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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.

1. Commonly used anthelmintic agents act on the muscle cells of parasitic nematodes to cause paralysis of the parasite and its expulsion from the host. 2. The motonervous system of nematodes contains neuropeptides, many of which are myoactive and elicit prolonged worm paralysis. Here we describe the actions of a novel peptide, KPNFIRFamide (Lys-Pro-Asn-Phe-Ileu-Arg-Phe-amide; PF4), which mediates relaxation of the somatic muscle of the parasitic nematode Ascaris suum. Its mechanism of action is compared to that of the inhibitory neuromuscular junction transmitter, gamma-aminobutyric acid (GABA), which gates a chloride channel on Ascaris muscle. 3. Both PF4 and GABA hyperpolarized the muscle cells (EC50 values 98 nM and 59 microM, respectively; n = 6) and this was accompanied by an increase in input conductance. 4. The increase in input conductance elicited by PF4 and a supramaximal concentration of GABA were additive (10 microM PF4, 7.78 +/- 1.88 microS; 10 mM GABA, 4.68 +/- 1.39 microS; 10 mM GABA and 10 microM PF4 12.05 +/- 2.6 microS, n = 6, P0.02 with respect to PF4 alone; P0.01 with respect to GABA alone). 5. The membrane potential response to 10 microM PF4 initially consisted of a fast hyperpolarization that occurred within 1 min of PF4 application. The reversal potential for this early response to PF4 (PF4-early) was determined at different extracellular chloride concentrations. Linear regression analysis of the natural logarithm of the extracellular chloride concentration against the reversal potential for PF4-early yielded a straight line with a slope of -29.6 +/- 2.4 (-34.4 to -24.9, 95% confidence limits; r2 = 0.82). This is close to the slope of -26.5 for a chloride-dependent event, as predicted by the Nernst equation. There was a significant correlation between the reversal potential for this event and the reversal potential for GABA (r = 0.94; P0.001; n = 12). 6. The late response to PF4 (PF4-late) appeared after 1 min and consisted of a slow reduction in the hyperpolarization to a plateau level, before the return of the membrane potential to the resting value. PF4-late is not likely to be a chloride-dependent event as during the hyperpolarization caused by a supramaximal concentration of GABA the muscle cells depolarized when a supramaximal concentration of PF4 was added to the perfusate. The membrane potential in the presence of 1 mM GABA was -61.8 +/- 4.8 mV and in the presence of 1 mM GABA with 10 microM PF4 was -47.5 +/- 1.5 mV (P0.02; n = 6). 7. The conductance increase elicited by 30 microM GABA was blocked by 10 microM ivermectin (before ivermectin 0.97 +/- 0.2 microS, after ivermectin 0.33 +/- 0.12 microS; n = 5; P0.05; Student's paired t test) but the conductance increase elicited by 1 microM PF4 was not (before ivermectin 0.96 +/- 0.14 microS, after ivermectin 1.07 +/- 0.19 microS; n = 0.34; Student's paired t test). 8. These data indicate that PF4 elicits a potent, inhibition of Ascaris muscle cells which is partially mediated by chloride and which is independent of the inhibitory GABA receptor.

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 Ca 2+ transient measured at a 2 kHz resolution, suggesting that modulation was not due to an acceleration in Ca 2+ 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.

We investigated the Ca2+channel-synaptic vesicle topography at the inhibitor of the crayfish ( Procambarus Clarkii) neuromuscular junction (NMJ) by analyzing the effect of different modes of Ca2+channel block on transmitter release. Initial identification of Ca2+channels revealed the presence of two classes, P and non-P-type with P-type channels governing ∼70% of the total Ca2+influx. The remaining Ca2+influx was completely blocked by Cd2+but not by saturating concentrations of ω-conotoxins MVIIC and GVIA, or nifedipine and SNX-482. To examine the relative spatial distribution of Ca2+channels with respect to synaptic vesicles, we compared changes in inhibitory postsynaptic current amplitude and synaptic delay resulting from different spatial profiles of [Ca2+]iaround release sites. Specifically, addition of either [Mg2+]o, which decreases single-channel current, or ω-Aga IVA, which completely blocks P-type channels, prolonged synaptic delay by a similar amount when Ca2+influx block was 2+influx was blocked by ∼50%, ω-Aga IVA increased delay significantly more than Mg2+, suggesting that P-type channels are located closer than non-P-type channels to synaptic vesicles. This distribution of Ca2+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.

The effect of partially replacing extracellular chloride by formate on the inhibitory junction of the crayfish opener muscle is investigated. Inhibitory postsynaptic potential (IPSP) amplitude, recorded in muscle fibers and presynaptic axons, increases significantly in the formate saline whereas input resistance and resting membrane potential of muscle fibers are not affected. The increase in IPSP amplitude is mainly due to an increase in IPSP driving force while the GABA mediated conductance change underlying IPSP is not altered. The waveform of presynaptic action potential is slightly altered by formate substitution where an after-depolarizing potential is decreased. This change does not seem to affect the probability of transmitter release because the magnitude of synaptic facilitation is unchanged. In conclusion, formate substitution significantly increases IPSP amplitudes by increasing its driving force without affecting presynaptic release mechanisms.

SUMMARYPF1 (SDPNFLRFamide) is a FMRFamide-like peptide extracted from the free-living nematodePanagrellus redivivus. Here we show that this peptide causes a hyperpolarization of somatic muscle cells of the parasitic nematodeAscaris suumand a relaxation of the somatic muscle strip preparation. We have assessed whether or not the relaxation ofAscarisdorsal muscle strip by PF1 is due to (i) inhibition of the release of the excitatory neuromuscular junction transmitter acetylcholine (ACh), (ii) potentiation of the release of the inhibitory neuromuscular junction transmitter γ-aminobutyric acid (GABA) or (iii) a direct inhibitory action of the peptide on the muscle cells. Under the experimental conditions described here, tonic ACh release does not seem to be involved in determining the resting membrane potential or resting tone of theAscarisdorsal muscle strip and thus inhibition of tonic ACh release is unlikely to explain the relaxation elicited by the peptide. Furthermore, PF1 (100 nM–1 μM) inhibited the contraction of the muscle strip elicited by bath application of ACh, suggesting either a direct inhibitory action of the peptide on the muscle cells or a potentiation of GABA release. In electrophysiological experiments, the reversal potential for the PF1 hyperpolarization was not the same as that for GABA. Thus, PF1 hyperpolarizesAscarismuscle by a mechanism that does not involve stimulation of GABA release from inhibitory pre-synaptic terminals.

GABA-evoked changes in membrane conductance of lobster muscle fibres were measured using the ‘short’ cable equations. In 79 fibres, the mean resting conductance (ℷmL) was 12.9μmho (S.D. ± 5.6 μmho). In the presence of GABA (5 μM to 640 μM) the membrane conductance increased with little change in resting potential and with no visible decline in response during exposure. In contrast with the non-cumulative GABA dose/conductance curves, cumulatively established curves attained an apparent maximum at GABA concentrations exceeding 80 μM. The non-cumulative dose/conductance curve was better fitted by a two independent binding-site receptor model than by a two-site high facilitation model or a single-site model. Values for the apparent GABA/receptor dissociation constant estimated by ‘least squares’ analysis (on the basis of the independent model) were similar to those estimated by double reciprocal plot (ca. 30 μM).

1. The inhibitory neuromuscular junction of the abductor muscle of the large claw of the hermit crab (Eupagurus bernhardus) was investigated using electrophysiological intracellular techniques in order to elucidate further the relative contributions of the pre- and post-synaptic mechanisms of action of GABA and of neural inhibition.2. The electrical constants of the post-synaptic membrane, calculated using the equations for a ;short cable' model, were characteristic of a poorly developed electrical excitability; the specific membrane resistance was usually1000 Omega cm(2) and the specific membrane capacitance was40 muF/cm(2).3. Stimulation of the excitatory axon to the abductor muscle of the large claw at a frequency of 20 Hz evoked highly facilitating excitatory junction potentials (e.j.p.s); stimulation of the inhibitory axon (60-220 Hz) during the excitatory train elicited inhibition which was manifest as an attenuation of the e.j.p.s.4. The addition of gamma-aminobutyric acid (GABA) to the bathing solution produced a dose-dependent reduction of e.j.p. amplitude and membrane resistance. The inhibitory effect of concentrations (5 x 10(-5) and 1 x 10(-4)M) which caused a 40-75% e.j.p. attenuation could largely be accounted for by a post-synaptic action on membrane conductance.5. Experiments with picrotoxin suggest that presynaptic inhibitory mechanisms have an important role in neurally evoked inhibition.6. Picrotoxin (1-5 x 10(-5)M) effectively blocked neural inhibition and the actions of GABA in this preparation, whereas bicuculline proved to be considerably less potent and therefore less useful as a physiological tool for studying GABA-mediated inhibition in crustacea.

The effect of tetanus toxin on inhibitory synaptic transmission in crayfish muscle, which seemed to be mediated by GABA, was investigated by means of intracellular recording. Tetanus toxin caused a gradual block of inhibitory synaptic transmission. The GABA sensitivity of the postsynaptic membrane and the electrical activity of the presynaptic nerve terminal remained unaffected. It is concluded that the toxin can abolish the inhibitory synaptic transmission by reducing the amount of GABA released from inhibitory nerve terminal, but the toxin does not interfere with the conduction of impulses along the inhibitory nerve axons up to the nerve terminal.

Log-dose/conductance curves for γ-aminobutyric acid (GABA), γ-amino-β-hydroxybutyric acid (GABOB), δ-aminovaleric acid (DAV) and guanidoacetic acid (GuAc) were compared at the lobster inhibitory neuromuscular junction. GABOB and DAV curves paralleled the GABA curves and could be fitted by the same two independent-binding site receptor model (KGABOB = 275 μM; KDAV = 945 μM: apparent dissociation constants estimated by double-reciprocal plot). GuAc and GABA curves were non-parallel, and showed different apparent maxima. The normalized GuAc dose/conductance curve could be fitted by a modified form of the general two binding-site model with K1 =7.4 μM and K2 = 92 μM. It is suggested that GABA, GABOB and DAV were agonists of comparable ‘intrinsic activity’ but differing receptor affinity whereas GuAc was behaving as a partial agonist.

SINCE it was reported that bicuculline is a specific antagonist of γ-aminobutyric acid (GABA)1–3, much attention has been paid to the action of this alkaloid on cortical neurones4–6. Thus it seemed worthwhile to test the mode of action of bicuculline on the inhibitory neuromuscular junction of the crayfish, where the action of GABA has been well documented7,8.

1. The effect of picrotoxin on the neuromuscular junction of the crayfish (Cambarus clarkii) was investigated. The potential changes were recorded intracellularly and extracellularly with micro-electrodes. The membrane conductance of the muscle fibre was also measured. 2. Picrotoxin depressed the amplitudes of the inhibitory junctional potentials and the potential changes produced by iontophoretically applied γ-aminobutyric acid (GABA), but had no appreciable effect on the excitatory junctional potentials and the potential changes produced by L-glutamate. 3. The presynaptic action of GABA and the neural transmitter was depressed by picrotoxin. The presynaptic action of β-guanidinopropionic acid was also depressed by picrotoxin. 4. The increase in the membrane conductance produced by the addition of GABA in the bath fluid was depressed by picrotoxin. The dose—response relation showed that picrotoxin depressed the conductance increase produced by GABA in a non-competitive manner. The action of picrotoxin on the conductance increase produced by GABA was more effective in low Cl- solution. 5. The analysis of the dose—response curves showed that the action of picrotoxin was well expressed by the Michaelis—Menten equation, but the slope of the dose—response curve of GABA was steeper than this relation. It is proposed that the conductance of the junctional membrane was increased by the combination of two molecules of GABA with a receptor, and the attachment of one molecule of picrotoxin to a specific site depressed the conductance increase.

1. Investigations on the inhibitory neuromuscular junction of crayfish stretch receptor muscles were performed electrophysiologically and electron microscopically.2. Fast receptor muscles responded to direct stimulation with a transient depolarization which exhibited the property of a propagated action potential, but did not show any overshoot. On the other hand, slow muscles did not generate an action potential but showed excitatory junction potentials of 10-30mV in amplitude. EJPs of fast receptor muscles were summated by repetitive stimulation until all-or-nothing spikes were generated.3. When a nerve bundle was carefully isolated and one of the isolated filaments were stimulated, inhibitory junction potentials could be obtained from fast stretch receptor muscles by the microelectrode inserted near the receptor neuron. They had a reversal potential approximately at-60mV. Inhibitory junction potentials completely inhibited excitatory junction potentials and spikes when inhibitory nerves were stimulated at high frequency of 100c/s for several seconds.4. Application of GABA of 10-4-10-5M showed similar effects to stimulation of inhibitory nerves. It's effect was due to the increase of postsynaptic membrane conductance.5. Two different types of synapses were identified electron microscopically at fast stretch receptor muscles. The one contained spherical synaptic vesicles, the other ellipsoidal ones. The latter was observed within the limit of the vicinity of the neuron soma. From UCHIZONO'S (1965, 1966) previous studies and the present electrophysiological experiments, it was concluded that the latter was the terminal of inhibitory neuromuscular junctions.