Spontaneous Quantal Transmitter Release Research Articles

ON THE FIRING ACTIVITY OF A STATE-DEPENDENT STOCHASTIC NEURONAL MODEL

We address the problem of studying the number of excitatory stimuli producing a spike for a Stein-type stochastic model which includes a multiplicative state-dependent effect. Some results on the probability distribution of the number M of excitatory stimuli triggering a spike are obtained. We also evaluate the distribution of M conditional on the firing time, and disclose some properties of its mode. Finally, some results on the probability generating function of the conditional distribution are given. 1 Introduction A mathematical model based on Stein stochastic differential equation has been recently proposed in (5) to describe the firing activity of a neuronal unit. Similarly to Stein approach (15), such model includes the arrival of excitatory stimuli according to a Poisson process with an exponential decay in absence of stimuli. As an innovative feature, the model is characterized by a multiplicative state-dependent effect, since the depolariza- tions are random and depend on the voltage level at the stimulus time. This state-dependent effect is suitable to describe a behavior sometimes observed in neuronal dynamics (see, for instance, (14) where noisy stochastic conductance components are multiplicatively coupled to the membrane potential). Closed-form expressions for the distribution of the membrane potential level and for the firing density (in the case of homogeneous Poisson inputs) have been recently obtained in (5) for our model. The state-dependent Stein-type model is considered here to study the distribution of the number of excitatory stimuli able to yield a neuronal firing. This provides a basic characterization of the relationship between environmental stimuli and neural response. For instance, it is well-known that spontaneous quantal transmitter release from the motor nerve endings follows the Poisson distribution and generates miniature potentials which contribute to the neuron postsynaptic potential (7). The study performed in the present paper can be useful to face the question of what determines the post-synaptic response to the release of the quantal transmitter (see, for instance, (6) where an analysis of how retinal ganglion cells re-encode the received information is present). More generally, the obtained results are suitable to characterize the firing activity of a postsynaptic cell in networks of Stein-type neuronal units under ad hoc assumptions about the dynamics of presynaptic neurons (see (2), (3) and (4) where a simulation-based approach is used to investigate the synchronization between interacting neurons). The mathematical background of the present contribution is that of classical treatises on stochastic neuronal models, such as (12) and (13).

Analysis of Caribbean ciguatoxin-1 effects on frog myelinated axons and the neuromuscular junction

Caribbean ciguatoxin-1 (C-CTX-1) induced, after about 1 h exposure, muscle membrane depolarisation and repetitive post-synaptic action potentials (APs) in frog neuromuscular preparations. This depolarising effect was also observed in a Ca 2+-free medium with a strong enhancement of spontaneous quantal transmitter release, compared with control conditions. The ciguatoxin-induced increase in release could be accelerated when Ca 2+ was present in the extracellular medium. C-CTX-1 also enhanced nerve-evoked quantal acetylcholine (ACh) release. At normal neuromuscular junctions loaded with the fluorescent dye FM1-43, C-CTX-1 induced swelling of nerve terminals, an effect that was reversed by hyperosmotic d-mannitol. In myelinated axons, C-CTX-1 increased nodal membrane excitability, inducing spontaneous and repetitive APs. Also, the toxin enlarged the repolarising phase of APs in control and tetraethylammonium-treated axons. Overall, our data suggest that C-CTX-1 affects nerve excitability and neurotransmitter release at nerve terminals. We conclude that C-CTX-1-induced up-regulation of Na + channels and the inhibition of K + channels, at low nanomolar concentrations, produce a variety of functional dysfunctions that are in part responsible for the human muscle skeletal symptoms observed in ciguatera. All these dysfunctions seem to result from the subtle balance between ionic currents, intracellular Na + and Ca 2+ concentrations, and engaged second messengers.

Cholesterol and synaptic transmitter release at crayfish neuromuscular junctions

During exocytosis of synaptic transmitters, the fusion of highly curved synaptic vesicle membranes with the relatively planar cell membrane requires the coordinated action of several proteins. The role of membrane lipids in the regulation of transmitter release is less well understood. Since it helps to control membrane fluidity, alteration of cholesterol content may alter the fusibility of membranes as well as the function of membrane proteins. We assayed the importance of cholesterol in transmitter release at crayfish neuromuscular junctions where action potentials can be measured in the preterminal axon. Methyl-beta-cyclodextrin (MbetaCD) depleted axons of cholesterol, as shown by reduced filipin labelling, and cholesterol was replenished by cholesterol-MbetaCD complex (Ch-MbetaCD). MbetaCD blocked evoked synaptic transmission. The lack of postsynaptic effects of MbetaCD on the time course and amplitude of spontaneous postsynaptic potentials or on muscle resting potential allowed us to focus on presynaptic mechanisms. Intracellular presynaptic axon recordings and focal extracellular recordings at individual boutons showed that failure of transmitter release was correlated with presynaptic hyperpolarization and failure of action potential propagation. All of these effects were reversed when cholesterol was replenished with Ch-MbetaCD. However, focal depolarization of presynaptic boutons and administration of a Ca2+ ionophore both triggered transmitter release after cholesterol depletion. Therefore, both presynaptic Ca2+ channels and Ca2+-dependent exocytosis functioned after cholesterol depletion. The frequency of spontaneous quantal transmitter release was increased by MbetaCD but recovered when cholesterol was reintroduced. The increase in spontaneous release was not through a calcium-dependent mechanism because it persisted with intense intracellular calcium chelation. In conclusion, cholesterol levels in the presynaptic membrane modulate several key properties of synaptic transmitter release.

Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity.

We report here the purification of glycerotoxin from the venom of Glycera convoluta, a novel 320 kDa protein capable of reversibly stimulating spontaneous and evoked neurotransmitter release at the frog neuromuscular junction. However, glycerotoxin is ineffective at the murine neuromuscular junction, which displays a different subtype of voltage- dependent Ca(2+) channels. By sequential and selective inhibition of various types of Ca(2+) channels, we found that glycerotoxin was acting via Ca(v)2.2 (N-type). In neuroendocrine cells, it elicits a robust, albeit transient, influx of Ca(2+) sensitive to the Ca(v)2.2 blockers omega-conotoxin GVIA and MVIIA. Moreover, glycerotoxin triggers a Ca(2+) transient in human embryonic kidney (HEK) cells over-expressing Ca(v)2.2 but not Ca(v)2.1 (P/Q-type). Whole-cell patch-clamp analysis of Ca(v)2.2 expressing HEK cells revealed an up-regulation of Ca(2+) currents due to a leftward shift of the activation peak upon glycerotoxin addition. A direct interaction between Ca(v)2.2 and this neurotoxin was revealed by co-immunoprecipitation experiments. Therefore, glycerotoxin is a unique addition to the arsenal of tools available to unravel the mechanism controlling Ca(2+)-regulated exocytosis via the specific activation of Ca(v)2.2.

Temperature effect on carbachol-induced depression of spontaneous quantal transmitter release in frog neuromuscular junction.

The effects of carbachol (CCh) on the frequency (f) of the miniature endplate potentials were tested at temperatures between 5 and 30 degrees C. Higher CCh concentrations, 1 x 10(-5) and 5 x 10(-6) M, reduced the f to 60% and the temperature dependence was negligible. However, an inverse temperature dependence was found when low concentrations 3 x 10(-7) and 6 x 10(-7) M were applied. The depression of f was 40-50% in 5-10 degrees C but only 10-20% of the control in the 25 and 30 degrees C. During application of CCh, the new steady of f was reached at temperatures between 5 and 30 degrees C within 17-20 min (Q10 = 1.07). Much greater temperature dependence of recovery was observed during washing out CCh (Q10 = 1.6). The temperature-independence of the steady state effects of CCh, good agreement with Langmuir adsorption-desorption theory and non-steady kinetics indicate that physical rather than receptor-mediated events are responsible for the depression of f.

Trachynilysin mediates SNARE-dependent release of catecholamines from chromaffin cells via external and stored Ca2+.

Trachynilysin, a 159 kDa dimeric protein purified from stonefish (Synanceia trachynis) venom, dramatically increases spontaneous quantal transmitter release at the frog neuromuscular junction, depleting small clear synaptic vesicles, whilst not affecting large dense core vesicles. The basis of this insensitivity of large dense core vesicles exocytosis was examined using a fluorimetric assay to determine whether the toxin could elicit catecholamine release from bovine chromaffin cells. Unlike the case of the motor nerve endings, nanomolar concentrations of trachynilysin evoked sustained Soluble N-ethylmaleimide-sensitive fusion protein Attachment Protein REceptor-dependent exocytosis of large dense core vesicles, but only in the presence of extracellular Ca2+. However, this response to trachynilysin does not rely on Ca2+ influx through voltage-activated Ca2+ channels because the secretion was only slightly affected by blockers of L, N and P/Q types. Instead, trachynilysin elicited a localized increase in intracellular fluorescence monitored with fluo-3/AM, that precisely co-localized with the increase of fluorescence resulting from caffeine-induced release of Ca2+ from intracellular stores. Moreover, depletion of the latter stores inhibited trachynilysin-induced exocytosis. Thus, the observed requirement of external Ca2+ for stimulation of large dense core vesicles exocytosis from chromaffin cells implicates plasma membrane channels that signal efflux of Ca2+ from intracellular stores. This study also suggests that the bases of exocytosis of large dense core vesicles from motor nerve terminals and neuroendocrine cells are distinct.

Sodium-dependent increase in quantal secretion induced by brevetoxin-3 in Ca2+-free medium is associated with depletion of synaptic vesicles and swelling of motor nerve terminals in situ

Brevetoxin-3 at nanomolar concentrations markedly enhanced spontaneous quantal transmitter release from neuromuscular junctions equilibrated in a Ca2+-free EGTA medium. After about 3h, the sustained increase in miniature endplate potential frequency led to an exhaustion of transmitter release. This increase still occurred after loading the nerve terminals with the Ca2+ chelator bis-(aminophenoxy)ethanetetra-acetate or after pretreatment with various pharmacological agents known to prevent Ca2+ release from intracellular pools, but was completely prevented by the Na+ channel blocker tetrodotoxin. Brevetoxin-3 also increased miniature endplate potential frequency from junctions treated with botulinum type-A toxin, but to a smaller extent than at normal junctions. At normal junctions, brevetoxin-3 exposure for 2h increased the three-dimensional projected area of living motor nerve terminals in situ by about 74% while at botulinum type-A poisoned junctions a similar toxin exposure caused only a 29% increase. Tetrodotoxin prevented such effects, indicating that they are related to both Na+ entry into the terminals and increased quantal transmitter release. Ultrastructural examination of nerve terminals from junctions exposed for 3h to brevetoxin-3 revealed profound depletions of clear and large dense core synaptic vesicles and an increase in coated vesicles and axolemma infoldings. These results indicate that brevetoxin-3 impairs the recycling of clear synaptic vesicles and are consistent with our immunofluorescent observations showing that synaptophysin epitopes can be revealed without nerve terminal permeabilization. In contrast, no such changes were detected in nerve terminals poisoned with botulinum type-A toxin which, after 3h exposure to brevetoxin-3, retained their synaptic vesicles and had a normal appearance.We conclude that tetrodotoxin-sensitive Na+ entry into motor nerve terminals induced by brevetoxin-3 triggers external Ca2+-independent asynchronous quantal transmitter release, blocks synaptic vesicle recycling and induces swelling of the terminals. We suggest that an excess of cytoplasmic Na+per se can activate the asynchronous neurotransmitter release process.

The effects of the muscle relaxant, CS-722, on synaptic activity of cultured neurones.

1. The pharmacological properties of the centrally acting muscle relaxant, CS-722, were studied in cultured hippocampal cells and dorsal root ganglion cells of the rat using the whole-cell variation of the patch clamp technique. 2. CS-722 inhibited the occurrence of spontaneous excitatory and inhibitory postsynaptic currents in hippocampal neurones at concentrations of 100-300 microM, but had no effect on postsynaptic currents evoked by the application of glycine, gamma-aminobutyric acid, glutamate or N-methyl-D-aspartate. 3. CS-722 reduced voltage-gated sodium currents, while shifting the sodium channel inactivation curve to more negative membrane potentials. This effect is similar to that reported for local anaesthetics. Voltage-gated potassium currents were decreased by CS-722 by approximately 20%, whereas voltage-activated calcium currents were inhibited by about 25%. 4. CS-722 inhibited evoked inhibitory postsynaptic currents. However, the spontaneous quantal release of inhibitory transmitter was not affected. 5. The inhibitory effect of CS-722 on spontaneous inhibitory postsynaptic currents and excitatory postsynaptic currents in hippocampal cultures probably results from an inhibition of both sodium and calcium currents. This inhibitory effect is likely to be amplified in polysynaptic neuronal circuits.

Effects of lanthanum at snake twitch and tonic muscle fibre endplates.

1. The effects of 1 mM lanthanum on miniature endplate current (MEPC) frequency, amplitude, and decay time course were studied in voltage-clamped twitch and tonic muscle fibres in the garter snake, Thamnophis. 2. Lanthanum produced a marked increase in MEPC frequency in both fibre types. The maximum frequency in lanthanum was greater at twitch endplates than at tonic endplates although the increase in frequency relative to control levels was as great in tonic fibres as in twitch fibres. 3. In twitch fibres continually exposure to lanthanum, the frequency of MEPCs reached a peak value and then declined progressively until, after approximately 6 h, no MEPCs were recorded. In contrast, at tonic endplates exposed to 1 mM lanthanum, MEPC frequency remained elevated above control levels for periods greater than 20 h. 4. Lanthanum decreased the mean amplitude of MEPCs, skewed the amplitude distribution and increased MEPC duration at both twitch and tonic fibre endplates. 5. Ultrastructural analysis showed that after a 15 min exposure to 1 mM lanthanum, approximately half of the boutons innervating a twitch fibre contained fewer synaptic vesicles than boutons at control endplates, whereas nerve terminals innervating tonic fibre endplates were similar in appearance to those in control preparations. At endplates on both fibres, the postsynaptic membrane was more electron dense than that of control preparations. 6. Following a 6 h exposure to lanthanum, all nerve terminals innervating twitch endplates contained only a few synaptic vesicles and numerous intracellular deposits of electron dense material. The nerve terminals innervating tonic endplates still contained many synaptic vesicles, but the number appeared to be less than that of tonic terminals in untreated preparations. 7. The results demonstrate that lanthanum stimulates spontaneous quantal transmitter release from nerve terminals innervating either twitch or tonic fibres. However, the terminals innervating twitch fibres become depleted of synaptic vesicles, whereas this does not occur as readily in nerve terminals innervating tonic fibres.

Sulfonylurea-sensitive K+ channels and their probable role for the membrane potential of mouse motor nerve endings.

We studied the effect of the KATP channel blockers tolbutamide and glibenclamide on presynaptic membrane currents in the mouse M. triangularis sterni preparation using the perineural recording technique. Both sulfonylureas blocked part of the fast K+ component within 2 min after application. The block was much more pronounced under glucose-free conditions and was completely reversible by washing. Addition of glucose to glucose-free bath solution also reduced the K+ component. A further effect of the sulfonylureas was observed under glucose-free conditions. With a delay of 5 to 10 min, the nodal Na+ component began to diminish and disappeared within 30 min. This was associated with a dramatic increase in spontaneous quantal transmitter release suggesting that the block of sulfonylurea-sensitive K+ channels causes depolarization of motor nerve terminals and fibres thus inactivating Na+ channels. Tetraethylammonium (TEA) which blocks ATP-dependent K+ channels in high concentrations caused, under glucose-free conditions, the same delayed effect as the sulfonylureas. This delayed effect was fully reversible by washing with glucose-containing, but not with glucose-free solution. Our findings strongly suggest that KATP channels exist in mammalian motor nerve endings and that under hypoglycemic conditions these channels open and become essential for the maintenance of the membrane potential.

Regulation of single quantal efficacy at the snake neuromuscular junction.

1. Postsynaptic responses to spontaneous quantal transmitter release have been compared among neuromuscular junctions in a thin snake muscle. For each junction the type, diameter, and input conductance, G(in) of the postsynaptic muscle fibre were determined. Particularly among fibres of a given type, G(in) was directly correlated with fibre diameter. 2. Miniature endplate potentials (MEPPs) were recorded intracellularly near endplates visualized with Nomarski optics. Mean MEPP amplitude decreased with increasing G(in) among fibres in one muscle. However, the dependence of mean amplitude upon G(in) was not ohmic, as would be expected if the underlying single quantal currents (miniature endplate currents, MEPCs) were of similar amplitude at all junctions. Instead, the relation between MEPPs and G(in) suggested that mean MEPC amplitudes, calculated as mean MEPP amplitude x G(in), increased with increasing G(in). 3. MEPCs were recorded directly using the two-microelectrode voltage clamp technique. Mean MEPC amplitudes depended systematically on G(in), again such that MEPCs were on average larger in fibres with higher G(in). 4. MEPCs were recorded extracellularly from small regions of endplates (underlying a few nerve terminal boutons). Amplitudes of MEPCs depended on G(in) or fibre diameter in the same manner as amplitudes of MEPCs recorded by intracellular voltage clamp. 5. When the anticholinesterase agent neostigmine was added to the bath, amplitude and duration of MEPPs, MEPCs, and extracellular MEPCs increased. However, the systematic dependence of mean MEPC amplitude on G(in) or fibre diameter remained. 6. Evoked subthreshold endplate potentials (EPPs) were recorded under conditions of low extracellular Ca2+. Endplate currents (EPP amplitudes x G(in)) were systematically larger in fibres with larger G(in), indicating regulation of evoked synaptic current in the muscle. The regulation was found to be due to a combination of increased quantal content and larger single quantal currents in larger (higher G(in)) fibres. 7. Synaptic size, assessed either by area of cholinesterase staining or number of terminal boutons, increased with increasing fibre diameter. Assuming that quantal content is proportional to synaptic size, this relation was sufficient to account for the observed increase in quantal content with increasing G(in) among fibres in the muscle, but was not alone sufficient to account for the observed regulation of evoked current. 8. It is concluded that the efficacy of individual transmitter quanta released at the snake neuromuscular junction is regulated such that large muscle fibres receive larger single quantal currents. Regulation of single quantal current contributes substantially to overall regulation of synaptic strength in the muscle.

Ultrastructure of botulinum type-A poisoned frog motor nerve terminals after enhanced quantal transmitter release caused by carbonyl cyanide m-chlorophenylhydrazone

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on spontaneous quantal transmitter release and nerve terminal ultrastructure were studied on isolated cutaneous pectoris nerve-muscle preparations from frogs that were completely paralysed by a single sublethal dose of Clostridium botulinum type A toxin (BoTx). CCCP enhanced miniature endplate potential frequency at poisoned junctions and caused a reduction in the density of clear synaptic vesicles and of large dense core vesicles in motor nerve terminals. However, the intensity of these effects was much less important than that previously reported at unpoisoned junctions. The moderate depletion of synaptic vesicles can be related to the low levels of transmitter release detected with CCCP at BoTx-poisoned terminals.

An electrophysiological study of the actions of angiotensin II at the sympathetic neuroeffector junction in the guinea‐pig vas deferens

1. The effects of angiotensin II on sympathetic neuroeffector transmission in the guinea-pig vas deferens have been investigated by the use of intracellular and focal extracellular recording techniques to measure indirectly, the release of adenosine 5'-triphosphate (ATP). 2. Angiotensin II (10-100 nM) did not alter the amplitude of the first excitatory junction potential (e.j.p.) in a train but increased the amplitude of subsequent e.j.ps. There was a corresponding increase in the probability of occurrence of extracellularly recorded evoked excitatory junction currents (e.j.cs). Spontaneous quantal transmitter release was unaffected by angiotensin II. 3. The enhancement of transmitter release produced by angiotensin II was prevented by the angiotensin receptor antagonist, saralasin. 4. The increase in transmitter release produced by angiotensin II was due to an increase in the probability of transmitter release from individual varicosities and not due to any detectable change in the configuration of the nerve terminal impulse or to the induction of repetitive firing. 5. There was no overall enhancement of e.j.ps or e.j.cs by angiotensin II in reserpinized tissues. Surprisingly, the predominant effect of angiotensin II in reserpinized vasa deferentia was to inhibit evoked transmitter release, an effect reversed by indomethacin (3 microM). 6. The results show that angiotensin II increases the release of sympathetic transmitter by activating prejunctional angiotensin II receptors. However, when the co-transmitter noradrenaline was depleted, angiotensin II now inhibited transmitter release indirectly, presumably by stimulating prostaglandin formation in the smooth muscle cells which then inhibited release transjunctionally.

K+ and Ca2+ channel blocking agents increase or decrease stimulus‐evoked but not spontaneous quantal transmitter release in sympathetic nerve terminals

Etude visant a mettre en evidence l'influence du tetraethylammonium et de la 4-aminopyridine (agents bloquant les canaux K + et Ca 2+ ) sur l'impulsion nerveuse induite et sur la liberation quantique d'ATP au niveau des nerfs sympathiques du canal deferent de souris. Utilisation de methodes d'enregistrement extracellulaire

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.

Adenosine effects upon the spontaneous quantal transmitter release at the frog neuromuscular junction in the presence of protein kinase C-blocking and -activating agents

This paper gives experimental evidence involving protein kinase C (PKC) in the inhibitory effects of adenosine (ADO) upon the spontaneous transmitter release at the frog neuromuscular junction. In the presence of two PKC inhibitors--polymyxin B (5 x 10(-6) mol/l) and H-7 (10(-5) mol/l), both adenosine (5 x 10(-5) mol/l) and its stable analogue 1-PIA (5 x 10(-8) mol/l), significantly increased the rate of the spontaneous release of acetylcholine quanta. Even when PKC was activated with OAG (5 x 10(-6) mol/l) or TPA (162 x 10(-9) mol/l) and quantal release was increased greatly, ADO still inhibited release. ADO deaminase increased the PKC-induced activation of the transmitter release significantly.

Transduction mechanism involving the presynaptic adenosine receptor at mouse motor nerve terminals

The inhibitory effect of 2-chloroadenosine on spontaneous quantal release of transmitter at the mouse neuromuscular junction was abolished after pretreating tissues either with pertussis toxin (PTX), or with H 7, a protein kinase inhibitor. H 7 alone caused a fall in miniature endplate potential (MEPP) frequency, but PTX did not. The results are consistent with the hypothesis that rates of neurotransmitter release are directly related to intraterminal cyclic AMP levels, and that these can be reduced by A 1 adenosine receptor agonists through the mediation of a G i protein.

Immunoglobulins from amyotrophic lateral sclerosis patients enhance spontaneous transmitter release from motor-nerve terminals.

Amyotrophic lateral sclerosis (ALS) is an incapacitating neuromuscular disease of unknown etiology. Although laboratory evidence is lacking, circumstantial evidence supports the importance of immune factors in the pathogenesis of ALS. In the present study immunoglobulins from 4 of 8 ALS patients induced a significant increase in spontaneous quantal transmitter release as monitored by miniature end-plate potential (MEPP) frequency in mouse phrenic nerve-diaphragm preparations at 23 degrees C, whereas immunoglobulins from normal individuals and from patients with other neuromuscular diseases had no effect. At 32 degrees C neither normal nor disease control immunoglobulins influenced MEPP frequency, but 8 of 11 ALS immunoglobulin samples produced a significant increase in spontaneous quantal transmitter release. The enhancing effect could be prevented by 10 mM Mg2+. No effects were noted on MEPP amplitude and muscle resting potential. These findings suggest that the presynaptic terminal of the motor neuron may be an early target and that immunological factors may play an important role in the disease process.

Effects of carbonyl cyanide m-chlorophenylhydrazone on quantal transmitter release and ultrastructure of frog motor nerve terminals

The quantal acetylcholine release and the ultrastructural effects of the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone have been examined at frog neuromuscular junctions. Carbonyl cyanide m-chlorophenylhydrazone (2 μM) caused a temperature-dependent block of evoked quantal transmitter release accompanied by an increase in the rate of spontaneous quantal release. The carbonyl cyanide m-chlorophenylhydrazone-induced increase in miniature endplate potential frequency was neither antagonized nor prevented by tetrodotoxin. It also occurred in a Ca 2+-free medium and after replacement of Ca 2+ by Sr 2+, indicating that it does not depend upon a Na + or Ca 2+ influx from the external medium but may act by releasing Ca 2+ from intraterminal stores. Spontaneous quantal transmitter release was exhausted irreversibly within 4 h of carbonyl cyanide m-chlorophenylhydrazone (2 μM) action, during which time an average of 4.7 × 10 5 acetylcholine quanta were released per junction. The morphologic analysis revealed a significant temperature and time-dependent reduction in the number of synaptic vesicles with swelling and dispersion of mitochondria within the motor nerve terminals. Changes in synaptic vesicle number appear to be directly related to the intensity of transmitter release. The good correlation observed between the number of quanta secreted and the number of vesicles lost by nerve terminals in the absence of vesicle recycling provides an estimate of the initial store of transmitter quanta.

Bay K 8644 increases release of acetylcholine at the murine neuromuscular junction

In this study we describe the stimulatory effects of submicrolomar of BAY K 8644 on neurally evoked and spontaneous release of transmitter at the vertebrate neuromuscular junction, a model cholinergic synapse. BAY K 8644 increases mean quantal content. This effectmis blocked by pretreatment with the dihydropyridine (DHP) antagonist, nimodipine, but nimodipine itself had no effect on quantal content. Furthermore, BAY K 8644 induces a marked increase in spontaneous quantal release of transmitter as measured by miniature endplate potentional frequency. These results are important in that htey indicate that DGP-sensitive Ca 2+ channels exist motor nerve terminals and when activated can participate in transmitter release; second, they imply that these elements do not normally participate in trnasmitter release; third, they indicate a functional effect of DHPs on neurons under somewhat more relevant physiological than those imposed by prolonged K +-induced edpolarization, a technique used to evoked transmitter release in subcellular systems anf fourth, they indicate an effect of BAY K 8644 on spontaneous release of transmiiter, an effect not reported in previous neuochemical ezperiments.