Miniature End-plate Potentials at Mammalian Neuromuscular Junctions Poisoned by Botulinum Toxin

IT is generally accepted that botulinum toxin entirely blocks transmitter release from motor nerve terminals without affecting nerve conduction or the sensitivity of the muscle membrane to acetylcholine. In particular, it has been reported that with both acute and chronic intoxication with type A botulinum, miniature end-plate potentials (m.e.p.p.s.) disappear completely from a muscle at about the time that transmission is blocked^1,2. The action of botulinum toxin has been reinvestigated following acute application of toxin to the rat diaphragm in vitro and chronic paralysis of rat soleus muscle following a single intramuscular injection of toxin; miniature potentials have been observed to persist following blockade of neuromuscular transmission.     

Calcium and facilitation at two classes of crustacean neuromuscular synapses

The closer muscle of the crab, Chionoecetes, has at least two classes of excitatory neuromuscular synapses. In one class of synapses an action potential depolarizing the synaptic region releases much more transmitter if it has been preceded recently by another action potential. The other class of synapses shows this property, called facilitation, to a far lesser extent. Immediately after one conditioning stimulus the level of facilitation is similar in both classes. The rate of the ensuing decay of the facilitation is the critical factor differentiating the two classes of synapses. The relationship between external Ca⁺⁺ concentration and transmitter release is similar for both classes of synapses. The slope of a double logarithmic plot of this relationship varies from 3.1 between 5 and 10 mM Ca⁺⁺ to 0.9 between 30 and 40 mM Ca⁺⁺. Facilitation does not significantly change when tested in external Ca⁺⁺ concentrations ranging from 7 to 30 mM. The extracellularly recorded nerve terminal action potential does not increase in amplitude during facilitation. The results suggest that the mechanism of synaptic facilitation is similar for both classes of synapses and occurs after the stage in transmitter release involving Ca⁺⁺.     

Glutamate uptake by a stimulated insect nerve muscle preparation

Recent reports suggest that glutamate may be the excitatory neuromuscular transmitter in insects. In this study, glutamate uptake by isolated cockroach nerve muscle preparations was investigated by means of chemical and electron microscope radioautographic techniques. We found that the preparation had a high affinity for glutamate and that nerve stimulation enhanced glutamate uptake. Chemical studies showed that the average tissue concentration of glutamate bound during a 1 hr incubation period in 10^-5 M glutamate-³H after nerve stimulation was 2.8 x 10^-5 M. Less than 1% of the radioactivity was present in the perchloric acid-precipitated protein fraction. Using electron microscope radioautography, we observed that sheath cells showed the highest glutamate concentration of all cellular compartments. Uptake was greater at neuromuscular junctions than in other regions of the tissue. The data suggest a possible mechanism for transmitter inactivation and protection of synapses from high blood glutamate.     

Changes in the Asynchronicity of Transmitter Release at the Neuromuscular Junction during Prolonged High-Frequency Stimulation

In experiments on neuromuscular junctions in the frog m. cutaneous-pectoris, changes in the intensity and asynchronicity of transmitter release during high-frequency (10 and 50 sec^-1) rhythmic stimulation of the motor nerve were investigated using extracellular recording. At low extracellular Ca²⁺ concentrations, rhythmic stimulation resulted in a gradual enlargement of the quantum content of end-plate currents (EPC), the so-called facilitation. The latter phenomenon was accompanied by an increase in the average value and variance of synaptic delays of single-quantum EPC, a shift of the main mode of their distribution towards greater values, and an increase in the latency of the nerve ending responses. The above-described changes reduce the magnitude of facilitation in the neuromuscular synapse.     

Effects of carbamazepine on nerve activity and transmitter release in neuroblastoma-glioma hybrid cells and the frog neuromuscular junction

Effects of the antiepileptic drug carbamazepine on nerve action potential and transmitter release in mouse neuroblastoma-glioma hybrid cells (NG108-15) and the frog neuromuscular junction were studied. Carbamazepine within a concentration range of 0.1–0.5 mmol/L reduced the peak height of the action potential of the NG108-15 cells, whereas the membrane potential and membrane resistance were unaffected. Voltage clamp revealed that the decrease in the action potential was due to the blockage of the Na⁺, delayed K⁺ and transient Ca²⁺ currents. Carbamazepine did not affect Ca²⁺-activated and A type K⁺ currents and long-lasting Ca²⁺ current. In the frog neuromuscular junction, carbamazepine decreased the mean quantal content by a parallel shift in the frequency augmentation–potentiation (FAP) relation. It is concluded that carbamazepine blocks the voltage-dependent Na⁺, delayed K⁺, and transient Ca²⁺ currents and quantal transmitter release through a decrease of nerve excitation.     

Changes in Electrogenesis in the Motor Nerve Terminal with Long-Lasting High-Frequency Activation as a Factor of the Control of Transmitter Release

Using the technique of extracellular recording from the region of the neuromuscular junction in the cutaneous-sternal muscle in the frog under conditions of a reduced concentration of Ca²⁺ in the surrounding milieu, we demonstrated that long-lasting (10 min) rhythmic stimulation of the motor nerve with a frequency of 10 sec^{− 1} leads to a gradual increase in the evoked transmitter release. These changes are accompanied by a decrease in the amplitude of electrical responses of the nerve terminal (NT) and by a retardation of its second phase, as well as by a diminution of the third phase. Under conditions of long-lasting (5 min) stimulation with a frequency of 50 sec⁻¹, we observed a two-phase change in the intensity of transmitter release: on the 2nd min, the initial rise was replaced by inhibition. Modifications of the response of the NT with different stimulation frequencies were qualitatively similar, but with a frequency of 10 sec⁻¹ they were clearly expressed. Mathematical simulation of ion currents in the NT demonstrated that voltage-dependent potassium and sodium channels are inactivated in the course of long-lasting high-frequency excitation; the shape of the action potential is modified with changes in the rate of such inactivation. This leads to either an increase or a decrease of the inward calcium current. We conclude that the change in electrogenesis in the NT with long-lasting high-frequency activation of neuromuscular junctions exerts a significant influence on the dynamics of transmitter release. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 108–115, March–April, 2005.     

Cyclic AMP,Transmitter Release and the Effect of Adenosine on Neuromuscular Transmission

THE high activities of the enzymes in nervous tissue regulating the metabolism of 3',5' adenosine monophosphate (cyclic AMP) and its apparent involvement in secretion from glands have prompted considerable speculation about its possible role in the release of transmitters from nerve endings^1–4. There is some evidence that cyclic AMP is concerned in the release of acetylcholine from motor nerve endings^1,3. It has been found, for example, that neuromuscular transmission is facilitated by catecholamines^5,6 and methylxanthines^1,3; noradrenaline is known to increase both the quantal content of the endplate potential⁶ and the level of cyclic AMP in nervous tissue^7,8 and the increase in the quantal content is more marked in the presence of theophylline³. It was also found that increases in cyclic AMP concentration of up to thirty-fold occur in brain slices in the presence of 0.1-1 mM adenosine. We have therefore examined the effect of this substance on transmitter release.     

Reduced transmitter release conferred by mutations in theslowpoke-encoded Ca2+-activated K+ channel gene ofDrosophila

Potassium channels control the repolarization of nerve terminals and thus play important roles in the control of synaptic transmission. Here we describe the effects of mutations in theslowpoke gene, which is the structural gene for a calcium activated potassium channel, on transmitter release at the neuromuscular junction inDrosophila melanogaster. Surprisingly, we find that theslowpoke mutant exhibits reduced transmitter release compared to normal. Similarly, theslowpoke mutation significantly suppresses the increased transmitter release conferred either by a mutation inShaker or by application of 4-aminopyridine, which blocks theShaker-encoded potassium channel at theDrosophila nerve terminal. Furthermore, theslowpoke mutation suppresses the striking increase in transmitter release that occurs following application of 4-aminopyridine to theether a go-go mutant. This suppression is most likely the result of a reduction of Ca²⁺ influx into the nerve terminal in theslowpoke mutant. We hypothesize that the effects of theslowpoke mutation are indirect, perhaps resulting from increased Ca²⁺ channel inactivation, decreased Na⁺ or Ca²⁺ channel localization or gene expression, or by increases in the expression or activity of potassium channels distinct fromslowpoke.     

Transmitter release in proximal and distal parts of a nerve terminal of the frog sartorius muscle

Evoked synaptic potential were recorded extracellularly in experiments on a nervemuscle preparation of the frog sartorius muscle. A decrease in evoked transmitter release was found from the proximal to the distal parts of the nerve ending, due to a decrease in the probability of transmitter quantum release. The terminal portions of the synapse are less sensitive than the proximal parts to changes in Ca⁺⁺ concentration, they show less marked facilitation of transmitter release during paired and repetitive stimulation, and exhibit deeper and more rapidly developing depression. It is concluded that differences in transmitter release in the terminal parts of the synapse are due to the low reserves of transmitter and the lower premeability of the presynaptic membrane to Ca⁺⁺.     

Action of thiamine on neuromuscular transmission in the frog

The action of thiamine on neuromuscular transmission in the frog sartorius muscle was investigated. It was found that thiamine at a concentration of 1×10^–14 to 1×10^–4 M increases transmitter secretion at the nerve endings. This is demonstrated by the increased frequency, amplitude, and quantal content of miniature endplate potentials, and is due to the enhanced likelihood of transmitter release. The role of thiamine in regulating synaptic transmission and the mechanism of its interaction with thiamine-sensitive receptors are examined.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 794–800, November–December, 1985.     

Long term facilitation of tension in crustacean muscle and its modulation by temperature,activity and circulating amines

Summary Prolonged stimulation of the motor axon of the opener and stretcher muscles of the crayfish claw leads to long-term facilitation (LTF) of transmitter release at the neuromuscular junction. This facilitation is correlated with enhancement of tension development. Factors shown to enhance LTF of transmitter release, such as increased frequency of excitation, lower temperature, and exposure to ouabain also enhance tension development (Figs. 1, 2 and 4). Prolonged stimulation delivered in a bursting pattern enhances the development of tension more than an equivalent amount of stimulation delivered in a regular pattern (Fig. 3).Two circulating neurohormones, serotonin and octopamine, were examined for their effect on the development of tension during short and long periods of muscle activation. Serotonin and LTF of transmitter release appear to have an additive effect on the development of tension. The threshold for a detectable serotonin effect is 10^–10 M. The effect of octopamine on the development of tension appears to be enhanced by longer periods of maintained muscle activation. LTF of transmitter release resulting from 5 min of continuous activation at 15 Hz is accompanied by a drop in the threshold of an observable octopamine effect on tension from 10^–9Mto 10^–10 M. It is proposed that octopamine's trophic effects on metabolism in muscle act to sustain muscular performance during maintained activity.Abbreviations LTF long term facilitation - ec Membrane potential threshold for contraction - STF short term facilitation - e.j.p. excitatory junction potential This work was supported by a N.S.E.R.C. grant to H.L.A.     

Effects of Noradrenaline on End-Plate Currents and the Kinetics of Transmitter Release under Long-Lasting Repetitive Stimulation

Noradrenaline causes a significant increase in the amplitude of multiquantum end-plate currents (EPC) and also diminishes the EPC rising phase vs the rising phase of the miniature EPC ratio in the frog neuromuscular junction under conditions of low-frequency long-lasting stimulation of the motor nerve. Noradrenaline changes the kinetics of transmitter release due to synchronization of the quantum transmitter secretion. The synchronizing action of noradrenaline can underlie its de-fatiguing effect in the neuromuscular junction.     

Drosophila larval neuromuscular junction's responses to reduction of cAMP in the nervous system

We investigated the effects of chronically lowered cyclic adenosine monophosphate (cAMP) on the morphology and physiology of the Drosophila larval neuromuscular junction, using two fly lines in which cAMP was significantly lower than normal in the nervous system: (a) transgenic flies in which the dunce (dnc) gene product was overexpressed in the nervous system, and (b) flies mutant for the rutabaga gene (rut¹) which have reduced adenylyl cyclase activity. In comparison with controls, larvae with reduced cAMP exhibited a smaller number of synaptic varicosities. This effect was more pronounced in transgenic larvae, in which the reduction of neural cAMP was more pronounced. Synaptic transmission was also reduced in both cases, as evidenced by smaller excitatory junctional potentials (EJPs). Synaptic currents recorded from individual synaptic varicosities of the neuromuscular junction indicated almost normal transmitter release properties in transgenic larvae and a modest impairment in rut¹ larvae. Thus, reduction in EJP amplitude in transgenic larvae is primarily due to reduced innervation, while in rut¹ larvae it is attributable to the combined effects of reduced innervation and a mild impairment of transmitter release. We conclude that the major effect of chronically lowered cAMP is reduction of innervation rather than impairment of transmitter release properties. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 1–13, 1999     

Inhibitory Miniature Potentials in the Stretch Receptor Neurons of Crayfish

Intracellular microelectrodes inserted into the soma of crayfish stretch receptor neurons record frequent fluctuations of the membrane potential. Time course, amplitude, and interval distribution indicate that they are miniature potentials. At the average resting potential the polarity of the miniature potentials depends on the anion used in the microelectrode: KCl electrodes record depolarizing, K citrate or K₂SO₄ electrodes, hyperpolarizing miniature potentials. The inhibitory postsynaptic potentials (i.p.s.p.'s) show a similar polarity change. The reversal potentials of i.p.s.p.'s and miniature potentials are equal and within 10 mv of the resting potential, more negative with K citrate (or K₂SO₄), less negative with KCl electrodes. Reversal can be accomplished by changing the membrane potential by stretching or by current passing. Injection of Cl^- into the soma or replacement of external Cl by propionate results in an abrupt increase of the amplitude of the miniature potentials lasting for several minutes. The miniature potentials like the i.p.s.p.'s are reversibly abolished by the application of picrotoxin and γ-aminobutyric acid. They are not affected by tetrodotoxin, nor by acetylocholine, eserine, or atropine. It is concluded that the miniature potentials represent a spontaneous quantal release of transmitter substance from inhibitory nerve terminals, and that the transmitter substance predominantly increases the Cl^- permeability of the postsynaptic membrane. The effect of the spontaneously released transmitter on the behavior of the receptor neuron is considerable. The membrane conductance is increased by up to 36% and the excitability is correspondingly depressed.     

Kinetic analyses of transmitter release in neuromuscular transmission

A majority of transmitter quanta in the nerve terminal is in a large pool of store (S), which can be utilized for release only after transformation into activated quanta (n) via two intermediate states called available quanta (A) and releasable quanta (N). Mobilization is a collective term applicable for aggregates of S → A, A → N and N → n conversions. In the present article, various electrophysiological procedures for kinetic analyses of the transmitter release in neuromuscular transmission were discussed to elucidate this not well understood process of mobilization. Especially, frequency augmentation, tetanic rundown and depression, and recovery from depression experiments were proposed to be very useful tools in identifying the drug effect on the mobilization process. since d-tubocurarine, 2-(4-phenylpiperidino) cyclohexanol (AH5183) and Ba ions satisfied the above three criteria of drug action on mobilization, these were concluded to affect the mobilization of transmitter in neuromuscular transmission.     

Role of Potassium Channels in Facilitation of Transmitter Release from Frog Motor Nerve Ending (Electrophysiology and Mathematical Simulation)

In experiments on neuromuscular junctions in the frog m. thoraco-cutaneous, we studied changes in the transmitter release and shape of the nerve ending (NE) response related to high-frequency (10 or 50 sec^-1) rhythmic stimulation of the motor nerve; an extracellular recording technique was used. At a low extracellular Ca²⁺ concentration, rhythmic stimulation resulted in a gradual increase in the quantum content of end-plate currents, i.e., in facilitation. Simultaneously, the third (positive) phase of the NE response became smaller, the amplitude of the second (negative) phase of this response also decreased, while the duration of this phase increased. Modifications of the NE response upon stimulation with a 10 sec^-1 frequency were more clearly expressed than those at 50 sec^-1 stimulation. In Ca²⁺-free solutions, rhythmic stimulation was accompanied by analogous modifications of the shape of NE responses, and the dynamics of these changes were the same at both the above-mentioned stimulation frequencies. When 0.5-1.0 mM tetraethylammonium was applied, 10 sec^-1 stimulation was accompanied by no facilitation of transmitter release; at 50 sec^-1 stimulation, this phenomenon was observed but was weaker than in the control, and the shape of NE responses underwent only mild changes. Simulation of electrogenesis in the studied structure showed that modifications of the NE response shape related to rhythmic 10 sec^-1 stimulation can develop in the case of a gradual decrease in the voltage-dependent potassium membrane conductivity, which results in prolongation of the de- and repolarization phases of action potentials and increases in the amplitude and duration of the inward calcium current. At higher stimulation frequencies (50 sec^-1), this mechanism is accompanied by a gradual increase in the Ca²⁺-dependent potassium conductivity, due to an increase in the intracellular Ca²⁺ concentration. These data allow us to conclude that the intensity of facilitation of transmitter release from the frog motor NE is related not only to accumulation of residual calcium, but also to changes of presynaptic calcium current due to modification of the kinetics of functioning of the potassium channels.     

Presynaptic mechanisms of zinc effects on the neuromuscular transmission]

The effect of zinc ions on presynaptic currents and transmitter release was studied at the neuromuscular junction of the frog cutaneous pectoris muscle preparation with using an extracellular microelectrode. It has been shown that zinc (100 mkM) amplified MEPP frequency at first, but suppressed it later. Zinc affected the presynaptic spike waveform and transmitter release in a concentration-dependent manner. Depending on concentration and time of exposure zinc increased or suppressed transmitter release. Increase of transmitter release was shown to be resulted by blockade voltage gated and calcium activated potassium channels in nerve ending, leading to broad of both presynaptic spike and action potential. Strong change of presynaptic spike waveform after high concentration zinc treatment supposed that under this condition zinc depressed voltage gated calcium and sodium channel leading to decrease of transmitter release. It was concluded that the final and irreversible depression of acetylcholine release by zinc was due to alteration of whole ion conductances in nerve ending and to change of configuration of proteins included in structure of ion channels. It is discussed possible mechanisms of various effects of zinc ions at the neuromuscular synapse.     

Prostaglandin controls Neuromuscular Transmission in Guinea-pig Vas Deferens

PROSTAGLANDINS of the E type (PGE₁, PGE₂) inhibit sympathetic neurotransmission in several tissues and species^1–4. On the basis of their natural occurrence and availability for release, as well as observations on the pharmacological actions of the PGs, endogenous PGE₁ and PGE₂ are postulated to operate on sympathetic neurotransmission by a feedback mechanism and thereby modulate the effector responses to nerve activity^{1, 5}. Inhibition by 5,8,11,14-eicosatetraynoic acid (ETA) of PG synthesis in the cat spleen and in the rabbit heart increases the release of noradrenaline (NA) in response to nerve stimulation, thus strongly supporting the hypothesis^{6, 7}. We report here that guinea-pig vas deferens releases PG in response to nerve stimulation and that the neuromuscular transmission is facilitated after inhibition of PG synthesis. PG synthesis was irreversibly inhibited using ETA⁸.     

Changes in the asynchronism of transmitter release in a neuromuscular synapse and the time course of evoke postsynaptic signals during growth and branching og frog nerve ending

The influence of both growth and branching of a nerve terminal on the asynchronism of transmitter release and the time-course of evoked postsynaptic responses was investigated using a model of a frog neuromuscular synapse in which the nerve terminal represents a population of spatially isolated active zones. It was shown that the appearance of additional branching in proximal parts of the nerve ending leads to decrease in the asynchronism of transmitter release, an increase in quantum content and the amplitude of the postsynaptic signal, and the shortening of its phase of growth. It was found that the asynchronism of transmitter release has a much stronger influence on the time-course of end plate currents compared with end plate potentials. The factors strengthening and weakening the asynchronism of transmitter release in a neuromuscular synapse and the reasons for various length and branching of vertebrate nerve terminals are considered.