Effect Of Tetanus Toxin Research Articles

The effectiveness of tetanus toxin on sciatic nerve regeneration: A preliminary experimental study in rats

The purpose was to investigate the effects of local tetanus toxin (TeTx) application on sciatic nerve regeneration following a rat model of transection injury. After both sciatic nerves were transected and repaired with three epineural sutures, 12 male Wistar albino rats were divided into two groups. 0.25 ml (2.5 flocculation units) TeTx was injected into a piece of absorbable gelatin sponge in TeTx group. In controls, 0.25 ml saline injected. Assessments were performed by using climbing degrees, compound muscle action potentials (CMAPs) and histological parameters (axon number and axonal diameter) 12th week. CMAPs amplitudes were 11.6 ± 4.7 mV and 1.4 ± 1.3 mV in gastrocnemius and interdigital muscles in TeTx group (5.8 ± 2.4 mV and 0.2 ± 0.1 mV, P < 0.05). Climbing degrees were significantly different (61.6 ± 1.7 vs. 38.3 ± 2.6, P < 0.05). Total axon numbers were higher (1341.1 ± 57.3 vs. 877.5 ± 34.9, P < 0.05) and the mean axon diameter was smaller (4.2 ± 2.1 vs. 2.5 ± 1.9, P < 0.05) in the TeTx group. This preliminary study firstly demonstrated the effectiveness of TeTx on nerve repair in experimental sciatic rat model based on functional, electromyographic and histological parameters.

Structural and functional substrates of tetanus toxin in an animal model of temporal lobe epilepsy

The effects of tetanus toxin (TeNT) both in the spinal cord, in clinical tetanus, and in the brain, in experimental focal epilepsy, suggest disruption of inhibitory synapses. TeNT is a zinc protease with selectivity for Vesicle Associated Membrane Protein (VAMP; previously synaptobrevin), with a reported selectivity for VAMP2 in rats. We found spatially heterogeneous expression of VAMP1 and VAMP2 in the hippocampus. Inhibitory terminals in stratum pyramidale expressed significantly more VAMP1 than VAMP2, while glutamatergic terminals in stratum radiatum expressed significantly more VAMP2 than VAMP1. Intrahippocampal injection of TeNT at doses that induce epileptic foci cleaved both isoforms in tissue around the injection site. The cleavage was modest at 2 days after injection and more substantial and extensive at 8 and 16 days. Whole-cell recordings from CA1 pyramidal cells close to the injection site, made 8–16 days after injection, showed that TeNT decreases spontaneous EPSC frequency to 38 % of control and VAMP2 immunoreactive axon terminals to 37 %. In contrast, TeNT almost completely abolished both spontaneous and evoked IPSCs while decreasing VAMP1 axon terminals to 45 %. We conclude that due to the functional selectivity of the toxin to the relative sparing of excitatory synaptic transmission shifts the network to pathogenically excitable state causing epilepsy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00429-013-0697-1) contains supplementary material, which is available to authorized users.

EFFECTS OF COMBINING A CRYPTOCHROME MUTATION WITH OTHER VISUAL-SYSTEM VARIANTS ON ENTRAINMENT OF LOCOMOTOR AND ADULT-EMERGENCE RHYTHMS IN DROSOPHILA

“For every behavioral observation, there is an equal and opposite observation.”S. BenzerPhotoreception is an important component of rhythm systems and is involved in adjusting circadian clocks to photic features of daily cycles. In Drosophila, it has been suggested that there are three light input pathways to the clock that underlie rhythms of adult behavior: One involves the eyes; the other two extraocular photoreception through a structure called the Hofbauer-Buchner (H-B) eyelet and light reception carried out by pacemaker neurons themselves, mediated by a substance called cryptochrome. All photoreceptor cells including the H-B eyelet have been surmised to be removed by glass-null mutations. Mutations in the no-receptor-potential-A(norpA) gene cause the compound eyes and ocelli to be non-functional and may also affect the eyelet's function. The one cryptochromemutant known (cryb) harbors an amino-acid substitution in the blue-light absorbing protein encoded by this gene. With regard to adult locomotor rhythms, all single mutants (gl60j, norpAP41, and cryb) re-entrained to altered light:dark (LD) cycles in which the L phase involved relatively intense light. Dropping light levels ca. 10 or ca. 30-fold permitted small percentages of doubly-mutant gl60jcrybflies clearly to re-synchronize their behavior. The marginal re-entrainability in the lowest-light situation nevertheless involved superior responsiveness of the gl60jcrybtype, compared with that observed previously using a different re-entrainment protocol. Furthermore, transgenic types in which rhodopsin-expressing cells within the H-B eyelet were ablated or suffered from the effects of tetanus-toxin also entrained with behavior similar or superior to that of gl60jcrybat a low light level. Light inputs that are necessary to synchronize periodic adult emergence can be inferred (from previous studies) to involve a cry-dependent pathway and perhaps also a norpA-dependent one, so that combining mutations in these two genes would cause cultures to be unentrainable. The current results were that each singly-mutant type eclosed rhythmically; flies emerging from norpAP41;crybcultures also (on balance) exhibited solid eclosion rhythmicity. The ensemble of these behavioral and adult-emergence results suggest that additional light-to-clock pathways function within the system; alternatively, that rhythm assays employed here have teased out residual function of the mutated CRY protein.

A mechanism of the thearubigin fraction of black tea (camellia sinensis) extract protecting against the effect of tetanus toxin.

The aim of the present study was to elucidate the mechanism of the protective effect of black tea extract's thearubigin fraction against the action of tetanus toxin. The effects of thearubigin fraction extracted from a black tea infusion were examined for neuromuscular blocking action on tetanus toxin in mouse phrenic nerve-diaphragm preparations and on the binding of this toxin to the synaptosomal membrane preparations of rat cerebral cortices. The interaction between tetanus toxin and thearubigin fraction was also investigated. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in mouse phrenic nerve-diaphragm preparations within 150 min. Thearubigin fraction mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing iodinated toxin with thearubigin fraction inhibited the specific binding of [125I]tetanus toxin to the synaptosomal membrane preparation. The effects of thearubigin fraction were dose-dependent. The elution profile of [125I]tetanus toxin on Sephadex G-50 column chromatography was different from that of toxin mixed with thearubigin fraction. These findings indicate that thearubigin fraction protects against the action of tetanus toxin by binding with the toxin.

Effects of tetanus toxin on functional inhibition after injection in separate cortical areas in rat

Tetanus Toxin is widely used as a model of chronic focal epilepsy and is assumed to act by blocking neurotransmitter release with high selectivity for inhibitory synapses. However, the exact mechanisms are not fully understood, since, e.g., GABA release is only temporarily decreased although epileptiform activity persists pointing towards a change in the interplay of excitation and inhibition. Furthermore there have been reports about different effects of tetanus toxin after injection in separate brain areas. Therefore, we investigated the functional inhibition after injecting tetanus toxin either in the motor or sensory cortex of adult rats by using a paired-pulse paradigm as a measure of excitatory and inhibitory drive. Tetanus toxin injection into the motor cortex ( n=10) induced a marked, long-lasting reduction in inhibition which was highly significant in most parts of the injected cortical area. Injections into the sensory cortex, however, showed less marked changes in inhibition which were more widespread and significant only in 3 of 14 animals injected. These results give further evidence for a prominent effect of tetanus toxin on functional inhibition and strengthen the idea of a differential effect in separate cortical areas. They may be accounted for by the different cytoarchitecture of cortical areas with variable inhibitory and excitatory intracortical connections.

Unforgettable tetanus

Unforgettable tetanus

Effect of tetanus toxin on basal and evoked release of 5-hydroxytryptamine and dopamine in rat hippocampus in vivo

Tetanus toxin is a potent neurotoxin which produces seizures and usually death in mammals. In the present study, we have investigated the effect of tetanus toxin on extracellular basal and evoked release of 5-hydroxytryptamine (5-HT), dopamine (DA) and their metabolites in vivo, 7 days after toxin injection into the hippocampus of rats. Tetanus toxin decreased both basal and evoked release of 5-HT and DA. The 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) was not significantly decreased in the extracellular space. Of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) concentration was decreased while that of homoillic acid (HVA) was unchanged. These findings are considered in light of the possible contributory role that a decrease in monoamine concentration in the hippocampus might have in the effects of tetanus toxin.

Tetanus toxin inhibits depolarization-induced [ 3H]serotonin release from rat brain cortex synaptosomes

The effect of tetanus toxin on depolarization-induced [ 3H]serotonin release from superfused rat brain cortex synaptosomes was investigated. Two hours' preincubation of the synaptosomes with tetanus toxin resulted in a concentration-dependent decrease of K +-stimulated release, with an IC 50 of about 30 nM (4.5 μg/ml); this inhibitory effect was blocked by a previous incubation of the tetanus toxin with antitoxin serum. Tetanus toxin had no effect on reserpine-induced release, a model of Ca 2+-independent release. These results indicate that tetanus toxin is able to alter the exocytotic machinery of serotoninergic terminals, in agreement with results obtained with other neurotransmitters. They also indicate that serotoninergic terminals possess the receptor for tetanus toxin. These findings are in line with in vivo observations suggesting a role for serotoninergic system in tetanus intoxication.

Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin

Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by μM concentrations of free Ca 2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.

The light chain of tetanus toxin inhibits calcium-dependent vasopressin release from permeabilized nerve endings

The effects of tetanus toxin and its light and heavy chain subunits on vasopressin release were investigated in digitonin-permeabilized neurosecretory nerve terminals isolated from the neural lobe of the rat pituitary gland. Exocytosis was induced by challenging the permeabilized nerve endings with micromolar calcium concentrations. Tetanus toxin inhibited vasopressin release only in the presence of the reducing agent dithiothreitol. This effect was irreversible. The purified light chain of tetanus toxin strongly inhibited exocytosis in a dose-dependent manner with half-maximal effect at c. 10 nM. The action of the light chain was observed after only 2.5 min of preincubation. Separated heavy chain subunit had no effect on hormone secretion. Inhibition of vasopressin release could be prevented by preincubating the light chain of tetanus toxin with an immune serum against tetanus toxin. The data clearly demonstrate that in mammalian neurosecretory nerve endings tetanus toxin acts at a step downstream from the activation by Ca 2+ of the exocytotic machinery and that the functional domain of this toxin is confined to its light chain.

L- and T-type calcium channels in cultured neuronal cell lines are insensitive to tetanus toxin

Possible effects of tetanus toxin (TeTox) on voltage-activated Ca 2+ channels of the mouse neuroblastoma cell line N1E-115 and of the neuroblastoma x glioma hybrid cell line NG108-15 have been investigated using the whole-cell voltage clamp technique. Similar to N1E-115 cells, differentiated NG108-15 cells express transient (T-type) as well as long-lasting (L-tyoe) Ca 2+ channels. Using various treatment protocols N1E-115 and NG108-15 cells were exposed to TeTox externally and by internal dialysis. In the cells treated with TeTox normal Ca 2+ channel activity was present, as measured by the voltage-activated Ba 2+ currents under voltage clamp conditions. In addition, intracellular microelectrode recordings showed that TeTox did not block the Ca 2+ action potential in N1E-115 cells. It is concluded that TeTox, in contrast to previously reported results 20,21, does not affect voltage-activated T- and L-type Ca 2+ channels in cultured neuronal cell lines. The results also indicate that Ca 2+ channel block is unlikely to be an explanation for the block of neurotransmitter release by TeTox in vivo.

Chronic epileptic foci in neocortex: in vivo and in vitro effects of tetanus toxin.

Injection of 0.2 - 3.0 ng of tetanus toxin into rat parietal neocortex resulted in permanent (> 7 months) changes in the local circuit properties of this tissue. It caused excessive synchronization of neuronal activity. This was seen as spontaneous paroxysmal field potentials and/or evoked all-or-none population burst discharges. Such activity was recorded widely over the parietal and temporal areas of both the injected and the contralateral hemispheres from as little as 16 h after injection up to the maximum survival time of 7 months. Several observations suggest that the speed with which the hypersynchronous activity spread to the opposite hemisphere reflects transport of the toxin through corticocortical axons, and consequent blockade of synaptic inhibition. However, from what is known of the half life of the peptide in brain, it is unlikely that the persistent, widespread distribution of epileptiform discharge several months after injection was due to the continued presence of toxin. Thus, intracortical application of tetanus toxin provides a good experimental model of chronic focal epilepsies, and raises fundamental questions regarding the long term regulation of local circuit properties in the neocortex.

Tetanus toxin inhibits neurotensin-induced mobilization of cytosolic protein kinase C activity in NG-108 cells

There is considerable literature on the pathogenesis of tetanus toxin poisoning; however, the mechanism of action and intracellular substrate of this toxin have not been defined. It was demonstrated that the NG-108 neuroblastoma × glioma cell line is a suitable model in which to study the mechanism of tetanus toxin action, from binding of the toxin to inhibition of transmitter release. Further, it has been shown that tetanus toxin pretreatment attenuates the ability of phorbol myristate acetate to mobilize cytosolic protein kinase C (PKC) in this cell line. In the present study a 4-hr tetanus toxin pretreatment (10 −10–10 −13 M) completely inhibited the mobilization of cytosolic PKC induced by a 30-min exposure to 10 μM neurotensin. Pretreatment with 10 −10 M tetanus toxin for periods as short as 1 hr was sufficient to attenuate the ability of neurotensin to mobilize cytosolic PKC; however, a 30-min pretreatment had no significant effect. At a concentration of 10 −11 M, it was necessary to pretreat the cells for greater than 1 hr to significantly attenuate neurotensin-mobilized PKC activity. The exact role that PKC plays in the secretory process is not yet known; however, these findings suggest that the effect of tetanus toxin on neurotransmitter release is accompanied by an alteration in PKC metabolism in differentiated NG-108 cells.

The effect of tetanus toxin on in vitro synaptogenesis

Cultures of spinal cord neurons and cocultures of rat embryo neurons and muscle cells have been studied in the presence of tetanus toxin (TT) at a concentration of 40 μg/ml of medium. TT strongly stimulated neurite outgrowth, notably branching from the cell bodies. In addition it induced a marked, overall increase in acetylcholine receptor (AChR), but inhibited focalisation of AChR and acetylcholinesterase (AChE) at the synaptic sites. TT seems to act on neurite emergence, on the neuronal factor(s) controlling AChE and AChR concentrations, and on the factor(s) modulating degradation and/or synthesis of AChR.

Effect of Tetanus Toxin on Oxytocin and Vasopressin Release from Nerve Endings of the Neurohypophysis

The effect of tetanus toxin on neuropeptide hormone release from isolated nerve endings of the neural lobe of rat pituitaries (neurosecretosomes) was measured in a perfusion system. Tetanus toxin inhibited depolarization-evoked release of oxytocin and vasopressin in a time- and dose-dependent manner. At 1 microgram/ml, tetanus toxin blocked stimulated release by 85%. Tetanus toxin that was preincubated with a neutralizing monoclonal antibody or heated to 100 degrees C had no effect on hormone release. The ionophores A23187 and ionomycin were potent stimulators of hormone release in control nerve endings, but were not able to overcome the effect of tetanus toxin in intoxicated nerve endings. 8-Bromo-cyclic GMP, which has been reported to reverse the action of tetanus toxin in PC12 cells, had no effect on the action of tetanus toxin in neurosecretosomes. Neurosecretosomes are the first system in which tetanus toxin has been shown to block release from peptidergic nerve terminals. They appear to be a valuable in vitro system for studying the biochemical mechanism of tetanus toxin action.

Tetanus toxin produces neuronal loss and a reduction in GABAA but not GABAB binding sites in rat hippocampus

The neuropathological effects of tetanus toxin, microinjected in the rat CA1 hippocampal area, were studied by using a microscopical and autoradiographical approach. Tetanus toxin produced a dose- and time-dependent neuronal loss in the CA1 area accompanied by a reduction in the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to GABAA but not GABAB sites in the pyramidal cell layer.

Effect of tetanus toxin on synaptosomal protein phosphorylation: Peter R. Dunkley, Peter P. Presek1, F. Dreyer1 and Paula E. Jarvie (The Neuroscience Group, Faculty of Medicine, The University of Newcastle, Shortland, 2308, New South Wales, Australia, and 1Rudolf-Bucheim Institute for Pharmacology, Justus-Liebig University, Giessen, D-6300, F.R.G.)

Effect of tetanus toxin on synaptosomal protein phosphorylation: Peter R. Dunkley, Peter P. Presek1, F. Dreyer1 and Paula E. Jarvie (The Neuroscience Group, Faculty of Medicine, The University of Newcastle, Shortland, 2308, New South Wales, Australia, and 1Rudolf-Bucheim Institute for Pharmacology, Justus-Liebig University, Giessen, D-6300, F.R.G.)

Effect of tetanus toxin and its fragment A–B on the excitatory synapses of the spinal motor-cell of the cat: K. Takano (Abteilung Pathoneurophysiologie, Universität Göttingen, Humboldtallee 23, D-3400 Göttingen, F.R.G.)

Effect of tetanus toxin and its fragment A–B on the excitatory synapses of the spinal motor-cell of the cat: K. Takano (Abteilung Pathoneurophysiologie, Universität Göttingen, Humboldtallee 23, D-3400 Göttingen, F.R.G.)

Endogenous Amino Acid Release from Cultured Cerebellar Neuronal Cells: Effect of Tetanus Toxin on Glutamate Release

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.

Effects of tetanus toxin on catecholamine release from intact and digitonin-permeabilized chromaffin cells.

Tetanus exotoxin inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner in digitonin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca2+. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca2+ concentrations. Tetanus toxin also inhibited catecholamine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca2+-dependent exocytotic pathway to inhibit secretion.