Joro Spider Research Articles

Activated PKA and PKC, but not CaMKIIα, are required for AMPA/Kainate-mediated pain behavior in the thermal stimulus model

Secondary mechanical allodynia resulting from a thermal stimulus (52.5 °C for 45 s) is blocked by intrathecal (i.t.) pretreatment with calcium-permeable AMPA/KA receptor antagonists, but not NMDA receptor antagonists. Spinal sensitization is presumed to underlie thermal stimulus-evoked secondary mechanical allodynia. We investigated whether this spinal sensitization involves activation and phosphorylation of calcium-dependent protein kinases (PKA, PKC and CaMKIIα), and examined if the noxious stimulus increases phosphorylated AMPA GLUR1 (pGLUR1 Ser-845 and pGLUR1 Ser-831). Secondary mechanical allodynia after thermal stimulation was not altered by i.t. pretreatment with control vehicles (saline or 5% DMSO). Comparable allodynia was observed after pretreatment with a selective CaMKIIα inhibitor (17 and 34 nmol KN-93). In marked contrast, pretreatment with either a PKA (10 nmol H89) or PKC (30 nmol chelerythrine) inhibitor blocked allodynia. Western immunoblot analyses supported behavioral findings and revealed a thermal stimulus-evoked increase in spinal phosphorylated PKA and PKC, but not CaMKIIα. There was no increase in any of the total protein kinases. Although thermal stimulation did not change either pGLUR1 Ser-845 or pGLUR1 Ser-831, it was associated with an increase in cytosolic total GLUR1. Pretreatment with a selective calcium-permeable AMPA/KA receptor antagonist (5 nmol joro spider toxin), but not an NMDA receptor antagonist (25 nmol d-2-amino-5-phosphonovalerate, AP-5), blocked thermal stimulus-evoked increases in phosphorylated PKA and PKC, in addition to increased cytosolic GLUR1. These findings indicate that spinal sensitization in the thermal stimulus model does not involve CaMKIIα activation or AMPA GLUR1 receptor phosphorylation, and differs from that occurring in NMDAr-dependent pain states.

Antiepileptic effect of acylpolyaminetoxin JSTX-3 on rat hippocampal CA1 neurons in vitro

The Joro spider toxin (JSTX-3), derived from Nephila clavata, has been found to block glutamate excitatory activity. Epilepsy has been studied in vitro, mostly on rat hippocampus, through brain slices techniques. The aim of this study is to verify the effect of the JSTX-3 on the epileptiform activity induced by magnesium-free medium in rat CA1 hippocampal neurons. Experiments were performed on hippocampus slices of control and pilocarpine-treated Wistar rats, prepared and maintained in vitro. Epileptiform activity was induced through omission of magnesium from the artificial cerebrospinal fluid (0-Mg 2+ ACSF) superfusate and iontophoretic application of N-methyl- d-aspartate (NMDA). Intracellular recordings were obtained from CA1 pyramidal neurons both of control and epileptic rats. Passive membrane properties were analyzed before and after perfusion with the 0-Mg 2+ ACSF and the application of toxin JSTX-3. During the ictal-like activity, the toxin JSTX-3 was applied by pressure ejection, abolishing this activity. This effect was completely reversed during the washout period when the slices were formerly perfused with artificial cerebrospinal fluid (ACSF) and again with 0-Mg 2+ ACSF. Our results suggest that the toxin JSTX-3 is a potent blocker of induced epileptiform activity.

Characterization of arazyme, an exocellular metalloprotease isolated from Serratia proteamaculans culture medium

We investigated the biochemical properties of a 51.5 kDa metalloprotease (arazyme, 3.4.24.40) secreted into the culture medium by Aranicola proteolyticus, a symbiotic bacterium of the spider Nephila clavata. The enzyme was purified to apparent homogeneity by ion exchange chromatography in a Resource Q column (FPLC system). The substrate specificity requirements of purified arazyme were examined using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = ethylenediaminedinitrophenyl). Three series of peptides were assayed to map the S 2, S 1 and S ′ 1 subsites: Abz-KXRFSKQ-EDDnp, Abz-KLXFSKQ-EDDnp and Abz-KLRXSKQ-EDDnp (X are natural amino acids). The results indicated that S 1 subsite has a broad specificity, being Gly the preferred amino acid for this subsite followed by positively charged residues (Arg and His). The S 2 and S ′ 1 subsites accommodated better hydrophobic residues with aliphatic or aromatic side chains (Leu, Phe). The pH effect on hydrolysis of Abz-KLFFSKQ-EDDnp indicated that optimal hydrolysis occurred at pH 8.0 or higher. The effect of NaCl on the arazyme activity depends on the substrate, but in general the activity was reduced with this salt. The temperature did not affect the enzyme from 10 to 45 °C, after which activity decreased sharply. Arazyme presented high hydrolytic activity on substance P and peptides related to bradykinin. In addition, arazyme activity was resistant to the treatment by pepsin, trypsin and chymotrypsin. In conclusion, arazyme has a broad hydrolytic profile and works in very aggressive conditions, which justify its potential use in therapeutics and biotechnological applications.

AMPA/Kainate Receptor-Mediated Downregulation of GABAergic Synaptic Transmission by Calcineurin after Seizures in the Developing Rat Brain

Hypoxia is the most common cause of perinatal seizures and can be refractory to conventional anticonvulsant drugs, suggesting an age-specific form of epileptogenesis. A model of hypoxia-induced seizures in immature rats reveals that seizures result in immediate activation of the phosphatase calcineurin (CaN) in area CA1 of hippocampus. After seizures, CA1 pyramidal neurons exhibit a downregulation of GABA(A) receptor (GABA(A)R)-mediated inhibition that was reversed by CaN inhibitors. CaN activation appears to be dependent on seizure-induced activation of Ca2+-permeable AMPA receptors (AMPARs), because the upregulation of CaN activation and GABA(A)R inhibition were attenuated by GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride] or Joro spider toxin. GABA(A)R beta2/3 subunit protein was dephosphorylated at 1 h after seizures, suggesting this subunit as a possible substrate of CaN in this model. Finally, in vivo administration of the CaN inhibitor FK-506 significantly suppressed hypoxic seizures, and posttreatment with NBQX (2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline) or FK-506 blocked the hypoxic seizure-induced increase in CaN expression. These data suggest that Ca2+-permeable AMPARs and CaN regulate inhibitory synaptic transmission in a novel plasticity pathway that may play a role in epileptogenesis in the immature brain.

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as γ-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4–100 kDa. A biological assay using Joro spiders ( Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.

Ca2+-permeable and Ca2+-impermeable AMPA receptors coexist on horizontal cells

Fura-2 fluorescent calcium imaging was used for analyzing the subtype of AMPA receptors in freshly dissociated horizontal cells of carp retina. Exogenous application of AMPA induced an increase of intracellular concentration of free Ca2+ ([Ca2+]i) in horizontal cells, while the [Ca2+]i increase was partly inhibited by nifedipine. The residual [Ca2+]i increase was completely eliminated by joro spider toxin-3, a blocker of Ca2+-permeable AMPA receptors. On the other hand, the application of pentobarbital, which blocked Ca2+-impermeable AMPA receptors, could also partly inhibit the increase of [Ca2+]i, implying that the application of AMPA induced the activation of both Ca2+-permeable and Ca2+-impermeable AMPA receptors and the consequent activation of voltage-gated Ca2+ channels. Taken together, these results suggested that Ca2+-permeable and Ca2+-impermeable AMPA receptors were coexpressed on horizontal cells.

Spider Polyamine Toxin

In the early 1980s, a new type of polyamine toxins was found in the venom of several orb-web spiders. Joro spider toxins (JSTXs) in the venom of Nephila clavata and Nephila spider toxins (NSTXs) derived from Nephila maculata blocked postsynaptic glutamate receptors in the invertebrate and vertebrate nervous system. Subsequently, chemical characterization and synthesis of JSTXs and NSTXs were carried out. These toxins and a synthetic analog, 1-naphthylacetylated spermine (Naspm), effectively suppressed glutamate channel responses of AMPA/KA type in mammalian central neurons. By use of recombinant subunit receptors expressed in Xenopus oocytes, JSTX was found to cause subunit specific block of the Ca2+-permeable AMPA receptors. This specific nature of JSTX was utilized to identify Ca2+-permeable AMPA receptors in various neurons and glial cells. The JSTXs strongly suppressed excitatory postsynaptic currents (EPSCs) in the hippocampal CA1 neurons after transient brain ischemia. The results indicate that JSTXs are effective at blocking abnormal EPSCs that may induce Ca2+ accumulation leading to delayed neuronal death after transient ischemic insult. Recent evidence shows that Ca2+-permeable AMPA receptors are involved in a variety of nervous diseases including amyotrophic lateral sclerosis (ALS) and allodynia. The JSTXs are potentially useful to understand pathogenesis of these diseases.

Transient Ca2+‐permeable AMPA receptors in postnatal rat primary auditory neurons

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynaptically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.

Evaluation of the Resistance of Spider Silk to Ultraviolet Irradiation

The effects of ultraviolet (UV) irradiation on spider silk were studied in Nephila clavata draglines collected during different developmental stages in order to clarify the resistance of the draglines to sunlight. Electron spin resonance measurements revealed that photo-irradiation produced C-centered radicals of the protein molecules constituting the draglines. This was attributed to the photo-induced cleavage of chemical bonds. Greater numbers of radicals were produced in the draglines of mature spiders than in those of juveniles, and in the silks of silkworm com- pared with the draglines of spider. Spider silk is therefore more resistant to UV irradiation, which might be a conse- quence of the outside environment in which it functions. The spider silk is expected to be a useful next-generation ma- terial for textiles. (DOI 10.1295/polymj.36.623)

Mode of action of novel polyamines increasing the permeability of bacterial outer membrane

We have clarified how the polyamines naphthylacetylspermine and methoctramine ( N,N′ -bis[6-[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine), originally developed as a synthetic analogue of joro spider toxin and a muscarinic receptor antagonist, respectively, can increase the permeability of the outer membrane of Escherichia coli. These polyamines were recently found to be outer membrane permeabilisers, based on investigations of the structure-activity relationship using ion-selective electrodes. In a standard microbiological assay examining membrane-permeabilising ability, these polyamines enhanced the action of hydrophobic antibiotics such as novobiocin and erythromycin, which ineffectively traverse the outer membrane of E. coli, to inhibit the growth of E. coli. This result substantiated the outer membrane-permeabilising ability of these polyamines demonstrated by using ion-selective electrodes. We observed the release of lipopolysaccharide from the outer membrane in the concentration range causing permeabilisation, showing that the action of the polyamines is attributable to disruption of the outer membrane structure.

日本産ジョロウグモ Nephila clavata の横糸遺伝子の大腸菌における発現

日本産ジョロウグモ Nephila clavata の横糸遺伝子の大腸菌における発現

Calcium-Permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/Kainate Receptors Mediate Development, but Not Maintenance, of Secondary Allodynia Evoked by First-Degree Burn in the Rat

Intrathecal pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists blocks development of spinal sensitization in a number of pain models. In contrast, secondary mechanical allodynia evoked by thermal injury (52.5 degrees C for 45 s) applied to the hind paw of the rat is not blocked by intrathecal pretreatment with NMDA receptor antagonists. It is, however, blocked by antagonists to the non-NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/KA) and calcium-permeable AMPA/KA receptors. These findings suggest a role for these receptors in the development of spinal sensitization. The present study used the same thermal injury model to assess the effects of the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and specific calcium-permeable AMPA/KA receptor antagonists philanthotoxin (PHTx) and joro spider toxin (JST) when given as postinjury treatments. Intrathecal saline injection at 5 and 30 min postinjury had no effect on thermal injury-evoked allodynia as measured by calibrated von Frey filaments. In contrast, 36 nmol of CNQX given at either time point reversed allodynia. Intrathecal 13 nmol of PHTx or 9 nmol of JST (higher doses than that required for pretreatment) reversed allodynia at the 5-min time point, but neither drug was antiallodynic at the 30-min time point. Thus, secondary mechanical allodynia in this model is not maintained by calcium-permeable AMPA/KA receptors, but instead requires activation of calcium-impermeable AMPA/KA receptors. This finding supports a role for AMPA/KA receptor function in responses occurring during spinal sensitization.

Spinal glutamate receptor antagonists differentiate primary and secondary mechanical hyperalgesia caused by incision

Secondary mechanical hyperalgesia has been demonstrated in postoperative patients indicating that central sensitization occurs after surgery. However, the underlying mechanisms are unknown. Here, we studied the role of spinal N-methyl- d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate receptors for pain behaviors indicating secondary hyperalgesia caused by gastrocnemius incision in the rat. We further determined if Ca 2+ permeable AMPA/kainate receptors are important for secondary hyperalgesia after gastrocnemius incision and for pain behaviors indicating primary hyperalgesia and guarding behavior after plantar incision. Withdrawal thresholds (WTs) to punctate mechanical stimuli were assessed by applying calibrated monofilaments to the plantar hind paw before gastrocnemius incision. WTs were tested again 2 h after gastrocnemius incision and again after intrathecal (IT) injection of either dizocilpine maleate (MK-801), 2-amino-5-phosphonovaleric acid (AP5), 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo[f]quinoxaline-7-sulfonamide (NBQX), or Joro spider toxin (JSTX). The doses used were: MK-801 (vehicle, 15, 30, 40 nmol), AP5 (vehicle, 10, 30 nmol), NBQX (vehicle, 5, 10 nmol), and JSTX (vehicle, 2, 5, 9 nmol). In the same rats, WTs were tested on postoperative day 2 before and after the same drugs were injected again. In other rats, WTs to monofilaments and response frequencies to a non-punctate mechanical stimulus or guarding behaviors were determined before, 1 h after plantar incision was made, and assessed again after JSTX (9 nmol or vehicle) was administered IT. Secondary mechanical hyperalgesia after gastrocnemius incision was dose-dependently blocked by NBQX but was only marginally affected by AP5 or MK-801. Only secondary mechanical hyperalgesia was reversed by JSTX; primary mechanical hyperalgesia and guarding behavior were unchanged. These results indicate that spinal sensitization contributing to behaviors for secondary hyperalgesia after incision requires Ca 2+ permeable AMPA/kainate receptors. The data further demonstrate that behaviors for secondary mechanical hyperalgesia after incision can be inhibited without affecting behaviors for primary mechanical hyperalgesia and guarding. Mechanisms for central sensitization causing secondary hyperalgesia in postoperative patients may therefore be seperated from spontaneous pain and hyperalgesia that arises adjacent to the area of the incision.

Calcium-permeable AMPA/kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors.

Hypoxic-ischemic brain injury in premature infants results in cerebral white matter lesions with prominent oligodendroglial injury and loss, a disorder termed periventricular leukomalacia (PVL). We have previously shown that glutamate receptors mediate hypoxic-ischemic injury to oligodendroglial precursor cells (OPCs) in a model of PVL in the developing rodent brain. We used primary OPC cultures to examine the mechanism of cellular toxicity induced by oxygen-glucose deprivation (OGD) to simulate brain ischemia. OPCs were more sensitive to OGD-induced toxicity than mature oligodendrocytes, and OPC toxicity was attenuated by nonselective [2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX), 6-cyano-7-nitroquinoxaline-2,3-dione], alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring (GYKI 52466), kainate-preferring (gamma-d-glutamylaminomethanesulfonic acid), or Ca2+-permeable AMPA/kainate receptor antagonists (joro spider toxin, JSTx) administered either during or after OGD. Furthermore, NBQX or JSTx blocked OGD-induced Ca2+ influx. Relevant to recurrent hypoxic-ischemic insults in developing white matter, we examined the effects of sublethal OGD preconditioning. A prior exposure of OPCs to sublethal OGD resulted in enhanced vulnerability to subsequent excitotoxic or OGD-induced injury associated with an increased Ca2+ influx. AMPA/kainate receptor blockade with NBQX or JSTx either during or after sublethal OGD prevented its priming effect. Furthermore, OGD preconditioning resulted in a down-regulation of the AMPA receptor subunit GluR2 on cell surface that increased Ca2+ permeability of the receptors. Overall, these data suggest that aberrantly enhanced activation of Ca2+-permeable AMPA/kainate receptors may be a major mechanism in acute and repeated hypoxic-ischemic injury to OPCs in disorders of developing cerebral white matter, such as PVL.

Uptake of atmospheric carbon dioxide into silk fiber by silkworms.

The relation between the uptake of atmospheric CO(2) and insect's production of silk fiber has not yet been reported. Here, we provide the first quantitative demonstrations that four species of silkworms (Bombyx mori, Samia cynthia ricini, Antheraea pernyi, and Antheraea yamamai) and a silk-producing spider (Nephila clavata) incorporate atmospheric CO(2) into their silk fibers. The abundance of (13)C incorporated from the environment was determined by mass spectrometry and (13)C NMR measurements. Atmospheric CO(2) was incorporated into the silk fibers in the carbonyl groups of alanine, aspartic acid, serine, and glycine and the C(gamma) of aspartic acid. We show a simple model for the uptake of atmospheric CO(2) by silkworms. These results will demonstrate that silkworm has incorporated atmospheric CO(2) into silk fiber via the TCA cycle; however, the magnitude of uptake into the silk fibers is smaller than that consumed by the photosynthesis in trees and coral reefs.

Fine Structure of the Ovarian Development in the Orb‐web Spider, Nephila clavata

ABSTRACT Fine structural changes of the ovary and cellular composition of oocyte with respect to ovarian development in the orb‐web spider, Nephila clavata were examined by scanning and transmission electron microscopy. Unlike the other arthropods, the ovary of this spider has only two kinds of cells‐follicle cells and oocytes. During the ovarian maturation, each oocyte bulges into the body cavity and attaches to surface of the elongated ovarian epithelium through its peculiar short stalk attachments. In the cytoplasm of the developing oocyte two main types of yolk granules, electron‐dense proteid yolk and electron‐lucent lipid yolk granules, are compactly aggregated with numerous glycogen particles. The cytoplasm of the developing oocyte contains a lot of ribosomes, poorly developed rough endoplasmic reticulum, mitochondria and lipid droplets. These cell organelles, however, gradually degenerate by the later stage of vitellogenesis. During the active vitellogenesis stage, the proteid yolk is very rapidly formed and the oocyte increases in size. However, the micropinocytosis invagination or pinocytotic vesicles can scarcely be recognized, although the microvilli can be found in some space between the oocyte and ovarian epithelium. During the vitellogenesis, the lipid droplets in the cytoplasm of oocytes increase in number, and become abundant in the peripheral cytoplasm close to the stalks. On completion of the yolk formation the vitelline membrane, which is composed of an inner homogeneous electron‐lucent component and an outer layer of electron‐dense component is formed around the oocyte.

Safety Coefficient of the Mechanical Lifeline of Spiders

I studied the safety coefficient of the Nephila clavata spider’s dragline, which was defined as the ratio of the elastic limit strength of the dragline to the spider’s weight, at all stages of the spider’s growth. It was found that the dragline gave a safety coefficient of about 3 for young or immature spiders and that of about 2 for mature spiders. It became clear that a high safety coefficient of about 3 could be ascribed to the large cross section area per spider’s weight. With respect to the mechanical safety coefficient, the draglines may be overly designed to protect immature spiders surrounded by various dangers while they may be designed to give the most efficient safety for mature spiders. The concept of safety obtained for immature and mature spiders may be applied to the development of a new type of industrial materials and also to animals and humans.

Neurohormonal and glutamatergic neuronal control of the cardioarterial valves in the isopod crustacean Bathynomus doederleini.

The heart of Bathynomus doederleini gives rise to an anterior median artery (AMA), one pair of anterior lateral arteries (ALAs) and five pairs of lateral arteries (LAs). Cardioarterial valves are located at the junctions between the heart and arteries, each composed of a pair of muscular flaps. All valves of the AMA and the ALAs receive valve excitatory (constrictor) nerves (VEs). The valves of the ALAs receive dual innervation from both constrictor and inhibitor (dilator) nerves, while the valves of the AMA receive innervation from a constrictor nerve alone. The effects of candidate neurohormones on cardioarterial valves were examined by measuring the pressure in each artery at which haemolymph flows out of the heart through the valve. Serotonin, octopamine, norepinephrine, glutamate (Glu) and proctolin constricted the cardioarterial valves and thus decreased the arterial pressure in all the arteries. Dopamine also decreased the arterial pressure of arteries except for the ALAs, in which pressure was increased. Among the neurohormones exerting excitatory effects on the valves, only Glu depolarized the membrane potential of valve muscle cells. The glutamatergic agonists kainate and quisqualate also depolarized the valve muscle cells of the AMA. Excitatory junctional potentials produced in the valves of the AMA in response to the stimulation of a VE were blocked by the glutamatergic antagonists Joro spider toxin and MK-801. Glu is the likeliest candidate for a neurotransmitter for the VEs.

Ionotropic glutamate receptors: still a target for neuroprotection in brain ischemia? insights from in vitro studies

Although experimental studies have widely shown that the pharmacological blockade of ionotropic glutamate receptors reduces ischemic damage, clinical trials with classical AMPA and NMDA glutamate receptor antagonists have provided negative results. To address the involvement of ionotropic glutamate receptors in ischemic damage, corticostriatal brain slices were prepared from adult rats. Extracellular recordings were performed in the striatum after stimulation of the glutamatergic corticostriatal fibres. In vitro ischemia was induced for a 10-min period by omitting oxygen and glucose from the external medium. Under control conditions, ischemia produced an irreversible loss of the corticostriatal field potential amplitude, AP5, a competitive NMDA receptor antagonist, induced a slight rescue of the potential, while ifenprodil, a positive modulator of the proton sensor of the NMDA receptors, allowed a complete recovery from the ischemic insult. Similar neuroprotection was achieved by utilizing either CNQX, a broad spectrum AMPA receptors antagonist, or Joro spider toxin, a selective blocker of calcium permeable AMPA receptors. Interestingly, while CNZX also fully suppressed physiological excitatory transmission, Joro spider toxin was ineffective on this parameter. Finally, lamotrigine and remacemide, two antiepileptic drugs that differentially affect excitatory transmission, exerted neuroprotective effects against ischemia. Noticeably, a combination of low concentrations of these two drugs exerted a stronger neuroprotection than a single drug given in isolation. Thus, it might be possible to reach a neuroprotective action by utilizing doses of these compounds low enough to avoid side effects. Our experimental data still support the idea that a negative modulation of excitatory transmission can be neuroprotective against ischemia. In addition, our findings support the concept that it is possible to produce a significant neuroprotective action in the absence of a relevant interference with normal synaptic transmission.

Tension sensitivity of the heart pacemaker neurons in the isopod crustacean Ligia pallasii.

In the crustacean neurogenic heart, the cardiac ganglion (CG) acts as a peripherally located central pattern generator (CPG) by producing rhythmic motor output that initiates the heartbeat. In the isopod Ligia, the CG consists of six electrically coupled neurons that all function both as endogenous oscillators and as glutamatergic motoneurons innervating heart muscle. In the present study, we present several lines of evidence to suggest that the CG neurons are sensitive to passive stretch and active tension of the heart muscle. Stretching the heart wall caused a sustained decrease in the burst frequency of the CG neuron. Releasing from the stretch caused a rebound increase in burst frequency above the control rate. A brief stretch (200-300 ms duration) caused either phase advance or phase delay of the following CG bursts, depending on the timing at which the stretch was applied. Repeated brief stretches could entrain the CG bursts to either higher or lower frequencies than the free-run burst frequency. Intracellular recording from one of the CG neurons revealed that it exhibited hyperpolarization during the stretch. The stretch-induced hyperpolarization was followed by a burst discharge upon release from the stretch. With increased stretch amplitude, the amplitude of hyperpolarizing response increased and the timing of the following burst was advanced. When the myogenic activity of the heart muscle was pharmacologically isolated from the ganglionic drive by applying a glutamatergic antagonist, Joro spider toxin (JSTX), the spontaneous muscle contraction caused a hyperpolarizing deflection in the CG neuron. Under specific conditions made by JSTX and tetrodotoxin, the CG burst became entrained to the myogenic rhythm. These results suggest that the Ligia CG neurons have tension sensitivity in addition to their pacemaker and motoneuronal functions. Such multifunctional neurons may form a single neuron reflex arc inside the heart.