Single glutamate-gated synaptic channels at the crayfish neuromuscular junction

Single, glutamate activated ionic channel currents were recorded from crayfish muscle in the cell attached mode. Different concentrations of glutamate were present in the patch clamp pipette. Bursts of openings were observed with a concentration dependent number of short gaps per burst. Also the mean burst length was concentration dependent and varied between 0.3 ms (100 microM) and 1.3 ms (20 mM). Even with the highest concentrations of glutamate the channel activations were well separated and the beginning and the end of a burst could be defined. The distributions of open times and of burst lengths could be fitted well with a single exponential component for all studied concentrations of glutamate. The distributions of closed times were composed of two or three exponential components (with possibly more than one channel contributing). The mean burst length was compared with the time constants of decay of synaptic currents (0.8-3.0 ms at 19 degrees C) which were measured either with the same pipette as the single channel currents or with a macro patch technique. An estimation of the glutamate concentration at the receptors during synaptic transmission gave values in the millimolar range. The most simple model of glutamate-receptor interaction contains two binding sites for glutamate but no singly liganded open states. Rate constants were estimated for this model.     

Single glutamate-gated synaptic channels at the crayfish neuromuscular junction

Single glutamate activated ionic channels were recorded with the patch clamp technique from untreated crayfish muscle fibres with M seals, and after treatment with collagenase, with G seals. In regions with single channel activity spontaneous synaptic currents could also be recorded, and the channels were threrefore identified as synaptic. The single channel current amplitude was –7 to –8 pA at the resting potential of –70 mV, representing a conductance of 100 pS. The amplitudes decreased by a factor of two when the temperature was lowered by 10°C. Openings occurred in bursts, and the mean burst length varied between 0.3 ms (50 M glutamate in the pipette) and 0.8 ms (1 mM glutamate in the pipette). After treatment with collagenase, G seals could be formed. The conductance of the channel and the mean burst length was not affected by the enzyme, but after treatment active spots could be found easier and they were distributed more uniformly along the fibre. After treatment the concentrations of glutamate necessary to elicit channel openings were higher (100 M compared to 20–50 M) and simultaneous openings of two or more channels were observed very rarely. Synaptic currents could not be recorded from preparations cleaned by collagenase (2 mg/ml) for longer than 60 min.Supported by the Deutsche Forschungsgemeinschaft     

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.     

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.     

Phosphatidylinositol system's role in serotonin-induced facilitation at the crayfish neuromuscular junction

1. In a crustacean neuromuscular preparation, the walking leg opener muscle of the freshwater crayfish Procambarus clarkii, application of serotonin (1 microM) produces presynaptic depolarization and long-lasting facilitation of excitatory postsynaptic potentials (EPSPs). The frequency of spontaneously released transmitter quanta also increases. Facilitation of evoked EPSPs declines after serotonin application in two phases. 2. Serotonin-induced facilitation was examined using simultaneous pre- and postsynaptic intracellular microelectrode recording. A presynaptic microelectrode recorded action potentials and membrane potential of a presynaptic axonal branch, and one or more postsynaptic microelectrodes recorded EPSPs in muscle fibers innervated by the excitatory motor axon. Components of the phosphatidylinositol second messenger system and pharmacologic agents affecting this system were injected through the presynaptic electrode, and changes in synaptic transmission were measured. 3. Presynaptic injection of inositol 1,4,5-triphosphate (IP3) causes presynaptic depolarization, increases the frequency of spontaneously released transmitter quanta, and promotes a relatively short-lasting facilitation of evoked EPSPs. These actions are consistent with elevation of intracellular Ca2+ and resemble the early phase of serotonin-induced facilitation. 4. Application of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), that activates protein kinase C (C-kinase), produces a long-lasting, low-level facilitation of evoked EPSPs. Application of another phorbol ester, phorbol-12-monoacetate (PTMA), which does not activate C-kinase has no effect. 5. Presynaptic injection of RA 233, a phospholipase C (PLP-C) inhibitor, blocks all aspects of serotonin-induced facilitation. This compound was found to have no general deleterious effects on synaptic transmission and does not block other forms of synaptic facilitation in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in statistical parameters during facilitation at the crayfish neuromuscular junction

1. Transmitter release at excitatory neuromuscular junctions of the crayfish was studied at different frequencies of stimulation ranging from 1/sec to 20/sec.2. Over this frequency range the average number of quanta released per stimulus (m) increased with frequency by a factor of 6-7.3. Analysis of the fluctuations in quantal release using binomial statistics indicated that the increase in m was associated with increase in the average quantal release probability (p) at stimulation frequencies between 5/sec and 20/sec. Between 1/sec and 5/sec there was an apparent increase in the number of quanta available for release (n).     

Modulation of neurotransmission by reciprocal synapse-glial interactions at the neuromuscular junction

Perisynaptic Schwann cells are glial cells that are closely associated with pre- and postsynaptic elements of the neuromuscular junction. Recent evidence shows that these cells detect and modulate neurotransmission in an activity-dependent fashion. Through G-protein signalling and Ca(2+) released from internal stores they can decrease or increase neurotransmitter release, respectively. Thus, they help to establish the level of neurotransmission associated with activity dependent short-term synaptic plasticity. We discuss evidence implicating perisynaptic Schwann cells as being active partners in neurotransmission at the neuromuscular junction, with emphasis on the modulation of short-term plasticity and potential implications for long-term changes.     

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.     

Long-term facilitation alters transmitter releasing properties at the crayfish neuromuscular junction

Synaptic transmission at the neuromuscular junction of the excitatory axon supplying the crayfish opener muscle was examined before and after induction of long-term facilitation (LTF) by a 10-min period of stimulation at 20 Hz. Induction of LTF led to a period of enhanced synaptic transmission, which often persisted for many hours. The enhancement was entirely presynaptic in origin, since quantal unit size and time course were not altered, and quantal content of transmission (m) was increased. LTF was not associated with any persistent changes in action potential or presynaptic membrane potential recorded in the terminal region of the excitatory axon. The small muscle fibers of the walking-leg opener muscle were almost isopotential, and all quantal events could be recorded with an intracellular microelectrode. In addition, at low frequencies of stimulation, m was small. Thus it was possible to apply a binomial model of transmitter release to events recorded from individual muscle fibers and to calculate values for n (number of responding units involved in transmission) and p (probability of transmission for the population of responding units) before and after LTF. In the majority of preparations analyzed (6/10), amplitude histograms of evoked synaptic potentials could be described by a binomial distribution with a small n and moderately high p. LTF produced a significant increase in n, while p was slightly reduced. The results can be explained by a model in which the binomial parameter n represents the number of active synapses and parameter p the mean probability of release at a synapse. Provided that a pool of initially inactive synapses exists, one can postulate that LTF involves recruitment of synapses to the active state.     

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.     

Anion interaction at the inhibitory post-synaptic membrane of the crayfish neuromuscular junction

1. (24)Na, (36)Cl and (35)S thiourea were infused I.V. in rabbits according to schedules designed to yield approximately level activity in plasma for periods up to 5 hr. Cerebrospinal fluid was sampled before ending the experiment by decapitation and the radioactivities in cerebrospinal fluid and in homogenized brain were compared in each case to a time weighted mean value for plasma.2. The results are considered in terms of a simplified model which specifically acknowledges the continuity of the extracellular and cerebrospinal fluids and thus the coupling between processes which occur at the interfaces bordering those fluids.3. From the rate constants for exchange across the blood-brain interface that were necessary for simulation of the observed behaviours, permeability coefficients for that interface were estimated for the materials studied and, from experiments of others, for (42)K.     

Effects of glycine on the crayfish neuromuscular junction

Glycine applied in the bathing medium at concentrations exceeding 0.1 mol/l elicited high rates of spontaneous inhibitory postsynaptic currents (sIPSCs) in crayfish neuromuscular junctions. This effect of glycine was reversible within seconds. In several experiments on application of 0.5 mol/l glycine the rate of sIPSCs immediately increased to about 10 kHz and thereafter declined exponentially with time constants of between 10 and 20 s. This resulted in a release of about 140,000-200,000 inhibitory quanta per trial. When the readily releasable pool of transmitter had been so depleted by glycine, it was necessary to superfuse the preparation with normal solution for 5-10 min in order to be able to again evoke a high rate of sIPSCs. A similar effect of glycine on spontaneous release was also observed in some preparations which had been previously bathed in zero Ca2+ solution for up to 45 min. Addition of 25 mmol/l Mg2+ to the bathing fluid did not block the glycine evoked release of transmitter. However, in sodium-free superfusions the increase in the rate of sIPSCs induced by glycine was reduced. In the presence of 0.5 mol/l glycine no excitatory miniature currents (sEPSCs) were observed, in fact, glycine depressed excitatory synaptic transmission. In addition to the increasing the rate of sIPSCs, high concentrations of glycine evoked 'giant' sIPSCs (gsIPSCs). They were about 10-15 times larger than the normal sIPSCs and occurred at rates lower than 3 Hz, irrespective of whether the bathing medium contained sodium or not. However, in sodium-free superfusions the time constants of the decay of gsIPSCs were prolonged by a factor 2-3. These results suggest that glycine elicited sIPSCs and gsIPSCs by different mechanisms. Possible mechanisms which might explain the effects of glycine on release of inhibitory transmitter are discussed.     

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.     

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

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

Potentiation and desensitization after glutamate induced postsynaptic currents at the crayfish neuromuscular junction

Glutamate was applied iontophoretically in short (10 to 20 ms) pulses through micropipettes of 10 to 30 M Omega resistance to synaptic spots on crayfish muscle fibres. The resulting glutamate induced postsynaptic currents (gEPSCs) were measured using a voltage clamp of the muscle fiber. Dose-response curves for the action of glutamate were constructed summing g-EPSCs elicited by two separate iontophoretic pipettes at the same synapse. The dose-response curves showed a log log slope of 2, the g-EPSC rising with the second power of the glutamate concentration. Following a small conditioning g-EPSC with delay of up to 1 s, a second g-EPSC was potentiated. For short delays maximum potentiation was 3-fold. For large g-EPSCs potentiation gave way to desensitization: following a large conditioning gEPSC a second one was reduced for delays of up to several seconds. Possible mechanisms of potentiation and desensitization are discussed.