Mepp Amplitude Research Articles

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.

Muscle BDNF: a potential therapeutic target for Kennedy's disease.

Muscle BDNF: a potential therapeutic target for Kennedy's disease.

Evaluation of the neuromuscular junction in a middle-aged mouse model of congenital myasthenic syndrome.

Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.

Restoration of Motor Defects Caused by Loss of Drosophila TDP-43 by Expression of the Voltage-Gated Calcium Channel, Cacophony, in Central Neurons.

Defects in the RNA-binding protein, TDP-43, are known to cause a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. A variety of experimental systems have shown that neurons are sensitive to TDP-43 expression levels, yet the specific functional defects resulting from TDP-43 dysregulation have not been well described. Using the Drosophila TDP-43 ortholog TBPH, we previously showed that TBPH-null animals display locomotion defects as third instar larvae. Furthermore, loss of TBPH caused a reduction in cacophony, a Type II voltage-gated calcium channel, expression and that genetically restoring cacophony in motor neurons in TBPH mutant animals was sufficient to rescue the locomotion defects. In the present study, we examined the relative contributions of neuromuscular junction physiology and the motor program to the locomotion defects and identified subsets of neurons that require cacophony expression to rescue the defects. At the neuromuscular junction, we showed mEPP amplitudes and frequency require TBPH. Cacophony expression in motor neurons rescued mEPP frequency but not mEPP amplitude. We also showed that TBPH mutants displayed reduced motor neuron bursting and coordination during crawling and restoring cacophony selectively in two pairs of cells located in the brain, the AVM001b/2b neurons, also rescued the locomotion and motor defects, but not the defects in neuromuscular junction physiology. These results suggest that the behavioral defects associated with loss of TBPH throughout the nervous system can be associated with defects in a small number of genes in a limited number of central neurons, rather than peripheral defects.SIGNIFICANCE STATEMENT TDP-43 dysfunction is a common feature in neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar dementia, and Alzheimer's disease. Loss- and gain-of-function models have shown that neurons are sensitive to TDP-43 expression levels, but the specific defects caused by TDP-43 loss of function have not been described in detail. A Drosophila loss-of-function model displays pronounced locomotion defects that can be reversed by restoring the expression levels of a voltage-gated calcium channel, cacophony. We show these defects can be rescued by expression of cacophony in motor neurons and by expression in two pairs of neurons in the brain. These data suggest that loss of TDP-43 can disrupt the central circuitry of the CNS, opening up identification of alternative therapeutic targets for TDP-43 proteinopathies.

P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction

It is known that adenosine 5′-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12–13 receptors, 2-methylthioadenosine 5′-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12–13 antagonists AR-C69931MX or 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5′-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.

Homocysteine aggravates ROS-induced depression of transmitter release from motor nerve terminals: potential mechanism of peripheral impairment in motor neuron diseases associated with hyperhomocysteinemia.

Homocysteine (HCY) is a pro-inflammatory sulphur-containing redox active endogenous amino acid, which concentration increases in neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). A widely held view suggests that HCY could contribute to neurodegeneration via promotion of oxidative stress. However, the action of HCY on motor nerve terminals has not been investigated so far. We previously reported that oxidative stress inhibited synaptic transmission at the neuromuscular junction, targeting primarily the motor nerve terminals. In the current study, we investigated the effect of HCY on oxidative stress-induced impairment of transmitter release at the mouse diaphragm muscle. The mild oxidant H2O2 decreased the intensity of spontaneous quantum release from nerve terminals (measured as the frequency of miniature endplate potentials, MEPPs) without changes in the amplitude of MEPPs, indicating a presynaptic effect. Pre-treatment with HCY for 2 h only slightly affected both amplitude and frequency of MEPPs but increased the inhibitory potency of H2O2 almost two fold. As HCY can activate certain subtypes of glutamate N-methyl D-aspartate (NMDA) receptors we tested the role of NMDA receptors in the sensitizing action of HCY. Remarkably, the selective blocker of NMDA receptors, AP-5 completely removed the sensitizing effect of HCY on the H2O2-induced presynaptic depressant effect. Thus, at the mammalian neuromuscular junction HCY largely increases the inhibitory effect of oxidative stress on transmitter release, via NMDA receptors activation. This combined effect of HCY and local oxidative stress can specifically contribute to the damage of presynaptic terminals in neurodegenerative motoneuron diseases, including ALS.

Differential Effects of Mefloquine Enantiomers on a Cholinergic Synapse

Racemic mefloquine activates spontaneous vesicle release (SVR) from motor nerve endings and inhibits endplate acetylcholinesterase (AChE). Such cholinergic effects may reduce mefloquine's safety as an antimalarial drug. We tested the hypothesis that (+) and (‐) mefloquine differentially affect the neuromuscular junction (NMJ). Intracellular recordings at NMJs of mouse Triangularis sterni muscles suggested that 30 min exposure to 10 μM (+) or (‐) enantiomer alters the frequency of miniature endplate potentials (mEPPs). Quantitative comparisons were not possible since (+) reduced mEPP amplitude. Therefore, we recorded miniature endplate currents (mEPCs). (+) and (‐) significantly (p<0.01) increased mEPC frequency to 59.7 ± 20.05 Sec‐1 (mean ± SEM, N=21 NMJs) and 17.2 ± 1.98 Sec‐1 (N=14), respectively, from the control value of 5.6 ± 0.76 Sec‐1 (N=25). (+) also reduced amplitude (p<0.01) to 2.9 ± 0.18 nAmps (N=21) from the control value of 3.4 ± 0.18 nAmps (N=25); (‐) did not affect mEPC amplitude. (+), but not (‐), significantly (p<0.01) prolonged the endplate current decay time constant to 2.5 ± 0.16 mSec (N=12) from the control value of 1.1 ± 0.05 mSec (N=16). Thus, (+) may reduce conductance and prolong open time of the endplate acetylcholine receptor (AChR) as well as activate SVR. Neither (+) nor (‐) altered stimulus‐evoked quantal release. Assay of whole muscle homogenates revealed that while both enantiomers (1 to 100 μM) inhibit AChE, (‐) is more potent. The data suggest that (‐) is a less potent activator of SVR but a more potent inhibitor of AChE than (+) mefloquine. Supported by the Kirby Foundation.

Gender-Specific Mechanism of Synaptic Impairment and Its Prevention by GCSF in a Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motoneurons which progresses differentially in males and females for unknown reason. Here we measured gender differences in pre- and post-synaptic parameters of the neuromuscular transmission in a mutant G93A-SOD1 mouse model of ALS. Using intracellular microelectrode technique we recorded miniature and evoked end-plate potentials (MEPPs and EPPs) in the diaphragm muscle of G93A-SOD1 mice at early symptomatic stage. While no evident alterations in the amplitude of MEPPs was observed in male or female G93A-SOD1 mice, G93A-SOD1 mice displayed dramatically reduced probability of spontaneous acetylcholine release. In contrast, the EPPs evoked by single nerve stimulation had unchanged amplitude and quantal content. In males, but not females, this was accompanied by reduced readily releasable transmitter pool. Transmitter release in both sexes was sensitive to the inhibitory action of reactive oxygen species (ROS), but the production of ROS was increased in the spinal cords of male but not female G93A-SOD1 mice. Treatment with granulocyte colony stimulating factor (GCSF), which we previously found to be beneficial in males, attenuated the increased ROS production indicating involvement of the antioxidant mechanisms and improved ALS-induced synaptic dysfunctions only in males being ineffective in females. Consistent with our findings at synaptic level, GCSF did not change the survival rate or motor performance of female ALS mice. In summary, neuromuscular transmission in ALS mice is impaired at early symptomatic stage when a dramatic presynaptic decline of spontaneous release occurs. Beneficial effects of GCSF treatment on survival in males may be explained by GCSF-improved presynaptic functions in male G93A-SOD1 mice. Development of efficient treatment strategies for ALS may need to be directed in a gender-specific manner.

Influence of acute inspiratory loading upon diaphragm motor-evoked potentials in healthy humans

Acute prior activity of the inspiratory muscles can enhance inspiratory muscle strength and reduce effort perception during subsequent inspiratory efforts. However, the mechanisms subserving these changes are poorly understood. Responses to magnetic stimulation in 10 subjects were studied after an acute bout of nonfatiguing inspiratory muscle loading (IML), corresponding to 40% of subjects' initial maximal inspiratory pressure (MIP), and after an acute bout of nonloaded, forced inspiration (NLF). Motor-evoked potentials elicited by cortical stimulation (MEP(c)) and by phrenic nerve stimulation (MEP(p)) were recorded transcutaneously from the diaphragm before, immediately after, and 15 min after two sets of 30 inspiratory efforts, at rest and during an MIP effort. After IML, MIP increased to 113 +/- 3% (SE) of baseline and diaphragm MEP(p) (during MIP) significantly increased (129 +/- 10% of baseline). Diaphragmatic MEP(c) (during MIP), expressed as a percentage of maximal MEP(p), decreased after IML (from 29 +/- 9% to 20 +/- 6%; P = 0.017) and after NLF (from 43 +/- 5% to 31 +/- 5%; P = 0.032). Observations from the biceps brachi demonstrated that changes after IML and NLF were specific to the inspiratory muscle, since no significant changes were observed in biceps force generation or in MEP(p) or MEP(c) amplitudes. These data indicate that after IML increased global inspiratory strength is accompanied by increased peripheral excitability and by a dampening of corticospinal excitability of the diaphragm.

Shp2 Is Dispensable in the Formation and Maintenance of the Neuromuscular Junction

SHP2, a protein tyrosine phosphatase with two SH2 domains, has been implicated in regulating acetylcholine receptor (AChR) gene expression and cluster formation in cultured muscle cells. To understand the role of SHP2 in neuromuscular junction (NMJ) formation in vivo, we generated mus cle-specific deficient mice by using a loxP/Cre strategy since Shp2 null mutation causes embryonic lethality. Shp2^{floxed/floxed} mice were crossed with mice expressing the Cre gene under the control of the human skeletal α-actin (HSA) promoter. Expression of SHP2 was reduced or diminished specifically in skeletal muscles of the conditional knockout (CKO) mice. The mutant mice were viable and fertile, without apparent muscle defects. The mRNA of the AChR α subunit and AChR clusters in CKO mice were localized in a narrow central region surrounding the phrenic nerve primary branches, without apparent change in intensity. AChR clusters colocalized with markers of synaptic vesicles and Schwann cells, suggesting proper differentiation of presynaptic terminals and Schwann cells. In comparison with age-matched littermates, no apparent difference was observed in the size and length of AChR clusters in CKO mice. Both the frequency and amplitude of mEPPs in CKO mice were similar to those in controls, suggesting normal neurotransmission when SHP2 was deficient. These results suggest that Shp2 is not required for NMJ formation and/or maintenance.

Long‐term in vivo modulation of synaptic efficacy at the neuromuscular junction of Rana pipiens frogs

Prolonged changes in motor neurone activity can result in long-term changes in synaptic transmission. We investigated whether mechanisms commonly thought to be involved in determining synaptic efficacy of vertebrate motor neurones are involved in these long-term changes. The nerve supplying the cutaneous pectoris muscle was chronically stimulated via skin surface electrodes in freely moving frogs for 5-7 days. Chronic stimulation induced a 50% reduction in evoked endplate potential (EPP) amplitude at stimulated neuromuscular junctions (NMJs). These changes appear to be presynaptic since miniature EPP (mEPP) amplitude was unchanged while mEPP frequency was decreased by 46% and paired-pulse facilitation was increased by 26%. High frequency facilitation (40 Hz, 2 s) was also increased by 89%. Moreover, stimulated NMJs presented a 92% decrease in synaptic depression (40 Hz, 2 s). An increase in mitochondrial metabolism was observed as indicated by a more pronounced labelling of active mitochondria (Mitotracker) in stimulated nerve terminals, which could account for their greater resistance to synaptic depression. NMJ length visualized by alpha-bungarotoxin staining of nAChRs was not affected. Presynaptic calcium signals measured with Calcium Green-1 were larger in stimulated NMJs at low frequency (0.2 Hz) and not different from control NMJs at higher frequency (40 Hz, 2 s and 30 s). These results suggest that some mechanisms downstream of calcium entry are responsible for the determination of synaptic output, such as a down-regulation of some calcium-binding proteins, which could explain the observed results. The possibility of a change in frequenin expression, a calcium-binding protein that is more prominently expressed in phasic synapses, was, however, refuted by our results.

The effect of N‐ethylmaleimide on transmitter release from the skeletal neuromuscular junction of Bufo marinus

N-ethylmaleimide (NEM) has been used extensively in biochemical assays as an inhibitor of the NEM sensitive fusion protein (NSF). However, examination of the effect of NEM on transmitter release in more physiologically relevant preparations has proved inconclusive. In the present study, we have examined the effect of low concentrations of NEM on synaptic transmission in intact nerve-muscle preparations from toads (Bufo marinus). Under conditions of low transmitter release probability (0.3 mM calcium, 1 mM magnesium), treatment with NEM (10 microM) caused a significant increase in the amplitude of stimulus-evoked endplate potentials (EPPs) and a significant increase in the frequency of spontaneously occurring miniature EPPS (MEPPS) without affecting the amplitude of MEPPs. When the calcium concentration in the bath was raised to 4 mM, 10 microM NEM had no effect on EPP amplitude. Under these conditions, NEM treatment reduced paired pulse facilitation and increased depression during stimulus trains. Treatment with NEM also resulted in a significant decrease in the synaptic delay. The effects of NEM on transmitter release in the present study were not due to inactivation of G-proteins. The results of the present study show a calcium-dependent facilitation of stimulus-evoked transmitter release by NEM. These results are discussed in terms of the possible sites of NEM action leading to the observed changes in transmitter release.

Morphometric characterization of the neuromuscular junction of rodents intoxicated with 2,4-dithiobiuret: evidence that nerve terminal recycling processes contribute to muscle weakness

2,4-Dithiobiuret (DTB) causes ascending motor weakness when given chronically to rodents. In muscles of animals with DTB-induced weakness, quantal release of acetylcholine (ACh) is impaired. We examined in detail the structural changes that occurred at neuromuscular junctions and their associated Schwann cells of extensor digitorum longus (EDL) muscles of male rats treated with DTB to the onset of muscle weakness, 5–8 days. Our objective was to assess the involvement of the Schwann cells and to determine the most likely primary targets of DTB. At the onset of muscle weakness, nerve terminals exhibited some enlarged regions, but did not sprout. Terminal Schwann cells became flatter and expanded to cover most of the endplate. The extent of invasion of the synaptic cleft by Schwann cell processes was not significantly different from controls; extension of Schwann cell sprouts away from the junction was not seen. Thus, the morphology of the Schwann cells, although clearly affected by DTB, does not suggest that they contribute directly to the physiological defects of DTB-treated terminals. Abnormal tubulovesicular structures or tangles of neurofilaments were clustered in the centers of about 25% of treated terminals. Fewer synaptic vesicles occupied the region opposite the postsynaptic folds. Vesicle volumes were variable and included some very large vesicles, corresponding with the variable MEPP amplitudes reported previously for terminals of DTB-treated rodents. The postsynaptic area stained by rhodamine-labeled α-bungarotoxin expanded with terminal swelling, apparently by unpleating of the postsynaptic folds. No loss of ACh receptors or spread of ACh receptors beyond terminal boundaries was detected. Morphometric data are consistent with the conclusion that DTB affects, either directly or indirectly, vesicular release of ACh and the subsequent vesicular recycling process.

Increased quantal size in transmission at slow but not fast neuromuscular synapses of apolipoprotein E deficient mice

Uncertainties from the literature concerning the role of apolipoprotein E (apoE) in central cholinergic function prompted us to investigate what effect apoE may have on transmission at the neuromuscular junction. Both spontaneous and evoked release were measured in isolated extensor digitorum longus (edl) and soleus muscles from both wild-type and apoE-deficient mice. Miniature endplate and nerve-evoked endplate potentials (MEPPs and EPPs, respectively) were indistinguishable in edl muscles in both groups of mice; however, MEPP amplitudes in soleus muscles were significantly larger (by an average of 23%) in apoE-deficient mice compared with 5- to 7-week-old age-matched wild-type mice. The EPP amplitudes were also larger in soleus muscles in the mutant mice, but this was a reflection of the larger quantal size in this muscle because quantal content, determined from the ratio of the average EPP amplitude to average MEPP amplitude, was unchanged from normal in the mutant mice. The MEPP frequency and the percent of nerve stimulations failing to produce an EPP were unchanged from normal in both muscle types in the mutant mice. The difference in quantal size in soleus muscle transmission between mutant and wild-type mice was abolished in the presence of neostigmine, an acetylcholinesterase inhibitor. The results suggest that apoE normally associates with acetylcholinesterase in the synaptic cleft of slow muscles, modulating the activity of the enzyme and therefore quantal size.

Neuromuscular Transmission and Muscle Contractility in SNAP-25-deficient Coloboma Mice

Synaptosomal associated protein of 25 kDa (SNAP-25) is a cytoplasmic protein that participates in the docking and fusion of synaptic vesicles with the nerve terminal in preparation for neurotransmitter release. SNAP-25 is also a substrate for three of the seven serotypes of botulinum neurotoxin (BoNT). Intoxication by BoNT/A, /C1 or /E results in weakness and paralysis of skeletal muscle due to cleavage of SNAP-25 (and syntaxin 1a in the case /C1) at discrete serotype-specific sites. To elucidate the role of SNAP-25 in muscle function in more detail, contractility and neuromuscular transmission were studied in a mutant mouse model termed coloboma. The coloboma mutation results from a contiguous deletion of 1–2 centiMorgans on chromosome 2, which includes the entire SNAP-25 locus and three other identified genes. Homozygotes do not survive beyond gestation day 6; heterozygotes (C m/+) have a normal life-span but express reduced levels of SNAP-25 mRNA and protein in the brain. The consequences of the C m/+ mutation on twitch and tetanic tension, quantal release of neurotransmitter and spinal motoneuron expression of SNAP-25 were examined in the present study. Contrary to expectations, C m/+ mice exhibited no alteration in twitch tension and generated normal tetanic tension even at the highest frequency examined (800 Hz). Microelectrode recordings revealed that MEPP amplitude and frequency were both within control limits. The ventral spinal cord of C m/+ mice showed no deficiency in SNAP-25 content and immunohistochemical examination of nerve terminals in C m/+ mice disclosed that SNAP-25 levels and distribution were similar to those of control mice. It is concluded that spinal motor neurons up-regulate SNAP-25 to preserve vital neuromuscular function.

Spontaneous Activity of Murine Neuromuscular Junctions in the Presence of Dantrolene

By recording miniature end-plate potentials (mEPP), the effects of dantrolene (10-100 μM), a blocker of ryanodine receptors, were studied on the isolated diaphragm of mice. The effects to be studied were as follows: on spontaneous secretion of acetylcholine quanta and on the pattern of interaction with ryanodine effects. Two-hour-long application of dantrolene to the muscle caused no significant changes in the amplitude and dispersion (σ2) of mEPP, nor on its time course. In the presence of 100 μM dantrolene, the mean frequency of mEPP increased, on average, by 58.3 ± 5.9% (P < 0.05). Dantrolene suppressed in a dose-dependent manner a number of ryanodine effects (this agent was used in a concentration of 0.5 μM as an intensifier of intracellular Ca2+ mobilization): it completely prevented the appearance of the population of high-amplitude (the so-called giant) mEPP, reduced by 50-80% the increment of the mEPP amplitude dispersion, and increased by 25-45% the mEPP mean amplitude; the above effects were induced by ryanodine application for 120 min. After preliminary application of dantrolene (10-100 μM), ryanodine caused an effect not observable in the absence of dantrolene: mEPP became more frequent (140-210%). Thus, when acting on motor synapses, dantrolene behaves as a nontoxic agent, inducing only a presynaptic effect – a moderate increase in the mEPP frequency. The dual character of interaction between dantrolene and ryanodine in motor synapses was observed: on the one hand, dantrolene acts as a physiological antagonist of ryanodine by reducing the ryanodine-induced increase of dispersion and mEPP amplitude; on the other hand, dantrolene unmasks the ability of ryanodine to increase the mEPP frequency.

Physiological properties of zebrafish embryonic red and white muscle fibers during early development.

The zebrafish is a model organism for studies of vertebrate muscle differentiation and development. However, an understanding of fish muscle physiology during this period is limited. We examined the membrane, contractile, electrical coupling, and synaptic properties of embryonic red (ER) and white (EW) muscle fibers in developing zebrafish from 1 to 5 days postfertilization. Resting membrane potentials were -73 mV in 1 day ER and -78 mV in 1 day EW muscle and depolarized 17 and 7 mV, respectively, by 5 days. Neither fiber type exhibited action potentials. Current-voltage relationships were linear in EW fibers and day 1 ER fibers but were outwardly rectifying in some ER fibers at 3 to 5 days. Both ER and EW fibers were contractile at all ages examined (1 to 5 days) and could follow trains of electrical stimulation of up to 30 Hz without fatiguing for up to 5 min. Synaptic activity consisting of miniature endplate potentials (mEPPs) was observed at the earliest ages examined (1.2-1. 4 days) in both ER and EW fibers. Synaptic activity increased in frequency, and mEPP amplitudes were larger by 5 days. Miniature EPP rise times and half-widths decreased in ER fibers by 5 days, while EW fiber mEPPs showed fast kinetics as early as 1.2-1.4 days. ER and EW muscle fibers showed extensive dye coupling but not heterologous (red-white) coupling. Dye coupling decreased by 3 days yet remained at 5 days. Somites were electrically coupling, and this allowed filtered synaptic potentials to spread from myotome to myotome. It is concluded that at early developmental stages the physiological properties of ER and EW muscle are similar but not identical and are optimized to the patterns of swimming observed at these stages.

Increased quantal release of acetylcholine at the neuromuscular junction following scald injury in the rat.

Following severe burns, patients frequently develop a profound resistance to nondepolarizing neuromuscular blockers. Several mechanisms have been proposed to account for this, including upregulation of nicotinic acetylcholine receptors. We investigated the effects of a 30% body surface area (BSA) scald on neuromuscular transmission in slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) of rats. Rats were sacrificed 72 h after the injury, a time at which sepsis is unlikely and body weight gain and core temperature have returned to normal. Further groups of rats were sham operated and either pair fed to the scalded rats or freely fed to assess the influence of food restriction. When compared with muscle from pair-fed control rats, scald resulted in an almost 50% increase in miniature endplate potential (mEPP) frequency in both SOL and EDL. However, scald did not increase mean mEPP amplitude in SOL, although it did cause a 10% increase in EDL. Scald injury did produce a significant increase in the size of the evoked endplate potential in SOL (33%) and EDL (37%). These data indicate that a significant increase in the quantal content of evoked transmitter released in SOL (38%) and EDL (30%) occurred by 72 h after scald. Such an increase may contribute to the resistance to nondepolarizing neuromuscular blockers documented in patients following thermal injury.

The unitary evoked potential at the frog nerve-muscle junction results from synchronous gating of fusion pores at docked vesicles.

Exocytosis of a single vesicle has been proposed as the mechanism which determines quantal size by releasing a prepackaged and standard amount of acetylcholine. As first described by del Castillo and Katz (1954) the endplate potential is composed of 100 unitary events and the small variance suggests a binomial release from 100 "discrete patches of membrane". However, exocytosis of 100 vesicles selected randomly from 5000 docked vesicles would yield a variance that is 7 times greater than observed values. We propose that the presynaptic ridge with its compliment of docked vesicles functions as the "discrete patch of membrane" such that arrays of calcium activated fusion pores meter transmitter to form the unit of release. A model based on the synchronous flicker of a large number of fusion pores produces the small variance of both miniature end plate potentials and unitary end plate potentials. Release from a single locus (fusion pore) would generate the sub-MEPP. This model permits vesicle trafficking and vesicular content depletion during tetanic stimulation and explains the frequency dependency of MEPP amplitudes and changes in sub-MEPP to bell-MEPP class ratios.

Gender does not influence neuromuscular properties in dimorphic skeletal muscles of the toad

The aim of this work was to study gender differences on the physiology of the dimorphic brachial musculature involved in the clasp reflex of the toad ( Bufo marinus L.). The neuromuscular transmission, the sensitivity to acetylcholine (ACh) and the cholinesterase activity were compared on the forelimb sternoradialis muscles (SR) from male and female toads. The interosseous muscles of the first finger were used to compare the properties of the nicotinic receptor/ionic channel complex (AChR). All the muscles studied were dimorphic, i.e. significantly smaller in the female than in the male frog in otherwise similar size animals. The SR of either sex contracted to bath application of ACh with similar EC50. In physiological solution the frequency of the miniature end-plate potentials (mepps) was very low (0.1 s −1) and no gender difference was detected. The mepp amplitudes were 0.62±0.03 and 0.58±0.03 mV in SR from male and female toads, respectively. To increase exocytosis the muscles were incubated in hypertonic solution (158 mM NaCl). Under this condition mepp frequency was increased by five and seven times and mepp amplitude increased by 1.3 and 1.6 times in SR from male and female toads, respectively. The cholinesterase activity measured by the colorimetric method, did not differ in SR from male and female toads. In muscle fibers dissociated from the dimorphic interosseous muscles of male and female toads, the ionic channel conductance was 43±5.3 and 44±4.5 pS, respectively. The mean channel open time was voltage-dependent and not significantly different in preparations from both genders. These observations indicate that neither the ACh-nicotinic receptor interaction, nor the AChR complex kinetics and the nicotinic excitation–contraction coupling or the cholinesterase activity differ in dimorphic muscles from Bufo genders. No gender difference was detected in neuromuscular transmission of the studied muscle. Only a slight increase in mepp frequency and amplitude could be detected when the muscles were incubated in hypertonic solution.