both in vivo and in vitro suggests that theproliferation of myoblasts during metamorphosis is dependentupon innervation.     

Ultrastructural Evidence for Neuromuscular Systems in Coelenterates

Cellular interrelationships and synaptic connections in tentaclesof several species of coelenterates were examined by means ofelectron microscopy to determine if neuromuscular pathways werepresent. The presence of sensory cells, ganglion cells, epitheliomuscularcells, interneuronal synapses, and neuromuscular junctions suggeststhat neuromuscular pathways are present in coelenterates. Nakedaxons without sheath cells form several synapses en passantwith the same and with different epitheliomuscular cells aswell as with nematocytes and other neurons. Interneuronal synapsesand neuromuscular and neuronematocyte junctions have clear ordense-cored vesicles (700–1500 Å in diameter) associatedwith a dense cytoplasmic coat on the presynaptic membrane, acleft (100–300 Å in width) with intracleft filaments,and a subsynaptic membrane with a dense cytoplasmic coat. Atscyphozoan neuromuscular junctions there is a subsurface cisternaof endoplasmic reticulum, which is separated from the epitheliomuscularcell membrane by a narrow cytoplasmic gap (100–300 Åin width) . Neuromuscular junctions in coelenterates resembleen passant axonal junctions with smooth muscle in higher animals. Morphological evidence is presented for a simple reflex involvinga two-cell (sensory or ganglion-epitheliomuscular cell) or three-cell(sensory-ganglion-epitheliomuscular cell) pathway that may resultin the coordinated contraction of the longitudinal muscle intentacles of coelenterates.     

Degeneration and Regeneration in Crustacean Neuromuscular Systems

Crayfish motor neurons seem to repair damage to peripheral axonsby selective fusion of outgrowing proximal stumps with severeddistal processes that can survive morphologically and physiologicallyintact for over 200 days. Survival of isolated motor and CNSgiant axons is associated with much hypertrophy of their glialsheath. The severed stumps of peripheral sensory neurons oftendegenerate within 21 days and their glial sheath does not hypertrophy.Denervation and immobilization produce relatively little changein the morphology and physiology of the opener muscle, whereastenotomy produces much atrophy within 30-60 days. Crayfish motor and CNS giant neurons show no capability forregenerating ablated cell bodies, whereas peripheral sensorysomata regenerate after limb autotomy. An entire opener musclecan be replaced after limb autotomy but the organism shows littleor no ability to redifferentiate an entire muscle in the absenceof body part regeneration. However, a few opener muscle fiberscan be regenerated if the bulk of the muscle mass remains intact.The significance of all these findings are interpreted withrespect to the developmental capabilities and environmentaladaptations of the crayfish together with the evolution of regenerativeabilities in anthropods and vertebrates.     

The Phylogeny of the Extant Chelicerate Orders

The phylogeny of the extant chelicerate orders is examined in the light of morphological and molecular evidence. Representatives from each of the chelicerate ‘orders’ and mandibulate and onychophoran outgroups are examined. Molecular (small and large ribosomal subunit DNA) and morphological information is combined in a total evidence regime to determine the most consistent picture of extant chelicerate relationships for these data. Multiple phylogenetic analyses are performed with variable analysis parameters yielding largely consistent results. A normalized incongruence length metric is used to assay the relative merit of the multiple analyses. The combined analysis with lowest character incongruence yields the scheme of relationships (Pycnogonida+ (Xiphosura+((Opiliones+((Solifugae+Pseudoscorpiones)+Scorpiones))+((Ricinulei+Acari)+(Palpigradi+ ((Thelyphonida+Schizomida=Uropygi)+(Amblypygi+ Araneae))))))). This result is fairly robust to variation in analysis parameters, with the placement of solifugids and the status of the pedipalps responsible for most disagreement.     

Morphine impairs Acetylcholine Release but facilitates Acetylcholine Action at a Skeletal Neuromuscular Junction

THERE is considerable evidence that morphine impairs the release of acetylcholine (ACh) at cholinergic synapses in the brain^1–5, although there are considerable problems in determining the exact site and mechanism of this action. A simple synaptic model would be useful for pursuing this problem and the question arises whether this action of morphine is universal for cholinergic synapses or is restricted to particular sites. Morphine impairs the release of ACh at peripheral muscarinic sites^6–8 but there are no reports about the effects of morphine on ACh release at nicotinic neuromuscular sites. We have reported that both morphine and nalorphine block neuromuscular transmission in amphibian and mammalian skeletal neuromuscular preparations^9,10, apparently as a result of impairment of ACh release. We have now determined by direct measurement that morphine impairs ACh release at a skeletal neuromuscular junction.     

Wnt Signaling in Skeletal Muscle Dynamics: Myogenesis,Neuromuscular Synapse and Fibrosis

The signaling pathways activated by Wnt ligands are related to a wide range of critical cell functions, such as cell division, migration, and synaptogenesis. Here, we summarize compelling evidence on the role of Wnt signaling on several features of skeletal muscle physiology. We briefly review the role of Wnt pathways on the formation of muscle fibers during prenatal and postnatal myogenesis, highlighting its role on the activation of stem cells of the adult muscles. We also discuss how Wnt signaling regulates the precise formation of neuromuscular synapses, by modulating the differentiation of presynaptic and postsynaptic components, particularly regarding the clustering of acetylcholine receptors on the muscle membrane. In addition, based on previous evidence showing that Wnt pathways are linked to several diseases, such as Alzheimer's and cancer, we address recent studies indicating that Wnt signaling plays a key role in skeletal muscle fibrosis, a disease characterized by an increase in the extracellular matrix components leading to failure in muscle regeneration, tissue disorganization and loss of muscle activity. In this context, we also discuss the possible cross-talk between the Wnt/β-catenin pathway with two other critical profibrotic pathways, transforming growth factor β and connective tissue growth factor, which are potent stimulators of the accumulation of connective tissue, an effect characteristic of the fibrotic condition. As it has emerged in other pathological conditions, we suggests that muscle fibrosis may be a consequence of alterations of Wnt signaling activity.     

Cellular Interactions in the Development of Annelid Neuromuscular Systems

The ontogeny of muscles, motor neurons, and the central circuitryinvolved in producing patterned motor outputs is often thoughtof as a series of relatively independent and stereotyped events.Although mature neuromuscular systems are indeed often highlystereotyped, there is mounting evidence that the developmentalmechanisms that give rise to such stereotypy may often be interactive.The medicinal leech, Hirudo medicinalis, has stereotyped neuronsand muscles, yet at least some of the neuromuscular componentsseem to depend upon a particular sequence of cell-cell interactionsto differentiate, express identifiable phenotypes, or selectsynaptic partners. Three examples are summarized to illustratethese possibilities. First, a pattern-forming cell (the C-cell) develops at an earlystage and projects parallel processes that are used as a scaffoldupon which myocytes assemble. In the absence of the C-cell,oblique muscle fascicles never become organized. Second, atleast one way to match neuronal phenotype to that appropriatefor a segment-specific target is for homologous neurons to receivea signal from that target locally to partially respecify furtherdifferentiation of that neuron. Third, how do identified motorneurons select appropriate target muscles and how do interneuronalcircuits become matched to particular muscles when an interposedmotor neuron disallowsdirect interactions? A well-defined pattern-generatingsystem driving the muscular heart tubes in this leech is beginningto provide insights into these issues.     

Functional and Structural Parallels in Crustacean and Drosophila Neuromuscular Systems

Comparison of morphological and physiological phenotypes ofrepresentative crustacean motor neurons, and selected motorneurons of Drosophila larval abdominal muscles, shows severalfeatures in common. Crustacean motor nerve terminals, and thoseof Drosophila, possess numerous small synapses with well-definedactive zones. In crustaceans, neurons that are more tonicallyactive have markedly varicose terminals; synapses and mitochondriaare selectively localized in the varicosities. Phasic motoraxons have filiform terminals, sometimes with small varicosities;mitochondrial content is less than for tonic axons, and synapsesare distributed along the terminals. Tonic axons generate smallexcitatory potentials which facilitate strongly at higher frequencies,and which are resistant to depression. Thephasic neurons generatelarge excitatory potentials which exhibit relatively littlefrequency facilitation, and depress rapidly. In Drosophila,counterparts of crustacean phasic and tonic motor neurons havebeen found, but the differentiation is less pronounced. It isinferredthat cellular factors regulating the number of participatingsynapses and the probability of quantal release are similarin crustaceans and Drosophila, and that advantage can be takenof this in future to develop experiments addressing the regulationof synaptic plasticity.     

Staining OF Embryonic and Small Mammalian Skeletal Systems

The methods commonly used for staining developing bone (Lundvall, 1905; Spalteholz, 1914; Batson, 1921; Dawson, 1926; Richmond and Bennett, 1938; Cumley, Crow, and Griffin, 1939; Williams, 1941), have involved the use of 1% or 2% Aqueous KOH without employing any bleaching agent. The introduction of hydrogen peroxide into the technic proves to be a distinct advantage, and in the concentration here proposed, does not result in the production of fine bubbles. The maceration process can be hastened through the use of an increased concentration of potash in the absence of a fixative, although this in itself is one of the hazards of the technic. However, if the preparations are carefully watched, this difficulty may be largely avoided. The method works admirably and gives a very clear muscle tissue so that the skeletal elements are very sharply outlined. The proposed method (arranged in the form adopted in Staining Procedures) is herewith described.     

Chelicerate Hox genes and the homology of arthropod segments

Genes of the homeotic complex (HOM-C) in insects and vertebrates are required for the specification of segments along the antero-posterior axis. Multiple paralogues of the Hox genes in the horseshoe crab Limulus poliphemus have been used as evidence for HOM-C duplications in the Chelicerata. We addressed this possibility through a limited PCR survey to sample the homeoboxes of two spider species, Steatoda triangulosa and Achaearanea tepidariorum. The survey did not provide evidence for multiple Hox clusters although we have found apparent duplicate copies of proboscipedia ( pb ) and Deformed ( Dfd   ). In addition, we have cloned larger cDNA fragments of pb, zerknullt ( zen / Hox3 ) and Dfd. These fragments allowed the determination of mRNA distribution by in situ hybridization. Our results are similar to the previously published expression patterns of Hox genes from another spider and an oribatid mite. Previous studies compared spider/mite Hox gene expression patterns with those of insects and argued for a pattern of segmental homology based on the assumption that the co-linear anterior boundaries of the Hox domains can be used as markers. To test this assumption we performed a comparative analysis of the expression patterns for UBX/ABD-A in chelicerates, myriapods, crustaceans, and insects. We conclude that the anterior boundary can be and is changed considerably during arthropod evolution and, therefore, Hox expression patterns should not be used as the sole criterion for identifying homology in different classes of arthropods.     

Neuromuscular Electrical Stimulation as a Method to Maximize the Beneficial Effects of Muscle Stem Cells Transplanted into Dystrophic Skeletal Muscle

Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES) for 1 or 4 weeks following muscle-derived stem cell (MDSC) transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation) presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle.