Spontaneous Muscle Action Potentials Research Articles

Effects of IgG anti-GM1 monoclonal antibodies on neuromuscular transmission and calcium channel binding in rat neuromuscular junctions.

Guillain-Barré syndrome is a type of acute inflammatory neuropathy that causes ataxia and is associated with the IgG anti-GM1 antibody. However, the pathogenic role of the IgG anti-GM1 antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. Thus, the aim of the present study was to investigate the effects of the IgG anti-GM1 monoclonal antibody (mAb) on spontaneous muscle action potentials (SMAPs), and the effects of calcium channel blockers, in a rat spinal cord-muscle co-culture system. In addition, the binding of IgG anti-GM1 mAb to calcium channels was investigated in the rat hemidiaphragm. The frequency of SMAPs in the innervated muscle cells was acutely inhibited by the IgG anti-GM1 mAb; however, this effect was blocked by the N-type calcium channel blocker, ω-conotoxin GVIA (30 nM). Furthermore, the P/Q-type calcium channel blocker, ω-agatoxin IVA (10 nM), was found to partially block the IgG anti-GM1 mAb-induced inhibitory effect in the spinal cord-muscle co-culture system. Immunohistochemical analysis of the rat hemidiaphragm indicated that IgG anti-GM1 mAb binding overlapped with anti-Cav2.2 (α1B) antibody binding in the nerve terminal. In addition, IgG anti-GM1 mAb binding partially overlapped with anti-Cav2.1 (α1A) antibody binding. Thus, the results demonstrated that the IgG anti-GM1 mAb binds to calcium channels in the nerve terminals of NMJs. Therefore, the inhibitory effect of IgG anti-GM1 mAb on SMAPs may involve N-type and P/Q-type calcium channels in motor nerve terminals at the NMJ.

Effects of IgM Anti-Galnac-GD1a Monoclonal Antibody on Neuromuscular Transmission and Calcium Channel Binding in the Rat Neuromuscular Junction

Guillain-Barre syndrome with antibodies against ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a) is characterized by a rapid onset of predominantly distal pure motor neuropathy. However, the pathogenic role of anti- GalNAc-GD1a antibody and calcium channels in neuromuscular junctions (NMJs) remains unclear. We investigated the effects of IgM anti-GalNAc-GD1a monoclonal antibody (IgM anti-GalNAc-GD1a mAb) on spontaneous muscle action potentials (SMAP) in a rat spinal cord–muscle co-culture system and the localization of IgM anti-GalNAc- GD1a mAb and calcium channel binding in the rat hemi-diaphragm. Immunohistochemistry of the rat hemidiaphragm showed that IgM anti-GalNAc-GD1a mAb binding overlapped with anti-neurofilament 200 antibody and α- bungarotoxin staining, demonstrating that IgM anti-GalNAc-GD1a mAb was localized at the motor nerve terminal. Moreover, IgM anti-GalNAc-GD1a mAb binding overlapped with anti-Cav2.1 antibody in the nerve terminal. We suggest that the inhibitory effect of IgM anti-GalNAc-GD1a mAb on SMAP is related to the GalNAc-GD1a epitope on P/Q-type calcium channels in motor nerve terminals at NMJs.

Neurophysiological and immunohistochemical studies of IgG anti-GM1 monoclonal antibody on neuromuscular transmission: effects in rat neuromuscular junctions

Guillain-Barré syndrome, which is a variant of acute inflammatory neuropathy, is associated with anti-GM1 antibodies and causes ataxia. We investigated the effects of IgG anti-GM1 monoclonal antibody (IgG anti-GM1 mAb) on spontaneous muscle action potentials in a rat spinal cord-muscle co-culture system and the localization of IgG anti-GM1 mAb binding in the rat hemi-diaphragm. The frequency of spontaneous muscle action potentials in innervated muscle cells was acutely inhibited by IgG anti-GM1 mAb. When cultures were pretreated with GM2 synthase antisense oligodeoxynucleotide, IgG anti-GM1 mAb failed to inhibit spontaneous muscle action potentials, demonstrating the importance of the GM1 epitope in the action of IgG anti-GM1 mAb. Immunohistochemistry of rat hemi-diaphragm showed that IgG anti-GM1 mAb binding overlapped with neurofilament 200 (NF200) antibodies staining, but not α-bungarotoxin (α-BuTx) staining, demonstrating that IgG anti-GM1 mAb was localized at the presynaptic nerve terminal. IgG anti-GM1 mAb binding overlapped with syntaxin antibody and S-100 antibody in the nerve terminal. After collagenase treatment, IgG anti-GM1 mAb and NF200 antibodies did not show staining, but α-BuTx selectively stained the hemi-diaphragm. IgG anti-GM1 mAb binds to the presynaptic nerve terminal of neuromuscular junctions. Therefore, we suggest that the inhibitory effect of IgG anti-GM1 mAb on spontaneous muscle action potentials is related to the GM1 epitope in presynaptic motor nerve terminals at the NMJs.

Effect of sera from seronegative myasthenia gravis patients on neuromuscular junctions

About 85% of patients with generalized myasthenia gravis (MG) have anti-nicotinic acetylcholine receptor (nAChR) antibodies in their sera (seropositive MG; SPMG). The other 15% (seronegative MG; SNMG) are also considered to have antibody-mediated disease, but the nature of the antibodies in SNMG is not fully understood. We investigated the effect of sera from patients with MG on spontaneous muscle action potentials and acetylcholine (ACh)-induced potentials, and we examined the localization of epitopes recognized by SPMG sera or SNMG sera. SPMG sera and SNMG sera inhibited spontaneous muscle action potentials and ACh-induced potentials in the spinal-muscle co-culture system. However, spontaneous muscle action potentials and ACh-induced potentials in the neuromuscular junctions were strongly blocked by SPMG serum, whereas they were weakly blocked by SNMG serum. Both types of sera reacted strongly with the neuromuscular junctions in normal rat muscles, as shown by double immunostaining with serum and α-bungarotoxin. The SPMG epitope remained in the neuromuscular junctions, whereas that of SNMG disappeared after denervation of the sciatic nerve. Therefore, we suggest that the skeletal muscle weakness in SNMG may be due to an interaction with presynaptic neuromuscular transmission and nAChR.

Ca2+ Channel Currents Inhibited by Serum from Select Patients with Guillain-Barré Syndrome

We performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a coculture of rat muscle and spinal cord by exposure to serum, as well as purified IgG, from patients with the acute motor axonal neuropathy (AMAN) variant of Guillain-Barré syndrome (GBS). However, exposure to serum from two patients with the acute inflammatory demyelinating polyneuropathy (AIDP) form of GBS had no effect. Using a whole-cell recording technique, we then investigated the effects of serum and purified IgG from patients with GBS on voltage-dependent calcium channel (VDCC) currents in nerve growth factor-differentiated PC12 cells. Serum from patients with GBS (AMAN) inhibited VDCC currents in PC12 cells, which was fully reversible by washing with the bath solution. Similarly, purified IgG from the serum of two patients with GBS (AMAN) also inhibited VDCC currents in PC12 cells. In contrast, sera from patients with AIDP and healthy volunteers did not affect VDCC currents in PC12 cells. These results suggest that muscle weakness in some patients with GBS might be induced by inhibition of Ca²⁺ channel currents within motor nerve terminals.

Spontaneous muscle action potentials are blocked by N-type and P/Q-calcium channels blockers in the rat spinal cord–muscle co-culture system

This study investigated the effects of the calcium channel blockers nicardipine, calcicludine, ω-conotoxin GVIA, ω-agatoxin IVA, SNX-482, and NiCl on spontaneous muscle action potential of a rat spinal cord–muscle co-culture system. Spontaneous muscle action potential of the innervated muscle cells was blocked by d-tubocurarine (1 μM), but was not significantly affected by the L-type channel blocker nicardipine (100 nM). The neuronal L-type calcium channel blocker, calcicludine (50 and 100 nM), also had no effect on the frequency of spontaneous muscle action potential. However, nicardipine (100 nM) and calcicludine (100 nM) significantly increased the amplitude of muscle action potential. Application of the N-type calcium channel blocker, ω-conotoxin GVIA (50 and 100 nM), and the P/Q-type calcium channel blocker, ω-agatoxin IVA (10, 30, 50, and 100 nM), blocked the frequency and amplitude of spontaneous muscle action potential of the spinal cord–muscle co-cultured cells. In contrast, spontaneous muscle action potential was not affected by the R-type calcium channel blockers SNX-482 (100 nM) or NiCl (500 nM). These results indicate that blockers of N- and P/Q-type voltage-dependent calcium channels inhibit transmitter release at neuromuscular junctions in the rat spinal cord–muscle co-culture system.

Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors.

In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: alpha2betagammadelta (gamma-AChR) or alpha2betaepsilondelta (epsilon-AChR). After birth, gamma-AChRs are replaced by epsilon-AChRs (gamma/epsilon-switch). The two receptors have different conductances and open times. During perinatal period, the long open time gamma-AChRs generate random myofiber action potentials from uniquantal miniature end-plate potentials (mEPPs). epsilon-AChRs are suitable for strong adult muscle activities. Since the effect of the gamma/epsilon-switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate gamma-subunit-deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included epsilon-subunits, and muscle fibers had multiple neuromuscular junctions. Both pre- and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for gamma-AChRs in mediating synaptically-induced action potential activity critical for neuromuscular development.

Flight muscle resting potential and species-specific differences in chill-coma

Resting potentials in Apis mellifera and Drosophila melanogaster flight muscles decrease with falling temperatures. When resting potentials fall to between −37 and −45 mV they activate a final burst of spontaneous muscle action potentials (MAPs). This final burst of MAPs marks the beginning of chill-coma for each species. The temperature at which the final burst occurs for D. melanogaster (7.0±0.9°C) is significantly lower than that of A. mellifera workers (10.6±1.2°C), queens (10.2±0.8°C), and drones (12.8±0.8°C). Prior to chill-coma, MAP amplitudes decrease and durations increase with falling temperatures in both A. mellifera and D. melanogaster. The rate of these changes and the temperatures at which they occur appear to be related to the rate of decline in each species' resting potential. These results suggest that insect chill-coma varies with a species' ability to maintain its resting potential at low temperatures.

Characteristic features of inhibitory junction potentials evoked by single stimuli in the guinea‐pig isolated taenia caeci.

1. Changes in membrane potential of the guinea-pig isolated taenia caeci evoked by single stimuli have been investigated using intracellular recording techniques. Nifedipine (10 microM) was used to arrest spontaneous muscle action potentials. Single stimuli elicited complex junction potentials which consisted of both excitatory and inhibitory components. 2. The excitatory component of the compound junction potential was unaffected by hexamethonium (100 microM) but abolished by atropine (1 microM) and omega-conotoxin GVIA (10-100 nM). 3. In the presence of atropine, single stimuli elicited fast inhibitory junction potentials (IJPs). IJPs were sometimes biphasic during repolarization with a noticeable 'slow tail'. Apamin (30-100 nM) potently inhibited the fast IJP and revealed an underlying slow IJP. 4. The fast IJP was also abolished by omega-conotoxin GVIA (100 nM). However, the slow IJP was insensitive to omega-conotoxin GVIA but was abolished by cadmium (30 microM). 5. Guanethidine (3 microM) and N omega-nitro-L-arginine (10-100 microM) had no detectable effects on either of the IJPs. The dye Reactive Blue 2 reduced the amplitude of the fast IJP but this reduction was associated with a membrane hyperpolarization. 6. The existence of two distinct IJPs in the guinea-pig taenia caeci has been demonstrated. The ability of omega-conotoxin GVIA to selectively abolish the fast IJP leaving the slow IJP intact suggests that separate nerves are involved in mediating these responses.

Ciguatoxin enhances quantal transmitter release from frog motor nerve terminals

1. Ciguatoxin (CTX), a marine toxin produced by the benthic dinoflagellate Gambierdiscus toxicus, is responsible for a complex endemic disease in man known as ciguatera fish poisoning. In the present study we have investigated the effects of purified CTX extracted for Gymnothorax javanicus moray-eel liver on frog isolated neuromuscular preparations with conventional electrophysiological techniques. 2. CTX (1-2.5 nM) applied to cutaneous pectoris nerve-muscle preparations induced, after a short delay, spontaneous fibrillations of the muscle fibres that could be suppressed with 1 microM tetrodotoxin (TTX) or by formamide to uncouple excitation-contraction. 3. In preparations treated with formamide, CTX (1-2.5 nM) caused either spontaneous or repetitive muscle action potentials (up to frequencies of 60-100 Hz) in response to a single nerve stimulus. Recordings performed at extrajunctional regions of the muscle membrane revealed that during the repetitive firing a prolongation of the repolarizing phase of the action potential occurred. At junctional sites the repetitive action potentials were triggered by repetitive endplate potentials (e.p.ps). 4. CTX (2.5 nM) caused a TTX-sensitive depolarization of the muscle membrane. 5. In junctions equilibrated in solutions containing high Mg2+ + low Ca2+, addition of CTX (1.5 nM) first induced an average increase of 239 +/- 36% in the mean quantal content of e.p.ps. Subsequently CTX reduced and finally blocked nerve-evoked transmitter release irreversibly. 6. CTX (1.5-2.5 nM) increased the frequency of miniature endplate potentials (m.e.p.ps) in junctions bathed either in normal Ringer, low Ca2(+)-high Mg2+ medium or in a nominally Ca2(+)-free solution containing EGTA.2+ Extensive washing with toxin-free solutions did not reverse the effect. Furthermore, Cd2 + (0.1 mM), a potent calcium channel blocker, neither antagonized nor abolished the increase in transmitter release caused by CTX. 7. TTX (1 microM) completely prevented the effect of CTX (2.5nM) on m.e.p.p. frequency. This effect was independent of the presence of extracellular Ca2 +. TTX, when added after CTX (2.5 nM) exposure, antagonized the increase in m.e.p.p. frequency. The antagonism was complete in Ca2 +-free medium. These results strongly suggest that increased permeability of the nerve terminal to Na+ is responsible for the increase in m.e.p.p. frequency caused by CTX. It is likely that CTX may trigger calcium release from internal stores due to an increase of intraterminal Na+ concentration. 8. It is concluded that CTX exerts, in the nanomolar concentration range, a selective action on sodium channels of the neuromuscular junction causing both pre- and postsynaptic effects.

Measurements of the membrane potential of arterial smooth muscle in anesthetized animals and its relationship to changes in artery diameter

Intracellular recordings of membrane potential were made from the smooth muscle of small (90–200 μm i.d.) arteries in the ears of anaesthetized guinea pigs and rabbits. Perivascular nerve stimulation evoked excitatory junction potentials and action potentials in the smooth muscle. No spontaneous muscle action potentials were recorded. Local block of perivascular nerve activity with tetrodotoxin caused a vasodilatation and a small hyperpolarization of the smooth muscle. Prazosin caused a smaller vasodilatation and no hyperpolarization. It was concluded that some of the nerve-mediated arterial tone may be caused by noradrenaline acting α 1-receptors, and some by some other mechanism.

In vivo development of cholinesterase at a neuromuscular junction in the absence of motor activity in Xenopus laevis.

Embryos of Xenopus laevis were selected prior to the onset of innervation and were raised for 2 days in the anaesthetic tricaine methanesulphonate (200 micrograms/ml). The gross development of these tricaine-reared animals appeared normal despite the absence of spontaneous motor activity and the lack of motor responses to prodding with a pin. Motor activity quickly appeared when the anaesthetic was withdrawn. Intracellular recording from the myotomes of intact, tricaine-maintained animals failed to reveal any spontaneous muscle action potentials. Synaptic potentials increased in frequency and amplitude upon withdrawing tricaine, but resting potentials remained unchanged. Cholinesterase activity, detected histochemically, was observed at the ends of the myotomes, the main site of innervation. The intensity of the histochemical reaction product at these sites appeared to be about as great in the myotomes of tricaine-reared animals as in control myotomes. Miniature end-plate currents (m.e.p.c.s), examined by focal external recording, declined with a time constant of 2.9 +/- 0.2 ms (mean +/- S.E. of mean) in the myotomes of tricaine-reared animals (stages 40-41). The time constants in the myotomes of control animals were 1.8 +/- 0.1 ms at stages 40-41 and 8.7 +/- 0.7 ms at stages 24-26 (shortly after the onset of innervation). The anticholinesterase neostigmine doubled m.e.p.c. time constants in the myotomes of tricaine-reared animals as well as in control myotomes at stages 40-41. It is concluded that motor activity is not required for the in vivo development of physiological levels of synaptic cholinesterase in Xenopus myotomal muscle.

Electrophysiological studies of thymectomized and nonthymectomized acetylcholine receptor-immunized animal models of myasthenia gravis

White New Zealand rabbits were immunized with acetylcholine receptor (AChR) purified from the electric organs of either the Electrophorous electricus (eel) or Torpedo californica (torpedo). Approximately 4 days after a second injection of 100 μg AChR in complete Freund's adjuvant, the immunized animals developed weakness of the peripheral muscles which was aggravated by repetitive activity and was partially alleviated by anticholinesterases such as edrophonium or pyridostigmine. Electromyograms taken from the anterior tibialis muscle demonstrated a decrement of the compound action potentials at a stimulation frequency of 5 Hz which was reversed by the intravenous administration of 1 mg pyridostigmine. If the animals were not treated with anticholinesterases they died within a couple of days. Biopsies of the intercostal muscle were done under Nembutal anesthesia, and intracellular recordings revealed subliminal end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) of greatly diminished amplitudes. At some neuromuscular junctions no MEPPs were detected, and in other junctions MEPPs were seen but no EPP was evoked upon nerve stimulation. Muscles from two immunized rabbits showed spontaneous muscle action potentials occurring at 1 to 2 Hz. Serum from the rabbits immunized with torpedo or eel AChR, when applied to in vitro preparations of the eel electroplaque or frog cutaneous pectoris muscle, caused a decrease in postjunctional membrane sensitivity to carbamylcholine and reduced the MEPP and EPP amplitudes even after heat inactivation of the serum complement (56°C for 30 min). In addition to a decrease in chemosensitivity, an enhancement of receptor desensitization occurred in the neuromuscular junctions of serum-treated frog muscles. Thymectomies on adult rabbits prior to the immunization had the effect of delaying the onset of myasthenia in two of six rabbits, and in three of six rabbits only mild myasthenia occurred. The blood antibody titers to AChR were similar in both the thymectomized and nonthymectomized rabbits.