tetrodotoxin and Paralysis

Saxitoxins (STX) and tetrodotoxins (TTX) are a group of chemical compounds produced by certain species of marine algae and fish. Lethal dose for a human is about 0.5-2.0 mg when the toxin enters the body via food, and 0.05 mg of poisoning at the time of injection. In the case of aerosol the lethal dose for human being is 5 mg/min/m(3). STX and TTX poisoning cause mostly symptoms from the nervous system in the form of: paresthesia around the lips, tongue, gums, distal segments of the limbs, headache, dysphonia, astigmatism, floating feeling, muscle weakness, paralysis of cranial and peripheral nerves. There is no specific antidote for STX and TTX. It is recommended supportive treatment.

Topics: Administration, Inhalation; Administration, Oral; Aerosols; Animals; Astigmatism; Biological Warfare Agents; Dysphonia; Headache; Humans; Lethal Dose 50; Muscle Weakness; Paralysis; Paresthesia; Saxitoxin; Tetrodotoxin

Seafood poisoning has been recognized as a problem in both coastal and inland populations for millennia. Many types of sea creatures from shellfish to the largest fish have been implicated. Severe cases of many different types of seafood poisonings can result in fatalities. While the pathophysiology of the toxins is well known in some cases, others, like ciguatera, remain somewhat confusing. As a result, the treatment of these conditions remains controversial, although supportive care continues to be the mainstay of therapy. In this manuscript, we review the pathophysiology, clinical presentation, and treatment of some of the most common and toxic varieties of seafood poisoning resulting from toxins.

Topics: Animals; Ciguatera Poisoning; Fishes; Humans; Kainic Acid; Marine Toxins; Paralysis; Seafood; Shellfish Poisoning; Tetrodotoxin

Three conditions that may occur after consumption of seafood--puffer fish poisoning, ciguatera, and paralytic shellfish poisoning--are caused by a group of poisons that block voltage-gated sodium channels in myelinated and non-myelinated nerves. The conditions cannot be distinguished clinically and so constitute an entity for which the name pelagic paralysis is proposed. Variations in the clinical features can be accounted for by large differences in the amount of toxin present in the seafood.

Topics: Animals; Ciguatera Poisoning; Fishes, Poisonous; Foodborne Diseases; Humans; Marine Toxins; Mollusca; Paralysis; Shellfish Poisoning; Terminology as Topic; Tetrodotoxin

Tetrodotoxin (TTX) is an extremely toxic marine compound produced by different genera of bacteria that can reach humans through ingestion mainly of pufferfish but also of other contaminated fish species, marine gastropods or bivalves. TTX blocks voltage-gated sodium channels inhibiting neurotransmission, which in severe cases triggers cardiorespiratory failure. Although TTX has been responsible for many human intoxications limited toxicological data are available. The recent expansion of TTX from Asian to European waters and diversification of TTX-bearing organisms entail an emerging risk of food poisoning. This study is focused on the acute toxicity assessment of TTX administered to mice by oral gavage following macroscopic and microscopic studies. Necropsy revealed that TTX induced stomach swelling 2 h after administration, even though no ultrastructural alterations were further detected. However, transmission electron microscopy images showed an increase of lipid droplets in hepatocytes, swollen mitochondria in spleens, and alterations of rough endoplasmic reticulum in intestines as hallmarks of the cellular damage. These findings suggested that gastrointestinal effects should be considered when evaluating human TTX poisoning.

Topics: Administration, Oral; Animals; Brain; Endoplasmic Reticulum, Rough; Female; Intestines; Kidney; Liver; Lung; Mice; Microscopy, Electron, Transmission; Mitochondria; Myocardium; Neurotoxins; Paralysis; Seizures; Spleen; Stomach; Tetrodotoxin; Toxicity Tests, Acute

The high medical importance of Crotalus snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the Bothrops genus, the Crotalus genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all Crotalus snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10-25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed in vitro and in vivo assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, ex vivo assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine in vivo. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (ip) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.

Topics: 4-Aminopyridine; Animals; Crotalid Venoms; Dose-Response Relationship, Drug; Hindlimb; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Pain Measurement; Paralysis; Potassium Channels, Voltage-Gated; Tetrodotoxin; Voltage-Gated Sodium Channel Blockers; Voltage-Gated Sodium Channels

We test previous claims that the bacteria Vibrio alginolyticus produces tetrodotoxin (TTX) when living in symbiosis with the nemertean Lineus longissimus by a setup with bacteria cultivation for TTX production. Toxicity experiments on the shore crab, Carcinus maenas, demonstrated the presence of a paralytic toxin, but evidence from LC-MS and electrophysiological measurements of voltage-gated sodium channel-dependent nerve conductance in male Wistar rat tissue showed conclusively that this effect did not originate from TTX. However, a compound of similar molecular weight was found, albeit apparently non-toxic, and with different LC retention time and MS/MS fragmentation pattern than those of TTX. We conclude that C. maenas paralysis and death likely emanate from a compound <5 kDa, and via a different mechanism of action than that of TTX. The similarity in mass between TTX and the Vibrio-produced low-molecular-weight, non-toxic compound invokes that thorough analysis is required when assessing TTX production. Based on our findings, we suggest that re-examination of some published claims of TTX production may be warranted.

Topics: Animals; Brachyura; Chromatography, Liquid; Helminths; Male; Molecular Weight; Paralysis; Rats; Rats, Wistar; Symbiosis; Tandem Mass Spectrometry; Tetrodotoxin; Vibrio alginolyticus; Voltage-Gated Sodium Channels

Paralytic shellfish toxins (PST) traditionally have been analyzed by liquid chromatography with either pre- or post-column derivatization and always with a silica-based stationary phase. This technique resulted in different methods that need more than one run to analyze the toxins. Furthermore, tetrodotoxin (TTX) was recently found in bivalves of northward locations in Europe due to climate change, so it is important to analyze it along with PST because their signs of toxicity are similar in the bioassay. The methods described here detail a new approach to eliminate different runs, by using a new porous graphitic carbon stationary phase. Firstly we describe the separation of 13 PST that belong to different groups, taking into account the side-chains of substituents, in one single run of less than 30 min with good reproducibility. The method was assayed in four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Pecten maximus), scallop (Ruditapes decussatus) and oyster (Ostrea edulis). The results for all of the parameters studied are provided, and the detection limits for the majority of toxins were improved with regard to previous liquid chromatography methods: the lowest values were those for decarbamoyl-gonyautoxin 2 (dcGTX2) and gonyautoxin 2 (GTX2) in mussel (0.0001 mg saxitoxin (STX)·diHCl kg(-1) for each toxin), decarbamoyl-saxitoxin (dcSTX) in clam (0.0003 mg STX·diHCl kg(-1)), N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2) in scallop (0.0001 mg STX·diHCl kg(-1) for each toxin) and dcSTX (0.0003 mg STX·diHCl kg(-1) ) in oyster; gonyautoxin 2 (GTX2) showed the highest limit of detection in oyster (0.0366 mg STX·diHCl kg(-1)). Secondly, we propose a modification of the method for the simultaneous analysis of PST and TTX, with some minor changes in the solvent gradient, although the detection limit for TTX does not allow its use nowadays for regulatory purposes.

Topics: Animals; Bivalvia; Chromatography, High Pressure Liquid; Fluorometry; Food Contamination; Graphite; Limit of Detection; Mytilus; Ostrea; Oxidation-Reduction; Paralysis; Pecten; Pectinidae; Porosity; Reproducibility of Results; Saxitoxin; Seafood; Shellfish Poisoning; Tetrodotoxin

The first European case of tetrodotoxin intoxication is reported in a patient who ingested a trumpet shellfish from the Atlantic Ocean in Southern Europe. He suffered general acute paralysis with respiratory failure necessitating ventilation. Early neurophysiologic studies showed complete peripheral nerve inexcitability, with no recordable sensory or motor responses, and normal electroencephalography. Tetrodotoxin was detected in high quantities in the patient's blood and urine through high performance liquid chromatography-mass spectrometry analysis. Seventy-two hours after admission the patient recovered normal strength, reflexes and sensation.

Topics: Electroencephalography; Foodborne Diseases; Humans; Male; Middle Aged; Paralysis; Seafood; Tetrodotoxin

Poisoning after eating puffer fish containing highly lethal tetrodotoxin (TTX) is widespread in Asia. In 2008, naïve inland populations in Bangladesh were exposed to cheap puffer fish sold on markets. In three outbreaks, 141 patients with history of puffer fish consumption were hospitalized. Symptoms of poisoning included perioral paraesthesia, tingling over the entire body, nausea and vomiting, dizziness, headache, abdominal pain and muscular paralysis of the limbs. Seventeen patients (12%) died from rapidly developing respiratory arrest. Blood and urine samples from 38 patients were analyzed using a TTX-specific enzyme-linked immunoassay (ELISA). Medium to high TTX levels were detected (1.7-13.7 ng/ml) in the blood of 27 patients. TTX was below detection level (< 1.6 ng/ml) in 11 blood samples but the toxin was detected in urine. Ten patients had blood levels above 9 ng/ml and developed paralysis; seven of these died. The remaining patients recovered with supportive treatment. High concentrations of TTX and its analogues 4-epiTTX and 4,9-anhydroTTX were also found in cooked puffer fish by post-column liquid chromatography-fluorescence detection. To prevent future instances of puffer fish poisoning of this magnitude, measures should be implemented to increase awareness, to control markets and to establish toxicological testing. To improve the management of this and other poisoning in Bangladesh, facilities for life-saving assisted ventilation and related training of healthcare personnel are urgently needed at all levels of the health system.

Topics: Adolescent; Adult; Animals; Bangladesh; Child; Child, Preschool; Chromatography, Liquid; Disease Outbreaks; Enzyme-Linked Immunosorbent Assay; Female; Fishes, Poisonous; Foodborne Diseases; Humans; Male; Middle Aged; Paralysis; Poverty Areas; Respiratory Insufficiency; Tetraodontiformes; Tetrodotoxin; Young Adult

Tetrodotoxin is considered the most lethal toxin in the marine environment. Prior cases of intoxication previously described correspond to consumption of tetrodotoxin in tropical or subtropical regions of Asia or the Pacific Islands.. We present the first European case of tetrodotoxin intoxication in a patient who ingested part of a trumpet shellfish (Charonia sauliae) from the Atlantic Ocean in Southern Europe.. Our patient suffered general paralysis, including the respiratory muscles, a few minutes after the consumption of a few grams of C. sauliae. Intubation and mechanical ventilation were necessary for 52 h after the intoxication. The corresponding electrophysiologic studies showed complete non-excitability, with no recordable sensory or motor nerve conduction. We detected the presence of tetrodotoxin in the mollusk and the patient's blood and urine by means of high-performance liquid chromatography-mass spectrometry analysis technique. A previous bioassay showed extremely high quantities of the toxin in the mollusk.. This case alerts us to the possibility of a very harmful biotoxin in European coastal waters. This now should be included in the differential diagnosis of similar cases in Europe, and we must be vigilant for its possible presence in Europe.

Topics: Chromatography, Liquid; Electroencephalography; Europe; Humans; Male; Mass Spectrometry; Middle Aged; Mollusk Venoms; Paralysis; Respiration, Artificial; Respiratory Muscles; Shellfish Poisoning; Sodium Channel Blockers; Tetrodotoxin

On 24 July 2005, six members of a single family were admitted to the Medicine and Pediatrics Department of Khulna Medical College Hospital, Khulna, Bangladesh, with a history of ingestion of puffer fish. All patients developed toxic manifestations. The cases were clinically analysed with successful outcomes.

Topics: Adolescent; Adult; Animals; Bangladesh; Child; Child, Preschool; Female; Fishes, Poisonous; Foodborne Diseases; Humans; Male; Neostigmine; Paralysis; Parasympathomimetics; Respiratory Muscles; Tetraodontiformes; Tetrodotoxin; Treatment Outcome

Bivalve molluscs, the primary vectors of paralytic shellfish poisoning (PSP) in humans, show marked inter-species variation in their capacity to accumulate PSP toxins (PSTs) which has a neural basis. PSTs cause human fatalities by blocking sodium conductance in nerve fibres. Here we identify a molecular basis for inter-population variation in PSP resistance within a species, consistent with genetic adaptation to PSTs. Softshell clams (Mya arenaria) from areas exposed to 'red tides' are more resistant to PSTs, as demonstrated by whole-nerve assays, and accumulate toxins at greater rates than sensitive clams from unexposed areas. PSTs lead to selective mortality of sensitive clams. Resistance is caused by natural mutation of a single amino acid residue, which causes a 1,000-fold decrease in affinity at the saxitoxin-binding site in the sodium channel pore of resistant, but not sensitive, clams. Thus PSTs might act as potent natural selection agents, leading to greater toxin resistance in clam populations and increased risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to communities and ecosystems.

Topics: Amino Acid Sequence; Animals; Bivalvia; Cell Line; Ciguatera Poisoning; Drug Resistance; Electric Conductivity; Humans; Molecular Sequence Data; Mutation; Paralysis; Risk; Saxitoxin; Selection, Genetic; Sodium; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Unlike predators, which immediately consume their prey, parasitoid wasps incapacitate their prey to provide a food supply for their offspring. We have examined the effects of the venom of the parasitoid wasp Ampulex compressa on the metabolism of its cockroach prey. This wasp stings into the brain of the cockroach causing hypokinesia. We first established that larval development, from egg laying to pupation, lasts about 8 days. During this period, the metabolism of the stung cockroach slows down, as measured by a decrease in oxygen consumption. Similar decreases in oxygen consumption occurred after pharmacologically induced paralysis or after removing descending input from the head ganglia by severing the neck connectives. However, neither of these two groups of cockroaches survived more than six days, while 90% of stung cockroaches survived at least this long. In addition, cockroaches with severed neck connectives lost significantly more body mass, mainly due to dehydration. Hence, the sting of A. compressa not only renders the cockroach prey helplessly submissive, but also changes its metabolism to sustain more nutrients for the developing larva. This metabolic manipulation is subtler than the complete removal of descending input from the head ganglia, since it leaves some physiological processes, such as water retention, intact.

Topics: Anesthetics, Local; Animals; Behavior, Animal; Body Mass Index; Body Water; Cockroaches; Food Preservation; Host-Parasite Interactions; Hymenoptera; Larva; Life Cycle Stages; Life Expectancy; Mortality; Neck Injuries; Oxygen Consumption; Paralysis; Predatory Behavior; Survival Rate; Tetrodotoxin; Time Factors; Wasp Venoms

Outbreaks of paralytic snail poisoning have recently occurred in Asia, especially in China. The epidemiological characteristics of this disease from an outbreak in Zhoushan City, China, were recorded. Forty-two outbreaks of paralytic snail poisoning, involving 309 cases of illness, occurred from 1977 to 2001. Sixteen people (5.2%) died, 48 people (15.5%) required intubations, and 140 people (45.3%) required emergency hospital treatment as a result of these outbreaks. Outbreaks involved multiple marine snail species and occurred primarily during the summer (from June to August) on 11 islands with high population densities. Peak numbers of outbreaks and amounts of snail toxicity occurred from 1978 to 1979, from 1985 to 1987, and from 1992 to 1994. Toxicity varied depending on specimen, region, and season. The toxin involved was identified as tetrodotoxin. The data obtained in this study suggest that snails should not be eaten unless they are certified to be nontoxic.

Topics: Animals; China; Disease Outbreaks; Foodborne Diseases; Humans; Paralysis; Population Density; Retrospective Studies; Seasons; Snails; Tetrodotoxin

Paralytic toxicity of ribbon worms ("himomushi" in Japanese), identified as undescribed species of the genus Cephalothrix, found on the surface of the shells of cultured oysters in Hiroshima Bay, Hiroshima Prefecture was examined between April 1998 and December 2001. The toxicity study showed that all of specimens were found to contain toxins with strong paralytic action in mice; the highest toxicity (as tetrodotoxin, TTX) was 25,590 mouse units (MU) per gram for whole body throughout the monitoring period. The main toxic component of this himomushi toxin (HMT) was isolated from a pooled specimen (390 g; total toxicity 2,897,000MU) by a method that consisted of treatment with activated charcoal, chromatography on Bio-Gel P-2 and Bio-Rex 70 (H+ form), and finally crystallization from an acidified methanolic solution. The recrystallized toxin showed a specific toxicity of 3520MU/mg. This toxin showed (M+H)+ and (M+H-H(2)O)+ ion peaks at m/z 320 and 302, respectively, by electrospray ionization-mass spectrometry (ESI-MS). The absorption band at 3353, 3235, 1666, 1612 and 1076 cm(-1) were observed in infrared spectrum of this toxin. This spectrum was indistinguishable from that of TTX. The 1H-NMR spectrum for the recrystallized toxin was the same as that for TTX. The pair of doublets centered at 2.33 (J=10.0Hz) and 5.48 ppm (J=10.0Hz) which are characteristic of TTX, were shown to be coupled by double irradiation. Furthermore, by gas chromatography-mass spectrometry (GC-MS) of the alkali-hydrolyzate of this toxin indicated the presence of quinazoline skeleton (C9-base) specific to TTX.

Topics: Animals; Gas Chromatography-Mass Spectrometry; Invertebrates; Japan; Magnetic Resonance Spectroscopy; Male; Marine Toxins; Mice; Ostreidae; Paralysis; Seasons; Seawater; Symbiosis; Tetrodotoxin

Cyclin-dependent kinase 5 (Cdk5) was originally identified as a serine/threonine kinase and subsequently demonstrated to play a critical role in the development of CNS. We recently reported the novel function of Cdk5 in the neuregulin signaling pathway during the development of neuromuscular junction (NMJ). Here, we report the regulation of Cdk5 and p35 in rat skeletal muscle after nerve injury. Northern blot analysis revealed that Cdk5 and p35 transcripts were up-regulated in muscle after nerve denervation. The temporal profiles for the regulation of Cdk5 and p35 transcripts were different, suggesting that these changes in gene transcription might be regulated by different mechanism. Our finding on the ability of tetrodotoxin to induce p35 transcript in muscle suggested that electrical activity could regulate p35 expression. In addition to the induction of mRNA expression, the total Cdk5 and p35-associated kinase activity in muscle increased prominently after nerve denervation. Taken together, our findings suggest that Cdk5 and p35 may play important physiological roles in muscle regeneration following nerve injury.

Topics: Animals; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Denervation; Muscle, Skeletal; Nerve Tissue Proteins; Neuromuscular Junction; Paralysis; Rats; RNA, Messenger; Sciatic Nerve; Tetrodotoxin

The expression of five myosin heavy chain (MHC) isoforms was analyzed in the rat soleus (Sol) and the deep and superficial medial gastrocnemius (dGM, sGM) muscle after 2 and 4 wk of TTX paralysis by using immunohistochemical techniques. In Sol, after 4 wk of paralysis, fibers containing type I MHC were either pure type I (14%) or also contained developmental (D; 76%), IIa (26%), or IIx (18%) MHC. Values for corresponding fibers in dGM were 8.5, 65, 38, and 22%. Also, by 4 wk an increase was seen in the proportions of fibers expressing IIa MHC in Sol (from 16 to 38%) and dGM (from 24 to 74%). In a region of sGM in control muscles containing pure IIb fibers, a major proportion (86%) remained pure after 4 wk of paralysis, with the remainder coexpressing IIb and IIx. The results indicate that TTX-induced muscle paralysis results in an increase in fibers containing multiple MHC isoforms and that the D isoform appears in a major proportion of these hybrid fibers.

Topics: Animals; Body Weight; Female; Immunohistochemistry; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Myosin Heavy Chains; Organ Size; Paralysis; Protein Isoforms; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors

In 1998, during the surveillance of the toxicity of various marine fouling organisms in Hiroshima Bay, Hiroshima Prefecture, Japan, specimens of the ribbon worm, "himomushi" Cephalothrix sp. (Nemertean) adherent to the shells of cultured oysters hanging onto floating culture rafts were found to contain toxins which showed strong paralytic action in mice throughout the survey period, February to May. The maximum toxicity (as tetrodotoxin, TTX) was 14,734 MU/g whole body. Attempts were made to identify the paralytic toxins in this worm. The "himomushi" toxin (HMT) was extracted from the worm with 80% methanol acidified with acetic acid and the extract defatted with dichloromethane. The aqueous layer was chromatographed on activated charcoal and the unbound and bound toxic fractions were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. It was rather unexpectedly revealed from these results that HMT was comprised of TTX, 4-epiTTX, anhydroTTX and three unidentified toxins. To our knowledge, this is the first report of the occurrence of toxic organisms, containing a high concentration of TTX, adherent to cultured bivalves such as oysters.

Topics: Animals; Annelida; Gas Chromatography-Mass Spectrometry; Japan; Male; Marine Toxins; Mice; Ostreidae; Paralysis; Symbiosis; Tetrodotoxin

Considerably high toxicity was detected in marine puffers collected from Masinloc Bay, Philippines. The toxicity was detected in the liver, intestine, muscle and skin. Noteworthy, the specimens, the muscle of which showed high toxicity, appeared in high frequency, indicating that puffers from this area is not safe for human consumption. These puffer specimens contained paralytic shellfish poisoning (PSP) toxins, often as major toxin components, the profile of which was similar to that of freshwater puffers reported from tropical areas. These results indicate that PSP toxins are common in tropical puffers both from marine and freshwater.

Topics: Animals; Chromatography, High Pressure Liquid; Fishes; Hydrogen-Ion Concentration; Male; Marine Toxins; Mice; Paralysis; Shellfish; Skin; Species Specificity; Tetrodotoxin

The peptide LqhalphaIT is an alpha-scorpion toxin that shows significant selectivity for insect sodium channels over mammalian channels. We examined the symptoms of LqhalphaIT-induced paralysis and its neurophysiological correlates in the house fly (Musca domestica). Injection of LqhalphaIT into fly larvae produced hyperactivity characterized by continuous, irregular muscle twitching throughout the body. These symptoms were correlated with elevated excitability in motor units caused by two physiological effects of the toxin: 1) increased transmitter release and 2) repetitive action potentials in motor nerves. Increased transmitter release was evident as augmentation of neurally evoked synaptic current, and this was correlated with an increased duration of action potential-associated current (APAC) in loose patch recordings from nerve terminals. Repetitive APACs were observed to invade nerve endings. The toxin produced marked inhibition of sodium current inactivation in fly central neurons, which can account for increased duration of the APAC and elevated neurotransmitter release at the neuromuscular junction. Steady-state inactivation was shifted significantly to more positive potentials, whereas voltage-dependent activation of the channels was not affected. The shift in steady-state inactivation provides a mechanism for inducing repetitive activity in motoneurons. The effects of LqhalphaIT on sodium channel inactivation in motor nerve endings can account both for increased transmitter release and repetitive activity leading to hyperactivity in affected insects.

Topics: Action Potentials; Animals; Behavior, Animal; Electrophysiology; Excitatory Postsynaptic Potentials; Houseflies; In Vitro Techniques; Larva; Membrane Potentials; Motor Activity; Neurotoxins; Paralysis; Patch-Clamp Techniques; Scorpion Venoms; Sodium Channel Blockers; Stimulation, Chemical; Synapses; Synaptic Transmission; Tetrodotoxin

In this study, we test the hypothesis that 4 weeks tetrodotoxin (TTX) paralysis altered the passive membrane properties of rat tibial motoneurones. Impulse activity along the sciatic nerve was blocked for 4 weeks using TTX delivered by an osmotic minipump to a Silastic cuff placed around the nerve. That portion of the sample exhibiting the 20% slowest After-hyperpolarization (AHP) decay time (AHPd), and which therefore included presumptive type S motoneurons, demonstrated responses (reduced AHPd, increased rheobase and rheobase voltage), which were not evident in the rest of the sample (presumptive fast motoneurons), in which an increased AHPd, in fact, was found. The results thus support the hypothesis that retrograde signals from inactive slow and fast muscle fibers have different effects on their innervating motoneurones.

Topics: Animals; Cell Communication; Cell Membrane; Motor Neurons; Muscle Fibers, Skeletal; Neuromuscular Junction; Paralysis; Rats; Signal Transduction; Tetrodotoxin; Time Factors

The muscle-specific helix-loop-helix (HLH) transcription factors myoD, myogenin, MRF4, and myf-5 are called the muscle regulatory factor family (MRF). Levels of MRFs are strongly regulated by muscle electrical activity and are thought to control downstream genes that are important for muscle phenotype such as the acetylcholine receptor (AChR) and possibly genes connected to muscle metabolic properties. The MRFs interact with ubiquitously expressed HLH factors such as E-proteins and Id-proteins to form heterodimers. In the present paper, we report the effects of paralysis obtained by nerve impulse block with tetrodotoxin (TTX) and denervation on messenger ribonucleic acid (mRNA) levels for Id-1, E47, myogenin, AChR alpha-subunit and beta-actin. Both Id-1 and E47 showed twofold increases in absence of nerve evoked electrical activity. These changes in the ubiquitously expressed HLH factors might have important functional implications for downstream gene expression, but in comparison, myogenin mRNA was increased 10-fold. We conclude that myogenin and the other muscle-specific MRFs remain the transcription factors with the strongest activity dependence that has so far been described in muscle.

Topics: Animals; Helix-Loop-Helix Motifs; Male; Muscle Denervation; Muscle, Skeletal; Paralysis; Rats; Rats, Wistar; RNA, Messenger; Tetrodotoxin; Transcription Factors

Muscle-specific kinase (MuSK) is part of the receptor complex that is involved in the agrin-induced formation of the neuromuscular junction. In the rodent, prominent mRNA expression of MuSK is restricted to skeletal muscle while the expression of agrin can also be detected in brain and certain nonneuronal tissues. The recent identification of Xenopus MuSK reveals that MuSK can be detected in tissues other than skeletal muscle, such as the neural tube, eye vesicles, and spleen. In this study, we describe the cloning and characterization of the chicken ortholog of MuSK and demonstrate that the regulation of MuSK expression in muscle is conserved from avian to rodent. Abundant mRNA expression of MuSK can be detected in early embryonic chick muscle and is up-regulated after nerve injury. More importantly, we also demonstrate that, in the chicken, MuSK mRNA is expressed during development in brain and liver, suggesting possible novel functions for MuSK. Furthermore, the regulatory profile of MuSK expression in chick muscle closely parallels that observed for acetylcholine receptor, in terms of both mRNA expression and protein localization. Finally, studies with paralyzed chicken muscle as well as with cultured chick myotubes demonstrate the dependence of MuSK on both electrical activity and trophic factors.

Topics: Animals; Cells, Cultured; Chick Embryo; Chickens; Cloning, Molecular; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Muscle Denervation; Muscle Fibers, Skeletal; Muscle, Skeletal; Nerve Crush; Paralysis; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Rats; Receptor Protein-Tyrosine Kinases; Receptors, Cholinergic; Sciatic Nerve; Sequence Homology, Amino Acid; Species Specificity; Tetrodotoxin; Transcription, Genetic

Topics: Animals; Cell Membrane; Glycerolphosphate Dehydrogenase; Histocytochemistry; Muscle, Skeletal; Oxidoreductases; Paralysis; Rats; Succinate Dehydrogenase; Tetrodotoxin; Time Factors

Recent evidence suggests that the high content of acetylcholinesterase (AChE) globular form G4, characteristic of fast muscles, is controlled by phasic high-frequency activity performed by these muscles. This indicates that inactive, though still innervated, fast muscles should be devoid of their characteristic G4 pool. Accordingly, in the absence of phasic activity, both fast and slow muscles should exhibit a common basic profile of AChE molecular forms of the slow type. We first tested this hypothesis by examining the AChE content in cultures of myotubes obtained from the fusion of satellite cells originating from fast and slow muscles. These two cell populations produced AChE molecular-form profiles of the slow type characterized by modest levels of G4 together with an increased proportion of the asymmetric forms A8 relative to A12. Second, we determined the impact of muscle paralysis on the specific content of AChE molecular forms of adult rat fast and slow muscles. Complete paralysis of hindlimb muscles was achieved by chronic superfusion of tetrodotoxin (TTX) onto the sciatic nerve. Ten days after TTX inactivation, the distributions of AChE molecular forms of both fast extensor digitorum longus (EDL) and plantaris muscles were transformed into ones resembling the slow soleus, the latter showing no significant modifications in its AChE profile. Finally, we investigated the impact of nerve-mediated phasic high-frequency stimulation of TTX-inactivated fast and slow muscles on the content of AChE molecular forms. This stimulation produced a profile of AChE molecular forms similar to that observed in control EDL muscles, indicating that phasic activation counteracted the TTX-induced transformation in the distribution of AChE molecular forms in fast EDL muscles. Together, these results are consistent with the proposal that adult fast muscles constitutively express a basic profile of AChE molecular forms of the type displayed by slow muscles, onto which varying levels of G4 are added according to the amount of phasic activity performed by the muscles.

Topics: Acetylcholinesterase; Animals; Cells, Cultured; Electric Stimulation; Enzyme Activation; Female; Hindlimb; Molecular Conformation; Muscle Denervation; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Paralysis; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tetrodotoxin

1. Although the inactivity of a slow muscle (cat soleus) induced via nerve impulse blockade has been demonstrated to have some axotomy-like effects (decreased after-hyperpolarization (AHP) duration) on its innervating motoneurons, the reported effects of inactivity on motoneurons which innervate fast muscles containing mixtures of motor unit types are equivocal. This study was designed to determine the effect of a period (2 weeks) of complete hindlimb muscle paralysis, via tetrodotoxin (TTX) blockade of sciatic nerve impulses, on the contractile (muscle units) and electrophysiological (motoneurons) properties of motor units in the rat gastrocnemius. Motoneuron properties were also compared with those of rats subjected to sciatic nerve axotomy 2 weeks earlier. 2. At the time of the terminal experiment (24 h after the removal of the TTX delivery system) in anaesthetized animals, properties of tibial motoneurons (i.e. rheobase current, input resistance, time course of after-potentials) were determined using conventional microelectrode techniques. For those tibial motoneurons innervating the gastrocnemius, muscle unit responses (i.e. twitch force and time course, maximum tetanic tension, fatigability) were also recorded in response to current injection. 3. Consistent with previously reported whole-muscle responses to TTX-induced disuse, the TTX-treated gastrocnemius muscle units showed weaker tetanic forces, prolonged twitches and elevated twitch/tetanic ratios. These effects were similar for motor units classified as small, medium and large according to their tetanic tension-generating capacities. Muscle unit fatigue resistances appeared to be unchanged. 4. The mean values, distributions and ranges of tibial motoneuron properties were similar between control and TTX-treated groups for rheobase, input resistance and AHP half-decay time. In the case of the latter, the proportion of motoneurons possessing "slow' AHP half-decay times (> 20 ms) was not significantly different in control (17%) and TTX-treated groups (11%). 5. Motoneurons axotomized 2 weeks earlier had a significantly higher (42-69%) mean input resistance and a longer (34-42%) mean AHP half-decay time when compared with the control and TTX-treated groups. 6. It appears that, for fast muscles containing several different motor unit types, TTX-induced axon blockade does not produce similar effects on motoneuron intrinsic properties to those evoked by axotomy. This lack of effect on the distribution a

Topics: Animals; Male; Motor Neurons; Muscle Fibers, Skeletal; Paralysis; Rats; Rats, Sprague-Dawley; Tetrodotoxin

The effects of long lasting (4-5 weeks) nerve conduction block and denervation were compared by investigating contractile, morphological and histochemical properties of slow (soleus) and fast (EDL) rat skeletal muscles. The block was based on improved perfusion techniques of the sciatic nerve with a tetrodotoxin (TTX) solution delivered at doses adequate to obtain maximal effects in the muscles. The TTX-inactivated axons retained normal histological and physiological properties such as the ability to evoke full contractile responses, to regenerate, and to completely reinnervate muscle. In spite of their intact innervation or of their full reinnervation, the TTX-paralysed muscles underwent weight loss, fibre atrophy and reduction in force, output quantitatively indistinguishable from those following denervation. The same was true for all other contractile parameters tested, that is, twitch speed, twitch to tetanus ratio, post-tetanic potentiation, endurance, and fibre type composition. The results indicate the fundamental role of activity as a regulatory signal for muscle contractile properties, while they do not support the notion of a participation of chemical, activity-independent factors in this regulation.

Topics: Animals; Axons; Dose-Response Relationship, Drug; Electric Stimulation; Male; Muscle Contraction; Muscle Denervation; Muscle, Skeletal; Paralysis; Rats; Rats, Wistar; Tetrodotoxin

The extent to which myosin profiles within adult fast and slow muscles are altered by short-term paralysis remains equivocal. We used an array of specific antibodies to identify adult and developmental MHC isoforms within EDL and soleus muscle fibers, and show a marked multiple expression of MHCs with a general shift towards slower and more energy efficient MHC profiles after 2 weeks of denervation or TTX nerve conduction block. Paralysis also induced marked expression of an embryonic MHC within most EDL cell types, and a subtle, paralysis-sensitive, expression of alpha-cardiac MHC within specific EDL and soleus extrafusal fibers. Comparison of treatment groups also permitted assessment of the relative influence of neural activity versus trophic factors on these isoforms, and confirmed activity as a major, but not sole, regulator of MHC expression.

Topics: Animals; Animals, Newborn; Embryo, Mammalian; Female; Gene Expression Regulation; Hindlimb; Immunohistochemistry; Muscle Denervation; Muscle Fibers, Fast-Twitch; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; Myosin Light Chains; Paralysis; Rats; Rats, Sprague-Dawley; Tetrodotoxin

A recently discovered spider toxin (DTX9.2) induced a rapid paralysis when injected into insects. In neurophysiological experiments, DTX9.2 elevated spike discharges in sensory nerves and at the neuromuscular junction, and also caused a depolarization of the membrane potential in cockroach giant axons. All these effects were reversed by treatment with the specific sodium channel blocker, tetrodotoxin. These findings suggest that DTX9.2 acts upon the voltage-dependent sodium channels of insect nerve membrane.

Topics: Action Potentials; Animals; Axons; Cockroaches; Drug Interactions; Insecticides; Neuromuscular Junction; Paralysis; Peptides; Sodium Channels; Spider Venoms; Tetrodotoxin

1. Do motoneurons regulate muscle extrajunctional membrane properties through chemical (trophic) factors in addition to evoked activity? We addressed this question by comparing the effects of denervation and nerve conduction block by tetrodotoxin (TTX) on extrajunctional acetylcholine (ACh) sensitivity and action potential resistance to TTX in adult rats. 2. We applied TTX to sciatic or tibial nerves for up to 5 weeks using an improved blocking technique which completely suppresses conduction but avoids nerve damage. 3. Reinnervation by TTX-blocked axons had no effect on the high ACh sensitivity and TTX resistance induced by nerve crush. 4. Long-lasting block of intact nerves (up to 38 days) induced extrajunctional changes as pronounced as after denervation. At shorter times (3 days), however, denervation induced much larger changes than TTX block; such a difference is thus only transiently present in muscle. 5. The effects of long-lasting block were dose dependent. Dose levels (6.6 micrograms day-1) corresponding to those used in the literature to block the rat sciatic nerve induced muscle effects much smaller than those induced by denervation, confirming published data. Our novel finding is that equal effects are obtained using doses substantially higher (up to 10.5 micrograms day-1). For the soleus it was necessary in addition to apply the TTX directly to the smaller tibial nerve. 6. The TTX-blocked nerves were normal in their histological appearance and capacity to transport anterogradely 3H-labelled proteins, to release ACh in quantal and non-quantal form or cluster ACh receptors and induce functional ectopic junctions on denervated soleus muscles. 7. We conclude that muscle evoked activity is the physiological regulator of extrajunctional membrane properties. Chemical factors from the nerve do not appear to participate in this regulation. The stronger response to denervation at short times only is best accounted for by factors produced by degenerating nerves.

Topics: Acetylcholine; Animals; Axonal Transport; Axons; Dose-Response Relationship, Drug; Electrophysiology; Fluorescent Dyes; Male; Membrane Potentials; Motor Neurons; Muscle, Skeletal; Nerve Degeneration; Nerve Endings; Nerve Regeneration; Neural Conduction; Neuromuscular Junction; Paralysis; Rats; Rats, Wistar; Receptors, Cholinergic; Sciatic Nerve; Sodium Channels; Tetrodotoxin; Time Factors; Tritium

1. The number of acetylcholine receptors (AChRs) per neuromuscular junction in soleus muscles of adult rats was estimated from counts of 125I-alpha-bungarotoxin binding sites. The muscles were either denervated, denervated and electrically stimulated, paralysed by botulinum toxin (BoTX), or paralysed by tetrodotoxin (TTX). 2. After denervation, the number of junctional AChRs was normal after 18 days and then fell to 54 and 35% of normal after 33 and 57 days, respectively. 3. Direct high frequency muscle stimulation (100 Hz) maintained a normal number of junctional AChRs for at least 2 months when the stimulation started on the day of denervation. When the stimulation was started progressively later, the effect of the stimulation on AChR number disappeared within about a week. The disappearance was gradual and appeared to affect all the muscle fibres equally. 4. Stimulation at 100 Hz, starting on the day of denervation and stopping after 18 days, did not prevent the endplates from losing AChRs during the subsequent 15 days without stimulation. Thus 100 Hz stimulation and innervation are not equivalent in their effects on junctional AChR number. 5. Direct low frequency muscle stimulation from the day of denervation did not maintain a normal number of junctional AChRs, as the number of AChRs fell to 70 and 62% of normal after 33 days of stimulation at 20 and 10 Hz, respectively. 6. Endplates paralysed by BoTX or TTX for 33 days lost about as many junctional AChRs (54 and 55%) as endplates denervated for 33 days (46%). Direct stimulation at 100 Hz during the last 15 days of BoTX treatment reduced but did not prevent this AChR loss (36% loss at 33 days). 7. The results show that when motor nerve terminals in rat soleus muscles are removed by axotomy, they leave a 'trace' which, in conjunction with appropriate muscle stimulation, can maintain a normal number of AChRs in the postsynaptic region. In non-stimulated muscles the trace responsible for this maintenance disappears within about a week. In stimulated muscles it persists for at least 2 months. From indirect evidence it appears that the trace is a factor, or the postsynaptic effect of a factor, released by impulse activity in the nerve, and that its degradation after denervation is accelerated by the acute effects of nerve degeneration.

Topics: Animals; Botulinum Toxins; Bungarotoxins; Denervation; Evoked Potentials; Male; Muscle Fibers, Skeletal; Muscle, Skeletal; Nerve Fibers; Neuromuscular Junction; Paralysis; Rats; Rats, Wistar; Receptors, Cholinergic; Tetrodotoxin; Time Factors

Paralytic toxicity was detected by tetrodotoxin (TTX) bioassay in all 15 specimens of the xanthid crab Lophozozymus pictor collected from northern Taiwan in 1993. The average toxicity of crab specimens was 921 +/- 231 (mean +/- S.E.) mouse units. The toxin of crab was partially purified and then identified. It was found that the crab toxin contained TTX and gonyautoxin. The ratio of TTX to gonyautoxin for crab toxin was about 9:1.

Topics: Animals; Brachyura; Chromatography, High Pressure Liquid; Dinoflagellida; Electrophoresis, Polyacrylamide Gel; Fish Venoms; Food Contamination; Foodborne Diseases; Gas Chromatography-Mass Spectrometry; Marine Toxins; Paralysis; Saxitoxin; Shellfish; Tetrodotoxin

Some controversy exists as to whether alpha-motoneurons adapt their oxidative metabolism to changes in chronic activity levels and to altered status of their end organs, as occurs in other neuron types in the central nervous system. We measured, using a personal computer-based image analysis system, succinate dehydrogenase (SDH) activity in rat hindlimb motoneurons under conditions of increased activity (daily voluntary exercise plus treadmill endurance training, the latter 2 h/day, 4 days/wk, for 12 wk) and in a condition of muscle disuse (tetrodotoxin-induced paralysis for 2 wk) in which muscle oxidative enzymes are reduced. Although exercise-trained medial gastrocnemius showed significant adaptations (increased mean SDH activity of type I and increased proportion and total SDH activity of type I and combined I + IIa fibers), SDH activity of innervating motoneurons (identified by retrograde tracing using fast blue) was unchanged. In addition, tetrodotoxin-induced disuse, which results in hindlimb atrophy and SDH decreases (30% decrease measured in medial gastrocnemius muscle homogenates), failed to alter soma SDH or size in unspecified lumbar motoneurons. These results, obtained over a wider range of activity levels than in previous reports, suggest that the oxidative enzymes of motoneurons do not change despite clear adaptations in the muscles they innervate.

Topics: Adaptation, Physiological; Animals; Disease Models, Animal; Male; Motor Neurons; Muscle Fibers, Skeletal; Muscle, Skeletal; Paralysis; Physical Exertion; Rats; Rats, Sprague-Dawley; Spectrophotometry; Succinate Dehydrogenase; Tetrodotoxin

Selected enzymes were measured in mixed-fiber bundles and individual fibers from rat plantaris (PL) and soleus (Sol) muscles that had undergone either 2 wk of tetrodotoxin (TTX) inactivation of the sciatic nerve, a sham operation, or were contralateral to the TTX limb. TTX disuse caused severe wasting of PL (46%) and Sol (26%) muscles and of single fibers (50% and 40%, respectively). TTX PL and Sol also had reduced (50%) glycogen content. In TTX, PL, and Sol macro samples and single fibers, the activities (mol.h-1.kg dry wt-1) of hexokinase, glycogen phosphorylase, and lactate dehydrogenase were higher, lower, and unchanged, respectively, compared with controls. Single-fiber data showed that these changes occurred in all fibers. In TTX PL macro samples, activities of glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase (PK), malate dehydrogenase (MDH), citrate synthase (CS), beta-hydroxyacyl-CoA dehydrogenase (BOAC), and thiolase were, or tended to be, lower. Single-fiber data showed a disappearance of high-oxidative moderate glycolytic fibers (i.e., usually fast-twitch oxidative in control) and the appearance of more fibers with a metabolic enzyme profile approaching that of control slow-oxidative fibers. In TTX Sol macro samples, GPDH and PK tended to be higher, and thiolase, BOAC, CS, and MDH lower. Single-fiber data corroborated these findings and suggested the appearance of fast fibers with downregulated oxidative enzyme profiles. Our results suggest that neuromuscular activity is a major, but not the sole, determinant of the size and metabolic heterogeneity that exists in muscle cells.

Topics: Animals; Female; Hindlimb; Muscles; Paralysis; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tetrodotoxin

Topics: Adult; Animals; Fishes, Poisonous; Foodborne Diseases; Headache; Humans; Male; Paralysis; Respiratory Insufficiency; Sensation Disorders; Tetrodotoxin

A case of severe puffer fish poisoning was reported. After ingestion of puffer fish, symptoms develop rapidly with paralysis of the whole body, respiratory distress and nonreactive dilated pupils. After ventilatory support, the patient gradually recovered to normal activity within 48 hrs.

Topics: Adult; Animals; Fishes, Poisonous; Foodborne Diseases; Humans; Male; Paralysis; Tetrodotoxin

Our aim was to determine the role of fetal breathing movements (FBM) in the maintenance of fetal lung liquid volume. Experiments were performed in 14 chronically catheterized fetal sheep. FBM were selectively abolished for 48 h by the infusion of tetrodotoxin (TTX) onto the phrenic nerves of five fetuses. Lung liquid volumes and secretion rates were measured before each treatment, 46-48 h after the start of the TTX infusion, and 22-24 h after the end of the infusion. Blockade of the phrenic nerves reduced fetal lung liquid volumes from 27.6 +/- 1.9 to 21.8 +/- 2.6 ml/kg and increased lung liquid secretion rates from 3.8 +/- 0.6 to 6.2 +/- 1.1 ml.h-1.kg-1. Control experiments confirmed the lack of effect of TTX infused intravenously and saline infused intrapleurally on changes in fetal lung liquid volume and secretion rate. To measure the static relaxation volume of the fetal lung, in six fetuses we combined skeletal muscle paralysis with bypass of the upper airway for 48 h. This reduced fetal lung liquid volume from 39.1 +/- 3.1 to 23.0 +/- 2.5 ml/kg and increased lung liquid secretion rates from 4.1 +/- 0.7 to 5.8 +/- 0.9 ml.h-1.kg-1. This experiment demonstrates that the fetal lung is normally maintained at a level of expansion that is much greater than its static relaxation volume. We conclude that the volume of luminal liquid in the fetal lungs is dependent on the diaphragmatic contractions associated with FBM. Their effect is to resist the elastic recoil of the fetal lungs, thereby reducing the loss of liquid from the lungs via the trachea.

Topics: Animals; Blood Gas Analysis; Electromyography; Female; Infusions, Intravenous; Injections; Lung; Lung Volume Measurements; Nerve Block; Paralysis; Phrenic Nerve; Pleura; Pregnancy; Respiratory Mechanics; Respiratory Muscles; Sheep; Sodium Chloride; Tetrodotoxin

A recently described disorder in certain registered Quarter horses bears many clinical similarities to the muscle disease identified as hyperkalemic periodic paralysis (HPP) in humans. Pathological changes in membrane permeability or Na(+)-K+ pump activity have been proposed to produce the muscle depolarization and inexcitability that characterize the condition in humans. Biopsies of external intercostal muscle from normal and affected horses were used to determine whether alterations in either permeability and/or pump activity could be linked to the pathology in horses. Affected horse muscle is approximately 16 mV more depolarized than normal muscle at rest, and the muscle membrane potential of HPP horses is less responsive to changes in extracellular K+. Calculation of the relative membrane permeabilities of Na+ and K+ (PNa/PK) indicates that this ratio is significantly increased in HPP muscle. The increase is probably due to an increase in PNa rather than to a decrease in PK, since addition of 10(-6) M tetrodotoxin produces an approximately 14-mV membrane hyperpolarization in HPP fibers but is without effect in normal fibers. The clinical similarities between HPP in horses and humans suggest a common genetic defect in the two species.

Topics: Aging; Animals; Body Water; Epinephrine; Horse Diseases; Horses; Hyperkalemia; Membrane Potentials; Muscle Development; Muscles; Ouabain; Paralysis; Potassium; Reference Values; Temperature; Tetrodotoxin

Increased accumulation of muscle-specific isozyme (MSI) of creatine kinase (CK), lactate dehydrogenase (LDH), glycogen phosphorylase (GP), and phosphoglycerate mutase (PGAM) occurs with development and indicates muscle fiber maturation. The expression of MSIs of those four enzymes is greatly enhanced in innervated-contracting as compared to noninnervated and noncontracting cultured human muscle fibers. We have now studied the effect of contractile activity on developmental accumulation of MSIs in innervated-contracting, innervated-paralyzed (2 microM tetrodotoxin for 30 days), and noninnervated-noncontracting cultured human muscle fibers. Muscle acetylcholinesterase (AChE) and total enzyme activities were also studied under the same conditions. We observed a different dependency on contractile activity between total enzymatic activities of CK, LDH, and AChE, which were substantially reduced after paralysis, and GP and PGAM, which were unchanged. The expression of MSIs of CK, GP, PGAM, and LDH was always significantly increased in innervated as compared to noninnervated fibers. While the expression of MSIs of GP and PGAM was the same in contracting-innervated and paralyzed-innervated muscle fibers, the expression of MSIs of CK and LDH in paralyzed-innervated muscle fibers was very slightly decreased as compared to their contracting-innervated controls. Our studies demonstrate that in human muscle: (1) total enzymatic activities and the expression of MSIs of GP and PGAM are regulated by neuronal effect(s); (2) total enzymatic activities of CK, LDH, and AChE depend mainly on muscle contractile activity; and (3) MSIs of CK and LDH are regulated predominantly by neuronal factors and to a much lesser degree by muscle contractile activity.

Topics: Acetylcholinesterase; Bisphosphoglycerate Mutase; Cells, Cultured; Creatine Kinase; Humans; Isoenzymes; Kinetics; L-Lactate Dehydrogenase; Models, Biological; Muscle Contraction; Muscles; Paralysis; Phosphorylases; Phosphotransferases; Tetrodotoxin

Developmental aspects of the neuromuscular system in mouse embryos chronically paralyzed in utero with tetrodotoxin (TTX) between embryonic days 14 and 18 were studied using biochemical and histological methods. The number of lumbar spinal motoneurons (MNs) was higher in inactive embryos than in controls suggesting a decreased motoneuron cell death. In association with the increase in MN number, choline acetyltransferase activity was significantly increased in both spinal cord and peripheral synaptic sites. Paralyzed muscles exhibited a decreased number of mature myofibers and the nuclei were centrally located. Creatine kinase activity was greatly decreased and total acetylcholine receptor and receptor cluster numbers per myofiber were significantly increased in paralyzed muscles. A similar pattern of changes occurs in the neuromuscular system of the mutant mouse muscular dysgenesis (mdg). However, in contrast to the mdg mutant, tetrodotoxin-treated muscles were similar to controls in their innervation pattern, in the ultrastructural aspects of the excitation-contraction coupling system (i.e., dyads and triads) and in the extent of dihydropyridine binding. Thus, neuromuscular inactivity is not sufficient to impair the pattern of muscle innervation or the appearance of either the triadic junctions or dihydropyridine receptors. These results indicate that alterations of dihydropyridine binding sites and triads in muscular dysgenesis cannot be accounted for by inactivity but rather must reflect a more primary defect involving the structural gene(s) regulating the development of one or more aspects of muscle differentiation.

Topics: Animals; Cell Differentiation; Cell Survival; Embryo, Mammalian; Mice; Mice, Inbred Strains; Motor Neurons; Muscle Development; Muscles; Nervous System; Paralysis; Parasympathetic Nervous System; Tetrodotoxin

The necessity of innervation and/or neural activity for the formation of muscle spindles was investigated by treating fetal rats with neurotoxins on embryonic day 16 or 17 (E16-17), one or two days prior to the onset of spindle assembly. The neurotoxin-treated soleus muscles were examined on E21 for the presence of spindles and immunocytochemical expression of the slow-tonic myosin heavy-chain (MHC) isoform, which is characteristic of intrafusal fibers. Irreversible destruction of sensory and motor nerves by beta-bungarotoxin prevented the formation of spindles and expression of the slow-tonic MHC. Abolishment of nerve and muscle activity by tetrodotoxin did not block the spindle assembly or expression of the slow-tonic MHC. Thus, the formation and differentiation of spindles is dependent on innervation, but is independent of activity in nerve fibers or muscle cells.

Topics: Animals; Bungarotoxins; Female; Fetus; Hindlimb; Muscles; Neurotoxins; Paralysis; Pregnancy; Rats; Rats, Inbred Strains; Tetrodotoxin

To examine the role of muscle activity in the expression of fetal- and adult-type acetylcholine receptors (AChRs), we studied the effects of muscle stimulation in cell culture and of tetrodotoxin (TTX)-induced paralysis and denervation in adult rat muscles. The AChR content of these muscles was determined using [125I]alpha-bungarotoxin and the proportion of fetal-type receptors was estimated using a radioimmunoprecipitation assay with a myasthenic serum that was highly specific for fetal-type receptors. We found that both stimulated, aneural muscle cells in vitro and inactive muscles in vivo produced predominantly fetal-type AChRs. However the TTX-paralysed muscles had a lower proportion of fetal-type receptors than the denervated muscles. We conclude that neither muscle activity nor innervation alone, but a combination of both, is required for full regulation of AChR antigenicity.

Topics: Animals; Bungarotoxins; Cells, Cultured; Electric Stimulation; Embryo, Mammalian; Muscle Denervation; Muscles; Paralysis; Radioimmunoassay; Rats; Receptors, Cholinergic; Tetrodotoxin

Calcitonin gene-related peptide (CGRP) coexists with acetylcholine (ACh) in motor nerve terminals. Externally applied CGRP has been shown to increase the synthesis of ACh receptors in cultured myotubes by a mechanism independent of muscle activity. Thus, CGRP is suggested to be a neurotrophic factor that may regulate the expression of several long-term events occurring at the neuromuscular junction. We have examined the effect of CGRP on the sprouting of motor nerve terminals induced by chronic block of nerve-muscle activity in adult rats. Daily treatment with CGRP suppressed the disuse-induced terminal sprouting in a dose-dependent manner, whereas the morphology of motor nerve terminals in active muscles was unaffected by CGRP. CGRP may be a possible candidate for an antisprouting agent which has been postulated to exist in nerve terminals. The disuse-induced outgrowth of terminal sprouts was accompanied by an increase in the mean quantum content of end-plate potentials, as well as in the frequency of spontaneous miniature end-plate potentials. This increased transmitter release was still maintained at the junctions in which disuse-induced terminal sprouting had been suppressed by CGRP. It is suggested that the formation of terminal sprouts per se is not responsible for the plastic change of transmitter release induced by prolonged disuse of the neuromuscular junction.

Topics: Animals; Calcitonin Gene-Related Peptide; Motor Endplate; Neuromuscular Junction; Neuronal Plasticity; Neuropeptides; Neurotransmitter Agents; Paralysis; Rats; Rats, Inbred Strains; Tetrodotoxin

Using immunofluorescence procedures with specific polyclonal and monoclonal antimyosin antibodies we have found that embryonic and neonatal myosin heavy chains (MHCs), which in rat skeletal muscle disappear during the first weeks after birth, are reexpressed in adult muscle after denervation. Reactivity for embryonic and neonatal MHCs was detected in some fibers as early as 3 days after denervation, became more evident by 7 days, and occurred exclusively in the type 2A fiber population. Paralysis of innervated muscles by tetrodotoxin block of the sciatic nerve also resulted in the reappearance of embryonic and neonatal MHCs in type 2A fibers. Significant variation in the degree of immunoreactivity was observed in different segments of the same muscle fiber, suggesting that coordination of muscle fiber nuclei in the control of myosin heavy chain gene expression is partially lost following denervation.

Topics: Aging; Animals; Animals, Newborn; Fetus; Fluorescent Antibody Technique; Muscle Denervation; Muscles; Myosins; Paralysis; Rats; Rats, Inbred Strains; Sciatic Nerve; Tetrodotoxin

Topics: Adolescent; Animals; Fishes, Poisonous; Foodborne Diseases; Humans; Male; Paralysis; Paresthesia; Tetrodotoxin

The intrathecal injection of gentamicin into a human patient with gram-negative bacterial meningitis as well as its intracisternal injection into rabbits caused spongy-like lesions in the gray matter and tetraplegia in rabbits. To characterize this neurotoxic effect, gentamicin was injected into the subarachnoid space of the lumbar spinal cord of the rat. A biphasic hindlimb paralysis ensued which consisted at first of a transient flaccid paralysis lasting 1 to 5 hr followed by a permanent flaccid paralysis which developed after 24 to 36 hr. The initial paralysis occurred simultaneously with the transient loss of reflex transmission through the cord but in the absence of lesions in the spinal cord or physiological alterations of neuromuscular transmission and muscle contraction. The onset of the second phase of paralysis occurred concomitant with changes in reflex transmission and appearance of lesions. Loss of neuromuscular transmission and appearance of signs of denervation (e.g., depolarization, alteration in action potential parameters, and chemosensitivity) appeared after the second phase of paralysis was established. Both the initial transient and late permanent paralysis originated in the spinal cord. The early transient paralysis appears to be due to a central block of transmission while the late paralysis apparently resulted from neuronal damage. The neurotoxic effects of aminoglycosides on neuronal elements in the spinal cord resulted in secondary effects (signs of denervation) in hindlimb muscles.

Topics: Action Potentials; Aminoglycosides; Animals; Anti-Bacterial Agents; Female; Gentamicins; Hindlimb; Membrane Potentials; Motor Endplate; Muscle Contraction; Paralysis; Rats; Rats, Inbred Strains; Spinal Cord; Tetrodotoxin

Chronic paralysis of rat embryos during the last 4 to 6 prenatal days causes a diminution in skeletal muscle fiber numbers but inhibits motoneuron death. Consequently, as paralyzed muscles develop, an increased number of motoneurons attempts to form synapses at a reduced number of synaptic sites. Paralyzed muscle fibers receive their synapses at a single endplate, as in control muscles, but these endplates are hyperinnervated, with about twice the normal number of inputs. Counts of axons, synaptic inputs, and muscle units showed that motoneurons normally contact a maximum number of muscle fibers shortly before birth, and this number remains stable for several days postnatal until it finally is reduced to the adult number. The average motor unit size in paralyzed embryos at the time of birth was the same as in controls. We suggest that it is not necessary to postulate the existence of competition between embryonic nerve terminals in order to explain regulation of the number of muscle fibers initially contacted by a motoneuron. Motoneuron death was not immediately affected by paralysis, but paralysis "rescued" all motoneurons whose death normally would have occurred 24 hr or more after the time when paralysis was initiated, regardless of when this was. This implies that the peak period for determination to die is during embryonic day 14, when myotube formation is just beginning and no recognizable endplate structures are present in muscles. When paralyzed, motoneurons normally destined to die are capable of forming a normal number of functional nerve-muscle contacts.

Topics: Animals; Embryo, Mammalian; Kinetics; Motor Endplate; Motor Neurons; Muscle Denervation; Muscles; Neuromuscular Junction; Paralysis; Phrenic Nerve; Rats; Spinal Cord; Synapses; Tetrodotoxin

Resting membrane potentials (RMP) and resistance to tetrodotoxin (TTX) have been compared in denervated rat soleus muscles and muscles reinnervated with tetrodotoxin-inactive nerves for periods of 15-18 days. RMP's of the two muscle groups exhibited the same low values typical of denervated muscles. Similarly, comparable values of TTX-resistance were found in the two muscle groups, although exceptions with slightly lower values in the innervated-paralyzed muscles were noted. It is concluded that muscle reinnervation restores to normal the membrane properties altered by denervation essentially through the return of muscle activity.

Topics: Action Potentials; Animals; Male; Membrane Potentials; Muscle Denervation; Muscles; Paralysis; Rats; Rats, Inbred Strains; Tetrodotoxin

For many years students of Haitian society have suggested that there is an ethnopharmacological basis for the notorious zombies, the living dead of peasant folklore. The recent surfacing of three zombies, one of whom may represent the first potentially verifiable case, has focused scientific attention on the reported zombi drug. The formula of the poison was obtained at four widely separated localities in Haiti. The consistent ingredients include one or more species of puffer fish (Diodon hystrix, Diodon holacanthus or Sphoeroides testudineus) which contain tetrodotoxins, potent neurotoxins fully capable of pharmacologically inducing the zombi state. The ingredients, preparation and method of application are presented. The symptomology of tetrodotoxication as described in the biomedical literature is compared with the constellations of symptoms recorded from the zombies in Haiti. The cosmological rationale of zombies within the context of Voodoo theology is described. Preliminary laboratory tests are summarized.

Topics: Amphibians; Animals; Culture; Fishes; Haiti; Humans; Paralysis; Plants, Toxic; Poisons; Reptiles; Tetrodotoxin

Topics: Animals; Cells, Cultured; Chick Embryo; Cnidarian Venoms; Membrane Potentials; Muscles; Nucleotides, Cyclic; Paralysis; Receptors, Cholinergic; Tetrodotoxin; Veratrine

Topics: Action Potentials; Animals; Drosophila melanogaster; Ion Channels; Membrane Proteins; Mutation; Neural Conduction; Neurons; Paralysis; Temperature; Tetrodotoxin

A technique is described for the slow-release of tetrodotoxin in peripheral nerves using a constriction capillary. The capillary, which was implanted under the epineurium of the sciatic nerve, released tetrodotoxin from a 25 micrometer pore. Nerve block was complete after approximately 20 min and lasted 6--9 days. Replacement of the capillary enabled paralysis of the rat hindlimb to be maintained for periods of 21 days and longer. Studies with radioactively labelled compounds demonstrated that the efflux rate from the capillary was dependent on the size of the pore and the relative molecular mass of the compound.

Topics: Animals; Hindlimb; Methods; Paralysis; Rats; Tetrodotoxin; Time Factors

Topics: Action Potentials; Animals; Female; Male; Mice; Motor Neurons; Nerve Endings; Neuromuscular Junction; Paralysis; Tetrodotoxin; Time Factors

Topics: Animals; Axonal Transport; Bungarotoxins; Female; Muscle Denervation; Muscles; Nerve Degeneration; Paralysis; Rats; Receptors, Cholinergic; Tetrodotoxin

Topics: Animals; Axonal Transport; Bungarotoxins; Female; Muscles; Paralysis; Rats; Receptors, Cholinergic; Sciatic Nerve; Tetrodotoxin

Topics: Adolescent; Animals; Arrhythmias, Cardiac; Consciousness; Edrophonium; Fishes, Poisonous; Foodborne Diseases; Glucose; Humans; Infusions, Parenteral; Intubation, Intratracheal; Male; Paralysis; Plasma Substitutes; Potassium Chloride; Respiration, Artificial; Sodium Chloride; Tetrodotoxin

Topics: Animals; Arrhythmias, Cardiac; Blood Pressure; Contracture; Electrocardiography; Heart; Male; Paralysis; Rats; Respiratory Insufficiency; Respiratory Paralysis; Scorpions; Tetrodotoxin; Venoms

1. The electric properties of the giant synapse in the stellate ganglion of the squid have been further investigated.2. During tetrodotoxin (TTX) paralysis, a local response can be elicited from the terminal parts of the presynaptic axons after intracellular injection of tetraethyl ammonium ions (TEA).3. The response is characterized by an action potential of variable size and duration, whose fall is often preceded by a prolonged plateau. The response, especially the duration of the plateau, is subject to ;fatigue' during repetitive stimulation.4. The TTX-resistant form of activity is localized in the region of the synaptic contacts, and shows a marked electrotonic decrement even within less than 1 mm from the synapse. It is found only on the afferent, not on the efferent, side of the synapse.5. During the plateau of the response, the membrane resistance is greatly reduced below its resting value.6. The response depends on presence of external calcium and increases in size and duration with the calcium concentration. Strontium and barium substitute effectively for calcium. Manganese and, to a lesser extent, magnesium, counteract calcium and reduce the response. The response also declines, and ultimately disappears, if sodium is withdrawn for long periods.7. The relation of the local TTX-resistant response to the influx of calcium ions and to the release of the synaptic transmitter is discussed.

Topics: Action Potentials; Animals; Barium; Calcium; Electric Stimulation; Electrophysiology; Magnesium; Manganese; Mollusca; Nerve Endings; Paralysis; Sodium; Stellate Ganglion; Strontium; Synapses; Synaptic Transmission; Tetraethylammonium Compounds; Tetrodotoxin

Topics: Animals; Blood Pressure; Cats; Dogs; Hypotension; Muscles; Paralysis; Peripheral Nerves; Respiration; Respiratory Insufficiency; Tetrodotoxin; Toxins, Biological

Topics: Anesthesia; Anesthesia, Local; Animals; Antihypertensive Agents; Antitoxins; Chemical Phenomena; Chemistry; Fishes; Muscles; Nervous System; Paralysis; Pharmacology; Tetrodotoxin; Toxicology; Toxins, Biological; Urodela; Venoms

Topics: Animals; Antitoxins; Hypotension; Nerve Tissue; Neurotoxins; Nictitating Membrane; Paralysis; Tetrodotoxin; Toxins, Biological

Topics: Fishes; Paralysis; Poisons; Tetrodotoxin