Transmitter release in botulinum-poisoned muscles

Examination of miniature end-plate potentials (m.e.p.ps) in rat skeletal muscle poisoned in vivo by botulinum toxin type A reveals the presence of two populations of potentials. One population which corresponds to m.e.p.ps in unpoisoned muscles and to quantal end-plate potentials. The frequency of these m.e.p.ps is greatly reduced by botulinum toxin. The second population of m.e.p.ps has quite different characteristics. These m.e.p.ps have a more variable, but generally much larger amplitude, and their time to peak is longer than normal m.e.p.ps. The frequency of these m.e.p.ps increases during poisoning and reaches 0.3-1 Hz after 10-14 days. In addition to the variability in amplitude and time-to-peak these m.e.p.ps differ from those at unpoisoned junctions by being unaffected by procedures which alter extra- or intracellular Ca2+ concentrations. The appearance of this Ca2+-insensitive spontaneous quantal secretion of acetylcholine is apparently not a direct effect of the toxin but secondary to blockade of impulse transmission since it also appears at unpoisoned end-plates when transmission is impaired for other reasons. Procedures which increase the intracellular Ca2+ concentration in nerve terminals restore transmitter release from botulinum toxin poisoned nerves. Furthermore, the block caused by the toxin is very temperature-dependent, a reduction in temperature relieving the block. Since presynaptic Ca2+ currents are unaltered by the toxin it is proposed that the block of transmission is due to a reduction in the calcium content of the nerve terminal to a level where the amount of Ca2+, which normally enters, is insufficient to activate transmitter release.     

Estimates of quantal content during 'chemical potentiation' of transmitter release.

The number of quantal transmitter packets (m), released from motor nerve terminals in response to a single stimulus, has been estimated from the ratio of the amplitudes of endplate currents (e.p.c.) to spontaneous miniature endplate currents (m.e.p.c.), in voltage-clamped endplates of the frog. At 6 degrees C, the average value of m at normal nerve-muscle junctions was about 300. If allowance is made for the temporal dispersion of quantal transmitter release during the e.p.c., this value is increased by about 30%. After treatment with diaminopyridine or tetraethylammonium, transmitter release in response to a nerve stimulus is greatly enhanced and values of m exceeding 10(4) are frequently found. Moreover, the duration of the e.p.c. becomes much longer than that of the m.e.p.cs. The number of packets then liberated during the e.p.c. is much larger than the number of 'active zones' of the endplate and may even exceed the total number of vesicles lined up in twin-files adjacent to the presynaptic membrane.     

Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions

Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat. At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps. At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal. The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates. At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced. The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps. To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps. Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.     

Effect of hyaluronidase on the quantum content and binomial p and n parameters of neuromuscular transmission in frogs

In the frog dermo-sternal muscle hyaluronidase (0.01--0.1%) caused e.p.p. amplitude and quantum content of transmission to fall when acting in a solution containing 1 mM Ca2+ and 4 mM Mg2+. The change in the quantum content was related to a decrease in binomial parameter n, the probability of mediator p release increased in the first moments of the enzyme action. With the prolongation of hyaluronidase action, negative values of p appeared. Hyaluronidase caused an increase in e.p.p. amplitude and in quantum content of transmission when acting in a solution containing 8 mM Ca2+ and d-tubocurarine. It was suggested that hyaluronidase modifies the normal calcium exchange between the bulk solution and the unstirred layer near nerve terminal membrane thus affecting the transmission.     

Evidence for a presynaptic action of chlordimeform at the insect neuromuscular junction

The mechanism of action of chlordimeform on the mealworm nerve-muscle preparation was studied with microelectrodes. Chlordimeform affected neither the mean amplitude nor the frequency of spontaneous miniature excitatory postsynaptic potentials. Extracellular focal recordings show that in the presence of 0.8 mM chlordimeform the presynaptic spike is almost unchanged, but the quantal content for evoked transmitter release is reduced. It is suggested that chlordimeform decreases the influx of calcium at the presynaptic terminal during the active phase of the nerve terminal action potential, thereby inhibiting evoked transmitter release.     

Changes in the synaptic activity in sciatic nerve-extensor digitorum longus (preparations induced by ethanol)

The aim of this research is the study of the modification of synaptic activity caused by ethanol in the rat sciatic nerve-extensor digitorum longus (EDL) muscle preparation. For such a purpose, intracellular recordings have been carried out, keeping the muscle immersed in normal Ringer solution and in Ringer solutions containing ethanol at different concentrations up to 0,8 M. Therefore, the resting potential of muscle cells and the frequency of m.e.p.p.s were measured. Qualitative observations of m.e.p.p.s shape were also carried out. Ethanol increases the frequency of m.e.p.p.s in the rat sciatic nerve - EDL muscle preparation. The logarithm of relative frequency (frequency in Ringer solution with ethanol/frequency in normal Ringer solution) is linear with respect to the concentration of ethanol, with a slope of 1.44. Furthermore, ethanol increases the amplitude and lengthens the time course of m.e.p.p.s. The muscle cells undergo a hyperpolarization of about 2-3% at the lowest concentrations of ethanol tested.     

Effect of prostaglandins E1 and F2 alpha on neuromuscular transmission in the frog sartorius muscle.

End plate potentials (e.p.p.s.) and miniature end plate potentials (m.e.p.p.s.) were recorded intracellularly at the neuromuscular junction of the frog sartorius muscle. Addition of as little as 8.5 x10(-8)M PGE1 reduced the mean m.e.p.p. frequency. The mean amplitude of m.e.p.p.s was not changed, the mean amplitude of the e.p.p.s and the quantum content of the transmitter released by a nerve impulse was slightly reduced. A decrease in mean m.e.p.p. frequency was also seen in response to the administration of 8.5 x 10(-8)M PG2 alpha. The mean amplitude of e.p.p.s and m.e.p.p.s and the quantum content remained unchanged. The possible presynaptic mode of action of PGs in the preparation of discussed.     

Effect of ethanol on young nerve endings

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

Some statistical properties of the miniature endplate potentials

I will demonstrate that series of miniature endplate potentials (m.e.p.ps) showing a high proportion of so-called giant m.e.p.ps (g.m.e.p.ps) have different statistical structures from series where the proportion of g.m.e.p.ps is low. The nature of the different structures will be discussed on the basis of two statistical models, one for the distribution of the m.e.p.p. amplitudes and one for the series of point events occurring in time.     

Action of botulinum A toxin and tetanus toxin on synaptic transmission

Intracellular recordings of the spontaneous activity from mammalian spinal cord neurons in culture demonstrated different sensitivities of excitatory and inhibitory synaptic transmission for the action of tetanus toxin (Tetx) and botulinum toxin type A (Botx). The effects of Tetx and Botx on spontaneous and nerve-evoked transmitter release were compared under identical experimental conditions in experiments on in vitro poisoned mouse diaphragms. At 37 degrees C completely paralyzed endplates are characterized by a very low frequency of spontaneous miniature endplate potentials (m.e.p.p.s) and by a 100% failure to evoke endplate potentials (e.p.p.s) in response to single nerve stimuli. Striking differences in the action of both toxins have been observed when the very low transmitter release probabilities of paralyzed nerve-muscle preparations were increased by tetanic nerve stimulation and/or application of potent K+-channel blockers and/or by reduction of temperature to 25 degrees C. While Botx did not change the short latency between nerve impulse and postsynaptic response, Tetx produced a temporal dispersion of the quantal release suggesting that the toxins act at different sites in the chain of events that result in transmitter release. To find further evidence to support the different actions of the toxins the spontaneous transmitter release was studied in more detail. Tetx blocked preferentially the release of so-called large mode m.e.p.p.s without affecting the frequency of the small mode ones. In contrast, Botx strongly inhibited both the small and large mode m.e.p.p.s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual action of intracellularly released calcium on the quantal mediator secretion

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

The time course of the evoked secretion of the mediator quanta in various regions of the frog motor nerve ending

Apart from the fact that the gradient of the velocity of the AP propagation along the nerve terminal and the intensity of secretion do exist, the kinetics of a quanta transmitter release may also be revealed in different parts of the terminal. The velocity of the propagation and the minimum sympatric delay tend to diminish along with moving away from the myelinated part of axon, whereas the synchronicity of the quanta release rises. The distinctions in the time course of secretion in different parts of the terminal were amplified when the calcium ion concentration in the medium was enhanced. The observed peculiarities of the secretion kinetics in different regions of nerve ending seem to compensate for diminishing of the amplitude of multiquantal endplate current.     

Depolarization affects neuromuscular junction of streptozotocin-diabetic mice.

The effect of increasing extracellular calcium concentration on spontaneous transmitter release was studied at both soleus (slow) and fast extensor digitorum longus (EDL) nerve terminals of control and streptozotocin-induced diabetic (STZ-D) young C57 BL mice (7 months old) depolarized by high (20 mM) extracellular potassium [K]o. Diabetes was induced by i.p. injection with a single dose of streptozotocin (200 mg/kg) at the age 5 months and the electrophysiological studies were carried out after 8 more weeks. By using intracellular recording, miniature endplate potentials (MEPPs) were first recorded in a normal [K]o Krebs solution. Subsequently, MEPPs were recorded in high [K]o Krebs solution with 4 different Ca concentrations: Ca-free/ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA), 0.5, 1.5 and 2 mM Ca. MEPP frequency was lower at STZ-D than control nerve terminals in EDL but not soleus. However, MEPP frequency was progressively higher at both EDL and soleus of STZ-D than control with increasing Ca concentration in Krebs that contained 20 mM [K]o. In STZ-D slow soleus muscle, depolarization produced 0.7, 4.3, 41.6 and 62.7 vs 1.4, 2.8, 20.7 and 31.6 Hz for control in the 4 different Ca concentrations. In STZ-D fast EDL muscle, depolarization produced 0.5, 4.9, 48.2 and 66.8 vs 1.2, 2.5, 27 and 35.4 Hz for control in the 4 different Ca concentrations. Bimodal and unimodal MEPP amplitude were present at both slow and fast nerve terminals. However, depolarization increased the percentage of bimodal MEPP amplitude in STZ-D compared to control (p<0.01) mice in EDL but not soleus. The results revealed that these changes in muscle firing pattern may provide a protective effect against diabetes-induced neuropathy at the neuromuscular junction.     

Pre-junctional inhibitory action of prostaglandin E2 on excitatory neuro-effector transmission in the human bronchus

The effects of prostaglandin E2 (PGE2) and indomethacin on excitatory neuro-effector transmission in the human bronchus were investigated by tension recording and microelectrode methods. PGE2 (10(-10)-10(-9)M) suppressed the amplitude of twitch contractions and excitatory junction potentials (e.j.ps) evoked by field stimulation at a steady level of basal tension obtained by the combined application of indomethacin (10(-5) M) and FPL55712 (10(-6) M). In doses over 10(-8)M, PGE2 reduced the muscle tone and dose-dependently suppressed the amplitude of twitch contractions. Indomethacin (10(-5) or 5 x 10(-5) M) reduced the muscle tone and enhanced the amplitude of twitch contractions and e.j.ps evoked by field stimulation in the presence of FPL55712. PGE2 (10(-9) M) had no effect on the post-junctional response of smooth muscle cells to exogenously applied acetylcholine (ACh) (4 x 10(-7) M). However, indomethacin (10(-5) M) significantly enhanced the ACh-induced contraction of the human bronchus. These results indicate that PGE2 in low concentrations has a pre-junctional action to inhibit excitatory neuro-effector transmission in addition to a post-junctional action, presumably by suppressing transmitter release from the vagus nerve terminals in the human bronchial tissues.     

Functioning of catfish electroreceptors: relation between skin potential and receptor activity

Properties of catfish electroceptors were investigated by simultaneous recording of the skin potential and the activity of an afferent nerve. 1. The normal threshold stimulus intensity induces a potential amplitude of about 10 to 30 muV across the skin (Table I). 2. The average spike frequency in the nerve increases approximately with the logarithm of the stimulus intensity (Fig. 1). 3. The direct current restoring the receptor activity in calcium deficient media makes the skin potential more negative. 4. Presumably, not the skin potential itself but a difference between the skin potential and the e.m.f. generated by the receptor epithelium influences receptor functioning. 5. Amplitude and phase characteristics can be described by a filter circuit (Fig. 4 and 5).     

Stimulatory effect of trans-cinnamaldehyde on noradrenaline secretion in guinea-pig ileum myenteric nerve terminals

The effect of trans-cinnamaldehyde (CNMA) on the release of noradrenaline (NA) from nerve terminal was investigated using isolated ileal synaptosomes of guinea-pig. Release was determined as the amount of NA, quantified by h.p.l.c.-electrochemical detection, from samples incubated with CNMA minus that in parallel blanks treated with same volume of vehicle. CNMA stimulated the secretion of NA in a concentration-dependent manner from 5 microM to 50 microM, while the value of lactate dehydrogenase in the incubated medium was not influenced by CNMA. However, trans-cinnamic acid, cinnamoyl chloride and cinnamamide failed to produce similar effect. Specific action of CNMA can thus be considered. Guanethidine inhibited the release of NA by CNMA in a concentration- dependent manner. Saxitoxin attenuated the action of CNMA at concentrations sufficient to block sodium channels. The depolarizing effect of CNMA on the membrane potential was also illustrated by a concentration-dependent increase in the fluorescence of bisoxonol, a potential sensitive dye. The NA releasing action of CNMA was deleted by removal of calcium chloride from the bathing medium. This action of CNMA was also attenuated by Rp-cAMP at concentrations sufficient to inhibit the action of cyclic AMP. These findings suggest that CNMA can depolarize the membrane to result in a calcium-dependent and cyclic AMP-related release of NA from noradrenergic terminals.     

The mechanism of β-bungarotoxin action. I. Modification of transmitter release at the neuromuscular junction

The protein, β-bungarotoxin, a presynaptic neurotoxin isolated from the venom of the snake Bungarus multicinctus, is known to inhibit mitochondrial function. Within 30 min after adding the toxin to a rat diaphragmphrenic nerve preparation, the quantal content increased tenfold and the frequency of miniature endplate potentials increased fourfold. No increase in miniature endplate potential frequency was seen in the absence of extracellular calcium. Since mitochondria may be involved in regulating intracellular calcium levels, the rate at which the transmitter release is turned off was studied by measuring delayed release in the presence and absence of toxin. Delayed release is elevated about eightfold by the toxin. If delayed release is due to residual calcium, as has been hypothesized, these data may be explained if the toxin does not alter the amount of calcium which enters the terminal, but rather the rate at which that calcium is removed. Alternatively, a calcium-dependent modification of the release process itself might be produced. The eventual reduction in transmitter output did not appear to result from depletion of the terminal of releaseable packets of transmitter, but does require extracellular calcium.     

Muscarinic M1 acetylcholine receptors regulate the non-quantal release of acetylcholine in the rat neuromuscular junction via NO-dependent mechanism

Nitric oxide (NO), previously demonstrated to participate in the regulation of the resting membrane potential in skeletal muscles via muscarinic receptors, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh release was estimated by the amplitude of endplate hyperpolarization (H-effect) following a blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine. The muscarinic agonists oxotremorine and muscarine lowered the H-effect and the M1 antagonist pirenzepine prevented this effect occurring at all. Another muscarinic agonist arecaidine but-2-ynyl ester tosylate (ABET), which is more selective for M2 receptors than for M1 receptors and 1,1-dimethyl-4-diphenylacetoxypiperidinium (DAMP), a specific antagonist of M3 cholinergic receptors had no significant effect on the H-effect. The oxotremorine-induced decrease in the H-effect was calcium and calmodulin-dependent. The decrease was negated when either NO synthase was inhibited by N(G)-nitro-L-arginine methyl ester or soluble guanylyl cyclase was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The target of muscle-derived NO is apparently nerve terminal guanylyl cyclase, because exogenous hemoglobin, acting as an NO scavenger, prevented the oxotremorine-induced drop in the H-effect. These results suggest that oxotremorine (and probably also non-quantal ACh) selectively inhibit the non-quantal secretion of ACh from motor nerve terminals acting on post-synaptic M1 receptors coupled to Ca(2+) channels in the sarcolemma to induce sarcoplasmic Ca(2+)-dependent synthesis and the release of NO. It seems that a substantial part of the H-effect can be physiologically regulated by this negative feedback loop, i.e., by NO from muscle fiber; there is apparently also Ca(2+)- and calmodulin-dependent regulation of ACh non-quantal release in the nerve terminal itself, as calmidazolium inhibition of the calmodulin led to a doubling of the resting H-effect.     

The effect of calcium ions on the secretion of quanta evoked by an impulse at nerve terminal release sites

A study has been made of the effects of calcium ions on the number of quanta secreted from all the release sites at an amphibian motor nerve terminal recorded with an intracellular microelectrode (m) compared with the number secreted simultaneously from a small number of release sites recorded with an extracellular microelectrode (me). If the endplate potential was made subthreshold by lowering the external calcium concentration ([Ca]o less than or equal to 0.4 mM), it was possible to find small groups of release sites for which me was comparable to m, indicating considerable nonuniformity in the probability of release of a quantum at different groups of release sites (Pe) in a given [Ca]o. Increasing [Ca]o in the range from 0.25 to 0.4 mM increased the probability of release of a quantum at groups of release sites (Pe), independent of the initial value of Pe, and the dependence of Pe on [Ca]o followed a fourth power relationship. A conditioning impulse enhanced the probability of release of a quantum by a subsequent test impulse at release sites, if Pe was less than 1.0 during the conditioning impulse. It is shown that the present observations regarding the dependence of Pe on [Ca]o and on conditioning impulses can be quantitatively predicted from previous observations regarding the dependence of the binomial parameters m, p, and n on [Ca]o and on conditioning impulses determined with intracellular electrodes, if the probability of secretion of a quantum at a release site (Pj) is different for different release sites and Pj is distributed as a beta random variable.     

Divalent cations and transmitter release at low concentration of tetrodotoxin

Transmitter release from frog motor terminals was studied in the presence of very low concentrations of tetrodotoxin (TTX, 4.10(-10)--6.10(-9) g/ml). TTX reversibly reduced the amplitude of the end-plate potential (epp), while leaving the amplitude distribution to follow Poisson's law. The effects of a number of divalent cations were studied in the presence of TTX. It was found that after the addition of TTX there was an increase in the constant of dissociation of calcium and strontium from a hypothetical membrane "release site," while the dissociation constants of magnesium and manganese remained unaltered. It is concluded that the release site is probably intracellular and that a reduced presynaptic spike amplitude, as well as magnesium and manganese ions, decrease the access of calcium and strontium to the site.