Dietary NMN supplementation enhances motor and NMJ function in ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that causes the degeneration of motor neurons in the motor cortex and spinal cord. Patients with ALS experience muscle weakness and atrophy in the limbs which eventually leads to paralysis and death. NAD+ is critical for energy metabolism, such as glycolysis and oxidative phosphorylation, but is also involved in non-metabolic cellular reactions. In the current study, we determined whether the supplementation of nicotinamide mononucleotide (NMN), an NAD+ precursor, in the diet had beneficial impacts on disease progression using a SOD1G93A mouse model of ALS. We found that the ALS mice fed with an NMN-supplemented diet (ALS+NMN mice) had modestly extended lifespan and exhibited delayed motor dysfunction. Using electrophysiology, we studied the effect of NMN on synaptic transmission at neuromuscular junctions (NMJs) in symptomatic of ALS mice (18 weeks old). ALS+NMN mice had larger end-plate potential (EPP) amplitudes and maintained better responses than ALS mice, and also had restored EPP facilitation. While quantal content was not affected by NMN, miniature EPP (mEPP) amplitude and frequency were elevated in ALS+NMN mice. NMN supplementation in diet also improved NMJ morphology, innervation, mitochondrial structure, and reduced reactive astrogliosis in the ventral horn of the lumbar spinal cord. Overall, our results indicate that dietary consumption of NMN can slow motor impairment, enhance NMJ function and improve healthspan of ALS mice.

Molecular and neural adaptations to neuromuscular electrical stimulation; Implications for ageing muscle.

One of the most notable effects of ageing is an accelerated decline of skeletal muscle mass and function, resulting in various undesirable outcomes such as falls, frailty, and all-cause mortality. The loss of muscle mass directly leads to functional deficits and can be explained by the combined effects of individual fibre atrophy and fibre loss. The gradual degradation of fibre atrophy is attributed to impaired muscle protein homeostasis, while muscle fibre loss is a result of denervation and motor unit (MU) remodelling. Neuromuscular electrical stimulation (NMES), a substitute for voluntary contractions, has been applied to reduce muscle mass and functional declines. However, the measurement of the effectiveness of NMES in terms of its mechanism of action on the peripheral motor nervous system and neuromuscular junction, and multiple molecular adaptations at the single fibre level is not well described. NMES mediates neuroplasticity and upregulates a number of neurotropic factors, manifested by increased axonal sprouting and newly formed neuromuscular junctions. Repeated involuntary contractions increase the activity levels of oxidative enzymes, increase fibre capillarisation and can influence fibre type conversion. Additionally, following NMES muscle protein synthesis is increased as well as functional capacity. This review will detail the neural, molecular, metabolic and functional adaptations to NMES in human and animal studies.

Physical Activity-Dependent Regulation of Parathyroid Hormone and Calcium-Phosphorous Metabolism.

Exercise perturbs homeostasis, alters the levels of circulating mediators and hormones, and increases the demand by skeletal muscles and other vital organs for energy substrates. Exercise also affects bone and mineral metabolism, particularly calcium and phosphate, both of which are essential for muscle contraction, neuromuscular signaling, biosynthesis of adenosine triphosphate (ATP), and other energy substrates. Parathyroid hormone (PTH) is involved in the regulation of calcium and phosphate homeostasis. Understanding the effects of exercise on PTH secretion is fundamental for appreciating how the body adapts to exercise. Altered PTH metabolism underlies hyperparathyroidism and hypoparathyroidism, the complications of which affect the organs involved in calcium and phosphorous metabolism (bone and kidney) and other body systems as well. Exercise affects PTH expression and secretion by altering the circulating levels of calcium and phosphate. In turn, PTH responds directly to exercise and exercise-induced myokines. Here, we review the main concepts of the regulation of PTH expression and secretion under physiological conditions, in acute and chronic exercise, and in relation to PTH-related disorders.

The Impact of Kinases in Amyotrophic Lateral Sclerosis at the Neuromuscular Synapse: Insights into BDNF/TrkB and PKC Signaling.

Brain-derived neurotrophic factor (BDNF) promotes neuron survival in adulthood in the central nervous system. In the peripheral nervous system, BDNF is a contraction-inducible protein that, through its binding to tropomyosin-related kinase B receptor (TrkB), contributes to the retrograde neuroprotective control done by muscles, which is necessary for motor neuron function. BDNF/TrkB triggers downstream presynaptic pathways, involving protein kinase C, essential for synaptic function and maintenance. Undeniably, this reciprocally regulated system exemplifies the tight communication between nerve terminals and myocytes to promote synaptic function and reveals a new view about the complementary and essential role of pre and postsynaptic interplay in keeping the synapse healthy and strong. This signaling at the neuromuscular junction (NMJ) could establish new intervention targets across neuromuscular diseases characterized by deficits in presynaptic activity and muscle contractility and by the interruption of the connection between nervous and muscular tissues, such as amyotrophic lateral sclerosis (ALS). Indeed, exercise and other therapies that modulate kinases are effective at delaying ALS progression, preserving NMJs and maintaining motor function to increase the life quality of patients. Altogether, we review synaptic activity modulation of the BDNF/TrkB/PKC signaling to sustain NMJ function, its and other kinases' disturbances in ALS and physical and molecular mechanisms to delay disease progression.

Hypothalamic Sirt1 protects terminal Schwann cells and neuromuscular junctions from age-related morphological changes.

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age-related changes. Aging effects on the NMJ components-motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)-have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild-type mice, all NMJ components showed age-associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain-specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic-specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age-related changes via sympathetic innervation.

High-Frequency Neuromuscular Electrical Stimulation Increases Anabolic Signaling.

PURPOSE: Neuromuscular electrical stimulation (NMES) is commonly used in rehabilitation settings to increase muscle mass and strength. However, the effects of NMES on muscle growth are not clear and no human studies have compared anabolic signaling between low-frequency (LF) and high-frequency (HF) NMES. The purpose of this study was to determine the skeletal muscle anabolic signaling response to an acute bout of LF- and HF-NMES. METHODS: Eleven young healthy volunteers (6 men, 5 women) received an acute bout of LF-NMES (20 Hz) and HF-NMES (60 Hz). Muscle biopsies were obtained from the vastus lateralis muscle before the first NMES treatment and 30 min after each NMES treatment. Phosphorylation of the following key anabolic signaling proteins was measured by Western blot, and proteins are expressed as a ratio of phosphorylated to total: mammalian target of rapamycin, p70-S6 kinase 1, and eukaryotic initiation factor 4E binding protein 1. RESULTS: Compared with pre-NMES, phosphorylation of mammalian target of rapamycin was upregulated 40.2% for LF-NMES (P = 0.018) and 68.4% for HF-NMES (P < 0.0001), and HF-NMES was 29.3% greater than LF-NMES (P = 0.026). Phosphorylation of p70-S6 kinase 1 after HF-NMES was 96.6% higher than pre-NMES (P = 0.001) and was not different between pre-NMES and LF-NMES (although it was 50.4% higher after LF-NMES) or LF- and HF-NMES (P > 0.05). There were no differences between treatment conditions for eukaryotic initiation factor 4E binding protein 1 phosphorylation (P > 0.05). CONCLUSIONS: An acute bout of LF- and HF-NMES upregulated anabolic signaling with HF-NMES producing a greater anabolic response compared with LF-NMES, suggesting that HF stimulation may provide a stronger stimulus for processes that initiate muscle hypertrophy. In addition, the stimulation frequency parameter should be considered by clinicians in the design of optimal NMES treatment protocols.

Mechanism of Rhinella icterica (Spix, 1824) toad poisoning using in vitro neurobiological preparations.

The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heart́s tissue and mice hippocampal slices. At chick biventer cervicis preparation, RITS (5, 10 and 20µg/mL) produced a concentration-independent irreversible neuromuscular blockade, which was preceded by a transitory increase of muscle twitch tension with the lowest concentration, in 120min recordings. In this set of experiments, RITS incubation partially prevented the curare neuromuscular blockade. The assessment of chick biventer cervicis muscle acetylcholinesterase (AChE) in the presence of RITS showed a significant inhibition of the enzyme, similarly to neostigmine. The incubation of muscles with digoxin or ouabain mimicked the poison activity by increasing the amplitude of the twitches followed by a progressive depression of the muscle strength. In addition, RITS demonstrated a digitalic-like activity, by inhibiting significantly the cardiac Na+, K+-ATPase. When the central nervous system was accessed, RITS induced an increase in the cell viability, in the lowest concentration. In addition, the poison protected slices subject to oxygen/glucose deprivation. Altogether, these data indicate that the poisonous extract of R. icterica is able to interfere with peripheral and central neurotransmission, probably due to a direct interaction with AChE, calcium channels and Na+, K+-ATPase. A further investigation upon the poison toxic components will unveil the components involved in such a pharmacological activity and the potential biotechnological application of this poison.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Neuromuscular Electrical Stimulation and Anabolic Signaling in Patients with Stroke.

INTRODUCTION: Stroke results in limited ability to produce voluntary muscle contraction and movement on one side of the body, leading to further muscle wasting and weakness. Neuromuscular electrical stimulation is often used to facilitate involuntary muscle contraction; however, the effect of neuromuscular electrical stimulation on muscle growth and strengthening processes in hemiparetic muscle is not clear. This study examined the skeletal muscle anabolic response of an acute bout of neuromuscular electrical stimulation in individuals with chronic stroke and healthy older adults. METHODS: Eleven individuals (59.8 ± 2.7 years old) were divided into a chronic stroke group (n = 5) and a healthy older adult control group (n = 6). Muscle biopsies were obtained before and after stimulation from the vastus lateralis of the hemiparetic leg for the stroke group and the right leg for the control group. The neuromuscular electrical stimulation protocol consisted of a 60-minute, intermittent stimulation train at 60 Hz. Phosphorylation of mammalian target of rapamycin and ribosomal protein S6 kinase beta-1 were analyzed by Western blot. FINDINGS: An acute bout of neuromuscular electrical stimulation increased phosphorylation of mammalian target of rapamycin (stroke: 56.0%; control: 51.4%; P = .002) and ribosomal protein S6 kinase beta-1 (stroke: 131.2%; control: 156.3%; P = .002) from resting levels to post-neuromuscular electrical stimulation treatment, respectively. Phosphorylated protein content was similar between stroke and control groups at both time points. CONCLUSION: Findings suggest that paretic muscles of patients with chronic stroke may maintain ability to stimulate protein synthesis machinery in response to neuromuscular electrical stimulation.

The Power of Human Protective Modifiers: PLS3 and CORO1C Unravel Impaired Endocytosis in Spinal Muscular Atrophy and Rescue SMA Phenotype.

Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

New insights into the acute actions from a high dosage of fluoxetine on neuronal and cardiac function: Drosophila, crayfish and rodent models.

The commonly used mood altering drug fluoxetine (Prozac) in humans has a low occurrence in reports of harmful effects from overdose; however, individuals with altered metabolism of the drug and accidental overdose have led to critical conditions and even death. We addressed direct actions of high concentrations on synaptic transmission at neuromuscular junctions (NMJs), neural properties, and cardiac function unrelated to fluoxetine's action as a selective 5-HT reuptake inhibitor. There appears to be action in blocking action potentials in crayfish axons, enhanced occurrences of spontaneous synaptic vesicle fusion events in the presynaptic terminals at NMJs of both Drosophila and crayfish. In rodent neurons, cytoplasmic Ca(2+) rises by fluoxetine and is thapsigargin dependent. The Drosophila larval heart showed a dose dependent effect in cardiac arrest. Acute paralytic behavior in crayfish occurred at a systemic concentration of 2mM. A high percentage of death as well as slowed development occurred in Drosophila larvae consuming food containing 100µM fluoxetine. The release of Ca(2+) from the endoplasmic reticulum in neurons and the cardiac tissue as well as blockage of voltage-gated Na(+) channels in neurons could explain the effects on the whole animal as well as the isolated tissues. The use of various animal models in demonstrating the potential mechanisms for the toxic effects with high doses of fluoxetine maybe beneficial for acute treatments in humans. Future studies in determining how fluoxetine is internalized in cells and if there are subtle effects of these mentioned mechanisms presented with chronic therapeutic doses are of general interest.

21 days of mammalian omega-3 fatty acid supplementation improves aspects of neuromuscular function and performance in male athletes compared to olive oil placebo.

BACKGROUND: Omega-3 polyunsaturated fatty acids (N-3) are essential nutrients for human health and integral components of neural tissues. There is evidence that N-3 supplementation may benefit exercise performance, however, no study has investigated the ergogenic potential of N-3 supplementation. Our objective was to determine the effect of short-term N-3 supplementation on neuromuscular-function and physical-performance in well-trained athletes. METHODS: Male athletes (n = 30), 25 years (SD 4.6), training 17 h(.)wk(-1) (SD 5) completed this randomized, placebo-controlled, parallel-design study. At baseline a blood sample was collected, maximal voluntary isometric contractions (MVC) with electromyography (EMG) recordings were measured, and participants underwent various performance tests including a Wingate test and 250 kJ time trial (TT) followed by repeated MVC and EMG measurement. Participants were then randomly assigned to receive N-3 (5 ml seal oil, 375 mg EPA, 230 mg DPA, 510 mg DHA) or placebo (5 ml olive oil) for 21-days after which baseline testing was repeated. The magnitude-based inference approach was used to estimate the probability that N-3 had a beneficial effect on neuromuscular-function and performance of at least ±1%. Data are shown as mean ± 90% confidence-interval. RESULTS: Plasma EPA was higher on N-3 than placebo (p = 0.004) but the increases in DPA and DHA were not significant (p = 0.087, p = 0.058). N-3 supplementation had an unclear effect on MVC force (4.1 ± 6.6%) but increased vastus lateralis EMG by 20 ± 18% vs placebo (very likely beneficial). N-3 supplementation reduced Wingate percent power drop by 4.76 ± 3.4 % vs placebo (very likely beneficial), but the difference in TT performance was unclear (-1.9 ± 4.8%). CONCLUSION: Our data indicates N-3 PUFA supplementation improved peripheral neuromuscular function and aspects of fatigue with an unclear effect on central neuromuscular function. Clinical trial registration NCT0201433.

Effects of electroacupuncture on recovery of the electrophysiological properties of the rabbit gastrocnemius after contusion: an in vivo animal study.

BACKGROUND: Our preliminary studies indicated that electroacupuncture (EA) at the ST36 and Ashi acupoints could promote regeneration of the rabbit gastrocnemius (GM) by improving microcirculation perfusion, promoting the recovery of myofiber structures, and inhibiting excessive fibrosis. However, the effects of EA on recovery of the electrophysiological properties of the GM after contusion are not yet clear. Thus, the purpose of this study was to investigate the effects of EA at the Zusanli (ST36) and Ashi acupoints with regard to recovery of the electrophysiological properties of the rabbit GM after contusion. METHODS: Forty-five rabbits were randomly divided into three groups: normal, contusion, and EA. After an acute GM contusion was produced (in rabbits in the contusion and EA groups), rabbits in the EA group were treated with electrostimulation at the ST36 and Ashi acupoints with 0.4 mA (2 Hz) for 15 min. The contusion group received no EA treatment. At different time points (7, 14, and 28 days) after contusion, we performed surface electromyography (EMG) and measured the nerve conduction velocity (NCV) of the GM and the GM branch of the tibial nerve. We also examined acetylcholinesterase (AchE) and Agrin expression in the neuromuscular junction (NMJ) via immunohistochemistry. RESULTS: Compared with the contusion group, the EMG amplitude and NCV in rabbits in the EA group were significantly higher at all time points after contusion. AchE and Agrin expression in the EA group were significantly higher than those in the contusion group. CONCLUSIONS: Our results showed that EA at the ST36 and Ashi acupoints effectively promoted recovery of the electrophysiological properties of the rabbit GM after contusion. The effects of EA were realized by promotion of the regeneration of myofibers and nerve fibers, as well as acceleration of NMJ reconstruction by upregulation of AchE and Agrin expression in the motor endplate area.

Protective effect of Sijunzi decoction on neuromuscular junction ultrastructure in autoimmune myasthenia gravis rats.

OBJECTIVE: To investigate the protective role of Sijunzi decoction in neuromuscular junction (NMJ) and muscle cell mitochondria ultrastructure; as well as its effects on the amount of adenosine triphosphate (ATP) and the activities of mitochondrial respiratory chain complexes I, II, III, and IV in autoimmune myasthenia gravis rats. METHODS: An experimental autoimmune myasthenia gravis (EAMG) rat model was established by inoculating rats with acetylcholine receptors extracted from Torpedo. Rats were divided into three groups: model, prednisone, and Sijunzi decoction, and were fed physiological saline, prednisone, or Sijunzi decoction, respectively. NMJ and muscle cell mitochondria ultrastructure were observed by transmission electron microscope. The amount of ATP was assessed by high performance liquid chromatography. The activities of mitochondrial respiratory chain complexes I, II, III, and IV was determined using the Clark oxygen electrode method. RESULTS: In the model group, there were sparse muscle fibers, with decreased mitochondria, and sparse, diffluent, or absent NMJ folds. After intervention with Sijunzi decoction, the above pathology changes were improved: muscle fiber structure was clear and complete; the mitochondria count was higher; and the NMJ structure was close to normal. Gastrocnemius muscle mitochondria in the model group produced significantly less ATP than those in the prednisone group (P < 0.01). Conversely, the ATP of Sijunzi decoction group was significantly higher than prednisone group (P < 0.01). The activities of gastrocnemius muscle mitochondrial respiratory chain complexes I, II, III, and IV in both the prednisone and Sijunzi decoction groups was dramatically higher compared with the model group (P < 0.05). The activities of complexes I and III in the Sijunzi decoction group were significantly higher than those in the prednisone group (P < 0.05), but there was no obvious difference in complex II or IV activities between the two groups (P > 0.05). CONCLUSION: Sijunzi decoction improved pathological changes in muscle mitochondria and NMJ, enhanced the amount of ATP in gastrocnemius muscle mitochondria, and improved the activities of respiratory chain complexes I, II, III, and IV (especially I and III) of the EAMG rats.

RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction.

Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca(2+) channels and proteins that influence Ca(2+) channel accumulation at release sites. Here we identify Drosophila RIM (Rab3 interacting molecule) and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission because of a significant reduction in both the clustering of Ca(2+) channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Because RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild-type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca(2+) channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca(2+) channel accumulation but is not a Rab3 effector for release machinery distribution across release sites.

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects.

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freeze-thaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A(2) were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 ± 34.7% of control twitch height, which were blocked with either 100 µ m of phentolamine or 1 m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-N(G) -Nitroarginine methyl ester (0.5 m m) or 1 m m of lidocaine. PHY-N (1, 3 and 10 µg ml(-1) ) induced an increase in tonus of the biventer-cervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 µ m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release.

Low-intensity electrical stimulation ameliorates disruption of transverse tubules and neuromuscular junctional architecture in denervated rat skeletal muscle fibers.

We determine the effects of direct electrical stimulation (ES) on the histological profiles in atrophied skeletal muscle fibers after denervation caused by nerve freezing. Direct ES was performed on the tibialis anterior (TA) muscle after denervation in 7-week-old rats divided into groups as follows: control (CON), denervation (DN), or denervation with direct ES (subdivided into a 4 mA (ES4), an 8 mA (ES8), or a 16 mA stimulus (ES16). The stimulation frequency was set at 10 Hz, and the voltage was set at 40 V (30 min/day, 6 days/week, for 3 weeks). Ultrastructural profiles of the membrane systems involved in excitation-contraction coupling, and four kinds of mRNA expression profiles were evaluated. Morphological disruptions occurred in transverse (t)-tubule networks following denervation: an apparent disruption of the transverse networks, and an increase in the longitudinal t-tubules spanning the gap between the two transverse networks, with the appearance of pentads and heptads. These membrane disruptions seemed to be ameliorated by relatively low intensity ES (4 mA and 8 mA), and the area of longitudinally oriented t-tubules and the number of pentads and heptads decreased significantly (P < 0.01) in ES4 and ES8 compared to the DN. The highest intensity (16 mA) did not improve the disruption of membrane systems. There were no significant differences in the (alpha1s)DHPR and RyR1 mRNA expression among CON, DN, and all ES groups. After 3 weeks of denervation all nerve terminals had disappeared from the neuromuscular junctions (NMJs) in the CON and ES16 groups. However, in the ES4 and ES8 groups, modified nerve terminals were seen in the NMJs. The relatively low-intensity ES ameliorates disruption of membrane system architecture in denervated skeletal muscle fibers, but that it is necessary to select the optimal stimulus intensities to preserve the structural integrity of denervated muscle fibers.

Purinergic and nitrergic junction potential in the human colon.

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 microM). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 microM), MRS 2179 (1 microM), and NF023 (10 microM). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).

Neurotization improves contractile forces of tissue-engineered skeletal muscle.

Engineered functional skeletal muscle would be beneficial in reconstructive surgery. Our previous work successfully generated 3-dimensional vascularized skeletal muscle in vivo. Because neural signals direct muscle maturation, we hypothesized that neurotization of these constructs would increase their contractile force. Additionally, should neuromuscular junctions (NMJs) develop, indirect stimulation (via the nerve) would be possible, allowing for directed control. Rat myoblasts were cultured, suspended in fibrin gel, and implanted within silicone chambers around the femoral vessels and transected femoral nerve of syngeneic rats for 4 weeks. Neurotized constructs generated contractile forces 5 times as high as the non-neurotized controls. Indirect stimulation via the nerve elicited contractions of neurotized constructs. Curare administration ceased contraction in these constructs, providing physiologic evidence of NMJ formation. Histology demonstrated intact muscle fibers, and immunostaining positively identified NMJs. These results indicate that neurotization of engineered skeletal muscle significantly increases force generation and causes NMJs to develop, allowing indirect muscle stimulation.