Frog Nerve-muscle Preparations Research Articles

Purification of a post-synaptic neurotoxic phospholipase A 2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A 2 (PLA 2) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD 50 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA 2 by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A 2 activity of NN-XIa-PLA 2, isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA 2: WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA 2 isoforms from the venom to varying extent. The interaction of the WSG with the PLA 2 is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA 2 using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA 2 has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.

A chloride channel blocker reduces acetylcholine uptake into synaptic vesicles at the frog neuromuscular junction

A key mechanism for loading acetylcholine (ACh +) into synaptic vesicles uses energy to transport H + into the vesicle interior and then exchanges H + for ACh +. This mechanism requires anions to follow the H + into the vesicles to prevent the building up of an overwhelming electrical gradient across the vesicle membrane. Frog nerve-muscle preparations were treated with hypertonic solution in which sodium gluconate was the major constituent, which substantially increases the sizes of the quanta by increasing their ACh + content. The Cl − channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) antagonized the increases in quantal size, so it seems likely that Cl − follows H + to prevent the buildup of a potential gradient across the vesicular membrane.

The influence of quantal content on the time course of the endplate current in frogs.

The aim of this study was to find out whether control and facilitated endplate current (EPC) of a curarized frog nerve-muscle preparation stimulated with paired impulses decay at the same time constant. The experiments revealed that the decay of the greater second EPC was slower in nearly half of the cases. This half of the results suggests that a larger quantity of the released transmitter and, consequently, a higher density of open channels promote a cooperative effect among them. The end effect can be a longer mean lifetime of open acetylcholine channels. In the other half of the experiments, the mean lifetime of open channels seems to be independent of the agonist concentration.

The strategy used by some piscivorous cone snails to capture their prey: the effects of their venoms on vertebrates and on isolated neuromuscular preparations

Three piscivorous Conus species, C. ermineus, C. consor and C. catus were acclimatized in aquaria. The study of their strategy to capture the prey and details of their radula's morphology revealed that all of them used a `hook and line' strategy which consists of immobilizing the prey rapidly before engulfing it. The venoms from these piscivorous species clearly elicit, when injected into fish, an excitotoxic shock characterized by a sudden tetanus of the prey. In mammals, the venoms induce both flaccid paralysis via i.p. injection and seizures via i.c.v. injection. Intracellular recordings from frog nerve-muscle preparations revealed that the venoms from these Conus species first caused spontaneous synaptic potentials which in turn triggered muscle action potentials. Such spontaneous activity is due to an increased nerve terminal excitability. In addition, the venoms suppressed neuromuscular transmission probably by blocking postsynaptic nicotinic acetylcholine receptors. No direct effect of these Conus venoms was observed on the membrane of skeletal muscle fibres. In conclusion, C. ermineus, C. consor and C. catus, which have not securely tethered their prey used a mixture of toxins which target both pre-and postsynaptic elements of the neuromuscular junction and which produce rapid immobilization of their prey.

An O-glycosylated neuroexcitatory conus peptide.

We purified and characterized a novel peptide from the venom of the fish-hunting cone snail Conus striatus that inhibits voltage-gated K+ channels. The peptide, kappaA-conotoxin SIVA, causes characteristic spastic paralytic symptoms when injected into fish, and in frog nerve-muscle preparations exposed to the toxin, repetitive action potentials are seen in response to a single stimulus applied to the motor nerve. Other electrophysiological tests on diverse preparations provide evidence that is consistent with the peptide blocking K+ channels. The peptide has three disulfide bonds; the locations of Cys residues indicate that the spastic peptide may be the first and defining member of a new family of Conus peptides, the kappaA-conotoxins, which are structurally related to, but pharmacologically distinct from, the alphaA-conotoxins. This 30 AA tricyclic toxin has several characteristics not previously observed in Conus peptides. In addition to the distinctive biological and physiological activity, a novel biochemical feature is the unusually long linear N-terminal tail (11 residues) which contains one O-glycosylated serine at position 7. This is the first evidence for O-glycosylation as a posttranslational modification in a biologically active Conus peptide.

The effect of antibodies directed against the crotoxin on the frog nerve-muscle preparation

The effect of antibodies directed against the crotoxin on the frog nerve-muscle preparation

Illumination partly reverses the postsynaptic blockade of the frog neuromuscular junction by the styryl pyridinium dye RH414.

The styryl pyridinium dye RH414, which we have used recently for optical monitoring of synaptic vesicle membrane trafficking in frog motor nerve terminals, reversibly inhibited contractions in the frog nerve-muscle preparations used for those studies. We report here the results of experiments into the basis of this inhibition and how the blocking effects can be modulated by incident light, by using conventional intracellular recording and iontophoretic techniques. Bath application of 5-42.5 microM RH414 blocked nerve-evoked twitches in frog cutaneus pectoris muscles, although subthreshold endplate potentials and miniature endplate potentials could still be recorded. In the presence of the dye, illumination of the recording area with light from a mercury arc lamp over a wide range of wavelengths (340-560 nm) potentiated the amplitude of endplate potentials, miniature endplate potentials, and depolarizations resulting from iontophoretic application of acetylcholine. The magnitude of both the light-induced disinhibition of endplate and iontophoretic potentials increased with the intensity of the illumination and developed exponentially with a time constant of several hundred milliseconds. Both the dye-induced inhibition and the light-induced disinhibition disappeared after washing in dye-free physiological saline. Muscle fibre resting membrane potential, input conductance and miniature endplate potential frequency, however, were not affected by these manipulations. These data are consistent with a specific, curare-like interaction of the dye with acetylcholine receptors which can be modulated by light.

P-143 - Propylene glycol-induced skeletal muscle excitation.

The effects of propylene glycol (PG) on frog nerve-muscle preparations were examined to determine whether it has an effect on neuromuscular transmission and, if so, to elucidate the mode of the action. PG (5%, v/v) increased the twitch tension to over twice the control value. PG at concentrations above 0.2% significantly increased the amplitude of the endplate potential. PG (1%) raised the frequency of the miniature endplate potentials and increased their amplitude. These results show that PG both facilitates transmitter release from the nerve terminals and raises the acetylcholine sensitivity of the muscle endplate.

Propylene glycol-induced skeletal muscle excitation

The effects of propylene glycol (PG) on frog nerve-muscle preparations were examined to determine whether it has an effect on neuromuscular transmission and, if so, to elucidate the mode of the action. PG (5%, v/v) increased the twitch tension to over twice the control value. PG at concentrations above 0.2% significantly increased the amplitude of the endplate potential. PG (1%) raised the frequency of the miniature endplate potentials and increased their amplitude. These results show that PG both facilitates transmitter release from the nerve terminals and raises the acetylcholine sensitivity of the muscle endplate.

Presynaptic localization of ω-conotoxin-sensitive calcium channels at the frog neuromuscular junction

By the use of anti-ω-CTx antibodies in indirect immunofluorescence we demonstrated the presence of ω-CTx binding sites in the presynaptic compartment of frog nerve-muscle preparations. The images we obtained indicate that ω-CTx-sensitive channels are clustered at discrete sites corresponding in distribution to active zones.

O-269 - Contractile suppression induced by glucose in frog nerve-muscle preparations.

O-269 - Contractile suppression induced by glucose in frog nerve-muscle preparations.

Effect of electromagnetic induction on impulse conduction in the frog nerve-muscle preparation

Conference Article| December 01 1989 Effect of electromagnetic induction on impulse conduction in the frog nerve-muscle preparation F. A. WALI; F. A. WALI *Respiratory Laboratory, The Hospital for Sick Children, Great Ormond Street, London WC1N Search for other works by this author on: This Site PubMed Google Scholar A. I. J. BRAIN A. I. J. BRAIN †Department of Anaesthetics, St Andrews Hospital, London E3, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1989) 17 (6): 1088–1089. https://doi.org/10.1042/bst0171088 Article history Received: April 05 1989 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation F. A. WALI, A. I. J. BRAIN; Effect of electromagnetic induction on impulse conduction in the frog nerve-muscle preparation. Biochem Soc Trans 1 December 1989; 17 (6): 1088–1089. doi: https://doi.org/10.1042/bst0171088 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: ES, electrical stimulation, EMI, electromagnetic induction This content is only available as a PDF. © 1989 Biochemical Society1989 Article PDF first page preview Close Modal You do not currently have access to this content.

Constraints on the interpretation of nonquantal acetylcholine release from frog neuromuscular junctions.

In the frog nerve-muscle preparation, there is evidence for nonquantal release of acetylcholine (ACh) at a level 100 times that attributable to spontaneous quantal events (miniature endplate potentials). It is widely assumed that nonquantal release occurs near the sites of quantal release (active zones) in the nerve terminal, close to the postsynaptic muscle membrane. This high level of nonquantal ACh release has led to the suggestion that it may serve a trophic function at the nerve-muscle junction. However, the precise origin and mechanism of nonquantal release have not been determined. We have used outside-out patches of ACh receptor-rich membrane as a sensitive technique for the direct measurement of ACh release from highly localized regions of enzymatically treated nerve terminals and have found little detectable ACh "leakage" from active-zone regions. If all of the nonquantal ACh release were localized to the under surface of the nerve terminal, we estimate that we would have detected more than 10 times the low level detected with our patch probe. Furthermore, although quantal release was easily measurable, vesicular exocytosis (hypothesized to insert ACh transport proteins into the plasma membrane, thereby producing the leak) did not increase single-channel activity in the patch probe above that attributable to quantal release. We conclude that, at rest, the active-zone region of nerve terminals is not a major source of nonquantal ACh release and that vesicular exocytosis does not noticeably increase the level of nonquantal release from the nerve terminal. Thus, with biochemical measurements that indicate that spontaneous ACh release is relatively unchanged by prior denervation, these results question the assumed source and mechanism of nonquantal release and the suggestion that leakage of ACh from active-zone regions plays a trophic role in nerve-muscle interaction.

Localized stimulation of neural tissues in the brain by means of a paired configuration of time-varying magnetic fields

A method of localized stimulation of the human brain is proposed. The basic idea is to concentrate induced eddy currents locally in the vicinity of a target in the cortex by a pair of coils which are positioned outside the head so that time-varying magnetic fields pass through the head in the opposite directions around a target. The eddy currents induced at the target are expected to flow together, which results in an increased current flow at the target. Spatial distributions of induced eddy currents are calculated in cubical and spherical volume conductor models by a finite element method. The results show that the current vectors make themselves two vortexes which flow together at the target. The current density at the target makes a peak which is higher by 2–3 times than current densities at nontarget regions. The validity of the proposed method is demonstrated by experiments using frog nerve-muscle preparations.

A further study of the neuromuscular effects of vesamicol (AH5183) and of its enantiomer specificity.

1. The effects of vesamicol (2-(4-phenylpiperidino) cyclohexanol), an inhibitor of acetylcholine storage, and its two optical isomers have been studied on neuromuscular transmission in rat and frog muscle, and on nerve conduction in frog nerve. 2. Racemic vesamicol produced a pre-block augmentation of twitch tension that also occurred in directly-stimulated muscle. This effect is thus at least partially due to an increase in muscle contractility. 3. (-)-Vesamicol was approximately 20 times more potent than (+)-vesamicol in blocking twitches elicited at 1 Hz. This degree of stereoselectivity is similar to that measured for inhibition of acetylcholine uptake by isolated synaptic vesicles. Both enantiomers were equally weak in reducing nerve action potential amplitude in frog nerve. 4. Further studies with the active isomer, (-)-vesamicol, showed that, like that produced by racemic vesamicol, the neuromuscular block was highly frequency-dependent. The block was not reversed by choline or neostigmine, but was partially reversed by 4- or 3,4-aminopyridine. 5. Preliminary electrophysiological studies showed that vesamicol reduced miniature endplate potential amplitude in rapidly-stimulated frog nerve-muscle preparations. Addition of lanthanum ions increased the frequency of miniature endplate potentials and led to the appearance of apparently normal-sized potentials amongst those of reduced amplitude. 6. The results show the close agreement between pharmacological and biochemical observations indicating the suitability of the rat diaphragm as a test model for substances of this nature. The degree of reversibility of the vesamicol-induced neuromuscular block by aminopyridines was unexpected, and it is suggested that in the presence of a drug which greatly increases release, a pool of acetylcholine is capable of being released which is not normally releasable after block of storage by vesamicol. It is also considered possible that the results from the intracellular recording studies may be explained in these terms.

Effects of changes in tonicity of the extracellular solution on the size of vesicles in frog motor nerve terminals.

Frog nerve-muscle preparations were soaked and then fixed in solutions roughly isotonic to frog plasma, or in solutions that were markedly hypertonic or hypotonic. The hypertonic solution decreased the cross-sectional area of the muscle fibers but not of the synaptic vesicles. The hypotonic solution increased the cross-sectional area of the muscle fibres but did not produce comparable increases in the areas of the synaptic vesicles. Apparently the vesicles in situ do not behave as simple osmometers. This fact is significant for theories of exocytosis and for the mechanism of transmitter packaging in the vesicles.

Frequency-dependent effects of phenytoin on frog junctional transmission: Presynaptic mechanisms

The action of the antiepileptic drug, phenytoin, on junctional transmission at various frequencies of synaptic activation was studied in frog nerve-muscle preparations. Intracellular recordings were made from muscle end-plates, and extracellular focal and subendothelial recordings were obtained from motor nerve terminals and their parent axons, respectively. When the motor nerve was stimulated at 100–200 Hz, exposure to the drug (0.1–0.3 mM) induced intermittent failures of junctional transmission which appeared faster as the rate of stimulation was increased. At these and at lower stimulation frequencies (30–50 Hz), in which failures of transmission occurred only rarely, phenytoin markedly limited the buildup of end-plate potential amplitude during the period of repetitive nerve stimulation (tetanic potentiation). Several lines of evidence suggest that both drug effects are consequent to a frequency-dependent depression of the action potential at motor axons and terminals, which could lead to an intermittent conduction block at the higher rates of stimulation. The selective action of phenytoin on high frequency synaptic transmission may contribute to the specificity shown by this drug in suppressing epileptic seizures while sparing normal neuronal activity.

Magnetic nerve stimulation without interlinkage between nerve and magnetic flux

A new method of magnetic stimulation of nerves is proposed. Nerves are located on a core aperture outside the core which is implanted in the body. Nerves can be stimulated by the secondary currents which flow in the body fluids around the core when the magnetic flux in the core is changed. One of the advantages in this method is to be able to avoid the interlinkage between the core and nerves. The equivalent resistance of tissues around the core is calculated, and current density for nerve excitation is estimated. The validity of the new method is demonstrated by experiments using frog nerve-muscle preparations. The results show that the nerve can be excited by a change of magnetic flux which generates an EMF of 0.8-volts peak amplitude and 0.8-ms duration in a monitor wire. The current density in the vicinity of the core aperture for nerve excitation is 3.2 mA/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> .

Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.

The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation

The effect of Bothrops jararacussu venom was studied in cutaneous pectoris nerve muscle preparations and in the desheathed sciatic nerve of the frog. The venom rapidly inhibited muscle twitch-tension, evoked either directly or indirectly through the motor nerve and abolished the compound action potential of the muscle and of the sciatic nerve. After fractionation of the venom by Sephadex G-50 column chromatography, all the activity was recovered in a fraction containing 30% of the total venom protein and highly enriched in two polypeptides with apparent M r of 13–15,000, as revealed by two-dimensional polyacrylamide gel electrophoresis. The concentration of active subfraction required to obtain 50% paralysis in 1h was 8 μg protein/ml. The active subfraction contained low levels of phospholipase A activity, whereas no proteolytic activity was detected. The paralyzing activity of the active subfraction on nerve-musle preparations was not dependent on the presence of Ca 2+, suggesting that phospholipase A activity is not required for the toxic effect. The active subfraction was found to cause an initial spontaneous contracture and fasciculation of the nerve-muscle preparation, and a rapid depolarization of the muscle membrane. The frequency of miniature endplate potentials was normal throughout the period of exposure to the active subfraction, although occasionally initial transient bursts were observed. At the end of the incubation, nerve endings still responded to high [K +] and to black widow spider venom. The exposure (1–2 h) to blocking concentrations of venom active subfraction provoked different degrees of morphological alteration of the muscle fibers. In contrast, no ultrastructural alterations were observed in nerve terminals, giving further support to the idea that terminals are not a prime site of the venom action. In addition to its effect on the nerve muscle-preparation, the active subfraction at higher concentrations, showed a Ca 2+-dependent hemolytic activity. In the light of these results, the properties of the active subfraction of B. jararacussu venom are compared with those of other known membrane-active toxins.