Joro Spider Toxin Research Articles

Graded neuromuscular transmission in the heart of the isopod crustacean Ligia exotica.

We present several lines of evidence for the occurrence of graded synaptic transmission in addition to impulse-mediated transmission at the neuromuscular junction between cardiac ganglion (CG) neurones and the myocardium in the isopod crustacean Ligia exotica. In the heart of adult Ligia exotica, the CG acts as a primary pacemaker for the heartbeat by generating periodic bursts of impulses and entrains the myogenicity of the myocardium via impulse-mediated excitatory junctional potentials. When impulse generation was blocked by tetrodotoxin (TTX; 50 nmol l(-)(1)), the CG neurones and the myocardium periodically exhibited synchronized slow depolarizing potentials. The association between the slow depolarizing potentials in the neurone and the myocardium was eliminated by application of Joro spider toxin (JSTX), a specific glutamate antagonist. When the CG neurone was made quiescent by a higher dose of TTX (1.0 micromol l(-)(1)), sinusoidal current injected into the CG neurone induced similar sinusoidal membrane potential responses in the myocardium. The sinusoidal muscle responses were eliminated by application of either JSTX or low-Ca(2+) saline. Under voltage-clamp conditions, the myocardium exhibited periodic inward current responses to sinusoidal current stimuli applied to the CG neurone. The reversal potential for the current response of the myocardium was similar to that of the impulse-mediated excitatory junctional current (EJC). Extracellular macropatch recordings of EJCs made at the neuromuscular junctional site revealed the spontaneous appearance of miniature EJCs asynchronous with the CG spikes in addition to large spike-evoked EJCs. The miniature EJCs were present in saline containing TTX, and their frequency was strongly affected by the slow membrane potential change in the CG neurone. These results suggest that the CG neurones drive the myocardium by graded neuromuscular transmission in addition to impulse-mediated transmission in the heart of Ligia exotica.

Contrasting molecular composition and channel properties of AMPA receptors on chick auditory and brainstem motor neurons.

1. Neurons in the brainstem auditory pathway exhibit a number of specializations for transmitting signals reliably at high rates, notably synaptic AMPA receptors with very rapid kinetics. Previous work has not revealed a common structural pattern shared by the AMPA receptors of auditory neurons that could account for their distinct functional properties. 2. We have used whole-cell patch-clamp recordings, mRNA analysis, immunofluorescence, Western blots and agonist-evoked cobalt uptake to compare AMPA receptors on the first-, second- and third-order neurons in the chick ascending auditory pathway with those on brainstem motor neurons of the glossopharyngeal/vagal nucleus, which have been shown to have very slow desensitization kinetics. 3. The results indicate that the AMPA receptors of the cochlear ganglion, nucleus magnocellularis and nucleus laminaris share a number of structural and functional properties that distinguish them from the AMPA receptors of brainstem motor neurons, namely a lower relative abundance of glutamate receptor (GluR)2 transcript and much lower levels of GluR2 immunoreactivity, higher relative levels of GluR3 flop and GluR4 flop, lower relative abundance of the C-terminal splice variants GluR4c and 4d, less R/G editing of GluR2 and 3, greater permeability to calcium, predominantly inwardly rectifying I-V relationships, and greater susceptibility to block by Joro spider toxin. 4. We conclude that the AMPA receptors of auditory neurons acquire rapid kinetics from their high content of GluR3 flop and GluR4 flop subunits and their high permeability to Ca2+ from selective post-transcriptional suppression of GluR2 expression.

Mechanisms of Ionotropic Glutamate Receptor-Mediated Excitotoxicity in Isolated Spinal Cord White Matter

Spinal cord injury involves a component of glutamate-mediated white matter damage, but the cellular targets, receptors, and ions involved are poorly understood. Mechanisms of excitotoxicity were examined in an in vitro model of isolated spinal dorsal columns. Compound action potentials (CAPs) were irreversibly reduced to 43% of control after 3 hr of 1 mM glutamate exposure at 37 degrees C. AMPA (100 microM) and kainate (500 microM) had similar effects. Antagonists (1 mM kynurenic acid, 10 microM NBQX, 30 microM GYKI52466) were each equally protective against a glutamate challenge, improving mean CAP amplitude to approximately 80% versus approximately 40% without antagonist. Joro spider toxin (0.75 microM), a selective blocker of Ca(2+)-permeable AMPA receptors, was also protective to a similar degree. Ca(2+)-free perfusate virtually abolished glutamate-induced injury ( approximately 90% vs approximately 40%). MK-801 (10 microM) had no effect. Glutamate caused damage (assayed immunohistochemically by spectrin breakdown products) to astrocytes and oligodendrocytes consistent with the presence of GluR2/3 and GluR4 in these cells. Myelin was also damaged by glutamate likely mediated by GluR4 receptors detected in this region; however, axon cylinders were unaffected by glutamate, showing no increase in the level of spectrin breakdown. These data may guide the development of more effective treatment for acute spinal cord injury by addressing the additional excitotoxic component of spinal white matter damage.

Contribution of Ca(2+)-permeable AMPA/KA receptors to glutamate-induced Ca(2+) rise in embryonic lumbar motoneurons in situ.

Intracellular Ca(2+) ([Ca(2+)](i)) was fluorometrically measured with fura-2 in lumbar motoneurons of acutely isolated spinal cord slices from embryonic rats. In ester-loaded cells, bath-applied glutamate (3 microM to 1 mM) evoked a [Ca(2+)](i) increase by up to 250 nM that was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 2-amino-5-phosphonovalerate (APV). CNQX or APV alone reduced the response by 82 and 25%, respectively. The glutamatergic agonists kainate (KA), quisqualate (QUI), and S-alpha-amino-3-hydroxy-5-methyl-4-isoxalone (S-AMPA) evoked a similar [Ca(2+)](i) transient as glutamate. N-methyl-D-aspartate (NMDA) was only effective to increase [Ca(2+)](i) in Mg(2+)-free saline, whereas [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD) had no effect. The glutamate-induced [Ca(2+)](i) rise was suppressed in Ca(2+)-free superfusate. Depletion of Ca(2+) stores with cyclopiazonic acid (CPA) did not affect the response. Thirty-six percent of the [Ca(2+)](i) increase in response to membrane depolarization induced by a 50 mM K(+) solution persisted on combined application of the voltage-gated Ca(2+) channel blockers nifedipine, omega-conotoxin-GVIA and omega-agatoxin-IVA. In fura-2 dialyzed motoneurons, the glutamate-induced [Ca(2+)](i) increase was attenuated by approximately 70% after changing from current to voltage clamp. Forty percent of the remaining [Ca(2+)](i) transient and 20% of the concomitant inward current of 0.3 nA were blocked by Joro spider toxin-3 (JSTX). The results show that voltage-gated Ca(2+) channels, including a major portion of R-type channels, constitute the predominant component of glutamate-induced [Ca(2+)](i) rises. NMDA and Ca(2+)-permeable KA/AMPA receptors contribute about equally to the remaining component of the Ca(2+) rise. The results substantiate previous assumptions that Ca(2+) influx through JSTX-sensitive KA/AMPA receptors is involved in (trophic) signaling in developing motoneurons.

Characterization of the AMPA-activated receptors present on motoneurons.

Motoneurons have been shown to be particularly sensitive to Ca2+-dependent glutamate excitotoxicity, mediated via AMPA receptors (AMPARs). To determine the molecular basis for this susceptibility we have used immunocytochemistry, RT-PCR, and electrophysiology to profile AMPARs on embryonic day 14.5 rat motoneurons. Motoneurons show detectable AMPAR-mediated calcium permeability in vitro and in vivo as determined by cobalt uptake and electrophysiology. Motoneurons express all four AMPAR subunit mRNAs, with glutamate receptor (GluR) 2 being the most abundant (63.9+/-4.8%). GluR2 is present almost exclusively in the edited form, and electrophysiology confirms that most AMPARs present are calcium-impermeant. However, the kainate current in motoneurons was blocked an average of 32.0% by Joro spider toxin, indicating that a subset of the AM PARs is Ca2+-permeable. Therefore, heterogeneity of AMPARs, rather than the absence of GluR2 or the presence of unedited GluR2, explains AMPAR-mediated Ca2+ permeability. The relative levels of flip/flop isoforms of each subunit were also examined by semiquantitative PCR. Both isoforms were present, but the relative proportion varied for each subunit, and the flip isoform predominated. Thus, our data show that despite high levels of edited GluR2 mRNA, some AMPARs are Ca2+-permeable, and this subset of AMPARs can account for the AMPAR-mediated Ca2+ inflow inferred from cobalt uptake and electrophysiology studies.

Long-term potentiation in the presence of NMDA receptor antagonist arylalkylamine spider toxins.

The role of the NMDA receptor (NMDAR) in long-term potentiation (LTP) is now well established. All potent NMDAR antagonists known to date inhibit the induction of LTP at the Schaffer collateral-CA1 pyramidal cell synapse in rat hippocampus, regardless of their site and mechanism of action. Arylalkylamine toxins are noncompetitive NMDAR antagonists in the mammalian central nervous system (CNS). The synthetic toxins argiotoxin-636 (Arg-636), Joro spider toxin (JSTX-3), alpha-agatoxin-489 and -505 (Agel-489 and Agel-505) and philanthotoxin-433 (delta-PhTX) were found in the present study to have no effect on the induction of LTP in the Schaffer collateral-CA1 pyramidal cell pathway in rat hippocampal slices maintained in vitro. Arylalkylamine toxins represent a class of potent NMDAR antagonists that fail to affect hippocampal LTP, and thus provide novel structural leads for the development of NMDAR antagonists that do not impair cognition.

Mechanical allodynia in rats is blocked by a Ca2+ permeable AMPA receptor antagonist.

Mild thermal injury to the hindpaw induces tactile allodynia distal to the injury. The allodynia is blocked by non-NMDA, but not NMDA, antagonists. The calcium permeable subtype of non-NMDA receptors is blocked by Joro spider toxin (JSTX). We injected JSTX or saline intrathecally followed after 5 min, 6 or 24 h by thermal injury. Rats receiving saline had decreased mechanical thresholds. Rats receiving 3 microg JSTX 5 min or 6 h prior to burn showed no allodynia. JSTX had no prominent side effects at doses between 1 and 5 microg. JSTX (5 microg) had no effect on thermal threshold. These results are consistent with the hypothesis that spinal mechanisms leading to tactile allodynia in this injury model act via a calcium permeable AMPA linkage.

Characterisation of kainate receptor mediated whole-cell currents in rat cultured cerebellar granule cells

Whole-cell voltage clamp recordings have been used to identify and characterise inward currents mediated by native kainate receptors in rat cultured cerebellar granule cells. While the selective AMPA receptor antagonist GYKI 53655 (50 μM) completely abolished inward currents evoked by AMPA (10–100 μM) in the presence of cyclothiazide (100 μM), kainate evoked currents in cells pretreated with concanavalin A (Con A) always showed a component (35–140 pA, n=13) resistant to blockade. The majority (73±7%, n=5) of GYKI 53655-resistant kainate-evoked inward currents remained in the presence of 100 μM AMPA. However, these currents were reversibly blocked by the competitive AMPA/kainate receptor antagonist NBQX (100 μM). (2 S, 4 R)-4-methylglutamate (SYM 2081, 10 μM) evoked inward currents in Con A treated cells (15–60 pA, n=7), which were resistant to complete blockade by GYKI 53655 (50 μM) but antagonised by NBQX (100 μM). Kainate-evoked responses in the presence of GYKI 53655 (50 μM) had linear or slightly outwardly rectifying current–voltage (I–V) relationships in all cells examined ( n=5) and were resistant to blockade by Joro spider toxin (JsTx, 1 μM; n=5). These results provide evidence that rat cultured cerebellar granule cells express functional kainate receptors made up of subunits which are edited at the Q/R site, and that SYM 2081 is an agonist at these native kainate receptors with a greater selectivity than kainate itself.

Glutamate Potentiates the Toxicity of Mutant Cu/Zn-Superoxide Dismutase in Motor Neurons by Postsynaptic Calcium-Dependent Mechanisms

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a subset of familial cases of amyotrophic lateral sclerosis. Using a primary culture model, we have demonstrated that normally nontoxic glutamatergic input, particularly via calcium-permeable AMPA/kainate receptors, is a major factor in the vulnerability of motor neurons to the toxicity of SOD-1 mutants. Wild-type and mutant (G41R, G93A, or N139K) human SOD-1 were expressed in motor neurons of dissociated cultures of murine spinal cord by intranuclear microinjection of plasmid expression vector. Both a general antagonist of AMPA/kainate receptors (CNQX) and a specific antagonist of calcium-permeable AMPA receptors (joro spider toxin) reduced formation of SOD-1 proteinaceous aggregates and prevented death of motor neurons expressing SOD-1 mutants. Partial protection was obtained by treatment with nifedipine, implicating Ca2+ entry through voltage-gated calcium channels as well as glutamate receptors in potentiating the toxicity of mutant SOD-1 in motor neurons. Dramatic neuroprotection was obtained by coexpressing the calcium-binding protein calbindin-D28k but not by increasing intracellular glutathione levels or treatment with the free radical spin trap agent, N-tert-butyl-alpha-phenylnitrone. Thus, generalized oxidative stress could have contributed in only a minor way to death of motor neurons expressing the mutant SOD-1. These studies demonstrated that the toxicity of these mutants is calcium-dependent and provide direct evidence that calcium entry during neurotransmission, coupled with deficiency of cytosolic calcium-binding proteins, is a major factor in the preferential vulnerability of motor neurons to disease.

ChemInform Abstract: Total Synthesis of NPTX‐5 and NPTX‐6, Joro Spider (Nephila clavata) Toxins Having a Unique Putreanine Trimer and Putreanine Tetramer Acylpolyamine Chain.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Effect of a plant-derived spider toxin analogue on crayfish neuromuscular junctions

N8-Coumaroyl spermidine (N8-CS) is an example of hydroxycinnamic acid - polyamine conjugates found in certain plants. Because of its structural similarity to some spider toxins, N8-CS was tested for its ability to block arthropod neuromuscular synapses. It inhibited chemical synaptic transmission at crayfish (Procambarus clarkii) neuromuscular junctions, the IC50 being approximately 200 µM. Its effect was at least partially reversed by washing with physiological saline. Joro spider toxin, a structurally similar compound, also blocked crayfish neuromuscular synapses but its effect was irreversible. These results suggest that plant-derived cinnamoyl spermidines might have paralytic properties similar to those of spider toxins.

Glutamatergic neuromuscular transmission in the heart of the isopod crustacean Ligia exotica.

Neuromuscular transmission between the cardiac ganglion (CG) and the myocardium was examined in the adult heart of the isopod crustacean Ligia exotica. Intracellular injection of neurobiotin into the CG neurones revealed that all six CG neurones send their axons onto the cardiac muscle, where they form axon terminals bearing varicosities. All the CG neurones and their processes exhibited glutamate-like immunoreactivity. The cardiac muscle showed depolarizing membrane potential responses to glutamate applied focally to sites close to axon terminals bearing varicosities. Both the glutamate-induced response and the excitatory junctional potential (EJP) showed desensitization in response to the repeated application of glutamate. Under voltage-clamp conditions, the cardiac muscle produced inward current responses to focally applied glutamate. The reversal potential for the glutamate-induced current estimated from extrapolation of the linear current/voltage relationship was similar to that of the excitatory junctional current evoked by ganglionic nerve stimulation. Both the glutamate-induced response and the EJP were blocked by a glutamate-specific antagonist, Joro spider toxin. These results led us to conclude that the CG neurones of Ligia exotica are glutamatergic motoneurones.

Dissociation between the Joro spider toxin sensitivity of recombinant a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and their ability to increase intracellular calcium

We compared the toxin sensitivity, Ca 2+ flux response and rectification properties of recombinant α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors obtained by transfecting human embryonic kidney (HEK) 293 cells with different ratios of GluR1 and GluR2 cDNAs (10:1 to 1:10). Simultaneous measurements of kainate-activated Ca 2+ fluxes and inward currents, using fura-2 microfluorimetry under voltage clamp conditions, suggested the existence of GluR2 containing channels which are permeable to Ca 2+ and insensitive to Joro spider toxin (JSTx). Imaging experiments showed that JSTx inhibition of the Ca 2+ response induced by kainate was reduced by increasing the relative amount of GluR2. However, even at GluR1/GluR2(R) ratios of 1:1 and 1:4, cells were still able to flux Ca 2+ when stimulated by kainate. GluR2 similarly inhibited the ability of JSTx to reduce kainate-evoked inward currents in whole cell patch-clamp experiments. Variations in the rectification properties of the AMPA currents, induced by changes in the cDNA ratio, were not always correlated with the changes in toxin sensitivity and [Ca 2+] i response . Thus, cells with almost linear I– V relationships were partially blocked by JSTx and still Ca 2+ permeable. Our results indicate a dissociation between the toxin sensitivity and Ca 2+ flux through GluR2 containing AMPA receptors and suggest that receptors with diverse Ca 2+ permeabilities are generated by the expression of variable amounts of GluR2.

Isolation of Neurogenic and Myogenic Activities by Joro Spider Toxin in the Adult Heart of the Isopod Crustacean Ligia exotica

Abstract The effects of Joro spider toxin (JSTX), a specific glutamate antagonist, on the adult heart of the isopod crustacean Ligia exotica were examined. By application of JSTX, excitatory junctional potentials (EJPs) caused by the cardiac ganglion activity in the myocardium were gradually abolished. Subsequently, the cardiac ganglion and myocardium exhibited independent activities with their respective rhythms. In saline containing JSTX, no changes were observed in the muscle activity when the ganglionic activity was changed by current injection into the cardiac ganglion neuron. These results indicate that two pacemaker sites, the cardiac ganglion and cardiac muscle, are present in the adult heart of Ligia exotica and suggest glutamatergic neuromuscular transmission between them.

Total synthesis of NPTX-5 and NPTX-6, Joro spider ( Nephila clavata) toxins having a unique putreanine trimer and putreanine tetramer acylpolyamine chain

The first total synthesis of NPTX-5 and NPTX-6, Joro spider ( Nephila clavata) toxins having a 4-hydroxyindole nucleus and a unique putreanine trimer and tetramer acylpolyamine chain, respectively, has been achieved by the iterative use of a key azide compound.

Neurotransmitters of motor neurons in the stomatogastric ganglion of an isopod crustacean, Ligia exotica

Neurotransmitters of motor neurons in the foregut muscles of an isopod Ligia exotica were identified by recording changes in membrane potential to exogenously applied glutamate and acetylcholine. The effects of antagonists, tubocurare and joro spider toxin, on excitatory junctional potentials evoked by nerve stimulation and by iontophoretic application of glutamate and acetylcholine provided additional evidence for identification. The junctional receptors were desensitized by putative neurotransmitters. Glutamate is a candidate as an excitatory neurotransmitter at the neuromuscular junctions in intrinsic muscles of the gastric mill and pylorus, and acetylcholine is a candidate in the extrinsic muscles of the gastric mill and cardiopyloric valve.

Ca 2+ permeability and Joro spider toxin sensitivity of AMPA and kainate receptors on cerebellar granule cells

We have investigated the Ca 2+ permeability of native kainate- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate- (AMPA) receptors in cultured rat cerebellar granule cells. Intracellular Ca 2+ ([Ca 2+] i) increases and Mn 2+ quench of fura-2 (a measure of Ca 2+ entry) mediated by kainate receptors were completely dependent on the presence of extracellular Na +. Kainate receptor-mediated [Ca 2+] i rises were reduced 37% by the L-type voltage-gated Ca 2+ channel blocker nifedipine (1 μM). AMPA receptor-mediated [Ca 2+] i rises observed in Na +-free buffer were sensitive to Joro spider toxin (500 nM) blockade showing a 65% reduction, while kainate receptor-mediated [Ca 2+] i responses were largely insensitive. These results suggest that a component of AMPA receptor-mediated [Ca 2+] i increases occurs through Ca 2+ permeable receptors which lack the GluR2 subunit and are Joro spider toxin sensitive. In contrast, kainate receptors do not appear to directly gate significant Ca 2+ but raise [Ca 2+] i through activation of voltage-gated Ca 2+ channels and seem largely insensitive to Joro spider toxin.

L-glutamate as an Excitatory Transmitter of Motor Giant Neurons in the Crayfish Procambarus Clarkii

Motor giant neurons (MoGs) of the crayfish are identifiable motor neurons that innervate abdominal fast flexor muscles. The role of L-glutamate as a neurotransmitter at the neuromuscular junction between MoG and the abdominal fast flexor muscles was investigated using electrophysiological and pharmacological techniques. Local application of L-glutamate to a fast flexor muscle induced a depolarization of the muscle fibers. This glutamatergic depolarization was dose-dependent and was associated with a conductance increase. Bath application of L-glutamate caused a reversible reduction in the amplitude of the excitatory junctional potentials (EJPs) of the fast flexor muscles evoked by MoG stimulation. Twenty micromole L-glutamate was sufficient to reduce the amplitude of MoG-evoked EJP. The effect of depression was dose-dependent and MoG stimulation elicited almost no response in flexor muscle fibers under 1 mM L-glutamate application. Joro spider toxin, an L-glutamate antagonist, reduced the amplitude of EJPs elicited by MoG stimulation at a concentration of 0.1 tAM. The response of the flexor muscle to MoG stimulation did not recover completely after washing. When 5 jAM Joro spider toxin was applied, the EJP evoked by MoG was completely blocked and no recovery was observed after washing. These results strongly suggest that L-glutamate is the neurotransmitter at the neuromuscular junction between MoG and the abdominal fast flexor muscle in the crayfish.

Effect of Glutamate on Dendritic Growth in Embryonic Rat Motoneurons

Glutamate is a major excitatory neurotransmitter for spinal motoneurons. We have investigated its effect on survival and neurite formation in cultures of highly enriched motoneurons from 15-d-old rat embryos. Whereas the survival of these neurons was not reduced by this treatment, a distinct and specific effect on dendrite outgrowth could be observed. Axon outgrowth was not affected by glutamate. Our data suggest that calcium influx via ionotropic AMPA/kainate (AMPA/KA) receptors is responsible for the regulation of dendrite outgrowth by excitatory neurotransmission. This was shown by the use of specific inhibitors for the different classes of glutamate receptors. The effect was reduced by continuous depolarization at 35 mM KCl and by treatment with joro spider toxin (JSTX-3, 3 microM), a blocker of Ca2+-conducting AMPA receptors. Removal of glutamate after 5 d of culture led to increased dendrite growth during the following culture period, and delayed addition resulted in a reduction in the length of already existing dendrites. Our observation that the effect is dose-dependent and reversible reflects a potential physiological function of excitatory neurotransmission on dendrite growth and morphology during a developmental period when synaptic contacts from afferent neurons to motoneurons are made in the spinal cord.

ChemInform Abstract: Total Synthesis of Nephilatoxin‐1 (NPTX‐1), a Joro Spider (Nephila clavata) Toxin Having a 4‐Hydroxyindole Nucleus.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.