Characterization of the actions of botulinum neurotoxin type E at the rat neuromuscular junction

Botulinum neurotoxin (BoTx) serotype E blocks spontaneous and evoked quantal release of acetylcholine at the rat neuromuscular junction. Increasing extracellular Ca2+ to 8 mmol l-1 or substituting Ca2+ with La3+ (0.1 and 1.0 mmol l-1) or depolarizing the nerve terminals by 20 mmol l-1 K+ markedly increases miniature end-plate potential frequency in normal muscle, but in BoTx-E poisoned preparations none of these ions, with the exception of 1 mmol l-1 La3+, was able to restore spontaneous quantal transmitter release to levels recorded at unpoisoned junctions. In absolute values the enhancement with La3+ was much less than that reported at normal junctions. Nerve stimulation in the presence of 3,4-diaminopyridine (10-20 mumol l-1) and high calcium (8 mmol l-1) evoked multiquantal end-plate potentials and muscle twitches. We conclude that the neuromuscular block produced by BoTx serotype E is similar to that previously described for BoTx serotype A but differs from that produced by BoTx serotypes B, D and F in not causing desynchronization of nerve impulse-evoked transmitter release. 3,4-Diaminopyridine might be useful in the treatment of poisoning by BoTx serotype E since it markedly enhanced synchronous transmitter release from poisoned motor nerve terminals.     

G protein betagamma subunits modulate the number and nature of exocytotic fusion events in adrenal chromaffin cells independent of calcium entry

G-protein-coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca(2+) channels (Ca(2+) channels) by G protein betagamma subunits (Gbetagamma) is one prominent mechanism, but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca(2+)-channel-independent effects of Gbetagamma on transmitter release, so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of Gbetagamma on Ca(2+) entry, we stimulated secretion using ionomycin (a Ca(2+) ionophore) or direct intracellular application of Ca(2+) through a patch pipette. Activation of endogenous GPCR or transient transfection with exogenous Gbetagamma significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge ("quantal size") and amplitude of the amperometric spikes were also significantly reduced by GPCR/Gbetagamma. We conclude that independent from effects on calcium entry, Gbetagamma can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which Gbetagamma might exert these novel effects including interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.     

Potency of depolarization-induced transmitter release is determined by divalent cation influx in PC 12 cells.

Evoked release of [3H]dopamine ([3H]DA) from pheochromocytoma cells (PC 12) is dependent on extracellular calcium ([Ca2+]ex), but it can take place if calcium ions (Ca2+) are substituted by other divalent ions such as strontium (Sr2+) and barium (Ba2+). The potency of the divalent cations at supporting release varies with the cell type; in PC 12 cells the order of potency is Ba2+ > Sr2+ > Ca2+. The close correlation between depolarization-evoked Ca2+ entry and depolarization-evoked transmitter release prompted us to examine whether the higher evoked transmitter release in the presence of Sr2+ correlates with an increased evoked Sr2+ influx. Influx studies were conducted on PC12 cells using a radioactive tracer (45Ca2+ or 85Sr2+, < 1 microM) in the presence of either Sr2+ (0.5 mM) or Ca2+ (0.5 mM). Depolarization with K Cl (60 mM) increased evoked 45Ca2+ influx 2-fold when Ca2+ was substituted with Sr2+. Similarly, evoked 85Sr2+ influx increased 1.87-fold by substituting Ca2+ for Sr2+. Thus the amount of evoked cation influx is determined by the type of divalent ion which is accessible in the extracellular medium, independently of the radioactive tracer used. Increased evoked transmitter release in the presence of Sr2+ was associated with increased evoked Sr2+ influx. This suggests that the potency of evoked transmitter release is determined predominantly by the influx of divalent cations. Furthermore, the steps subsequent to cation influx in the release process are equally efficient for both cations.     

Quantal release and facilitation at frog neuromuscular junctions at about 0 degrees C.

1. It has been reported that at the frog neuromuscular junction at temperatures around 0 degrees C the release of transmitter quanta following nerve stimulation becomes disrupted, and the facilitation obtained after a second stimulus is no longer detectable. We thought that further investigation might give insight into the mechanism of quantal release, so we undertook experiments on Rana pipiens and Rana berlanieri. 2. In these species neuromuscular transmission occurs at temperatures as low as -0.8 degrees C. As the temperature is decreased further, transmission fails, apparently by a block in nerve conduction. The number of quanta released per stimulus decreases as temperature is lowered, with a Q10 of approximately 2.4. Owing to the decrease in the quantal output and the probabilistic nature of the release process, in occasional single records of an end-plate current (EPC), the pattern of release appeared disrupted. The kinetics of quantal release was studied by the use of a deconvolution method, which requires recording of EPCs and miniature EPCs (MEPCs) in preparations in high Mg(2+)-low Ca2+ solution. At approximately 0 degrees C the pattern of quantal release was similar to that at higher temperatures, although with a slower time course. At 0 degrees C the peak of release occurred approximately 3.5 ms after onset. 3. In our experiments there was almost no difference in the frequency of MEPCs at 22 degrees C and at 0 degree C. 4. We observed as much facilitation to a second stimulus at 0 degree C as at 10 degrees C. The Q10 for the decay of facilitation with time was between 1.9 and 2.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.     

A study of the mechanism of quantal transmitter release at a chemical synapse

1. The nerve-muscle preparation of the cutaneous pectoris of the frog has been used to study quantal transmitter release.2. When the osmotic pressure of the external solution is raised 1.5-2 fold, the frequency of miniature end-plate potentials (m.e.p.p.s) rises by 1.5-2 orders of magnitude. This effect is independent of the presence of Ca(2+) ions and of the nature of the substances by which the osmotic pressure has been increased.3. In Ca(2+) free hypertonic solution the nerve impulse still invades the nerve terminals but does not alter the frequency of the m.e.p.p.s.4. The arrival of the impulse in the terminals causes an immediate increase in the rate of quantal release, provided divalent cations are present whose passage through the axon membrane is facilitated by excitation (Ca(2+), Sr(2+), Ba(2+)).5. Divalent cations which penetrate only slightly (Mg(2+), Be(2+)) lower the frequency of m.e.p.p.s and suppress the end-plate potential (e.p.p.) evoked by an impulse, in the presence of Ca(2+) ions. Be(2+) is a more effective inhibitor than Mg(2+).6. In Ca(2+) free solutions, adding Mg(2+) causes an increase in the frequency of m.e.p.p.s evoked by depolarization of the nerve endings or by treatment with ethanol.7. The trivalent cation La(3+) is more effective than divalent cations are in increasing the frequency of m.e.p.p.s. The tetravalent cation Th(4+) also raises the m.e.p.p. frequency.8. The observations summarized in paragraphs 2-7 indicate that the frequency of m.e.p.p.s at a constant temperature depends only on the concentration of uni-, di- and trivalent cations inside the nerve ending. It is suggested that the internal cation concentration influences the adhesion between synaptic vesicles and the membrane of the nerve ending.9. For a model experiment, artificial phospholipid membranes have been used to study the effect of uni-, di-, tri- and tetravalent cations on the adhesion process. At pH 7-7.4, the time required for adhesion to take place decreases with increasing cation concentration in the bath. Ca(2+) ions are 100-1000 times more effective than K(+) ions; La(3+) and Th(4+) ions are still more effective. The ;adhesion time' decreases when the pH is lowered; it increases greatly with lowering of temperature.10. The hypothesis is put forward that the mutual adhesion of artificial vesicles made of phospholipid membranes, and the adhesion between synaptic vesicles and the membrane of the nerve ending arise by a common mechanism. In both cases, the important factor is the influence of cations on the electric double layer at the membrane surface.     

Inositol trisphosphate and cyclic adenosine diphosphate-ribose increase quantal transmitter release at frog motor nerve terminals: possible involvement of smooth endoplasmic reticulum

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+. The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid or release of Ca2+ from intracellular stores such as mitochondria. Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose, so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10(-4)M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40 min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10(-4)M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.     

Lambert-Eaton myasthenic syndrome

The Lambert-Eaton myasthenic syndrome is associated in about 65% of cases with small cell carcinoma, a tumour of neurosecretory origin. It is characterised physiologically by a decrease in the nerve evoked quantal release of acetylcholine, and in the resting non-quantal release ("molecular leakage"). The associations with autoimmune disease, with other autoantibodies, with HLA-B8, and with the IgG heavy chain marker Glm (2) are consistent with an autoimmune aetiology. Clinical and electromyographic responses to plasma exchange point to a humorally mediated disorder. This has been substantiated by passive transfer of the the main electrophysiological features of LEMS to mice by daily injections of LEMS IgG. Plasma was no more effective in inducing the electrophysiological changes than the IgG fraction. The decrease in quantal content appeared closely to follow the level of human IgG in the mouse serum and complement (C5) deficient mice were as susceptible as normal controls. The principal physiological abnormalities are both Ca2+ dependent processes, suggesting that a defect in Ca2+ transport may underlie the disorder. Preliminary studies of quantal content at low Ca2+ concentrations in mice injected with LEMS IgG suggest the functional loss of 40% of Ca2+ channels. Electron microscopic freeze fracture studies in such animals show, as in the human disease, a significant reduction in the number of active zone particles which are believed to represent Ca2+ channels. Thus it seems likely that the disorder of acetylcholine release is due to an IgG antibody directed to nerve terminal determinants that include the Ca2+ channels or structures closely related to them. In cancer-associated LEMS, the autoantibody response may initially be made to similar determinants on the tumour cell membrane, cross-reactivity of the antibody with nerve terminal determinants leading to the disorder of transmitter release.     

Co-operative action a calcium ions in transmitter release at the neuromuscular junction

1. The quantitative dependence of transmitter release on external calcium concentration has been studied at the frog neuromuscular junction, using intracellular recording and taking the amplitude of the end-plate potential (e.p.p.) as an index of the number of packets released.2. The relation between [Ca] and the e.p.p. is highly non-linear. The initial part of this relation on double logarithmic co-ordinates gives a straight line with a slope of nearly four (mean 3.78 +/- 0.2 S.D. in 28 experiments). Addition of a constant amount of Mg reduces the e.p.p. without altering the slope of the log e.p.p./log Ca relation.3. The slope of this logarithmic relation diminishes as [Ca] is raised towards the normal level.4. The results are explained quantitatively on the hypothesis that Ca ions combine with a specific site X on the nerve terminal forming CaX, and that the number of packets of acetylcholine released is proportional to the fourth power of [CaX].5. The analysis suggests that a co-operative action of about four calcium ions is necessary for the release of each quantal packet of transmitter by the nerve impulse.     

Zinc competitively inhibits calcium-dependent release of transmitter at the mouse neuromuscular junction

The effect of zinc on the release of transmitter was investigated in preparations of mouse diaphragm by conventional microelectrode techniques. The frequency (FP of miniature end-plate potentials (MEPPs), elevated by Ca²⁺ in high K⁺ medium, was reduced by zinc in a concentration-dependent fashion. When the extracellular concentration of Ca²⁺ ([Ca²⁺]_o) was varied in the absence of zinc, a linear relationship between log(F) and log([Ca²⁺]_o) was obtained. When the effect of zinc was depicted graphically, it was found that zinc shifted the relationship between log(F) and log([Ca²⁺]_o) to the right, with respect to the control in the absence of zinc, without altering the slope. Zinc also reduced the quantal content (m) of end-plate potentials (EPPs). As [Ca²⁺]_o was varied in the absence of zinc, a linear relationship between ln(m) and ln([Ca²⁺]_o) was observed. Zinc shifted this linear relationship between ln(m) and ln([Ca²⁺]_o) to the right, with respect to the control, without altering the slope. Thus, zinc reduced both the asynchronous and the phasic release of transmitter. These results suggest that zinc competes with Ca²⁺, and this conclusion is confirmed by examination of a modified Lineweaver-Burk plot of the data. Zinc probably inhibits the entry of Ca²⁺ into the nerve terminals, thereby inhibiting transmitter release.     

Kinetic differences in the effect of calcium on quantal and non-quantal acetylcholine release at the murine diaphragm

The effects of Ca2+ withdrawal on non-quantal, evoked quantal and spontaneous quantal release of acetylcholine (ACh) from the motor nerve terminals were studied with standard intracellular recording techniques. Anticholinesterase-treated mouse diaphragms were used. In a Ca2(+)-free solution all forms of ACh release decreased, but with different kinetics. As expected, evoked quantal release declined to zero within a few minutes. Spontaneous quantal release, i.e. the frequency of the miniature end-plate potentials (MEPPs), decreased to 15% of the control within 20 min after calcium withdrawal. The slowest decay was that of non-quantal release which declined very slowly and reached zero after 45-50 min. Following Ca2+ re-admission, both quantal types of ACh release were rapidly restored (evoked in 10 min, spontaneous in 20 min). However, recovery of non-quantal release did not occur until after 50 to 60 min.     

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.     

Facilitation of neurotransmitter release in mouse motor synapses in different modes of protein kinase C activation

Protein kinase C blocker chelerythrine prevented the increase in quantal content of single and rhythmic evoked end-plate potentials after disinhibition of L-type Ca(2+)-channels with paxillin. Phorbol ester increased quantal content of single end-plate potentials and changed rhythmic activity of mouse motor synapses. The effects of phorbol ester were to a great extent neutralized by L-type Ca(2+)-channel blocker nitrendipine and were completely abolished by K(+)-channel blocker 4-aminopyridine. Thus, we discovered different facilitations of transmission after protein kinase C activation with calcium current through L-type channels and with phorbol ester.     

Effect of hypertonicity on augmentation and potentiation and on corresponding quantal parameters of transmitter release

Augmentation and (posttetanic) potentiation are two of the four components comprising the enhanced release of transmitter following repetitive nerve stimulation. To examine the quantal basis of these components under isotonic and hypertonic conditions, we recorded miniature endplate potentials (MEPPs) from isolated frog (Rana pipiens) cutaneous pectoris muscles, before and after repetitive nerve stimulation (40 s at 80 Hz). Continuous recordings were made in low Ca2+ high Mg2+ isotonic Ringer solution, in Ringer that was made hypertonic with 100 mM sucrose, and in wash solution. Estimates were obtained of m (no. of quanta released), n (no. of functional release sites), p (mean probability of release), and vars p (spatial variance in p), using a method that employed MEPP counts. Hypertonicity abolished augmentation without affecting potentiation. There were prolonged poststimulation increases in m, n, and p and a marked but transient increase in vars p in the hypertonic solution. All effects were completely reversed with wash. The time constants of decay for potentiation and for vars p were virtually identical. The results are consistent with the notion that augmentation is caused by Ca2+ influx through voltage-gated calcium channels and that potentiation is due to Na+-induced Ca2+ release from mitochondria. The results also demonstrate the utility of this approach for analyzing the dynamics of quantal transmitter release.     

Transmitter release at frog end-plate loaded with a Ca2+-chelator, BAPTA: hypertonicity and erythrosin B augment the release independently of internal Ca2+

A Ca2+-chelator, bis-(aminophenoxy)ethane-tetraacetic acid (BAPTA) was loaded into the presynaptic nerve terminal of frog end-plate. The BAPTA-loaded preparation showed little facilitation. However, the facilitation reappeared upon addition of an ionophore, X-537A, supporting the view that the loss of facilitation was due to the Ca2+-buffering action of BAPTA. Both hypertonic conditions and erythrosin B increased both the size of end-plate potentials and frequency of miniature end-plate potentials without any recovery of facilitation at the BAPTA-loaded end-plate. This suggested that transmitter release was increased by both conditions with little change in internal Ca2+ concentration.     

The role of calcium in depolarization—secretion coupling at the motor nerve terminal

1. The relation between m.e.p.p. frequency (F) and [Ca] was studied at the mouse neuromuscular junction, at varied concentrations of K(+) and at nerve terminals depolarized by focal depolarization.2. Under all conditions the relation between log F and log [Ca] was sigmoid, with a maximum slope that increased with depolarization or raised [K(+)]. In addition, depolarization or raised K(+) caused a progressive shift of the sigmoid curve upward and to the left (to reduced log [Ca]) and increased the range over which log F could be altered by [Ca].3. Reduction of osmotic pressure changed the relation between log F and log [Ca] in the same way as increase of depolarization, while increase of osmotic pressure did the opposite.4. Raised [Mg] acted in two ways: (a) to shift the curve of log F vs. log [Ca] to the right and (b) to reduce maximum Delta log F/Delta log [Ca] without altering the range of log F sensitive to [Ca].5. The relation between log quantal content of e.p.p.s and log [Ca] was similar to that between log m.e.p.p. frequency and log [Ca].6. Individual nerve terminals varied in both Ca-dependent and Ca-independent fractions of log F; a large Ca-independent portion appears to be associated with a low Ca-dependent portion and vice versa. With large prolonged depolarization the Ca-independent portion was increased, apparently at the expense of the Ca-dependent portion.7. The results of all experiments were summarized in terms of parameters found by fitting the observed log release -log [Ca] curves to two theoretical equations, each derived on the basis of a model: (a) all-or-nothing activation of release probability by Ca-complex(es) and (b) graded activation of release probability by Ca complex(es).8. On the basis of the all-or-nothing model, from which follows alinear relation between F and amounts of Ca complex(es), the number of Ca(2+) atoms that ;cooperate' to mediate release appeared to increase progressively with presynaptic depolarization, to a value of 4 or more with a presynaptic action potential.9. On the basis of the graded activation model, which predicts an exponential relation between F and amount of Ca complex, the number of Ca(2+) atoms that combine with Ca receptor appears to be independent of presynaptic depolarization.10. Various models which could account for the data are discussed. It was concluded that all the data are consistent with a model in which:(i) quantal release probability is continuously graded with the amount of a simple Ca complex (CaX) inside the nerve terminal.(ii) Ca entry is governed by presynaptic membrane potential (increasing exponentially with depolarization) and by the amount of a Ca complex (Ca(2)Y) on or in the membrane.(iii) Mg(2+) competes with Ca(2+) at both receptors, X and Y.(iv) The internal Ca receptor X is also increased by presynaptic depolarization.     

Presynaptic calcium channels and the depletion of synaptic cleft calcium ions

The entry of calcium ions (Ca(2+)) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca(2+) into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca(2+) depletion. The terminal was voltage clamped at a holding potential (V(H)) of -80 mV and a depolarizing pulse was applied to a range of potentials (-60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V(H), by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca(2+) electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V(1/2) of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V(1/2) of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba(2+) is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca(2+) depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.     

Inhibitory effect of intraterminal lithium on asynchronous release of excitatory quanta induced by veratridine in nerve-muscle synapses of crayfish

Crayfish muscle fibres were voltage-clamped at E = -80 mV membrane potential and superfused for about 10 min with Li+ saline (Na+ replaced by Li+) which contained picrotoxin to block inhibitory post-synaptic currents. Addition of veratridine (100 mumol/l) caused intense fluctuations in the voltage clamp current within 20-60 s due to vigorous asynchronous quantal release of excitatory transmitter from the nerve terminals distributed over the muscle fibre surface. Most likely, this quantal release resulted from loading the nerve terminals with Li+ via voltage-gated Na+ channels activated by veratridine. However, in the presence of Li+ quantal release was only transient; the quantal release rate, ?, attained a maximum of congruent to 10,000 quanta/s and then declined exponentially with tau congruent to 10 to 20 s. Removal of Li+ and reapplication of normal Na+ increased ? a second time. The amount of quanta released in the presence of Na+ was about an order of magnitude larger than that released previously in the presence of Li+. In preparations pretreated with Li+ superfusate for t greater than 45 min no marked quantal release could be elicited by veratridine. The experiments suggest an inhibitory effect of intraterminal Li+ on the quantal release process.     

Quantal independence and uniformity of presynaptic release kinetics at the frog neuromuscular junction

1. Amplitude and latency fluctuations of the end-plate potential at the frog neuromuscular junction were studied simultaneously at low temperatures, using intracellular or focal extracellular recording techniques and average quantal contents between 0.5 and 3.2. At the release rates studied, the evoked release of one quantum has in most cases no significant effect on the probability of subsequent quantal release to the same stimulus, confirming the mutual independence of quantal releases in this preparation.3. An equation derived from Poisson's law was applied to a histogram of the latencies of the first quantum released on each of a series of trials, to predict the average quantal content of end-plate responses originating at various times after nerve stimulation. The shape of the predicted time distribution of quantal contents usually agreed closely with that of the experimentally observed time distribution of end-plate response amplitudes. This agreement demonstrates that both the amplitude and the latency fluctuations of the end-plate response result from one presynaptic stochastic process that is uniform in magnitude and time course after each stimulus.4. Analysis of extracellular records from synaptic regions with a history of extensive activity often suggested the existence of depressive interaction among quantal releases, perhaps caused by depletion of the supply of releasable quanta.