Modification of Synaptic Transmission and Sodium Channel Inactivation by the Insect-Selective Scorpion Toxin Lqhalpha IT

Daewoo Lee,1 Michael Gurevitz,2 and Michael E. Adams1

 1Departments of Entomology and Neuroscience, University of California at Riverside, Riverside, California 92521; and  2Department of Botany, Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lee, Daewoo, Michael Gurevitz, and Michael E. Adams. Modification of Synaptic Transmission and Sodium Channel Inactivation by the Insect-Selective Scorpion Toxin Lqhalpha IT. J. Neurophysiol. 83: 1181-1187, 2000. The peptide Lqhalpha IT is an alpha -scorpion toxin that shows significant selectivity for insect sodium channels over mammalian channels. We examined the symptoms of Lqhalpha IT-induced paralysis and its neurophysiological correlates in the house fly (Musca domestica). Injection of Lqhalpha IT into fly larvae produced hyperactivity characterized by continuous, irregular muscle twitching throughout the body. These symptoms were correlated with elevated excitability in motor units caused by two physiological effects of the toxin: 1) increased transmitter release and 2) repetitive action potentials in motor nerves. Increased transmitter release was evident as augmentation of neurally evoked synaptic current, and this was correlated with an increased duration of action potential-associated current (APAC) in loose patch recordings from nerve terminals. Repetitive APACs were observed to invade nerve endings. The toxin produced marked inhibition of sodium current inactivation in fly central neurons, which can account for increased duration of the APAC and elevated neurotransmitter release at the neuromuscular junction. Steady-state inactivation was shifted significantly to more positive potentials, whereas voltage-dependent activation of the channels was not affected. The shift in steady-state inactivation provides a mechanism for inducing repetitive activity in motoneurons. The effects of Lqhalpha IT on sodium channel inactivation in motor nerve endings can account both for increased transmitter release and repetitive activity leading to hyperactivity in affected insects.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Scorpions have evolved a diversity of peptide toxins comprising an effective biochemical strategy for prey capture. These toxins modify ion channels in nerve membranes, producing physiological changes leading to various types of excitatory or flaccid paralysis. A primary target of scorpion venom toxins is the voltage-sensitive sodium channel, and an extensive literature is available on their modification of mammalian (Couraud and Jover 1984; Martin-Eauclaire and Couraud 1995; Strichartz et al. 1987) and insect channels (Zlotkin et al. 1994). alpha -Scorpion toxins were some of the first to be described in detail, both with respect to biochemical characteristics and neurophysiological actions. To a rather surprising degree, the alpha -scorpion toxins are characterized by their specific modification of vertebrate sodium channels, despite the fact that scorpions prey primarily on insects. Indeed, the binding site for alpha -scorpion toxins (site 3) (Catterall et al. 1992) is defined exclusively by characteristic epitopes on the mammalian sodium channel (Rogers et al. 1996).

A variety of scorpion toxins specific for insect sodium channels have been identified (Zlotkin et al. 1971, 1994), but only recently was an alpha -scorpion toxin-like peptide with potency against insects discovered. This toxin, Lqhalpha IT has high sequence-similarity to alpha -scorpion toxins and prolongs action potentials in a manner characteristic of this class of peptides (Adam et al. 1966; Eitan et al. 1990; Wang and Strichartz 1985). This effect results from inhibition of sodium channel inactivation (Catterall et al. 1992; Strichartz et al. 1987). Although also active against mammalian sodium channels, Lqhalpha IT differs from previously described alpha -scorpion toxins in that it shows preference for insect channels (Eitan et al. 1990; Gordon et al. 1996; Gordon and Zlotkin 1993). Structural correlates of the toxin responsible for insect activity recently have been defined (Zilberberg et al. 1996, 1997).

In some respects, the alpha -scorpion toxins are among the most well-characterized elements of the scorpion venom cocktail. Many electrophysiological studies have documented their inhibition of mammalian sodium channel inactivation (Adam et al. 1966; Martin-Eauclaire and Couraud 1995; Wang and Strichartz 1985), and the characteristic alpha -scorpion toxin binding site (site 3) has been pinpointed through site-directed mutagenesis and photoaffinity labeling (Rogers et al. 1996). However, few studies have documented the symptoms associated with alpha -scorpion toxin action in the intact animals, and little has been done to relate these to their effects on synapses. It is interesting to note that alpha -scorpion toxins do not alter the sodium channel activation mechanism, nor are they associated with persistent depolarization of nerve membranes (Catterall 1980). Nevertheless, Lqhalpha IT is reported to cause hyperexcitation in treated insects, the natural prey animals of scorpions (Eitan et al. 1990).

Here we provide a detailed examination of alpha -scorpion toxin action on an insect model at three levels: behavioral, synaptic, and sodium channel modification. We find that hyperexcitability produced in treated fly larvae is likely caused by two consequences of toxin on nerve terminals: increased transmitter release and repetitive firing of action potentials. These effects in turn can be attributed to prolongation of inward sodium current in nerve terminals and a shift of steady-state sodium channel inactivation to more positive potentials.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Scorpion toxin

In most experiments, native Lqhalpha IT (MW = 7,258) purified from the venom of Leiurus quinquestriatus hebraeus was used (Eitan et al. 1990). In some experiments (Figs. 4 and 5), the recombinant toxin produced in Escherichia coli was employed (Zilberberg et al. 1996).

Bioassay

House flies (Musca domestica; NAIDM strain) were obtained from laboratory colonies maintained in the Department of Entomology, University of California, Riverside. For toxicity assays, third instar house fly larvae were injected with various concentrations of Lqhalpha IT dissolved in insect saline through an abdominal intersegmental membrane. All larvae used in this bioassay weighed ~20 mg.

Intracellular recording

House fly prepupae were dissected, pinned in silicone elastomer (Sylgard) dishes, and flooded with physiological saline. The saline formulation is (in mM) 135 NaCl, 5 KCl, 0.75 CaCl2, 1 MgCl2, 5 NaHCO3, and 5 HEPES, adjusted to pH 7.2. Muscles for recordings were obtained from the longitudinal ventrolateral muscles 6A and 7A of house fly (Bindokas and Adams 1989; Irving and Miller 1980). Excitatory junctional potentials (EJPs) were evoked by a suction electrode attached to the cut ends of motor nerves. Isolated voltage pulses at a rate of 0.4 Hz were generated by a Grass S88 stimulator. EJPs were measured intracellularly with glass microelectrodes (5-10 MOmega ) filled with 3 M KCl. Voltages were amplified with an Axoclamp 2A (Axon Instruments) amplifier, and signals were digitized and processed with Data 6000A waveform analyzer (Analogic Instruments, Woburn, MA).

Two electrode voltage clamp

Synaptic currents from house fly muscles were evoked by stimulation of a motor nerve via the suction electrode and recorded with an Axoclamp 2A in two-electrode voltage-clamp mode. Tip resistance of current passing and recording electrodes were around 2 and 5-10 MOmega , respectively. Output signals were filtered with a Warner Instruments LPF-100 low-pass filter at 0.5 kHz.

Loose patch-clamp recordings

Miniature excitatory junctional currents (MEJCs), excitatory junctional currents (EJCs), and action potential-associated currents (APACs) were measured extracellularly from house fly neuromuscular junctions by patch pipettes that were pulled and fire-polished to achieve an internal tip diameter of 10-15 µm as previously described (Bindokas and Adams 1989). The saline formulation for loose patch-clamp recordings is the same as that for intracellular recordings except for the addition of 4 mM MgCl2 to minimize minute muscle movements. Saline-filled pipettes were placed over neuromuscular junctions, located by manipulation of pipette tips to sites producing maximum amplitude records after nerve stimulation. Once a large amplitude synaptic current was obtained, slight downward force was applied to produce approximate seal resistance of ~0.5 MOmega . The loose seal permitted relatively rapid access to bath-applied toxins. Preparations were allowed to equilibrate for at least 20 min after positioning the pipette to minimize the effects of mechanical disturbance. To monitor APACs at high gain, saline containing 2-5 mM CoCl2 or CdCl2 (see figure captions) was perfused to block EJCs. MEJCs, EJCs, and/or APACs were amplified with an Axopatch 1B (Axon Instruments), and output was filtered at 1 kHz through a four-pole low-pass Bessel filter and processed with a Data 6000A. Recordings usually were made in an AC-coupled, "auto-track" mode, which often caused a small amount of overshoot in current traces.

Preparation of house fly central neurons

Neurons from adult house fly thoracic and abdominal ganglia were dissociated and cultured for 1-2 days. Short-term culturing promotes expression of functional sodium channels in neuronal cell bodies that are normally inexcitable due to low expression of these channels. Ganglia were dissected in ice-cold house fly saline containing (in mg/100 ml) 800 NaCl, 20 KCl, 5 NaH2PO4, 100 NaHCO3, and 100 glucose, pH 7.2 (Wu et al. 1983). Ganglia were treated for 1 h at room temperature with 1 mg/ml collagenase (Type IA, Sigma). After washing, ganglia were gently triturated in Schneider's Drosophila medium (GIBCO) supplemented with 15% fetal bovine serum, 50 mg/ml streptomycin, 50 unit/ml penicillin, and 0.05 mg/ml insulin (O'Dowd and Aldrich 1988; Wu et al. 1983). Dissociated neuronal cell bodies were plated onto poly-D-lysine-coated dishes and incubated for 1-2 days at room temperature.

Whole cell recordings of neuronal sodium currents

Neuronal sodium currents were recorded in whole cell configuration (Hamill et al. 1981) using 1-2 MOmega Sylgard-coated patch pipettes, manufactured from borosilicate glass (Boralex glass; Dynalab). Pipettes were filled with (in mM) 70 CsF, 70 CsCl, 2 MgCl2, 0.1 CaCl2, 1.1 EGTA, and 10 HEPES (pH 7.2), whereas neurons were bathed in a recording solution containing (in mM) 140 NaCl, 3 KCl, 4 MgCl2, 2 CaCl2, 1 CoCl2, 20 TEA-Cl, 1 4-aminopyridine (4-AP), and 5 HEPES (pH 7.2). Currents were recorded using an Axopatch 200A amplifier (Axon Instruments) and filtered at 2 kHz. Neurons were clamped at a holding potential of -105 mV. Currents were evoked by brief depolarizing steps to test potentials (VT). Eighty-five percent of series resistance was compensated, and leak currents were on-line subtracted by using a P/4 procedure (Bezanilla and Armstrong 1977). Data were compensated for liquid junctional potentials of +5 mV on average. Cell stimulation and data acquisition were performed using pCLAMP 5.5.1 software (Axon Instruments) in Dell 466/MX personal computer.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lqhalpha IT induces an excitatory paralysis

Injection of house fly larvae with microgram doses of Lqhalpha IT caused hyperactivity leading to paralysis and death. Larvae (~20 mg body wt) injected with 1.5 µg Lqhalpha IT exhibited moderate sporadic contractions; local twitching and rolling of the body occurred within 1-2 min of injection. Within 10 min intense hyperactivity, manifested as uncoordinated and continuous muscle twitches throughout the insect body, was observed. In the dose range 0.15-1.5 µg/larva, hyperactivity persisted for several days until death occurred. In contrast, larvae injected with an equivalent volume (1 µl) of distilled water (n = 3) or physiological saline (n = 7) showed none of the symptoms mentioned above.

Augmentation of synaptic responses and repetitive activity caused by Lqhalpha IT

We observed the effects of Lqhalpha IT on neuromuscular responses after bath application of toxin to the larval musculature in vitro. Body wall muscles of house fly prepupae typically respond to nerve stimulation with a single EJP, but do not contract (Fig. 1, dotted lines). At the lowest concentrations of Lqhalpha IT (300 pM), evoked synaptic responses increased in size (Fig. 1A), and visual inspection revealed appearance of a twitch contraction in the postsynaptic cell. A second effect that emerged at higher toxin concentrations (1.5 nM) was repetitive synaptic responses to individual stimuli (Fig. 1B). This resulted in even larger twitch contractions. This repetitive activity in response to toxin exposure only occurred after nerve stimulation; spontaneous repetitive activity in the absence of applied stimuli was not observed in any of the experiments conducted (n = 11). Both the increased amplitude of synaptic responses and repetitive activity resulting from Lqhalpha IT exposure are indicative of increased excitability in house fly motor units and provide a physiological basis for the symptoms observed in vivo.



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Fig. 1. Effects of Lqhalpha IT on neurally evoked excitatory junctional potentials (EJPs) recorded in vitro. Dotted traces (a) in A and B are control responses recorded before toxin exposure. Trace b in A: after exposure to 300 pM Lqhalpha IT, the synaptic response was increased. Higher concentrations of Lqhalpha IT (1.5 nM) caused repetitive EJPs (trace b in B).

Lqhalpha IT prolongs the duration of the presynaptic action potential

We investigated further the physiological basis of increased excitability by measuring synaptic currents. Using two electrode voltage clamp, we measured evoked synaptic currents in muscles 6A and 7A (Fig. 2A, trace a). As shown in Fig. 2A, trace b, application of 5 nM Lqhalpha IT caused a significant increase in the amplitude of the evoked EJC after 5 min of exposure. After 10 min., the EJC was increased by threefold, and repetitive responses were registered (Fig. 2A, trace c).



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Fig. 2. Effect of Lqhalpha IT on synaptic currents. A: evoked synaptic currents (EJCs) recorded in the body wall muscles of house fly using the 2-electrode voltage-clamp technique. The amplitude of the control EJC (trace a) was increased 5 min after application of 5 nM Lqhalpha IT (trace b). Trace c: after 10 min exposure, a further increase of the evoked EJC was evident as well as repetitive responses. B: averaged spontaneous miniature excitatory junctional currents (MEJCs) recorded using a loose patch-clamp technique before and after application of 2.5 nM toxin. Averaged MEJCs before and after toxin exposure are shown (224 events averaged over 4 experiments). C: the frequency of spontaneous MEJCs before (Control) and after (Lqhalpha IT) application of toxin (2.5 nM). Error bars depict standard deviation for 4 experiments.

Because alpha -scorpion toxins are known to affect neuronal sodium channels, we hypothesized that the increase in EJC amplitude resulted from a presynaptic action of Lqhalpha IT, probably through elevated transmitter release. However, augmented EJC amplitudes also could result from increased sensitivity of the postsynaptic cell to released neurotransmitter, a postsynaptic effect. The latter possibility would be reflected as larger amplitudes of spontaneous MEJCs after toxin exposure. In loose patch-clamp recordings from junctional areas under control conditions, we observed MEJCs, the amplitudes of which remained unchanged after exposure to the toxin (Fig. 2B). We averaged a total population of 224 MEJCs accumulated from 4 independent experiments before and after application of 2.5 nM Lqhalpha IT and found that the amplitudes remained unchanged. These data indicate that postsynaptic membrane sensitivity to released transmitter was unaltered by the toxin. We also observed no significant difference in the frequency of spontaneous MEJCs before and after toxin exposure (Fig. 2C).

We next analyzed the effect of Lqhalpha IT on nerve terminal action potentials. Simultaneous measurements of presynaptic currents and postsynaptic potentials are shown in Fig. 3. Under normal conditions, nerve stimulation led to single EJCs and EJPs. Preceding each EJC was a small current that appeared in all-or-none fashion as stimulus intensity was increased. This current, which we refer to as APAC, appeared abruptly at the same stimulus intensity as the EJC and EJP. After application of Lqhalpha IT (10 nM), two obvious changes in synaptic responses were observed: increased EJC and EJP amplitude, and repetitive activity. Notably, each EJC was preceded by an APAC, suggesting that repetitive postsynaptic events originated presynaptically.



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Fig. 3. Pre- and postsynaptic events after exposure to Lqhalpha IT. Action potential-associated currents (APACs, *), and EJCs were recorded with a loose patch pipette; the EJP was recorded using an intracellular micropipette. Control traces are dotted lines. After application of Lqhalpha IT (10 nM), repetitive EJCs and EJPs (solid lines) were observed. APACs denoted by asterisks precede each EJC in the top trace, indicating that the repetitive activity in postsynaptic muscle was caused by the repetitive activity of action potential invading presynaptic terminal.

To confirm that the APAC indeed represented the presynaptic action potential, we first blocked the EJC by application of 2 mM cadmium (Fig. 4A). No effect on the APAC was evident. Subsequent addition of tetrodotoxin (2 µM) to the bath abolished the APAC, confirming its dependence on voltage-activated sodium channels. We conclude from this experiment that the APAC represents the presynaptic action potential as it invades the nerve terminal.



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Fig. 4. APAC is cadmium insensitive, but TTX sensitive. A: nerve stimulation (stimulus artifact; SA) leads to APAC (curved arrow) and EJC under normal conditions. The large overshoot of the EJC is due to AC coupling in the recording caused by the amplifier tracking circuit, which stabilizes the current recording at high gain. B: addition of 2 mM CdCl2 to the bath abolishes the EJC. C: addition of TTX (2 µM) to the bath eliminates the APAC. Asterisks denote occurrence of APACs.

Exposure of the preparation to Lqhalpha IT (20 nM) led within 1 min to repetitive EJCs, each preceded by an APAC (Fig. 5, A-C). After addition of cadmium to the bath, repetitive APACs could be observed in the absence of EJCs. Again, APACs were abolished after TTX application (Fig. 5D). Under these conditions, we noted a significant increase in duration of the APAC. Further analysis of the APAC at high gain showed that Lqhalpha IT increased APAC duration by more than twofold (n = 5; Fig. 6). In some experiments, variability in the amplitude of the APAC was observed, probably due to slight changes in the position of the loose patch pipette caused by minute muscle movements. For this reason, APACs often were normalized for comparison. These results indicate that action potentials invading nerve terminals are prolonged after exposure to Lqhalpha IT. To verify that these changes originate at the level of the sodium channel, we recorded whole cell currents from dissociated central neurons.



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Fig. 5. Lqhalpha IT induces repetitive synaptic currents and prolongation of the APAC. A: normal response to nerve stimulation; SA, stimulus artifact; APAC, action potential-associated current; EJC, excitatory junctional current. B: repetitive currents recorded after application of 20 nM Lqhalpha IT. C: APACs (*) recorded in the presence of toxin and 2 mM CdCl2. D: all responses are abolished after exposure to 2 µM TTX.



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Fig. 6. Prolongation of APAC by Lqhalpha IT. Exposure to 20 nM toxin caused more than a doubling of the APAC duration. Each trace is the average of 10 events. These experiments were carried out in 5 mM CoCl2 to block postsynaptic currents. APACs are normalized for comparison (see text).

Slowing of sodium channel inactivation by Lqhalpha IT

We recorded voltage-dependent sodium currents from dissociated central neurons (20-30 µm diam) of M. domestica using whole cell patch-clamp recordings. Because recordings from freshly dissociated fly neurons produced only minute sodium currents, a protocol was developed for short-term cultured central neurons dissociated from thoracic and abdominal ganglia (Fig. 7A). Robust, voltage-dependent sodium currents were measured in cultured neurons within 1-2 days (Fig. 7B). Currents activated in the range of -30 and -40 mV and inactivated rapidly. Peak currents occurred at around -5 mV (Fig. 8, A and D). All currents were uniformly sensitive to TTX, and we observed no evidence of biophysical or pharmacological heterogeneity in any of the neurons sampled.



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Fig. 7. Sodium currents in central neurons of the house fly. A: photographs of short-term cultured neurons from thoracic and abdominal ganglia. Neuronal cell bodies were cultured for 1-2 days (left and middle panels) before recording. Monopolar neurons of this size and appearance were used for recording sodium currents. Cultured neuron attached to the tip of a patch pipette (right panel) placed at the exit of a glass perfusion pipe for rapid perfusion of the Lqhalpha IT. Scale bar in each photograph indicates 20 µm. B: sodium currents were completely abolished by 0.5 µM TTX. C: application of 20 nM Lqhalpha IT dramatically increased steady-state sodium current [INa(s-s)] while producing a slight increase in peak current amplitude. Subsequent application of 0.5 µM TTX abolished the current. D: steady-state sodium current was plotted over time before and after application of Lqhalpha IT. Application of 20 nM toxin (white bar) produced an increase in INa(s-s). The time course of this increase was best fitted with a double exponential. After a break in sampling from ~40 to 320 s, resumption of stimuli revealed level of INa(s-s) inactivation predicted by the exponential function. Subsequent addition of 40 nM LqhaIT (gray bar) produced no further increase in INa(s-s). All current was abolished by addition of 1 µM TTX (black bar).



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Fig. 8. Sodium current (I-V) relationships before (A) and after (B) application of Lqhalpha IT (20 nM) evoked by test potentials from -45 to -10 mV in 5-mV increments. The holding potential was -105 mV. C: steady-state sodium currents were sampled at points indicated in A and B by arrows [INa(s-s)], and plotted as a function of test potentials. D: typical I-V curves for peak currents before and after application of Lqhalpha IT.

Lqhalpha IT (20 nM) produced a large increase in steady-state sodium current without changing the rising phase of the current (Figs. 7C and 8B). Higher concentrations (40 nM) of Lqhalpha IT produced no further change (n = 3), indicating that the effect of the toxin at 20 nM was complete within 1 min of application (Fig. 7D). All neurons sampled had TTX-sensitive sodium currents, confirmed by application of this toxin at the conclusion of the experiment. Voltage-dependent activation was unaffected, but peak sodium currents were slightly augmented (Figs. 8D and 9A). Test potentials to activate 50% of sodium channels, were -19.6 ± 0.2 mV before and 20.8 ± 0.6 mV after application of the toxin, respectively. No changes in the reversal potential were evident.



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Fig. 9. Voltage-dependent activation (A) and inactivation (B) of sodium channels in house fly neurons before and after application of Lqhalpha IT (20 nM). A: normalized sodium conductance plotted as a function of test potential. Each value for sodium conductance (gNa) was calculated by an equation, gNa = INa/(VT - Erev) and normalized to maximum sodium conductance. INa is a peak current evoked by a test potential (VT), and Erev is the reversal potential of sodium current. B: double-pulse protocol (inset) used to plot sodium channel inactivation curves. Each peak sodium current was evoked by a test potential (VT; -10 mV) after various prepulses (VP). Holding potential was -105 mV. Peak sodium currents were normalized to the maximum peak sodium current and plotted as a function of prepulses. Bars indicate SE in A and B.

Modification of sodium channel inactivation by Lqhalpha IT was particularly obvious in comparisons of steady-state inactivation (Fig. 9B). For these curves, a double-pulse protocol was employed, consisting of 100-ms prepulses to various voltages followed by the test potential (-10 mV; Fig. 9B). Peak sodium currents evoked by the test potential were normalized to the maximum peak current and plotted as a function of prepulse potentials. The hinfinity curve was fitted by the Boltzman equation: hinfinity  = ((1 - C)/{1 + exp[(VP - V1/2)/k]}) + C, where V1/2 is the voltage at half-inactivation, VP and k are the prepulse potential and the slope factor, respectively, and C is the noninactivated fraction. Lqhalpha IT (20 nM) shifted the hinfinity curve to more positive potentials and produced a small percentage of steady-state current (~10%) that remained activated (Fig. 9B). We note in particular that, under normal conditions, a -20-mV prepulse inactivated virtually all available sodium channels in the absence of the toxin. After Lqhalpha IT treatment, ~40% of the current remained at this prepulse potential.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that the behavioral effects of Lqhalpha IT in the house fly are associated with modification of neuromuscular transmission. The early effects of Lqhalpha IT are characterized by local twitching of the body wall and rolling of the whole insect. Subsequently, the body wall musculature throughout the insect becomes progressively more hyperactive, culminating in lethality. Our observations indicate that these symptoms can be explained by increased excitability of efferent motor units through two main mechanisms: 1) elevation of evoked neurotransmitter release and 2) repetitive firing of motor axons.

Elevated levels of transmitter release, evidenced here as increases in EJP and EJC amplitudes, are likely the result of a prolonged action potential duration in motor neurons. Evidence for this comes from the increased durations of action potential-associated current invading motor nerve terminals. Sustained depolarization of the nerve terminal allows for increased Ca2+ influx and hence increased neurotransmitter release (Katz and Miledi 1967). The progressive increase in transmitter release caused by the toxin accounts for the gradual increases in twitch contractions observed in affected animals.

Repetitive activity caused by Lqhalpha IT was evident both presynaptically in nerve terminals (APAC, EJCs) and postsynaptically in body wall muscles (EJPs). This repetitive activity originates in the nerve terminal because it persists in the presence of extracellular cobalt, which abolishes postsynaptic responses through blockade of transmitter release. Furthermore, each postsynaptic event was found to be preceded by an action potential-associated current. Repetitive motor-unit activity combined with the appearance of muscle action potentials lead to intense and prolonged muscle contraction observed at the behavioral level.

The neuromuscular effects of Lqhalpha IT can be understood at the level of the sodium channel as a modification of the sodium channel inactivation process. We found that steady-state inactivation in house fly neurons is increased after exposure to 20 nM Lqhalpha IT. Furthermore, the steady-state inactivation curve is shifted markedly to more positive potentials and is incomplete even at test potentials greater than or equal to +20 mV. Similar concentrations of the toxin also inhibit inactivation of sodium channels in cockroach giant axons, mammalian muscle fibers (Eitan et al. 1990), and mammalian peripheral nerves (Martin-Eauclaire and Couraud 1995; Strichartz et al. 1987; Wang and Strichartz 1982, 1985). Such effects of the toxin are not observed in insect muscle, due to the absence of voltage-activated sodium current. Instead, calcium channels are responsible for excitatory electrogenesis in insect muscle.

We have shown that, under normal conditions, a significant portion of the hinfinity curve for housefly central neurons lies to the left of the minfinity curve. However, after toxin exposure, the steady-state inactivation curve shifted to the right of the minfinity curve, providing a plausible explanation for the appearance of repetitive firing in house fly motoneurons. The consequences of decreased steady-state sodium channel inactivation include changes in threshold and tendency to fire repetitively (Vallbo 1964). We have shown that the specific effects of Lqhalpha IT on house fly motor units include inhibition of sodium channel inactivation, prolonged action potential duration, and increased transmitter release. These observations provide a physiological basis for the in vivo effects of Lqhalpha IT.

Although Lqhalpha IT cannot be characterized as an "insect-specific" neurotoxin, it is reported to be more selective for insects (Eitan et al. 1990). These workers cited a 20-fold higher toxicity to insects over mammals. Data presented here show that 20 nM Lqhalpha IT produces maximal inhibition of sodium channel inactivation in house fly neurons. Much higher concentrations (>200 nM) are needed to elicit similar effects on sodium channels in rat dorsal root ganglion neurons (Norris et al. 1995; Zilberberg et al. 1996). In combination, these data provide a molecular basis for the selective toxicity of Lqhalpha IT in insects versus mammals.


    ACKNOWLEDGMENTS

We thank Dr. David N. Mbungu for valuable assistance during the early stages of this project.

This work was supported by Binational Agricultural Research and Development Fund Grants IS-1982-91 and IS-2486-94C.

Present address of D. Lee: Dept. of Anatomy and Neurobiology, University of California, Irvine, CA 92697.


    FOOTNOTES

Address for reprint requests: M. E. Adams, Dept. of Neuroscience, 5419 Boyce Hall, University of California, Riverside, CA 92521.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4 August 1999; accepted in final form 1 November 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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