Mouse Motor Nerve Research Articles

Multiple types of Ca2+ channels in mouse motor nerve terminals.

We measured neurotransmitter release and motor nerve terminal currents in mouse phrenic nerve-diaphragm and triangularis sterni preparations, to evaluate the role of Ca2+-channel subtypes in regulating transmitter release. Saturated concentrations of either omega-agatoxin IVA [omega-Aga-IVA (0.3 microM), a blocker of P-type Ca2+ channels] or omega-conotoxin MVIIC [omega-CTx-MVIIC (2 microM), a P- and Q-type Ca2+-channel blocker], inhibited nerve-evoked muscle contractions and the amplitude of endplate potentials respectively. In contrast, combined treatment with nifedipine (50 microM, a blocker of L-type Ca2+ channels) plus omega-conotoxin GVIA [omega-CTx-GVIA (2 microM), a blocker of N-type Ca2+ channels] did not elicit inhibitory effects on nerve-evoked muscle contractions, endplate potentials or nerve terminal waveforms. Because of the non-linear relationship between endplate potentials and Ca2+ signals, a small decrease in presynaptic Ca2+ entry can significantly reduce the amplitude of the endplate potential. Thus, we applied 3,4-diaminopyridine (3,4-DAP, a K+-channel blocker) or high Ca2+ (10 mM) to accelerate and amplify the endplate potentials and Ca2+ currents. The endplate potentials amplified by 3,4-DAP or by high Ca2+ correspondingly proved to be quite resistant to both omega-Aga-IVA and omgea-CTx-MVIIC; omega-Aga-IVA exerted only a partial inhibitory effect on endplate potentials, and the omega-Aga-IVA-resistant component was further inhibited by omega-CTx-MVIIC. The component that was resistant to the two toxins could be completely blocked by the non-selective Ca2+ channel blocker Cd2+ (300 microM). A combination of the two toxins had no significant effects on either spontaneous transmitter release or postsynaptic resting membrane potentials of the diaphragm preparation and the Na+ and K+ waveforms of the triangularis sterni preparations. This finding suggests a preferential inhibitory effect at a presynaptic site. Measuring the Ca2+ currents in the triangularis sterni also revealed partial inhibition by omega-CTx-MVIIC with further incomplete inhibition by omega-Aga-IVA. Cd2+ (300 microM) abolished the toxin-resistant component of the Ca2+ current. In contrast, a combination of nifedipine (50 microM) with omega-CTx-GVIA (2 microM) was without inhibitory effect. We conclude that multiple types of Ca2+ channels, i.e. omega-Aga-IVA-sensitive, omega-CTx-MVIIC-sensitive and toxin-resistant Ca2+ channels, coexist in mouse motor nerve terminals.

Intrinsic neuronal determinants locally regulate extrasynaptic and synaptic growth at the adult neuromuscular junction.

Long-term functional plasticity in the nervous system can involve structural changes in terminal arborization and synaptic connections. To determine whether the differential expression of intrinsic neuronal determinants affects structural plasticity, we produced and analyzed transgenic mice overexpressing the cytosolic proteins cortical cytoskeleton-associated protein 23 (CAP-23) and growth-associated protein 43 (GAP-43) in adult neurons. Like GAP-43, CAP-23 was downregulated in mouse motor nerves and neuromuscular junctions during the second postnatal week and reexpressed during regeneration. In transgenic mice, the expression of either protein in adult motoneurons induced spontaneous and greatly potentiated stimulus-induced nerve sprouting at the neuromuscular junction. This sprouting had transgene-specific features, with CAP-23 inducing longer, but less numerous sprouts than GAP-43. Crossing of the transgenic mice led to dramatic potentiation of the sprout-inducing activities of GAP-43 and CAP-23, indicating that these related proteins have complementary and synergistic activities. In addition to ultraterminal sprouting, substantial growth of synaptic structures was induced. Experiments with pre- and postsynaptic toxins revealed that in the presence of GAP-43 or CAP-23, sprouting was stimulated by a mechanism that responds to reduced transmitter release and may be independent of postsynaptic activation. These results demonstrate the importance of intrinsic determinants in structural plasticity and provide an experimental approach to study its role in nervous system function.

Polyamine FTX-3.3 and Polyamine Amide sFTX-3.3 Inhibit Presynaptic Calcium Currents and Acetylcholine Release at Mouse Motor Nerve Terminals

FTX-3.3 is the proposed structure of a calcium-channel blocking toxin that has been isolated from the funnel web spider ( Agelenopsis aperta). The effects of FTX-3.3 and one of its analogues, sFTX-3.3, on acetylcholine release, on presynaptic currents at mouse motor nerve terminals and on whole-cell sodium currents in SK.N.SH cells (a human neuroblastoma cell line) have been studied. FTX-3.3 (10–30 μM) and sFTX-3.3 (100–300 μM) reversibly reduced release of acetylcholine by approximately 70–90% and 40–60%, respectively. FTX-3.3 (10 μM) blocked the fast component of presynaptic calcium currents by approximately 60%. sFTX-3.3 (100 μM) reduced the duration of the slow component of presynaptic calcium currents by about 50% of the control and also reduced presynaptic sodium current by approximately 20% of the control. sFTX-3.3 (100 μM) reduced whole-cell sodium current recorded from SK.N.SH cells by approximately 15%, whereas FTX-3.3, even at 200 μM, did not affect this current. Since the only difference in chemical structures of these toxins is that sFTX-3.3 has an amide function which is absent in FTX-3.3, the amide function may be responsible for the reduced potency and selectivity of sFTX-3.3. This study also provides further support for the existence of P-type calcium channels at mouse motor nerve terminals. © 1997 Elsevier Science Ltd. All rights reserved.

Intramuscular nerve sprouting induced by CNTF is associated with increases in CGRP content in mouse motor nerve terminals

It is known that motor nerve terminal sprouting induced by either nerve injury or muscle paralysis is associated with an increase in calcitonin gene-related peptide (CGRP) content in the soma of motoneurons and in motor endplates. In the present study, CGRP-like immunoreactivity (CGRP-LI) was determined in motor endplates of animals in which nerve terminal sprouting had been induced by exogenous application of ciliary neurotrophic factor (CNTF). After 18 days of CNTF treatment we observed a significant increase in CGRP-LI in motor endplates. The results indicate that CGRP is upregulated when motor nerve outgrowth is induced, even in the absence of muscle paralysis or nerve lesion.

Upregulation of calcitonin gene-related peptide at mouse motor nerve terminals poisoned with botulinum type-A toxin.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity of motor nerve terminals was investigated at different times after local in vivo injection of botulinum type-A toxin (BoNT/A) close to the mouse levator auris longus muscle. CGRP expression in most of control nerve terminals was undetectable, but markedly increased during muscle paralysis and synaptic remodelling and, declined once functional recovery occurred.

Synaptotagmin II immunoreactivity in normal and botulinum type-A treated mouse motor nerve terminals.

The distribution of synaptotagmin II, a synaptic vesicle protein, was examined by immunohistochemistry at normal mouse motor nerve terminals and after botulinum type-A treatment. An immunoreactivity to synaptotagmin II was detected in both control and botulinum type-A treated motor nerve terminals including newly formed sprouts. These data, together with other reports showing the absence of synaptotagmin I at the neuromuscular junction, suggest that synaptotagmin II is the isoform involved in transmitter release at motor nerve terminals.

Reduction of Calcium Currents by Lambert–Eaton Syndrome Sera: Motoneurons Are Preferentially Affected, and L-Type Currents Are Spared

Previous work has demonstrated that Lambert-Eaton syndrome (LES) antibodies reduce calcium currents in nonneuronal cells and neurons and reduce the amplitude of extracellularly recorded currents at mouse motor nerve terminals. We compared effects of LES sera on whole-cell currents of cultured nerve and muscle. LES sera more strongly reduced calcium currents in motoneurons than in sensory neurons. Motoneuronal potassium currents were unaffected. The sera minimally affected calcium currents in skeletal and cardiac muscle. In motoneurons, both low voltage-activated (LVA) and high voltage-activated (HVA) components of calcium current were decreased, demonstrating that the sera targeted more than one calcium channel type. The HVA current remaining in LES-treated motoneurons was little affected by micromolar omega-conotoxin MVIIC but was reduced > 70% by micromolar nimodipine. This pharmacological profile contrasts with untreated cells and suggest that LES sera primarily spare L-type currents in motoneurons.

Ruthenium Red, a Novel Enhancer of K + Currents at Mouse Motor Nerve Terminals

The effects of ruthenium red (RR) on transmitter release and pre-synaptic currents were studied in the mouse neuromuscular junction. The action of RR (10 μM) was shown not only in the complete suppression of nerve-evoked muscle contractions associated with the depression of endplate potential amplitudes but also in the partial inhibition of the amplitude of miniature-endplate potentials. However, the other ruthenium compounds, ruthenium chloride and tris (2,2-bipyridyl) ruthenium chloride did not significantly affect the neuromuscular transmission. In pre-synaptic waveform studies, the fast K +-current [IK (f)] as well as the Ca 2+-activated K +-current [IK (ca)] was significantly enhanced by 10 μM RR. Furthermore, 10 μM RR antagonized the action of β-bungarotoxin (a blocker of slow K +-channel [IK (s)] in enhancing pre-synaptic Ca 2+ currents. In contrast, the typical Ca 2+-channel blockers, ω-agatoxin (0.5 μM), Gd 3 + (0.5 mM) and Cd 2 + (0.3 mM) all suppressed the IK (ca). Although RR (1–30 μM) inhibited the Ca 2+-currents of the nerve terminals induced by the combined treatment with the K +-channel blockers, 3,4-diaminopyridine plus tetraethylammonium chloride in a concentration-dependent manner, it is considered that RR-enhanced K + currents were responsible for, at least in part, the observed inhibition of the Ca 2+-current which led to the blockade of transmitter release. Copyright © 1996 Elsevier Science Ltd

Suppression of potassium currents by cyanide on the mouse motor nerve terminals

NaCn at low concentrations markedly depressed the potassium currents in the motor nerve terminal of mouse triangularis sterni neuromuscular junction pretreated with potassium channel blockers 4-aminopyridine (4-AP), tetraethylammonium (TEA) or glucose-free medium. Neither azide nor dinitrophenol nor ouabain mimicked the effect of cyanide. This inhibitory effect of cyanide on nerve terminal spikes was correlated to its dramatic increase in spontaneous transmitter release under glucose-free condition. These results suggest that the effect of cyanide on the electrogenesis of nerve terminals is due to the direct suppression of ATP-sensitive K + current since the effect was antagonized by ATP-sensitive K + channels opener diazoxide and this may modulate the transmitter release.

P-956 - Augmentation of calcium influx by propylene glycol at mouse motor nerve terminals.

P-956 - Augmentation of calcium influx by propylene glycol at mouse motor nerve terminals.

The effects of two phospholipase A 2 inhibitors on the neuromuscular blocking activities of homologous phospholipases A 2 from the venom of Pseudechis australis, the Australian king brown snake

Previous studies have shown that homologous phospholipases A 2 (PLA 2) (Pa-3, Pa-9C, Pa-10F and Pa-11) from the venom of the Australian king brown snake, Pseudechis australis, significantly reduce the resting membrane potentials and quantal contents of endplate potentials recorded from endplate regions of mouse triangularis sterni nerve-muscle preparations. It is not clear whether PLA 2 activity is essential for their neuromuscular activities. Therefore, pharmacological studies were carried out to determine whether neuromuscular activity of the toxins changed after treatment with the phospholipase A 2 inhibitors 7,7-dimethyl-eicosadienoic acid (DEDA) and manoalide. After incubation of the toxins with manoalide (120nM), or DEDA (50 μM), no PLA 2 activity against 1-stearoyl 2-[ 3H]arachidonoylglycerophosphocholine was detected. After incubation with manoalide and/or DEDA, the toxins did not depolarize muscle fibre membranes up to 60 min after administration. However, manoalide and DEDA had different influences on the inhibitory effect of these toxic enzymes on acetylcholine release from nerve terminals. Manoalide abolished the inhibitory effect of the toxins on evoked release of acetylcholine. In contrast, DEDA was not able to prevent the reduction of quantal content of endplate potentials induced by the toxins. This study provides evidence that the depolarizing action and the inhibitory effect on release of acetylcholine exerted by these toxic PLA 2 from king brown snake are independent phenomena. The evidence for this conclusion was that inhibition of enzymatic activity with an arachidonic acid analogue (DEDA) abolished the depolarizing effect of the toxins but not the effects on the quantal release of acetylcholine from mouse motor nerve terminals. The data suggest that the depolarizing effect of these toxins is probably due to the enzymatic activity. Since manoalide interacts with lysine residues of PLA 2 polypeptides, and, as shown here, manoalide prevented inhibition of neurotransmitter release, lysine residues may play an important role in the inhibitory activity of these toxins.

Functional assessment of Ca 2+-current in the mouse motor nerve terminals

The purpose of this study was to characterize voltage-gated Ca 2+ channels on the mouse motor nerve terminals. Mouse diaphragm and triangularis sterni preparations were used for this study in order to assess the functional Ca 2+ channels in the transmitter release. The results showed that ω-conotoxin MVIIC (CTx-MVIIC, 0.5–1 μM) but not ω-conotoxin GVIA (1 mM) markedly inhibits not only the nerve-evoked muscle contractions accompanied by a decrease in the amplitude of end plate potentials (epps) in the mouse phrenic-nerve diaphragm but also the Ca 2+-waveforms in the nerve terminals of triangularis sterni. The inhibitory effects of CTx-MVIIC were considered to be specifically presynaptic rather than myogenic, since none of the electrical properties of muscle fibers including action potentials, resting membrane potentials and the miniature endplate potential, were affected. Moreover, Na +- and K +-waveforms of the nerve terminals were unaffected by CTx-MVIIC. At a saturating concentration of 3–5 mM, CTx-MVIIC exerted a maximal inhibitory effect by 38% of 3,4-diaminopyridine-prolonged epps area and inhibited only the slow component of Ca 2+-current, respectively, and the remaining fast component could be inhibited by subsequent addition of cadmium chloride (Cd 2+). All of these findings indicate that at least two components (a slow CTx-MVIIC sensitive component and a fast Cd 2+ sensitive component) of the mouse motor nerve terminals would cooperate in the induction of the transmitter release from motor nerve endings.

Mouse motor nerve terminal immunoreactivity to synaptotagmin II during sustained quantal transmitter release

An antibody directed against the lumenal NH 2-terminus of synaptotagmin II was used to examine the distribution of this vesicular protein either after spontaneous acetylcholine release or after sustained release induced by La 3+ or α-latrotoxin, in conditions that prevent endocytosis. The detection of the epitope was examined in the presence or absence of Triton X-100. We show that, in resting conditions of transmitter release, permeabilization of nerve terminal membranes is required for obvious detection of synaptotagmin II immunoreactivity whereas during sustained rates of quantal release, permeabilization is not necessary. These data indicate that, in the latter conditions, synaptotagmin II is incorporated into the terminal axolemma and its intravesicular domain exposed at the extracellular nerve terminal surface.

Enhancement of a slow potassium current component by uranyl nitrate and its relation to the antagonism on β-bungarotoxin in the mouse motor nerve terminal

Uranyl nitrate (UO 2(NO 3) 2) has been shown to be capable of increasing transmitter release from the motor nerve accompanied by the potentiation of nerve evoked muscle contraction. In this paper, we have demonstrated that UO 2 2+ induced an initial twitch depression followed by a later twitch potentiation in low (0.35 mM) Ca 2+ medium. Although UO 2 2+ has been identified as a K +-channel blocker, we have found it only partially blocked the fast K +-current (IK (f)) as recorded in the perineurial sheath of the mouse triangularis sterni preparation. Increasing the concentration of UO 2 2+ to a high concentration of 0.4 mM did not further inhibit IK (f) but markedly prolonged the duration of the outward current of the nerve terminals. From the time course of its appearance together with the specific inhibition by 4-aminopyridine, dendrotoxin and β-bungarotoxin, which has been shown to be capable of blocking the K +-current of the motor nerve terminal, it was proposed that UO 2 2+ prolonged the duration of the nerve terminal spikes by an enhancement of an IK (s)-like current, which was further characterized by its susceptibility to be enhanced by low K +, low Ca 2+ and Cd 2+ but attenuated by high K + and high Ca 2+. These cation effects not only supported UO 2 2+-induced IK (s) current but also excluded the possibility of an enhancement of Ca 2+-activated K +-current induced by UO 2 2+ plus TEA. The significance of this enhancement of IK (s) induced by UO 2 2+ has been elucidated by the finding that dendrotoxin inhibited but tetraethylammonium potentiated not only UC 2 2+-induced IK (s) but also UO 2 2+-induced twitch depression. Therefore, it is concluded that in addition to the blockade of IK (f), UO 2 2+ can also enhance IK (s)-like current which accounts for presynaptic inhibition of the twitch contractions. Moreover, UO 2(NO 3) 2 has been found to be a selective potent antagonist of β-bungarotoxin (β-BuTX) in blocking the neuromuscular transmission of mouse phrenic nerve diaphragm. In accordance with their effects on UO 2 2+-induced IK (s), TEA markedly potentiated UO 2 2+ in antagonizing the neuromuscular blocking action of β-BuTX, while 4-AP, DTX, high K + and Ca 2+ all reversed the antagonistic action of UO 2 2+ plus TEA against β-BuTX. These findings provide more evidence that IK (s) plays a role in the regulation of transmitter release and UO 2 2+ antagonizes the neuromuscular blocking action of β-BuTX through their interaction at least in part on the slow K +-channels of the motor nerve terminals.

Inhibition of acetylcholine release from mouse motor nerve by a P-type calcium channel blocker, omega-agatoxin IVA.

1. The effects were studied of the central neurone P-type Ca2+ channel blockers, omega-agatoxin IVA, omega-conotoxin MVIIC (polypeptide toxins) and synthetic funnel-web spider polyamine toxin on acetylcholine release from mouse motor nerve. 2. omega-Agatoxin IVA decreased the quantal content of endplate potentials and blocked synaptic transmission in the nanomolar range in a reversible manner, whereas the other toxins depressed transmission in the hundred micromolar range. 3. The polyamine toxin, but not the polypeptide toxins, decreased the amplitude of the miniature endplate potential. The increase in the frequency of miniature endplate potentials evoked by high [K+], but not that evoked by alpha-latrotoxin, was effectively antagonized by omega-agatoxin IVA. 4. In the presence of omega-agatoxin IVA, high frequency nerve stimulation produced facilitation of endplate currents and tetanic contractions. 5. The results suggest that, under physiological conditions, the Ca2+ necessary for nerve action potential-evoked acetylcholine release is translocated via a subtype of the P-type Ca2+ channel sensitive to omega-agatoxin IVA.

Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus

D. L. Marshall, H. Vatanpour, A. L. Harvey, P. Boyot, S. Pinkasfeld, Y. Doljansky, F. Bouet and A. Ménez. Neuromuscular effects of some potassium channel blocking toxins from the venom of the scorpion Leiurus quinquestriatus hebreus. Toxicon 32, 1433–1443, 1994.—The scorpion venom Leiurus quinquestriatus hebreus was fractionated by chromatography in order to isolate toxins that affected binding of radiolabelled dendrotoxin to K + channel proteins on synaptosomal membranes and that facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations. In addition to the previously characterized charybdotoxin, three toxins were isolated: 14-2, 15-1 and 18-2. Toxin 14-2 has a blocked N-terminus and because of low quantities, it has not been sequenced; 15-1 is a newly sequenced toxin of 36 residues with some overall homology to charybdotoxin and noxiustoxin; 18-2 is identical to charybdotoxin-2. The apparent K i against dendrotoxin binding were: charybdotoxin, 3.8 nM; 14-2, 150 nM; 15-1, 50 nM; and 18-2, 0.25 nM. Toxin 14-2 (75 nM–1.5 μM) had a presynaptic facilitatory effect on neuromuscular preparations. Toxin 15-1 augmented responses to direct muscle stimulation, probably because it blocked Ca 2+-activated K + currents in muscle fibres. Toxin 18-2 (charybdotoxin-2) had a potent presynaptic facilitatory action, with less effect on direct muscle stimulation. This contrasts with the relatively weak neuromuscular effects of the highly homologous charybdotoxin. On a Ca 2+-activated K + current in mouse motor nerve endings, charybdotoxin and toxin 18-2 produced maximal block at around 100 nM, whereas 15-1 was inactive at 300 nM. Charybdotoxin can increase quantal content, but this is more likely to result from block of voltage-dependent K + channels than Ca 2+-activated channels: the increase in transmitter release occurred in conditions in which little I KCa would be present; higher concentration of charybdotoxin and longer exposure times were required to increase transmitter release than those needed to block I KCa, and the facilitatory effects of charybdotoxin and toxin 18-2 correlated more with their effects on dendrotoxin binding than on block of I KCa.

Inhibition by lead of the calcium plateau current at mouse motor nerve endings

The inhibition by lead of motor terminal calcium plateau signals has been studied using extracellular, perineuronal electrodes applied to the M. triangularis sterni preparation of the mouse. The long-lasting calcium pateau signal induced by 3,4-DAP (250 mumol) and TEA (10 mmol) blockade of presynaptic potassium channels was irreversibly blocked by micromolar lead concentrations in a concentration-dependent manner. These results suggest that lead antagonizes neuronal calcium channels involved in transmitter release from motor nerve terminals.

Inhibition by lead of the calcium plateau current at mouse motor nerve endings

The inhibition by lead of motor terminal calcium plateau signals has been studied using extracellular, perineuronal electrodes applied to theM. triangularis sternipreparation of the mouse. The long-lasting calcium pateau signal induced by 3,4-DAP (250 μmol) and TEA (10 mmol) blockade of presynaptic potassium channels was irreversibly blocked by micromolar lead concentrations in a concentration-dependent manner. These results suggest that lead antagonizes neuronal calcium channels involved in transmitter release from motor nerve terminals.

Effect of Pinellia ternata Lectin on Membrane Currents of Mouse Motor Nerve Terminals

Pinellia ternata lectin (PTL) extracted from the fresh juice of rhizome of pinellia ternata used as a traditional Chinese medicine facilitated the quantal release of acetylcholine (ACh) in the mouse motor nerve terminals and formed cation channels in artificial lipid bilayer. Here we report the action of PTL on presynaptic membrane currents of motor nerve terminals.The experiments were performed on the intercostal nerve triangularis sterni muscle preparations. By means of the perineurial recording, the effects of PTL on the sodium current in the preterminal part , three potassium currents and two calcium currents generated from the nerve terminals were investigated. The results show that PTL increases voltage-dependent fast Ca2+ current (ICa,f), Na+ current (INa) and Ca2+-acti-vated K+ current (IK,Ca) without action on either the voltage-dependent fast K+ current (IK,f) or the slow K+ current (IK,S). These effects are irreversible, but can be reversed by mannan, the specific binding sugar for PTL.The total action of PTL can be considered to raise [Ca2+], by promoting Ca2+ influx, and then facilitate the neurotransmitter release. To our knowledge, this is the first report about the influence of lectin on presynaptic membrane currents at mammalian motor nerve terminals.

Action of toosendanin on the membrane current of mouse motor nerve terminals

By perineurial recording, the actions of toosendanin on three potassium currents and two calcium currents in the nerve terminal were observed in the intercostal nerve triangularis stein muscle of the mouse. Toosendanin partially blocked the voltage-dependent fast K + current and irreversibly increased the voltage-dependent slow Ca 2+ current. The increase in the Ca 2+ current of the nerve terminal accounts for the facilitation of transmitter release and the antibotulismic effect of toosendanin.