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1.
BMC Genomics ; 23(1): 553, 2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35922761

RESUMO

Tetrodotoxin (TTX) is a deadly neurotoxin and usually accumulates in large amounts in the ovaries but is non-toxic or low toxic in the testis of pufferfish. The molecular mechanism underlying sexual dimorphism accumulation of TTX in ovary and testis, and the relationship between TTX accumulation with sex related genes expression remain largely unknown. The present study investigated the effects of exogenous TTX treatment on Takifugu flavidus. The results demonstrated that exogenous TTX administration significantly incresed level of TTX concentration in kidney, cholecyst, skin, liver, heart, muscle, ovary and testis of the treatment group (TG) than that of the control group (CG). Transcriptome sequencing and analysis were performed to study differential expression profiles of mRNA and piRNA after TTX administration of the ovary and testis. The results showed that compared with female control group (FCG) and male control group (MCG), TTX administration resulted in 80 and 23 piRNAs, 126 and 223 genes up and down regulated expression in female TTX-treated group (FTG), meanwhile, 286 and 223 piRNAs, 2 and 443 genes up and down regulated expression in male TTX-treated group (MTG). The female dominant genes cyp19a1, gdf9 and foxl2 were found to be up-regulated in MTG. The cyp19a1, whose corresponding target piRNA uniq_554482 was identified as down-regulated in the MTG, indicating the gene expression feminization in testis after exogenous TTX administration. The KEGG enrichment analysis revealed that differentially expressed genes (DEGs) and piRNAs (DEpiRNAs) in MTG vs MCG group were more enriched in metabolism pathways, indicating that the testis produced more metabolic pathways in response to exogenous TTX, which might be a reason for the sexual dimorphism of TTX distribution in gonads. In addition, TdT-mediated dUTP-biotin nick end labeling staining showed that significant apoptosis was detected in the MTG testis, and the role of the cell apoptotic pathways was further confirmed. Overall, our research revealed that the response of the ovary and testis to TTX administration was largely different, the ovary is more tolerant whereas the testis is more sensitive to TTX. These data will deepen our understanding on the accumulation of TTX sexual dimorphism in Takifugu.


Assuntos
Takifugu , Testículo , Animais , Feminino , Feminização , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Masculino , RNA Interferente Pequeno/metabolismo , Takifugu/genética , Takifugu/metabolismo , Testículo/metabolismo , Tetrodotoxina/metabolismo , Tetrodotoxina/toxicidade
2.
Science ; 377(6604): 411-415, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35862530

RESUMO

Tetrodotoxin (TTX) is a neurotoxic natural product that is an indispensable probe in neuroscience, a biosynthetic and ecological enigma, and a celebrated target of synthetic chemistry. Here, we present a stereoselective synthesis of TTX that proceeds in 22 steps from a glucose derivative. The central cyclohexane ring of TTX and its α-tertiary amine moiety were established by the intramolecular 1,3-dipolar cycloaddition of a nitrile oxide, followed by alkynyl addition to the resultant isoxazoline. A ruthenium-catalyzed hydroxylactonization set the stage for the formation of the dioxa-adamantane core. Installation of the guanidine, oxidation of a primary alcohol, and a late-stage epimerization gave a mixture of TTX and anhydro-TTX. This synthetic approach could give ready access to biologically active derivatives.


Assuntos
Tetrodotoxina , Bloqueadores do Canal de Sódio Disparado por Voltagem , Catálise , Reação de Cicloadição , Guanidina/química , Rutênio/química , Estereoisomerismo , Tetrodotoxina/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química
3.
Sci Rep ; 12(1): 11610, 2022 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-35803955

RESUMO

Neural networks tune synaptic and cellular properties to produce stable activity. One form of homeostatic regulation involves scaling the strength of synapses up or down in a global and multiplicative manner to oppose activity disturbances. In American bullfrogs, excitatory synapses scale up to regulate breathing motor function after inactivity in hibernation, connecting homeostatic compensation to motor behavior. In traditional models of homeostatic synaptic plasticity, inactivity is thought to increase synaptic strength via mechanisms that involve reduced Ca2+ influx through voltage-gated channels. Therefore, we tested whether pharmacological inactivity and inhibition of voltage-gated Ca2+ channels are sufficient to drive synaptic compensation in this system. For this, we chronically exposed ex vivo brainstem preparations containing the intact respiratory network to tetrodotoxin (TTX) to stop activity and nimodipine to block L-type Ca2+ channels. We show that hibernation and TTX similarly increased motoneuron synaptic strength and that hibernation occluded the response to TTX. In contrast, inhibiting L-type Ca2+ channels did not upregulate synaptic strength but disrupted the apparent multiplicative scaling of synaptic compensation typically observed in response to hibernation. Thus, inactivity drives up synaptic strength through mechanisms that do not rely on reduced L-type channel function, while Ca2+ signaling associated with the hibernation environment independently regulates the balance of synaptic weights. Altogether, these results point to multiple feedback signals for shaping synaptic compensation that gives rise to proper network function during environmental challenges in vivo.


Assuntos
Hibernação , Animais , Neurônios Motores/fisiologia , Plasticidade Neuronal/fisiologia , Rana catesbeiana , Sinapses/fisiologia , Tetrodotoxina/farmacologia
4.
J Org Chem ; 87(14): 9023-9033, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35765754

RESUMO

The collective synthesis of the four spiro-cyclic guanidines Tb-210B, Tb-226, Tb-242C, and Tb-258, all of which have been isolated from puffer fish and are considered possible biosynthetic intermediates of tetrodotoxin, has been achieved. Our synthesis is based on the stepwise deoxygenation or hydroxylation of a common intermediate, prepared from a known oxazoline.


Assuntos
Tetraodontiformes , Animais , Guanidina , Guanidinas , Hidroxilação , Tetraodontiformes/metabolismo , Tetrodotoxina/metabolismo
5.
J Headache Pain ; 23(1): 73, 2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35764917

RESUMO

BACKGROUND: Growing evidence supports the important role of persistent sodium currents (INaP) in the neuronal excitability of various central neurons. However, the role of tetrodotoxin-resistant (TTX-R) Na+ channel-mediated INaP in the neuronal excitability of nociceptive neurons remains poorly understood. METHODS: We investigated the functional role of TTX-R INaP in the excitability of C-type nociceptive dural afferent neurons, which was identified using a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchloride (DiI), and a whole-cell patch-clamp technique. RESULTS: TTX-R INaP were found in most DiI-positive neurons, but their density was proportional to neuronal size. Although the voltage dependence of TTX-R Na+ channels did not differ among DiI-positive neurons, the extent of the onset of slow inactivation, recovery from inactivation, and use-dependent inhibition of these channels was highly correlated with neuronal size and, to a great extent, the density of TTX-R INaP. In the presence of TTX, treatment with a specific INaP inhibitor, riluzole, substantially decreased the number of action potentials generated by depolarizing current injection, suggesting that TTX-R INaP are related to the excitability of dural afferent neurons. In animals treated chronically with inflammatory mediators, the density of TTX-R INaP was significantly increased, and it was difficult to inactivate TTX-R Na+ channels. CONCLUSIONS: TTX-R INaP apparently contributes to the differential properties of TTX-R Na+ channels and neuronal excitability. Consequently, the selective modulation of TTX-R INaP could be, at least in part, a new approach for the treatment of migraine headaches.


Assuntos
Neurônios Aferentes , Canais de Sódio , Animais , Potenciais da Membrana/fisiologia , Neurônios Aferentes/metabolismo , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Tetrodotoxina/farmacologia
6.
Neuropharmacology ; 215: 109156, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35691365

RESUMO

Prostaglandin E2 (PGE2) is an important inflammatory mediator for the initiation and maintenance of inflammatory and neuropathic pain. The acute effect of PGE2 on sodium currents has been widely characterized in sensory neurons; however, the prolonged effect of PGE2 remains to be determined. Here, we performed patch clamp recordings to evaluate the acute and prolonged effects of PGE2 on sodium currents in trigeminal ganglionic (TG) neurons from male Sprague-Dawley rats. We found that 24-h treatment with PGE2 (10 µM) increased the peak sodium current density by approximately 31% in a voltage-dependent manner and shifted the activation curve in a hyperpolarized direction but did not affect steady-state inactivation. Furthermore, treatment with PGE2 for 24 h increased the current density of tetrodotoxin-sensitive (TTX-S) but not TTX-resistant (TTX-R) channels significantly. Interestingly, TTX-S current was increased mostly in medium-sized, but not in small-sized, neurons after 24 h of treatment with PGE2. Moreover, the mRNA level of TTX-S Nav1.1 but not TTX-R Nav1.8 or Nav1.9 was significantly increased after 24 h of treatment with PGE2. In contrast, 5-min treatment with PGE2 (10 µM) increased the peak sodium current density by approximately 29% and increased TTX-R sodium currents, but not TTX-S currents, in both small- and medium-sized TG neurons. Our results presented a differential regulation of subtypes of sodium channels by acute and prolonged treatments of PGE2, which may help to better understand the mechanism of PGE2-mediated orofacial pain development.


Assuntos
Dinoprostona , Sódio , Animais , Dinoprostona/farmacologia , Gânglios Espinais , Masculino , Potenciais da Membrana , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais , Tetrodotoxina/farmacologia
7.
Chemosphere ; 303(Pt 1): 134962, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35580645

RESUMO

Tetrodotoxin (TTX) is a potent marine neurotoxin that exists in a variety of aquatic and terrestrial organisms. Pufferfish in different habitats show great variation in their TTX contents. Exploring the genes involved in TTX metabolism could contribute to our understanding of the molecular mechanisms underlying TTX accumulation, translocation, and detoxification in pufferfish. In this study, transcriptomic analysis was used to identify the functional genes related to TTX metabolism in the blood, liver, and muscle of the toxic and non-toxic tiger puffer (Takifugu rubripes). A total of 6101 differentially expressed genes (DEGs) were obtained after transcriptomic analysis; of these, 2401 were identified in the blood, 2262 in the liver, and 1438 in the muscle. After enrichment analysis, fourteen genes encoding glutathione S-transferases (GSTs), glutathione peroxidase (GPx), thioredoxins (TXNs), superoxide dismutase (SOD), ATP-binding cassettes (ABCs), apolipoproteins (APOs), inhibitors of apoptosis protein (IAP), and solute carrier (SLC), which are mainly antioxidant enzymes, membrane transporters, or anti-apoptotic factors, were revealed in the blood. Thirty-six genes encoding SLCs, ABCs, long-chain-fatty-acid-CoA ligases (ACSLs), interleukin 6 cytokine family signal transducer (IL6ST), endoplasmic reticulum (ER), and heat shock protein family A (Hsp70) were involved in transmembrane transporter activity and innate immune response. Notably, a large number of slc genes were found to play critical and diverse roles in TTX accumulation and translocation in the liver of T. rubripes. Nine genes from the slc, hsp70, complement C5 (c5), acsl, er, and serpin peptidase inhibitor (serpin) gene families were found to participate in the regulation of protein processing and anti-apoptosis. These results reflect the diverse functions of genes closely related to TTX accumulation, translocation, and detoxification in T. rubripes.


Assuntos
Takifugu , Transcriptoma , Animais , Perfilação da Expressão Gênica , Fígado/metabolismo , Takifugu/genética , Takifugu/metabolismo , Tetrodotoxina/metabolismo
8.
Chem Senses ; 472022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-35580574

RESUMO

Toxic puffers accumulate their defense substance (tetrodotoxin; TTX) through the food chain. Although the previous study suggests that 5,6,11-trideoxyTTX, a nontoxic TTX analog detected simultaneously with TTX in toxic puffers or their prey, acts as an olfactory chemoattractant for grass puffers, it is unclear whether toxic puffers are commonly attracted to 5,6,11-trideoxyTTX, and which types of olfactory sensory neurons (OSNs) detect 5,6,11-trideoxyTTX. Here, we demonstrated that green spotted puffer, a phylogenetically distant species from the grass puffer, is attracted to 5,6,11-trideoxyTTX. 5,6,11-TrideoxyTTX administration made green spotted puffers stay longer at the administered site, whereas a food odor (l-Arg) made them actively swim throughout the aquarium. Attractive responses were not observed when TTX or its vehicle was administered, nor when 5,6,11-trideoxyTTX was administered to anosmic fish. Furthermore, double immunohistochemistry with activity marker and crypt OSN marker antibodies labeled oval cells with apical invagination on the olfactory epithelium surface treated with 5,6,11-trideoxyTTX. These results suggest that 5,6,11-trideoxyTTX acts as an olfactory chemoattractant detected by crypt OSNs, and attraction to 5,6,11-trideoxyTTX odor appears to be a trait shared by toxic puffers for social communication or effective toxification.


Assuntos
Neurônios Receptores Olfatórios , Tetraodontiformes , Animais , Fatores Quimiotáticos , Odorantes , Mucosa Olfatória , Tetrodotoxina/química , Tetrodotoxina/farmacologia
9.
Spectrochim Acta A Mol Biomol Spectrosc ; 279: 121275, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-35605417

RESUMO

Tetrodotoxin (TTX) is one of the most lethal neurotoxins, so the reliable quantitative analysis of TTX is crucial for food and environmental safety monitoring. Herein, a novel dual-molecule calibration aptasensor was developed for detection of TTX based on Surface-enhanced Raman scattering (SERS). The adaptive surface has high affinity recognition sites for the target of interest, which ensures the high specificity and stability of the aptasensor. In addition, the uniquely labeled signal molecules located in the Raman silent region (1800-2400 cm-1) can avoid the interference of other exogenous biological signal molecules. Meanwhile, in quantitative analysis, the SERS signal generated by the reporter is calibrate in real time using the second-order peak of silicon molecule (Si). The detection linear range of the aptasensor was 0.0319 ng/mL-319.27 ng/mL, with a limit of detection (LOD) of 0.024 ng/mL and the excellent uniformity (RSD = 8.8%) for TTX detection. As a promising and versatile detection candidate, the ultra-sensitive quantitative detection aptasensor of TTX had important practical significance, which can offer more favorable persuasion for TTX analysis in real seafood samples.


Assuntos
Nanopartículas Metálicas , Tetraodontiformes , Animais , Calibragem , Ouro , Limite de Detecção , Análise Espectral Raman , Tetrodotoxina
10.
Toxins (Basel) ; 14(5)2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35622597

RESUMO

Tetrodotoxin is one of the most potent neurotoxins in the aquatic world. This review of published and unpublished reports aims to describe the poisoning cases that have occurred in mainland France and overseas territories. Six articles were included, with 13 poisoning events, individuals or collective (number (n) = 53 patients). Moreover, 13 unpublished poisoning events from toxicovigilance networks were found (n = 17). All cases happened in overseas territories: French Guyana (n = 7), New Caledonia (n = 11), Reunion (n = 35) and French Polynesia (n = 17). The median age was 36 years. The most frequent signs were neurological (81.8%), digestive (54.5%) and general (52.3%). Three cases of dysgueusia and nine cases of urogenital discomfort were observed in French Polynesia. Twelve severe cases were reported, including seven deaths. Only three events (11.5%) were documented by a tetrodotoxin assay. Two families of fish accounted for 91.6% of the poisonings: 33.3% due to the Diodontidae family and 58.3% to the Tetraodontidae family. Although rare, information and collection campaigns on tetrodotoxin poisoning are, therefore, essential.


Assuntos
Intoxicação por Ciguatera , Síndromes Neurotóxicas , Tetraodontiformes , Animais , França , Humanos , Tetrodotoxina/toxicidade
11.
Mol Ecol ; 31(14): 3827-3843, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35596742

RESUMO

The repeated evolution of tetrodotoxin (TTX) resistance provides a model for testing hypotheses about the mechanisms of convergent evolution. This poison is broadly employed as a potent antipredator defence, blocking voltage-gated sodium channels (Nav ) in muscles and nerves, paralysing and sometimes killing predators. Resistance in taxa bearing this neurotoxin and a few predators appears to come from convergent replacements in specific Nav residues that interact with TTX. This stereotyped genetic response suggests molecular and phenotypic evolution may be constrained and predictable. Here, we investigate the extent of mechanistic convergence in garter snakes (Thamnophis) that prey on TTX-bearing newts (Taricha) by examining the physiological and genetic basis of TTX resistance in the Sierra garter snake (Th. couchii). We characterize variation in this predatory adaptation across populations at several biological scales: whole-animal TTX resistance; skeletal muscle resistance; functional genetic variation in three Nav encoding loci; and levels of gene expression for one of these loci. We found Th. couchii possess extensive geographical variation in resistance at the whole-animal and skeletal muscle levels. As in other Thamnophis, resistance at both levels is highly correlated, suggesting convergence across the biological levels linking organism to organ. However, Th. couchii shows no functional variation in Nav loci among populations or difference in candidate gene expression. Local variation in TTX resistance in Th. couchii cannot be explained by the same relationship between genotype and phenotype seen in other taxa. Thus, historical contingencies may lead different species of Thamnophis down alternative routes to local adaptation.


Assuntos
Colubridae , Adaptação Fisiológica/genética , Animais , Colubridae/genética , Comportamento Predatório/fisiologia , Salamandridae/fisiologia , Tetrodotoxina/química , Tetrodotoxina/toxicidade
12.
Prog Chem Org Nat Prod ; 118: 101-130, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35416518

RESUMO

The North American newt genera Taricha and Notophthalmus (order Caudata) are well known for the combination of potent toxicity, aposematic coloration, and striking defense postures that protects these animals from predation. This suite of traits is centered around the neurotoxin tetrodotoxin, which causes paralysis and death in metazoans by disrupting the initiation and propagation of electrical signals in the nerves and muscles. Tetrodotoxin defends newts from predation across multiple life history stages and its role in generating arms-race coevolution between Taricha newts and garter snake (genus Thamnophis) predators is well studied. However, understanding the broader picture of chemical defenses in Taricha and Notophthalmus requires an expanded comprehension of the defensive chemical ecology of tetrodotoxin that includes possible coevolutionary interactions with insect egg predators, protection against parasites, as well as mimicry complexes associated with tetrodotoxin and aposematic coloration in both genera. Herein the authors review what is known about the structure, function, and pharmacology of tetrodotoxin to explore its evolution and chemical ecology in the North American newt. Focus is made specifically on the origin and possible biosynthesis of tetrodotoxin in these taxa as well as providing an expanded picture of the web of interactions that contribute to landscape level patterns of toxicity and defense in Taricha and Notophthalmus.


Assuntos
Colubridae , Notophthalmus , Animais , Colubridae/fisiologia , América do Norte , Salamandridae/fisiologia , Tetrodotoxina/toxicidade
13.
Leg Med (Tokyo) ; 57: 102078, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35483105

RESUMO

Identifying poisonous plants and animals is very important because they not only can cause food poisoning, but also can be eaten for the purpose of suicide. A pufferfish is a poisonous fish that contains tetrodotoxin. In Japan, 136 pufferfish poisoning cases occurred from 2015 to 2019, but in many cases, the specific species involved was unidentified. To address this, we focused on a rapid and simple DNA chromatography technology called Single-stranded Tag Hybridization (STH). We collected seven pufferfish species of the genus Takifugu and designed species-specific primers as target regions of cytochrome c oxidase subunit I (COI) and cytochrome b with a specific base sequence at the 3' end. STH-PCR was performed in two separate reactions. After mixing the PCR products, a developing solution was added to perform chromatograph development and the results were visually analyzed. Specific lines were detected in all seven species. The pufferfish species could be properly determined using between 0.1 ng and 50 ng of template DNA. The PCR product length was 85-149 bp, making it very resistant to degradation. The species could be properly identified even in a mixture of multiple pufferfish species DNA. Furthermore, verification was performed using the supernatants of digested samples with artificial gastric juice and processed foods. Extracted DNA was obtained in all but the highly roasted fins, enabling discrimination. Overall, we applied a novel DNA chromatography detection system capable of discriminating seven species of the genus Takifugu, which have closely related DNA sequences.


Assuntos
DNA , Takifugu , Animais , Cromatografia , DNA/metabolismo , Humanos , Reação em Cadeia da Polimerase , Takifugu/genética , Takifugu/metabolismo , Tetrodotoxina/análise , Tetrodotoxina/metabolismo
14.
Life Sci ; 298: 120524, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35367467

RESUMO

AIM: To investigate the effect of nerve growth factor (NGF) neutralization on Na+ channel plasticity of bladder afferent neurons in mice with spinal cord injury (SCI). MAIN METHODS: Female C57/BL6 mice were randomly divided into spinal intact (SI) group, SCI group and SCI + NGF-Ab group. SCI was induced by spinal cord transection at the Th8/9 level. In SCI + NGF-Ab group, anti-NGF antibodies (10 µg·kg-1 per hour) were continuously administered for 2 weeks using osmotic pumps. Bladder afferent neurons were labelled with Fluoro­gold (FG) injected into the bladder wall. L6-S1 dorsal root ganglion (DRG) neurons were dissociated and whole-cell patch clamp recordings were performed on FG-labelled neurons. Expression of Nav1.7 and Nav1.8 was examined by immunofluorescent staining. KEY FINDINGS: Whole-cell patch clamp recordings showed that TTX only partially inhibited action potentials (AP) and Na+ currents of bladder afferent neurons in SI mice, but it almost completely inhibited them in SCI mice. Total and TTX-sensitive Na+ currents were increased and TTX-resistant currents were decreased in bladder afferent neurons from SCI mice vs. SI mice. These changes in SCI mice were significantly reversed by NGF-antibody treatment. Immunostaining results showed the increased and decreased levels of Nav1.7 and Nav1.8, respectively, in FG-labelled bladder afferent neurons in SCI mice vs. SI mice, which was significantly reversed in SCI + NGF-Ab mice. SIGNIFICANCE: NGF mediates the Na+ channel plasticity with a shift from TTX-resistant Nav1.8 to TTX-sensitive Nav1.7 in bladder afferent neurons, which could be a possible underlying mechanism of bladder afferent hyperexcitability and detrusor overactivity after SCI.


Assuntos
Traumatismos da Medula Espinal , Bexiga Urinária , Animais , Feminino , Gânglios Espinais/metabolismo , Camundongos , Fator de Crescimento Neural/metabolismo , Neurônios Aferentes , Traumatismos da Medula Espinal/metabolismo , Tetrodotoxina/farmacologia , Bexiga Urinária/metabolismo
15.
Toxicon ; 213: 7-12, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35421438

RESUMO

The origin and biogenesis of tetrodotoxin (TTX) is one of the most interesting and perplexing questions remaining for TTX researchers. Newts can possess extreme quantities of TTX and are one of the most well-studied of all TTX-bearing organisms, yet seemingly conflicting results between studies on closely related species continues to generate debate. In this study, eggs from 12 female newts (Taricha granulosa) were reared in captivity and the metamorphosed juveniles were fed a TTX-free diet for 3 years. Using a non-lethal sampling technique, we collected skin samples from each individual each year. Wild-caught juveniles from the same population were also sampled for TTX. In lab-reared juveniles, mass increased rapidly, and after only 2 years individuals approached adult body mass. TTX levels increased slowly during the first two years and then jumped considerably in year three when fed a diet free of TTX. However, wild-caught juvenile newts of unknown age were more toxic than their lab-reared counterparts. These results, coupled with additional data on the long-term production and synthesis of TTX in adult newts suggest that TTX is unlikely to come through dietary acquisition, but rather newts may be able to synthesize their own toxin or acquire it from symbiotic bacteria.


Assuntos
Bactérias , Salamandridae , Animais , Feminino , Humanos , Simbiose , Tetrodotoxina/toxicidade
16.
Anesth Analg ; 134(6): 1140-1152, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35436248

RESUMO

BACKGROUND: Cholinergic stimulation of prefrontal cortex (PFC) can reverse anesthesia. Conversely, inactivation of PFC can delay emergence from anesthesia. PFC receives cholinergic projections from basal forebrain, which contains wake-promoting neurons. However, the role of basal forebrain cholinergic neurons in arousal from the anesthetized state requires refinement, and it is currently unknown whether the arousal-promoting effect of basal forebrain is mediated through PFC. To address these gaps in knowledge, we implemented a novel approach to the use of chemogenetic stimulation and tested the role of basal forebrain cholinergic neurons in behavioral arousal during sevoflurane anesthesia. Next, we investigated the effect of tetrodotoxin-mediated inactivation of PFC on behavioral arousal produced by electrical stimulation of basal forebrain during sevoflurane anesthesia. METHODS: Adult male and female transgenic rats (Long-Evans-Tg [ChAT-Cre]5.1 Deis; n = 22) were surgically prepared for expression of excitatory hM3D(Gq) receptors or mCherry in basal forebrain cholinergic neurons, and activation of these neurons by local delivery of compound 21, an agonist for hM3D(Gq) receptors. The transgenic rats were fitted with microdialysis probes for agonist delivery into basal forebrain and simultaneous prefrontal acetylcholine measurement. Adult male and female Sprague Dawley rats were surgically prepared for bilateral electrical stimulation of basal forebrain and tetrodotoxin infusion (156 µM and 500 nL) into PFC (n = 9) or bilateral electrical stimulation of piriform cortex (n = 9) as an anatomical control. All rats were implanted with electrodes to monitor the electroencephalogram. Heart and respiration rates were monitored using noninvasive sensors. A 6-point scale was used to score behavioral arousal (0 = no arousal and 5 = return of righting reflex). RESULTS: Compound 21 delivery into basal forebrain of rats with hM3D(Gq) receptors during sevoflurane anesthesia produced increases in arousal score (P < .001; confidence interval [CI], 1.80-4.35), heart rate (P < .001; CI, 36.19-85.32), respiration rate (P < .001; CI, 22.81-58.78), theta/delta ratio (P = .008; CI, 0.028-0.16), and prefrontal acetylcholine (P < .001; CI, 1.73-7.46). Electrical stimulation of basal forebrain also produced increases in arousal score (P < .001; CI, 1.85-4.08), heart rate (P = .018; CI, 9.38-98.04), respiration rate (P < .001; CI, 24.15-53.82), and theta/delta ratio (P = .020; CI, 0.019-0.22), which were attenuated by tetrodotoxin-mediated inactivation of PFC. CONCLUSIONS: This study validates the role of basal forebrain cholinergic neurons in behavioral arousal and demonstrates that the arousal-promoting effects of basal forebrain are mediated in part through PFC.


Assuntos
Anestesia , Prosencéfalo Basal , Acetilcolina/metabolismo , Animais , Nível de Alerta , Prosencéfalo Basal/metabolismo , Colinérgicos/farmacologia , Eletroencefalografia , Feminino , Imidazóis , Masculino , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Sevoflurano/farmacologia , Sulfonamidas , Tetrodotoxina/metabolismo , Tiofenos
17.
Elife ; 112022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35441593

RESUMO

Resurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing and contribute to neuronal hyperexcitability and disease pathophysiology. Here, we show, for the first time, that INaR can be reconstituted in a heterologous system by coexpression of sodium channel α-subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin (TTX)-resistant neuronal channels, and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.


Assuntos
Células Receptoras Sensoriais , Canais de Sódio Disparados por Voltagem , Potenciais de Ação/fisiologia , Gânglios Espinais/metabolismo , Isoformas de Proteínas/metabolismo , Células Receptoras Sensoriais/fisiologia , Tetrodotoxina/farmacologia , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo
18.
Ann Neurol ; 92(1): 45-60, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35467038

RESUMO

OBJECTIVE: Infantile spasms are associated with a wide variety of clinical conditions, including perinatal brain injuries. We have created a model in which prolonged infusion of tetrodotoxin (TTX) into the neocortex, beginning in infancy, produces a localized lesion and reproduces the behavioral spasms, electroencephalogram (EEG) abnormalities, and drug responsiveness seen clinically. Here, we undertook experiments to explore the possibility that the growth factor IGF-1 plays a role in generating epileptic spasms. METHODS: We combined long-term video EEG recordings with quantitative immunohistochemical and biochemical analyses to unravel IGF-1's role in spasm generation. Immunohistochemistry was undertaken in surgically resected tissue from infantile spasms patients. We used viral injections in neonatal conditional IGF-1R knock-out mice to show that an IGF-1-derived tripeptide (1-3)IGF-1, acts through the IGF-1 receptor to abolish spasms. RESULTS: Immunohistochemical methods revealed widespread loss of IGF-1 from cortical neurons, but an increase in IGF-1 in the reactive astrocytes in the TTX-induced lesion. Very similar changes were observed in the neocortex from patients with spasms. In animals, we observed reduced signaling through the IGF-1 growth pathways in areas remote from the lesion. To show the reduction in IGF-1 expression plays a role in spasm generation, epileptic rats were treated with (1-3)IGF-1. We provide 3 lines of evidence that (1-3)IGF-1 activates the IGF-1 signaling pathway by acting through the receptor for IGF-1. Treatment with (1-3)IGF-1 abolished spasms and hypsarrhythmia-like activity in the majority of animals. INTERPRETATION: Results implicate IGF-1 in the pathogenesis of infantile spasms and IGF-1 analogues as potential novel therapies for this neurodevelopmental disorder. ANN NEUROL 2022;92:45-60.


Assuntos
Espasmos Infantis , Animais , Modelos Animais de Doenças , Eletroencefalografia/métodos , Humanos , Lactente , Fator de Crescimento Insulin-Like I , Camundongos , Ratos , Espasmo/induzido quimicamente , Espasmos Infantis/induzido quimicamente , Espasmos Infantis/tratamento farmacológico , Tetrodotoxina/farmacologia
19.
J Physiol ; 600(10): 2499-2513, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35413129

RESUMO

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50  = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Rabdomiossarcoma , Linhagem Celular , Humanos , Canal de Sódio Disparado por Voltagem NAV1.4 , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor , Rabdomiossarcoma/genética , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia
20.
Cells ; 11(7)2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35406789

RESUMO

B cells express various ion channels, but the presence of voltage-gated sodium (NaV) channels has not been confirmed in the plasma membrane yet. In this study, we have identified several NaV channels, which are expressed in the human B cell membrane, by electrophysiological and molecular biology methods. The sensitivity of the detected sodium current to tetrodotoxin was between the values published for TTX-sensitive and TTX-insensitive channels, which suggests the co-existence of multiple NaV1 subtypes in the B cell membrane. This was confirmed by RT-qPCR results, which showed high expression of TTX-sensitive channels along with the lower expression of TTX-insensitive NaV1 channels. The biophysical characteristics of the currents also supported the expression of multiple NaV channels. In addition, we investigated the potential functional role of NaV channels by membrane potential measurements. Removal of Na+ from the extracellular solution caused a reversible hyperpolarization, supporting the role of NaV channels in shaping and maintaining the resting membrane potential. As this study was mainly limited to electrophysiological properties, we cannot exclude the possible non-canonical functions of these channels. This work concludes that the presence of voltage-gated sodium channels in the plasma membrane of human B cells should be recognized and accounted for in the future.


Assuntos
Canais de Sódio Disparados por Voltagem , Membrana Celular/metabolismo , Humanos , Potenciais da Membrana/fisiologia , Sódio/metabolismo , Tetrodotoxina , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo
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