Levator Auris Longus Muscle Research Articles

Activation of TRPV1 Channels Inhibits the Release of Acetylcholine and Improves Muscle Contractility in Mice.

TRPV1 represents a non-selective transient receptor potential cation channel found not only in sensory neurons, but also in motor nerve endings and in skeletal muscle fibers. However, the role of TRPV1 in the functioning of the neuromuscular junction has not yet been fully established. In this study, the Levator Auris Longus muscle preparations were used to assess the effect of pharmacological activation of TRPV1 channels on neuromuscular transmission. The presence of TRPV1 channels in the nerve terminal and in the muscle fiber was confirmed by immunohistochemistry. It was verified by electrophysiology that the TRPV1 channel agonist capsaicin inhibits the acetylcholine release, and this effect was completely absent after preliminary application of the TRPV1 channel blocker SB 366791. Nerve stimulation revealed an increase of amplitude of isometric tetanic contractions upon application of capsaicin which was also eliminated after preliminary application of SB 366791. Similar data were obtained during direct muscle stimulation. Thus, pharmacological activation of TRPV1 channels affects the functioning of both the pre- and postsynaptic compartment of the neuromuscular junction. A moderate decrease in the amount of acetylcholine released from the motor nerve allows to maintain a reserve pool of the mediator to ensure a longer signal transmission process, and an increase in the force of muscle contraction, in its turn, also implies more effective physiological muscle activity in response to prolonged stimulation. This assumption is supported by the fact that when muscle was indirect stimulated with a fatigue protocol, muscle fatigue was attenuated in the presence of capsaicin.

Synaptic retrograde regulation of the PKA-induced SNAP-25 and Synapsin-1 phosphorylation

BackgroundBidirectional communication between presynaptic and postsynaptic components contribute to the homeostasis of the synapse. In the neuromuscular synapse, the arrival of the nerve impulse at the presynaptic terminal triggers the molecular mechanisms associated with ACh release, which can be retrogradely regulated by the resulting muscle contraction. This retrograde regulation, however, has been poorly studied. At the neuromuscular junction (NMJ), protein kinase A (PKA) enhances neurotransmitter release, and the phosphorylation of the molecules of the release machinery including synaptosomal associated protein of 25 kDa (SNAP-25) and Synapsin-1 could be involved.MethodsAccordingly, to study the effect of synaptic retrograde regulation of the PKA subunits and its activity, we stimulated the rat phrenic nerve (1 Hz, 30 min) resulting or not in contraction (abolished by µ-conotoxin GIIIB). Changes in protein levels and phosphorylation were detected by western blotting and cytosol/membrane translocation by subcellular fractionation. Synapsin-1 was localized in the levator auris longus (LAL) muscle by immunohistochemistry.ResultsHere we show that synaptic PKA Cβ subunit regulated by RIIβ or RIIα subunits controls activity-dependent phosphorylation of SNAP-25 and Synapsin-1, respectively. Muscle contraction retrogradely downregulates presynaptic activity-induced pSynapsin-1 S9 while that enhances pSNAP-25 T138. Both actions could coordinately contribute to decreasing the neurotransmitter release at the NMJ.ConclusionThis provides a molecular mechanism of the bidirectional communication between nerve terminals and muscle cells to balance the accurate process of ACh release, which could be important to characterize molecules as a therapy for neuromuscular diseases in which neuromuscular crosstalk is impaired.

Catecholamine-dependent hyperpolarization of the junctional membrane via β2- adrenoreceptor/Gi-protein/α2-Na-K-ATPase pathway

We investigated the effects of catecholamines, adrenaline and noradrenaline, as well as β-adrenoceptor (AR) modulators on a resting membrane potential at the junctional and extrajunctional regions of mouse fast-twitch Levator auris longus muscle. The aim of the study was to find which AR subtypes, signaling molecules and Na,K-ATPase isoforms are involved in the hyperpolarizing action of catecholamines and whether this action could be accompanied by changes in the pump abundance on the sarcolemma. Adrenaline, noradrenaline and specific β2-AR agonist induced hyperpolarization of both junctional and extrajunctional membrane, but the underlying mechanisms were different. In the junctional membrane the hyperpolarization depended on α2 isoform of the Na,K-ATPase and Gi-protein, whereas in the extrajunctional regions the hyperpolarization mainly relied on α1 isoform of Na,K-ATPase and adenylyl cyclase activities. In both junctional and extrajunctional regions, AR activation caused an increase in Na,K-ATPase abundance in the plasmalemma in a protein kinase A-dependent manner. Thus, the compartment-specific mechanisms are responsible for catecholamine-mediated hyperpolarization in the skeletal muscle.

Dry Needling Produces Mild Injuries Irrespective to Muscle Stiffness and Tension in Ex Vivo Mice Muscles.

Numerous studies have suggested that the myofascial trigger points are responsible for most of the myofascial pain syndrome, so it seems reasonable that its destruction is a good therapeutic solution. The effectiveness of dry needling (DN) has been confirmed in muscles with myofascial trigger points, hypertonicity, and spasticity. The objective of this study is to analyze the need of repetitive punctures on muscles in different situations. The levator auris longus (LAL) muscle and gastrocnemius muscle from adult male Swiss mice were dissected and maintained alive, while being submerged in an oxygenated Ringer's solution. DN was evaluated under four animal models, mimicking the human condition: normal healthy muscles, muscle fibers with contraction knots, muscles submerged in a depolarizing Ringer solution (KCl-CaCl2), and muscles submerged in Ringer solution with formalin. Thereafter, samples were evaluated with optical microscopy (LAL) and scanning electron microscopy (gastrocnemius). Healthy muscles allowed the penetration of needles between fibers with minimal injuries. In muscles with contraction knots, the needle separated many muscle fibers, and several others were injured, while blood vessels and intramuscular nerves were mostly not injured. Muscles submerged in a depolarizing solution inducing sustained contraction showed more injured muscular fibers and several muscle fibers separated by the needle. Finally, the muscles submerged in Ringer solution with formalin showed a few number of injured muscular fibers and abundant muscle fibers separated by the needle. Scanning electron microscopy images confirm the optical analyses. In summary, dry needling is a technique that causes mild injury irrespective of the muscle tone.

The Mouse Levator Auris Longus Muscle: An Amenable Model System to Study the Role of Postsynaptic Proteins to the Maintenance and Regeneration of the Neuromuscular Synapse.

The neuromuscular junction (NMJ) is the peripheral synapse that controls the coordinated movement of many organisms. The NMJ is also an archetypical model to study synaptic morphology and function. As the NMJ is the primary target of neuromuscular diseases and traumatic injuries, the establishment of suitable models to study the contribution of specific postsynaptic muscle-derived proteins on NMJ maintenance and regeneration is a permanent need. Considering the unique experimental advantages of the levator auris longus (LAL) muscle, here we present a method allowing for efficient electroporation-mediated gene transfer and subsequent detailed studies of the morphology and function of the NMJ and muscle fibers. Also, we have standardized efficient facial nerve injury protocols to analyze LAL muscle NMJ degeneration and regeneration. Our results show that the expression of a control fluorescent protein does not alter either the muscle structural organization, the apposition of the pre- and post-synaptic domains, or the functional neurotransmission parameters of the LAL muscle NMJs; in turn, the overexpression of MuSK, a major regulator of postsynaptic assembly, induces the formation of ectopic acetylcholine receptor clusters. Our NMJ denervation experiments showed complete reinnervation of LAL muscle NMJs four weeks after facial nerve injury. Together, these experimental strategies in the LAL muscle constitute effective methods to combine protein expression with accurate analyses at the levels of structure, function, and regeneration of the NMJ.

Tumor necrosis factor alpha mediates neuromuscular synapse elimination

During the development of mammalian neuromuscular junction (NMJ), the original supernumerary axon inputs are gradually eliminated, finally leaving each muscle fiber innervated by a single axon terminal. However, the molecular cues that mediate the elimination of redundant axon inputs remain unclear. Here we show that tumor necrosis factor-α (TNFα) expressed in postsynaptic muscle cells plays an important role in presynaptic axonal elimination at the NMJ. We found that intramuscular injection of TNFα into the levator auris longus (LAL) muscles caused disassociation of presynaptic nerve terminals from the postsynaptic acetylcholine receptor (AChR) clusters. By contrast, genetic ablation of TNFα globally or specifically in skeletal muscle cells, but not in motoneurons or Schwann cells, delayed the synaptic elimination. Moreover, ablation of TNFα in muscle cells attenuated the tendency of activity-dependent competition in a motoneuron–muscle coculture system. These results suggest a role of postsynaptic TNFα in the elimination of redundant synaptic inputs.

Dry needling in situations with varying degrees of muscle tone: an ex vivo study

Abstract Introduction Numerous studies support the fact that myofascial trigger points are responsible for the clinical findings in myofascial pain syndrome, therefore it is reasonable to believe that the destruction of the same via dry needling may be an optimal solution. However, this technique has not been assessed in relation to muscle tone. Methods The experiments were performed with the levator auris longus (LAL) muscle of Swiss adult mice. The muscles, once extracted from the animal, were maintained ex vivo using normal oxygenated Ringer solution. Once these were extracted, they were extended upon a Sylgard® surface, maintained in vivo at all times. In these conditions, 15 insertions were performed with dry needling needles (0.25 mm X 25mm; AguPunt) in the attempt to avoid repeating needling on the same site. Different degrees of muscle tone were obtained as follows (from lesser to greater tone): healthy animals; animal model of myofascial trigger points (PGM); muscles which, once extracted were treated with ClK 30 mM and ClCa2 5mM; formaldehyde. Immediately after treatment, the muscles were dyed with methylene blue. Results The muscle fibers of healthy muscles are mostly pushed aside by the needle, practically without injury. Muscles with MTrPs are more injured than healthy muscles because of the use of dry needling, albeit only partially. The tone acquired with ClK and ClCa2 is more powerful and generalized, therefore, dry needling creates a greater injury. Lastly, formaldehyde provokes a powerful increase of muscle tension, however the injury produced by dry needling is not so noteworthy, due to the fact that the muscle fibers fixed with formaldehyde are extremely resistant. Conclusions Dry needling is a technique which, overall, provokes limited lesions, independent of the muscle tone.

Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCβI and nPKCε) in Neuromuscular Developmental Synapse Elimination.

Background: During neuromuscular junction (NMJ) development, synapses are produced in excess. By sensing the activity-dependent release of ACh, adenosine, and neurotrophins, presynaptic receptors prompt axonal competition and loss of the unnecessary axons. The receptor action is mediated by synergistic and antagonistic relations when they couple to downstream kinases (mainly protein kinases A and C (PKA and PKC)), which phosphorylate targets involved in axonal disconnection. Here, we directly investigated the involvement of PKA subunits and PKC isoforms in synapse elimination. Methods: Selective PKA and PKC peptide modulators were applied daily to the Levator auris longus (LAL) muscle surface of P5–P8 transgenic B6.Cg-Tg (Thy1-YFP) 16 Jrs/J (and also C57BL/6J) mice, and the number of axons and the postsynaptic receptor cluster morphology were evaluated in P9 NMJ. Results: PKA (PKA-I and PKA-II isozymes) acts at the pre- and postsynaptic sites to delay both axonal elimination and nAChR cluster differentiation, PKC activity promotes both axonal loss (a cPKCβI and nPKCε isoform action), and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Moreover, PKC-induced changes in axon number indirectly influence postsynaptic maturation. Conclusions: PKC and PKA have opposed actions, which suggests that changes in the balance of these kinases may play a major role in the mechanism of developmental synapse elimination.

Experimental myofascial trigger point creation in rodents.

Myofascial pain syndrome is one of the most common forms of muscle pain. In this syndrome, pain is originated by the so-called trigger points, which consists of a set of palpable contraction knots in the muscle. It has been proposed that a high, spontaneous neurotransmission may be involved in the generation of these contraction knots. To confirm this hypothesis, we exposed mouse muscles to an anticholinesterasic agent to increase the neurotransmision in the synaptic cleft in two different conditions, in vivo and ex vivo experiments. Using intracellular recordings, a sharp increase in the spontaneous neurotransmission in the levator auris longus muscle and a lower increase in the diaphragm muscle could be seen. Likewise, electromyography recordings reveal an elevated endplate noise in gastrocnemius muscle of treated animals. These changes are associated with structural changes such as abundant neuromuscular contracted zones observed by rhodaminated α-bungarotoxin and the presence of abundant glycosaminoglycans around the contraction knots, as shown by Alcian PAS staining. In a second set of experiments, we aimed at demonstrating that the increases in the neurotransmission reproduced most of the clinical signs associated to a trigger point. We exposed rats to the anticholinesterase agent neostigmine, and 30 min afterward we observed the presence of palpable taut bands, the echocardiographic presence of contraction knots, and local twitch responses upon needle stimulation. In summary, we demonstrated that increased neurotransmission induced trigger points in both rats and mice, as evidenced by glycosaminoglycans around the contraction zones as a novel hallmark of this pathology. NEW & NOTEWORTHY In rodents, when neostigmine was injected subcutaneously, the neuromuscular neurotransmission increased, and several changes can be observed: an elevated endplate noise compared with normal endplate noise, as evidenced by electromyographyc recording; many muscular fibers with contraction knots (narrower sarcomeres and locally thickened muscle fiber) surrounded by infiltration of connective tissue like glycosaminoglycans molecules; and palpable taut bands and local twitch responses upon needle stimulation. Several of these signs are also observed in humans with muscle pain.

Postnatal Development and Distribution of Sympathetic Innervation in Mouse Skeletal Muscle.

Vertebrate neuromuscular junctions (NMJs) have been conceived as tripartite synapses composed of motor neuron, Schwann cell, and muscle fiber. Recent work has shown the presence of sympathetic neurons in the immediate vicinity of NMJs and experimental and clinical findings suggest that this plays an eminent role in adult NMJ biology. The present study examined the postnatal development and distribution of sympathetic innervation in different muscles using immunofluorescence, confocal microscopy, and Western blot. This demonstrates the proximity of sympathetic neurons in diaphragm, extensor digitorum longus, tibialis anterior, soleus, and levator auris longus muscles. In extensor digitorum longus muscle, sympathetic innervation of NMJs was quantified from perinatal to adult stage and found to increase up to two months of age. In diaphragm muscle, an extensive network of sympathetic neurons was prominent along the characteristic central synapse band. In summary, these data demonstrate that an elaborate sympathetic innervation is present in several mouse skeletal muscles and that this is often next to NMJs. Although the presence of sympathetic neurons at the perisynaptic region of NMJs increased during postnatal development, many synapses were already close to sympathetic neurons at birth. Potential implications of these findings for treatment of neuromuscular diseases are discussed.

The Levator Auris Longus (LAL) muscle as an accessible system to study the effects of Botulinum Toxins in vivo

The Levator Auris Longus (LAL) muscle as an accessible system to study the effects of Botulinum Toxins in vivo

Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions

Huntington's disease (HD) is a progressive and fatal degenerative disorder that results in debilitating cognitive and motor dysfunction. Most HD studies have focused on degeneration of the CNS. We previously discovered that skeletal muscle from transgenic R6/2 HD mice is hyperexcitable due to decreased chloride and potassium conductances. The progressive and early onset of these defects suggest a primary myopathy in HD. In this study, we examined the relationship between neuromuscular transmission and skeletal muscle hyperexcitability. We used an ex vivo preparation of the levator auris longus muscle from male and female late-stage R6/2 mice and age-matched wild-type controls. Immunostaining of the synapses and molecular analyses revealed no evidence of denervation. Physiologically, we recorded spontaneous miniature endplate currents (mEPCs) and nerve-evoked EPCs (eEPCs) under voltage-clamp, which, unlike current-clamp records, were independent of the changes in muscle membrane properties. We found a reduction in the number of vesicles released per action potential (quantal content) in R6/2 muscle, which analysis of eEPC variance and morphology indicate is caused by a reduction in the number of vesicle release sites (n) rather than a change in the probability of release (prel). Furthermore, analysis of high-frequency stimulation trains suggests an impairment in vesicle mobilization. The depressed neuromuscular transmission in R6/2 muscle may help compensate for the muscle hyperexcitability and contribute to motor impersistence.SIGNIFICANCE STATEMENT Recent evidence indicates that Huntington's disease (HD) is a multisystem disorder. Our examination of neuromuscular transmission in this study reveals defects in the motor nerve terminal that may compensate for the muscle hyperexcitability in HD. The technique we used eliminates the effects of the altered muscle membrane properties on synaptic currents and thus provides hitherto the most detailed analysis of synaptic transmission in HD. Clinically, the striking depression of neurotransmission we found may help explain the motor impersistence in HD patients. Therapies that target the highly accessible peripheral nerve and muscle system provide a promising new avenue to lessen the debilitating motor symptoms of HD.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M₁, M₂, and M₄ subtypes) and adenosine receptors (A₁ and A_2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A_2A, M₁, M₄, and A₁ receptors (in this order of ability) delayed axonal elimination at P7. M₄ produces some occlusion of the M₁ pathway and some addition to the A₁ pathway, which suggests that they cooperate. M₂ receptors may modulate (by allowing a permissive action) the other receptors, mainly M₄ and A₁. The continued action of these receptors (now including M₂ but not M₄) finally promotes axonal loss at P9. All 4 receptors (M₂, M₁, A₁, and A_2A, in this order of ability) are necessary. The M₄ receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M₁, A₁, and A_2A receptors, which show an additive effect, whereas the potent M₂ effect is largely independent of the other receptors (though can be modulated by M₄). At P9, there is a full mutual dependence between the A₁ and A_2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development.

The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M1-, M2- and M4-subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo. Our previous results show that M1, M2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M2 receptor is largely independent of both M1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.

Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

BackgroundThe development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses.ResultsHere we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses.ConclusionThe three receptor sets considered (mAChR, AR and TrkB receptors) intervene in modulating the conditions of the competition between nerve endings, possibly helping to determine the winner or the lossers but, thereafter, the final elimination would occur with some autonomy and independently of postsynaptic maturation.

Sod1 gene ablation in adult mice leads to physiological changes at the neuromuscular junction similar to changes that occur in old wild-type mice

Reactive oxygen species (ROS) are believed to be important mediators of muscle atrophy and weakness in aging and many degenerative conditions. However, the mechanisms and physiological processes specifically affected by elevated ROS in neuromuscular units that contribute to muscle weakness during aging are not well defined. Here we investigate the effects of chronic oxidative stress on neurotransmission and excitation–contraction (EC) coupling mechanisms in the levator auris longus (LAL) muscle from young (4–8 months) and old (22–28 months) wild-type mice and young adult Cu-Zn superoxide dismutase 1 knockout (Sod1−/−) mice. The frequency of spontaneous neurotransmitter release and the amplitude of evoked neurotransmitter release in young Sod1−/− and old wild-type LAL neuromuscular junctions were significantly reduced from the young wild-type values, and those declines were mirrored by decreases in synaptic vesicle pool size. Presynaptic cytosolic calcium concentration and mitochondrial calcium uptake amplitudes showed substantial increases in stimulated young Sod1−/− and old axon terminals. Surprisingly, LAL muscle fibers from old mice showed a greater excitability than fibers from either young wild-type or young Sod1−/− LAL. Both evoked excitatory junction potential (EJP) and spontaneous mini EJP amplitudes were considerably higher in LAL muscles from old mice than in fibers from young Sod1−/− LAL muscle. Despite a greater excitability, sarcoplasmic calcium influx in both old wild-type and young Sod1−/− LAL muscle fibers was significantly less. Sarcoplasmic reticulum calcium levels were also reduced in both old wild-type and young Sod1−/− mice, but the difference was not statistically significant in muscle fibers from old wild-type mice. The protein ratio of triad calcium channels RyR1/DHPR was not different in all groups. However, fibers from both young Sod1−/− and old mice had substantially elevated levels of protein carbonylation and S-nitrosylation modifications. Overall, our results suggest that young Sod1−/− recapitulate many neuromuscular and muscle fiber changes seen in old mice. We also conclude that muscle weakness in old mice might in part be driven by ROS-mediated EC uncoupling, while both EC uncoupling and reduced neurotransmitter release contribute to muscle weakness in Sod1−/− mice.

Chronic proximal axonopathy in rats is associated with long-standing neurofilament depletion in neuromuscular junctions and behavioral deficits.

In rodents exposed to 3,3'-iminodipropionitrile (IDPN), neurofilaments (NFs) accumulate in swollen proximal axon segments; this also occurs in motor neurons of patients with amyotrophic lateral sclerosis. We hypothesized that early loss of NFs in neuromuscular junctions (NMJs) in IDPN proximal neuropathy would result in neuromuscular dysfunction and lead to neuromuscular detachment. Adult male rats were given 0 or 15 mmol/L IDPN in drinking water for up to 1 year. The IDPN-exposed rats dragged their tails and had impaired endurance in a grip test. Neuromuscular junctions and distal axons were examined in the levator auris longus muscle after 3, 6, 9, and 12 months. Neuromuscular junctions showed a progressive reduction in NF immunolabeling, which became undetectable in up to 70% of the NMJs after 12 months. Neurofilament labeling was also reduced in preterminal axons and in a more proximal axon level within the muscle. Triple-label analysis with antisyntaxin demonstrated that the terminals remained in place and usually contained a few minute NF bundles. Electron microscopy revealed the disappearance of terminal NFs, reduced content in synaptic vesicles, and accumulation of multilamellar bodies, but scant degeneration. Thus, IDPN proximal neurofilamentous axonopathy is associated with NF depletion in motor terminals; motor weakness and structural changes in the NMJs suggest impaired synaptic function despite long-term preservation of the NMJs.

The interaction between tropomyosin-related kinase B receptors and serine kinases modulates acetylcholine release in adult neuromuscular junctions

We conducted an electrophysiological study of the functional link between the tropomyosin-related kinase B (trkB) receptor signaling mechanism and serine-threonine kinases, both protein kinase C (PKC) and protein kinase A (PKA). We describe their coordinated role in transmitter release at the neuromuscular junction (NMJ) of the Levator auris longus muscle of the adult mouse. The trkB receptor normally seems to be coupled to stimulate ACh release because inhibiting the trkB receptor with K-252a results in a significant reduction in the size of EPPs. We found that the intracellular PKC pathway can operate as in basal conditions (to potentiate ACh release) without the involvement of the trkB receptor function, although the trkB pathway needs an operative PKC pathway if it is to couple to the release mechanism and potentiate it. To actively stimulate PKA (which also results in ACh release potentiation), the operativity of trkB is a necessary condition, and one effect of trkB may be PKA stimulation.

Neuromuscular damage and repair after dry needling in mice.

Objective. Some dry needling treatments involve repetitive and rapid needle insertions into myofascial trigger points. This type of treatment causes muscle injury and can also damage nerve fibers. The aim of this study is to determine the injury caused by 15 repetitive punctures in the muscle and the intramuscular nerves in healthy mouse muscle and its ulterior regeneration. Methods. We repeatedly needled the levator auris longus muscle of mice, and then the muscles were processed with immunohistochemistry, methylene blue, and electron microscopy techniques. Results. Three hours after the dry needling procedure, the muscle fibers showed some signs of an inflammatory response, which progressed to greater intensity 24 hours after the procedure. Some inflammatory cells could still be seen when the muscle regeneration was almost complete seven days after the treatment. One day after the treatment, some changes in the distribution of receptors could be observed in the denervated postsynaptic component. Reinnervation was complete by the third day after the dry needling procedure. We also saw very fine axonal branches reinnervating all the postsynaptic components and some residual sprouts the same day. Conclusion. Repeated dry needling punctures in muscle do not perturb the different stages of muscle regeneration and reinnervation.

Exogenous ciliary neurotrophic factor (CNTF) reduces synaptic depression during repetitive stimulation

It has been shown that ciliary neurotrophic factor (CNTF) has trophic and maintenance effects on several types of peripheral and central neurons, glia, and cells outside the nervous system. Both CNTF and its receptor, CNTF-Rα, are expressed in the muscle. We use confocal immunocytochemistry to show that the trophic cytokine and its receptor are present in the pre- and post-synaptic sites of the neuromuscular junctions (NMJs). Applied CNTF (7.5-200 ng/ml, 60 min-3 h) does not acutely affect spontaneous potentials (size or frequency) or quantal content of the evoked acetylcholine release from post-natal (in weak or strong axonal inputs on dually innervated end plates or in the most mature singly innervated synapses at P6) or adult (P30) NMJ of Levator auris longus muscle of the mice. However, CNTF reduces roughly 50% the depression produced by repetitive stimulation (40 Hz, 2 min) on the adult NMJs. Our findings indicate that, unlike neurotrophins, exogenous CNTF does not acutely modulate transmitter release locally at the mammalian neuromuscular synapse but can protect mature end plates from activity-induced synaptic depression.