RESUMEN
Neurotoxins pose significant challenges in defense and healthcare due to their disruptive effects on nervous tissues. Their extreme potency and enormous structural diversity have hindered the development of effective antidotes. Motivated by the properties of cell membrane-derived nanodiscs, such as their ultrasmall size, disc shape, and inherent cell membrane functions, here, we develop neuronal membrane-derived nanodiscs (denoted "Neuron-NDs") as a countermeasure nanomedicine for broad-spectrum neurotoxin detoxification. We fabricate Neuron-NDs using the plasma membrane of human SH-SY5Y neurons and demonstrate their effectiveness in detoxifying tetrodotoxin (TTX) and botulinum toxin (BoNT), two model toxins with distinct mechanisms of action. Cell-based assays confirm the ability of Neuron-NDs to inhibit TTX-induced ion channel blockage and BoNT-mediated inhibition of synaptic vesicle recycling. In mouse models of TTX and BoNT intoxication, treatment with Neuron-NDs effectively improves survival rates in both therapeutic and preventative settings. Importantly, high-dose administration of Neuron-NDs shows no observable acute toxicity in mice, indicating its safety profile. Overall, our study highlights the facile fabrication of Neuron-NDs and their broad-spectrum detoxification capabilities, offering promising solutions for neurotoxin-related challenges in biodefense and therapeutic applications.
Asunto(s)
Membrana Celular , Nanoestructuras , Neuronas , Neurotoxinas , Tetrodotoxina , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Animales , Ratones , Tetrodotoxina/química , Tetrodotoxina/farmacología , Neurotoxinas/química , Neurotoxinas/toxicidad , Neurotoxinas/farmacología , Nanoestructuras/química , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Toxinas Botulínicas/química , Toxinas Botulínicas/farmacología , Toxinas Botulínicas/metabolismo , Inactivación MetabólicaRESUMEN
Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.
Asunto(s)
Analgésicos , Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.7 , Neuronas , Tetrodotoxina , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/citología , Neuronas/efectos de los fármacos , Ratones , Tetrodotoxina/farmacología , Analgésicos/farmacología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Venenos de Crotálidos/toxicidad , Venenos de Crotálidos/farmacología , Masculino , Crotalus , Potenciales de la Membrana/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , PéptidosRESUMEN
Gut motility undergoes a switch from myogenic to neurogenic control in late embryonic development. Here, we report on the electrical events that underlie this transition in the enteric nervous system, using the GCaMP6f reporter in neural crest cell derivatives. We found that spontaneous calcium activity is tetrodotoxin (TTX) resistant at stage E11.5, but not at E18.5. Motility at E18.5 was characterized by periodic, alternating high- and low-frequency contractions of the circular smooth muscle; this frequency modulation was inhibited by TTX. Calcium imaging at the neurogenic-motility stages E18.5-P3 showed that CaV1.2-positive neurons exhibited spontaneous calcium activity, which was inhibited by nicardipine and 2-aminoethoxydiphenyl borate (2-APB). Our protocol locally prevented muscle tone relaxation, arguing for a direct effect of nicardipine on enteric neurons, rather than indirectly by its relaxing effect on muscle. We demonstrated that the ENS was mechanosensitive from early stages on (E14.5) and that this behaviour was TTX and 2-APB resistant. We extended our results on L-type channel-dependent spontaneous activity and TTX-resistant mechanosensitivity to the adult colon. Our results shed light on the critical transition from myogenic to neurogenic motility in the developing gut, as well as on the intriguing pathways mediating electro-mechanical sensitivity in the enteric nervous system. HIGHLIGHTS: What is the central question of this study? What are the first neural electric events underlying the transition from myogenic to neurogenic motility in the developing gut, what channels do they depend on, and does the enteric nervous system already exhibit mechanosensitivity? What is the main finding and its importance? ENS calcium activity is sensitive to tetrodotoxin at stage E18.5 but not E11.5. Spontaneous electric activity at fetal and adult stages is crucially dependent on L-type calcium channels and IP3R receptors, and the enteric nervous system exhibits a tetrodotoxin-resistant mechanosensitive response. Abstract figure legend Tetrodotoxin-resistant Ca2+ rise induced by mechanical stimulation in the E18.5 mouse duodenum.
Asunto(s)
Canales de Calcio Tipo L , Calcio , Sistema Nervioso Entérico , Motilidad Gastrointestinal , Neuronas , Tetrodotoxina , Animales , Canales de Calcio Tipo L/metabolismo , Tetrodotoxina/farmacología , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/metabolismo , Sistema Nervioso Entérico/fisiología , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Calcio/metabolismo , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiología , Ratones Endogámicos C57BL , Bloqueadores de los Canales de Calcio/farmacología , Femenino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Nicardipino/farmacología , Compuestos de BoroRESUMEN
An accumulation of reactive oxygen species (ROS) in cardiomyocytes can induce pro-arrhythmogenic late Na+ currents by removing the inactivation of voltage-gated Na+ channels including the tetrodotoxin (TTX)-resistant cardiac α-subunit Nav1.5 as well as TTX-sensitive α-subunits like Nav1.2 and Nav1.3. Here, we explored oxidant-induced late Na+ currents in mouse cardiomyocytes and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in HEK 293 cells expressing Nav1.2, Nav1.3, or Nav1.5. Na+ currents in mouse cardiomyocytes and hiPSC-CMs treated with the oxidant chloramine T (ChT) developed a moderate reduction in peak current amplitudes accompanied by large late Na+ currents. While ChT induced a strong reduction in peak current amplitudes but only small persistent currents on Nav1.5, both Nav1.2 and Nav1.3 produced increased peak current amplitudes and large persistent currents following oxidation. TTX (300 nM) blocked ChT-induced late Na+ currents significantly stronger as compared to peak Na+ currents in both mouse cardiomyocytes and hiPSC-CMs. Similar differences between Nav1.2, Nav1.3, and Nav1.5 regarding ROS sensitivity were also evident when oxidation was induced with UVA-light (380 nm) or the cysteine-selective oxidant nitroxyl (HNO). To conclude, our data on TTX-sensitive Na+ channels expressed in cardiomyocytes may be relevant for the generation of late Na+ currents following oxidative stress.
Asunto(s)
Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Oxidación-Reducción , Tetrodotoxina , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Humanos , Animales , Tetrodotoxina/farmacología , Ratones , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células HEK293 , Cloraminas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Sodio/metabolismo , Potenciales de Acción/efectos de los fármacos , Compuestos de TosiloRESUMEN
A large body of evidence has shown that treatments that interfere with memory consolidation become ineffective when animals are subjected to an intense learning experience; this effect has been observed after systemic and local administration of amnestic drugs into several brain areas, including the striatum. However, the effects of amnestic treatments on the process of extinction after intense training have not been studied. Previous research demonstrated increased spinogenesis in the dorsomedial striatum, but not in the dorsolateral striatum after intense training, indicating that the dorsomedial striatum is involved in the protective effect of intense training. To investigate this issue, male Wistar rats, previously trained with low, moderate, or high levels of foot shock, were used to study the effect of tetrodotoxin inactivation of dorsomedial striatum on memory consolidation and subsequent extinction of inhibitory avoidance. Performance of the task was evaluated during seven extinction sessions. Tetrodotoxin produced a marked deficit of memory consolidation of inhibitory avoidance trained with low and moderate intensities of foot shock, but normal consolidation occurred when a relatively high foot shock was used. The protective effect of intense training was long-lasting, as evidenced by the high resistance to extinction exhibited throughout the extinction sessions. We discuss the possibility that increased dendritic spinogenesis in dorsomedial striatum may underly this protective effect, and how this mechanism may be related to the resilient memory typical of post-traumatic stress disorder (PTSD).
Asunto(s)
Reacción de Prevención , Cuerpo Estriado , Extinción Psicológica , Ratas Wistar , Tetrodotoxina , Animales , Masculino , Extinción Psicológica/efectos de los fármacos , Extinción Psicológica/fisiología , Ratas , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Cuerpo Estriado/fisiología , Cuerpo Estriado/efectos de los fármacos , Tetrodotoxina/farmacología , Consolidación de la Memoria/efectos de los fármacos , Consolidación de la Memoria/fisiología , Amnesia/fisiopatología , Amnesia/prevención & control , ElectrochoqueRESUMEN
Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.
Asunto(s)
Empalme Alternativo , Canal de Sodio Activado por Voltaje NAV1.6 , Isoformas de Proteínas , Canal de Sodio Activado por Voltaje NAV1.6/genética , Humanos , Isoformas de Proteínas/genética , Empalme Alternativo/genética , Tetrodotoxina/farmacología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Mutación , Línea Celular , AnimalesRESUMEN
Toxic puffers accumulate tetrodotoxin (TTX), a well-known neurotoxin, by feeding on TTX-bearing organisms and using it to defend themselves from predators. Our previous studies have demonstrated that toxic puffers are attracted to 5,6,11-trideoxytetrodotoxin (TDT), a nontoxic TTX analog that is simultaneously accumulated with TTX in toxic puffers and their prey. In addition, activity labeling using immunohistochemistry targeting neuronal activity marker suggests that TDT activates crypt olfactory sensory neurons (OSN) of the green spotted puffer. However, it remains to be determined whether individual crypt OSNs can physiologically respond to TDT. By employing electroporation to express GCaMP6s in OSNs, we successfully identified a distinct group of oval OSNs that exhibited a specific calcium response when exposed to TDT in green spotted puffers. These oval OSNs showed no response to amino acids (AAs), which serve as food odor cues for teleosts. Furthermore, oval morphology and surface positioning of TDT-sensitive OSNs in the olfactory epithelium closely resemble that of crypt OSNs. These findings further substantiate that TDT is specifically detected by crypt OSNs in green spotted puffer. The TDT odor may act as a chemoattractant for finding conspecific toxic puffers and for feeding TTX-bearing organisms for effective toxification.
Asunto(s)
Odorantes , Neuronas Receptoras Olfatorias , Tetraodontiformes , Tetrodotoxina , Animales , Tetrodotoxina/farmacología , Neuronas Receptoras Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/metabolismo , Odorantes/análisis , Calcio/metabolismoRESUMEN
Fermented foods play a significant role in the human diet for their natural, highly nutritious and healthy attributes. Our aim was to study the effect of yeast extract, a fermented substance extracted from natural yeast, on colonic motility to better understand its potential therapeutic role. A yeast extract was given to rats by gavage for 3 days, and myogenic and neurogenic components of colonic motility were studied using spatiotemporal maps made from video recordings of the whole colon ex vivo. A control group received saline gavages. The yeast extract caused excitation of the musculature by increasing the propagation length and duration of long-distance contractions, the major propulsive activity of the rat colon. The yeast extract also evoked rhythmic propulsive motor complexes (RPMCs) which were antegrade in the proximal and mid-colon and retrograde in the distal colon. RPMC activity was evoked by distention-induced neural activity, but it was myogenic in nature since we showed it to be generated by bethanechol in the presence of tetrodotoxin. In conclusion, ingestion of yeast extract stimulates rat colon motility by exciting neurogenic and myogenic control mechanisms.
Asunto(s)
Colon , Motilidad Gastrointestinal , Animales , Colon/efectos de los fármacos , Colon/fisiología , Motilidad Gastrointestinal/efectos de los fármacos , Ratas , Masculino , Levaduras , Ratas Sprague-Dawley , Tetrodotoxina/farmacologíaRESUMEN
Vincristine-induced peripheral neuropathy is a common side effect of vincristine treatment, which is accompanied by pain and can be dose-limiting. The molecular mechanisms that underlie vincristine-induced pain are not well understood. We have established an animal model to investigate pathophysiological mechanisms of vincristine-induced pain. Our previous studies have shown that the tetrodotoxin-sensitive voltage-gated sodium channel Nav1.6 in medium-diameter dorsal root ganglion (DRG) neurons contributes to the maintenance of vincristine-induced allodynia. In this study, we investigated the effects of vincristine administration on excitability in small-diameter DRG neurons and whether the tetrodotoxin-resistant (TTX-R) Nav1.8 channels contribute to mechanical allodynia. Current-clamp recordings demonstrated that small DRG neurons become hyper-excitable following vincristine treatment, with both reduced current threshold and increased firing frequency. Using voltage-clamp recordings in small DRG neurons, we now show an increase in TTX-R current density and a -7.3 mV hyperpolarizing shift in the half-maximal potential (V1/2) of activation of Nav1.8 channels in vincristine-treated animals, which likely contributes to the hyperexcitability that we observed in these neurons. Notably, vincristine treatment did not enhance excitability of small DRG neurons from Nav1.8 knockout mice, and the development of mechanical allodynia was delayed but not abrogated in these mice. Together, our data suggest that sodium channel Nav1.8 in small DRG neurons contributes to the development of vincristine-induced mechanical allodynia.
Asunto(s)
Ganglios Espinales , Hiperalgesia , Canal de Sodio Activado por Voltaje NAV1.8 , Neuronas , Vincristina , Animales , Vincristina/toxicidad , Vincristina/farmacología , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8/genética , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Masculino , Ratones Noqueados , Tetrodotoxina/farmacología , Potenciales de Acción/efectos de los fármacos , Ratones Endogámicos C57BL , Antineoplásicos Fitogénicos/toxicidad , Técnicas de Placa-ClampRESUMEN
Long-term potentiation (LTP) is a synaptic mechanism involved in learning and memory. Experiments have shown that dendritic sodium spikes (Na-dSpikes) are required for LTP in the distal apical dendrites of CA1 pyramidal cells. On the other hand, LTP in perisomatic dendrites can be induced by synaptic input patterns that can be both subthreshold and suprathreshold for Na-dSpikes. It is unclear whether these results can be explained by one unifying plasticity mechanism. Here, we show in biophysically and morphologically realistic compartmental models of the CA1 pyramidal cell that these forms of LTP can be fully accounted for by a simple plasticity rule. We call it the voltage-based Event-Timing-Dependent Plasticity (ETDP) rule. The presynaptic event is the presynaptic spike or release of glutamate. The postsynaptic event is the local depolarization that exceeds a certain plasticity threshold. Our model reproduced the experimentally observed LTP in a variety of protocols, including local pharmacological inhibition of dendritic spikes by tetrodotoxin (TTX). In summary, we have provided a validation of the voltage-based ETDP, suggesting that this simple plasticity rule can be used to model even complex spatiotemporal patterns of long-term synaptic plasticity in neuronal dendrites.
Asunto(s)
Potenciales de Acción , Región CA1 Hipocampal , Dendritas , Potenciación a Largo Plazo , Modelos Neurológicos , Células Piramidales , Dendritas/fisiología , Potenciación a Largo Plazo/fisiología , Células Piramidales/fisiología , Animales , Región CA1 Hipocampal/fisiología , Región CA1 Hipocampal/citología , Potenciales de Acción/fisiología , Plasticidad Neuronal/fisiología , Tetrodotoxina/farmacología , Simulación por ComputadorRESUMEN
Tetrodotoxin (TTX) is a neurotoxic molecule used by many animals for defense and/or predation, as well as an important biomedical tool. Its ubiquity as a defensive agent has led to repeated independent evolution of tetrodotoxin resistance in animals. TTX binds to voltage-gated sodium channels (VGSC) consisting of α and ß subunits. Virtually all studies investigating the mechanisms behind TTX resistance have focused on the α subunit of voltage-gated sodium channels, where tetrodotoxin binds. However, the possibility of ß subunits also contributing to tetrodotoxin resistance was never explored, though these subunits act in concert. In this study, we present preliminary evidence suggesting a potential role of ß subunits in the evolution of TTX resistance. We gathered mRNA sequences for all ß subunit types found in vertebrates across 12 species (three TTX-resistant and nine TTX-sensitive) and tested for signatures of positive selection with a maximum likelihood approach. Our results revealed several sites experiencing positive selection in TTX-resistant taxa, though none were exclusive to those species in subunit ß1, which forms a complex with the main physiological target of TTX (VGSC Nav1.4). While experimental data validating these findings would be necessary, this work suggests that deeper investigation into ß subunits as potential players in tetrodotoxin resistance may be worthwhile.
Asunto(s)
Canales de Sodio Activados por Voltaje , Animales , Tetrodotoxina/farmacología , Funciones de Verosimilitud , Canales de Sodio Activados por Voltaje/genética , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
Adiponectin (ADPN) has been reported to induce inhibitory effects on gastric motor activity, which, being a source of peripheral satiety signals, would contribute to the central anorexigenic effects of the hormone in rodents. However, peripheral satiety signals can also originate from the small intestine. Since there are no data on the effects of ADPN in this gut region, the present study aimed to investigate whether ADPN affects murine ileal contractility. Immunofluorescence experiments and Western blot were also performed to reveal the expression of ADPN receptors. Mechanical responses of ileal preparations were recorded in vitro via force-displacement transducers. Preparations showed a tetrodotoxin- and atropine-insensitive spontaneous contractile activity. Electrical field stimulation (EFS) induced tetrodotoxin- and atropine-sensitive contractile responses. ADPN induced a decay of the basal tension and decreased the amplitude of either the spontaneous contractility or the EFS-induced excitatory responses. All ADPN effects were abolished by the nitric oxide (NO) synthesis inhibitor NG-nitro l-arginine. The expression of the ADPN receptor, AdipoR1, but not AdipoR2, was also revealed in enteric glial cells. The present results offer the first evidence that ADPN acts on ileal preparations. The hormone exerts inhibitory effects, likely involving AdipoR1 on enteric glial cells and NO. From a physiological point of view, it could be hypothesized that the depressant action of ADPN on ileal contractility represents an additional peripheral satiety signal which, as also described for the ileal brake, could contribute to the central anorexigenic effects of the hormone.NEW & NOTEWORTHY This study provides the first evidence that adiponectin (ADPN) is able to act on ileal preparations. Functional results demonstrate that the hormone, other than causing a slight decay of the basal tension, depresses the amplitude of both spontaneous contractility and neurally induced excitatory responses of the mouse ileum through the involvement of nitric oxide. The expression of the ADPN receptor AdipoR1 and its localization on glial cells was revealed by Western blot and immunofluorescence analysis.
Asunto(s)
Adiponectina , Óxido Nítrico , Animales , Ratones , Adiponectina/farmacología , Atropina/farmacología , Íleon/metabolismo , Contracción Muscular/fisiología , Óxido Nítrico/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
<b>Background and Objective:</b> Functional Voltage-Gated Sodium Channels (VGSCs) are expressed in metastatic prostate cancer (PCa) cells. A number of <i>in vitro</i> studies have evaluated the effect of functional VGSC expression on the metastatic cell behavior of PCa cells. This study aimed to evaluate the effect of VGSC inhibition on metastatic cell behavior in PCa cells by meta-analysis. <b>Materials and Methods:</b> Meta-analysis was performed on data taken from 13 publications that examined the effect of VGSC inhibitors on the metastatic cell behavior of metastatic PCa cells expressing functional VGSCs. The measure of effect was calculated according to the random effects model using mean differences and presented with a forest plot graph. Heterogeneity was checked using the Cochran's Q Test (Chi-square statistic) and the I<sup>2</sup> test statistic. In order to evaluate the objectivity, the funnels-plot graph was used. <b>Results:</b> The g value showing the effect size was calculated as 4.49 (95% CI = 5.35-3.62) in the experiments where Tetrodotoxin (TTX) was used, which has a very high specificity for VGSCs but is not licensed for clinical use. In experiments using licensed inhibitors Lamotrigine, Oxcarbazepine, Phenytoin, Ranolazine, Riluzole and Lidocaine, the g value was 1.37 (95 % CI = 2.02-0.71). Suppression of metastatic cell behavior in both subgroups is statistically significant (p<0.00001). <b>Conclusion:</b> Meta-analysis confirmed that VGSCs are an enhancing factor in the metastasis of PCa cells. The VGSCs appear to be an important target in the diagnosis and development of new treatment options in PCa.
Asunto(s)
Neoplasias de la Próstata , Bloqueadores del Canal de Sodio Activado por Voltaje , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Ranolazina/farmacología , Canales de Sodio/metabolismo , Tetrodotoxina/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
Tetrodotoxin (TTX) is an exceedingly toxic non-protein biotoxin that demonstrates remarkable selectivity and affinity for sodium channels on the excitation membrane of nerves. This property allows TTX to effectively obstruct nerve conduction, resulting in nerve paralysis and fatality. Although the mechanistic aspects of its toxicity are well understood, there is a dearth of literature addressing alterations in the neural microenvironment subsequent to TTX poisoning. In this research endeavor, we harnessed human pluripotent induced stem cells to generate cerebral organoids-an innovative model closely mirroring the structural and functional intricacies of the human brain. This model was employed to scrutinize the comprehensive transcriptomic shifts induced by TTX exposure, thereby delving into the neurotoxic properties of TTX and its potential underlying mechanisms. Our findings revealed 455 differentially expressed mRNAs (DEmRNAs), 212 differentially expressed lncRNAs (DElncRNAs), and 18 differentially expressed miRNAs (DEmiRNAs) in the TTX-exposed group when juxtaposed with the control cohort. Through meticulous Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction (PPI) analysis, we ascertained that these differential genes predominantly participate in the regulation of voltage-gated channels and synaptic homeostasis. A comprehensive ceRNA network analysis unveiled that DEmRNAs exert control over the expression of ion channels and neurocytokines, suggesting their potential role in mediating apoptosis.
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MicroARNs , Síndromes de Neurotoxicidad , Humanos , Tetrodotoxina/farmacología , Transcriptoma , MicroARNs/genética , MicroARNs/metabolismo , Perfilación de la Expresión Génica , Canales de Sodio/genética , Canales de Sodio/metabolismo , Síndromes de Neurotoxicidad/genética , Redes Reguladoras de GenesRESUMEN
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to relieve various symptoms such as headache, arthralgia, and dental pain. While the primary mechanism of NSAID-based pain relief is the inhibition of cyclooxygenase-2, several NSAIDs also modulate other molecular targets related to nociceptive transmission such as voltage-gated Na+ channels. In the present study, we examined the effects of NSAIDs on persistent Na+ current (INaP) mediated by tetrodotoxin-resistant (TTX-R) Na+ channels in small-to medium-sized trigeminal ganglion neurons using a whole-cell patch-clamp technique. At clinically relevant concentrations, all propionic acid derivatives tested (ibuprofen, naproxen, fenoprofen, and flurbiprofen) preferentially inhibited the TTX-R INaP. The inhibition was more potent at acidic extracellular pH (pH 6.5) than at normal pH (pH 7.4). Other NSAIDs, such as ketorolac, piroxicam, and aspirin, had a negligible effect on the TTX-R INaP. Ibuprofen both accelerated the onset of inactivation and retarded the recovery from inactivation of TTX-R Na+ channels at acidic extracellular pH. However, all NSAIDs tested in this study had minor effects on voltage-gated K+ currents, as well as hyperpolarization-activated and cyclic nucleotide-gated cation currents, at both acidic and normal extracellular pH. Under current-clamp conditions, ibuprofen decreased the number of action potentials elicited by depolarizing current stimuli at acidic (pH 6.5) extracellular pH. Considering that extracellular pH falls as low as 5.5 in inflamed tissues, TTX-R INaP inhibition could be a mechanism by which ibuprofen and propionic acid derivative NSAIDs modulate inflammatory pain.
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Ibuprofeno , Ganglio del Trigémino , Ratas , Animales , Tetrodotoxina/farmacología , Ibuprofeno/farmacología , Canales de Sodio , Bloqueadores de los Canales de Sodio/farmacología , Ratas Sprague-Dawley , Potenciales de la Membrana , Antiinflamatorios no Esteroideos/farmacología , Neuronas , Dolor , Ácidos , Concentración de Iones de HidrógenoRESUMEN
Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell levels; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.
Asunto(s)
Células Madre Pluripotentes Inducidas , Plasticidad Neuronal , Humanos , Ratas , Animales , Células Cultivadas , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Técnicas de Cocultivo , Tetrodotoxina/farmacologíaRESUMEN
Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5-5 µM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100 µM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1 µM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100 µM riluzole or 100 µM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1 µM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca2+/high Mg2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment.
Asunto(s)
Aconitina , Tronco Encefálico , Animales , Ratas , Animales Recién Nacidos , Aconitina/farmacología , Tetrodotoxina/farmacología , Ratas Wistar , Bulbo Raquídeo/fisiología , Médula Espinal , Lidocaína/farmacologíaRESUMEN
Action potential (AP) conduction depends on voltage-gated sodium channels, of which there are nine subtypes. The vagus nerve, comprising sensory afferent fibers and efferent parasympathetic fibers, provides autonomic regulation of visceral organs, but the voltage-gated sodium channels (NaV1) subtypes involved in its AP conduction are poorly defined. We studied the A- and C-waves of electrically stimulated compound action potentials (CAPs) of the mouse and rat vagus nerves with and without NaV1 inhibitor administration: tetrodotoxin (TTX), PF-05089771 (mouse NaV1.7), ProTX-II (NaV1.7), ICA-121341 (NaV1.1, NaV1.3, and NaV1.6), LSN-3049227 (NaV1.2, NaV1.6, and NaV1.7), and A-803467 (NaV1.8). We show that TTX-sensitive NaV1 channels are essential for all vagal AP conduction. PF-05089771 but not ICA-121341 inhibited the mouse A-wave, which was abolished by LSN-3049227, suggesting roles for NaV1.7 and NaV1.2. The mouse C-wave was abolished by LSN-3049227 and a combination of PF-05089771 and ICA-121341, suggesting roles for NaV1.7 and NaV1.6. The rat A-wave was inhibited by ProTX-II, ICA-121341, and a combination of these inhibitors but only abolished by LSN-3049227, suggesting roles for NaV1.7, NaV1.6, and NaV1.2. The rat C-wave was abolished by LSN-3049227 and a combination of ProTX-II and ICA-121341, suggesting roles for NaV1.7 and NaV1.6. A-803467 also inhibited the mouse and rat CAP suggesting a cooperative role for the TTX-resistant NaV1.8. Overall, our data demonstrate that multiple NaV1 subtypes contribute to vagal CAPs, with NaV1.7 and NaV1.8 playing predominant roles and NaV1.6 and NaV1.2 contributing to a different extent based on nerve fiber type and species. Inhibition of these NaV1 may impact autonomic regulation of visceral organs.NEW & NOTEWORTHY Distinct NaV1 channels are involved in action potential (AP) initiation and conduction from afferent terminals within specific organs. Here, we have identified the NaV1 necessary for AP conduction in the entire murine and rat vagus nerve. We show TTX-sensitive channels are essential for all AP conduction, predominantly NaV1.7 with NaV1.2 and NaV1.6 playing lesser roles depending on the species and fiber type. In addition, we show that NaV1.8 is also essential for most axonal AP conduction.
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Canales de Sodio Activados por Voltaje , Ratones , Ratas , Animales , Potenciales de Acción/fisiología , Canales de Sodio Activados por Voltaje/fisiología , Tetrodotoxina/farmacología , Nervio Vago/fisiologíaRESUMEN
In isolated segments of the rat proximal colon, the dopamine reuptake inhibitor GBR 12909 (GBR) causes a dilatation, while the D1-like receptor antagonist SCH 23390 (SCH) induces a tonic constriction, suggesting that neurally released dopamine tonically stimulates enteric inhibitory efferent neurons. Here, the targets of the enteric dopaminergic neurons were investigated. Cannulated segments of rat proximal colon were bathed in physiological salt solution and luminally perfused with 0.9% saline, while all drugs were applied to the bath. Spatio-temporal maps of colonic motility were constructed from video recordings of peristaltic contractions, and the maximum diameter was measured as an index of colonic contractility. GBR (1 µM)-induced dilatations of colonic segments were prevented by SCH (5 µM), L-nitro arginine (L-NA; 100 µM), a nitric oxide synthase inhibitor, or tetrodotoxin (0.6 µM). In contrast, constrictions induced by a higher concentration of SCH (20 µM) were unaffected by either L-NA or tetrodotoxin. The vasoactive intestinal peptide (VIP) receptor antagonist VIP10-28 (3 µM) or P2Y1 receptor antagonist MRS 2500 (1 µM) had no effect on either the GBR-induced dilatation or the SCH-induced constriction. In colonic segments that had been pretreated with 6-hydroxydopamine (100 µM, 3 h) to deplete enteric dopamine, GBR failed to increase the colonic diameter, while SCH was still capable of constricting colonic segments. Enteric dopaminergic neurons appear to project to nitrergic neurons to dilate the proximal colon by activating neuronal D1-like receptors. In addition, constitutively activated D1-like receptors expressed in cells yet to be determined may provide a tonic inhibition on colonic constrictions.
Asunto(s)
Dopamina , Neuronas , Ratas , Animales , Dopamina/farmacología , Tetrodotoxina/farmacología , Peristaltismo/fisiología , Arginina/farmacología , Colon , Motilidad GastrointestinalRESUMEN
Very little is known about the physiological role of nicotinic receptors in canine bladders, although functional nicotinic receptors have been reported in bladders of many species. Utilizing in vitro methods, we evaluated nicotinic receptors mediating bladder function in dogs: control (9 female and 11 male normal controls, 5 sham operated), Decentralized (9 females, decentralized 6-21 mo), and obturator-to-pelvic nerve transfer reinnervated (ObNT-Reinn; 9 females; decentralized 9-13 mo, then reinnervated with 8-12 mo recovery). Muscle strips were collected, mucosa-denuded, and mounted in muscle baths before incubation with neurotransmitter antagonists, and contractions to the nicotinic receptor agonist epibatidine were determined. Strip response to epibatidine, expressed as percent potassium chloride, was similar (â¼35% in controls, 30% in Decentralized, and 24% in ObNT-Reinn). Differentially, epibatidine responses in Decentralized and ObNT-Reinn bladder strips were lower than controls after tetrodotoxin (TTX, a sodium channel blocker that inhibits axonal action potentials). Yet, in all groups, epibatidine-induced strip contractions were similarly inhibited by mecamylamine and hexamethonium (ganglionic nicotinic receptor antagonists), SR 16584 (α3ß4 neuronal nicotinic receptor antagonist), atracurium and tubocurarine (neuromuscular nicotinic receptor antagonists), and atropine (muscarinic receptor antagonist), indicating that nicotinic receptors (particularly α3ß4 subtypes), neuromuscular and muscarinic receptors play roles in bladder contractility. In control bladder strips, since tetrodotoxin did not inhibit epibatidine contractions, nicotinic receptors are likely located on nerve terminals. The tetrodotoxin inhibition of epibatidine-induced contractions in Decentralized and ObNT-Reinn suggests a relocation of nicotinic receptors from nerve terminals to more distant axonal sites, perhaps as a compensatory mechanism to recover bladder function.