Two classes of channel-specific toxins from funnel web spider venom

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Division of Toxicology and Physiology, Department of Entomology, ... Synaptic toxins from spider venom ... Milked venom from Agelenopsis aperta spiders was.
Joumal of Sensory, Comparative 9

J Comp Physiol A (1989) 164:333-342

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Physiology A

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9 Sprin0er-Verlag 1989

Two classes of channel-specific toxins from funnel web spider venom Michael E. Adams, Edward E. Herold, and Virginia J. Venema Division of Toxicology and Physiology, Department of Entomology, University of California, Riverside, California 92521, USA Accepted October 11, 1988

Summary. 1. The paralytic effects and neuromuscular actions of Agelenopsis aperta venom on insects were analyzed biochemically and electrophysiologically. 2. Paralysis caused by Agelenopsis venom is correlated with two effects on neuromuscular transmission: postsynaptic inhibition and presynaptic excitation. These effects are explained by the actions of two classes of toxins purified by RPLC, the y and ~t-agatoxins. 3. The y are low molecular weight, acylpolyamines which cause rapid, reversible paralysis correlated with use-dependent postsynaptic block of EPSPs and ionophoretic glutamate potentials. The g-agatoxins are cysteine-rich polypeptides which cause irreversible paralysis and repetitive action potentials originating in presynaptic axons or nerve terminals. 4. The joint actions of the y and tx-agatoxins lead to significantly higher rates of paralysis than are obtained by either toxin class alone, and this may relate to enhancement by excitatory ~t-agatoxins of use-dependent block caused by y

Introduction Ion channel proteins are primary targets for paralytic venom toxins, many of which show extraordinary binding affinities and specificities. Consequently, toxins from a variety of animals continue to serve as key molecular probes for the characterAbbreviations : E P S P excitatory postsynaptic potential; R P L C reversed phase liquid chromatography; T T X tetrodotoxin; TFA trifluoroacetic acid; HFBA heptafluorobutyric acid; A G489 ~agatoxin 489; g-Aga I g-agatoxin I, AR659 argiotoxin 659

ization and classification of both voltage- and ligand-sensitive ion channels. For instance, scorpion toxins show selectivity for at least four distinct binding sites on voltage-sensitive sodium channels (Catterall 1984) and as well detect differences between insect, crustacean or mammalian sodium channels (Zlotkin 1983). Some venoms, e.g. those of fish-hunting cone snails, contain multiple ctasses of 'conotoxins' affecting different types of ion channels involved in sequential steps of synaptic transmission (Olivera et al. 1985). Conotoxins show specificity for nicotinic acetylcholine receptor, distinguish between nerve and muscle sodium channels (Cruz et al. 1985), and between different classes of neuronal calcium channels (Olivera et al. 1987). Spider venoms are increasingly recognized as important sources of toxins acting specifically on functionally distinct types of ion channels operating both pre- and postsynaptically. Inhibition of transmitter release at the Drosophila neuromuscular junction by polypeptide toxins from Hololena curta and Plectruerys tristis spider venoms (Bowers et al. 1987; Branton et al. 1987), appears to involve blockade of presynaptic calcium channels. While Hololena and Plectruerys toxins are reported to be specific for insects, toxins from the venom of Agelenopsis aperta, a funnel web spider, are reported to block calcium channels in the avian cochlear nucleus (Jackson et al. 1986) and guinea pig cerebellum (Sugimori and Llin 8 1987). Toxins acting postsynaptically are now known to occur in orb weaver spider venoms (genera Argiope, Araneus and Nephila). These are low molecular weight acylpolyamine/amino acid hybrids (Grishin et al. 1986; Aramaki et al. 1986; Adams et al. 1987) which block glutamatergic transmission at insect and crustacean nerve-muscle synapses (Usherwood

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et al. 1984; Magazanik et al. 1987; Miwa et al. 1987; Adams et al. 1987; Budd et al. 1988), avian cochlear nucleus (Jackson et al. 1985), mammalian hippocampus (Saito et al. 1985; Ashe et al., in press) and amphibian spinal cord (Antonov et al. 1987). Here, we report the isolation and characterization of two classes of channel-specific toxins from Agelenopsis aperta venom, which cause distinct paralytic and neuromuscular effects in insects. The 'y are acylpolyamines causing reversible paralysis and use-dependent block of postsynaptic glutamate-sensitive receptor channels. 'gagatoxins' are polypeptide excitatory toxins causing spastic paralysis and activation of presynaptic voltage-sensitive ion channels. Evidence presented hefe indicates that the combined actions of these two types of toxins result in enhancement of paralysis caused by either class of toxin alone. A note dealing with preliminary stages of this work has appeared previously (Adams et al. 1986). Materials and methods Materials. Milked venom from Agelenopsis aperta spiders was purchased from Spider Pharm (P.O. Box 339, Black Canyon City, AZ 85324). Male and female adult spiders were collected from the field at various sites in southern California and held in individual containers for repeated milking at approximately 10-14 day intervals. The milking technique involves direct electrical stimulation of the venom apparatus in vivo and is designed to minimize contamination from digestive enzymes. Purification. Whote venom was dissolved in 1% aqueous trifluoroacetic acid (TFA) and fractionated by reversed-phase liquid chromatography (RPLC). Separations were performed using a Perkin Elmer 410 solvent delivery system equipped with a Brownlee RP-8 reversed-phase cartridge column (15 x 0.46 cm; 10 gm particle size; 300 A pore size). Peak integrations and spectral analyses were accomplished with a Hewlett-Packard series 1040A photodiode-array detector and Model 300 workstation. Venom toxins were purified in three steps according to the following solvent conditions: 1. acetonitrile/water gradient in constant 0.1% TFA, 2. acetonitrile/water gradient in constant 0.1% heptafluorobutyric acid (HFBA), and 3. l-propanol/water gradient in constant 1.0% TFA. All gradients were linear at 0.25%/min; the flow rate was 1.0 ml/min throughout. Fractions were evaporated to dryness in a Speed-Vac concentrator (Savant). Stability problems encountered with c~-agatoxins were eliminated by storage of dry samples under argon at - 8 0 ~ Such precautions were not necessary for g-agatoxins, which were stored at either - 20 or - 80 ~ Molar concentrations for y were determined by comparison of their UV absorbance peaks from RPLC to those of appropriate standard chromophores. Two of the c~-agatoxins (AG489 and AG,88) contain UV chromophores and molar extinction coefficients almost identical to the 2[Trp]-proctolin (Arg-Trp-Leu-Pro-Thr; Fig. 5), whereas the chromophores and extinction coefficients of AG 87 and AG87 match those of argiotoxin-659 (AR659) (Adams et al. 1987). Quantification of the g-agatoxins was accomplished as follows: the UV absorb-

M.E. Adams et al. : Synaptic toxins from spider venom ance peak of each g-agatoxin was integrated at 220 nm, after which aliquots were subjected to hydrolysis and amino acid analysis. Amino acid analyses were performed on a Beckman amino acid analyzer using external standards. Based on stoichiometry obtained from sequencing (Skinner et al., in press), we used molar values for arginine and serine to quantify each toxin.

Paralysis assays. Adult house flies (Musca domestica, N A I D M strain) were injected with crude venom or purified fractions from RPLC chromatography. Samples were dissolved in insect saline (in mM: NaC1, 140; KC1, 5; CaCI~, 5; MgClz, 1; NaHCO3, 4; HEPES, 5; pH=7.2) and were injected with a Hamilton syringe fitted with a 30-gauge hypodermic needle. Three-day old adult, female house flies were injected intra-abdominally with i lal volumes of test solutions. Each fraction from RPLC of whole venom (Figs. 1, 2) was evaluated (3 flies per fraction) and scored for loss of righting response, which was an unambiguous criterion for 'paralyzed'. Potency values for y shown in Table 1 are expressed as EDso values (effective dose: loss of righting response in 50% of treated insects at one hour post-injection). Because the g-agatoxins produce irreversible paralysis, biological activity is expressed as LDso (lethal dose: 50% mortality at 24 h post-injection). Relative potency data (Table 1) were obtained by probit analysis (Finney 1971) according to the method of Raymond (1985), using 10-30 flies per dose. ED87 and LD87 values were computed from log-dose-probit lines fitted to a minimum of four points. In assays measuring the joint actions of toxins (Figs. 9, 10), insects were treated as groups of 25 and scored as percent paralyzed at hourly intervals.

Neuromuscular assays. Synaptic actions of whole Agelenopsis venom and purified toxins were investigated using the longitudinal ventrolateral muscles 6A and 7A (Irving and Miller 1980) of pre-pupal Musca domestica. Muscles 6A and 7A are rectangular (300 x 700 I~m), segmentally-repeated single cell pairs, each of which receives innervation from a segmental nerve containing fast and slow motoneurons. Neuromuscular junetions in cyclorrhaphan dipteran larvae are formed on the surface of each cell and are essentially devoid of glial wrapping which normally surrounds synaptic junctions in adult insect muscle (Hardie 1976; Irving and Miller 1980). Pre-pupal muscle does not contract upon nerve stimulation, while normal electrical properties remain intact. Excitatory postsynaptic potentials (EPSPs) were evoked by applying 0.5 ms current pulses to segmental nerves via a polyethylene suction electrode. Intracellular muscle recordings were made with glass microelectrodes filled with a mixture 2 M potassium chloride and 2 M potassium acetate; tip resistances measured 15-20 Mt2. Ionophoretic glutamate potentials were obtained by passing 10 nA 10 ms negative current pulses through microelectrodes filled with 0.5 M L-glutamate (sodium salt) adjusted to pH 8.0. Electrode impedances of glutamate electrodes measure 70-100 Mf2. An Axoclamp 2 (Axon Instruments, Burlingame, CA) served as a preamplifier for intracellular recordings (bridge mode) and as the constant current source for ionophoretic pulses. Data were digitized and processed using a Data 6000 waveform analyzer (Data Precision, Danvers, MA). Alternatively, long term recordings were initially stored in analog form on a Racal Store 4 FM tape recorder for later processing. Toxins were dissolved in physiological saline (same composition as above, except that CaC12 was adjusted to 0.75 m M in order to maximize the amptitude of ionophoretic glutamate potentials) and applied by continuous superfusion via a 400 gm pipette directed at the muscle. Synthetic AR659 and Z[Trp]-proctolin (Arg-Trp-Leu-ProThr-OH) were provided by the Zoecon Research Institute, Sandoz Crop Protection, Palo Alto, CA.

M.E. Adams et al. : Synaptic toxins from spider venom

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Results

Effects of whole venom Insects injected with whole Agelenopsis venom become ataxic, exhibit tremor and develop irreversible paralysis. The time course of paralysis is dose dependent; injection of whole venom at 1:1000 dilution causes paralysis in 50% of treated flies within 30 min. Lethal paralysis is observed at dilutions as high as 1:5000, which corresponds to an equivalent dose of 0.2 nl of whole venom per fly. Whole venom causes two distinct effects on neuromuscular transmission: reduction in EPSP amplitude and initiation of repetitive activity in motor units. Perfusion of venom at 1 : 20 000 dilution leads to almost immediate reduction in EPSP amplitudes (Fig. I), followed shortly thereafter by initiation of repetitive EPSPs, which are corretated with repetitive action potentials in the segmental nerve (Fig. 2). Repetitive activity occurs spontaneously or in response to nerve stimulation and can continue uninterrupted for minutes. The repetitive burst shown in Fig. 1, (1 min) proceeded continuously for about 45 s. The onset of repetitive activity is followed by virtual blockade of EPSPs (Fig. 1, 5 min). This suppression is reversible upon removal of venom from the bath, but repetitive activity continues for hours (Fig. 2) and is essentially irreversible. The two synaptic effects, reversible EPSP suppression and repetitive activity, are related to the actions of two classes of synaptic toxins the isolation of which is described in the hext section.

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Fig. 2. Persistent repetitive activity after removal of whole venom from the bath. Partial recovery of EPSPs is observed, but repetitive activity persists for hours and is essentially irreversible. EPSPs (top) are correlated with recurrent action potentials recorded in the segmental nerve (bottom). Calibration marks: ]0mV, 100 ms

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Fig. 3. RPLC fractionation of crude Agelenops~s venom, using a stepwise series of linear gradients of acetonitrile/water in constant 0.1% TFA. Scale bar corresponds to UV absorbance at 220 nm. Multiple y elute as a large, single peak between 20 and 30 min. Biological activity associated with these fractions involves reversible paralysis in injected flies and suppression of EPSP amplitudes in neuromuscular preparations. A series of g-agatoxins eluting later produce gradual, but irreversible paralysis characterized by excitation. The g-agatoxins cause repetitive activity in neuromuscular preparations

d 20 msec Fig. 1. Synaptic actions of crude Agelenopsis venom. The larval house fly longitudinal ventrolateral muscle 6A is perfused with 1:20000 dilution of whole venom dissolved in physiological saline. Excitatory postsynaptic potentials (EPSPs) are evoked by single stimuli applied to the segmental nerve (control). Treatment with Agelenopsis venom leads to suppression of the synaptic potential within 30 s followed 1 min later by repetitive EPSP activity. This repetitive activity is followed by virtual block of EPSPs amplitudes within 5 min

lsolation of synaptic toxins Initial fractionation of whole venom using reversed-phase liquid chrom atography (RPLC) yields two groups of toxins (Fig. 3) distinguished by different column retention times and biological actions. The first group of toxins eluting between 20-30 min as a composite peak (Fig. 3, '~-agatoxins ') cause flaccid paralysis within minutes of injec-

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M.E. Adams et al. : Synaptic toxins from spider venom O~-AGATOXlNS

Hydrolysis and amino acid composition analyses of each c~-agatoxin failed to detect conventional amino acids, but a strong ammonia peak was ob489 served. We also noted a significant increase in coli488 i I u m n retention for all of the ~-agatoxins when 40 0 switching from acetonitrile/TFA to acetonitrile/ Z // H F B A solvent conditions, indicating that the toxins possess a basic character. The UV absorbance spectra of AG489 and AG488 show a tryptophan20 like pattern which closely resembles that of 2[Trp]proctolin (Fig. 5). A second type of UV spectrum t~ is observed for AGso5 and AGso4, which virtually matches that of AR659 (Fig. 5), characterized by o 70 4'0 6'o 8'0 loo its 4-hydroxyindole-3-acetic acid chromophore TIME (min) (Adams et al. 1987). Additional mass spectral and Fig. 4. Repurification of the ~-agatoxin material from Fig. 3 N M R data to be reported elsewhere (Skinner et al., resolves 4 discrete peaks using a gradient of acetonitrile/water in constant 0.1% H F B A acid. The toxins are labelled according in press) show that the y are acylpolyato their molecular weights as AGs0y AG488, AG 87 and AG~89 mines bearing structural resemblance to the indolic argiotoxins AR659 and AR673, recently isolated from venom of the orb weaver spider Argiope aurantia (Adams et al. 1987). The absorbance spectra loo 1 ~~--------.>~~ .~--,~,. presented here indicate that hydroxylation of the aromatic chromophore observed for argiotoxins and AG 87 and AGso4 is abseht in AG489 and < 6o r .~--.~ AG488. Using 2[Trp]-proctolin and AR659 as refer!// 2,TRP}-PROCTOUN '~k'~~ 4o 11" ence chromophores for mass determinations, we find that the y are present at millimolar ~2o concentrations in whole Agelenopsis venom (fange: 32 m M for AGy to 3 m M for AG 87 Table 1). 0 260 270 280 290 300 The biological potencies of the y deWAVELENGTH (nm) fined as ability to cause paralysis in 50% of inFig. 5. UV absorbance spectra of the y and related jected flies (ED87 range from 30 to 200 pmol/mg chromophores. AG488 and AG489 show tryptophan-like spectra similar to that of 2[Trp]-proctolin. AGso4 and AG 87 spec(15-100 Ixg/g; Table 1), compared to 0.8 pmol/mg tra are essentially identical to the argiotoxin AR659, which confor A R 6 5 9 . tains 4-hydroxyindole-3-acetic acid The second class of paralytic toxins, the 'txagatoxins', appear as a series of discrete peaks numbered 'lx-Aga I - V I ' according to elution order tion, which disappears within a few hours. A secunder acetonitrile/TFA solvent conditions. The IXond group of toxins (Fig. 3, 'g-agatoxins') eluting agatoxins elute in essentialty pure form as shown between 55-80 min are slow-acting, causing spasin Fig. 3, except for tx-Aga IV and V, which comodic movements, ataxia and eventual irreversible elute as a single peak under these conditions. Reparalysis. purification using an acetonitrile/water gradient of Repurification of the early-eluting composite 0.25%/min in constant 0.1% H F B A achieves compeak resolves four individual ' y plete separation of Ix-Aga IV and V. Amino acid (Fig. 4), all of which cause a rapid but transient analysis reveals the Ix-agatoxins to be cysteine-conparalysis (Figs. 9, 10). Fast atom bombardment taining peptides having relative molecular masses mass spectroscopy of each peak yielded protonated of approximately 4000. Each toxin contains eight ions (MH ยง corresponding to molecular weights cysteines, suggesting the presence of four disulfide of 489, 505, 504, and 488 (Skinner et al., in press). bridges. The Ix-agatoxins show extensive sequence homology to each other (Skinner et al., in press) The toxins are abbreviated using the first two letand all cause a similar type of paralysis and repetiters of the genus name (AG), subscripted by the molecular weight: AG489, AG488, AG 87187AG 87171 tive activity in motor units (see below). Detailed amino acid composition and sequence analyses of (Fig. 4), a nomenclature consistent with that rethe Ix-agatoxins will appear in a companion paper ported for multiple argiotoxins identified from orb (Skinner et al., in press). The Ix-agatoxins are about weaver spider venom (Adams et al. 1987). 60

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M.E. Adams et al. : Synaptic toxins from spider venom Table 1. In vivo biological potencies of agatoxins in Musca domestiea a

Toxin

Abundance (raM) b

AG489 AGs05 AG488 AGso4

32 5.3 8.7 3

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0.41 0.8 0.6 1.3 1.3 1.6

0.075_+0.005 1.38 _+0.08 0.58 + 0.06 0.03 + 0.003 0.08 _+0.02 0.15 _+0.005

a Whole venom lethal dose (LDso): 2 pL/mg (M. domestica) b Molar quantities are based on molecular weights of the free bases 3-day old adult female M. domestica. EDs0 values for c~agatoxins and AR6s9 are effective doses to paralyze 50% of treated insects at I h post-injection~ LDso values for the gagatoxins are lethal doses at 24 h post-injection a Variability in values given as _+half of the 95% fiducial limit ~ AR659 values from Adams et al. (1987)

10-fold less abundant than the ~-agatoxins, occurring in the concentration range of 0.4-1.6 mM. These toxins produce a gradual but irreversible spastic paralysis at doses down to 30 fmol/mg (Table 1), corresponding to less than 1 pmol per individual fly (average adult fly weight: 18-20 mg). Synaptic actions of o~- and p-agatoxins

Individual e-agatoxins cause dose-dependent, reversible suppression of neurally-evoked EPSPs and ionophoretic glutamate potentials in the pre-pupal neuromuscular junction preparation. Bath application of the major 0~-agatoxin, A G 4 8 9 (0.8 ~ M ) , produces 50% block of the EPSP and complete suppression of the ionophoretic glutamate potential (Fig. 6). Both responses return following 10-15 min of wash in toxin-free saline. Inhibition of both EPSPs and glutamate potentials by AG489 suggests blockade of postsynaptic glutamate-sensitive receptor channels. Neuromuscular block by AG4s9 proceeds at two distinct rates. Superfusion of toxin during continuous stimulation at 0.4 Hz leads to immediate reduction of EPSP amplitude according to a time constant of approximately 1.4 min (Fig. 7A). The fast block subsides after about 2 rain, after which a slower rate of block, described by a time constant of about 21 min, continues. Control experiments

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Fig. 6. AG~89 inhibits both the neurally-evoked EPSP and the ionophoretic glutamate potential (L-Glu). The toxin was superfused at a concentration of 0.8 g M for 10 rain, leading to a 50% reduction of EPSP amplitude and complete block of the ionophoretic glutamate potential. Saline washout of the toxin leads to almost complete recovery of both responses within 15 min (not shown). Synaptic responses were evoked at 0.4 Hz throughout the experiment

conducted over the same time flame (20 min) indicate that the slow rate of block is not the result of run-down artifact in the preparation. Removal of toxin after 10 rain of exposure leads to essentially complete recovery of the evoked EPSP. Neuromuscular block by ~-agatoxins is use-dependent. Whereas toxin perfusion during continuous stimulation causes immediate block (Fig. 7 A), exposure to toxin in the absence of stimulation has no apparent effect on EPSP amplitude until stimulation is resumed (Fig. 7 B). Upon re-application of stimuli, a progressive reduction of EPSP amplitudes is observed. Note that the first postsynaptic response is of normal amplitude, but successive stimuli produce EPSPs of progressively reduced amplitude, demonstrating use-dependence of synaptic block by AG489. The magnitude of block is essentially the same as that observed when stimuli and exposure to toxin were coupled (Fig. 7A), although the fast time course of block may be more rapid. Recovery of the EPSP also is observed upon removal of toxin. Synaptic actions of the g-agatoxins result in excitation rather than the inhibition just described for the c~-agatoxins. Perfusion of neuromuscular preparations with submicromolar concentrations of la-agatoxins leads to repetitive action potentials and EPSPs. Repetitive EPSPs and corresponding recurrent spikes in the segmental nerve (Fig. 8) are similar to the effects generated by exposure to crude venom (Figs. I, 2). The repetitive activity induced by the g-agatoxins persists for many hours after washing with toxin-free saline. Repetitive activity caused by crude venom (Fig. 2) and g-agatoxins (Fig. 8) originates presyn-

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M.E. Adams et al. : Synaptic toxins from spider venom

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Fig. 7A, B. Suppression of the excitatory junction potentials (EJP) by gM AG4a9 follows two time courses and is stimulus dependent. EJPs were neurally evoked at 0.4 Hz. Representative EJP waveforms are shown before (left), during (middle) and after (right) exposure to the toxin. Successive neurally evoked EJP amplitudes were subjected to trend analysis, which plots the time course of block and recovery as a series of dots from left to right. A Trend analysis of EJP amplitudes (dotted line) shows that toxin application (beginning at the solid triangle and ending with the open triangle) accompanied by continuous stimulation (S!) results in almost immediate suppression of the EJP. After an initial 2 min period corresponding to a fast time constant, a slower rate of block ensues, lasting for about 8 min. Saline wash (open triangle) leads to 90% recovery of the EJP amplitude. B The experiment shown in A is repeated on a different muscle, but stimuli are discontinued just prior to toxin application (arrow) and re-introduced after a 4 min pre-incubation with toxin in the absence of stimuli. Note that block of the EJP is delayed until stimulation (S.0 resumes. Scaling: vertical mV scales at the left refer both to the amplitudes of the individual waveforms as well as to each point in the trend progressions. Time calibrations correspond to the EJP (ms) and Trend (min)

aptically and appears to involve voltage-sensitive c h a n n e l s e i t h e r in t h e a x o n s o r p r e s y n a p t i c n e r v e t e r m i n a l s o f t h e m o t o n e u r o n s ( F i g s . 2, 8). If, a f t e r i n d u c t i o n o f r e p e t i t i v e a c t i v i t y b y ~t-Aga I, t h e p r e p a r a t i o n is t r e a t e d w i t h s a l i n e c o n t a i n i n g c o b a l t in p l a c e o f c a l c i u m , E P S P s d i s a p p e a r w h i l e p r e s y n aptic action potentials persist, indicating that they o r i g i n a t e p r e s y n a p t i c a l l y . R e p e t i t i v e a c t i v i t y rec o r d e d in t h e s e g m e n t a l n e r v e c a n b e a b o l i s h e d by exposure of a toxin-treated preparation to 50 n M T T X ( F i g . 8). S i n c e e x t e r n a l s o d i u m i o n s are required to generate repetitive action potentials in t h e s e g m e n t a l n e r v e , it s e e m s l i k e l y t h a t v o l t a g e

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