A study of excitatory neuromuscular transmission in the bovine trachea

1. The excitatory innervation of bovine tracheal smooth muscle has been studied with the sucrose-gap apparatus.2. Single 2 ms electrical stimuli applied to the whole tissue excited intrinsic nerves, and produced a small transient depolarization of the smooth muscle, the excitatory junction potential (e.j.p.). The e.j.p. caused a twitch-type contraction; twitches and e.j.p.s summated during repetitive stimulation but facilitation was not observed, and action potentials were never elicited.3. The effects of electrical stimulation could be abolished by atropine (5 x 10(-7) mol/l) and augmented by neostigmine (4 x 10(-6) mol/l), and were mimicked by exogenous acetylcholine (1.0 mug/ml).4. With the electron microscope, the density of innervation was found to be low (one axon per ninety smooth muscle cells). Axons were found in small groups in the clefts between bundles of cells, but no axons penetrated within the muscle bundles. Naked axon varicosities containing agranular vesicles were seen, but no axon approached within 200 nm of a smooth muscle cell.5. It is difficult to reconcile the sparsity of innervation with the dependence of the tissue on nerve excitation to initiate activity.     

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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This work was supported by the Deutsche Forschungsgemeinschaft.     

Ionic mechanism of the excitatory synaptic membrane of the crayfish neuromuscular junction.

1. The reversal potential for the excitatory neuromuscular junction of the crayfish (Cambarus clarkii) was measured using the voltage clamp method. The potential change was recorded with an intracellular microcapillary and the negative phase of the output of the feed-back amplifier was connected to the stainless-steel wire which was inserted longitudinally into the muscle fibre. 2. When the excitatory nerve was stimulated, a transient feed-back current flowed inwardly through the membrane. This current was called the excitatory junctional current (e.j.c.). 3. Reversal potentials were determined by extrapolating the e.j.c.s measured at different membrane potentials. They were about 10-20 mV positive with respect to the bath solution (11-5 +/- 1-2 mV, mean +/- S.E.). 4. The reversal potential for the iontophoretically applied glutamate was identical with that for the e.j.c. 5. In hypertonic solutions, the reversal potentials for e.j.c. and glutamate became more negative. 6. When the sodium concentration of the bath solution was decreased, the reversal potential became more negative. 7. When the chloride and potassium concentration were altered, little, if any, change was observed in the reversal potential. 8. It was concluded that the e.j.c. was carried mainly by sodium ions. Contribution of other ions, possibly calcium ions, was discussed.     

Facilitation of transmission at the crayfish neuromuscular synapse by a convulsant phenol

The effects of the convulsant drug 4-Cl phenol on synaptic transmission were studied in the opener muscle of the crayfish walking leg. 4-Cl phenol was found to increase the amplitude of the excitatory postsynaptic potential without affecting the resting potential or input resistance of the muscle fiber. The drug did not change the frequency of spontaneous miniature postsynaptic potentials in K+-depolarized fibers. The postsynaptic voltage response to bath-applied glutamate (the excitatory transmitter compound) was decreased while the Cl(-) -conductance increase related to the action of bath-applied gamma-aminobutyric acid (the inhibitory transmitter) was not affected. In the light of previous results obtained on crayfish axons it is concluded that convulsant phenols induce an increase in the evoked release of transmitter by increasing the duration of the presynaptic depolarization through a block of voltage-dependent potassium channels.     

Enhanced release of inhibitory and excitatory transmitter quanta in the crayfish neuromuscular junction by glycine and GABA

High concentrations of glycine (greater than or equal to 0.1 mol/l) applied to the bathing medium of voltage-clamped muscle fibres elicited high rates of spontaneous inhibitory miniature currents (sIPSCs) which were identified by means of the noise analysis technique. The rate of spontaneous excitatory miniature currents (sEPSCs) was not raised appreciably in presence of these high concentrations of glycine. Contrary to the effect of glycine, high concentrations of GABA (greater than or equal to 0.1 mol/l) desensitized inhibitory postsynaptic receptors and induced high rates of sEPSCs. When, in addition to GABA, glycine was also applied at high concentrations, the discharge of sEPSCs was suppressed. Moreover, glycine reduced significantly the current noise intensity elicited by activation of excitatory postsynaptic membrane channels with 1.10(-4) mol/l glutamate. The experiments suggest an inhibitory action of glycine on the excitatory receptor--channel complex in the postsynaptic membrane.     

The timing of calcium action during neuromuscular transmission

1. When a nerve-muscle preparation is paralysed by tetrodotoxin, brief depolarizing pulses applied to a motor nerve ending cause packets of acetylcholine to be released and evoke end-plate potentials (e.p.p.s), provided calcium ions are present in the extracellular fluid.2. By ionophoretic discharge from a 1 M-CaCl(2) pipette, it is possible to produce a sudden increase in the local calcium concentration at the myoneural junction, at varying times before or after the depolarizing pulse.3. A brief application of calcium facilitates transmitter release if it occurs immediately before the depolarizing pulse. If the calcium pulse is applied a little later, during the period of the synaptic delay, it is ineffective.4. It is concluded that the utilization of external calcium ions at the neuromuscular junction is restricted to a brief period which barely outlasts the depolarization of the nerve ending, and which precedes the transmitter release itself.5. The suppressing effect of magnesium on transmitter release was studied by a similar method, with ionophoretic discharges from a 1 M-MgCl(2)-filled pipette. The results, though not quite as clear as with calcium, indicate that Mg pulses also are only effective if they precede the depolarizing pulses.     

The effects of calcium and magnesium on statistical release parameters at the crayfish neuromuscular junction

1. Transmitter release at excitatory neuromuscular junctions of crayfish muscle was studied at low temperature by recording synaptic potentials with extracellular micro-electrodes.2. Increasing the Ca concentration in the bathing solution produced an increase in the average number of quanta released per nerve stimulus (m). Increasing the Mg concentration resulted in a decrease in m.3. Statistical analysis of fluctuations in the quantal release from trial to trial, assuming binomial statistics, indicated that both the changes in m were due to changes in the average quantal release probability (p).     

Effect of repetitive stimulation on the frog neuromuscular transmission

The elimination rate constant, half-life and turnover time of dopamine (DA) and norepinephrine (NE) were determined, after inhibiting their biosynthesis by -methyl-para-tyrosine (MT), in the hypothalamus, striatum and the remainder of the brain of rats exposed to different degrees of hypobaric hypoxia, corresponding to altitudes of 1,800, 3,800, 5,200 and 7,000 meters. The effects varied as a function of the degree of hypoxia and the brain region studied. The turnover time of NE in the hypothalamus remained unchanged, regardless of the altitude, while in the rest of the brain the rate constant of neurotransmitter elimination decreased inversely as a linear function of the degree of hypoxia. On the contrary, the changes of the DA turnover time in the striatum and the rest of the brain, were biphasic, being accelerated by moderate altitudes (1,800 m) and retarded by the two highest simulated altitudes studied as a function of hypoxia. The differential effects of hypoxia on NE and DA turnovers are attributed to different sensitivities of the respective enzyme systems.A preliminary report of this work was presented at the Fourth International Catecholamine Symposium, Pacific Grove, Sept. 1978     

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.     

The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction

Excitatory postsynaptic currents (EPSCs) were recorded extracellularly from large muscle fibers by means of patch clamp electrodes. Compared to usual extracellular recordings, better signal/noise ratio and temporal stability were achieved. In the range of extracellular calcium concentrations [Ca]₀ between 2.7 and 13.5 mmol/l (normal), the average amplitude of the EPSC increased more than proportional to [Ca]₀. The unit quantum current,C ₁, and the average release rate,m, were determined from EPSCs and also from spontaneous sEPSCs, using both Poisson and binomial statistics. The main effect of [Ca]₀ was onm: at different synaptic sitesm depended on the second to fourth power of [Ca]₀. In terms of binomial parameters, the release probabilityp is the [Ca]₀-dependent one. In addition, reduction of [Ca]₀ from 13.5 to 2.7 mmol/l decreased the unit quantumC ₁ consistently to 60%; simultaneously the rise and decay of EPSCs and sEPSCs were shortened by 10–20%. [Ca]₀ thus has strong presynaptic effects on the release probability, but in addition smaller ones on teh postsynaptic channel characteristics.This investigation was supported by the Deutsche Forschungsgemeinschaft