Effects of pindolol,sotalol and the optical isomers of propranolol on muscle action potentials and depolarization-secretion coupling in the rat

Muscle action potentials and miniature end-plate potential frequency were studied in different concentrations of pindolol, d-and l-propranolol and sotalol using intracellular microelectrode recording from rat phrenic nerve-diaphragm preparations, d-and l-propranolol at concentrations of 10 to 40 mg/1 and pindolol at 20 to 100 mg/1 slowed down the rate of rise, prolonged rise and half-decay times and reduced the overshoot of the muscle action potentials. Sotalol had similar effects but only at higher concentrations (100 to 200 mg/1). The drugs had no effect on the increase in the miniature end-plate potential frequency obtained with depolarization by increased extracellular potassium concentration.     

Depression, facilitation, and mobilization of transmitter at the rat diaphragm neuromuscular junction.

The characteristics of depression, facilitation, and mobilization of transmitter were examined at the rat diaphragm neuromuscular junction. Intracellular recording techniques were used to monitor end-plate potentials (EPPs), miniature end-plate potentials (MEPPs) and the muscle resting potentials. The cut-muscle technique was used to prevent muscle action potentials. Quantal release was determined by the direct method. The binomial statistical parameters, releasable store (n) and probability of release (p), were examined under various stimulating conditions to determine the basis for depression and facilitation. The present experiments demonstrate that p remains unchanged during repetitive nerve stimulation at low or moderately high frequencies. The experiments demonstrate that depression is due to a decrease in n and facilitation is due to an elevation in n. It is suggested that the increase in n during facilitation is due to a transient recruitment of inactive releasing sites. Substantial replenishment of n by mobilization occurs within a few ms after a stimulus but a slow residual rate of mobilization is needed to replenish n to resting levels.     

Estimates of quantal-release and binomial statistical-release parameters at rat neuromuscular junction.

Quantal-release and binomial statistical-release parameters were examined in the isolated rat diaphragm phrenic nerve preparation. The muscle resting potentials were reduced by cutting the muscle fibers to prevent muscle action potentials and contractions. The cutting technique was modified to allow persistent observation of miniature end-plate potentials (MEPPs) and end-plate potentials (EPPs) with intracellular recording techniques. Direct estimates of quantal release were examined and compared with predicted binomial and Poisson distributions. The results indicate that release is binomial when the nerve is stimulated with low- or high-frequency stimuli. The indirect method of estimating quantal release (variance method), which assumes release is described by a Poisson distribution, seriously overestimates quantal release. The binomial analysis indicates that the statistical store is small (less than 90 quanta) and that most of these quanta are released with each nerve impulse.     

A study of frog muscle maintained in organ culture

1. Frog muscles are isolated and maintained in organ culture conditions for periods of up to 2 months. During the first 2 weeks, muscle fibres have normal resting membrane and action potentials. Subsequently the potentials decline in amplitude.2. Slow muscle fibres also survive in culture and retain their ability to give maintained contractures.3. Muscle sensory receptors continue to function in culture until the axon terminals degenerate at about 2 weeks.4. Neuromuscular transmission is normal during the first few days of culture, after which the motor endings degenerate. Transmission persists longer (up to 17 days) if a long segment of nerve is left attached to the muscle. With short-nerve preparations failure of transmission in vivo occurs at about the same time as in culture. With long-nerve preparations failure of transmission is delayed even further in culture.5. In short-nerve preparations miniature end-plate potentials disappear, in general, at about the time that transmission fails. In long-nerve preparations some end-plates continue to have miniature end-plate potential activity for a short time after nerve impulses cease to evoke any response; but eventually miniature potential activity disappears from all end-plates.6. After a few days of electrical silence, miniature end-plate potentials reappear at some of the denervated end-plates. The proportion of denervated end-plates which show miniature end-plate potentials in culture is smaller than in muscles denervated in situ.7. Electron microscopy shows that muscle structure is well preserved in culture, that the axons degenerate and that the Schwann cells move to occupy the space vacated by the axons. The Schwann cells are very probably the source of the acetylcholine which evokes miniature potentials in the denervated end-plates.     

Some effects of nerve stimulation and hemicholinium on quantal transmitter release at the mammalian neuromuscular junction

1. Rat phrenic nerve-diaphragm preparations have been used to assess some effects of prolonged nerve stimulation on transmitter release.2. The amplitude of the end-plate potentials evoked by prolonged repetitive nerve stimulation fell gradually during stimulation. Most of this fall was due to a reduction in the number of transmitter quanta released by each nerve impulse; however there was also a small reduction in the muscle cell depolarization produced by each quantum of transmitter.3. Repetitive nerve stimulation also produced a small reduction in the amplitude of the miniature end-plate potentials. Recovery of amplitude occurred within about 7-8 min of ceasing stimulation.4. A much greater reduction in miniature end-plate potential amplitude accompanied prolonged nerve stimulation if hemicholinium was present in the bathing solution.5. Estimates of the ;readily available transmitter' (Elmqvist & Quastel, 1965b) were made at intervals following prolonged nerve stimulation. Readily available transmitter was reduced, and recovered over approximately 15 min.6. The relationship of these changes to the changes in nerve terminal synaptic vesicle numbers and volumes induced by similar prolonged nerve stimulation (Jones & Kwanbunbumpen, 1970) is discussed.     

Immobilization atrophy and membrane properties in rat skeletal muscle fibres

Wet mass, resting membrane potential, frequency of miniature end-plate potentials and the concentration of [³H]ouabain-binding sites were studied after 7 days' immobilization of the rat soleus and extensor digitorum longus (EDL) muscles in the shortened or stretched position and after 3 and 7 days of remobilization. We observed that the loss of muscle mass by 37% in the rat soleus immobilized for 7 days in the shortened position is accompanied by a membrane depolarization of about 5 mV, a decrease in frequency of miniature end-plate potentials by 60 % and a decrease of [³H]ouabain binding by 25%. Only minor changes were found in stretched soleus and in shortened and stretched EDL. After 3 days of remobilization of stretched soleus the muscle mass, [³H]ouabain binding and miniature end-plate potential frequency recovered to control values but the resting membrane potential continued to decrease. All changes induced by immobilization disappeared on day 7 of remobilization.     

Characteristics of transmitter release at regenerating frog neuromuscular junctions

1. A study was made of the onset of transmission and the characteristics of transmitter release from regenerating nerve terminals in frog muscle fibres.

2. Soon after transmission had been restored, some junctions were found which responded to nerve stimulation with only subthreshold end-plate potentials.

3. The evoked transmitter release had a non-linear dependence on the external calcium concentration, like that seen at normal junctions.

4. The synaptic delay was only slightly longer than normal, and the amplitudes of single quantum potentials evoked by nerve stimulation seemed to have a normal distribution.

5. The mean amplitude of the spontaneous miniature end-plate potentials was often substantially smaller than the mean amplitude of the evoked quantal potentials at a given end-plate. Some of these small spontaneous potentials were due to transmitter release from the axon terminal. Possible explanations for this discrepancy in size of spontaneous and evoked potentials are discussed. Two to three weeks after reinnervation began, the amplitude of the spontaneous miniature end-plate potentials returned to normal.

     

Influence of presynaptic receptors on neuromuscular transmission in rat

The presence and physiological significance of acetylcholine (ACh) receptors on motor nerve terminals was examined at the rat diaphragm neuromuscular junction. Intracellular recording techniques were used to monitor end-plate potentials (EPP), miniature end-plate potentials (MEPP), and resting potentials of the muscle fibers. Muscle action potentials were blocked by the cut-muscle technique. Quantal release was determined by the ratio EPP/MEPP, after correcting for nonlinear summation. Blockade of acetylcholinesterase with eserine and neostigmine was tested to determine the influence of residual ACh on transmitter release. Partial blockade of ACh receptors with curare was examined to further clarify the role of these presynaptic receptors. The experiments demonstrate that residual ACh inhibits transmitter release and that blockade of ACh receptors enhances transmitter release. It is concluded that presynaptic ACh receptors exist and that they serve an important physiological function. It is suggested that the presynaptic ACh receptors normally serve to limit transmitter release in a negative feedback pathway.     

Non-equivalence of impulse blockade and denervation in the production of membrane changes in rat skeletal muscle.

1. A complete and long lasting blockade of nerve impulses was established in the sciatic nerve of rats, by implanting silastic cuffs of critical internal diameters. Either marcaine-impregnated or plain cuffs were used. The contralateral sciatic nerve was sectioned. 2. At various days after the initial procedures, the extensor digitorum longus muscles of the two sides were examined with intracellular electrodes. 3. Decrease in resting membrane potential, fibrillatory activity and resistance of the action potential to tetrodotoxin developed not only in the denervated but also in the impulse-blocked muscles. In the latter, the fibres were normally innervated since they displayed miniature end-plate potentials and were excitable by nerve stimulation distal to the blocking cuff. 4. However, all of the above mentioned denervation-like changes were significantly less pronounced in the blocked muscles than in the denervated ones. 5. It is concluded that in addition to loss of nerve impulses, some other neural factor must be taken into account to explain the origin of muscle changes induced by denervation. The possible relation of this additional factor with nerve degeneration is discussed.     

Electrophysiological studies of neuromuscular transmission in hereditary `motor end-plate disease' of the mouse

1. Studies have been made on isolated nerve-muscle preparations from mice with hereditary ;motor end-plate disease'.2. Spontaneous fibrillation was observed in the isolated preparation.3. Muscles gave only a weak twitch or failed to contract in response to nerve stimulation. Direct stimulation of muscles caused a twitch response which had a slower time course than normal. Peripheral nerve conduction was normal.4. Intracellular recording from single muscle fibres showed that with longer survival of the animal an increasing proportion of fibres failed to show end-plate potentials or action potentials in response to nerve stimulation.5. Miniature end-plate potentials (m.e.p.p.s) were recorded in almost all muscle fibres including those in which neuromuscular transmission had failed. The frequency of m.e.p.p.s was greater than normal, was not increased by tetanic stimulation of the nerve but was increased by a raised external potassium concentration.6. Muscle fibres were supersensitive to acetylcholine.7. The results suggest that the muscular weakness in this disease is due to the failure of nerve action potentials to invade motor nerve terminals so that muscle fibres become ;functionally denervated'.     

The effect of type D botulinum toxin on frog neuromuscular junctions

1. Botulinum toxin type D blocks neuromuscular transmission in frogs. Motor nerve impulses continue to invade the nerve terminals but cease to evoke the phasic release of transmitter normally associated with them.2. Sensory receptors in the muscle continue to generate impulses even after 4 days of continuous exposure to botulinum toxin.3. Contrary to expectations, spontaneous miniature end-plate potentials did not disappear completely after botulinum intoxication; they still occurred, although with reduced frequency, in most end-plates. These miniature potentials had a skew amplitude distribution instead of the bell-shaped distribution of normal end-plates.4. The electron-microscopic appearance of botulinum-poisoned end-plates was not obviously altered.5. Even though after botulinum nerve impulses fail to release transmitter, tetanic nerve stimulation causes an increase in the frequency of miniature end-plate potentials. Many of the unit potentials which appear during the tetanic period are of an amplitude which is larger than that of spontaneous potentials, indicating that a different class of unit is being released preferentially. Some implications of this finding are briefly discussed.     

An electrophysiological investigation of the slow fibre system in the frog rectus abdominis muscle

1. Junctional potentials were recorded with a micro-electrode inserted into muscle fibres of the rectus abdominis muscle of the frog. Large and small nerve fibres were stimulated separately, using a selective stimulation technique. In a few muscle fibres rectangular current pulses were applied through a second micro-electrode to examine current-voltage relations.2. Two groups of muscle fibres could be distinguished: (a) Muscle fibres with resting potentials less negative than -75 mV, being localized at the ventral surface only and representing about 9% of the superficial fibres. These fibres were found to be innervated by small (high-threshold) nerve fibres and responded to single indirect stimuli with a typical s.j.p. (long latency, multiple components, after-hyperpolarization); upon repetitive stimulation summation of s.j.p.s and a slowly increasing tension could be recorded. The fibres showed a slow time course of electrotonic potentials, relatively high membrane resistance and ;delayed rectification'. (b) Muscle fibres with generally much higher resting potentials, being innervated by large (low-threshold) nerve fibres only. Upon indirect stimulation a short-latency end-plate or action potential (followed by a twitch) was recorded. The membrane time constant and input resistance of these fibres were similar to those of twitch fibres in other frog muscles.3. No evidence was found in the present work for the existence of an intermediate type of muscle fibre.4. It is concluded that the rectus muscle, similar to other frog muscles, contains two distinct types of muscle fibres, twitch and slow.     

Pre- and post- junctional effects of 1-fluoro-2,4-dinitrobenzene at the frog neuromuscular junction

1. FDNB increased by 60-90% the depolarization of the end-plate produced by applied carbachol in frog sciatic nerve-sartorius muscle preparations.2. In partially curarized preparations, FDNB (0.4 mM) increased the amplitude of the end-plate potential by a factor of 1.8.3. The quantal content of end-plate potentials was increased by FDNB (2 mM) as determined by the method of failures.4. After approximately 25-35 min, neuromuscular transmission was blocked by 0.4 mM-FDNB, as evidenced by abolition of neurally elicited end-plate potentials. At this stage miniature end-plate potentials could still be recorded, which indicates that the neuromuscular block was presynaptic.5. FDNB (0.4 mM) increased miniature end-plate potential frequency several hundred-fold when the Ringer solution contained normal calcium concentration (1.8 mM) or 0.45 mM calcium and 5.4 mM magnesium.6. During the first 60 min of exposure to 0.4 mM-FDNB there was a slight drop (4-6 mV) in resting potentials of muscle fibres. During this period directly initiated action potentials showed a marked decrease in the rate of repolarization and a small decrease in the amplitude and rate of rise.7. Using the technique of point voltage clamping in tetrodotoxin-treated muscles, it has been found that FDNB almost completely abolished the active increase in g(K) during stepwise depolarization of the nonjunctional muscle fibre membrane from -90 to 0 mV. The passive outward leakage current appeared unaffected by FDNB.     

Early membrane depolarization of the fast mammalian muscle after denervation

Summary The first alteration noted after denervation of the extensor digitorum longus muscles of rats was a decrease in resting membrane potential (RMP) which occurred at about 2h. The exact time course of this membrane depolarization was dependent upon the intramuscular length of the degenerating nerve stump. The decrease in RMP occurred prior to any detectable alteration in adenosine triphosphate and phosphocreatine content of the muscle. Prior to failure of spontaneous transmitter release, which occurred 10 h after denervation, some muscle fibres showed an increase in frequency of the miniature end-plate potentials (m.e.p.p.) with no alterations in amplitude and shape of the single potentials. Appearance of areas sensitive to acetylcholine (ACh) on the extra-junctional membrane occurred at 24 h after crushing the motor nerve. At 48 h after denervation a high ACh-sensitivity appeared at the muscle-tendon region, but was not detected in the majority of the muscle fibres studied midway between end-plate region and muscle-tendon area. The transverse resistance of a unit area of the muscle membrane was increased 3 days after denervation.These results provide evidence that the processes of post-denervation changes occur in the following temporal sequence: a) partial depolarization of the postsynaptic muscle membrane; b) a decrease and subsequent cessation of the spontaneous transmitter release preceded in some fibres by a transient increase in m.e.p.p. frequency; c) appearance of extrajunctional ACh sensitivity; d) increase in the transverse resistance of a unit area of the muscle membrane. It is suggested that the motor nerve releases more than one neurotrophic substance.Recipient of a Buswell Fellowship from the State University of New York at Buffalo and on leave of absence from the Christian-Albrechts-Universität, Kiel, West Germany.     

Dual end-plate potentials at the single neuromuscular junction of the adult frog

Summary Electrophysiological evidence is presented that at least 30 percent of sartorius muscle fibres of adult frogs are innervated by two or more axons at a single end-plate zone. In these fibres, increasing stimulation of the common sartorius nerve led to the appearance of two or more distinct levels of end-plate potentials (e.p.p.) (or currents, measured by the voltage clamp technique). They had an identical time course, reversal potential and delay to nerve stimulation. When the recording microelectrode was moved along the same fibre and reinserted, both components of e.p.p. decreased proportionally. This indicated that both components of e.p.p. originated very closely to each other on the muscle fibre, presumably in one end-plate zone. Many fibres of the sartorius muscle of adult frogs therefore possess polyneural innervation of a single end-plate zone, which is otherwise typical for early stages of ontogenesis.     

Control of ACh sensitivity by muscle activity in the rat

1. Rat soleus and extensor digitorum longus (EDL) muscles were examined following complete blockade of sciatic nerve impulses with anaesthetics or diphtheria toxin for periods up to 14 days.2. Muscles showed atrophy equivalent to that seen after similar periods of denervation.3. Nerve blockade appeared to have little or no effect on neuromuscular transmission when tested by stimulation beyond the block. Normal spontaneous miniature end-plate potentials were present.4. Nerve impulse blockade caused the entire muscle membrane to become sensitive to iontophoretically applied acetylcholine.5. The increase in sensitivity in soleus could be prevented by chronic nerve stimulation distal to the region of block.6. Tenotomy, of 5-12 days duration, which produced atrophy, had no effect on the sensitivity of soleus to acetylcholine.7. Chronic direct stimulation of denervated soleus or EDL muscles could prevent the usual denervation supersensitivity, or cause it to decline towards normal once it had appeared. However, the sensitivity of the end-plate region remained normal.     

Membrane and biochemical alterations after denervation and during reinnervation of mouse skeletal muscle

Denervation of the extensor digitorum longus (EDL) muscle of the mouse by either nerve crush or nerve section produced: a reduction of the resting membrane potential (E_m), alterations in the properties of muscle fibre action potentials and the development of tetrodotoxin (TTX)-resistant action potentials. These changes in membrane electrical properties were accompanied by an increase in the endocytic activity of the muscle and an increase in the activities of the lysosomal enzymes cathepsin D and N-acetylglucos-aminidase (NAGA). Reinnervation of muscle was indicated at 9 days after nerve crush by the presence of miniature end-plate potentials. The recovery of membrane electrical properties, beginning with the onset of reinnervation, were not temporally related. The E_m increased in two stages: an early rapid repolarization and a later slower repolarization. The muscle fibers were sensitive to the blocking action of TTX by 12 days after nerve crush, whereas the rate of rise (dV/dt) of the action potential did not approach values of innervated muscles until 21 days. Reinnervation resulted in a decrease in endocytosis and a decrease in the activities of cathepsin D and NAGA toward innervated values by 21 days after nerve crush. The results suggest that membrane alterations after denervation and during reinnervation may occur by endo- and exocytosis of membrane constituents and that the lysosomal system may play a role in the breakdown and/or recycling of these structures.     

A note on the interaction of spontaneous and evoked release at the frog neuromuscular junction

1. The interaction between spontaneous miniature end-plate potentials and evoked end-plate potentials was investigated at the frog neuromuscular junction using focal extracellular recording techniques.2. End-plate potentials evoked immediately after a spontaneous miniature potential were facilitated by up to 20%. The percentage facilitation was negatively correlated with the average quantal content of the end-plate potential.     

On the role of mitochondria in transmitter release from motor nerve terminals.

1. The changes in transmitter release produced by mitochondrial inhibitors has been studied at the frog neuromuscular junction using conventional electrophysiological techniques for stimulation and intracellular recording. 2. Inhibitors of the electron transport chain and inhibitors of oxidative phosphorylation produce an increase in the frequency of appearance of the miniature end-plate potentials. This increase in frequency is observed also in calcium-free media. Mitochondrial inhibitors also augment the amount of transmitter liberated by a nerve impulse. 3. Ruthenium red, which is an inhibitor of calcium uptake by mitochondria, increases the spontaneous transmitter release but decreases the quantal content. The latter effect of Ruthenium red is antagonized by calcium. 4. The mitochondrial content of the motor nerve terminals is, on the average, 6.59%. 5. The experimental results are explained on the hypothesis that spontaneous release of transmitter reflects the resting level of intracellular free calcium and the evoked release reflects the sum of the resting calcium and the calcium brought in by the action potential. The mitochondria play a role in transmitter release by participating in the regulation of the intracellular free Ca.     

Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog

1. End-plate potentials were recorded intracellularly at the frog neuromuscular junction bathed in a solution containing a low concentration of calcium and a high concentration of magnesium.2. The muscle was subsequently subjected to ;cholinesterase staining', and the area of the individual end-plates, studied with intracellular electrodes, was measured.3. A positive correlation was found between the end-plate area and the diameter of muscle fibres.4. The mean quantum content (m) showed a positive correlation with the size of end-plates.5. The frequency of spontaneous miniature end-plate potentials was positively correlated with m as well as with end-plate area.6. It is concluded that the amount of transmitter released following nerve stimulation is related to the size of nerve endings.