Mepp Frequency Research Articles

Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity.

Botulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery. Mice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8-12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size. In addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases.

Stress increases the spontaneous release of ACh and may be involved in the generation and maintenance of myofascial trigger points in mouse

An increase in spontaneous neurotransmission may be related to myofascial pain. Sympathetic neurons innervate most of the neuromuscular junction sand are involved in the modulation of synaptic transmission. Therefore, a direct action of stress on acetylcholine release is expected. For this reason, this study aims to evaluate the relationship between stress and spontaneous neurotransmission. Five acute stressors (immobilization, forced swimming, food and water deprivation, social isolation and ultrasound) were tested in 6 weeks adult Swiss male mice. Subsequently, these types of stress were combined to generate a model of chronic stress. The study of ACh release was evaluated before and after the application of stress by intracellular recording of spontaneous neurotransmission (mEPPs). In each one of the stressors, an increase in the frequency of mEPPs was obtained immediately after treatment, which remained elevated for 5 days and thereafter returned to control values after a week. With chronic stress, a much higher increase in the frequency of mEPPs was obtained and it was maintained for 15 days. In summary, stress, both in its acute and chronic forms, increased spontaneous neurotransmission significantly. There is a possibility that chronic stress is related with the genesis or maintenance of myofascial pain.

Development of abnormalities at the neuromuscular junction in the SOD1-G93A mouse model of ALS: dysfunction then disruption of postsynaptic structure precede overt motor symptoms.

The ultimate deficit in amyotrophic lateral sclerosis (ALS) is neuromuscular junction (NMJ) loss, producing permanent paralysis, ultimately in respiratory muscles. However, understanding the functional and structural deficits at NMJs prior to this loss is crucial for therapeutic strategy design. Should early interventions focus on reversing denervation, or supporting largely intact NMJs that are functionally impaired? We therefore determined when functional and structural deficits appeared in diaphragmatic NMJs relative to the onset of hindlimb tremor (the first overt motor symptoms) in vivo in the SOD1-G93A mouse model of ALS. We employed electrophysiological recording of NMJ postsynaptic potentials for spontaneous and nerve stimulation-evoked responses. This was correlated with fluorescent imaging microscopy of the postsynaptic acetylcholine receptor (AChR) distribution throughout the postnatal developmental timecourse from 2 weeks to early symptomatic ages. Significant reduction in the amplitudes of spontaneous miniature endplate potentials (mEPPs) and evoked EPPs emerged only at early symptomatic ages (in our colony, 18-22 weeks). Reductions in mEPP frequency, number of vesicles per EPP, and EPP rise time were seen earlier, at 16weeks, but this reversed by early symptomatic ages. However, the earliest and most striking impairment was an inability to maintain EPP amplitude during a 20 Hz stimulus train, which appeared 6 weeks before overt in vivo motor symptoms. Despite this, fluorescent α-bungarotoxin labelling revealed no systematic, progressive changes in 11 comprehensive NMJ morphological parameters (area, shape, compactness, number of acetylcholine receptor, AChR, regions, etc.) with disease progression. Rather, while NMJs were largely normally-shaped, from 16 weeks there was a progressive and substantial disruption in AChR concentration and distribution within the NMJ footprint. Thus, NMJ functional deficits appear at least 6 weeks before motor symptoms in vivo, while structural deficits occur 4 weeks later, and predominantly within NMJs. These data suggest initial therapies focused on rectifying suboptimal NMJ function could produce effective relief of symptoms of weakness.

Shock waves as treatment of mouse myofascial trigger points.

An abnormal increase in spontaneous neurotransmission can induce subsynaptic knots in the myocyte called myofascial trigger points. The treatment of choice is to destroy these trigger points by inserting needles. However, 10% of the population has a phobia of needles, blood, or injuries. Therefore, the objective of this study is to verify the usefulness of shock waves in the treatment of myofascial trigger points. Two groups of mice have been developed for this: healthy muscles treated with shock waves; trigger points affected muscles artificially generated with neostigmine and subsequently treated with shock waves. Muscles were stained with methylene blue, PAS-Alcian Blue, and labeling the axons with fluorescein and the acetylcholine receptors with rhodamine. Using intracellular recording the frequency of miniature endplate potentials (mEPPs) was recorded and endplate noise was recorded with electromyography. No healthy muscles treated with shock waves showed injury. Twitch knots in mice previously treated with neostigmine disappeared after shock wave treatment. Several motor axonal branches were retracted. On the other hand, shock wave treatment reduces the frequency of mEPPs and the number of areas with endplate noise. Shock waves seem to be a suitable treatment for myofascial trigger points. In the present study, with a single session of shock waves, very relevant results have been obtained, both functional (normalization of spontaneous neurotransmission) and morphological (disappearance of myofascial trigger points). Patients with a phobia of needles, blood, or injuries who cannot benefit from dry needling may turn to noninvasive radial shock wave treatment.

Взаимодействие механизмов угнетения квантового выделения ацетилхолина при активации ваниллоидных (TRPV1) и пуриновых рецепторов в нервно-мышечном синапсе мыши

The main goal of the study was to analyze the relationship between the signaling pathways of the regulation of acetylcholine (ACh) quantal release in the peripheral synapse triggered by the activation of vanilloid (TRPV1) and purine receptors. In electrophysiological experiments carried out at the neuromuscular synapses of the m. Levator Auris Longus, it was found that the frequency of miniature endplate potentials (mEPPs) and the quantal content of endplate potentials (EPPs) decrease in the presence of TRPV1 agonist capsaicin. This effect was completely reversed by SB366791, a specific competitive antagonist of TRPV1 receptors. ATP, like capsaicin, decreased the frequency of mEPPs and the EPP quantal content. Against the background of the TRPV1 antagonist, the inhibitory effect of ATP on ACh secretion was realized in full. At the same time, against the background of TRPV1 channels activation by capsaicin, the effect of ATP on both spontaneous and evoked ACh release was absent. It was suggested that the mechanisms of action of ATP and capsaicin may be associated with a change in Ca2+ entry into the nerve ending. To test this hypothesis, experiments were carried out to assess the changes in the presynaptic calcium level (Ca2+ transient) using a fluorescent calcium dye upon nerve stimulation. The amplitude of the calcium transient did not change either with the application of ATP or with the addition of capsaicin. Thus, in the neuromuscular synapse of mammals, along with purinergic pathway of ACh secretion regulation, there is also a mechanism of neurosecretion modulation mediated by the activation of TRPV1 channels. The triggering of these mechanisms leads to the suppression of the processes of both spontaneous and evoked release of ACh quanta from the motor nerve endings. It was demonstrated that both pathways of regulation are not accompanied by a change in the Ca2+ transient, but have a common link of regulation of the quantal neurosecretion.

Percutaneous Application of Galvanic Current in Rodents Reverses Signs of Myofascial Trigger Points.

An increase in the spontaneous release of acetylcholine (ACh) at the motor endplate is directly related to the generation of myofascial trigger points (MTrPs). In this study, percutaneous electric fields were applied to an animal model of MTrPs with high levels of spontaneous ACh release. All experiments were performed on Swiss mice and Sprague Dawley rats. For evaluating the spontaneous neurotransmission, intracellular recordings were performed, and the frequency of miniature endplate potentials was evaluated. Electromyographic recordings were also conducted to evaluate the endplate noise. Finally, the number and strength of local twitch responses (LTR) were evaluated using ultrasound recordings. The protocols used for the electric currents were 0.4 mA for five seconds and four repetitions (protocol 1), 1.5 mA for five seconds and three repetitions (protocol 2), and 3 mA for three seconds and three repetitions (protocol 3). After a subcutaneous injection of neostigmine (NTG), a great increase was observed in the frequency of mEPPs, together with an elevated endplate noise. Protocols 2 and 3 were the most effective. Protocol 3 could completely reverse the action of NTG at both three hours and 24 hours, respectively. The application of percutaneous currents produced both an increase in the number (144%) and in the speed (230% faster) of LTR compared with dry needling. In conclusion, higher doses of electrical current are more effective for decreasing MTrPs findings in an animal model.

Methanolic extract of Rhinella schneideri (cururú toad) poison crude cause ultrastructural changes in nerve terminals of mouse phrenic nerve-diaphragm preparations

Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison.

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.

Adenosine A1 and A2A receptor‐mediated modulation of acetylcholine release in the mice neuromuscular junction

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 μm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity.

Pharmacological study of a new Asp49 phospholipase A2 (Bbil-TX) isolated from Bothriopsis bilineata smargadina (forest viper) venom in vertebrate neuromuscular preparations

The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve–diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5–10 μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4–6) for 1, 5 and 10 μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 μg/ml) BC preparations. However, Bbil-TX (10 μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 μg/ml)-induced blockade was markedly inhibited at 22–24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3–30 μg/ml, n = 4–6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 μg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from −80 ± 1 mV (basal) to −66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.

Quantal release of acetylcholine in mice with reduced levels of the vesicular acetylcholine transporter

Mammalian motor nerve terminals contain hundreds of thousands of synaptic vesicles, but only a fraction of these vesicles is immediately available for release, the remainder forming a reserve pool. The supply of vesicles is replenished through endocytosis, and newly formed vesicles are refilled with acetylcholine through a process that depends on the vesicular acetylcholine transporter (VAChT). During expression of short-term plasticity, quantal release can be increased, but it is unknown whether this reflects enhanced recruitment of vesicles from the reserve pool or rapid recycling. We examined spontaneous and evoked release of acetylcholine at endplates from genetically modified VAChT KD(HOM) mice that express approximately 30% of the normal level of VAChT to determine steps rate-limited by synaptic vesicle filling. Quantal content and quantal size were reduced in VAChT KD(HOM) mice compared with wild-type controls. Although high-frequency stimulation did not reduce quantal size further, the post-tetanic increase in end-plate potential amplitude or MEPP frequency was significantly smaller in VAChT KD(HOM) mice. This was the case even when tetanic depression was eliminated using an extracellular solution containing reduced Ca(2+) and raised Mg(2+). These results reveal the dependence of short-term plasticity on the level of VAChT expression and efficient synaptic vesicle filling.

Abnormal development of the neuromuscular junction in Nedd4-deficient mice

Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. Here we show that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5–E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms.

3P-193 カエル神経筋接合部シナプスにおけるMg^&lt;2+&gt;を含んだCa^&lt;2+&gt;フリー溶液中での連続刺激による自発放出頻度増大現象の主要因はCa^&lt;2+&gt;チャネルを通じてのシナプス前末端内へのMg^&lt;2+&gt;蓄積である(神経・感覚(細胞・膜蛋白質・分子),第47回日本生物物理学会年会)

3P-193 カエル神経筋接合部シナプスにおけるMg^&lt;2+&gt;を含んだCa^&lt;2+&gt;フリー溶液中での連続刺激による自発放出頻度増大現象の主要因はCa^&lt;2+&gt;チャネルを通じてのシナプス前末端内へのMg^&lt;2+&gt;蓄積である(神経・感覚(細胞・膜蛋白質・分子),第47回日本生物物理学会年会)

Effect of Vitamin В1 Alimentary Deficiency on Spontaneous and Evoked Transmitter Release in Murine Neuromuscular Synapses

Using a microelectrode technique, we studied the effects of alimentary vitamin В 1 deficiency on synaptic transmission in isolated phrenico-hemidiaphragmatic murine preparations. Animals of group І (control) were on a standard thiamine-controlled diet (16 mg/kg thiamine) with no limitations. Animals of group II (control with alimentary limitation) were on the same diet, but daily consumption in these animals was limited and made similar to the amount of food consumed by the animals of group ІІІ within idential periods of cage housing (for differentiation of the effects of anorexia related to the thiamine-deficient state in group III and proper effects of В 1 hypovitaminosis). Animals of group ІІІ (thiamine-deficient) were on a standard diet (with no limitations) mostly analogous to that in group І but containing no thiamine. In phrenicohemidiaphragmatic preparations obtained from animals of group ІІІ, the amplitude of end-plate potentials (EPPs) and miniature EPPs (mEPPs) on the 10th day of consumption of the thiamine-defficient diet and the quantum composition of EPPs on the 20th day became significantly (P < 0.01) smaller than in preparations obtained from animals of both groups І and ІІ. The frequency of mEPPs and membrane potential of muscle fibers in group ІІІ remained unchanged. Two processes, a decrease in the dimension of the transmitter quantum (which is observed within rather early stages of the development of thiamine-defficient state) and a decrease in the quantum composition of evoked EPPs (at later stages) underlie a gradual decrease in the amplitude of EPPs related to the development of alimentary vitamin В 1 deficiency.

Differential effect of serum from bipolar versus schizophrenic patients on spontaneous acetylcholine release at mammalian neuromuscular junction

Objective The diagnosis of bipolar disease frequently requires a long time since the age of onset, especially because the disease is misdiagnosed with schizophrenia. The aim of the present work was to investigate whether sera from bipolar patients have an active substance that allows making a fast identification of the disease. Methods Sera from healthy volunteers, euthymic and non-stabilized bipolar patients, and schizophrenic patients were passively transferred into CF1 mice and after 2 day injections, MEPP frequency from diaphragm muscles was recorded. The same procedure was performed with sera fraction of high and low MW (cut-off 3000). Results Sera from non-stabilized bipolar patients induced a decreased MEPP frequency and occluded the presynaptic inhibitory effect of the specific adenosine A 1 receptor agonist 2-chloro- N 6-cyclopentyl-adenosine (CCPA) in the recipient mice, while in the euthymic bipolar group spontaneous secretion reached control values although the action of CCPA was still prevented. Similar results were obtained with low MW sera fraction from euthymic and non-stabilized bipolar patients. The addition of adenosine deaminase to the sera fraction prevented the modification of spontaneous ACh release. In mice injected with sera from schizophrenic patients, MEPP frequency was within control values and CCPA induced its typical inhibitory action. Conclusions These results indicate that bipolar patients contain in their blood an active substance compatible with adenosine, which was able to modify spontaneous ACh release in the recipient mice. This effect was not observed with sera from healthy volunteers and schizophrenic patients. The increase of adenosine concentration may result from synaptic hyperactivity that presumably plays a role in the symptoms of bipolar disorder and/or may derive from peripheral cells through a more general mechanism. Significance The different results obtained with bipolar and schizophrenic sera raise the possibility that the passive transfer model could be used as a diagnostic test in the future.

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.

Mefloquine selectively increases asynchronous acetylcholine release from motor nerve terminals

Effectiveness against chloroquine-resistant Plasmodia makes mefloquine a widely used antimalarial drug. However, mefloquine's neurologic effects offset this therapeutic advantage. Cellular actions which might contribute to the neurologic effects of mefloquine are not understood. Structural similarity to tacrine suggested that mefloquine might alter cholinergic synaptic transmission. Therefore, we examined mefloquine's effects at a model cholinergic synapse. Triangularis sterni nerve–muscle preparations were isolated from adult mice and examined with sharp electrode current clamp technique. Within 30 min of exposure to 10 μM mefloquine, miniature endplate potentials (mepps) occurred in summating bursts and their mean frequency increased 10-fold. The threshold concentration for the increase of mean mepp frequency was 0.6 μM mefloquine. Mefloquine continued to increase mean mepp frequency for preparations bathed in extracellular solution lacking Ca 2+. In contrast, mefloquine no longer increased mean mepp frequency for preparations pre-treated with the intracellular Ca 2+ buffer BAPTA-AM. Although mefloquine disrupts a thapsigargin-sensitive neuronal Ca 2+ store, pre-treatment with thapsigargin did not alter the mefloquine-induced alterations of mepps. Since mefloquine, like oligomycin, inhibits mitochondrial F OF 1H +ATP synthase we tested the interaction between these two chemicals. Like mefloquine, oligomycin induced bursts and increased mean frequency of mepps. Furthermore, pre-treatment with oligomycin precluded the mefloquine-induced alterations of asynchronous transmsitter release. These data suggest that mefloquine inhibits ATP production which increases the concentration of Ca 2+ within the cytosol of nerve terminals. This elevation of Ca 2+ concentration selectively increases asynchronous transmitter release since 10 μM mefloquine did not alter stimulus-evoked transmsitter release.

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.