Effect Of Tetanus Toxin Research Articles

Botulinum A neurotoxin unlike tetanus toxin acts via a neuraminidase sensitive structure

H. Bigalke, H. Müller and F. Dreyer. Botulinum A neurotoxin unlike tetanus toxin acts via a neuraminidase sensitive structure. Toxicon 24, 1065 – 1074, 1986. — The binding and effects of tetanus and botulinum A neurotoxins were studied on mouse spinal cord cultures treated with neuraminidase. In untreated cultures both neurotoxins blocked synaptic transmission. Treatment of the cell cultures with neuraminidase, 25 mU/ml for 24 hr, decreased the potency of botulinum A neurotoxin. At 7 × 10 −11 M no toxin effect on inhibitory or excitatory synapses was observed, whereas at higher concentrations of the toxin the concentration - response curve was shifted to the right by a factor of about 30. Surprisingly, the action of tetanus toxin over a large concentration range was unaffected by pretreatment of the neurones with the enzyme. Accordingly, neurones treated with neuraminidase failed to bind 125I-botulinum A neurotoxin, whereas labelled tetanus toxin was still fixed by cell bodies, as well as by neurites, as shown by histoautoradiography. Chromatographic extraction of gangliosides from cultures prelabelled with 14C-glucosamine showed a dramatic loss in the contents of polysialogangliosides following treatment with neuraminidase. Our results indicate that neuraminidase-sensitive structures might be important for the action of botulinum A neurotoxin. The effect of tetanus toxin appears to be mediated by a different site which is insensitive to neuraminidase.

Human lymphoblastoid cell line producing protective monoclonal IgG1, kappa anti-tetanus toxin.

Peripheral blood lymphocytes from an individual, recently boosted with tetanus toxoid (TT), were transformed with Epstein-Barr virus. No antigen-specific selection nor stimulation of B cells was performed prior to transformation. One stable cell line, designated CLB-Hu-TT-1, was established. This cell line has a doubling time of 24 h and yields 10 micrograms/ml of a monoclonal IgG1, kappa anti-TT antibody in bulk cultures. The antibody is biologically active in that it can protect mice against the effects of tetanus toxin. The cell line has been characterized with regard to some cytoplasmic and membrane markers.

Transmitter release in tetanus and botulinum A toxin-poisoned mammalian motor endplates and its dependence on nerve stimulation and temperature.

The effects of tetanus toxin (TeTx) and botulinum A toxin (BoTx) on spontaneous and nerve-evoked transmitter release have been compared in mouse hemidiaphragms poisoned in vitro. At 37 degrees C endplates poisoned with either of these agents were characterized by (1) a decrease of miniature endplate potential (m.e.p.p.)-frequency to less than 30/min for TeTx and 3/min for BoTx, (2) reduced mean m.e.p.p.-amplitude and (3) 100% failure to show endplate potentials (e.p.p.s) in response to single nerve stimuli. In addition (4) tetanic nerve stimulation and/or reduction of temperature to about 20 degrees C caused a remarkable increase in the nerve-evoked transmitter release, but did not affect the low frequency of spontaneous m.e.p.p.s. However, several important differences exist between the effects of both toxins. (1) At room temperature even single nerve stimuli could elicit e.p.p.s in BoTx-muscles the failure rate being about 80%. For TeTx the failure was 100%. However, if the nerve was stimulated with higher frequencies (greater than 5 Hz), the probability of quantal release increased, the delay for release from the onset of stimulation being several seconds and similar to that observed at 37 degrees C. (2) TeTx distorted the synchronous release of quanta increasing the distribution of their synaptic delays. BoTx did not influence the time course of the phasic secretion process in response to nerve action potentials. (3) TeTx preferentially blocked the release of spontaneous m.e.p.p.s of large amplitude without affecting the frequency of the small amplitude ones, while BoTx inhibited both the small and large amplitude m.e.p.p.s. The distribution of the amplitudes of the nerve-evoked m.e.p.p.s were similar to those of spontaneous m.e.p.p.s before the blockade with the toxins.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of tetanus toxin on the excitatory and the inhibitory post-synaptic potentials in the cat motoneurone.

Tetanus toxin (100 mouse minimal lethal doses per kilogram) was injected into the medial gastrocnemius muscle of the cat. At various times thereafter, homonymous and heteronymous group Ia excitatory post-synaptic potentials (e.p.s.p.s), disynaptic reciprocal Ia inhibitory post-synaptic potentials (i.p.s.p.s) and post-synaptic potentials (p.s.p.s) produced by sural nerve stimulation were recorded in the medial gastrocnemius motoneurones. The duration of the after-hyperpolarization, the input resistance and the axonal conduction velocity of motoneurones were also measured. Homonymous Ia e.p.s.p.s remained normal until 72 h after toxin injection. However, 5 days after toxin injection, the amplitudes of Ia e.p.s.p.s. were significantly smaller than those in control animals (1.5 +/- 1.0 mV versus 5.6 +/- 2.7 mV; t test, P less than 0.001). Heteronymous Ia e.p.s.p.s produced by stimulation of the lateral gastrocnemius-soleus nerve 5 days after toxin injection were also significantly smaller than those in control animals (0.6 +/- 0.6 mV versus 2.5 +/- 1.5 mV; P less than 0.001). However, these heteronymous Ia e.p.s.p.s remained normal when the lateral gastrocnemius-soleus nerve was ligated and sectioned at the entry to those muscles just before the toxin injection. The ascending volleys, which are supposed to represent mainly the action potentials of the dorsal spinocerebellar tract and to be elicited monosynaptically by collaterals of group I afferents, were essentially the same in the left tetanic and right control sides up to 5 days after toxin injection. Ia i.p.s.p.s and the hyperpolarizing component of sural p.s.p.s could not be produced or were very small in motoneurones sampled later than 30 h after toxin injection. The duration of the after-hyperpolarization and the input resistance of motoneurones remained normal. Axonal conduction velocity of motoneurones measured 5 days after toxin injection was 89.4 +/- 12.7 m/s, and was significantly slower than that of control motoneurones (94.1 +/- 15.4 m/s) (P less than 0.005). Differences in the amplitude of group I incoming volleys between tetanic leg and contralateral control leg were not observed. These results suggest that tetanus toxin blocks excitatory synapses in the central nervous system as well as inhibitory synapses.

Effect of tetanus toxin on K+ and Na+ concentrations in synaptosomes

A study was made of the effect of tetanus toxin (TT) in doses of 20-1000 MLD (for rats) on synaptosomes isolated from the rat brain cortex. TT produced a decrease in K+ content in synaptosomes. The effect depended on the dose of TT (I50=22 MLD). The content of Na+ remained unchanged. The action of TT depended on the time and the maximal effect was seen upon 60 minutes of incubation. TT inactivated by boiling or by antitoxin did not affect K+ content in synaptosomes. An increase of K+ concentration up to 17 mM in an incubation medium led to an increase in K+ content in TT-poisoned synaptosomes but not in the control ones. Possible changes in membrane electrogenesis in nerve terminals caused by TT are discussed.

Effect of tetanus toxin on mechanisms of regulation of transmitter secretion in neuromuscular junctions by calcium ions

Effect of tetanus toxin on mechanisms of regulation of transmitter secretion in neuromuscular junctions by calcium ions

Tetanus toxin and botulinum A toxin inhibit release and uptake of various transmitters, as studied with particulate preparations from rat brain and spinal cord.

The effects of tetanus toxin and botulinum A toxin on the uptake and evoked release of various neurotransmitters were studied using particles from rat forebrain, corpus striatum and spinal cord.

Effect of tetanus toxin on transmitter release from 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.

Effect of tetanus toxin on noradrenalin liberation from rat brain synaptosomes

A purified preparation of tetanus toxin (TT) (80–800 MLD/mg protein) was shown to induce liberation of both endogenous and exogenous (labeled with14C) noradrenalin (NA) from isolated nerve endings (synaptosomes) of the rat brain. Within the range of concentrations studied TT does not inhibit secretion of NA evoked by depolarization of synaptosomes by different methods in vitro.

Relations between the effect of tetanus toxin on the neuromuscular transmission and histological functional properties of various muscles of the rat.

Various doses of tetanus toxin were injected into three hind leg and two fore leg muscles of the rat. The neuromuscular transmission was tested by recording the mass action potential of the muscles elicited by a single electrical stimulus to the motor nerve after strong symptoms of local tetanus had developed. The muscle responses were depressed and blocked at lower toxin doses in the fast tibialis anterior than in the mixed gastrocnemius latemlis, while blocking of the slow soleus required the highest dose. The extensor carpi radialis and the flexor carpi ulnaris muscles showed medium sensitivity. In all five muscles the contraction time was measured and correlated with its individual minimal blocking dose. The more phasic (i.e., the faster) the muscle, the more sensitive its neuromuscular transmission was to tetanus toxin. The proportional distribution of red, white, and intermediate fibres, which are associated with specific end-plate types, was evaluated for the five muscles. The percentage of white fibres in the muscles displayed a very good negative correlation with the blocking dose. The relation between structures of end-plates and effects of tetanus toxin were analysed and it is suggested that the differences in sensitivity to tetanus toxin in the neuromuscular transmission in the five muscles is determined by a differential distribution of endplates with varying sensitivities to this toxin due to structural properties.

Effect of tetanus toxin on the accumulation of the permeant lipophilic cation tetraphenylphosphonium by guinea pig brain synaptosomes

Accumulation of the permeant lipophilic cation [(3)H]tetraphenylphosphonium (TPP(+)) by synaptosome preparations from guinea pig brain cerebral cortex is inhibited 1:10 by medium containing 193 mM K(+) and by veratridine. A further 1:10 to 1:15 decrease in TPP(+) uptake occurs under nitrogen and in the presence of mitochondrial inhibitors such as oligomycin, whereas starvation and succinate supplementation have no effect. These data indicate that, in analogy to intact neurons, there is an electrical potential (DeltaPsi, interior negative) of -60 to -80 mV across the synaptosomal membrane that is due primarily to a K(+) diffusion gradient (K(+) (in)-->K(+) (out)). The data also indicate that mitochondria entrapped within the synaptosome but not free mitochondria make a large contribution to the TPP(+) concentration gradients observed. Conditions are defined in which tetanus toxin binds specifically and immediately to synaptosomes in media used to measure TPP(+) uptake. Under these conditions tetanus toxin induces dose-dependent changes in TPP(+) uptake that are blocked by antitoxin and not mimicked by biologically inactivated toxin preparations. The effect of tetanus toxin on TPP(+) uptake is not evident in the presence of 193 mM K(+) or veratridine but remains under conditions known to abolish the mitochondrial DeltaPsi. Moreover, tetanus toxin has no effect on TPP(+) uptake by isolated synaptosomal mitochondria. The results thus define an in vitro action of tetanus toxin on the synaptosomal membrane that can be correlated with biological potency in vivo and is consistent with the in vivo effects of tetanus toxin on neuronal transmission.

Tetanus toxin and synaptic inhibition in the substantia nigra and striatum of the rat.

1. The effects of tetanus toxin were determined on GABA-mediated synaptic inhibition of substantia nigra neurones evoked by striatal stimulation and on the presumed dopamine- and 5-hydroxytryptamine-mediated synaptic inhibition of striatal neurones evoked by nigral and dorsal raphe nucleus stimulation, respectively, in the urethane-anaesthetized rat. 2. Following an intranigral injection of tetanus toxin, striatal-evoked inhibition of substantia nigra neurones, which is sensitive to bicuculline, was rapidly abolished. This effect was not accompanied by any significant change in the responses of nigral neurones to ionophoretically administered GABA or other putative neurotransmitters and thus indicates a presynaptic site of action of the toxin. 3. The rate of onset of action of the toxin in the substantia nigra was extremely rapid (1-4 min) and appeared to be related to the rate of activation of the inhibitory pathway. 4. Injections into the substantia nigra of tetanus toxin neutralized with antitoxin had no significant effect on striatal-evoked inhibition in the substantia nigra. 5. Injections of tetanus toxin into the striatum failed to influence the inhibition of striatal neurones evoked by stimulation of the ipsilateral substantia nigra or the dorsal raphe nucleus, suggesting that tetanus toxin does not impair monoamine-mediated inhibition in the central nervous system. 6. Synaptic excitation which preceded substantia-nigra-evoked inhibition in striatal neurones and which occasionally preceded striatal-evoked inhibition in nigral neurones was also unaffected by tetanus toxin. 7. It is suggested that tetanus toxin selectively abolishes GABA-mediated synaptic inhibition in the central nervous system and may be a useful tool in the identification of such synaptic inhibitory mechanisms.

Effect of tetanus toxin on the excitatory transmission of the motoneuroǹe and other synapses in the motor system: K. Takano, +K. Kanda, F. Kirchner, H. Kretzschmar, +M. Mizote and P. Terhaar Department of Physiology, University of Göttingen, D-3400 Göttingen, Germany +Department of Physiology, Chiba University, Chiba, Japan

Effect of tetanus toxin on the excitatory transmission of the motoneuroǹe and other synapses in the motor system: K. Takano, +K. Kanda, F. Kirchner, H. Kretzschmar, +M. Mizote and P. Terhaar Department of Physiology, University of Göttingen, D-3400 Göttingen, Germany +Department of Physiology, Chiba University, Chiba, Japan

Local tetanus in rats; concentration of amino acids as studied in spinal cord segments, spinal roots, and dorsal root ganglia.

1. The effect of tetanus toxin injected into one gastrocnemius muscle on the steady state concentration of several amino acids was investigated in spinal cord half segments, spinal roots and dorsal root ganglia of rats. Care was taken to ensure a symmetrical afferent input to the spinal cord and to localize the segment with the highest concentration of tetanus toxin. 2. In the spinal cord segments containing the highest concentration of tetanus toxin the steady state concentration of glycine was higher on the side of the tetanus than on the contralateral control side. Results obtained after intravenous injection of 14C-glycine do not indicate that the higher concentration of glycine on the side of the tetanus was due to a higher uptake of glycine. 3. The results obtained in the spinal cord contradict previous findings of other authors but lend support to the prevailing concept about the action of tetanus toxin in local and general tetanus.

Tetanus Toxin Interactions with the Thyroid: Decreased Toxin Binding to Membranes from a Thyroid Tumor with a Thyrotropin Receptor Defect andin VivoStimulation of Thyroid Function

Normal rat thyroid membranes adsorb neurotoxicity when incubated with purified tetanus toxin. Membranes from a rat thyroid tumor with a thyrotropin receptor defect adsorb very little neurotoxicity when similarly evaluated. This inability of the tumor membranes to adsorb neurotoxicity is correlated with a defect in their ability to bind both 125I-labeled tetanus toxin and [125I]iodothyrotropin. The effect of tetanus toxin on the release of radioiodine from the thyroids of appropriately prepared mice has been measured by adapting methods used for the bioassay of thyrotropin. One minimum lethal dose of tetanus toxin given sc caused a significant release of radioiodine into the blood of mice 48 h after injection. In mice subjected to the stress of prior bleedings or anesthesia, the release of radioiodine from the thyroid by tetanus toxin was accelerated, i.e., the increase in blood radioiodine could be measured 24 h after injection. These results again suggest that tetanus toxin may interact with thyrotropin receptors on thyroid plasma membranes. The "sympathetic overactivity syndrome" seen in some patients with tetanus and the syndrome characterized as "thyroid storm" in patients with Graves' disease are discussed as they may relate to these observations.

Effect of tetanus toxin on protein composition of synaptic structures of the rat brain and spinal cord

It was shown by electrophoresis on polyacrylamide gel that the content of proteins with low electrophoretic mobility rises in a Triton extract of the fractions of synaptic structures from the spinal cord tissue of rats with local tetanus, whereas no change was found in the protein spectrum in the dodecyl sulfate extract. In experiments in vitro tetanus toxin stimulated the incorporation of lysine-H3 into total proteins of cortical synaptosomes.

The effect of tetanus toxin on the extensor and flexor muscles of the hind leg of the cat: Takano, K., Department of Physiology, University of Göttingen, Göttingen, West Germany

The effect of tetanus toxin on the extensor and flexor muscles of the hind leg of the cat: Takano, K., Department of Physiology, University of Göttingen, Göttingen, West Germany

Effect of tetanus toxin on contractility of the actomyosin-like protein of rat brain

Preparation of purified actomyosin-like protein (ALP) was isolated from the coarse fraction of rat brain mitochondria. The ALP preparation contained Mg(Ca)-ATPase activity, inhibited by 0.1% deoxycholate, together with acetylcholinesterase activity and contractility (superprecipitation reaction). The contraction of ALP and ATPase activity were considerably reduced by tetanus toxin. Inactivated toxin had no effect. The possible functional role of ALP in the maintenance and secretion of mediators by nerve endings is discussed.

The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse.

1. A sublethal dose of tetanus toxin was injected into the muscles of one hind leg of the mouse and caused local tetanus which persisted for 4 weeks.2. Neuromuscular transmission was studied in vitro in nerve-muscle preparations of soleus, a slow muscle, and extensor digitorum longus (EDL), a fast muscle, from 1 day to 6 months after the injection of toxin.3. Soleus failed to respond to nerve stimulation, became supersensitive to acetylcholine and showed spontaneous fibrillations for several weeks before returning to normal. EDL did not show these changes. A higher dose of tetanus toxin, lethal within 24 hr, caused paralysis of EDL as well as soleus.4. In muscle fibres in which neuromuscular transmission was blocked spontaneous miniature end-plate potentials (m.e.p.p.s) were recorded. The frequency of m.e.p.p.s was increased by repetitive nerve stimulation but not by raising the external potassium concentration.5. The amplitude of spontaneous m.e.p.p.s showed a skew distribution because of a disproportionate number of potentials of less than 0.2 mV.6. Raising the external calcium concentration did not restore neuromuscular transmission.7. Histological examination of soleus showed atrophy of muscle fibres with normal preterminal axons. There was sprouting from motor nerve terminals and subsequently new motor end-plates were formed. These changes were not found in EDL.8. The results indicate that, in the mouse, tetanus toxin causes a presynaptic block of neuromuscular transmission and ;functional denervation' of muscle. Slow muscle is more sensitive to the effects of the toxin than fast.

Effect of tetanus toxin on the inhibitory neuromuscular junction of crayfish muscle

The effect of tetanus toxin on inhibitory synaptic transmission in crayfish muscle, which seemed to be mediated by GABA, was investigated by means of intracellular recording. Tetanus toxin caused a gradual block of inhibitory synaptic transmission. The GABA sensitivity of the postsynaptic membrane and the electrical activity of the presynaptic nerve terminal remained unaffected. It is concluded that the toxin can abolish the inhibitory synaptic transmission by reducing the amount of GABA released from inhibitory nerve terminal, but the toxin does not interfere with the conduction of impulses along the inhibitory nerve axons up to the nerve terminal.