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.     

Periodate-Modified Gangliosides Enhance Surface Binding of Tetanus Toxin to PC 12 Pheochromocytoma Cells

The interaction of 125I-labeled tetanus toxin with PC12 pheochromocytoma cells in monolayer cultures has been examined. Under regular growth conditions, the PC12 cells bind 125I-tetanus toxin to a limited degree compared with dissociated cerebral neuron cultures. After exposure to nerve growth factor for 2 days in low serum-containing media with growth factor supplements, binding of toxin increases over twofold compared with untreated PC12 cells. Binding can also be enhanced (greater than 2.5-fold) after treatment of cells with 2 mM sodium metaperiodate for 20 min. Dissociated cerebral neurons but not fibroblasts in cell culture bind more toxin after periodate treatment. The effect of periodate can be abolished by 5 mM sodium borohydride. A ganglioside isolated from periodate-treated PC12 cells and tentatively identified as GT1b [(N-acetylneuraminyl)galactosyl-N-acetylgalactosaminyl(N- acetylneuraminyl-N-acetylneuraminyl)-galactosyl-glucosylceramide] binds 125I-tetanus toxin on silica gel chromatoplates and on nitrocellulose paper. There are no indications to suggest binding to a polypeptide from treated cells after polyacrylamide gel electrophoresis. Cells artificially supplemented with GT1b and subsequently treated with periodate effectively bind the toxin. The data suggest that modified sialyl groups linked to gangliosides, and not to proteins, are preferential targets for tetanus toxin.     

Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells

Ganglioside expression and tetanus toxin binding were studied in the rat pheochromocytoma cell line PC12. Seven ganglioside species were readily detected in extracts of PC12 cells; two were identified as tri- and tetrasialogangliosides, which are common brain constituents but unusual components of neuronal cell lines. Carbohydrate composition, acid and enzyme hydrolyses, and mass spectral analysis revealed that the major species is GT 1b, a predominant mammalian brain ganglioside previously reported to support high affinity tetanus toxin binding (Rogers, T. B., and Snyder, S. H. (1981) J. Biol. Chem. 256, 2402-2407). Direct binding of 125I-tetanus toxin to PC12 gangliosides on TLC plates revealed selective binding to the tri- and tetrasialogangliosides. Radioiodinated toxin also bound with high affinity to intact PC12 cells or their isolated membranes. The binding affinity (Kd = 1.25 nM), density of receptors (Bmax = 238 pmol/mg of membrane protein), and dependence on pH, ionic strength, and temperature were similar to those previously reported for toxin binding to rat brain synaptic membranes. Differentiation of PC12 cells caused an increase in expression of the tri- and tetrasialogangliosides and a closely matched increase in tetanus toxin binding to cell membranes. These data provide evidence that complex gangliosides may act as tetanus toxin receptors, and demonstrate the utility of the PC12 cell line for studies of tetanus toxicity and complex ganglioside expression.     

Tetanus toxin and botulinum toxins type A and B inhibit glutamate, gamma-aminobutyric acid, aspartate, and met-enkephalin release from synaptosomes. Clues to the locus of action.

Tetanus toxin (100 nM) when preincubated with guinea pig cerebrocortical synaptosomes for 45 min reduces the final extent of the KCl-evoked, Ca(2+)-dependent, glutamate transmitter release to 30% of non-intoxicated controls. Similarly, 100 nM Botulinum neurotoxins, types A and B, preincubated for 90 min inhibit release to 45-60% of non-intoxicated controls. The toxins preferentially attenuate a slow phase of KCl-evoked glutamate release which may be associated with synaptic vesicle mobilization. Tetanus toxin additionally inhibits the release of aspartate, gamma-aminobutyric acid and met-enkephalin from the same preparation. Since amino acids and neuropeptides are released by distinct mechanisms, this indicates that the toxin affects a step common to both exocytotic pathways. When Ba2+ (which does not interact with calmodulin) is substituted for Ca2+, the control KCl-evoked release of each transmitter is unaffected and tetanus toxin is still inhibitory. Taken together these results implicate a calmodulin-independent locus (or loci) of action common to small- and large-dense-core vesicles and associated with vesicle transport.     

Tetanus toxin binds with high affinity to neuroblastoma x glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release

Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.     

Characterization of Curaremimetic Neurotoxin Binding Sites on Cellular Membrane Fragments Derived from the Rat Pheochromocytoma PC 12

Studies were conducted on the properties of 125I-labeled alpha-bungarotoxin binding sites on cellular membrane fragments derived from the PC12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d-tubocurarine are among the most potent inhibitors of high-affinity toxin binding. The affinity of high-affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N-ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high-affinity toxin binding to PC12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high-affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti-nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors.     

Affinity purified tetanus toxin binds to isolated chromaffin granules and inhibits catecholamine release in digitonin-permeabilized chromaffin cells

Tetanus toxin, a potent neurotoxin which blocks neurotransmitter release in the CNS, also inhibits Ca2+-induced catecholamine release from digitonin-permeabilized, but not from intact bovine chromaffin cells. In searching for intracellular targets for the toxin we studied the binding of affinity-purified tetanus toxin to bovine adrenal chromaffin granules. Tetanus toxin bound in a neuraminidase-sensitive fashion to intact granules and to isolated granule membranes, as assayed biochemically and visualized by electron microscopic techniques. The binding characteristics of the toxin to chromaffin granule membranes are very similar to the binding of tetanus toxin to brain synaptosomal membranes. We suggest that the toxin-binding site is a glycoconjugate of the G1b type (a polysialoganglioside or a glycoprotein-proteoglycan) which is localized on the cytoplasmic face of the granule membrane and might directly be involved in exocytotic membrane fusion.     

Action of tetanus toxin on cholinergic neuroblastoma X glioma hybrid cells: selective blockade of Ca spikes

We examined the effect of tetanus toxin on clonal neuroblastoma X glioma hybrid cells, NG108-15, by intracellular microelectrode studies of passive membrane electrical properties and action potentials generated under various conditions. Binding of tetanus toxin to the surface of the cells was demonstrated by indirect immunofluorescent staining but no morphological alteration was observed in tetanus toxin-treated cells under a phase contrast microscope. These is no significant difference between the tetanus toxin-treated and untreated cells in their passive electrical membrane properties, i.e. resting membrane potentials, input resistances, time constants and input capacities. Cells in 120 mM Na+, 2 mM Ca2+ salt solution showed Na spikes, and cells in high Ca2+ (30 mM), Na+-free salt solution showed Ca spikes in response to depolarizing current pulses. While the Na spike was not affected by tetanus toxin, the Ca spike was blocked by the toxin. The minimum dose of tetanus toxin for maximum suppression of the peak potential level of the Ca spike was 250 ng/ml. Addition of tetraethyl ammonium (TEA) to extracellular fluid enhanced the Ca spike in untreated cells. In toxin-treated cells, TEA did not alter the effect of tetanus toxin on the Ca spike. Blockade of the Ca spike by tetanus toxin could be detected even at low extracellular Ca2+ concentration (10 mM) by adding TEA to the extracellular fluid and adjusting the membrane potential to a steady hyperpolarized level (-80 mV) to ensure optimal and uniform electrical responses. The usefulness of NG108-15 hybrid cells for in vitro investigations on the mechanism of action of tetanus toxin was discussed.     

Tetanus and Botulinum Toxins Inhibit, and Black Widow Spider Venom Stimulates the Release of Methionine-Enkephalin-Like Material In Vitro

The actions of tetanus toxin, botulinum A toxin, and black widow spider venom on the release of methionine-enkephalin-like immunoreactivity have been studied; a particulate fraction prepared from rat striata was used. Depending on the duration of preincubation, tetanus toxin diminished the release evoked by veratridine (50 microM final concentration), and abolished it at final concentrations between 0.1 and 1 micrograms/ml. Botulinum A toxin was about 10 to 20 times less potent. Heating or pretreatment with antitoxin inactivated the clostridial toxins. The particulate fraction pretreated with V. cholerae neuraminidase retained its toxin sensitivity. Tetanus toxin also depressed the release due to sea anemone toxin II and high K+. Spider venom stimulated the release in a concentration-dependent manner and required the presence of Ca2+; its effects were depressed by tetanus toxin. These results support the view that both clostridial toxins and spider venom act as broad-range presynaptic neurotoxins on peptidergic transmitter systems.     

SV2 mediates entry of tetanus neurotoxin into central neurons

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.     

Maitotoxin, a Ca2+ channel activator candidate

Effects of maitotoxin, the most potent marine toxin, were studied using a rat pheochromocytoma cell line, PC12h. A low concentration (10(-8) g/ml) of maitotoxin induced a profound increase in CA2+ influx into PC12h cells and the Ca2+-dependent release of [3H]norepinephrine from them. The effects of maitotoxin were not affected by treatment with tetrodotoxin (10(-6) M) and were observed even in the absence of external Na+. Furthermore, these effects were markedly inhibited or abolished by treatment with verapamil (30-300 microM), Mn2+ (5 mM), or tetracaine (1 mM). These results suggest that maitotoxin activates the voltage-dependent calcium channels of PC12h cells.     

EFFECTS OF BOTULINUM AND TETANUS TOXINS ON CHOLINE ACETYLTRANSFERASE ACTIVITY IN SKELETAL MUSCLE IN THE MOUSE

Abstract— The effects of botulinum and tetanus toxins on the activity of choline acetyltransferase present in the motor nerve terminals of fast and slow skeletal muscle in the mouse were investigated. There was no change in the activities of choline acetyltransferase in either muscle after the injection of botulinum toxin but tetanus toxin caused a rise in the activity of the enzyme in fast muscle. Botulinum toxin is known to inhibit the release of acetylcholine and whilst neuromuscular transmission is blocked the motor nerves sprout and form new end-plates. Tetanus toxin has been shown to cause hyperactivity of motor neurons. The nerve growth caused by the botulinum toxin did not result in increased choline acetyltransferase levels in the muscles, whereas the synaptic hyperactivity caused by tetanus was associated with increased enzyme levels.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-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 catechol-amine 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.     

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.     

Tetanus Toxin Hc Fragment Induces the Formation of Ceramide Platforms and Protects Neuronal Cells against Oxidative Stress

Tetanus toxin (TeTx) is the protein, synthesized by the anaerobic bacteria Clostridium tetani, which causes tetanus disease. TeTx gains entry into target cells by means of its interaction with lipid rafts, which are membrane domains enriched in sphingomyelin and cholesterol. However, the exact mechanism of host membrane binding remains to be fully established. In the present study we used the recombinant carboxyl terminal fragment from TeTx (Hc-TeTx), the domain responsible for target neuron binding, showing that Hc-TeTx induces a moderate but rapid and sustained increase in the ceramide/sphingomyelin ratio in primary cultures of cerebellar granule neurons and in NGF-differentiated PC12 cells, as well as induces the formation of ceramide platforms in the plasma membrane. The mentioned increase is due to the promotion of neutral sphingomyelinase activity and not to the de novo synthesis, since GW4869, a specific neutral sphingomyelinase inhibitor, prevents neutral sphingomyelinase activity increase and formation of ceramide platforms. Moreover, neutral sphingomyelinase inhibition with GW4869 prevents Hc-TeTx-triggered signaling (Akt phosphorylation), as well as the protective effect of Hc-TeTx on PC12 cells subjected to oxidative stress, while siRNA directed against nSM2 prevents protection by Hc-TeTx of NSC-34 cells against oxidative insult. Finally, neutral sphingomyelinase activity seems not to be related with the internalization of Hc-TeTx into PC12 cells. Thus, the presented data shed light on the mechanisms triggered by TeTx after membrane binding, which could be related with the events leading to the neuroprotective action exerted by the Hc-TeTx fragment.     

Picrosides I and II,selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.     

Black tea extract, thearubigin fraction, counteracts the effect of tetanus toxin in mice

The aim of this study was to find an inactivating substance for tetanus toxin in natural foodstuff. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in In vitro mouse phrenic nerve-diaphragm preparations within 2.5 hr. Hot water infusion of black tea mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing the toxin with thearubigin fraction extracted from black tea infusion produced an identical result. Furthermore, thearubigin fraction mixed with the toxin protected against the in vivo paralytic effect of the toxin. Thearubigin fraction had no protective effect on other toxins, such as tetrodotoxin and saxitoxin. The specific binding of [125I]tetanus toxin to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. These results imply that thearubigin fraction counteracts the effect of tetanus toxin by binding with toxin, and also suggest that this fraction may be able to apply for prophylaxis of tetanus.     

Differential Effects of Tetanus Toxin on Inhibitory and Excitatory Neurotransmitter Release from Mammalian Spinal Cord Cells in Culture

The effect of tetanus toxin on depolarization-evoked and spontaneous synaptic release of inhibitory and excitatory neurotransmitters was examined in murine spinal cord cell cultures. Toxin action on the release of radiolabeled glycine and glutamate was followed over time intervals corresponding to the early phase of convulsant activity through the later phase of electrical quiescence. Tetanus toxin inhibited potassium-evoked release of [3H]glycine and [3H]glutamate in a time- and dose-dependent manner. Ninety minutes after the application of toxin (6 x 10(-10) M), the stimulated release of [3H]glycine was blocked completely, whereas stimulated release of [3H]glutamate was not blocked completely until 150-210 min after toxin application. Fragment C, the binding portion of the tetanus toxin molecule, had no effect on stimulated release of either transmitter. The spontaneous synaptic release of [3H]glycine was blocked totally within 90 min of toxin exposure. In contrast, the spontaneous release of [3H]glutamate, in toxin-exposed cultures, was elevated to nearly twice that of control cultures at this time. Thus, toxin-induced convulsant activity is characterized by a reduction in the spontaneous synaptic release of inhibitory neurotransmitter with a concomitant increase in the release of excitatory neurotransmitter, as well as the more rapid onset of blockade of depolarization-evoked release of inhibitory versus excitatory neurotransmitter.     

Tetanus Toxin Inhibition of K+ -Stimulated [3H]GABA Release from Developing Cell Cultures of the Rat Cerebellum

Abstract: The effect of tetanus toxin pretreatment on K⁺ -stimulated [³H]γ-aminobutyric acid release from neuron-enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1-3-day-old) cultures, but markedly reduced K⁺-evoked [³H]γ-aminobutyric acid release from 7- and 14-day-old cultures (∼80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin-sensitive transmitter release mechanisms similar to their in vivo counterparts.     

Oxygen or glucose deprivation-induced neuronal injury in cortical cell cultures is reduced by tetanus toxin.

We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.