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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.

A chronically implantable electrode design permitting alternate extracellular nerve recording and axon stimulation in freely behaving crayfish was developed. The electrode consists of a double hook made from 20 microm thin platinum wire that can be fitted to various nerve diameters, and is easily implantable. A fast curing, flexible two-component silicone was used for insulation. The double hook was connected to plugs and fixed on the carapace of a crayfish allowing the animals to roam freely. The setup also allows for repeated dis- and re-connection of the crayfish for alternating recording and stimulation. Two channel recordings were used to determine directionality and to discriminate between afferent activity of the two stretch receptor neurons and efferent activity of several motor neurons. In addition, they were also used to determine the conduction velocity of the recorded efferent activity. Stable two-channel recordings could be obtained for up to 5 months and 15 days without apparent effects on the animal. In vivo stimulation could be performed for at least 3 1/2 weeks. The implantable double hook is suitable for widespread use in invertebrate neurobiology., (Copyright 2002 Elsevier Science B.V.)

The characteristics of the medial and lateral superficial extensor muscles (sem and sel) in the crayfish Orconectes limosus abdomen and their developmental and activity-dependent plasticity were studied. It was shown that both muscles are innervated by at least five excitatory and one inhibitory motor neuron in a nonuniform pattern. The muscles are composed of at least three different mATPase histochemistry-based fiber types that are all different from a fourth type in the uniform deep extensor muscles. sem and sel are composed of different ratios of these fiber types but do not show a constant fiber type pattern between segments and even between hemisegments. The three histochemically defined superficial extensor-fiber types have characteristic electrophysiological properties. The fiber types were shown to develop successively during the first postembryonic stages of development without a change in the number of muscle fibers. Based on histochemical ATPase staining after 21 days of chronic stimulation by means of an implantable, double-hook electrode, we show preliminary evidence that the fiber composition in the sem can switch from the presumably fast fiber type III to an intermediate type II. Repeated axotomy up to 53 days had no effect on the fiber type composition of the muscles., (Copyright 2002 Wiley-Liss, Inc.)

Most crustacean muscle fibers receive double excitatory innervation by functionally different motor neurons termed slow and fast. By using specific omega-toxins we show that the terminals of the slow closer excitor (SCE) and the fast closer excitor (FCE) at a crab muscle are endowed with different sets of presynaptic Ca(2+) channel types. omega-Agatoxin, a blocker of vertebrate P/Q-type channels, reduced the amplitude of EPSCs by decreasing the mean quantal content of transmitter release in both neurons by 70-85%, depending on the concentration. We provide the first evidence that omega-conotoxin-sensitive channels also participate in transmission at crustacean neuromuscular terminals and are colocalized with omega-agatoxin-sensitive channels in an axon-type-specific distribution. omega-Conotoxin, a blocker of vertebrate N-type channels, inhibited release by 20-25% only at FCE, not at SCE endings. Low concentrations of Ni(2+), which block vertebrate R-type channels, inhibited release in endings of the SCE by up to 35%, but had little effects in FCE endings. We found that two neuropeptides, the FMRFamide-like DF(2) and proctolin, which occur in many crustaceans, potentiated evoked transmitter release differentially. Proctolin increased release at SCE and FCE endings, and DF(2) increased release only at FCE endings. Selective blocking of Ca(2+) channels by different omega-toxins in the presence of peptides revealed that the target of proctolin-mediated modulation is the omega-agatoxin-sensitive channel (P/Q-like), that of DF(2) the omega-conotoxin-sensitive channel (N-like). The differential effects of these two peptides allows fine tuning of transmitter release at two functionally different motor neurons innervating the same muscle.

In the isopod Idotea emarginata, the neuropeptide proctolin is contained in a single pair of motoneurones located in pereion ganglion 4. The two neurones supply dorsal extensor muscle fibres of all segments. Proctolin (1 micromoll(-1)) potentiates the amplitude of contractures of single extensor muscle fibres elicited by 10 mmoll(-1) caffeine. In western blots of myofibrillar proteins isolated from single muscle fibres and treated with an anti-phosphoserine antibody, a protein with an apparent molecular mass of 30 kDa was consistently found. The phosphorylation of this protein was significantly increased by treating the fibres with proctolin. After separation of myofibrillar filaments, a 30 kDa protein was found only in the thin filament fraction. This protein is phosphorylated and detected by an antiserum against crustacean troponin I.

The effect of the biogenic amines octopamine and serotonin, and of both amines combined (cocktails) on transmitter release at neuromuscular junctions of two crustaceans was studied. octopamine (10(-8) mol l(-1) to 10(-6) mol l(-1)) either enhanced or decreased evoked transmitter release through presynaptic effects. The results were identical for the slow and the fast excitor in the closer muscle of the crab, and for the excitor in the opener muscle of the crayfish. Application of serotonin always resulted in a strong increase of release. However, this potentiating effect of serotonin was reduced in strength by subsequent application of cocktails consisting of serotonin and octopamine. In all experiments, a cocktail of serotonin and octopamine was less effective than serotonin alone. The decrease in the mean quantal content m by octopamine was due to a reduction of the probability of release p. Since both amines are synthesized in the central nervous system and are released from neurohaemal organs into the haemolymph bathing the neuromuscular junctions, the results suggest that the two amines, when present together, modulate transmitter release in an antagonistic way, and that the level of the two determines synaptic efficacy.

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, I(Ca). Normally, the K+ currents which can be isolated by using K+ channel blockers, mask I(Ca). I(Ca) 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 I(Ca) characterise it as a high threshold L-type current. The outward current consists primarily of a fast, transient A current, I(K(A)) and a maintained, delayed rectifier current, I(K(V)). In some fibres, a small Ca2+-dependent K+ current is also present. I(K(A)) activates fast at depolarisation above -45 mV, shows pronounced inactivation and is almost completely inactivated at holding potentials more positive than -40 mV. I(K(A)) is half-maximally blocked by 70 microM 4-aminopyridine (4-AP), and 70 mM tetraethylammonium (TEA). I(K(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 I(Ca). The voltage dependence of peak tension corresponds directly to the voltage dependence of the integrated I(Ca). 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.

Presynaptic inhibition exerted by the common inhibitor on the closer and opener muscles and by the specific inhibitor on the opener muscle was investigated in the crab Eriphia spinifrons. In the closer muscle, activation of GABA(B) receptors by baclofen reduced the mean quantal content of excitatory junctional currents by about 25%. Blocking GABA(B) receptors with CGP 55845 diminished presynaptic inhibition at a similar percentage. GABA(B) receptor-mediated presynaptic inhibition is linked to G proteins. Application of pertussis toxin eliminated about 25% of the inhibition exerted by the common inhibitory neuron. GABA(B) receptors participate in presynaptic inhibition at release boutons of the slow and the fast closer excitor at a similar percentage. In the opener muscle, presynaptic inhibition of transmitter release from the same endings of the opener excitor was about 15% stronger with the specific inhibitor than with the common inhibitor. About 10% of the presynaptic inhibition produced by either one of the two inhibitors could be abolished by blocking GABA(B) receptors. The amplitudes of the excitatory junctional currents in the opener were reduced in the presence of baclofen by about 25%, suggesting that synaptic terminals of the opener excitor are endowed with a similar percentage of GABA(B) receptors as terminals of the slow and the fast closer excitors. Baclofen had no effect on postsynaptic inhibition, indicating that GABA(B) receptors are not involved in postsynaptic neuromuscular inhibition.

The capacity of the larval insect nervous system to compensate for the permanent loss of one of the two excitatory motoneurons innervating a leg muscle was investigated in the locust (Locusta migratoria). In the fourth instar, the fast extensor tibiae (FETi) motoneuron in the mesothoracic ganglion was permanently removed by photoinactivation with a helium-cadmium laser. Subsequently, the animals were allowed to develop into adulthood. When experimental animals were tested as adults after final ecdysis, fast-contracting fibers in the most proximal region of the corresponding extensor muscle, which are normally predominantly innervated by FETi only, uniformly responded to activity of the slow extensor tibiae (SETi) neuron. In adult operated animals, single pulses to SETi elicited large junctional responses in the fibers which resulted in twitch contractions of these fibers similar to the responses to FETi activity in control animals. The total number of muscle fibers, their properties as histochemically determined contractional types (fast and slow), and their distribution were not affected by photoinactivation of FETi. Possible mechanisms enabling the larval neuromuscular system to compensate for the loss of FETi through functionally similar innervation by a different motoneuron, i.e. SETi, are discussed., (Copyright 2000 John Wiley & Sons, Inc.)

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., (Copyright 1999 Wiley-Liss, Inc.)

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. The data show that allatostatin occurs in identified motor neurons of Idotea and exerts complementary pre- and postsynaptic modulatory effects which reduce muscle responses. Thus, allatostatin counteracts the effects of another neuropeptide, proctolin, which is also present in Idotea and causes potentiating effects on the same muscle fibres.

Single-channel currents through calcium channels in muscle of a marine crustacean, the isopod Idotea baltica, were investigated in cell-attached patches. Inward barium currents were strongly voltage-dependent, and the channels were closed at the cell's resting membrane potential. The open probability (Po) increased e-fold for an 8.2 mV (+/- 2.4, n = 13) depolarization. Channel opening were mainly brief (< 0.3 ms) and evenly distributed throughout 100-ms pulses. Averaged, quasimacroscopic currents showed fast activation and deactivation and did not inactivate during 100-ms test pulses. Similarly, channel activity persisted at steadily depolarized holding potentials. With 200 mM Ba2+ as charge carrier, the average slope conductance from the unitary currents between +30 and +80 mV, was 20 pS (+/- 2.6, n = 12). The proportion of long openings, which were very infrequent under control conditions, was greatly increased by preincubation of the muscle fibers with the calcium channel agonist, the dihydropyridine Bay K8644 (10-100 microM). Properties of these currents resemble those through the L-type calcium channels of mammalian nerve, smooth muscle, and cardiac muscle cells.

1. The common inhibitory motoneurone 1 (CI1) in the mesothoracic ganglion of the locust was photoinactivated using a helium-cadmium laser or a mercury lamp as light source. Treated animals showed no signs of abnormal locomotory behaviour over periods of up to 40 days. 2. Photoinactivation of part of the neurone in the ganglion, i.e. the soma and the primary neurite, is sufficient to cause irreversible degeneration of all the peripheral extensions of the neurone. Three weeks after photoinactivation, all GABA immunoreactivity had disappeared from the axon branches of the photoinactivated neurone and from their terminals on one of the target muscles investigated, the anterior coxa rotator M92, and inhibitory postsynaptic potentials could no longer be elicited through stimulation. This was taken as proof of functional denervation of the muscle with regard to its inhibitory input. By this time, the axon of CI1 in nerve N3C1, which supplies M92, had also disappeared. 3. Animals treated during the fourth or fifth instars showed a permanent loss of the photoinactivated mesothoracic CI1 neurone after moulting into adulthood. 4. Denervation of M92 in the middle legs of instars and adults by axotomy of N3 always led to rapid functional reinnervation of the muscle. The first sign of reinnervation (excitatory neuromuscular activity upon mechanical stimulation of the tarsi) was detected electrophysiologically as early as 8 days after severing the motor nerve. 5. The elimination of CI1 by photoinactivation for a period of up to 40 days did not influence parameters of the target muscle, such as size, number of fibres and phenotypes of fibres defined histochemically according to their myofibrillar ATPase isoforms, irrespective of whether the operation was performed in instars or adults. Similarly, the short period of denervation following axotomy before reinnervation took place did not affect the fibre type composition of the muscle.

The neuropeptide proctolin in nanomolar concentrations enhances the contraction of crustacean muscle fibers manyfold. The cellular mechanisms underlying this potentiation were investigated in single, isolated, fast-contracting abdominal extensor muscle fibers of a small crustacean, the marine isopod Idotea baltica. Force measurements and current-clamp experiments revealed two actions of proctolin on the muscle fibers. In half of the preparations, proctolin (10(-9)-10(-6) M) increased the fiber's input resistance by up to 25%. In about one-fourth of the preparations, proctolin induced all-or-none action potentials in response to depolarizing current pulses in muscle fibers that showed graded electric responses under control conditions. In both cases, proctolin potentiated the peak force of muscle contractions (between 1.5- and 18-fold for 5 x 10(-9) M proctolin). Proctolin affected neither the membrane resting potential nor the threshold for excitation-contraction coupling. Using cell-attached patches on the sarcolemmal membrane, we identified non-voltage-dependent ion channels which contribute to the passive membrane properties of the muscle fibers. A 53 +/- 6 pS channel had its reversal potential near rest and carried outward current at depolarized potentials with physiological saline in the recording pipette. With isotonic K+ saline in the patch pipette, the reversal potential was +85 +/- 12 mV depolarized from the resting potential and single-channel conductances ranged from 36 to 166 pS. Proctolin modulated the activity of all these putative K+ channels by reducing the number of functionally active channels. The effects of proctolin on force of contraction, input resistance, and single-channel activity were mimicked by a membrane-permeating analog of cAMP. Conversely, a monothio analog of cAMP (Rp-cAMPS), a blocker of protein kinase A activity, substantially decreased the membrane input resistance of the muscle fibers. The results suggest that activation of the cAMP signal pathway and phosphorylation of non-voltage-dependent K+ channels by protein kinase A are involved in the potentiation of contractions by proctolin in the muscle fibers of this crustacean.

The 'catch-like' effect, a hysteresis phenomenon in arthropod skeletal muscle contraction thought to be related to the catch of molluscan smooth muscle, was investigated in the closer muscle of the crab Eriphia spinifrons. Several parameters were varied to determine their influence on the catch-like effect. These parameters were (1) the frequency of repetitive stimulation of the slow excitatory neuron, (2) additional stimulation of the inhibitory neuron, (3) the amount of stretch applied to the muscle and (4) the stiffness of the mechano-electrical transducer. The results show that the catch-like effect is not related to the catch of molluscan smooth muscle but rather to the 'residual force enhancement' or 'creep' phenomenon described for vertebrate muscle. A hypothesis for residual force enhancement implies that the increase in force is caused by non-uniformity of sarcomere lengths along the muscle fibre. Based on this hypothesis and the actual force-length relationship of the crab muscle studied, calculations were carried out to determine, if the observed catch-like effect can be explained by such a model. The calculations corroborate the experimental evidence. The catch-like effect of arthropod muscles can thus be explained by the same mechanism responsible for residual force enhancement and creep in vertebrate muscle. A physiological relevance of the catch-like effect in arthropod muscle is inferred.

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 Ca(2+)-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.

(1) Intracellular activities of K+, Na+ and Cl- were measured with ion-sensitive microelectrodes in four different types of muscle fibers in the closer muscle of the crab Eriphia. (2) The membrane resting potentials of the tonic fibers were 9-15 mV more positive than those of phasic muscle fibers. This was due to higher permeability of the membranes of tonic fibers for Na+. (3) The intracellular Na+-activity of tonic fibers was 35-40% higher than that of phasic fibers. Also intracellular Cl- -activity was about 15-33% higher in tonic fibers. (4) No significant differences in K+ -activities were found between physiologically different muscle fiber types. The K+ -equilibrium potentials were always more negative than the resting potentials. In muscle fibers with inhibitory innervation, Cl- -equilibrium potentials were close to (phasic fibers) or slightly more negative (tonic fibers) than resting potentials.

The effect of anemone toxin (ATX II), which slows sodium channel inactivation at electrically excitable membranes, was investigated in the slowly adapting stretch receptor organ of crayfish. The toxin affected the action potentials, but produced no changes in the stretch induced receptor potentials. This finding is further proof for the high selectivity of this toxin molecule for sodium channels which are gated by membrane depolarissations.

The superficial muscle fibres in the proximal part of the closer muscle in the crab Eriphia can be separated into four fibre groups (I-IV) on the basis of electrophysiological and histochemical characteristics. The activity levels of glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), citrate synthase (CS), NADP-isocitrate dehydrogenase (IDH) and 3-hydroxyacyl-CoA dehydrogenase (HAD), determined in single electrophysiologically identified fibres, differed significantly among the different fibre groups. In addition, fibres belonging to the same group, with similar electrophysiological characteristics, demonstrated variability with regard to metabolic enzyme activities. Nevertheless, comparison of absolute enzyme activities and enzyme activity ratios permitted the discrimination of at least three groups. These groups corresponded with those defined according to electrophysiological and histochemical characteristics. The group I fibres (tonic fibres) are intermediate in oxidative potential and show the lowest values of glycolytic enzymes. The group II and group III fibres can be regarded as fast oxidative fibres. The high ratio between activity levels of enzymes for glycolytic and oxidative metabolism found for group IV fibres (fast fibres) demonstrated that this group depends strongly on anaerobic metabolism.

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na+ channel specific action of ATX II. Na+ current inactivation is slowed with the time constant tau h increasing towards positive membrane potentials. The steady state inactivation curve hoo was shifted to more positive potentials and its slope reduced.

A neurotoxin (ATX-II) extracted from the tentacles of Anemonia sulcata has been found to interact with the sodium channel of the nodal membrane in myelinated nerve fibres from Rana esculenta. If externally applied at low concentration (Kd = 20 muM), it reduces considerably the rate of inactivation of the sodium conductance without affecting the activation. At such concentrations, the potassium conductance is not affected. If internally applied ATX-II does not affect the membrane conductance.

Myofibrillar actomyosin ATPase activity has been studied histochemically in the closer muscle of the crab Eriphia spinifrons. Preincubation at pH 4.6 and 5.0 reveals differences in the lability of the ATPase. This permits the discrimination of four fibre types. Of these, three represent subgroups of rapidly contracting fibres. The histochemically defined fibre types correspond well with four groups defined according to electrophysiological criteria.

The effects of three toxins (ATX I, II, III) isolated from the sea anemone Anemonia sulcata were studied in the soma membrane of a crustacean neurone under voltage-clamp conditions. 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 microM. In the presence of ATX the Na+ inactivation was incomplete even with pulses of 700 ms length or strong depolarizing prepulses. Besides the effects on the inactivation process ATX affected also the activation of the Na+ current. In cells treated with ATX the negative resistance branch of the peak Na+ current voltage relation was shifted by -5 mV to -20 mV. The time to peak was increased for small depolarizations (up to -30 mV) and the rate of rise (delta I/delta t) was enlarged by ATX. A slow activating current component was also observed after depolarizing prepulses or if the Na+ current was outward. 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. 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.

The effects of two toxins extracted from the tentacles of Anemonia sulcata on ionic currents have been tested on the nodal membrane of myelinated nerve fibres from Rana esculenta. While external application of Toxin I at 100 muM leaves both specific ionic currents unmodified, Toxin II at 10 muM reacts with a receptor site associated with the sodium conductance inactivation gating. Since internal application by diffusion of Toxin II at a concentration of 700 muM leaves the ionic currents unchanged, the receptor site is most likely located on the external side of the nodal membrane. An equilibrium dissociation constant for the effects of Toxin II was estimated as 20 muM. The on-reaction is fast (rate constant for the on-reaction roughly equal to 3.103 M-1) suggesting a readily accesible receptor site for the toxin. The kinetics characteristics of the sodium currents recorded in the presence of Toxin II suggest that there are at least two steps in the reaction leading to Na+ -channels with the inactivation gate completely immobilized. The relatively fast reversibility of the intermediate stage of the reaction and the rather slow but, in the end, complete reversal of the toxin effects suggest that the toxin acts by modifying the energy profile for the transition "inactivation gate in the open configuration to inactivation gate in the closed configuration." Toxin II at higher concentrations (greater than 100 muM) also inhibits the potassium currents but these effects were not studied in any detail.

Spontaneous and evoked release of transmitter at neuromuscular junctions in three different leg muscles of a tarantula (Dugesiella hentzi) was investigated. In most cases the spontaneous miniature potentials were released independently, although bursts from single synaptic junctions occasionally occurred. In contrast to recent findings in other arthropod muscles, focal extracellular recording from junctional areas revealed that the evoked release of transmitter quanta followed Poisson's theorem at low quantal content synaptic junctions in arachnid muscles.