Crayfish Axons Research Articles

Ultrasound-Induced Membrane Hyperpolarization in Motor Axons and Muscle Fibers of the Crayfish Neuromuscular Junction.

Focused ultrasound (FUS) can modulate neuronal activity by depolarization or hyperpolarization. Although FUS-evoked depolarization has been studied extensively, the mechanisms underlying FUS-evoked hyperpolarization (FUSH) have received little attention. In the study described here, we developed a procedure using FUS to selectively hyperpolarize motor axons in crayfish. As a previous study had reported that these axons express mechano- and thermosensitive two-pore domain potassium (K2P) channels, we tested the hypothesis that K2P channels underlie FUSH. Intracellular recordings from a motor axon and a muscle fiber were obtained simultaneously from the crayfish opener neuromuscular preparation. FUSH was examined while K2P channel activities were modulated by varying temperature or by K2P channel blockers. FUSH in the axons did not exhibit a coherent temperature dependence, consistent with predicted K2P channel behavior, although changes in the resting membrane potential of the same axons indicated well-behaved K2P channel temperature dependence. The same conclusion was supported by pharmacological data; namely, FUSH was not suppressed by K2P channel blockers. Comparison between the FUS-evoked responses recorded in motor axons and muscle fibers revealed that the latter exhibited very little FUSH, indicating that the FUSH was specific to the axons. It is not likely that K2P channels are the underlying mechanism for FUSH in motor axons. Alternative mechanisms such as sonophore and axon-specific potassium channels were considered. Although the sonophore hypothesis could account for electrophysiological features of axonal recordings, it is not consistent with the lack of FUSH in muscle fibers. An axon-specific and mechanosensitive potassium channel is also a possible explanation.

Nerve Structure-Function: Unusual Structural Details and Unmasking of Sulfhydryl Groups by Electrical Stimulation or Asphyxia in Axon Membranes and Gap Junctions.

This review describes and discusses unusual axonal structural details and evidence for unmasking sulfhydryl groups (-SH) in axoplasmic membranes resulting from electrical stimulation or asphyxia. Crayfish axons contain fenestrated septa (FS) that, in phase contrast, micrographs appear as repeated striations. In the electron microscope, each septum is made of two cross-sectioned membranes containing ~55 nm pores, each occupied by a microtubule. Thin filaments, which we believe are made of kinesin, bridge the microtubule to the edge of the pore. FS are believed to play a role in axoplasmic flow. The axons also display areas in which axon and sheath glial cell plasma membranes are sharply curved and project into the axoplasm. In freeze-fractures, the protoplasmic leaflet (P-face) of the projections appears as elongated indentations containing parallel chains of particles. The sheath glial cell plasma membrane also contains particles, but they are irregularly aggregated. The axons also display areas where axonal and glial plasma membranes fuse, creating intercellular pores. In axons fixed during electrical stimulation, the plasma membrane, the outer membrane of mitochondria, membranes of other cytoplasmic organelles, and gap junctions increase in electron opacity and thickness, resulting from unmasking of sulfhydryl groups (-SH). Similar changes occur in asphyxiated nerve cords.

Potential Role of Fenestrated Septa in Axonal Transport of Golgi Cisternae and Gap Junction Formation/Function.

Crayfish axons contain a system of parallel membranous cisternae spaced by ~2 μm and oriented perpendicularly to the axon's long axis. Each cisterna is composed of two roughly parallel membranes, separated by a 150-400 Å wide space. The cisternae are interrupted by 500-600 Å pores, each occupied by a microtubule. Significantly, filaments, likely made of kinesin, often bridge the gap between the microtubule and the edge of the pore. Neighboring cisternae are linked by longitudinal membranous tubules. In small axons, the cisternae seem to be continuous across the axon, while in large axons they are intact only at the axon's periphery. Due to the presence of pores, we have named these structures "Fenestrated Septa" (FS). Similar structures are also present in vertebrates, including mammals, proving that they are widely expressed in the animal kingdom. We propose that FS are components of the "anterograde transport" mechanism that moves cisternae of the Golgi apparatus (GA) toward the nerve ending by means of motor proteins, likely to be kinesins. In crayfish lateral giant axons, we believe that vesicles that bud off FS at the nerve ending contain gap junction hemichannels (innexons) for gap junction channel and hemichannel formation and function.

Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model.

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

Focused ultrasound transiently increases membrane conductance in isolated crayfish axon.

We report a novel phenomenon produced by focused ultrasound (US) that may be important for understanding its effects on cell membranes. When a US burst (2.1 MHz, 1-mm focal diameter, 0.1-1 MPa) was focused on a motor axon of the crayfish neuromuscular junction, it consistently produced a fast hyperpolarization, which was followed or superseded by subthreshold depolarizations or action potentials in a stochastic manner. The depolarization persisted in the presence of voltage-gated channel blockers [1 µM TTX ( INa), 50 µM ZD7288 ( Ih), and 200 µM 4-aminopyridine ( IK)] and typically started shortly after the onset of a 5-ms US burst, with a mean latency of 3.35 ± 0.53 ms (SE). The duration and amplitude of depolarizations averaged 2.13 ± 0.87 s and 10.1 ± 2.09 mV, with a maximum of 200 s and 60 mV, respectively. The US-induced depolarization was always associated with a decrease in membrane resistance. By measuring membrane potential and resistance during the US-induced depolarization, the reversal potential of US-induced conductance ( gus) was estimated to be -8.4 ± 2.3 mV, suggesting a nonselective conductance. The increase in gus was 10-100 times larger than the leak conductance; thus it could significantly influence neuronal activity. This change in conductance may be due to stimulation of mechanoreceptors. Alternatively, US may perturb the lateral motion of phospholipids and produce nanopores, which then increase gus. These results may be important for understanding mechanisms underlying US-mediated modulation of neuronal activity and brain function. NEW & NOTEWORTHY We report a specific increase in membrane conductance produced by ultrasound (US) on neuronal membrane. When a 5-ms US tone burst was focused on a crayfish motor axon, it stochastically triggered either depolarization or a spike train. The depolarization was up to 60 mV in amplitude and 200 s in duration and therefore could significantly influence neuronal activity. Depolarization was still evoked by US burst in the presence of Na+ and Ca2+ channel blockers and had a reversal potential of -8.4 ± 2.3 mV, suggesting a nonselective permeability. US can be applied noninvasively in the form of a focused beam to deep brain areas through the skull and has been shown to modulate brain activity. Understanding the depolarization reported here should be helpful for improving the use of US for noninvasive modulation and stimulation in brain-related disease.

New insights into the acute actions from a high dosage of fluoxetine on neuronal and cardiac function: Drosophila, crayfish and rodent models

The commonly used mood altering drug fluoxetine (Prozac) in humans has a low occurrence in reports of harmful effects from overdose; however, individuals with altered metabolism of the drug and accidental overdose have led to critical conditions and even death. We addressed direct actions of high concentrations on synaptic transmission at neuromuscular junctions (NMJs), neural properties, and cardiac function unrelated to fluoxetine's action as a selective 5-HT reuptake inhibitor. There appears to be action in blocking action potentials in crayfish axons, enhanced occurrences of spontaneous synaptic vesicle fusion events in the presynaptic terminals at NMJs of both Drosophila and crayfish. In rodent neurons, cytoplasmic Ca2+ rises by fluoxetine and is thapsigargin dependent. The Drosophila larval heart showed a dose dependent effect in cardiac arrest. Acute paralytic behavior in crayfish occurred at a systemic concentration of 2mM. A high percentage of death as well as slowed development occurred in Drosophila larvae consuming food containing 100μM fluoxetine. The release of Ca2+ from the endoplasmic reticulum in neurons and the cardiac tissue as well as blockage of voltage-gated Na+ channels in neurons could explain the effects on the whole animal as well as the isolated tissues. The use of various animal models in demonstrating the potential mechanisms for the toxic effects with high doses of fluoxetine maybe beneficial for acute treatments in humans. Future studies in determining how fluoxetine is internalized in cells and if there are subtle effects of these mentioned mechanisms presented with chronic therapeutic doses are of general interest.

Dimethylsulfoxide potentiates the nerve conduction blocking effect of lidocaine without augmentation of the intracellular lidocaine concentration in the giant axon of crayfish in vitro

Dimethylsulfoxide potentiates the nerve conduction blocking effect of lidocaine without augmentation of the intracellular lidocaine concentration in the giant axon of crayfish in vitro

Dimethylsulfoxide potentiates the nerve conduction–blocking effect of lidocaine without augmentation of the intracellular lidocaine concentration in the giant axon of crayfish in vitro

The purpose of this study was to investigate how dimethylsulfoxide (DMSO) potentiates the blocking action of lidocaine. A giant axon removed from a crayfish was used to investigate nerve conduction and intracellular lidocaine concentration. The maximum values of the differential waveform (dV/dt max) calculated from evoked action potentials were used for evaluating an inhibition of nerve conduction. The inhibition of the dV/dt max in low-frequency stimulation (tonic block) and high-frequency stimulation (phasic block) after perfusion of 1 mm lidocaine with or without 0.2 vol % DMSO, in which the concentration of DMSO alone had no anesthetic effect, was measured to evaluate the potentiating action of DMSO. The intracellular lidocaine concentration was measured via a lidocaine-sensitive glass microelectrode during 30 min of perfusion of 1 mm lidocaine alone or in combination with DMSO. When applied without lidocaine, DMSO caused a dose-dependent nerve conduction block when used at concentrations >1 vol %. The dV/dt max in the tonic block was significantly decreased when 0.2 vol % DMSO was added to the lidocaine solution (P = 0.004). In the phasic block, there was no significant potentiating action of DMSO. There were no significant differences in the intracellular lidocaine concentrations with or without DMSO. The potentiating effects of DMSO were observed only in the condition of low-frequency stimulation and were not related to the intracellular lidocaine concentration in the giant axon of crayfish in vitro.

A Comparison of Intracellular Lidocaine and Bupivacaine Concentrations Producing Nerve Conduction Block in the Giant Axon of Crayfish In Vitro

Clinically, lidocaine requires a larger concentration than bupivacaine to block nerves. Bupivacaine has a higher lipid solubility, tissue permeability, and affinity for sodium channels than lidocaine, resulting in greater anesthetic potency. Local anesthetics require access to the sodium channel from the intracellular milieu. In this study, we sought to determine the intracellular concentration of lidocaine and bupivacaine when a nerve was blocked in the giant axon of a crayfish. A solution of lidocaine or bupivacaine was perfused, and a nerve block was determined as the absence of an evoked action potential after tonic or phasic electrical stimulation. The intracellular lidocaine or bupivacaine concentration was measured using a lidocaine- or bupivacaine-sensitive glass micro-electrode. A phasic block was more effectively and rapidly achieved with a smaller concentration of bupivacaine than with lidocaine. The intracellular concentration and intra- to extracellular ratios were significantly larger with lidocaine than with bupivacaine when nerve conduction was blocked. These findings suggest that bupivacaine has a higher potency than lidocaine, at least in the giant axon of a crayfish in vitro. The implications of the present results are that bupivacaine is a more potent nerve block and produces a use-dependent (phasic) block at smaller concentrations than lidocaine.

Questioning the Mechanism of Nerve Injury

We thank Lambert et al. for their interest in our article1and stimulating comments. We would like to take the opportunity to address the issues raised by their insights.Lambert et al. suggest that the mechanism of local anesthetic neurotoxicity is unrelated to membrane disruption caused by the detergent nature of highly concentrated local anesthetics, instead resulting from breakdown of the sodium conductance system (i.e. , absence of compound action potential generation) despite the presence of an intact membrane structure and normal ionic gradients and compound resting potential. The basis for their contention is that, although both compound action potential and compound resting potential in excised sciatic nerves of the bullfrog disappear permanently after bathing in sodium lauryl sulfate (typical detergent) solution,2compound resting potential recovers without compound action potential recovery after bathing in 5% lidocaine solution for 15 min, as noted in their earlier electrophysiologic study.3However, the study cited as grounds for their question about our conclusions seems to have an inherent limitation in that the period for bathing the specimen in 5% lidocaine is 15 min. Kanai et al. 4reported a similar study using a single crayfish axon and demonstrated that although resting potential recovers after bathing in 80 mm lidocaine solution for 15 min, resting potential permanently disappears after bathing for 30 min. They suggested that highly concentrated lidocaine causes membrane disruption and indicated in that time-dependent study that sufficient exposure (≥ 30 min) was needed for membrane disruption to be induced by lidocaine. Ready et al. 5also demonstrated successfully that lidocaine at a concentration of 4% or higher induces histopathologic changes in spinal nerves during a dose-dependent study of a spinal anesthesia model in rabbits. The fact that highly concentrated lidocaine causes membrane disruption is thus not in doubt.We advocated in our article that the mechanism of membrane disruption induced by highly concentrated local anesthetics would result from the detergent nature of these agents.1From the perspective of results from various investigators, including Lambert et al. , we speculate that an intermediate step may exist in the membrane disruption dynamics induced by 5% lidocaine, with resting membrane potential maintained despite permanent inhibition of action potential. The electrophysiologic phenomena described by Lambert et al. may represent an early phase in the sequence of neurotoxic dynamics induced by 5% lidocaine.* Saga Medical School, Nabeshima, Saga, Japan. kitagawa@mail.anes.saga-med.ac.jp

Presynaptic membrane of inhibitory crayfish axon terminals is stained by antibodies raised against mammalian GABA(A) receptor subunits alpha3 and beta(2/3).

The opener muscle of the dactyl of the walking leg of crayfish is innervated by one excitatory axon releasing glutamate and one inhibitory axon releasing GABA. Functional GABA(A) receptors are present postsynaptically on the muscle and presynaptically on terminals and release boutons of the excitatory axon, whereas presynaptic GABA(A) autoreceptors have not been reported on terminals or release boutons of the inhibitory axon. Using antibodies raised against mammalian GABA(A) receptor subunits alpha3 and beta(2/3), we obtained highly specific staining of the presynaptic membrane of the inhibitory bouton and of the postsynaptic membrane of the muscle. Using pre- and postembedding techniques, staining was localized to only presynaptic and postsynaptic membranes of synaptic active zones. We also found extrasynaptic receptor subunit immunoreactivity near (up to 100 nm) to the active zones. Staining with antibodies for the alpha3 and beta(2/3) subunits showed colocalization of particles of the two subunits. We suggest that presynaptic inhibitory boutons of the crayfish possess GABA(A)-like autoreceptors composed of at least the alpha3 and beta(2/3) subunits.

Glutamine uptake and metabolism to N-acetylaspartylglutamate (NAAG) by crayfish axons and glia

We have proposed that N-acetylaspartylglutamate (NAAG) or its hydrolytic product glutamate, is a chemical signaling agent between axons and periaxonal glia at non-synaptic sites in crayfish nerves, and that glutamine is a probable precursor for replenishing the releasable pool of NAAG. We report here, that crayfish central nerve fibers synthesize NAAG from exogenous glutamine. Cellular accumulation of radiolabel during in vitro incubation of desheathed cephalothoracic nerve bundles with [ 3H]glutamine was 74% Na +-independent. The Na +-independent transport was temperature-sensitive, linear with time for at least 4 h, saturable between 2.5 and 10 mM l-glutamine, and blocked by neutral amino acids and analogs that inhibit mammalian glutamine transport. Radiolabeled glutamine was taken up and metabolized by both axons and glia to glutamate and NAAG, and a significant fraction of these products effluxed from the cells. Both the metabolism and release of radiolabeled glutamine was influenced by extracellular Na +. The uptake and conversion of glutamine to glutamate and NAAG by axons provides a possible mechanism for recycling and formation of the axon-to-glia signaling agent(s).

Properties of glutaminase of crayfish CNS: implications for axon–glia signaling

Glutaminase of crayfish axons is believed to participate in recycling of axon–glia signaling agent(s). We measured the activity and properties of glutaminase in crude homogenates of crayfish CNS, using ion exchange chromatography to separate radiolabeled product from substrate. Crayfish glutaminase activity is cytoplasmic and/or weakly bound to membranes and dependent on time, tissue protein, and glutamine concentration. It resembles the kidney-type phosphate-activated glutaminase of mammals in being stimulated by inorganic phosphate and alkaline pH and inhibited by the product glutamate and by the glutamine analog 6-diazo-5-oxo- L-norleucine. During incubation of crayfish CNS fibers in Na +-free saline containing radiolabeled glutamine, there is an increased formation of radiolabeled glutamate in axoplasm that is temporally associated with an increase in axonal pH from about 7.1 to about 8.0. Both the formation of glutamate and the change in pH are reduced by 6-diazo-5-oxo- L-norleucine. Our results suggest that crayfish glutaminase activity is regulated by cellular changes in pH and glutamate concentration. Such changes could impact availability of the axon–glia signaling agents glutamate and N-acetylaspartylglutamate.

Barrier Permeability at Cut Axonal Ends Progressively Decreases until an Ionic Seal Is Formed

After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6–70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0–120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction.

Functional and immunocytochemical identification of glutamate autoreceptors of an NMDA type in crayfish neuromuscular junction. J. Neurophysiol. 80: 2893-2899, 1998. N-Methyl--aspartate (NMDA) reduces release from crayfish excitatory nerve terminals. We show here that polyclonal and monoclonal antibodies raised against the mammalian postsynaptic NMDA receptor subunit 1 stain specifically the presynaptic membrane of release boutons of the crayfish neuromuscular junction. In crayfish ganglionic membranes, the polyclonal antibody recognizes a single protein band that is somewhat larger (by approximately 30 kD) than the molecular weight of the rat receptor. Moreover, the monoclonal (but not the polyclonal) antibody abolishes the physiological effect of NMDA on glutamate release. The monoclonal antibody did not prevent the presynaptic effects of glutamate, which also reduces release by activation of quisqualate presynaptic receptors. Only when 6-cyano-7-nitroquinoxatine-2,3,dione (CNQX) was added together with the monoclonal antibody was the presynaptic effect of glutamate blocked. These results show that presynaptic glutamate receptors of the crayfish NMDA type are involved in the regulation of neurotransmitter release in crayfish axon terminals. Although the crayfish receptor differs in its properties from the mammalian NMDA receptor, the two receptors retained some structural similarity.

Elimination of sensory inputs induces growth and synaptic changes in crayfish motor axons

Damage to motor neurons induces regeneration processes including axonal growth and change of synaptic properties. Sensory axons that run along the motor axons are also damaged, but their possible role in the motor neuron's regeneration is generally ignored. Here, the effect of eliminating some sensory inputs from intact motor axons on the motor axon's properties was studied. Micro-dissecting one of the segmental, bilateral, sensory stretch receptor pairs of the crayfish abdomen induced the deep extensor abdominal motor axons to grow and changed their synaptic properties. The results demonstrate directly, probably for the first time, that change in sensory neuron activity can induce motor axons to grow, form new synapses, and change their synaptic properties.

Slow inhibition of Na+ current in crayfish axons by 2-(1non-8enyl)-5-(1non-8enyl)pyrrolidine (Pyr9), a synthetic derivative of an ant venom alkaloid

2,5-Dialkylpyrrolidines present in the venom of ants from the genus Monomorium are natural insecticides causing a flaccid paralysis. The mechanism of action of 2-(1non-8enyl)-5-(1non-8enyl)pyrrolidine (Pyr9), a synthetic derivative of 2,5-dialkylpyrrolidines, has been investigated in vitro on preparations of the ventral nerve cord of the crayfish Procambarus clarkii. Our results clearly indicate that Pyr9 blocks spike conduction without affecting the resting potential. Voltage-clamp experiments carried out on axons demonstrate that this blockade is due to a dual expression of Na+ current inhibition: a tonic inhibition developing slowly (90 % of inhibition within 20 min for a Pyr9 concentration of 50 &micro;mol l-1) and independently of stimulation, and a phasic inhibition developing during repetitive stimulation (5 Hz), the accumulation kinetics of which is 0.072 pulse-1 at 5 Hz, according to the Courtney model. These findings suggest that tonic and phasic inhibition are due to different mechanisms. In addition, Pyr9 induces a shift of the Na+ inactivation curve towards more hyperpolarized potentials, which is in agreement with a higher affinity of Pyr9 for inactivated than for resting Na+ channels.

Monocarboxylic acids enhance the anesthetic action of procaine by decreasing intracellular pH

Sodium monocarboxylates are known to enhance the anesthetic action of procaine, and also decrease intracellular pH (pH i). We studied the effect of 30 mM Na monocarboxylates (formate, acetate, propionate, butyrate, and salicylate) on the pH i and on the anesthetic action of procaine HCl using giant axons of crayfish ( Procambarus clarkii). The pH i was measured using pH sensitive microelectrode method and the anesthetic action was evaluated by the change in the action potential (AP) amplitude. The tested acids except for formate showed apparent decrease in pH i and enhancement of the action of 2 mM procaine. Other organic acids (maleate and benzensulfonate) did not affect pH i and anesthetic action of procaine. In the bicarbonate free solution, pH i increased and the anesthetic action was weakened. The EC 25 values (the concentration of procaine which depresses the AP amplitude by 25%) of acetate, propionate, and bicarbonate free solution were coincided with the predicted EC 25 values from the simple simulation on intracellular procaine increase according to the pH i change. But the EC 25 value of salicylate group was less than half of the predicted. These results suggested that the enhancing action of straight chain monocarboxylic acids is due to pH i decrease, and salicylate has other additional mechanisms.

Quantal release at visualized terminals of a crayfish motor axon: Intraterminal and regional differences

Synaptic transmission was measured at visualized terminal varicosities of the motor axon providing the sole excitatory innervation of the “opener” muscle in walking legs of crayfish (Procambarus clarkii Girard). Two questions were addressed: 1) How uniform is quantal emission at different locations along terminals innervating a single muscle fiber, and 2) can differences in quantal emission account for the different excitatory postsynaptic potential (EPSP) amplitudes generated by terminals localized in defined regions of the muscle? Extracellular “macropatch” electrodes were placed over individual varicosities, viewed after brief exposure to a fluorescent dye, and synaptic currents were recorded to determine quantal content of transmission. Along terminals supplying a single muscle fiber, nonuniform release was found: Varicosities closer to the point of origin of the terminal branch released more transmitter than those located more distally. Quantal content was higher for varicosities of the muscle's proximal region (where large EPSPs occur) than for varicosities of the central region (where small EPSPs occur). The probability of transmitter release per synapse is estimated to be greater for the proximal varicosities. At low frequencies of stimulation, quantal content per muscle fiber is two to four times larger in the proximal region. Taken in conjunction with a twofold higher mean input resistance for the proximal muscle fibers, the difference in quantal content can account for a four- to eightfold difference in EPSP amplitude. The observed mean EPSP amplitude is at least eight times larger in the proximal region. We discuss factors contributing to differences in EPSP amplitudes. © 1996 Wiley-Liss, Inc.

Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity

Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthus martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V 8 protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus,