Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

Glutamate and Quisqualate Regulate Expression of Metabotropic Glutamate Receptor mRNA in Cultured Cerebellar Granule Cells

Abstract: Metabotropic glutamate receptor (type 1; mGluR1 ) is expressed predominantly in the hippocampus and the cerebellum. Using cultured cerebellar granule cells, we investigated the regulation of the mGluR1 mRNA expression. Levels of mGluR1 mRNA were decreased to less than half by high potassium stimulation and by glutamate and quisqualate. Although these glutamate receptor agonists tested are also known to cause neuronal cell death in culture, the effect of cell death cannot explain the observed reduction in mGluR1 mRNA because of the following reasons: (a) antagonists of N -methyl-D-aspartate and non- N -methyl-D-aspartate receptors inhibited cell death, but not the reduction of the level of mGluR1 mRNA; (b) mGluR1 mRNA returned to its initial level 48 h after the agonist application; and (c) the mRNA level of one of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptors (GluR1) was not altered by these conditions. Therefore, we conclude that the glutamate or quisqualate stimulation can specifically inhibit the expression of mGluR1 mRNA. The dose response of quisqualate for the reduction in mGluR1 mRNA is consistent with that for inositol phosphate formation stimulated through the cloned mGluR1 . The mRNA reduction did not require extracellular calcium. Desensitization of mGluR1 with phorbol ester abolished the mRNA reduction. These results suggest that the reduction in mGluR1 mRNA is mediated by the activation of the metabotropic receptor itself.     

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low-affinity GABA receptors (K_D 546 ± 117 nM) in addition to the high-affinity receptors (K_D 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED₅₀ 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABA_A receptors.     

Release of Endogenous and Accumulated Exogenous Amino Acids from Slices of Normal and Climbing Fibre-Deprived Rat Cerebellar Slices

Efflux of various amino acids from slices of rat cerebellar hemispheres was determined under resting or depolarizing conditions. It was increased under high K+(50 mM) as compared to low K+ (5 mM) conditions by 1258 pmol/mg protein for aspartate, 478 for gamma-aminobutyric acid (GABA), 44,693 for glutamate, and 615 for glycine. These were significantly higher than the corresponding values obtained under low-Ca2+ (0.1 mM), high-Mg2+ (12 mM) conditions, whereas for 11 other amino acids the K+-induced efflux was similar under normal and low-Ca2+ concentrations. The K+-induced efflux of exogenously accumulated L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate was higher by factors of 2, 5.8, and 6.3, respectively, under normal Ca2+ conditions, as compared with low-Ca2+, high-Mg2+ conditions. After climbing fibre degeneration induced by destruction of the inferior olive with 3-acetylpyridine, release of endogenous aspartate and exogenous L-[3H]glutamate and D-[3H]aspartate was significantly reduced, by 26%, 38%, and 27%, respectively. These results support the hypothesis that climbing fibres may use aspartate or a related compound as a neurotransmitter. In rat cerebellar tissue, L-[3H]glutamate and L-[3H]aspartate differ in several aspects: (1) L-[3H]glutamate uptake was 4 times higher than that of L-[3H]aspartate; (2) fractional rate constant of K+- evoked release of L-[3H]aspartate was 7% X 2.5 min-1, and of L-[3H]glutamate 36% X 2.5 min-1; and (3) specific activity of L-[3H]glutamate in the eluate collected during K+ stimulation was 3.5 times the value in the tissue, whereas for L-[3H]aspartate, specific activities in the eluate and tissue were similar.     

Metabotropic Glutamate Receptors in Cultured Cerebellar Granule Cells: Developmental Profile

Abstract: Excitatory amino acid (EAA)-induced polyphosphoinositide (PPI) hydrolysis was studied during the development in culture of cerebellar granule cells. The developmental pattern was similar using metabotropic glutamate (Glu) receptor (mGluR) agonists, including L-Glu, quisqualate, and trans -(±)-1-amino-1,3-cyclopentanedicarboxylic acid: The stimulation of [³H]inositol monophosphate ([³H]-InsP) formation was low at 2 days in vitro (DIV), but the response increased steeply, reaching a peak at 4 DIV, followed by a progressive decline. In contrast, carbamylcholine-induced PPI hydrolysis exhibited a plateau after a pronounced increase during the first week in vitro. At 6 DIV, but not at 4 DIV, when the activity peaked, PPI hydrolysis elicited by Glu was reduced by the N -methyl- d -aspartate (NMDA) receptor antagonist MK-801, indicating that in cultured granule cells, NMDA receptors contribute to [³H]-InsP formation and that this component of the response develops relatively late. Accordingly, NMDA-induced [³H]-InsP formation, estimated under Mg²⁺-free conditions, increased markedly from very low values at 2 DIV to a plateau at 8–10 DIV. The developmental pattern of EAA-induced PPI hydrolysis was paralleled by changes in the level of an mRNA for a specific mGluR subtype ( mGluR1 mRNA). RNA blot analysis performed with the pmGR1 cDNA probe revealed that the hybridization signal in RNA extracts from cultures at 1 DIV was very weak, but mGluR mRNA levels increased dramatically between 1 and 3 DIV, followed by a progressive decrease, so that by 15 DIV the mRNA levels were only ∼10% of the values at 3 DIV. These observations indicate that the functional expression of the mGluR is subject to developmental regulation, which critically involves receptor mRNA 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.     

Glutamate Receptor Subtypes in Cultured Cerebellar Neurons: Modulation of Glutamate and γ-Aminobutyric Acid Release

Using cerebellar, neuron-enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D-[3H]aspartate release, whereas N-methyl-D-aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20-100 microM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 microM. Kainate and dihydrokainate (20-100 microM) inhibited the initial rate of D-[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N-methyl-DL-aspartate were ineffective. D-[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D-[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50-200 microM) and, to a lesser extent, 2,3-cis-piperidine dicarboxylic acid (100-200 microM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20-100 microM) also stimulated gamma-[3H]-aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D-[3H]aspartate neuronal uptake by interfering with the acidic amino acid high-affinity transport system.     

Stimulation of Cultured Cerebellar Granule Cells via Glutamate Receptors Induces TRE- and CRE-Binding Activities Mediated by Common DNA-Binding Complexes

By use of nuclear mini-extracts prepared from cultured cerebellar granule cells in a gel-mobility assay, exogenous N-methyl-D-aspartate (NMDA) or kainate was shown to increase both 12-O-tetradecanoylphorbol 13-acetate-responsive element (TRE)- and cyclic AMP-responsive element (CRE)-binding activity. These increases were specifically prevented by the NMDA receptor antagonist D,L-2-amino-5-phosphonovalerate and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. The increase of TRE-binding activity was dependent on de novo protein synthesis, and its inductions by both NMDA and kainate required extracellular Ca2+. TRE-binding activity was competitively inhibited by the CRE, and vice versa, showing higher DNA-binding affinity to the CRE than to the TRE. A proteolytic clipping bandshift assay demonstrated that the increase in CRE-binding activity could be mediated by the TRE-binding activity. Thus, the TRE-binding activity cross-binding to the CRE could be activated by NMDA or kainate stimulation. The involvement of c-Fos or Fos-related proteins in the TRE- and CRE-binding complexes was shown by a supershift gel-mobility assay using anti-c-Fos antiserum.     

Release by Electrical Stimulation of Endogenous Glutamate, γ-Aminobutyric Acid, and Other Amino Acids from Slices of the Rat Medulla Oblongata

Evidence was obtained for the release of amino acids by electrical stimulation of slices of regions of the rat medulla oblongata: rostral ventrolateral, caudal ventrolateral and caudal dorsomedial. There was a Ca2+-dependent, tetrodotoxin-sensitive increase in the efflux of aspartate, glutamate, gamma-aminobutyric acid (GABA), glycine, and beta-alanine in all regions examined. There were distinct regional differences in the relative amounts of amino acids released. These results provide evidence for the possible neurotransmitter role of aspartate, glutamate, GABA, glycine, and beta-alanine in these regions of the rat medulla oblongata.     

In Vitro Effect of Tetanus Toxin on GAB A Release from Rat Hippocampal Slices

Abstract Ca²⁺-dependent K⁺-stimulated γ-aminobutyric acid release from rat hippocampal slices was reduced about 30% by pre-incubation of the slices with 10⁴ mouse LD₅₀/ml tetanus toxin for 3 h at 37°C.     

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.     

Effects of Dehydroevodiamine Exposure on Glutamate Release and Uptake in the Cultured Cerebellar Cells

Dehydroevodiamine has been reported to have neuroprotective and antiamnesic effects. This study examined the effects of dehydroevodiamine on glutamate release and uptake in cultured cerebellar cells. Chronic dehydroevodiamine exposure decreased the viability of granule cells. The basal and N-methyl-D-aspartate (NMDA)-induced release of glutamate from granule cells were decreased (26 and 14%) by dehydroevodiamine. The NMDA-induced release of glutamate was concentration-dependently inhibited in the granule cells. The basal and NMDA-induced releases of glutamate in chronically dehydroevodiamine-preexposed granule cells were unaffected by dehydroevodiamine. Glutamate uptake in the glial cells incubated without and with cAMP was inhibited (31% and 8%, respectively) by dehydroevodiamine. In the chronically dehydroevodiamine-preexposed glial cells, glutamate uptake was increased (8%) in the cAMP-coexposed glial cells by dehydroevodiamine but was unaffected in the naive cells. In addition, dehydroevodiamine potentiated (from 20% to 34%) the inhibition of L-pyrollidine-2,4-dicarboxylic acid (PDC) on glutamate uptake in naive glial cells, but this inhibition was reduced (from 41% to 26%) in cAMP-coexposed glial cells. These results suggest that dehydroevodiamine inhibits glutamate uptake and release. Furthermore, the results suggest that the characteristics of glutamate release and uptake in granule and glial cells may be altered by chronic exposure to dehydroevodiamine.     

Tetanus Toxin-Induced Effects on Extracellular Amino Acid Levels in Rat Hippocampus: An In Vivo Microdialysis Study

Abstract: Tetanus toxin is a potent neurotoxin that is widely considered to produce its effect through impairment of inhibitory neurotransmission. We report the effect of a single unilateral intrahippocampal injection of tetanus toxin on extracellular levels of neuroactive amino acids in freely moving rats, at times ranging between 1 and 7 days posttreatment. Tetanus toxin treatment did not alter extracellular levels of aspartate, glutamate, and taurine at any time during the study. However, although extracellular GABA levels were unaffected by toxin injection 1, 2, and 3 days after treatment, they were reduced (45 ± 8% of contralateral vehicle-injected level) at day 7. Challenge with a high K⁺ concentration, 7 days after treatment, produced elevations in extracellular levels of taurine and GABA in both vehicle- and toxin-injected hippocampi, with evoked levels of GABA being lower in the toxin-treated side (39 ± 16% of contralateral vehicle-injected level). Aspartate and glutamate levels were not increased by high-K⁺ infusion. These findings are discussed in relation to the possible role that an imbalance in excitatory/inhibitory tone may play in the production of tetanus toxin-induced neurodegeneration.     

Glutamate Neurotoxicity in Rat Cerebellar Granule Cells: A Major Role for Xanthine Oxidase in Oxygen Radical Formation

Abstract: To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2',7'-dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 µ M glutamate pulse administered to 7-day-old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O₂^•−) production are completely inhibited by concomitant incubation of glutamate with MK-801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate-triggered, NMDA-mediated, massive Ca²⁺ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death.     

Effect of Tetanus Toxin on Transmitter Release from the Substantia Nigra and Striatum In Vitro

Abstract: The effect of tetanus toxin on the uptake and release of radiolabelled transmitters from slices prepared from substantia nigra (SN) and striatum of rats has been investigated. Tetanus toxin-500–750 mouse lethal doses (MLD)-injected into the SN 6 h before preparing the slices significantly reduced the calcium-dependent, potassium-evoked release of [³H]GABA. Endogenous GABA levels in the SN and [³H]GABA uptake by nigral slices were unaffected by pretreatment with the toxin. Injections of tetanus toxin (1000–2000 MLD) into the striatum significantly reduced the calcium-dependent, potassium-evoked release of [¹⁴C]GABA and also [³H]dopamine, but had no effect on the K⁺-evoked release of [³H]5-hydroxytryptamine or [¹⁴C]acetylcholine. It is concluded that tetanus toxin inhibits GABA release directly and not by interference with synthesis or inactivation processes.     

Exocytotic and Nonexocytotic Modes of Glutamate Release from Cultured Cerebellar Granule Cells During Chemical Ischaemia

Abstract: The mechanism of glutamate release from cultured cerebellar granule neurones in response to a chemical model of ischaemia (10 m M 2-deoxyglucose plus 1 m M sodium cyanide) was investigated. In the first 2 min of ischaemia, release of preloaded d -[³H]aspartate could be extensively attenuated by tetanus toxin and bafilomycin A₁ and was dependent on the activation of Ca²⁺ channels sensitive to the "Q" type Ca²⁺ channel antagonist, ω-conotoxin-MVIIC. During this period, ATP/ADP ratios fell rapidly. The extent of release in the first 2 min was comparable to that evoked by 2-min depolarization by 50 m M KCl. Free Ca²⁺ concentrations, determined in neurites and somata, did not increase until after 2 min. The neurite increase in cellular Ca²⁺ precedes that of the cell somata. Release of d -[³H]aspartate was partially inhibited by the NMDA receptor antagonist MK-801, which also delayed the increase in free Ca²⁺ concentration. Prolonging the period of ischaemia to 6 and 10 min produced no further increase in the apparently exocytotic component of release, but initiated an extensive nonexocytotic release of the amino acid. Studies with the synaptic vesicle membrane probe FM1-43 in which released amino acid was removed by superfusion indicated that Ca²⁺-dependent exocytosis was delayed in this system. It is concluded that chemical ischaemia initiates an initial exocytotic followed by nonexocytotic release and that the former is facilitated by NMDA receptor activation. These events occur in cells that are still able to exclude propidium iodide, indicating that cell death has not yet occurred.     

Magnesium Ions Inhibit the Stimulation of Inositol Phospholipid Hydrolysis by Endogenous Excitatory Amino Acids in Primary Cultures of Cerebellar Granule Cells

Omission of Mg2+ from the incubation buffer results in a six- to eightfold increase in [3H]inositol-1-phosphate ([3H]Ins-1-P) accumulation in primary cultures of cerebellar granule cells at 7-9 days in vitro. This increase is reversed by low concentrations of 2-amino-5-phosphono-valerate (APV), a result indicating that the absence of Mg2+ facilitates the activation of a specific receptor by the endogenous excitatory amino acids (presumably L-glutamate and L-aspartate) released from the granule cells. The absence of Mg2+ also potentiates the action of exogenously applied N-methyl-D-aspartate (NMDA), L-glutamate, L-aspartate, and kainate. In contrast, the action of quisqualate is virtually unaffected by Mg2+ and is resistant to APV inhibition. Addition of the depolarizing agent veratridine enhances the accumulation of [3H]Ins-1-P also in Mg2+-containing buffer. The action of veratridine is antagonized by APV, a result suggesting that, under depolarized conditions, the NMDA receptor can be activated by the endogenously released excitatory amino acids, despite the presence of Mg2+. Accordingly, in the presence of Mg2+, veratridine potentiates the action of exogenously applied NMDA but does not facilitate the action of quisqualate.     

Microtubules and Microfilaments Participate in the Inhibition of Synaptosomal Noradrenaline Release by Tetanus Toxin

Abstract: Tetanus toxin (TeTX) has been demonstrated to inhibit transmitter release by two mechanisms: Zn²⁺-dependent proteolytic cleavage of synaptobrevin and activation of a neuronal transglutaminase. Herein, attenuation of TeTX-induced blockade of noradrenaline release from synaptosomes was achieved by prior disassembly of microfilaments with cytochalasin D or breakdown of microtubules by colchicine or nocodazole. These drugs and monodansylcadaverine, a transglutaminase inhibitor, displayed some additivity in antagonizing the inhibitory effect of the toxin on synaptosomal transmitter release; as none of them reduced synaptobrevin cleavage, all appear to work independently of the toxin's proteolytic action. Prior stabilization of microtubules with taxol prevented the antagonism seen with colchicine, highlighting that this cytoskeletal component is the locus of the effect of colchicine. Replacement of Ca²⁺ with Ba²⁺ caused disappearance of the fraction of evoked secretion whose inhibition by TeTX is reliant on polymerized actin but did not alter the blockade by toxin that is dependent on microtubules. Two temporally distinguished phases of release were reduced by TeTX, and colchicine lessened its effects on both. Blockade of the fast phase (≤10 s) of secretion by TeTX was unaffected by cytochalasin D, but it clearly antagonized the toxin-induced inhibition of the slow (10-s to ≥5-min) component; it is notable that such antagonism was accentuated during a second bout of evoked release. These findings are consistent with sustained release requiring dissociation of synaptic vesicles from the microfilaments, a step that seems to be perturbed by TeTX.     

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.