Presynaptic membrane potential and transmitter release at the crayfish neuromuscular junction

Release of transmitter was evoked at neuromuscular junctions of the crayfish opener muscle by passage of current through an intracellular electrode impaling a branch of the motor axon close to a muscle fiber. Membrane-potential changes in the presynaptic axon branch were monitored, together with postsynaptic potentials. Depolarization of impaled secondary axonal branches by more than 10 mV led to an increase in asynchronous transmitter release. The release was facilitated by prolonged (50-500 ms) depolarizations and it decayed rapidly when depolarization was terminated. Ca2+ was essential for facilitated release; however, no indication of a Ca spike was found at the recording site. Input-output curves for the synapse were obtained by applying depolarizing pulses of varying amplitude to the axon branch. Transmitter output was strongly influenced by both amplitude and duration of the applied depolarization. During normal synaptic transmission, propagated Na+-dependent action potentials were recorded in the secondary axonal branches but there was no evidence for a calcium-dependent component for these action potentials. Evoked release was dependent on Ca2+ and was steeply dependent on the amplitude of the action potential, which could be made variable in size by application of tetrodotoxin (TTX). Prolonged depolarization of axonal branches resulted in enhancement of transmitter release evoked by an action potential. The enhancement occurred in spite of a simultaneous reduction of the amplitude of the action potential. Morphological features of the terminals were investigated after injection of lucifer yellow into the axon. An electrical model incorporating the morphological features suggests that membrane-potential changes set up in the main axon reach the nearest terminals with 30-40% attenuation, while events originating in the terminals would be severely attenuated in the main axon. Comparison of the crayfish synapse with other frequently studied synapses shows both similarities and differences, suggesting that it is not possible to apply findings made in one synapse to all others.     

Relative distribution of Ca2+ channels at the crayfish inhibitory neuromuscular junction

We investigated the Ca(2+) channel-synaptic vesicle topography at the inhibitor of the crayfish (Procambarus Clarkii) neuromuscular junction (NMJ) by analyzing the effect of different modes of Ca(2+) channel block on transmitter release. Initial identification of Ca(2+) channels revealed the presence of two classes, P and non-P-type with P-type channels governing approximately 70% of the total Ca(2+) influx. The remaining Ca(2+) influx was completely blocked by Cd(2+) but not by saturating concentrations of omega-conotoxins MVIIC and GVIA, or nifedipine and SNX-482. To examine the relative spatial distribution of Ca(2+) channels with respect to synaptic vesicles, we compared changes in inhibitory postsynaptic current amplitude and synaptic delay resulting from different spatial profiles of [Ca(2+)](i) around release sites. Specifically, addition of either [Mg(2+)](o), which decreases single-channel current, or omega-Aga IVA, which completely blocks P-type channels, prolonged synaptic delay by a similar amount when Ca(2+) influx block was <40%. Because non-P-type channels are able to compensate for blocked P-type channels, it suggests that these channels overlap considerably in their distribution. However, when Ca(2+) influx was blocked by approximately 50%, omega-Aga IVA increased delay significantly more than Mg(2+), suggesting that P-type channels are located closer than non-P-type channels to synaptic vesicles. This distribution of Ca(2+) channels was further supported by the observations that non-P-type channels are unable to trigger release in physiological saline and EGTA preferentially prolongs synaptic delay dominated by non-P-type channels when transmitter release is evoked with broad action potentials. We therefore conclude that although non-P-type channels do not directly trigger release under physiological conditions, their distribution partially overlaps with P-type channels.     

Intraterminal Ca(2+) and spontaneous transmitter release at the frog neuromuscular junction

We investigated the relationship between intraterminal Ca(2+) concentration ([Ca(2+)](i)) and the frequency of miniature end plate potentials (MEPPs) at the frog neuromuscular junction by use of ratiometric imaging of fura-2-loaded nerve terminals and intracellular recording of MEPPs. Elevation of extracellular [KCl] over the range of 2-20 mM resulted in increases in [Ca(2+)](i) and MEPP frequency. Loading terminals with the fast and slow Ca(2+)-buffers bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-acetoxymethyl (BAPTA-AM) and EGTA-AM resulted in equivalent reductions in the KCl-dependent increases in MEPP frequency. The [Ca(2+)](i) dependence of MEPP frequency determined by elevation of [Ca(2+)](i) due to application of 0.1-10 microM ionomycin was similar to that determined when [Ca(2+)](i) was raised by increasing extracellular KCl. Measurements in 10 mM extracellular KCl revealed that application of the phorbol ester phorbol 12 myristate 13-acetetate (PMA) caused an increase in MEPP frequency while the inactive analogue, 4 alpha-PMA, did not. PMA application also caused an increase in [Ca(2+)](i). The relationship between [Ca(2+)](i) and MEPP frequency in PMA was the same as was determined by the other methods of raising [Ca(2+)](i). Under all conditions tested, our data revealed a low [Ca(2+)](i) threshold for activation of transmitter release and are consistent with a K(d) for [Ca(2+)](i) on the order of 1 microM.     

alpha-latrocrustatoxin increases neurotransmitter release by activating a calcium influx pathway at crayfish neuromuscular junction

alpha-latrocrustatoxin (alpha-LCTX), a component of black widow spider venom (BWSV), produced a 50-fold increase in the frequency of spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their amplitude distribution. During toxin action, periods of high-frequency mEPSP discharge were punctuated by periods in which mEPSP frequency returned toward control levels. EPSPs were increased in amplitude during periods of enhanced mEPSP discharge. alpha-LCTX had no effect when applied in Ca(2+)-free saline, but subsequent addition of Ca(2+) caused an immediate enhancement of mEPSP frequency even when alpha-LCTX was previously washed out of the bath with Ca(2+)-free saline. Furthermore removal of Ca(2+) from the saline after alpha-LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus alpha-LCTX binding is insensitive to Ca(2+), but toxin action requires extracellular Ca(2+) ions. Preincubation with wheat germ agglutinin prevented the effect of alpha-LCTX but not its binding. These binding characteristics suggest that the toxin may bind to a crustacean homologue of latrophilin/calcium-independent receptor for latrotoxin, a G-protein-coupled receptor for alpha-latrotoxin (alpha-LTX) found in vertebrates. alpha-LCTX caused "prefacilitation" of EPSP amplitudes, i.e., the first EPSP in a train was enhanced in amplitude to a greater degree than subsequent EPSPs. A similar alteration in the pattern of facilitation was observed after application of the Ca(2+) ionophore, A23187, indicating that influx of Ca(2+) may mediate the action of alpha-LCTX. In nerve terminals filled with the Ca(2+) indicator, calcium green 1, alpha-LCTX caused increases in the fluorescence of the indicator that lasted for several minutes before returning to rest. Neither fluorescence changes nor toxin action on mEPSP frequency were affected by the Ca(2+) channel blockers omega-agatoxin IVA or Cd(2+), demonstrating that Ca(2+) influx does not occur via Ca(2+) channels normally coupled to transmitter release in this preparation. The actions of alpha-LCTX could be reduced dramatically by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid. We conclude that induction of extracellular Ca(2+) influx into nerve terminals is sufficient to explain the action of alpha-LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions.     

Presynaptic and postsynaptic effects of inhibitory drugs on the crayfish neuromuscular junction

Summary -aminobutyric acid (GABA) and related drugs reduce the size of the excitatory postsynaptic potential (e.p.s.p.) in the crayfish neuromuscular preparation. The effective inhibitory concentrations of these drugs were compared and the sites of action—postsynaptic and presynaptic—were determined.GABA, -amino--hydroxy-butyric acid, guanidino acetic acid, -alanine and others were found to increase the membrane conductance of the muscle fibers. These drugs also shift the membrane potential in the same direction as the inhibitory postsynaptic potential. It is concluded that this group of drugs imitates the postsynaptic action of the inhibitory transmitter substance.-guanidino-propionic acid (GP), -guanidino-butyric acid and others did not affect the membrane conductance of the muscle fibers. These drugs therefore do not inhibit through the conductance type of postsynaptic inhibition.Both groups of drugs mentioned above (GABA and GP) did reduce the amount of transmitter released from the excitatory nerve terminal per stimulus, they thus have a presynaptic inhibitory effect similar to neural inhibition. This was shown by analysis of the quantum content of the excitatory postsynaptic potential: The inhibitory drugs reduced the number of quanta liberated per stimulus, the size of the quantum remaining constant.Although GP has no direct effects on the membrane resistance of the muscle fiber, it reduces the inhibitory postsynaptic potential and diminishes the action of applied GABA on the membrane conductance. GABA and GP seem to compete for the inhibitory receptor site on the muscle membrane.

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Zusammenfassung -amino-Buttersäure (GABA) und verwandte Substanzen verkleinern das postsynaptische erregende Potential (e.p.s.p.) des Nerv-Muskel-Präparates des Krebses. Die wirksamen hemmenden Konzentrationen dieser Drogen wurden verglichen, und es wurde versucht, Art und Ort der Hemmungswirkungen — postsynaptisch oder präsynaptisch — festzustellen.GABA, -amino--hydroxy-Buttersäure, guanidino-Essigsäure, -Alanin und andere erhöhten die Leitfähigkeit der Muskelzellmembran. Bei Gabe dieser Drogen verschiebt sich auch das Membranpotential in derselben Richtung wie das hemmende postsynaptische Potential. Folglich hat diese Gruppe von Drogen dieselbe Wirkung auf die Muskel-Zell-Membran wie der hemmende neurale Überträgerstoff.-guanidino-Propionsäure (GP), -guanidino-Buttersäure und andere Guanidinosäuren haben keinen Einfluß auf die Membranleitfähigkeit der Muskelzellen. Der hemmende Effekt dieser Drogen ist also nicht vom Typ der postsynaptischen Leitfähigkeitshemmung.Beide oben erwähnten Gruppen von Substanzen (GABA und GP) setzen die Menge des pro Reiz von der erregenden Nervenendigung freigesetzten Überträgerstoffes herab, sie hemmen also präsynaptisch ebenso wie die neurale Hemmung. Diese präsynaptische Hemmung wurde bewiesen durch Analyse des Quantengehalts der erregenden postsynaptischen Potentiale: Die hemmenden Drogen verkleinerten die Zahl der pro Reiz ausgeschütteten Quanten, ohne die Größe des Quantums zu verändern.Obgleich GP keine direkten Effekte auf den Membranwiderstand der Muskelfasern hat, verkleinert es das postsynaptische hemmende Potential und setzt die Wirkung von GABA auf die Leitfähigkeit der Muskelzellmembran herab. GABA und GP verdrängen sich gegenseitig am Hemmungs-Receptor der Muskelmembran, so daß GP den Effekt von GABA kompetitiv hemmt.

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With 5 Figures in the Text

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This work was supported by the Deutsche Forschungsgemeinschaft.     

Probing the endogenous Ca2+ buffers at the presynaptic terminals of the crayfish neuromuscular junction

Ca2+ indicators of varying affinity and mobility were pressure injected into the presynaptic axon of the inhibitor of the crayfish neuromuscular junction (NMJ). Fluorescence transients recorded at a 2-kHz resolution were used to probe physiological parameters governing the decay of fluorescence transients within 100 ms after an action potential (early decay). Blocking Ca2+ extrusion or Ca2+ sequestration processes did not significantly alter early decay, arguing against a role for either mechanism. Fluorescence transients recorded with low mobility or fixed indicators exhibited early decay similar to that recorded with indicators of comparable affinity but high mobility, suggesting that early decay was not due to the rate of Ca2+-indicator diffusion. The extent of early decay correlated closely with the affinity, but not mobility, of the Ca2+ sensitive dyes tested. These results implicate intrinsic buffers with slow Ca2+ binding kinetics as the most likely determinants of early decay. However, computer simulations showed that intrinsic buffers with a slow binding rate are unlikely to be the only ones present in the system because the slow kinetics would be unable to buffer incoming Ca2+ during an action potential and would result in momentary indicator saturation. In fact, experimental data show that the peak amplitude of an action potential activated Ca+ transient is about 20% of the maximal fluorescence intensity activated by prolonged Ca2+ influx. We conclude that endogenous buffering at the crayfish NMJ includes both fast and slow components, the former being fast enough to compete with fast Ca2+ indicators, and the latter dictating the early decay.     

Effects of increasing Ca2+ channel-vesicle separation on facilitation at the crayfish inhibitory neuromuscular junction

We investigated the mechanism of facilitation at the crayfish inhibitory neuromuscular junction before and after blocking P-type Ca(2+) channels. P-type channels have been shown to be closer to releasable synaptic vesicles than non-P-type channels at this synapse. Prior to the block of P-type channels, facilitation evoked by a train of 10 action potentials at 100 Hz was increased by application of 40 mM [Mg(2+)](o), but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM omega-Aga IVA, which reduced total Ca(2+) influx and release to levels comparable to that recorded in 40 mM [Mg(2+)](o), did not change the magnitude of facilitation. We explored whether this observation could be attributed to the buffer saturation model of facilitation, since increasing the Ca(2+) channel-vesicle separation could potentially enhance the role of endogenous buffers. The characteristics of facilitation in synapses treated with omega-Aga IVA were probed with broad action potentials in the presence of K(+) channel blockers. After Ca(2+) channel-vesicle separation was increased by omega-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg(2+)](o). EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in omega-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca(2+) influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca(2+) channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after omega-Aga IVA poisoning. Finally, we sought correlation between residual Ca(2+) and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca(2+) indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca(2+)) to that evoked during action potential (local Ca(2+)). This ratio provides an estimate of relative changes between residual Ca(2+) and local Ca(2+) important for release. There is a significant increase in the ratio when Ca(2+) influx is reduced by 40 mM [Mg(2+)](o). The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca(2+) channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca(2+) and lends support to the residual Ca(2+) hypothesis of facilitation.     

Presynaptic inhibition of the excitatory nerve terminal in the neuromuscular junction of the crayfish

Summary If a capillary microelectrode is positioned near a nerve terminal, on stimulation of the excitatory nerve an excitatory nerve terminal potential (e.n.t.p.) is recorded. The e.n.t.p. can be triphasic (positive-negative-positive), diphasic (positive-negative) or monophasic positive.Monophasic positive e.n.t.p.s and diphasic e.n.t.p.s with a relatively large positive phase seem to be recorded from a position next to the terminal. This and other findings are interpreted to indicate a block of conduction of the action potential in the nerve fiber proximal to the terminal.During presynaptic inhibition some diphasic e.n.t.p.s are increased (by up to 14%), others are decreased (by up to 55%). Monophasic e.n.t.p.s always are decreased by inhibition (by up to more than 80%). The e.n.t.p.s that decrease on inhibition are presumably recorded from nerve portions nearer to the terminal than those giving rise to increased e.n.t.p.s.It is derived from the cable equations that, if the membrane resistance decreases in a nerve fiber, an extracellularly recorded electrotonic potential increases near the point of current injection, and decreases at more distant portions of the fiber. It is concluded that the results of inhibition on the e.n.t.p. indicate a decrease of membrane resistance in the nerve terminal during presynaptic inhibition.The possibility is discussed that the same inhibitory transmitter substance mediates pre- and postsynaptic inhibition. The mechanism of both kinds of inhibition may be a decrease of the membrane resistance, resulting in a reduced excitatory potential change.

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Zusammenfassung Am Öffnermuskel der Krebsschere wurde der Mechanismus der präsynaptischen Hemmung untersucht. Befindet sich die Spitze einer Mikroelektrode nahe einer Nervenendigung auf einer Muskelfaser, so wird nach Reiz des erregenden Nerven von der erregenden Nervenendigung eine Potentialschwankung, das e.n.t.p., abgeleitet. Die Form dieses e.n.t.p. kann triphasisch (positiv-negativ-positiv) diphasisch (positiv-negativ) oder monophasisch positiv sein.Monophasisch positive e.n.t.p. und diphasische e.n.t.p. mit relativ großer positiver Phase werden von der äußersten Nervenendigung abgeleitet. Dieser und weitere Befunde legen die Interpretation nahe, daß die aktive Fortleitung des Nervenaktionspotentials etwas proximal der Endigung aufhört, und nur passive, elektronische Potentialänderungen die Nervenendigung erreichen.Wird die neuromuskuläre Übertragung präsynaptisch gehemmt, so sind an einigen Endigungen die diphasischen e.n.t.p. vergrößert (um bis zu 14%), an anderen sind sie verkleinert (um bis zu 55%). Monophasische e.n.t.p. werden bei Hemmung immer kleiner (bis zu mehr als 80% Abnahme). Die sich bei der Hemmung verkleinernden e.n.t.p. werden an der äußersten Nervenendigung gemessen, während die sich bei der Hemmung vergrößernden e.n.t.p. von etwas mehr proximal abgeleitet werden.Aus den Kabelgleichungen läßt sich ableiten, daß bei einer Erniedrigung des Membranwiderstandes einer Nervenfaser das extracellulär registrierte elektrotonische Potential nahe der Stromquelle vergrößert wird, während es in entfernteren Faseranteilen stark abnimmt. Es kann deshalb aus den Effekten der Hemmung auf das e.n.t.p. auf eine Verringerung des Membranwiderstandes in der Nervenendigung während der präsynaptischen Hemmung geschlossen werden.In der Diskussion wird die Hypothese besprochen, daß die hemmende Nervenendigung einen Überträgerstoff ausschüttet, der sowohl die präsynaptische Hemmung der erregenden Nervenendigung wie auch die postsynaptische Hemmung der Muskelfaser bewirkt. Der Mechanismus beider Arten der Hemmung könnte der gleiche sein: der hemmende Überträgerstoff führt zu einer Abnahme des Membranwiderstandes, wodurch erregende Potentialänderungen verkleinert werden.

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With 6 Figures in the Text

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This work was supported by the Deutsche Forschungsgemeinschaft.     

Modulation of available vesicles and release kinetics at the inhibitor of the crayfish neuromuscular junction

We have investigated the effect of serotonin (5-HT) and okadaic acid (OA) on presynaptic processes at the crayfish inhibitory neuromuscular junction. Two different physiological parameters of transmitter release were examined: release kinetics and the size of the readily releasable pool of vesicles (RRP). Using a paired pulse stimulus and high frequency trains, we established that a single broad action potential, recorded in 20 mM tetraethylammonium and 1 mM 4-amino-pyridine, released the RRP in its entirety. Thus, by measuring the amplitude of inhibitory postsynaptic potential (IPSC) we were able to directly assess the effects of 5-HT and OA on the RRP. Serotonin at 200 nM and OA at 2.5 μM each significantly increased IPSC above control levels and the effects of these two modulators were comparable. Both modulators also induced a leftward shift in the rising phase of IPSC, i.e. an apparent acceleration in release kinetics. The shift caused by OA was significantly more pronounced than that induced by 5-HT. This apparent acceleration in release was not associated with a corresponding change in the presynaptic Ca²⁺ transient measured at a 2 kHz resolution, suggesting that modulation was not due to an acceleration in Ca²⁺ channel kinetics. In view of the comparable increase in the size of the RRP by the modulators, the differential modulation of release kinetics suggests that these two parameters may be modulated by separate biochemical processes.     

Role of NSF in neurotransmitter release: a peptide microinjection study at the crayfish neuromuscular junction

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 +/- 12% (mean +/- SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.     

The mechanism of presynaptic inhibition at the crayfish neuromuscular junction

Summary After stimulation of the excitatory nerve fiber potential changes (e.n.t.p.s) were recorded extracellularly from the excitatory nerve terminals on crayfish muscle. The influence of neural presynaptic inhibition on these e.n.t.p.s was analysed.Diphasic e.n.t.p.s recorded at some distance from the nerve terminal were increased by inhibition. Diphasic e.n.t.p.s recorded nearer to the terminal could be abolished by inhibition, their negative phase was more affected than the positive one and sometimes they were made monophasic positive. These findings were interpreted to represent a block of conduction or a central shift of a preexisting block of conduction by inhibition of the excitatory nerve terminal.Monophasic positive e.n.t.p.s recorded at the ultimate terminal were much reduced by inhibition. The release of the excitatory transmitter substance is not decreased proportional to the reduction of the positive e.n.t.p.s by presynaptic inhibition.All effects of presynaptic inhibition on shape and amplitude of the e.n.t.p. can be explained by an increased membrane conductance for chloride and/or potassium ions in the terminal.

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Zusammenfassung Am Krebsmuskel wurden mit Hilfe von extracellulären Mikroelektroden nach Reiz der erregenden Nervenfaser von ihren Endigungen Potentialänderungen (e.n.t.p.) abgeleitet. Die Effekte der neuralen präsynaptischen Hemmung auf diese e.n.t.p. wurden analysiert.Diphasische e.n.t.p., die in einiger Entfernung von der Endigung abgeleitet wurden, wurden durch die Hemmung vergrößert. Dagegen konnten diphasische e.n.t.p., die näher der Endigung gemessen wurden, durch die Hemmung sehr stark reduziert werden; ihre negative Phase wurde mehr betroffen als die positive, und diphasische e.n.t.p. konnten so zu monophasisch-positiven werden. Diese Befunde wurden als Anzeichen einer Blockade der Erregungsfortleitung, oder einer zentralen Verschiebung einer schon bestehenden solchen Blockade, durch die Hemmung interpretiert.An der äußersten Nervenendigung wurden monophasisch positive e.n.t.p. registriert. Diese wurden durch die Hemmung sehr stark verkleinert. Die Freisetzung des erregenden Überträgerstoffes durch die Nervenendigung wurde durch die Hemmung nicht proportional zur Amplitude der positiven e.n.t.p. herabgesetzt.Alle durch präsynaptische Hemmung bewirkten Veränderungen der Form und Amplitude der e.n.t.p. können durch eine Erhöhung der Membranleitfähigkeit der Nervenendigung für Chlorid- und/oder Kaliumionen erklärt werden.

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With 8 Figures in the Text

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This investigation was supported by grants from the Deutsche Forschungsgemeinschaft.     

Stochastic properties of spontaneous transmitter release at the crayfish neuromuscular junction

1. Miniature excitatory junctional potentials (min.e.j.p.s) were recorded with an intracellular electrode from the adductor muscle of the dactyl of the first or second walking leg of the crayfish, Orconectes virilis.

2. The intervals between the min.e.j.p.s were compared to the exponential prediction by five goodness of fit tests. The results indicate that the intervals are not exponentially distributed.

3. The autocorrelogram of intervals shows that the intervals are unlikely to be independent.

4. A stochastic analysis that includes the power spectrum of intervals, the variance—time curve, and the ln-survivor curve suggest that there is a clustering of min.e.j.p.s. The results are similar to those on the frog neuromuscular junction.

5. An autocorrelogram of the min.e.j.p. amplitudes suggests that sizes are not independently distributed.

6. These results, which are similar to those previously reported from the frog neuromuscular junction, support the use of the branching Poisson process as a theoretical model for the stochastic properties of spontaneous quantal release of transmitter.

     

The binomial nature of transmitter release at the crayfish neuromuscular junction

1. Transmitter release at excitatory junctions on the opener muscle of the crayfish dactyl was studied by recording junctional potentials with extracellular micro-electrodes.2. At low temperatures, evoked release was dispersed sufficiently in time for potentials produced by individual quanta to be counted, and the mean (m) and variance (sigma(2)) of the quantum content distribution for a series of trials measured directly. These values were used to calculate the average probability of quantal release (p), assuming a binomial distribution.3. For all values of m and p, the observed release pattern (number of 0, 1, 2, 3,... quantal releases during a series of trials) was approximated closely by the corresponding binomial distribution. However, Poisson predictions differed significantly from the observed quantal distribution for values of p > 0.2.