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.     

Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.     

Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, <Emphasis Type="Italic">Cyclorana alboguttata</Emphasis>

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.     

Elucidation of the mechanism and site of action of quinuclidinyl benzilate (QNB) on the electrical excitability and chemosensitivity of the frog sartorius muscle

The effects of the muscarinic antagonist quinuclidinyl benzilate (QNB) on transmission at the frog sartorius neuromuscular junction have been examined. QNB decreases endplate potential (EPP) amplitude without affecting miniature endplate (MEPP) frequency or resting potential. QNB also increased the latency of the EPP and the nerve terminal spike in a frequency dependent fashion, suggesting the site of action is the unmyelinated nerve terminal. Since the rate of rise and amplitude of muscle action are potentials decreased it is likely that QNB causes a blockade of electrically excitable sodium channels; the agent also blocks ionic channels associated with nicotinic acetylcholine receptors. It is possible that these effects of QNB may explain some of the behavioral disturbances produced by its administration.     

Acetaldehyde affects mammalian neuromuscular transmission without observable postsynaptic effects.

Acetaldehyde, the first metabolite of ethanol, caused a reversible block of the end-plate potential (EPP) in the rat and mouse phrenic nerve--hemidiaphragm preparation. Decrease and block of the EPP occurred over a bath concentration range from 3 to 25 mM. The phrenic nerve compound action potential was blocked along with the EPP, and this block was not reversed by high bath Ca2+ concentration. The muscle action potential was unaffected even at concentrations up to 50 mM. Over the same concentration range (3--25 mM), miniature end-plate potential (MEPP) frequency sometimes decreased a few minutes after application, and over the ensuing 10--20 min would steadily increase to as much as 11 times the base-line frequency, particularly with higher doses. However, the shape of averaged MEPPs remained unchanged after acetaldehyde application, suggesting that this aldehyde does not have post-synaptic effects.     

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.     

Extraction of the EPP Component from the Surface EMG

A surface electromyogram (EMG), especially when recorded near the neuromuscular junction, is expected to contain the endplate potential (EPP) component which can be extracted with an appropriate signal filter. Two factors are important: the EMG must be recorded in monopolar fashion, and the recording must be done so the low frequency signal corresponding the EPP is not eliminated. This report explains how to extract the EPP component from the EMG of the masseter muscle in a human subject. The surface EMG is recorded from eight sites using traditional disc electrodes aligned along over the muscle, with equal inter-electrode distance from the zygomatic arch to the angle of mandible in response to quick gum clenching. A reference electrode is placed on the tip of the nose. The EPP component is extracted from the raw EMGs by applying a high-cut digital filter (2nd dimension Butterworth filter) with a range of 10-35 Hz. When the filter is set to 10 Hz, the extracted EPP wave deflects either negative or positive depending on the recording site. The difference in the polarity reflects the sink-source relation of the end plate current, with the site showing the most negative deflection corresponding to the neuromuscular junction. In the case of the masseter muscle, the neuromuscular junction is estimated to be located in the inferior portion close to the angle of mandible. The EPP component exhibits an interesting oscillation when the cut-off frequency of the high-cut digital filter is set to 30 Hz. The EPP oscillation indicates that muscle contraction is adjusted in an intermittent manner. Abnormal tremors accompanying various sorts of diseases may be substantially due to this EPP oscillation, which becomes slower and is difficult to cease.Download video file.^{(79M, mp4)}     

Morphologic and histochemical characteristics of frog skeletal muscle following application of colchicine to a motor nerve]

Experiments with application of colchicine to the muscle motor nerve carried out; this was done for the purpose of disturbance of rapid axoplasmic transport. A reduction of the areas of transverse sections of the muscle fibers, an increase in the number of fibers with a low succinic dehydrogenase (SDH) activity a greater homogeneity of the muscle fibers by the degree of optic density in staining for detection of the SDH activity was noted. Analogous changes were revealed under conditions of section of the motor nerve. However, denervation was accompanied by the block of conductivity and by degenerative changes in the nerve endings. As to the preparations treated with colchicine, transmission of excitation in the nerve and through the synapse was retained and was recorded by the end plate miniature potentials, end plate potentials and the action potentials of the muscle fibers. A conclusion was drawn that rapid axoplasmic transport brought substances maintaining differentiated state of the muscle fibers.     

Dual action of intracellularly released calcium on the quantal mediator secretion

The mice diaphragm muscle and microelectrode technique were used to check the influence of ryanodine (0.5 mcM) on spontaneous and evoked mediator release under conditions of potassium depolarization (8-16 mM [K+]ex or rhythmic (4-100 Hz) stimulation of motor nerve terminals. Weak tonic calcium loading (by muscle exposition to 8 mM [K+]ex) caused a two-fold frequency increase if miniature and plate potentials (MEPPs), which was returned to the basal level by subsequent application of ryanodine. This inhibitory effect of ryanodine was blocked by apamin (500 nM) a blocker of K+(Ca)-channels. A greater calcium load of terminals (in solution with 16 mM [K+]ex) caused a 15-fold increase of MEPPs frequency. Subsequent ryanodine application caused an additional 2-3-fold increase of MEPPs frequency. During rhythmic activity of motor synapses, ryanodine was able to decrease the amplitude of EPP by 60% at plateau phase at short low frequency (4 Hz) of discharges and to increase the amplitude of EPP by 60-150% at high frequency (70-100 Hz) of discharges. It is concluded that rynodine induced calcium release from intraterminal Ca2+-stores can influence dual: excitatory or inhibitory, action on spontaneous and evoked mediator release, due to different intraterminal calcium loads and regimen of synaptic activity.     

A Study of the Reinnervation of Fast and Slow Mammalian Muscles

Miniature end plate potential (mepp) frequency in innervated extensor muscle is significantly higher than in soleus muscle. 9 days after nerve crush mepps of low amplitude and prolonged duration reappeared at a frequency of 2% of control and were similar to normal muscles after 35 days. Membrane potential began to increase 9–10 days after nerve crush and at 30 days was similar to controls. The region most sensitive to ACh in denervated and reinnervated muscles was the end plate. Caffeine (20 mM, 23°C) induced contracture in innervated soleus but not in extensor muscles. After denervation the extensor became sensitive to caffeine while the soleus muscles decreased in sensitivity to the drug; 4–5 days after reinnervation the effect of caffeine on these muscles was similar to control. The events during reinnervation are: (a) reappearance of mepps at the same time as end plate potential and muscle twitch; (b) partial restoration of the membrane potential; (c) return of caffeine-induced contracture to normal levels in the soleus and its absence in the extensor muscles; (d) return of membrane resistance to normal values in both muscles at about 25 days; and (e) return of ACh-sensitivity to control levels at about 30 days in both muscles. Although these results suggest that the membrane potential and sarcoplasmic reticulum are under neural influence, it remains to be established whether or not separate neurotrophic factors are involved.     

Interaction of glutamate- and adenosine-induced decrease of acetylcholine quantal release at frog neuromuscular junction

In a frog neuromuscular preparation of m. sartorius, glutamate had a reversible dose-dependent inhibitory effect on both spontaneous miniature endplate potentials (MEPP) and nerve stimulation-evoked endplate potentials (EPP). The effect of glutamate on MEPP and EPP is caused by the activation of metabotropic glutamate receptors, as it was eliminated by MCPG, an inhibitor of group I metabotropic glutamate receptors. The depression of evoked EPP, but not MEPP frequency was removed by inhibiting the NO production in the muscle by L-NAME and by ODQ that inhibits the soluble NO-sensitive guanylyl cyclase. The glutamate-induced depression of the frequency of spontaneous MEPP is apparently not caused by the stimulation of the NO cascade. The particular glutamate-stimulated NO cascade affecting the evoked EPP can be down-regulated also by adenosine receptors, as the glutamate and adenosine actions are not additive and application of adenosine partially prevents the further decrease of quantal content by glutamate. On the other hand, there is no obvious interaction between the glutamate-mediated inhibition of EPP and inhibitory pathways triggered by carbacholine and ATP. The effect of glutamate on the evoked EPP release might be due to NO-mediated modulation (phosphorylation) of the voltage-dependent Ca2+ channels at the presynaptic release zone that are necessary for evoked quantal release and open during EPP production.     

Effect of ethanol on young nerve endings

The ethanol changes the quantal spontaneous release of acetylcholine and it affects the reinnervation time course. The effects of ethanol on regenerated nerve endings have been tested. 20 days after crushing sciatic nerve, the m.e.p.p. frequency at the end plate of rat extensor digitorum longus muscle keep in Ringer solution without and with ethanol has been estimated by intracellular recordings. The increase of the m.e.p.p. frequency produced by ethanol is greater in immature, than in normal nerve endings.     

Effect of dimedrol on the function of the neuromuscular synapse and the generation of action potentials by motor nerve fibers

Microelectrode registration of synaptic potentials in the frog cutaneous-pectoris muscle has shown dimedrol (7.9 X 10(-5) M) to act on synaptic transmission decreasing the quantal content, estimated by mean EPP amplitude to mean miniature EPP amplitude ratio, the quantal content calculated by variation coefficient of EPP amplitude being unaffected. The data suggest possible transmitter release and depletion of mediator stock. The experiments on isolated motor nerve fibers have demonstrated dimedrol to cause the increase in transmitter release probability by widening the action potentials in the terminals and thus enhancing Ca2+ influx.     

Electrophysiological study of mediator secretion in the frog neuromuscular synapse under a colchicine block of axoplasmic transport

Two weeks after colchicine nerve treatment the evoked transmitter release was blocked in part of the frog sartorius synapses, with spontaneous activity being absent from some of them. In the synapses with evoked and spontaneous transmitter release preserved within this period of time, the magnitudes of the absolute refractory phase of nerve terminals were significantly higher than the control ones, while in part of synapses, the frequency of miniature end plate potentials (MEPP) was considerably increased. Nerve stimulation (5 imp.s-1) led to a rise of the amplitude of evoked potentials and of MEPP frequency followed by irreversible blockade of synaptic activity. It is concluded that substances transported by rapid axonal flow control the level of membrane potential of nerve terminals and are fairly important for presynaptic membrane integrity.     

Changes in miniature endplate potential frequency during repetitive nerve stimulation in the presence of Ca2+, Ba2+, and Sr2+ at the frog neuromuscular junction

Miniature endplate potentials (MEPPs) were recorded from frog sartorious neuromuscular junctions under conditions of reduced quantal contents to study the effect of repetitive nerve stimulation on asynchronous (tonic) quantal transmitter release. MEPP frequency increased during repetitive stimulation and then decayed back to the control level after the conditioning trains. The decay of the increased MEPP frequency after 100-to 200-impulse conditioning trains can be described by four components that decayed exponentially with time constants of about 50 ms, 500 ms, 7 s, and 80 s. These time constants are similar to those for the decay of stimulation-induced changes in synchronous (phasic) transmitter release, as measured by endplate potential (EPP) amplitudes, corresponding, respectively, to the first and second components of facilitation, augmentation, and potentiation. The addition of small amounts of Ca2+ or Ba2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with Sr2+, led to a greater increase in the stimulation-induced increases in MEPP frequency. The Sr-induced increase in MEPP frequency was associated with an increase in the second component of facilitation of MEPP frequency; the Ba-induced increase with an increase in augmentation. These effects of Sr2+ and Ba2+ on stimulation-induced changes in MEPP frequency are similar to the effects of these ions on stimulation- induced changes in EPP amplitude. These ionic similarities and the similar kinetics of decay suggest that stimulation induced changes in MEPP frequency and EPP amplitude have some similar underlying mechanisms. Calculations are presented which show that a fourth power residual calcium model for stimulation-induced changes in transmitter release cannot readily account for the observation that stimulation- induced changes in MEPP frequency and EPP amplitude have similar time- courses.     

Histochemical localization of folic acid in the motor end plate

Folic acid investigated in motor end plate by histochemical method appeared in the long nerve terminals of the motor axon, in the cytoplasm of Schwann cell. In muscle fibre opposite to the nerve arborizations, spaced granular lines perpendicular to the sarcolemma were observed.     

A Role of Cyclic AMP in a Neurotrophic Process

TROPHIC effects of neurones can be defined as long-term interactions between nerve cells and the tissues they innervate, which initiate or control molecular modifications in the target cells¹. An example of trophic activity is the maintenance of the motor end plate of skeletal muscle by the nerve fibre. The motor nerve appears to exert a morphogenetic effect on its formation, because both the structural specializations and Cholinesterase activity of the end plate develop only in relation to the nerve fibre^2–5. Conversely, denervation is followed usually by degeneration of the junction and loss of end plate Cholinesterase activity^6–8.     

Electrophysiologic, morphometric and histocytochemical characteristics of frog sartorius muscle fibers]

Three groups of muscle fibers (dark, light, and intermediate) were revealed in the fibers of the frog sartorius muscle in examination of the succinate dehydrogenase (SDH) activity. There was revealed a reverse relationship between the diameter of the muscle fibers an the SDH activity in them. The external surface of sartorius muscle is chiefly represented by dark muscle fibers, whereas the internal one--by light ones. Microelectrode study demonstrated that the fibers of the external surface were characterized, in comparison with those of the internal one, by lesser action potentials, prolonged trace negative potential, low quant composition of the end plate potentials, high amplitude and low frequency of the end plate miniature potentials. Analysis of the data obtained demonstrated definite interrelationship between the histochemical profile of the muscle fibers of the frog sartorius muscle and their electrophysiological characteristics.     

End plate spikes in the human electromyogram. Revision of the fusimotor theory.

'End plate spike' (EPS) is a spontaneous action potential of a normal striated muscle. EPSs are found in local 'active spots' of the muscle. The prevailing hypothesis about the origin of EPSs states that when a needle electrode affects a motor nerve branch near the neuromuscular junction at the end plate zone, an increased leakage of acetylcholine to the synaptic cleft ensues. This elicits postsynaptic action potentials of the muscle fibre which can be recorded as EPSs with the same needle electrode. Thus EPSs are thought to be caused by needle injury or irritation of the motor axon. We suggest that EPSs are action potentials of intrafusal muscle fibres and that 'active spots' are in fact muscle spindles. Waveform analysis reveals three types of EPSs: small EPSs, not propagated outside the active spot either: i) with negative onset; or ii) with short positive initial deflection; and iii) large EPSs resembling propagated motor unit potentials (MUPs) but with a typical EPS firing pattern, distinctly different from that of the MUPs. Study of EPS activation in different manoeuvres associates small EPSs with intrafusal gamma motor units and large MUP-like EPSs with beta motor units.     

Nonquantum release of acetylcholine in frog neuromuscular junction after blocking of axoplasmic transport with colchicine

Standard microelectrode techniques were used to evaluate the effect of d-tubocurarine chloride on membrane potential of junctional and extrajunctional areas of muscle fibers in a potassium-free Ringer solution. The experiments were made on frogs after inactivation of acetylcholinesterase. d-Tubocurarine chloride hyperpolarized the membrane of muscle fibers only in the junctional area. Blockade of axoplasmic transport with colchicine did not affect the magnitude of the hyperpolarization response of the membrane end plate to the presence of d-tubocurarine chloride, but at the same time it significantly reduced the membrane rest potential of muscle fibers, and gave rise to the appearance of extrajunctional sensitivity to acetylcholine. It is concluded that the blockade of axoplasmic transport does not affect the pattern of non-quantum acetylcholine release from nerve terminals. Therefore, this is unlikely to cause denervation-like changes in the muscle under the conditions described.