Werner Rathmayer

The effect ofHabrobracon venom on excitatory neuromuscular transmission in insects

Summary1.The action of the venom of the braconid waspHabrobraconhebetor was studied in nerve-muscle preparations of its host the mealmoth larva (Ephestia kühniella) and of the locust (Locusta migratoria) by comparing physiological properties of normal and paralysed preparations.2.The venom neither had an effect on nervous conduction nor on membrane potential, current-voltage relationship, graded electrogenesis and contraction of the muscle fibres.3.Inhibitory transmission was not blocked. Excitatory junction potentials were either greatly diminished or absent during paralysis. They could not be restored to normal size by increasing the external Ca concentration.4.The postjunctional sensitivity to L-glutamic acid was not significantly altered by the venom.5.In paralysed locust preparations miniature excitatory junction potentials were still observed, but their frequency was reduced to as little as 1% of controls. Their amplitude distributions were similar to controls, except that the proportion of very large mejps was somewhat higher.6.In weakly paralysed preparations tetanic nerve stimulation caused facilitation and posttetanic potentiation of the reduced ejps. With more extensive paralysis, tetanic stimulation increased the frequency of the mejps and a few of the stimuli were followed by mejp-like ejps.7.Raising the osmolarity of the saline increased the mejp-frequenoy of paralysed preparations significantly less than in control preparations.8.The thiol oxidizing compound diamide caused a large increase of mejpfrequency in controls but completely blocked spontaneous release in paralysed preparations. These effects could be quickly reversed by a subsequent application of the disulfide reducing agent dithiothreitol.9.It is unlikely that the purely presynaptic effect ofHabrobracon venom is on the electrical properties of the excitatory nerve terminals. It is discussed whether the release mechanism or the supply of transmitter are affected. There may be a specific affinity of the venom to glutaminergic synapses.

Allatostatin modulates skeletal muscle performance in crustaceans through pre‐ and postsynaptic effects

Allatostatins, originally identified in insects as peptide inhibitors of juvenile hormone biosynthesis, are regarded as potent inhibitory regulators of intestinal muscles in insects and crustaceans. However, accumulating data indicate that allatostatins might also be involved in modulation of skeletal neuromuscular events. We show that most ganglia of two isopod crustaceans (Idotea baltica and I. emarginata) contain pairs of large, allatostatin‐immunoreactive motor neurons which supply several segmental muscles. Among them are the dorsal extensor muscles, of which some fibres receive immunoreactive, varicose innervation. We demonstrate, on identified muscle fibres, that allatostatin exerts a twofold inhibitory effect: it reduces contractions of single voltage‐clamped fibres, and it decreases the amplitude of evoked excitatory junctional currents recorded from individual release boutons. No change in excitation‐contraction threshold or in passive membrane parameters was observed. As the amplitude of miniature currents generated by spontaneously released single transmitter quanta was not changed, the inhibitory effect of the peptide on junctional currents must be of presynaptic origin. Supportive results were obtained on leg muscles of the crab Eriphia spinifrons, where allatostatin decreased evoked synaptic currents by reducing the mean number of transmitter quanta released by presynaptic depolarization without affecting the amplitudes of currents generated by single quanta. This effect of allatostatin was similar for two functionally different neurons, the slow and the fast closer excitor.

Shortening velocity and force/pCa relationship in skinned crab muscle fibres of different types

Single fibres of three different types, which had been characterized histochemically with regard to differences in myofibrillar adenosine triphosphatase (ATPase) activity and its pH stability, were microdissected from freeze dried preparations of the closer muscle in walking legs of the crab Eriphia spinifrons. Shortening velocities were determined in slack tests and under constant load conditions in maximally Ca2+-activated skinned muscle fibres. Force/pCa relationships were also measured for the different types of fibres. Compared with data on vertebrate muscles, all crab muscle fibres required large length changes to reach zero force and showed low Ca2+ sensitivity for isometric force generation. The length/time relationship obtained from slack tests had a biphasic course. Maximal velocity of filament sliding differed in the three types of fibres investigated. The filament sliding of type IV fibres was about 3 times faster than that of type I fibres. The values obtained for type II fibres ranged in between. These data are positively correlated with myofibrillar ATPase activity determined histochemically. Ca2+ sensitivity of force generation was lowest in the fast type IV fibres. It was high in the slow type I and the faster contracting type II fibres. Ca2+ sensitivity in crab muscle seems not to be correlated with speed of shortening.

Anemonia sulcata toxins modify activation and inactivation of Na+ currents in a crayfish neurone.

Abstract1. The effects of three toxins (ATX I, II, III) isolated from the sea anemoneAnemonia sulcata were studied in the soma membrane of a crustacean neurone under voltage-clamp conditions. 2. All three toxins affected the action potentials and the Na+ currents in a similar manner. The lowest concentrations tested (10 nM, 20 nM and 50 nM for AtX I, II and III, respectively) had pronounced selective effects on the Na+ current. No effect on K+ or Ca2+ currents was observed with concentrations up to 5 μM. 3. In the presence of ATX the Na+ inactivation was incomplete even with pulses of 700 ms length or strong depolarizing prepulses. 4. Besides the effects on the inactivation process ATX affected also the activation of the Na+ current. 5. In cells treated with ATX the negative resistance branch of the peak Na+ current voltage relation was shifted by −5 mV to −20 mV. 6. The time to peak was increased for small depolarizations (up to −30 mV) and the rate of rise (ΔI/Δt) was enlarged by ATX. A slow activating current component was also observed after depolarizing prepulses or if the Na+ current was outward. 7. The decay of the Na+ tail currents was considerably prolonged after the application of ATX if the membrane was repolarized to potentials more positive than about −60 mV. 8. Repetitive stimulation led to a shortening of the action potential in ATX II treated neurones. A simultaneous and parallel decrement of the peak and plateau current was observed with depolarizing voltage steps.

Calcium action potentials in larval muscle fibres of the mothEphestia kühniella Z. (Lepidoptera)

Summary1.The ionic requirements for the production of action potentials in the ventral longitudinal muscle fibres of the flour moth larvaEphestia kühniella Zeller (Lepidoptera) were investigated.2.The amplitude and maximal rate of rise of the action potential evoked by indirect stimulation declined when the extracellular Ca-concentration was reduced (Fig. 1).3.Action potentials elicited by intracellularly applied current pulses could be generated in the absence of extracellular Na and Mg.4.Neither TTX (1.5×10−5 g/ml; Fig. 2) nor procaine (15 mM) blocked the action potentials.5.The generation of action potentials could be prevented by omitting all extracellular Ca (Fig. 3).6.The action potential overshoot varied with [Ca++]o, having a slope of 24 to 28 mV for a tenfold change in [Ca++]o (Fig. 5).7.La (1 mM) irreversibly blocked the action potentials (Fig. 7).8.Both Ba and Sr could replace extracellular Ca in the generation of action potentials (Fig. 8).

Presynaptic inhibition of long duration at crab neuromuscular junctions

SummaryIn the closer muscle of various decapod crustaceans interjection of a single train of stimuli applied to the inhibitory axon causes long lasting depression of the contractions elicited by subsequent stimulations of the “slow” excitatory axon. This phenomenon of postinhibitory depression (PID) is due to long lasting presynaptic action of the inhibitory transmitter. Its duration differs from that of the postsynaptic inhibitory effects by at least one order of magnitude: the conductance increase of the postsynaptic membrane may last at most a few sec after cessation of an I-train, the presynaptic reduction of excitatory transmitter output, however, up to several min.

A single allatostatin-immunoreactive neuron innervates skeletal muscles of several segments in the locust

In the nervous system of embryos and adult Locusta migratoria, somata, neurites within the ganglia, and axons leaving the thoracic ganglia show allatostatin immunoreactivity. The immunoreactive efferent axons divide to follow different nerve branches and form varicose terminals on skeletal muscles. In the adult locust, one pair of motor neurons is particularly prominent among the allatostatin‐immunoreactive neurons. The somata are located symmetrically in a lateral position in the first abdominal neuromere of the fused metathoracic ganglion. Each neuron gives rise to five axon branches projecting into ipsilateral nerves. Three axons project posteriorly and exit through the dorsal nerves of the abdominal neuromeres A1, A2, and A3. One axon extends into the metathoracic neuromere and exits through metathoracic nerve 1 (N1). The fifth axon extends anteriorly through the connective into the mesothoracic ganglion, where it leaves through the mesothoracic N1. The targets of this neuron, among them the mesothoracic and metathoracic muscles M87, M88, M116 and the dorsal longitudinal muscles M81 and M112, are located in five different segments. In addition to supplying skeletal muscles, the neuron forms neurohaemal‐like structures in the sheath of nerve branches. The authors call this neuron the common lateral neuron (CLN). The innervation of several muscles by Diploptera allatostatin 7‐immunoreactive axon branches with a common cellular origin and the anatomy of one of the corresponding motor neurons in adults, the CLN, suggest that allatostatin acts as a modulator of neuromuscular parameters in insects by multisegmental direct innervation of skeletal muscles. J. Comp. Neurol. 413:507–519, 1999.

Localization of a FMRFamide-related peptide in efferent neurons and analysis of neuromuscular effects of DRNFLRFamide (DF2) in the crustacean Idotea emarginata.

In the ventral nerve cord of the isopod Idotea emarginata, FMRFamide‐immunoreactive efferent neurons are confined to pereion ganglion 5 where a single pair of these neurons was identified. Each neuron projects an axon into the ipsilateral ventral and dorsal lateral nerves, which run through the entire animal. The immunoreactive axons form numerous varicosities on the ventral flexor and dorsal extensor muscle fibres, and in the pericardial organs. To analyse the neuromuscular effects of a FMRFamide, we used the DRNFLRFamide (DF2). DF2 acted both pre‐ and postsynaptically. On the presynaptic side, DF2 increased transmitter release from neuromuscular endings. Postsynaptically, DF2 depolarized muscle fibres by approximately 10 mV. This effect was not observed in leg muscles of a crab. The depolarization required Ca2+, was blocked by substituting Ca2+ with Co2+, but not affected by nifedipine or amiloride. In Idotea, DF2 also potentiated evoked extensor muscle contractions. The amplitude of high K+ contractures was increased in a dose dependent manner with an EC50 value of 40 nm. In current‐clamped fibres, DF2 strongly potentiated contractions evoked by current pulses exceeding excitation‐contraction threshold. In voltage‐clamped fibres, the inward current through l‐type Ca2+ channels was increased by the peptide. The observed physiological effects together with the localization of FMRFamide‐immunoreactive efferent neurons suggest a role for this type of peptidergic modulation for the neuromuscular performance in Idotea. The pre‐ and postsynaptic effects of DF2 act synergistically and, in vivo, all should increase the efficacy of motor input to muscles resulting in potentiation of contractions.

Antagonistic Modulation of Neuromuscular Parameters in Crustaceans by the Peptides Proctolin and Allatostatin, Contained in Identified Motor Neurons

Recent work on the effects of two peptides, proctolin and allatostatin, on neuromuscular parameters in an isopod crustacean (Idotea) and the crab Eriphia spinfrons is reviewed. In Idotea, both peptides are present in identified motor neurons which supply a number of muscles with peptidergic innervation. Both peptides exert pre- and postsynaptic effects which are synergistic for a given peptide, but opposite for the two peptides. Proctolin enhances muscle contractions by at least three mechanisms. Postsynaptically, proctolin increases the input resistance of muscle fibres by closing voltage-independent K channels and it increases the inward current through L-type Ca channels. Presynaptically, it increases the mean quantal content of evoked transmitter release of slow and fast excitatory axons. Allatostatin decreases muscle contractions by at least two mechanisms: postsynaptically, it reduces the voltage dependent Ca current and presynaptically, it reduces the mean quantal content of transmitter release. The presynaptic inhibitory effect is also present at neuromuscular endings where GABA effects are absent.

Voltage-clamp analysis of membrane currents and excitation-contraction coupling in a crustacean muscle.

A single fibre preparation from the extensor muscle of a marine isopod crustacean is described which allows the analysis of membrane currents and simultaneously recorded contractions under two-electrode voltage-clamp conditions. We show that there are three main depolarisation-gated currents, two are outward and carried by K+, the third is an inward Ca2+ current, ICa. Normally, the K+ currents which can be isolated by using K+ channel blockers, mask ICa. ICa activates at potentials more positive than −40 mV, is maximal around 0 mV, and shows strong inactivation at higher depolarisation. Inactivation depends on current rather than voltage. Ba2+, Sr2+ and Mg2+ can substitute for Ca2+. Ba2+ currents are about 80% larger than Ca2+ currents and inactivate little. The properties of ICa characterise it as a high threshold L-type current. The outward current consists primarily of a fast, transient A current, IK(A), and a maintained, delayed rectifier current, IK(V). In some fibres, a small Ca2+-dependent K+ current is also present. IK(A) activates fast at depolarisation above −45 mV, shows pronounced inactivation and is almost completely inactivated at holding potentials more positive than −40 mV. IK(A) is half-maximally blocked by 70 μM 4-aminopyridine (4-AP), and 70 mM tetraethylammonium (TEA). IK(V) activates more slowly, at about −30 mV, and shows no inactivation. It is half-maximally blocked by 2 mM TEA but rather insensitive to 4-AP. Physiologically, the two K+ currents prevent all-or-nothing action potentials and determine the graded amplitude of active electrical responses and associated contractions. Tension development depends on and is correlated with depolarisation-induced Ca2+ influx mediated by ICa. The voltage dependence of peak tension corresponds directly to the voltage dependence of the integrated ICa. The threshold potential for contraction is at about −38 mV. Peak tension increases with increasing voltage steps, reaches maximum at around 0 mV, and declines with further depolarisation.