Frequency Of Miniature End-plate Potentials Research Articles

Excitatory neurotoxic properties of pontamine sky blue make it a useful tool for examining the functions of focal brain parts.

Pontamine sky blue (PSB) is used in brain studies to mark the position of microelectrode and micropipette tips. However, few studies have been made on the effects of PSB on neurons; therefore we examined these effects. When puffed on isolated sensory ganglion cells of rats, PSB increased membrane conductance, depolarized membrane potential, and reduced the amplitude of action potentials. When dripped on frog sympathetic ganglion, much like hexamethonium, PSB decreased the amplitude of compound action potentials of the postganglionic strand. A bath application of PSB to sartorius muscle fibers that had been treated with tetrodotoxin depolarized the membrane potential and increased the frequency and amplitude of miniature end-plate potentials. All these effects were reversible. When injected into the rat's pontine part corresponding to the location of the canine pontine defecation reflex center, PSB produced repetitive colorectal contractions and irreversibly abolished them in response to anal-canal stimulation. The excitatory and blocking effects of PSB and its staining ability make it a useful tool for examining the functions of focal brain parts.

Ca2+ channels and synaptic transmission at the adult, neonatal, and P/Q-type deficient neuromuscular junction.

Different types of voltage-activated Ca(2+) channels have been established based on their molecular structure and pharmacological and biophysical properties. One of them, the P/Q-type, is the main channel involved in nerve-evoked neurotransmitter release at neuromuscular junctions and the immunological target in Eaton-Lambert Syndrome. At adult neuromuscular junctions, L- and N-type Ca(2+) channels become involved in transmitter release only under certain experimental or pathological conditions. In contrast, at neonatal rat neuromuscular junctions, nerve-evoked synaptic transmission depends jointly on both N- and P/Q-type channels. Synaptic transmission at neuromuscular junctions of the ataxic P/Q-type Ca(2+) channel knockout mice is also dependent on two different types of channels, N- and R-type. At both neonatal and P/Q knockout junctions, the K(+)-evoked increase in miniature endplate potential frequency was not affected by N-type channel blockers, but strongly reduced by both P/Q- and R-type channel blockers. These differences could be accounted for by a differential location of the channels at the release site, being either P/Q- or R-type Ca(2+) channels located closer to the release site than N-type Ca(2+) channels. Thus, Ca(2+) channels may be recruited to mediate neurotransmitter release where P/Q-type channels seem to be the most suited type of Ca(2+) channel to mediate exocytosis at neuromuscular junctions.

Inositol derivatives modulate spontaneous transmitter release at the frog neuromuscular junction

One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP 3 and its metabolites 1,3,4,5-tetrakisphosphate (IP 4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP 6). Previous reports indicate that high inositol polyphosphates (IP 4 and IP 6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP 4 and IP 6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K + at frog motor nerve endings. In resting conditions, IP 4 and IP 6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP 4-mediated MEPP frequency increase by 60% and abolished the IP 6-mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP 4 and IP 6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP 3, but not other second messengers releasing Ca 2+ from internal Ca 2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca 2+ release vs. metabolism to IP 4 and IP 6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP 3), which is not converted to IP 4 or IP 6. 3F-IP 3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K +-Ringer solution, IP 4 and IP 6 have similar effects, except decreasing MEPP frequency at a higher concentration (10 −4 M). We conclude that (1) high inositol polyphosphates may represent a link between IP 3 and cAMP pathways; (2) the IP 3-induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP 4 and IP 6-mediated MEPP frequency increases, but may be involved in MEPP size.

Immunoglobulins inhibit pathophysiological effects of anti-GQ1b-positive sera at motor nerve terminals through inhibition of antibody binding.

High-dose intravenous immunoglobulin (IVIg) is an effective treatment for many antibody-mediated neuromuscular diseases, suggesting that IVIg directly interferes with the pathways through which the pathogenic antibodies exert their effects. However, the precise mechanisms of action are unclear. Serum anti-GQ1b antibodies are strongly associated with ophthalmoplegia in patients with Miller Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS). They induce complement-mediated alpha-latrotoxin-like effects on mouse neuromuscular junctions (NMJs) ex vivo, comprising transient muscle fibre twitching, due to a dramatic increase in the frequency of miniature end-plate potentials (spontaneous quantal acetylcholine release), followed by transmission block. To clarify the mechanisms by which IVIg may act in MFS and GBS, we investigated its effects on the interaction of anti-GQ1b antibodies with GQ1b in vitro and on anti-GQ1b antibody-mediated NMJ injury ex vivo, using anti-GQ1b-positive serum samples from MFS/GBS patients. We show that IVIg inhibits the binding of anti-GQ1b antibodies to GQ1b, thereby preventing complement activation and subsequent pathophysiological effects in our ex vivo mouse NMJ model. These results provide further support for the hypothesis that anti-ganglioside antibodies are the pathogenic factors in MFS/GBS and show that this NMJ model provides a suitable system for investigating the therapeutic effects of IVIg in antibody-mediated neuromuscular diseases.

Extra- and intracellular sphingosylphosphorylcholine promote spontaneous transmitter release from frog motor nerve endings.

Similar to phosphatidylinositol bisphosphate, sphingomyelin breakdown generates several lipids, including sphingosylphosphorylcholine (SPC), that are putative signaling molecules. The present study was undertaken to evaluate the involvement of SPC in transmitter release process. Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of SPC on neurosecretion in the form of miniature endplate potentials (MEPPs) were assessed. Extracellular application of SPC mixture (D,L-SPC) at 1, 10, and 25 microM increased the MEPP frequency by 68, 96, and 127%, respectively. D-erythro-SPC (dissolved in dimethyl sulfoxide but not coupled to bovine serum albumin), but not L-threo-SPC, was active extracellular; the former (at 10 microM) increased the MEPP frequency by 143%. D-erythro-SPC treatment did not significantly change the median amplitude or frequency-distribution of MEPPs. Intracellular delivery via liposomes, in which 10, 100, or 1000 microM SPC mixture was entrapped in liposomal aqueous phase, induced a concentration-dependent increase in MEPP frequency of 45, 91, and 100%, respectively. D-erythro-SPC and L-threo-SPC at the concentration of 100 microM increased the MEPP frequency by 117 and 67%, respectively, or 91 and 61%, respectively, when coupled to bovine serum albumin. Pretreatment with thapsigargin significantly reduced but did not abolish the effects of extracellular D-erythro-SPC (10 microM) or liposomes containing 100 microM D-erythro-SPC. Liposomes filled with 100 microM D-myo-inositol 1,4,5-trisphosphate (IP3) enhanced the MEPP frequency to the same magnitude as 100 microM D-erythro-SPC entrapped in liposomes. However, administration of 100 microM D-erythro-SPC and IP3 entrapped in the same liposomes enhanced the MEPP frequency by 70%, which was less than that produced by these two compounds alone. The result provides the first electrophysiological evidence that SPC can modulate transmitter release by an extra- or intracellular action at the frog motor nerve ending.

Effects of ascorbic acid on lead induced alterations of synaptic transmission and contractile features in murine dorsiflexor muscle

Lead is a common environmental toxin that affects neuromuscular junction and potentially might cause muscle weakness. Antioxidants like ascorbic acid may protect against lead induced myopathy. The present study measured isometric twitch tensions (evoked either directly by muscle stimulation or indirectly by nerve stimulation) to study effects of ascorbic acid on lead induced alterations at murine dorsiflexor skeletal muscle. Resting membrane potentials (RMPs), endplate potentials (EPPs) and miniature endplate potentials (MEPPs) were also recorded. Forty animals were divided into four groups of n = 10 each. (10 control, 10 lead alone, 10 ascorbic acid alone, 10 lead treated plus ascorbic acid). Lead (1 mg/kg) i.p, was administered daily for 2 weeks before the recording day and ascorbic acid (200 mg/kg, i.p) was given daily for 3 weeks prior to the experiment day. Lead treatment reduced twitch tension significantly (from 4.3 ± 0.5 g to 2.7 ± 0.2 g) and delayed half time of decay compared to the control. Similarly MEPPs frequencies were reduced following lead treatment. Application of ascorbic acid prevented twitch tension reduction in lead treated mice (3.3 ± 0.3 g) and reversed lead induced delay in half time of decay. The negative actions of lead treatment on MEPPs frequencies were also modified with ascorbic acid. It appears that ascorbic acid exerts a protective role against lead induced peripheral nerve and muscle dysfunction. This effect of ascorbic acid on lead induced neuromyopathy is probably mediated via a free radical scavenging mechanism or modification of Ca 2+ homeostasis.

Nifedipine-mediated mobilization of intracellular calcium stores increases spontaneous neurotransmitter release at neonatal rat motor nerve terminals.

The modulation of spontaneous release of acetylcholine by specific Ca2+ channel blockers was studied at neonatal rat neuromuscular junction. During early postnatal periods (0-4 days), blockers of N- and P/Q-type Ca2+ channels did not affect miniature endplate potential (MEPP) frequency. Unexpectedly, treatment with the L-type Ca2+ channel antagonist nifedipine, although not when treated with isradipine, nitrendipine, or calciseptine, resulted in strong increase in MEPP frequency. The potentiation effect of nifedipine was dose-dependent with a 56-fold maximum effect with 15 microM. The effect decreased during the first two postnatal weeks and disappeared by the third. The effect of nifedipine was not dependent on extracellular Ca2+ and was not altered by the presence of other Ca2+ channel blockers. In contrast, it was abolished by depleting intracellular Ca2+ stores with 2 microM thapsigargin and was partially inhibited by 10 microM ryanodine. In conclusion, we report a new ryanodine receptor-mediated effect of nifedipine on neonatal neuromuscular junction that may indicate the developmental expression of a specific receptor channel that interacts with intracellular Ca2+ stores. This effect of nifedipine should also be considered when using this drug as either a therapeutic or a research tool.

The vasoactive peptide urotensin II stimulates spontaneous release from frog motor nerve terminals.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca(2+) three-fold had no effect on the MEPP frequency increase to 25 microM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca(2+) caused a 61% reduction in the MEPP frequency increase to 25 microM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 microM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP(3)) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 microM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP(3) and cyclic AMP/protein kinase A.

Iberiotoxin-induced block of Ca2+-activated K+ channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals.

The role which Ca(2+)-activated K(+) (K(Ca)) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca(2+) channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 microM nimodipine, a dihydropyridine L-type Ca(2+) channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 microM S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 +/- 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 +/- 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 +/- 10.4% of control. Coapplication of 1 microM Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional K(Ca) channels unmasks normally silent L-type Ca(2+) channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization.

Effects of Alpha‐Tocopherol on Diabetes‐Induced Alterations of Synaptic Transmission and Contractile Features in Murine Dorsiflexor Muscle

Diabetes mellitus affects skeletal muscle and free radicals may be implicated in the manifestation of diabetes complications. The present study investigated effects of alpha‐tocopherol on diabetic dorsiflexor muscle via recording resting membrane potentials (RMPs), endplate potentials (EPPs), miniature endplate potentials (MEPPs) and isometric twitch tensions. Forty mice were divided randomly into two groups (n = 20). One group served as control and the other was injected once with streptozotocin (STZ) solution (60 mg/kg, i.p) to induce diabetes. The animals were then divided further into two subgroups (n = 10). Alpha‐tocopherol (100 mg/kg, i.p) was administered daily to one control and one diabetic group for 3 weeks prior to recording day. Experiments were conducted 4 weeks following diabetes induction. Isometric twitch tension was measured in anaesthetized mice (2 mg/g urethane, i.p) via a transducer connected to a computer system. Resting membrane potentials and MEPPs were measured by utilizing the intracellular recording method. Compared to control, diabetic mice showed reduced twitch tension (4.4 ± 0.4 g control vs. 2.5 ± 0.3 g diabetic) and demonstrated delayed half time of decay. Diabetic flexor muscle also displayed significant reduction in MEPPs frequencies with no changes in RMPs. Alpha‐tocopherol reversed tension reduction in diabetic mice (from 2.5 ± 0.3 to 3.8 ± 0.4 g), impacted delayed half time of decay and reversed reduction in MEPPs frequencies. Alpha‐tocopherol exerts a protective role against diabetes‐induced peripheral muscle dysfunction. This effect is probably mediated via a free radical scavenging mechanism or modification of Ca2+ homeostasis.

Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity.

We report here the purification of glycerotoxin from the venom of Glycera convoluta, a novel 320 kDa protein capable of reversibly stimulating spontaneous and evoked neurotransmitter release at the frog neuromuscular junction. However, glycerotoxin is ineffective at the murine neuromuscular junction, which displays a different subtype of voltage- dependent Ca(2+) channels. By sequential and selective inhibition of various types of Ca(2+) channels, we found that glycerotoxin was acting via Ca(v)2.2 (N-type). In neuroendocrine cells, it elicits a robust, albeit transient, influx of Ca(2+) sensitive to the Ca(v)2.2 blockers omega-conotoxin GVIA and MVIIA. Moreover, glycerotoxin triggers a Ca(2+) transient in human embryonic kidney (HEK) cells over-expressing Ca(v)2.2 but not Ca(v)2.1 (P/Q-type). Whole-cell patch-clamp analysis of Ca(v)2.2 expressing HEK cells revealed an up-regulation of Ca(2+) currents due to a leftward shift of the activation peak upon glycerotoxin addition. A direct interaction between Ca(v)2.2 and this neurotoxin was revealed by co-immunoprecipitation experiments. Therefore, glycerotoxin is a unique addition to the arsenal of tools available to unravel the mechanism controlling Ca(2+)-regulated exocytosis via the specific activation of Ca(v)2.2.

Sphingosine 1-phosphate enhances spontaneous transmitter release at the frog neuromuscular junction.

Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of sphingosine 1-phosphate (S1-P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1-P (1 and 30 micro M) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10(-5), 10(-4) or 10(-3) M S1-P was associated with a concentration-dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of 'giant' MEPPs. Pre-exposure of the preparations to S1-P 10(-5) but not 10(-8) M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1-P (10(-4) M)-filled liposomes on MEPP frequency. Thus, intracellular S1-P receptors seem to undergo 'desensitization' to higher concentrations of S1-P. The result provides the first evidence that S1-P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

New evidence for a presynaptic action of prednisolone at neuromuscular junctions.

The action of prednisolone at the neuromuscular junction was studied in mouse isolated phrenic nerve-diaphragm and rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. Prednisolone (0.03 mM and 0.3 mM) did not alter the twitch-tension in phrenic nerve-diaphragm preparations after 120 min, but increased the frequency (170 +/- 4%) and amplitude (200 +/- 13%) of miniature end-plate potentials. Quantal content was not influenced by the glucocorticoid treatment. Prednisolone (400 microg/kg) did not change the twitch-tension in rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. However, this steroid (0.3 mM) prevented the neuromuscular blockade by d-tubocurarine (1.45 microM) in mouse preparations by 70 +/- 10% (P < 0.05). A similar effect (82 +/- 6% protection, P < 0.05) occurred in rats treated with prednisolone (400 microg/kg) before d-tubocurarine (225 microg/kg). In phrenic nerve-diaphragm preparations, prednisolone (0.3 mM) increased (13 +/- 4%, p < 0.05) the twitch-tension in the presence of beta-bungarotoxin (1 microM), and prevented the blockade produced by this toxin (0.15 microM) in its third phase of action. This presynaptic facilitatory effect may contribute to the usefulness of prednisolone in myasthenia gravis.

Neuromuscular action of Bothrops lanceolatus (Fer de lance) venom and a caseinolytic fraction

A protein capable of inducing neuromuscular blockade in avian preparations and of depolarizing mouse diaphragm muscle was isolated from Bothrops lanceolatus venom using gel filtration and ion-exchange chromatography. The purified protein was a single chain polypeptide with an estimated molecular mass of 27.5 kDa by SDS–PAGE and had caseinolytic activity (13.3 units/mg), but no phospholipase A 2. B. lanceolatus venom (50 μg/ml) and the caseinolytic protein (20 μg/ml) produced contracture and progressive irreversible blockade (50% in 25±5 min (SEM) and 45±15 min, respectively), in indirectly stimulated chick biventer cervicis preparations. The contractile responses to acetylcholine (ACh; 37 and 74 μM, n=6) were inhibited by venom and the caseinolytic protein, whereas those to potassium (13.4 mM, n=6) were not. Membrane resting potential measurements in mouse hemidiaphragm preparations showed that B. lanceolatus venom and the purified protein caused depolarization which was prevented by d-tubocurarine (14.6 mM). The venom produced a slight increase in the amplitude and frequency of miniature end-plate potentials, but this effect was not seen with the purified fraction. These results suggest that the purified protein acts exclusively post-synaptically.

Effect of amyotrophic lateral sclerosis serum on calcium channels related to spontaneous acetylcholine release.

The aim of this work was to further investigate the effect of sera from sporadic amyotrophic lateral sclerosis (ALS) patients on miniature end-plate potentials (MEPP) frequency, by the mouse passive transfer model, and to study whether the transferred serum induces any change in the sensitivity of the L-type voltage-dependent calcium channels (VDCC) to its specific blocker Nitrendipine. A total of 35 CF1 mice were divided into 3 groups: (a) ALS group receiving sera from 15 patients that had been clinically and electromyographycally diagnosed as having sporadic ALS; (b) normal group receiving sera from 13 healthy volunteers and from 3 disease control patients, and (c) control group, which was kept untreated. Animals in groups (a) and (b) received daily intraperitoneal injection of 0.5-1ml of serum for 3 days, and 24h later the left hemidiaphragm was excised for electrophysiological recordings. Analysis of MEPPs frequency recorded from ALS group showed that 3 of them induced an increase in spontaneous neurotransmitter release while in 4 a decrease was observed, suggesting that sera alter spontaneous secretion as result of an increased or decreased Ca(2+) influx through the normally involved N-type or L-type VDCC, respectively. When the effect of Nitrendipine, an L-type VDCC blocker, was studied on ALS sera-injected mice, we found variable responses to the drug: only two mice showed control sensitivity to Nitrendipine, while in 7 its action was lower and surprisingly in 4 was greater than that without the drug. These results suggest that ALS sera contain factor(s) that are able to modify spontaneous neurotransmitter release by altering calcium current through L-type and N-type VDCC, and even inducing changes in the sensitivity to the L-type VDCC blocker.

Adenosine depresses a Ca(2+)-independent step in transmitter exocytosis at frog motor nerve terminals.

The depressant action of adenosine on acetylcholine release at frog motor nerve terminals was studied by intracellular recording and Ca(2+)-imaging techniques. Adenosine (200 microm) quickly and reversibly decreased the amplitude and quantal content of end-plate potentials (EPPs) with no change in quantal size in a low-Ca(2+), high-Mg(2+) solution, and EPP amplitude in normal Ringer containing d-tubocurarine. Likewise, adenosine (200 microm) reduced miniature EPP (MEPP) frequency, but not amplitude, in a high-K(+) (6 mm) solution. Adenosine (40-200 microm), however, did not affect single or repetitive impulse(s)-induced rises in Ca(2+) in the nerve terminals or its basal level. Adenosine (100-200 microm) reduced the Ca(2+)-independent enhancement of MEPP frequency caused by hypertonicity. EPPs induced by tetanic stimulation (33 Hz) in Ringer with d-tubocurarine initially increased in amplitude within 10 stimuli and then declined to the minimum. Adenosine (200 microm) decreased EPP amplitude in the initial phase of the tetanus, but enhanced it in the middle phase, thus prolonging the decay of EPP amplitude. The total sum of these EPPs, reflecting the readily releasable pool of vesicles and its refilling, however, was not changed. The results suggest that adenosine inhibits a Ca(2+)-independent step of transmitter exocytosis at frog motor nerve terminals.

THE IMPACT OF VITAMIN C ON DIABETES INDUCED ALTERATIONS AT MURINE NEUROMUSCULAR JUNCTION

Physiological functions of skeletal muscle are compromised in diabetes. This may involve free radical mechanisms and may be reversed by antioxidants. We have studied effects of vitamin C on twitch tension, resting membrane potential (RMP) and miniature endplate potentials (MEPPs) frequencies in dorsiflexor muscle of diabetic murine. Forty mice were divided randomly into 2 groups (n=20 each). One group served as control and the other was injected once with streptozotocin (STZ) solution (60 mg/kg, i.p) to induce diabetes. The animals were then divided further into two subgroups (n=10 each). Vitamin C (200 mg/kg, i.p) was administered daily to one control and one diabetic group for three weeks prior to recording day. Experiments were conducted four weeks following diabetes induction. Isometric twitch tension (evoked directly by muscle stimulation and indirectly by nerve stimulation) was measured in urethane anesthetized (2 mg/g, i.p) mice via a transducer connected to computer system. Utilizing intracellular recording method, resting membrane potential RMP and MEPPs frequencies were also measured. Compared to control, diabetic mice showed reduced twitch tension (4.2±0.5 g control versus 2.6±0.2 g diabetic) and demonstrated delayed half time of decay. Diabetic flexor muscle also displayed significant reduction in MEPPs frequencies with no changes in RMP. Vitamin C reversed tension reduction in diabetic mice (from 2.6±0.2 g to 3.9±0.3 g), impacted delayed half time of decay and increased MEPPs frequencies. Vitamin C improves diabetes-induced nerve and muscle dysfunction possibly via a free radical scavenging mechanism.

The effects of glutamate on spontaneous acetylcholine secretion processes in the rat neuromuscular synapse.

Experiments on rat diaphragm muscles showed that glutamate (10 microM-1 mM) had no effect on the mean frequency, interspike intervals, and amplitude-time characteristics of miniature endplate potentials, but had a suppressive action on non-quantum secretion (the intensity of which was assessed in terms of the H effect). The effect of glutamate was markedly concentration-dependent and was completely overcome by blockade of NMDA receptors, inhibition of NO synthase, and by binding of NO molecules in the extracellular space by hemoglobin. It is suggested that glutamate can modulate the non-quantum release of acetylcholine, initiating the synthesis of NO molecules in muscle fibers via activation of NMDA receptors followed by the retrograde action of NO on nerve terminals.

IgM monoclonal antibody against terminal moiety of GM2, GalNAc-GD1a and GalNAc-GM1b from a pure motor chronic demyelinating polyneuropathy patient: effects on neurotransmitter release

We describe a patient with a pure motor chronic demyelinating polyneuropathy with an IgM monoclonal component showing anti-GM2, GalNAc-GD1a and GalNAc-GM1b reactivity whose common epitope appears to be -[GalNAcβ1-4Gal(3-2αNeuAc)β1]. We used intracellular recording to study how IgM from this patient affected neurotransmitter release in the mouse diaphragm in vitro. Adding serum (and specifically, the purified monoclonal IgM component) blocked the nerve-evoked response in both quantal content and evoked endplate potential (EPP) amplitude in a complement-independent and reversible manner. The IgM increased the frequency of spontaneous miniature endplate potentials (MEPPs) in a complement-dependent and reversible manner but had no effect on MEPP amplitude.

Presynaptic congenital myasthenic syndrome due to quantal release deficiency.

To provide clinical, electrophysiologic, and ultrastructural findings in three patients with a presynaptic congenital myasthenic syndrome (CMS). Familial infantile myasthenia and paucity of synaptic vesicles are the only two fully characterized CMS. We are describing here three patients with another form of presynaptic CMS characterized by deficiency of the action potential-dependent release without reduction of the spontaneous release of neurotransmitter from the nerve terminal. The authors performed electromyography and anconeus muscle biopsies that included intracellular recordings and electron microscopy of the neuromuscular junction in three patients with presynaptic CMS. They also sequenced part of the P/Q-calcium alpha(1)-subunit gene (CACNA1A) and the acetylcholine receptor subunit (AChR) genes in these patients. In these patients there were additional neurologic findings including nystagmus and ataxia. In all three patients the end-plate potential quantal content (m) was markedly reduced but neither the amplitudes nor the frequencies of miniature end-plate potentials were diminished. Ultrastructurally, postsynaptic end-plate folds, nerve terminal size, and synaptic vesicle number were normal but double-membrane-bound sacs containing synaptic vesicles were present in the nerve terminal of all three patients. The screening of reported pathogenic mutations in the CACNA1A and a mutational analysis of AChR subunit genes were negative. This form of CMS appears to result only from a deficiency of the quantal release of neurotransmitter that may be due to an abnormal calcium mechanism or impaired endocytosis and recycling of synaptic vesicles.