Termination of the presynaptic effect of carbacholine by tubocurarine]

The presynaptic action of carbacholine (Cch) was studied in experiments on the frog sartorius muscle neuromuscular preparation. Cch proved to decrease the quantum content of the end plate potential (EPP); this effect was induced by a direct Cch action on the motor nerve endings. d-tubocurarine decreased the sensitivity of the nerve endings to Cch significantly. Both d-tubocurarine and Cch had concurrent antagonistic interrelations in respect to their action on the quantum content of EPP. Atropine in low concentrations had no influence on the presynaptic effect of Cch. It was concluded that Cch decreased the quantum content of EPP through the N-cholinergic structures of the motor nerve endings.     

Characteristics of electrical activity in different sections of nerve endings in the frog

In experiments on frog sartorius neuromuscular preparations, the evoked electrical responses of nerve endings were recorded by extracellular microelectrodes. It was shown that in proximal parts of the nerve ending, the three-phase response (+ - +) with a high amplitude negative phase occurred due to motor nerve stimulation. With movement of the extracellular electrode in distal direction a certain increase of the initial positive phase and a significant decrease of the negative one were observed. At the end of the terminal that response transformed to the monophasic one (+). On local iontophoretic application of tetrodotoxin (TTX) to the recording site two components of the nerve ending response were revealed: TTX-insensitive and TTX-sensitive. A significant decrease of the TTX-sensitive component occurred along the course of the nerve ending. That component was absent from the distal synaptic areas. It is concluded that in frog nerve ending, the action potential propogates with decrement while depolarization of the end parts of the terminal is passive in nature.     

The positive inotropic action of transmural electrical stimulation of the heart ventricles in the ontogeny of the frog Rana temporaria

Studies have been made of the effect of transmural electrical stimulation on twitch tension produced by atropinized ventricular preparations from tadpoles and adult frogs. In preparations from tadpoles at stage 42 and all the following stages, as well as in adult frogs, transmural electrical stimulation evoked positive inotropic responses which consisted of a slow propranolol-sensitive component or of a slow and fast components. It is highly probable that the slow component is induced by adrenergic transmitter. The fast propranolol-resistant component appears at stage 43. It may be prevented by bretilium being probably induced by a comediator which is released together with the adrenergic transmitter from the sympathetic nerve endings.     

Comparative research on synaptic transmission in the sympathetic ganglia of rabbits and frogs during acetylcholinesterase inhibition

Organophosphorus inhibitor of acetylcholinesterase (AChE) armin (1 x 10(-6) M) induced a variety of pre- and postsynaptic effects resulting from the AChE inhibition and subsequent accumulation of acetylcholine (ACh) in the synaptic cleft. The intensity of postsynaptic effects (level of neuron depolarization, degree of action potential depression) was shown to be different in the ganglia of frog and rabbit. This could be explained by differences in the total amount of ACh released in response to nerve stimulation as well as at rest. Both muscarinic and nicotinic cholinoreceptors were involved in the process of sustained depolarization of the neurons in the rabbit superior cervical ganglion after AChE inhibition. In frog ganglion neurons the nicotinic receptors did not participate in depolarization evidently due to their fast desensitization. The activation of presynaptic muscarinic receptors resulted in decrease of ACh released by nerve stimulation seems to weaken depolarization and blockade of synaptic transmission in sympathetic ganglia treated by AChE inhibitors.     

Potentiation of cholecystokinin-induced exocrine secretion by either electrical stimulation of the vagus nerve or exogenous VIP administration in the guinea pig pancreas

In the anaesthetized guinea pig, the secretory responses (pancreatic juice flow and protein output) induced by electrical stimulation of the vagus nerve were not blocked by atropine but by hexamethonium. Excitation of the left vagus nerve induced by electrical stimulation significantly potentiated the C-terminal octapeptide of cholecystokinin (CCK-OP)-induced secretory responses. In the isolated perfused pancreas of guinea pig, the secretory responses induced by CCK-OP at concentrations in the physiological range were markedly potentiated by simultaneous stimulation with vasoactive intestinal peptide (VIP). However, the secretory responses induced by CCK-OP at higher concentrations were not potentiated but inhibited by simultaneous stimulation of VIP. The secretory responses induced by carbachol (CCh) at any concentrations were not potentiated but inhibited by simultaneous stimulation of VIP. These results support the view that VIP released from the nerve endings can potentiate the hormonal action of CCK-OP.     

Measurement of fasciculations as motor nerve ending discharges in the rat: a dose related effect of neostigmine

The muscle fasciculations caused by neostigmine and similar agents are the result of a primary drug action on motor nerve endings. Asynchronous, repetitive firing of action potentials are evoked at motor nerve endings which are then transmitted to muscle. A dose-response relationship between neostigmine dose and the rate of/or total neural activity has been established in the rat. This fasciculatory response to neostigmine can serve as an index of motor nerve ending excitability and may be useful in assessing the effects of certain pathological states or drug actions at the neuromuscular junction.     

The site of the neuromuscular block produced by polymyxin B and rolitetracycline.

The site of neuromuscular blockade induced by polymyxin B and rolitetracycline was studied on isolated nerve and nerve-muscle preparations. Polymyxin B (1.8 X 10(-4) M) was equipotent to lidocaine as a local anaesthetic on a frog desheathed nerve preparation, while rolitetracycline (up to 3.6 X 10(-3)M) had no local anaesthetic effect. Polymyxin B (6 X 10(-5) M) and rolitetracycline (7 X 10(-4) M) blocked by 50% the response of rat diaphragm induced by phrenic nerve stimulation, but did not decrease the amount of acetylcholine (ACh) released from this preparation during nerve stimulation. Both antibiotics depressed the response of the rat diaphragm to inject ACh, and this response was more sensitive to inhibition by the drugs than was the response to nerve stimulation. With rolitetracycline, a concentration that blocked the response to nerve stimulation by 50% inhibited the response to injected ACh by 85%, and this relationship was similar to that with d-tubocurarine; however, polymyxin B was relatively more effective than d-tubocurarine in inhibiting the effect of ACh. Polymyxin B (1-1.5 X 10(-4) M) but not rolitetracycline (1 X 10(-3) M) depressed the response of the diaphragm to direct muscle stimulation. It is concluded that polymyxin B and rolitetracycline block neuromuscular transmission predominatly by an effect to depress the muscle's sensitivity to ACh; polymyxin B probably acts by an effect similar to that of local anaesthetics, while rolitetracycline probably acts by an effect similar to that of d-tubocurarine.     

Obidoxime antagonizes the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate via different mechanisms

The efficacies and mechanisms of obidoxime in antagonizing the neuromuscular failure induced by neostigmine and diisopropyl fluorophosphate (DFP) were studied in mouse phrenic nerve/diaphragm preparations. Obidoxime antagonized neostigmine-induced tetanic fade (EC₅₀: 300 µM) by inhibiting the regenerative and sustained depolarization during repetitive stimulation. The antagonism was associated with a depression and shortening of single endplate potentials (EPPs) and miniature EPPs (MEPPs). In contrast, the neuromuscular failure induced irreversibly after treatment with DFP and followed by washout was restored by obidoxime at concentrations (EC₅₀: 0.6 µM) 500-fold lower than that against neostigmine. The regenerative depolarization was abolished with no depression of single EPPs and MEPPs, and the antagonistic action persisted after washout of obidoxime. The EC₅₀ of obidoxime was proportionately increased in the presence of increasing concentrations of DFP. Nevertheless, the EC₅₀ against DFP, at a concentration (30 µM) 15-fold in excess of that which caused tetanic fade, was still 10-fold lower than that which antagonized neostigmine. In both cases, the amplitudes of train EPPs were increased. It is concluded that obidoxime antagonizes neostigmine-induced neuromuscular failure by a curare-like action but antagonizes DFP by an enzyme reactivation.     

Voltage-dependent P/Q-type calcium channels at the frog neuromuscular junction

It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.     

Action of a beta-bungarotoxin on autonomic ganglia and adrenergic neurotransmission.

A beta-bungarotoxin was isolated from the venom of Bungarus multicinctus by column chromatography on Sephadex G-50 and SP-Sephadex. The toxin produced presynaptic effects on neuromuscular transmission with characteristics similar to those described by others. In a sympathetic ganglion, the toxin increased spontaneous acetylcholine (ACh) release and decreased ACh release evoked by preganglionic nerve stimulation. The toxin did not block the response of isolated ileum to cholinergic nerve stimulation, did not block the release of noradrenaline from the adrenergic nerve terminals of a nictitating membrane preparation, and did not alter the responses of smooth and cardiac muscle preparations to noradrenaline. It is suggested that the specificity of beta-bungarotoxin for certain nerve terminals is related either to selective binding of the toxin or to the selective presence of a necessary substrate for its action. An attempt to show selective binding of 125I-toxin to cholinergic nerve terminals in skeletal muscle was not successful.     

Effect of armin on synaptic transmission in frog neuromuscular preparations

The action of armin, an organophosphorus inhibitor of cholinesterases, on synaptic transmission parameters was studied by means of intracellular registration of end plate potentials and currents (EPP and EPC) in the frog. On 10-minute exposure the increase in the temporary parameters became manifest provided the drug was administered at a concentration of 10(-6) g/ml and over. EPC reversal potential and cholinoreceptor sensitivity to armin did not change substantially. At a concentration of 10-(-8) g/ml armin exerted a potentiating effect on the frequency of miniature EPP and quantum composition of EPP, while that effect was not related to armin anticholinesterase activity. The presynaptic acetylcholine release was suppressed by high concentration of armin (10(-5) g/ml). Under the conditions cited there was a decrease in the depot of the available transmitter quanta in nerve terminals.     

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.     

Nitric oxide modulates intensity of non-quantal acetylcholine release in myocardium of the right atrium of rat

The main parasympathetic neurotransmitter acetylcholine (ACh) is released in the myocardium from the intramural postganglionic parasympathetic nerve endings. The mechanism of non-quantal ACh release has been recently demonstrated in these neurons. Non-quantal ACh release does not depend on exocytosis of ACh-containing vesicles in response to nerve impulse activity but is assumed to be mediated by the high-affinity choline uptake system. The intensity of non-quantal ACh release in the myocardium correlates with the degree of manifestation of the effects of acetylcholinesterase inhibitors inducing the accumulation of non-quantal ACh in the myocardium. The present study deals with the influence of putative modulators of non-quantal ACh release: nitric oxide (NO) and ATP, on the intensity of cholinergic effects induced by organophosphorous acetylcholinesterase inhibitor paraoxon. Intracellular registration of bioelectrical activity in isolated right atrium preparations from rats was used. Under normal conditions, paraoxon (10^?7–10^?5 M) induced a marked decrease in the action potential (AP) duration at a level of 50 and 90% repolarization in the working right atrial myocardium and slowed down the sinus rhythm. ATP, which is known to suppress nonquantal ACh release in the neuromuscular junction, did not induce significant reduction or augmentation of the effects of paraoxon (5 × 10^?6 M). The NO donors, sodium nitroprusside (10^?5 M) and SNAP (10^?4 M), significantly reduced the paraoxon-induced AP shortening. Moreover, sodium nitroprusside decreased the negative chronotropic effect of paraoxon by 43.7%. On the contrary, NO synthase inhibitor L-NAME (10^?4 M), which is known to suppress endogenous NO production, augmented the AP shortening caused by paraoxon. It may be deduced that NO is a universal regulator of non-quantal ACh release intensity both in the myocardium and in the neuromuscular junction.     

Presynaptic nicotinic cholinoreceptors modulate velocity of the action potential propagation along the motor nerve endings at a high-frequency synaptic activity

Experiments on frog neuromuscular junctions have demonstrated that asynchrony of the acetylcholine quantal release forming the multi-quantal evoked response at high-frequency synaptic activity is caused, in particular, by a decrease in velocity of the action potential propagation along the non-myelinated nerve endings, which is mediated by activation of the α7 and α4β4 nicotinic cholinoreceptors.     

Characterization of the neuromuscular block produced by clindamycin and lincomycin.

The site of neuromuscular blockade induced by clindamycin and lincomycin was studied on isolated nerve and nerve-muscle preparations. Clindamycin (3.6 X 10(-3) M) but not lincomycin (up to 1.5 X 10(-2) M) had a local anaesthetic effect on a frog desheathed nerve preparation. Clindamycin (8 X 10(-4) M) and lincomycin (4 X 10(-3) M) depressed the response of the rat diaphragm to nerve stimulation and to direct muscle stimulation in parallel. This indicated that the predominant neuromuscular blocking effect of these antibiotics was due to an effect on the muscle. Clindamycin was fivefold more potent than lincomycin in this effect, and the unionized form of both drugs was the active form. Lincomycin (4 X 10(-3) M) but not clindamycin (8 X 10(-4) M) also had some depressant effect on nerve-muscle transmission as indicated by the interaction of the effects of the antibiotics and d-tubocurarine. The significance of these findings is discussed in relation to the acute clinical toxicity of these antibiotics.     

Post-tetanic potentiation of reciprocal Ia inhibition in human lower limb.

The purpose of this study was to investigate how reciprocal Ia inhibition is changed during muscle fatigue of lower limb muscle, induced with a voluntary contraction or height frequency electrical stimulation. Reciprocal Ia inhibition from ankle flexors to extensors has been investigated in 12 healthy subjects. Hoffmann reflex (H-reflex) in the soleus muscle was used to monitor changes in the amount of reciprocal Ia inhibition from common peroneal nerve as demonstrated during voluntary dorsi or planterflexion and 50 Hz electrical stimulation induced dorsi or planterflexion. The test soleus H-reflex was kept at 20-25% of maximum directly evoked motor response (M response) and the strength of the conditioning common peroneal nerve stimulation was kept at 1.0 x motor threshold. At rest, weak la inhibition was demonstrated in 12 subjects, maximal inhibition from the common peroneal nerve was 28.8%. During voluntary dorsiflexion and 50 Hz electrical stimulation induced dorsiflexion, there absolute amounts of inhibition increased as compared to at rest, and decreased or disappeared during voluntary planterflexion and 50 Hz electrical stimulation induced planterflexion as compared to at rest. During voluntary or electrical stimulation induced agonist muscle fatigue, the inhibition of the soleus H-reflex from the common peroneal nerve was greater during voluntary dorsiflexion (maximal, 11.1%) and 50 Hz (maximal, 6.7%) electrical stimulation induced dorsiflexion than at rest. The inhibition was decreased or disappeared during voluntary planterflexion 50 Hz electrical stimulation induced planterflexion. It was concluded that the results were considered to support the hypothesis that alpha-motoneurones and la inhibitory intemeurones link to antagonist motoneurones in reciprocal inhibition. The diminished reciprocal Ia inhibition of voluntary contraction during muscle fatigue as compared to electrical stimulation, is discussed in relation to its possible contribution to ankle stability.     

Functional topography and ultrastructure of periarticular mechanoreceptors in the lateral elbow region of the rat

The distribution and ultrastructure of sensory nerve endings were investigated in the deep lateral elbow region of the rat. Three zones of distribution of mechanoreceptors were distinguished, each in relation to the functional architecture of the connective and muscular tissue in that area: (1) a zone with muscle spindles, Golgi tendon organs, free nerve endings and single small lamellated corpuscles ('muscle-tendon spectrum'), situated in the middle third of the supinator muscle and its superficial aponeurosis; (2) a zone with small lamellated corpuscles and free nerve endings, situated pericapsularly to the humeroradial joint capsule ('shearing spectrum'): this moderately dense, irregular connective tissue is covered by the proximal continuation of the supinator's aponeurosis, and muscle fibers insert from beneath this aponeurosis, which displays, as a part of the joint capsule, a strong collagenous tissue plate; (3) a zone with only free nerve endings within the tendon-like, most proximal part of the supinator's aponeurosis, inserting into the periosteal layer of the lateral humeral epicondyle ('endotenonial spectrum'): it is part of the joint capsule. The ultrastructure of these sensory endings is described and the distribution pattern of the mechanoreceptors observed is discussed in relation to the classification into 'muscle receptors' and 'joint receptors'.     

Role of extracellular calcium in the metabolic and hemodynamic actions of sympathetic nerve stimulation, noradrenaline and prostaglandin F2 alpha in perfused rat liver. Differential inhibition by nifedipine and verapamil

In perfused rat liver hepatic nerve stimulation (10 Hz, 2 ms) caused an increase in glucose and lactate output, a decrease in flow and an overflow of noradrenaline into the hepatic vein. Noradrenaline (1 microM) (NA) and prostaglandin F2 alpha (5 microM) (PGF2 alpha), which are implicated as mediators of nerve action, elicited similar effects. 1) All actions of nerve stimulation and the hemodynamic but not the metabolic effects of noradrenaline and PGF2 alpha were largely dependent on extracellular calcium. 2) The dihydropyridine type calcium antagonist nifedipine (5 microM) inhibited the hemodynamic but not the metabolic actions of nerve stimulation, NA and PGF2 alpha, while the phenylalkylamine type calcium antagonist verapamil (5 microM) had no effect. These findings allow the following conclusions: Calcium influx into I nerve endings, necessary for the release of neurotransmitter, II parenchymal cells, for the display of metabolic effects induced by nerve stimulation, and III the actions of NA and PGF2 alpha, do not appear to be mediated by the normal affinity nifedipine- or the verapamil-sensitive channels. Calcium influx into vascular smooth muscle and/or endothelial cells for the display of hemodynamic action induced by nerve stimulation and the NA and PGF2 alpha effects, appear to occur through nifedipine-sensitive but verapamil-insensitive channels.     

Magnetic stimulation for non-homogeneous biological structures

Background  

Magnetic stimulation has gained relatively wide application in studying nervous system structures. This technology has the advantage of reduced excitation of sensory nerve endings, and hence results in quasi-painless action. It has become clinically accepted modality for brain stimulation. However, theoretical and practical solutions for assessment of induced current distribution need more detailed and accurate consideration.     

Trachynilysin,a neurosecretory protein isolated from stonefish (Synanceia trachynis) venom,forms nonselective pores in the membrane of NG108-15 cells

Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca2+ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced. The trachynilysin-induced current was carried by cations and reversed at about -3 mV in standard physiological solutions, which led to strong membrane depolarization and Ca2+ influx. La3+ blocked the trachynilysin current in a dose-, voltage-, and time-dependent manner, and antibodies raised against the toxin antagonized its effect on the cell membrane. The inside-out configuration of the patch clamp technique allowed the recording of single channel activity from which various multiples of 22 pS elementary conductance were resolved. These results indicate that trachynilysin forms pores in the NG108-15 cell membrane, and they advance our understanding of the toxin's mode of action on motor nerve endings and neurosecretory cells.