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     

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.     

The accumulative properties of facilitation at crayfish neuromuscular synapses

1. Synaptic facilitation was measured with intracellular recording at two classes of neuromuscular synapses in the opener muscle of the crayfish dactyl by placing a test stimulus at various intervals after either a single conditioning stimulus or a short conditioning train.2. The facilitative effect of one stimulus reaches approximately the same level with both the superficial central and superficial distal synapses. The facilitation decreases smoothly in two phases after the conditioning stimulus at superficial central synapses and in a more complex fashion at superficial distal synapses.3. The two synaptic types differ in the manner in which they add up the facilitative effects produced by each of the stimuli in a short train. With superficial distal synapses the facilitative effects of conditioning stimuli add linearly, while with superficial central synapses the facilitative effects accumulate exponentially.4. The linear addition of facilitation at superficial distal synapses is not altered when the quantal content is lowered by decreasing the external Ca concentration from 13.5 to 3 mM.5. The rate of decay of facilitation is the same following both one and three conditioning stimuli, even though the facilitation is nearly six times larger in the latter case.6. The results are discussed in terms of mechanisms for synaptic facilitation.     

Contribution of intrinsic properties and synaptic inputs to motoneuron discharge patterns: a simulation study

Motoneuron discharge patterns reflect the interaction of synaptic inputs with intrinsic conductances. Recent work has focused on the contribution of conductances mediating persistent inward currents (PICs), which amplify and prolong the effects of synaptic inputs on motoneuron discharge. Certain features of human motor unit discharge are thought to reflect a relatively stereotyped activation of PICs by excitatory synaptic inputs; these features include rate saturation and de-recruitment at a lower level of net excitation than that required for recruitment. However, PIC activation is also influenced by the pattern and spatial distribution of inhibitory inputs that are activated concurrently with excitatory inputs. To estimate the potential contributions of PIC activation and synaptic input patterns to motor unit discharge patterns, we examined the responses of a set of cable motoneuron models to different patterns of excitatory and inhibitory inputs. The models were first tuned to approximate the current- and voltage-clamp responses of low- and medium-threshold spinal motoneurons studied in decerebrate cats and then driven with different patterns of excitatory and inhibitory inputs. The responses of the models to excitatory inputs reproduced a number of features of human motor unit discharge. However, the pattern of rate modulation was strongly influenced by the temporal and spatial pattern of concurrent inhibitory inputs. Thus, even though PIC activation is likely to exert a strong influence on firing rate modulation, PIC activation in combination with different patterns of excitatory and inhibitory synaptic inputs can produce a wide variety of motor unit discharge patterns.     

Relaxation after a voltage step of inhibitory synaptic current elicited by nerve stimulation (crayfish neuromuscular junction)

The clamped membrane potential of small crayfish muscle fibers was shifted in rapid steps between potentials of about –60 and –120 mV, and the clamp currents measured after de-and hyperpolarizing steps were averaged. In addition, the inhibitory nerver fiber was stimulated either synchronously or asynchronously with the averaging. Synchronous stimulation yielded the usual IPSCs, and asynchronous stimulation a steady state inhibitory current which relaxed to a new level after a voltage step. Fast relaxations were observed in all fibers. Their time constants =15 to 20 ms at –60 mV (10°C) decreased on hyperpolarization and agreed with those of the decay of the IPSC at the respective potential. The relaxations could be described quantitatively by a model in which the synaptic current depends on membrane potential due to (1) the potential dependence of the life time of a synaptic channel, (2) to a constant channel permeability, and (3) to the potential difference from the equilibrium potentialE _Cl.In many fibers slow relaxations of the inhibitory synaptic current were observed also, with time constants to 150 ms at –60 mV (10°C), which decreased at more negative potentials. These slow relaxations correspond to a reported slow current noise component induced by GABA. Interpretations of this slow synaptic current component are discussed.This investigation was supported by 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.

     

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.     

Electrophysiological characteristics of crayfish inhibitory neuromuscular synapses at different rhythms of activity

Parameters of IPSPs and characteristics of inhibitory neuromuscular transmission in the dactylopodite-opener muscle of the crayfish pincer at various rhythms of activity were studied by intracellular recording. The presynaptic conduction nature of high-frequency (pessimal) blockade of the inhibitory system was demonstrated. Transformation of the rhythm of stimulation correlated with a decrease in conduction velocity along the inhibitory axon and (or) its branches. At high frequencies of stimulation the mean quantum composition of IPSPs of the potentiation plateau before the beginning of rhythm transformation was lower than at lower frequencies. No signs of exhaustion of mediator reserves were found in the course of high-frequency activity. It is concluded that the mechanism of secretion of inhibitory mediator is more reliable than the function of conduction of the presynaptic nervous impulse. The possible functional significance of the comparatively low reliability of function of crustacean nerve fibers is discussed.Translated from Fiziologicheskii Zhurnal SSSR imeni I. M. Sechenova, Vol. 63, No. 11, pp. 1535–1540, November, 1977.     

Analysis of postural motoneuron activity in crayfish abdomen. I. Coordination by premotoneuron connections.

The activity of the crayfish abdominal postural motoneurons and their associated neurons (the accessory neuron(s) and the MRO(1)) were examined with the aid of techniques for the analysis of simultaneously recorded spike trains. A means of reliably identifying the spikes of the individual motoneurons based on their relative axon conduction velocities is presented. The analyses show that: 1) the large, phasically active synergist motoneurons innervating muscles producing the same movement show a marked similarity in their average responses, which is independent of the input source; 2) the small, tonically active and the middle-sized, tonicphasic synergist motoneurons innervating the same muscle and similar synergist motoneurons innervating antagonistic muscles are coordinated entirely by premotoneuron connections; 3) the accessory neuron is coordinated in its activity with the phasically active flexor excitor motoneurons and the extensor inhibitor motoneuron and thereby functions as a flexor synergist; and 4) the simultaneous presentation of flexion-producing and extension-producing inputs to the postural system results in a reciprocal oscillation in flexor-extensor motoneuron output. The functional significance of these results with respect to the operation of the postural system are discussed.     

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.     

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.     

Analysis of postural motoneuron activity in crayfish abdomen. II. Coordination by excitatory and inhibitory connections between motoneurons.

The identified spike trains of the crayfish abdominal postural efferent neurons were recorded simultaneously from one or more segments. The efferent activity was analyzed using cross-correlation histograms, peristimulus time scatter diagrams, and specialized antidromic techniques. The analyses show that the larger, phasically active motoneurons are coordinated in their activity by cross connections made at the motoneuron level. The cross connections are both excitatory and inhibitor in nature and result in significant alterations in spike output. Further, the accessory neuron receives an inhibitory cross connection from a middle-sized extensor excitor motoneuron or motoneurons. In each case, the cross connections appear to be appropriate to the function of the postural system.