Effects of phorbol ester on spontaneous transmitter release at frog neuromuscular junction

Spontaneous transmitter release was studied at frog neuromuscular junctions exposed to the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PrkC). TPA at concentrations between 10^–7 and 10^–6 M induced a dose-dependent increase in miniature end-plate potential frequency. This frequency increase was enhanced by raising [Ca]₀, diminished by lowering the temperature of the bath and virtually abolished in Ca²⁺-free Ringer. The TPA effect was only poorly reversible after washing. TPA was ineffective in increasing spontaneous release of transmitter at phosphatidylcholine-pretreated neuromuscular junctions. It is suggested that PrkC might play a role in neuromuscular transmission processes.     

Transient elevation of spontaneous release at the frog neuromuscular junction following acetylcholine iontophoresis

Miniature end-plate currents (m.e.p.c.s) were recorded from frog neuromuscular junctions using a two-electrode voltage clamp. The m.e.p.c. frequency was temporarily elevated following 10 s iontophoretic applications of acetylcholine (ACh) when the junctions were clamped at 100 mV. This post-ACh burst of quanta was also observed at unclamped junctions. At –100 mV, the intensity of the burst was proportional to the amount of current flowing across the end-plate during ACh iontophoresis, but usually did not reach its peak until the end-plate receptor channels were almost completely closed. Addition of 0.5 M TTX to the Ringer's solution, or total replacement of sodium with quanidine, lithium, or methylamine did not inhibit the burst. No burst was observed in Ca²⁺-free, EGTA solutions, or in Ca²⁺-free solutions containing 2 mM Mn²⁺. Sr²⁺ effectively substituted for Ca²⁺. Addition of 2 mM Co²⁺ or Mn²⁺ to normal Ringer's did not inhibit the burst. Presynaptic muscarinic receptors did not obviously contribute to the burst, since it was not blocked by atropine, nor produced by oxotremorine or pilocarpine. The ACh analogs carbachol and acetyl--methylcholine also produced the burst. The burst was highly dependent on the muscle membrane potential during the period of ACh iontophoresis, becoming more intense at potentials negative to –100 mV and disappearing at –60 mV. The critical importance of the post-synaptic membrane potential suggests that the burst may be due to an action of the muscle end-plate on the motor nerve terminal, possibly by the movement of an anionic substance through open end-plate receptor channels, but this hypothesis does not account for the delay of the burst until near the end of the ACh-induced end-plate current.     

The mechanisms of multi-component paired-pulse facilitation of neurotransmitter release at the frog neuromuscular junction

We have studied the mechanisms of paired-pulse facilitation (PPF) of neurotransmitter release in isolated nerve-muscle preparations of the frog cutaneous pectoris muscle. In normal extracellular Ca²⁺ concentration ([Ca²⁺]_o, 1.8 mM), as the interpulse interval was increased from 5 to 500 ms, PPF decayed as a sum of two exponential components: a larger but shorter first component (F1) and a smaller but more prolonged second component (F2). In low [Ca²⁺]_o (0.5 mM), both F1 and F2 increased, and a third “early” component (Fe) appeared whose amplitude was larger and whose duration was shorter than F1 or F2. In the presence of the “fast” Ca²⁺ buffer BAPTA-AM, Fe disappeared, whereas F1 and F2 decreased in amplitude and duration. In contrast, the “slow” Ca²⁺ buffer EGTA-AM caused a decrease of Fe and reduction or complete blockade of F2, without any changes of F1. In solutions containing Sr²⁺ (1 mM), the magnitude of Fe was decreased, F1 was significantly reduced and shortened, but F2 was unaffected. Application of the calmodulin inhibitor W-7 (10 μM) at normal [Ca²⁺]_o produced a marked decrease of F2, and at low [Ca²⁺]_o, a complete blockade of Fe. These results suggest that PPF at frog motor nerve terminals is mediated by several specific for different PPF components intraterminal Ca²⁺ binding sites, which trigger neurotransmitter release. These sites have a higher affinity for Ca²⁺ ions and are located farther from the release-controlling Ca²⁺ channels than the Ca ²⁺ sensor that mediates phasic release.     

Facilitation,augmentation and potentiation of transmitter release at frog neuromuscular junctions poisoned with botulinum toxin

Botulinum toxin type A (Botx) is a potent neurotoxin which inhibits specifically cholinergic synaptic transmission by an unknown mechanism. In order to gain further insight into the mode of action of this toxin, the effect of conditioning nerve stimuli on neuromuscular transmission was studied at endplates of Botx-poisoned and umpoisoned control cutaneous pectoris muscles in the frog. Effects of single conditioning stimuli (facilitation) and multiple high-frequency stimuli (augmentation and potentiation) on epp amplitude and mepp frequency were studied. The main results were that initial facilitation was significantly increased and its decay time constant significantly decreased in Botx-poisoned muscles, while augmentation was unchanged and potentiation was abolished. These changes could be detected before the muscle became completely paralysed, suggesting that they reflect a primary disturbance in the Ca²⁺-dependent release process.     

A new type of transmitter release at the neuromuscular junction

Examination of spontaneous miniature endplate potentials (MEPPs) in murine skeletal muscle has revealed that in conditions such as botulinum poisoning, during nerve terminal regeneration or in the presence of the drug 4-aminoquinoline, two types of acetylcholine release are responsible for the MEPPs. In addition to the MEPPs which correspond to the quantal component of a nerve impulse-evoked endplate potential a second type of acetylcholine release occurs. The latter type of transmitter release gives rise to MEPPs with a more prolonged time-to-peak and frequently a larger than normal amplitude. It is unaffected by nerve terminal depolarization and transmembrane Ca2+ fluxes. The relationship between MEPP frequency and temperature has a Q10 of about 12 compared to 2-3 for normal MEPPs. In botulinum-poisoned muscles this secretory type of transmitter release dominates, being exclusively present in muscles where nerve stimulation fails to release transmitter. In normal muscle such a release is induced by 4-aminoquinoline which may cause up to 45% of all the spontaneous MEPPs to be of that kind. It is suggested that the described spontaneous secretion of acetylcholine serves in inductory and neurotrophic function.     

Opposing effects of cannabinoids and vanilloids on evoked quantal release at the frog neuromuscular junction

Cannabinoids and vanilloids are two distinct groups of substances that share some pharmacological targets. Here we report that two cannabinoid type 1 receptor (CB1) agonists, WIN 55212-2 (WIN) and arachidonyl-2′-chloroethylamide (ACEA) have opposing effects on evoked quantal acetylcholine release – WIN decreased quantal content while ACEA increased quantal content. The decrease in quantal content by WIN was blocked by the CB1 antagonist AM 251. The increase in quantal content by ACEA was not blocked by AM 251, indicating it acts through a receptor other than CB1. As ACEA is also an agonist for the vanilloid receptor (TRPV1) we tested the effect of vanilloids on quantal content. Similar to ACEA, the vanilloid agonist capsaicin increased quantal content, and this effect was blocked by capsazepine, a TRPV1 antagonist. Capsazepine also blocked the increase in quantal content by ACEA. Together these data show an inhibitory effect of CB1 activation on evoked acetylcholine release and the first evidence for the presence of a vanilloid receptor at the neuromuscular junction.     

Effects ofAnemonia sulcata toxin II on presynaptic currents and evoked transmitter release at neuromuscular junctions of the mouse

The effect ofAnemonia sulcata toxin II (ATX-II) on the amount of transmitter released by nerve impulses was investigated in motor end-plates of the mouse. ATX-II (80 nM) caused repetitive end-plate potentials in response to a single nerve stimulus and a 3- to 4-fold increase in the quantal content of the phasic end-plate potential. This increase is less than what would be expected if ATX-II induced plateau action potentials at the motor endings. To solve this discrepancy presynaptic currents were recorded by focal extracellular electrodes. It was found that the K current present at the endings is strong enough to prevent the development of presynaptic plateau action potentials, in contrast to what has been observed in other excitable membranes (unmyelinated axons, nodes of Ranvier and skeletal muscle fibres). By using tetraethylammonium and 3,4-diaminopyridine to block K channels and Co²⁺ to block Ca channels, ATX-II allowed the development of prolonged plateau responses at the endings upon motor nerve stimulation. These results suggest that the mouse motor endings are endowed with a relatively powerful K channel system, which effectively controls the amount of presynaptic depolarization.     

Spontaneous and evoked quantal neurotransmitter release at the neuromuscular junction of the larval housefly,Musca domestica

The release of neurotransmitter was monitored at the neuromuscular junctions of larval housefly ventrolateral muscles 6a and 7a, using intracellular recording, and a loose patch clamp technique to isolate discrete release sites. Transmitter release occurred spontaneously and could also be evoked by neural stimuli. Spontaneous discharges consisted of events which were randomly distributed in time and of bursts of temporally ordered events. Evoked and spontaneous release occurred in a quantal manner. The quantal content of evoked excitatory postsynaptic currents (EPSCs) was dependent upon the extracellular calcium concentration, increasing with a 3.8 power dependency. The relationship between the quantal content of a response and extracellular calcium concentration was offset by the presence of magnesium in the bathing saline. The rates of decay of miniature EPSCs (mEPSCs) and EPSCs were also found to increase with extracellular calcium concentration, consistent with a non-diffusion limited block of the glutamate receptor-channel complex by calcium ions (K _B 2.5×10⁴ s^–1 M^–1,P<0.01). The frequency of random mEPSCs (0.26±0.32 Hz,n=24 cells) was independent of the extracellular calcium concentration. Random mEPSCs were not inhibited by 1 M tetrodotoxin which blocked mEPSC bursts and neurally evoked responses. EPSCs evoked during mEPSC bursts had a significantly lower quantal content than those EPSCs recorded from the same nerve terminal between bursting, indicating that both of these forms of release recruited quanta from a common pool of transmitter. Following a neurally evoked EPSC the mEPSC frequency was potentiated severalfold, this delayed release was influenced by EPSCs with large quantal contents evoked in saline containing elevated calcium concentrations. The alpha-cyano pyrethroid insecticide, cypermethrin, when applied at 10 nM concentration to this NMJ, caused a sustained 10-fold increase of transmitter quanta (240% of control), and blocked neurally evoked EPSCs after ca. 20 min (22°C). mEPSC frequency remained elevated for several hours after treatment with cypermethrin (ca. 3 Hz), before declining below control levels (0.26 Hz). A model based upon the frequency of mEPSC discharges, the quantal content of evoked responses, and the association constants of calcium to the four binding sites of calmodulin was used to investigate a possible role for this metalloprotein in both the spontaneous and evoked release of neurotransmitter. The probable lack of involvement of calmodulin in the mechanism of neurotransmitter release at the larval housefly neuromuscular junction is discussed in the light of this model, as are the implications of these findings for vesicular amino acid release in relation to pyrethroid insecticide action.Abbreviations used [Ca] ionic calcium concentration - CaM calmodulin - NMJ neuromuscular junction - EPSC excitatory postsynaptic current (prefix m denotes miniature EPSC) A preliminary presentation of some of these results have been presented at Neurotox '88, Neuropharmacology and Pesticide Action, Nottingham, UK     

Depolarization dependence of the kinetics of phasic transmitter release at the crayfish neuromuscular junction

Quantal synaptic currents were recorded at nerve terminals on the crayfish opener muscle by means of a macro-patch-clamp electrode. Release could be elicited by graded depolarization pulses through the recording electrode. At low temperature, distributions of delays of single quantal currents from the onset of depolarization were determined for depolarizations varying from threshold to saturation range. This time course of release was little affected by the amplitude of depolarization: There was a tendency for release to start earlier and to rise faster for larger depolarizations, while the termination of release showed no significant variations. The time course of release after an action potential in the motor axon was similar to that of release after a depolarization pulse. It is concluded that the time course of quantal release is rather independent of amplitude of depolarization and of the amount of calcium (Ca) inflow, which seems to rule out the control of the release after a depolarization by the time course of [Ca]_i.     

Effects of dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparation

In order to elucidate the role of cyclic nucleotides in the suprachiasmatic nucleus, we examined the effect of dibutyryl cyclic AMP (c-AMP) and dibutyryl cyclic GMP (c-GMP) on single neuronal activity of the suprachiasmatic nucleus of the rat. A hypothalamic slice about 300 micron thick, including the suprachiasmatic nucleus, was cut coronally using a vibratome. By means of bath- as well as iontophoretic application, c-AMP and c-GMP caused mainly an inhibition and facilitation in suprachiasmatic neuronal activity, respectively. The present results suggest that c-AMP and c-GMP play an important role in regulation of suprachiasmatic neuronal activity as a second messenger and that these two nucleotides may function as a reciprocal messenger.     

Delayed release of transmitter at the frog neuromuscular junction

1. After the end-plate potential (e.p.p.) there is an increase in the frequency of the miniature e.p.p.s. This delayed release of transmitter was studied at the frog neuromuscular junction, using conventional intracellular and extracellular recording techniques. E.p.p. amplitude was kept subthreshold by subnormal concentrations of activating divalent ions.

2. The ratio delayed release: initial release had values between 2 and 140%, depending on the experimental conditions; it decreased with an increase in Ca concentration and quantal content.

3. Delayed release is larger at low temperature than at room temperature.

4. Delayed release is statistically independent of the amplitude of the preceding e.p.p.

5. The time course of the decay of the delayed release is monotonic when strontium is the activating divalent ion; it shows a `dip' in more than 50% of the cases when Ca activated release.

6. The results were discussed in terms of the residual Ca ion hypothesis.

     

Strontium and quantal release of transmitter at the neuromuscular junction

1. Previous work has shown that in calcium-free solutions nerve impulses invade the motor nerve terminals at the neuromuscular junction, but fail to release transmitter. In these conditions, strontium ions applied iontophoretically to a minute part of a junction, or to the whole muscle by bath application, restore to the nerve impulse its ability to release transmitter.2. As with calcium, the transmitter released in the presence of strontium is in the form of packages (quanta) whose release can be predicted from Poisson's Theorem.3. The mean number of quanta released by a nerve impulse increases with the concentration of strontium. Strontium is much less effective than calcium in equimolar concentrations.4. Transmitter quanta released in the presence of strontium evoke larger unit potentials than quanta released in the presence of calcium. The larger size of the Sr-unit potentials is caused by a prolongation of transmitter action, presumably due to a post-synaptic effect of strontium.5. Neuromuscular transmission was blocked in some fibres when the concentration of strontium was raised beyond 10 mM. This junctional block was presumably due to a failure in the propagation of nerve impulses.6. The post-stimulation increase in the frequency of miniature end-plate potentials, which is normally seen in calcium solutions, is also observed when calcium is substituted by strontium. The post-stimulation effect increases with the concentration of strontium.7. It is concluded that strontium can substitute for calcium in the process of quantal release of transmitter. The physico-chemical mechanism of this substitution remains unknown.     

Inhibition of quantal transmitter release by the polycationic ligand cationized ferritin

Summary Cationized ferritin (200 g ml^–1) causes a reduction in both spontaneous and evoked transmitter release at the frog neuromuscular junction. The reduction in EPP amplitude probably reflects reduced Ca²⁺-influx since full recovery can be achieved by elevation of [Ca²⁺]_o. The effect on MEPP frequency is independent of [Ca²⁺]_o and may well reflect a direct effect on exocytosis.     

Purinergic modulation of synaptic signalling at the neuromuscular junction

Purines have physiologically important functions throughout the nervous system. In both the central (CNS) and peripheral nervous systems (PNS), purines in the form of adenosine triphosphate and adenosine can play a number of roles in neuronal activation and inhibition. In addition, purines are known to be important for glial cell signaling in both the CNS and PNS. In the PNS, the neuromuscular junction (NMJ) is an excellent model for studying simple synaptic interactions. It is well suited to investigations of neuron-glia interactions because synaptic properties are well defined and perisynaptic Schwann cells (PSCs), glial cells at the NMJ, dynamically interact with the pre- and postsynaptic elements. At the NMJ, purines are critical for presynaptic modulation but also for neuron-glia interactions. Purines signal to PSCs through metabotropic and ionotropic receptors and activation of these receptors can have both modulatory and activating functions. This review will discuss recent developments in our understanding of purinergic modulation of the NMJ with an emphasis on the involvement of purines in neuron-glia interactions at this synapse.     

Neurotrophin-4 couples to locally modulated ACh release at the end of neuromuscular synapse maturation

We use immunocytochemistry to show that neurotrophin-4 (NT-4) and its receptor proteins (p75^NTR and tropomyosin-related tyrosine kinase B) are present in neonatal neuromuscular junctions (NMJ) colocalized with several synaptic markers. NT-4 incubation (1 h, in the range 2–12 nM) does not change the size of the endplate potential between P6 and P45. However, extended exposure (3 h) to a relatively low dose of NT-4 (2 nM) potentiates ACh release (approx. 70%) in adult but not in neonatal muscles. The present results suggest that the developmental mechanism of axonal competition and neonatal elimination of redundant synapses cannot be modulated by added NT-4. However, this neurotrophin was able to modulate synaptic transmission locally in the adult NMJ.     

On facilitation of transmitter release at the toad neuromuscular junction

1. The time dependence of the increase in amplitude (facilitation) of a second end-plate potential (e.p.p.) elicited within 10-100 msec of a preceding e.p.p. was examined at neuromuscular junctions in sartorius muscles of toads. Facilitation was defined by two characteristics, initial facilitation and the time constant of its exponential decay.2. The time constant of decay of facilitation was longer at lower temperatures and the Q(10) was 4.3 in the range 10-25 degrees C. There was no significant effect of temperature on initial facilitation.3. Ouabain (10(-4)-10(-3)M), lithium substitution for sodium, sodium azide (5 mM) and N-ethylmaleimide (NEM, 0.1 mM) initially had no effect on initial facilitation or the decay of facilitation. After some time, they all caused a longer time constant of decay of facilitation and a depression of initial facilitation.4. It was concluded that the decay of facilitation is not directly dependent on active transport of sodium ions, calcium efflux, ATP-dependent movements of calcium or mitochondrial uptake of calcium following an action potential.5. Ouabain, lithium, sodium azide, and NEM all caused an increase in transmitter release. This effect, and the late effects on facilitation, were thought to be due to an increase in intracellular calcium concentration in nerve terminals.6. No relationship was found between the quantal content of e.p.p.s (range, 0.8-100) and initial facilitation, or the time constant of decay of facilitation.7. Substitution of strontium for calcium ions caused a marked prolongation of the time constant of decay of facilitation, and a depression of initial facilitation.8. The results were consistent with the hypothesis that the time constant of decay of facilitation is related to the rate of disappearance of an ;active' complex of calcium (CaA) which, of itself, is not sufficient for transmitter release. It is suggested that an action potential produces CaA which decays with the time constant of facilitation and CaS, a short-life complex of calcium which decays with the time constant of the phasic release of transmitter. The release of transmitter is proportional to some function of [CaA] and [CaS].     

The kinetics of transmitter release at the frog neuromuscular junction

1. Fluctuations in the latency of focally recorded end-plate currents were analysed to determine the time course of the probabilistic presynaptic process underlying quantal release evoked after single nerve stimuli at the frog neuromuscular junction.

2. The early falling phase of the presynaptic probability function can be fitted by a single exponential over two orders of magnitude of quantal release rate. The time constant of the early falling phase is about 0·5 msec at 11° C, and increases with decreasing temperature with a Q₁₀ of at least 4 over the range 1-12° C.

3. After this early exponential fall, quantal release probability returns to control levels with a much slower time course.

4. Conditioning nerve stimuli increase the magnitude and slightly prolong the early time course of release evoked by a test stimulus. When facilitation is calculated for matched time intervals following the conditioning and testing stimuli, it is found that the magnitude of the small, late residual tail of release is facilitated by a greater percentage than the magnitude of larger, early portions of release.

5. These results are discussed in terms of the hypothesis (Katz & Miledi, 1968) that evoked release and facilitation are mediated by a common presynaptic factor which activates release in a non-linear manner.

     

Spontaneous synchronization by 4-aminopyridine of acetylcholine release in the neuromuscular junction

The effect of 4-aminopyridine (4AP) on the character of acetylcholine release was investigated by intracellular recording of spontaneous synaptic activity in an isolated rate phrenic nervediaphragm preparation. No significant changes were found in the mean frequency and amplitude of miniature end-plate potentials (MEPP) in response to 4AP in concentrations of 1·10^–6 to 1·10^–3M. Meanwhile, 4AP caused the appearance of large spontaneous EPP capable of inducing spreading action potentials. 4AP changed the character of distribution of MEPP amplitudes, converting it to polymodal; in some cases the principal mode was shifted into the region of lower values. It is concluded that 4AP changes the character of acetylcholine release and potentiates spontaneous synchronization.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Department of Pharmacology, Faculty of Internal Medicine and Hygiene, I. M. Sechenov First Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. M. Chernukh.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 9, pp. 259–262, September, 1979.