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'.     

Effects of botulinum toxin on neuromuscular transmission in the rat.

1. Botulinum toxin (BoTx) type A partially blocks spontaneous transmitter release from nerve terminals in the rat. Minature end-plate potentials (m.e.p.p.s) are present at all end-plates, initially with a low frequency but increasing with time after posoning. Their amplitude distribution is at first skew with a predominace of very small m.e.p.p.s but, after a few days, larger than normal m.e.p.p.s appear. 2. Tetanic nerve stimulation, Black Widow Spider Venom, the Caionophore A 23187 or mechanical damage to nerve terminals increases the frequency of m.e.p.p.s and alters the amplitude distribution of m.e.p.p.s towards a normal Gaussian one; the m.e.p.p. size approaches that seen at normal end-plates. This was seen at any time after poisoning. 3. Nerve stimulation gives rise to end-plate potentials (e.p.p.s) of low amplitude and high failure rate. Statistical analysis indicates that evoked release is quantal in nature and follows Poisson statistics, quantum size being initially very small, but after a few days approaching normal size. Short-term tetanic nerve stimulation reversibly increases the quantum content of e.p.p.s and during early stages of paralysis long-term (2 hr) stimulation causes an apparently permanent increase in quantum size. 4. Raising the extracellular Ca concentration from 2 to 16 mM increases the frequency of m.e.p.p.s in normal muscle but not in BoTx poisoned ones. K-free medium or ouabain, which are believed to raise the intracellular Ca concentration in nerve terminals, similarly increases m.e.p.p. frequency in normal but not in poisoned muscles. When the Ca-ionophore A 23187 is used together with high extracellular Ca (greater than 4 mM) massive release of transmitter occurs from poisoned terminals. 5. The extracellular Ca concentration which causes a certain level of transmitter release in reponse to nerve impulses is considerably higher at BoTx poisoned end-plates than at normal ones. The slope value for Ca dependence of transmitter release is about 1-5 compared with about 3 at normal end-plates. 6. Tetraethylammonium (TEA) greatly increases the amount of transmitter released by nerve impulses and restores neuromuscular transmission during all stages of poisoning, although it has not effect on spontaneous transmitter release. In the presence of TEA the power relation between Ca concentration and quantum content at the BoTx poisoned end-plate is similar to that seen at normal end-plates. 7. It is suggested that in BoTx poisoning the mechanism for transmitter release has a reduced sensitivity to Ca, and the level for activation by intracellular Ca is elevated. Once the intracellular concentration of Ca is raised to this level, by tetanic nerve stimulation, mechanical injury to nerve terminals, the Ca-ionophore or the prolongation of the nerve action potential with TEA, augmented transmitter release occurs, similar to that which occurs in normal nerve terminals at a lower level of Ca.     

Tetanic and post-tetanic rise in frequency of miniature end-plate potentials in low-calcium solutions

1. Miniature end-plate potentials (min.e.p.p.s) were recorded intracellularly from frog neuromuscular junctions.2. The ;phasic' release of transmitter which is directly related to nerve impulses was suppressed by withdrawal of Ca from the external medium plus addition of Mg.3. Under these conditions, min.e.p.p.s continued to be discharged even when EGTA was added, although in this case min.e.p.p. frequency appeared to decrease to about half the rate in normal Ringer.4. Tetanic stimulation of the nerve approximately doubled the rate of min.e.p.p.s even in Ca-free solutions with EGTA added.5. The tetanic increase in frequency was greater without EGTA and greater still with some Ca added. Therefore, it is concluded that the tetanic rise in min.e.p.p. frequency can occur even in the absence of the immediate ;phasic' release of transmitter normally induced by nerve impulses; and that the magnitude of the increase is related to Ca concentration.A possible relation between ;phasic' and ;residual' effects of nerve impulses is described.     

On the degeneration of rat neuromuscular junctions after nerve section

1. A study was made of functional and structural changes during degeneration of end-plates in the rat diaphragm after phrenic nerve section at two levels.2. For 8-10 hr after cutting the nerve in the neck, all end-plates retain the ability to transmit impulses. During the following 8-10 hr, an increasing number of end-plates lose this ability so that after a total of about 20 hr, no end-plates can transmit.3. Transmission failure occurs abruptly at most end-plates. This failure is usually accompanied by cessation of spontaneous miniature end-plate potentials (min.e.p.p.s), though in a few cases min.e.p.p.s persist after junctional transmission has failed. Several degenerating junctions were observed where the frequency of min.e.p.p.s was very low, suggesting an intermediate stage in min.e.p.p. failure.4. The time of junctional failure depends on the length of the degenerating nerve stump. For each additional centimetre of nerve, failure is delayed about 45 min.5. Changes in ultrastructure of nerve endings closely parallel those of function. For about 8-12 hr after cutting the nerve, nearly all end-plates appear normal. During the period when transmission is failing, some end-plates are clearly undergoing structural break-down. By the time functional failure is complete, all end-plates appear grossly abnormal.6. During degeneration, the contents of the axoplasm undergo disruption and the nerve terminal breaks up into small fragments. In contrast, the Schwann cell appears to become very active and its processes extend into the synaptic cleft to surround fragments of the nerve terminal. Ultimately, the Schwann cell completely replaces the axon at the end-plate.7. Increasing the length of the peripheral nerve stump delays the onset of structural break-down. Disruption of end-plates near the site of nerve entry into the muscle occurs before those farther away.8. It is suggested that end-plate degeneration is triggered by a signal which passes from the site of injury to the nerve terminal. The duration of the period after transection when end-plates appear to be normal would then reflect the time required for this signal to travel the length of the isolated nerve stump.     

Transmitter release in tetanus and botulinum A toxin-poisoned mammalian motor endplates and its dependence on nerve stimulation and temperature

The effects of tetanus toxin (TeTx) and botulinum A toxin (BoTx) on spontaneous and nerve-evoked transmitter release have been compared in mouse hemidiaphragms poisoned in vitro. At 37 degrees C endplates poisoned with either of these agents were characterized by (1) a decrease of miniature endplate potential (m.e.p.p.)-frequency to less than 30/min for TeTx and 3/min for BoTx, (2) reduced mean m.e.p.p.-amplitude and (3) 100% failure to show endplate potentials (e.p.p.s) in response to single nerve stimuli. In addition (4) tetanic nerve stimulation and/or reduction of temperature to about 20 degrees C caused a remarkable increase in the nerve-evoked transmitter release, but did not affect the low frequency of spontaneous m.e.p.p.s. However, several important differences exist between the effects of both toxins. (1) At room temperature even single nerve stimuli could elicit e.p.p.s in BoTx-muscles the failure rate being about 80%. For TeTx the failure was 100%. However, if the nerve was stimulated with higher frequencies (greater than 5 Hz), the probability of quantal release increased, the delay for release from the onset of stimulation being several seconds and similar to that observed at 37 degrees C. (2) TeTx distorted the synchronous release of quanta increasing the distribution of their synaptic delays. BoTx did not influence the time course of the phasic secretion process in response to nerve action potentials. (3) TeTx preferentially blocked the release of spontaneous m.e.p.p.s of large amplitude without affecting the frequency of the small amplitude ones, while BoTx inhibited both the small and large amplitude m.e.p.p.s. The distribution of the amplitudes of the nerve-evoked m.e.p.p.s were similar to those of spontaneous m.e.p.p.s before the blockade with the toxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the effect of calcium on the frequency of miniature end-plate potentials at the frog neuromuscular junction.

1. The effect of the extracellular Ca concentration on the frequency of miniature end-plate potentials (min. e.p.p.s) at the frog neuromuscular junction was studied. 2. In saline containing elevated K (5 or 11 mM), the frequency of min. e.p.p.s increased as Ca concentration was increased from 0-1 to 1-3 mM. However, with further increases of Ca concentration up to 10 mM, min. E.P.P. frequency declined. 3. In saline containing the normal concentration of K (2 mM), increasing Ca concentration from 0-1 to 10 mM produced a slight, monotonic increase in min. e.p.p. frequency. 4. The non-monotonic effect of Ca on min. e.p.p. frequency in preparations depolarized by elevated K is consistent with the existence of two opposing effects of Ca on transmitter release. Firstly, raising the external concentration of Ca increases the electrochemical potential for Ca entry, which tends to increase Ca influx and transmitter release. Secondly, increasing external Ca concentration increases electrostatic screening of fixed negative charges on the outer surface of the nerve terminal membrane. Such an increase in screening of charges near voltage-sensitive Ca gates would produce a hyperpolarization across the gates and they would tend to close, an effect which would tend to decrease Ca influx. The monotonic increase in min. e.p.p. frequency with increasing Ca concentration in 2 mM-K is consistent with the voltage insensitivity of the Ca gates at potentials close to the normal resting potential.     

Miniature potentials in denervated slow muscle fibres of the frog

1. Pyriformis and iliofibularis muscles of the frog were studied with micro-electrodes. Both muscles contain a mixture of fast and slow muscle fibres, which can be distinguished by differences in their miniature end-plate potentials (min.e.p.p.s).2. In addition, the membrane time constant of fast fibres is < 20 msec, while that of slow fibres is > 200 msec.3. The characteristic difference in time constant persisted after denervation and allowed the identification of fibre type.4. Denervated slow fibres had min.e.p.p.s of variable time courses and skew amplitude distributions. Their frequency was lower than in normally innervated fibres, and could be increased by hypotonic solutions.5. In analogy with similar observations made previously in fast muscle fibres, it is suggested that after nerve degeneration the Schwann cells of small motor nerve fibres release packages of transmitter which give rise to min.e.p.p.s.     

Effects of calcium and magnesium on the frequency of miniature end-plate potentials during prolonged tetanization

1. End-plate potentials (e.p.p.s) and miniature end-plate potentials (min.e.p.p.s) were recorded intracellularly from the cutaneous pectoris nerve-muscle preparation of the frog during prolonged stimulation at low frequencies (5/sec-50/sec).2. When Ca was present in the bathing solution, the quantum content of the e.p.p. and the frequency of occurrence of the min.e.p.p.s gradually increased during the period of stimulation. During the first few minutes of stimulation, the min.e.p.p. frequency increased linearly with time, and the rate of increase was dependent on the Ca concentration of the bathing solution. However, Mg had no effect on this Ca-dependent increase in min.e.p.p. frequency.3. A large maintained increase in min.e.p.p. frequency also occurred during prolonged stimulation in solutions that contained no added Ca and 1-2 mM-EGTA. Under these conditions the increase in min.e.p.p. frequency was dependent on the Mg concentration of the bathing solution and was exponential in time.4. It is suggested that the rise in min.e.p.p. frequency is caused by an accumulation of Ca or Mg ions in the nerve terminal, and it is suggested that these ions enter the terminal at relatively non-specific sites distinct from the Ca-specific sites that trigger the ;phasic' release of transmitter.     

Observations on the action of type A botulinum toxin on frog neuromuscular junctions

1. Progressive block of neuromuscular transmission in frog sartorius and gastrocnemius preparations by haemagglutinin-free crystalline Type A botulinum toxin (BTX) was investigated by in vitro application and by injection of the toxin into living animals.2. Neuromuscular block was characterized by (a) decline in amplitude of evoked twitch contractions, (b) decline in amplitudes of end-plate potentials (e.p.p.s) and (c) changes in statistical characteristics of spontaneous miniature end-plate potentials (m.e.p.p.s).3. Progress of the block was enhanced by nerve stimulation.4. A decrease in frequency to less than 0.1/sec and decreased average amplitudes of m.e.p.p.s preceded observable impairment of neuromuscular transmission. These changes occurred as early as 3 hr after injection of the toxin into dorsal lymph sacs.5. The amplitude distributions of m.e.p.p.s changed from a normal distribution to one that showed an increased skewness toward smaller amplitudes as the block progressed. These changes were first detectable as early as 75 min following addition of the toxin to the bath.6. At later stages of toxin action, e.p.p.s began to decrease in amplitude and eventually failed altogether. E.p.p.s showed a normal quantal variation at very early stages in the block in Mg(2+)-treated preparations. At later stages of the block, it was not possible to test the quantal make-up of the e.p.p.7. At all stages before complete failure it was possible to obtain normal or greater than normal degrees of synaptic facilitation with paired stimuli to the nerve. This aspect of the coupling of nerve terminal depolarization to transmitter release appears to be relatively unaffected by BTX.8. Electrical depolarization of nerve terminals in partially blocked preparations evoked a maintained discharge of m.e.p.p.s with an amplitude distribution similar to that of the spontaneous m.e.p.p.s; hyperpolarization of the terminals evokes a distinctly larger class of m.e.p.p.s. In fully blocked preparations, depolarization of the terminals does not evoke transmitter release whereas hyperpolarization continues to yield the larger class of m.e.p.p.s.9. It is proposed that the neuromuscular block caused by BTX is due to impairment of a process by which vesicles become charged with transmitter before release.     

The effects of hypoxia on neuromuscular transmission in a mammalian preparation

1. The rat diaphragm-phrenic nerve preparation in vitro failed to contract in response to nerve impulses after 10-20 min exposure to solutions containing 95% N(2) and 5% CO(2) (hypoxic solutions) at temperatures between 33 and 38 degrees C. Intracellular recording revealed that end-plate potential (e.p.p.) amplitudes fell below the firing threshold for muscle fibres and then disappeared probably because of block of intramuscular nerve conduction.2. In curarized and Mg-paralysed preparations the reduction in e.p.p. amplitudes was found to be due to a fall in their quantal content. In about half of the Mg-paralysed preparations, however, and in curarized preparations after repeated exposures, there were increases in quantal content of e.p.p.s during hypoxia.3. Miniature end-plate potential (m.e.p.p. frequency increased in a cyclic fashion during hypoxia and this increase was largely suppressed in the presence of a raised extracellular Mg concentration. M.e.p.p. amplitude increased (range 0-100% of control value) after about 20 min hypoxia.4. Post-tetanic potentiation of e.p.p. amplitudes and m.e.p.p. frequency was reduced after exposure to hypoxic solutions. During hypoxia the e.p.p. amplitude potentiation was reduced but the m.e.p.p. frequency potentiation was augmented.5. There was an increase in the post-synaptic sensitivity to carbamylcholine after 20 min hypoxia which was sufficient to explain the increase in m.e.p.p. amplitude. Other post-synaptic changes were a fall in membrane potential (average 6 mV after 20 min) and a fall in membrane resistance after 30-60 min exposure to hypoxia.6. The effects of hypoxia upon neuromuscular transmission were partially explained by reduction of active transport of sodium and potassium ions and consequent depolarization of nerve and muscle.     

Competition between sodium and calcium ions in transmitter release at mammalian neuromuscular junctions

1. Frequencies of miniature end-plate potentials (m.e.p.p.s) were recorded at neuromuscular junctions in rat diaphragm-phrenic nerve preparations in vitro.2. In the presence of raised [K] (15-20 mM) lowering [Na] caused a rapid increase in m.e.p.p. frequency whether [Ca] was low or normal. Raising [Na] towards the normal concentration (162 mM) caused a slow fall in frequency and raising [Ca] in the range 0.32-2 mM caused a slow increase in frequency. These effects were less in the normal [K] (5 mM).3. Mean m.e.p.p. frequencies were determined for solutions containing 15 mM-K and combinations of [Ca] and [Na]. M.e.p.p. frequency varied inversely with [Na] when [Ca] was constant. In each of the three Na concentrations used (162, 113 and 65 mM) raising [Ca] in the range 0.32-2 mM increased m.e.p.p. frequency but when raised above 2-3 mM, Ca depressed frequency.4. A model was proposed in which Ca affected transmitter release by changing the concentration in the presynaptic membrane of a complex CaX to which the rate of transmitter release was directly proportional. Higher concentrations of Ca depressed transmitter release by inactivating CaX. Sodium ions competitively depressed release either by competing with calcium ions for association with X or by reducing the affinity of X for Ca.5. When [Na] was lowered in solutions containing raised [Mg] and [Ca], the increase of mean m.e.p.p. frequency was greater than that observed in raised [Ca] and normal [Mg] and was of the same order as the increases seen in low [Ca]. The result was interpreted to indicate either that Na and Mg do not compete with Ca at the same site or that Mg affects the affinity of X for Ca and Na.6. The effect of lowering [Na] on m.e.p.p. frequency was a specific effect of Na ions. When LiCl was substituted for NaCl, the increase of m.e.p.p. frequency persisted. Changes in [Cl] had no effect on m.e.p.p. frequency.7. There was a linear relation between the mean logarithm of m.e.p.p. frequencies and [K], the slope of the relation increasing as [Na] was lowered. Conversely, lowering [Na] caused a greater increase in m.e.p.p. frequency as [K] was raised.8. The variation of m.e.p.p. frequencies in a diaphragm was roughly proportional to a second or higher power of [Na] and inversely proportion to [Ca]. It was thought that this could be due to differences in chelation of Ca which were more apparent at low Ca concentrations.9. The similarities between the effects of Na, Ca and K on m.e.p.p. frequency and the effects of these ions on Ca-influx in heart muscle led to the suggestion that transmitter release is proportional to the concentration of a negatively charged complex of a carrier X with one calcium ion (CaX) at the internal surface of the membrane and that changes in membrane potential affect transmitter release by changing the distribution or location of CaX in the membrane.     

Transmitter release from insect excitatory motor nerve terminals

1. Intracellular and extracellular electrodes were used to study spontaneous and impulse-linked release of transmitter at locust retractor unguis nerve-muscle synapses.2. At most extracellular recording sites the amplitude distributions of the excitatory post-synaptic potentials (e.p.s.p.s) were apparently non-Poisson. However, interpretation of these amplitude distributions was complicated by the effect on the extracellular recordings of the complex structural organization of the retractor unguis nerve terminal with its spatially distinct transmitter release sites extending over distances of 15-30 mum.3. The spontaneous miniature excitatory post-synaptic potentials (min e.p.s.p.s) did not occur at random intervals, bursts of min e.p.s.p.s being frequently recorded. As a result the spontaneous release of transmitter rarely approximated a Poisson process.4. For a period of at least 390 msec following a conditioning nerve impulse a test e.p.s.p. was facilitated and the probability of spontaneous transmitter release was enhanced. A large primary phase of facilitation of impulse-linked and spontaneous release was invariably followed by one or more secondary phases of smaller magnitude.     

Spontaneous subminature end-plate potentials in mouse diaphragm muscle: evidence for synchronous release.

1. Miniature end-plate potentials (min.e.p.p.s) were recorded from small muscle cells of mouse diaphragms. Min.e.p.p. amplitude histograms showed successive peaks which were integral multiples of the smallest peak. The smallest potentials (submin.e.p.p.s) averaged 0-3-0-6mV and the mean of the larger min.e.p.p.s averaged 3-7 mV, depending on the muscle cell diameter. There was a positive correlation between time-to-peak and min.e.p.p. amplitude. Time-to-peak of the submin.e.p.p.s fell slightly below the regression line through the larger min.e.p.p.s. 2. Sometimes min.e.p.p. amplitude distributions changed spontaneously such that the mean of the major mode min.e.p.p.s decreased twofold during which time the mean of the submin.e.p.p.s did not change. Spontaneous decreases were most pronounced during low frequencies of release (10/min) achieved at 32 degrees C. 3. Small changes in temperature (2 degrees C steps in the range 32-40 degrees C) greatly altered the number of peaks of min.e.p.p. amplitude histograms without noticeably changing the position of the submin.e.p.p. peak. At 32 degrees C submin.e.p.p.s composed 5-20% of the histograms and the amplitude of the major mode peak was twelve to fifteen-times that of the submin.e.p.p.s. Over-all bell-shaped distributions were obtained at 37 degrees C which showed up to eight peaks with the major peak at the fourth to sixth peak. Temperatures slightly above 37 degrees C gave a flat distribution with the mean amplitude at the third peak. Min.e.p.p. amplitude histograms were initially skewed (mostly small min.e.p.p.s) after a 40 degrees C heat challenge. 4. Two to eight-times the normal concentration of Ca2+ in the saline reversibly increased the min.e.p.p. frequency and also decreased the mean of the major mode min.e.p.p.s (two to nine-times) without noticeably changing the mean of the submin.e.p.p.s. 5. Botulinum toxin A, 10(5) X intraperitoneal median lethal dose (10(5)I.P.LD50)/ml., almost abolished min.e.p.p.s in 30-90 min. The relative proportion of submin.e.p.p.s increased and the mean of the major mode min.e.p.p.s usually did not change during the initial decrease in frequency. Major mode min.e.p.p.s essentially ceased after 200-1000 were generated and remaining min.e.p.p.s of some cells showed skewed distributions with three small peaks that were integral multiples of the submin.e.p.p. peak. Smaller min.e.p.p.s were more resistant to block than the larger min.e.p.p.s and, although frequencies were low, small min.e.p.p.s were recorded after 4 hr of botulinum toxin incubation. 6. Colchicine (5 X 10(-4)M) within minutes reduced the major mode min.e.p.p.s by half (mean of major peak reduced to sixth or seventh peak). Additional colchicine (10(-3)M reduced the major mode min.e.p.p. amplitude to a fifth of that of control (mean of major mode min.e.p.p.s at the third peak) with no change in position of the submin.e.p.p. peak. Min.e.p.p. amplitudes slowly recovered to half control values after washing. 7...     

The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse

1. A sublethal dose of tetanus toxin was injected into the muscles of one hind leg of the mouse and caused local tetanus which persisted for 4 weeks.2. Neuromuscular transmission was studied in vitro in nerve-muscle preparations of soleus, a slow muscle, and extensor digitorum longus (EDL), a fast muscle, from 1 day to 6 months after the injection of toxin.3. Soleus failed to respond to nerve stimulation, became supersensitive to acetylcholine and showed spontaneous fibrillations for several weeks before returning to normal. EDL did not show these changes. A higher dose of tetanus toxin, lethal within 24 hr, caused paralysis of EDL as well as soleus.4. In muscle fibres in which neuromuscular transmission was blocked spontaneous miniature end-plate potentials (m.e.p.p.s) were recorded. The frequency of m.e.p.p.s was increased by repetitive nerve stimulation but not by raising the external potassium concentration.5. The amplitude of spontaneous m.e.p.p.s showed a skew distribution because of a disproportionate number of potentials of less than 0.2 mV.6. Raising the external calcium concentration did not restore neuromuscular transmission.7. Histological examination of soleus showed atrophy of muscle fibres with normal preterminal axons. There was sprouting from motor nerve terminals and subsequently new motor end-plates were formed. These changes were not found in EDL.8. The results indicate that, in the mouse, tetanus toxin causes a presynaptic block of neuromuscular transmission and ;functional denervation' of muscle. Slow muscle is more sensitive to the effects of the toxin than fast.     

Physiological characteristics of re-innervation of skeletal muscle in the mouse

1. Re-innervation of soleus was studied in the mouse after either crushing the sciatic nerve or re-implanting the nerve to soleus outside the original end-plate region.2. During the early stages of re-innervation subthreshold end-plate potentials (e.p.p.s) were recorded in muscle fibres in response to nerve stimulation. Later the e.p.p.s became large enough to evoke action potentials in muscle fibres.3. The rate of recovery of the release of acetylcholine (ACh) as estimated from the quantal content of e.p.p.s was faster when nerves re-innervated the old end-plate region after nerve crush than after re-implantation so that new neuromuscular junctions were formed.4. During re-innervation soleus fatigued more rapidly than normal during repetitive nerve stimulation. The fatigue was due to failure of neuromuscular transmission associated with an impaired release of ACh.5. During re-innervation soleus was supersensitive to ACh until nerve stimulation was capable of evoking action potentials in muscle fibres.     

Transmitter release by mammalian motor nerve terminals in response to focal polarization

1. A method is described by which mammalian motor nerve terminals may be uniformly polarized by focally applied current, and the extra-cellular potential in the synaptic cleft, corresponding to any current, estimated.2. The relationship between log m.e.p.p. frequency and local extra-cellular field is flat for hyperpolarization and ascends linearly with depolarization. With depolarization, m.e.p.p. frequency is multiplied about tenfold for every - 18 mV. This characteristic becomes steeper the closer the polarizing electrode to the nerve terminal with a limiting value of ten-fold per - 15 mV.3. There exists a population of small m.e.p.p.s which are generated at the same end-plate as normal m.e.p.p.s.4. Following a prolonged depolarizing pulse there is an increase of m.e.p.p. frequency which continues for periods of up to several minutes.5. With hyperpolarizing pulses m.e.p.p. frequency may increase in a characteristic ;bursty' manner. Similar bursts of m.e.p.p.s also occur spontaneously, but far less frequently, without polarization.6. During a depolarizing pulse, m.e.p.p. frequency becomes maximal or near maximal within 2 sec. There is little subsequent alteration of m.e.p.p. frequency. Numbers of m.e.p.p.s occurring during depolarizing pulses follow the Poisson distribution.7. Following a depolarizing pulse, numbers of m.e.p.p.s released by a subsequent pulse may be either increased or diminished.8. Comparison of the response of m.e.p.p. frequency to raised [K] and to extrinsic presynaptic polarization leads to the conclusion that the presynaptic transmembrane potential change corresponding to any focal current pulse is about two thirds of the local extracellular potential field. Hence the slope of the linear portion of the presynaptic transfer function is about tenfold per 10 mV presynaptic depolarization.     

Cumulative and persistent effects of nerve terminal depolarization on transmitter release

1. Following focal depolarization of rat motor nerve terminals there could often be observed an ;after-discharge' of m.e.p.p.s with transient frequencies of up to 1000/sec. This after-discharge was graded with intensity and duration of the previous depolarization.2. Following pulses which were relatively short (about 1 sec) and not too large (< -100 mV local extracellular potential field) the logarithm of m.e.p.p. frequency fell exponentially. With larger or longer pulses there was a tail to the after-discharge which could persist for several minutes.3. M.e.p.p. frequency during an after-discharge was not inhibited appreciably by nerve terminal hyperpolarization, raised [Ca] (8 mM) or lowered pH.4. Measured as a multiplication of spontaneous m.e.p.p. frequency after-discharge was depressed in solution containing no Ca(2+) and added 1 mM-MgEDTA but equal in 0.125 mM-Ca(2+) or 2 mM-Sr(2+) to that in 2 mM-Ca(2+) or 8 mM-Ca(2+).5. During an after-discharge the multiplication of m.e.p.p. frequency by focal nerve terminal depolarization or raised K(+) was reduced. This phenomenon was termed ;uncoupling'.6. It was concluded that the after-discharge is not caused by a persistent rise of K(+) concentration in the synaptic cleft, nor by a maintained nerve terminal depolarization.7. In preparations depolarized by raised K(+) m.e.p.p. frequency during a relatively small focal depolarizing pulse rose continuously, after an initial rapid rise, and after the pulse there was a tail of increased m.e.p.p. frequency. The magnitude of the rise during the pulse and the tail after it were similar on, a logarithmic basis; during both processes the logarithm of m.e.p.p. frequency usually followed (approximately) an exponential time course.8. The relative magnitude of the slow effect of depolarization, as compared with the fast effect, was increased by lowering [Ca] or increasing [Mg], and the slow effect of depolarization in contrast to the fast effect was found to exist in the presence of Ca reduced to about 10(-7)M, but only during pulses. At this [Ca] there was no rapid response to depolarization. At [Ca] about 10(-10)M, there was no response at all of m.e.p.p. frequency to nerve terminal depolarization.9. The results are discussed, and compared with similar data referring to ;facilitation' and ;post-tetanic potentiation'. It is concluded that these and the slow effect of depolarization represent the same phenomenon, a response of the transmitter release system which can be distinguished from the fast response in terms of ionic requirement as well as time course.     

The effect of tetanus toxin at the neuromuscular junction in the goldfish

1. The effect of tetanus toxin on neuromuscular transmission in the abductor superficialis muscle of the goldfish fin has been investigated.2. The abductor superficialis muscle is multiply innervated and junction potentials (j.p.s) and miniature junction potentials (min.j.p.s) can be recorded with an intracellular micro-electrode at any point of impalement. Intracellular recordings have been made from muscles in which neuromuscular transmission has been blocked, either completely, or partially, by I.M. injection of tetanus toxin. In addition, the tension response of both acutely and chronically toxin-blocked muscles to carbachol (1 x 10(-4)M) has been investigated.3. As the neuromuscular block proceeds, the frequency of min.j.p.s falls, and some time after the muscle has stopped responding to nerve stimulation the min.j.p.s disappear.4. In muscles in which the block has not yet proceeded to completion, it has been found that the reduction in the frequency of the min.j.p.s is unaccompanied by any change in their range of amplitudes.5. The min.j.p. frequency can be greatly increased in such incompletely blocked preparations by repetitive stimulation of the nerve (at, for example, 100/sec for 10 sec). The min.j.p.s obtained are indistinguishable from those seen in the absence of stimulation. Additionally, min.j.p.s can be evoked by similar repetitive stimulation in muscles which are completely blocked, and in which no min.j.p.s are seen without stimulation, so long as the block has only been complete for a short time.6. The tension response to carbachol of muscles which have been paralysed by tetanus toxin for only a few days is identical with that of normal muscles. In contrast, chronically toxin-paralysed muscles contract more rapidly, usually more vigorously, and relax more rapidly than normal muscles.7. It is concluded that tetanus toxin prevents both the nerve-stimulated and spontaneous release of acetycholine from the presynaptic terminals in the abductor superficialis muscle of the goldfish fin.     

Site of adrenaline blockade in the superior cervical ganglion of the rabbit

1. The blocking action of adrenaline on the superior cervical ganglion of the rabbit was investigated with intracellular recording techniques.2. Adrenaline (10(-5)M) blocked initiation of post-synaptic action potentials and decreased the amplitude of excitatory post-synaptic potentials (e.p.s.p.s), but did not hyperpolarize the post-synaptic membrane.3. The depressant action of adrenaline was antagonized by phenoxy-benzamine and dihydroergotamine.4. Acetylcholine depolarizations from iontophoretic ACh were not affected by adrenaline.5. Adrenaline decreased the frequency of miniature excitatory post-synaptic potentials (m.e.p.s.p.s) and decreased the quantal content of e.p.s.p.s in a low [Ca(2+)]: [Mg(2+)] media.6. It was concluded that adrenaline blocks ganglionic transmission by acting at an alpha-adrenoceptive site in the presynaptic nerve terminals.     

Effects of guanidine on transmitter release and neuronal excitability.

1. Guanidine hydrochloride (CH5N3-HCl) was applied to frog neuromuscular junctions blocked by reduced external Ca2+, or increased external Mg2+ concentration, or by both. Guanidine produced a dose-dependent increase in the average number of quanta released by presynaptic action potentials, the threshold dose being 0-1-0-2 mM. No post-synaptic effects were observed. 2. Guanidine also increased the excitability of the motor nerve fibres, as evidenced by multiple firing to single electrical stimuli and finally by spontaneous action potentials. These effects were studied in greater detail in giant axons (Müller axons) in the spinal cord of lamprey. Exposure to guanidine produced in these axons a progressive increase in excitability, manifested by repetitive firing to a single electrical stimulus, spontaneous membrane potential oscillations and spontaneous bursts of action potentials. Guanidine had no effect on the resting potential. 3. The effect of guanidine on the excitability of Müller axons was mimicked in every detail simply by reducing the divalent cation concentration of the bathing solution. 4. Guanidine also produced dose-dependent increases in the duration of action potentials in Müller axons. This effect always preceded in time the appearance of the excitability effects and was not mimicked by reducing the divalent cation concentration. It is suggested that the broadening of the action potential is separate from the excitability effects and may reflect a decrease of delayed rectification. 5. Guanidine (0-3 mM) increased the frequency of miniature end-plate potentials (min. e.p.p.) in solutions containing 2-11 mM-K+ in such a way as to shift the relationship between min. e.p.p. frequency and extracellular K+ toward lower values of K+. This effect was interpreted to mean that guanidine produced a depolarization of the nerve terminal which summed with the depolarization produced by a given concentration of K+. The calculated depolarization produced by 0-3 mM guanidine was 5-7 mV. 6. The effects of guanidine on evoked transmitter release, excitability, and min. e.p.p. frequency are consistent with a hypothesis which states that guanidine binds at or near fixed negative changes on the outside of nerve membrane and reduces the screening effect of divalent cations.