Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

A Possible Role of Tetrodotoxin-Sensitive Na+ Channels for Oxidation-Induced Late Na+ Currents in Cardiomyocytes.

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.

Intense training prevents the amnestic effect of inactivation of dorsomedial striatum and induces high resistance to extinction.

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).

Molecular and cellular context influences SCN8A variant function.

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.

Distribution of pufferfish saxitoxin- and tetrodotoxin-binding protein homolog and tetrodotoxin in the brain and pituitary of juvenile tiger puffer Takifugu rubripes.

Pufferfish saxitoxin- and tetrodotoxin (TTX)-binding protein (PSTBP) is considered to transfer TTX between tissues. The immunohistochemical distribution of PSTBP-homolog (PSTBPh) and TTX in the brain and pituitary of hatchery-reared juvenile tiger puffer Takifugu rubripes was investigated. PSTBPh was observed mainly in the pars intermedia of the pituitary. TTX was only detected in a TTX-fed fish in the neurohypophysis of the pituitary and in several other brain regions. The relationship between PSTBPh and TTX is discussed.

Ingesting yeast extract causes excitation of neurogenic and myogenic colonic motor patterns in the rat.

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.

A smartphone-based fluorescent biosensor with metal-organic framework biocomposites and cotton swabs for the rapid determination of tetrodotoxin in seafood.

BACKGROUND: Tetrodotoxin (TTX) is a potent neurovirulent marine biotoxin that is present in puffer fish and certain marine animals. It is capable of causing severe neurotoxic symptoms and even death when consumed through contaminated seafood. Due to its high toxicity, developing an effective assay for TTX determination in seafood has significant benefits for food safety and human health. Currently, it remains challenging to achieve on-site determination of TTX in seafood. To facilitate mass on-site assays, more affordable technologies utilizing accessible equipment that require no skilled personnel are needed. RESULTS: A smartphone-based portable fluorescent biosensor is proposed for TTX determination by using metal-organic framework (MOF) biocomposites and cotton swabs. Oriented antibody (Ab)-decorated and fluorescent quantum dot (QD)-loaded MOF biocomposites (QD@MOF*Ab) are rapidly synthesized for binding targets and fluorescent responses by utilizing the tunability of zinc-based MOF. Moreover, facile Ab-immobilized household cotton swabs are utilized as TTX capture tools. TTX forms sandwich immune complexes with QD@MOF*Ab probes, achieving signal amplification. These probes are excited by a portable device to generate bright fluorescent signals, which can be detected by the naked eye, and TTX quantitative results are obtained using a smartphone. When observed with the naked eye, the limit of detection (LOD) is 0.4 ng/mL, while intelligent quantitation presents an LOD of 0.13 ng/mL at logarithmic concentrations of 0.2-400 ng/mL. SIGNIFICANCE: This biosensor is convenient to use, and an easy-to-operate analysis is completed within 15 min, thus demonstrating excellent performance in terms of detection speed and portability. Furthermore, it successfully determines TTX contents in puffer fish and clam samples, demonstrating its potential for monitoring seafood. Herein, this work provides a favorable rapid sensing platform that is easily portable.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography-mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

Temporal variation in the concentrations and profiles of paralytic shellfish toxins and tetrodotoxin in scallop (Mizuhopecten yessoensis) and bloody clam (Anadara broughtonii) collected from the coast of Miyagi Prefecture, Japan.

For food safety, the concentrations and profiles of paralytic shellfish toxins (PSTs) and tetrodotoxin were examined in economically important scallops and bloody clams collected from the coast of the Miyagi Prefecture, Japan. PSTs were the major toxins in both species. The tetrodotoxin concentration in scallops increased in summer, although the highest value (18.7 µg/kg) was lower than the European Food Safety Authority guideline threshold (44 µg/kg). This confirmed the safety for tetrodotoxin in this area.

Sudden peak in tetrodotoxin in French oysters during the summer of 2021: Source investigation using microscopy, metabarcoding and droplet digital PCR.

Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June-15 July 2021), with concentrations decreasing in the DG from 424 µg/kg for the first detection to 101 µg/kg (equivalent to 74 to 17 µg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix.

Tetrodotoxins in Tissues and Cells of Different Body Regions of Ribbon Worms Kulikovia alborostrata and K. manchenkoi from Spokoynaya Bay, Sea of Japan.

Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs functions and accumulation. In the present study, Kulikovia alborostrata and K. manchenkoi, two closely related species, were analyzed for TTXs distribution in the body using the HPLC-MS/MS and fluorescence microscopy methods. The abundance of TTXs-positive cells was determined in the proboscis, integument, and digestive system epithelium. As a result, six TTXs-positive cell types were identified in each species; however, only four were common. Moreover, the proportions of the toxins in different body parts were estimated. According to the HPLC-MS/MS analysis, the TTXs concentrations in K. alborostrata varied from 0.91 ng/g in the proboscis to 5.52 ng/g in the precerebral region; in K. manchenkoi, the concentrations ranged from 7.47 ng/g in the proboscis to 72.32 ng/g in the posterior body region. The differences observed between the two nemerteans in the distribution of the TTXs were consistent with the differences in the localization of TTXs-positive cells. In addition, TTXs-positive glandular cell types were found in the intestine and characterized for the first time. TTXs in the new cell types were assumed to play a unique physiological role for nemerteans.

Automated Patch Clamp for the Detection of Tetrodotoxin in Pufferfish Samples.

Tetrodotoxin (TTX) is a marine toxin responsible for many intoxications around the world. Its presence in some pufferfish species and, as recently reported, in shellfish, poses a serious health concern. Although TTX is not routinely monitored, there is a need for fast, sensitive, reliable, and simple methods for its detection and quantification. In this work, we describe the use of an automated patch clamp (APC) system with Neuro-2a cells for the determination of TTX contents in pufferfish samples. The cells showed an IC50 of 6.4 nM for TTX and were not affected by the presence of muscle, skin, liver, and gonad tissues of a Sphoeroides pachygaster specimen (TTX-free) when analysed at 10 mg/mL. The LOD achieved with this technique was 0.05 mg TTX equiv./kg, which is far below the Japanese regulatory limit of 2 mg TTX equiv./kg. The APC system was applied to the analysis of extracts of a Lagocephalus sceleratus specimen, showing TTX contents that followed the trend of gonads > liver > skin > muscle. The APC system, providing an in vitro toxicological approach, offers the advantages of being sensitive, rapid, and reliable for the detection of TTX-like compounds in seafood.

[Determination of tetrodotoxin in urine by liquid chromatography-tandem mass spectrometry with internal standard calibration].

OBJECTIVE: An analytical method was developed for tetrodotoxin(TTX) in urine by liquid chromatography-tandem mass spectrometry(LC-MS/MS) with internal standard calibration. METHODS: TTX in the sample was extracted with the mixture of acetic acid/methanol/acetonitrile(0.005 mL/0.8 mL/1.8 mL), cleaned by solid phase extraction(SPE) with cation exchange cartridge, eluted with 50% acetonitrile/water containing 0.3% hydrochloric acid, and neutralized with ammonia. The extract was separated by a Waters XBridge~(TM) BEH Amide column(150 mm×3.0mm, 1.7 µm) and measured by MS/MS. By optimizing sample extraction and SPE cleanup conditions, the problems of low recovery and strong suppression effects of MS signal for TTX in urine were resolved when cleaned with cation exchange cartridge. RESULTS: Quantitatively calibrated by the internal standard of Kasugamycin, good linear relationship was found for TTX in urine at the range of 0.2-200 µg/L with the correlation coefficient(r~2) of 0.997. The limits of detection and quantitation for TTX in sample matrix were 0.1 and 0.2µg/L, respectively. The average recoveries at three spiking levels(0.2, 10.0 and 200 µg/L) were 89.3%-95.3% with relative standard deviation(n=6) less than 5.1%. The concentrations of TTX in urine from 11 poisoning patients were 0.4-138 µg/L. The detection rate was 100% in urine collected within 3 days after poisoning. CONCLUSION: The established method was simple, accurate and sensitive. It can provide reliable technical support for the rapid treatment of TTX poisoning events and the study of toxin metabolism in vivo.

A voltage-based Event-Timing-Dependent Plasticity rule accounts for LTP subthreshold and suprathreshold for dendritic spikes in CA1 pyramidal neurons.

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.

Sodium Channel ß Subunits-An Additional Element in Animal Tetrodotoxin Resistance?

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.

Total syntheses of Tetrodotoxin and 9-epiTetrodotoxin.

Tetrodotoxin and congeners are specific voltage-gated sodium channel blockers that exhibit remarkable anesthetic and analgesic effects. Here, we present a scalable asymmetric syntheses of Tetrodotoxin and 9-epiTetrodotoxin from the abundant chemical feedstock furfuryl alcohol. The optically pure cyclohexane skeleton is assembled via a stereoselective Diels-Alder reaction. The dense heteroatom substituents are established sequentially by a series of functional group interconversions on highly oxygenated cyclohexane frameworks, including a chemoselective cyclic anhydride opening, and a decarboxylative hydroxylation. An innovative SmI2-mediated concurrent fragmentation, an oxo-bridge ring opening and ester reduction followed by an Upjohn dihydroxylation deliver the highly oxidized skeleton. Ruthenium-catalyzed oxidative alkyne cleavage and formation of the hemiaminal and orthoester under acidic conditions enable the rapid assembly of Tetrodotoxin, anhydro-Tetrodotoxin, 9-epiTetrodotoxin, and 9-epi lactone-Tetrodotoxin.

Tetrodotoxin and Its Analogues (TTXs) in the Food-Capture and Defense Organs of the Palaeonemertean Cephalothrix cf. simula.

Tetrodotoxin (TTX), an extremely potent low-molecular-weight neurotoxin, is widespread among marine animals including ribbon worms (Nemertea). Previously, studies on the highly toxic palaeonemertean Cephalothrix cf. simula showed that toxin-positive structures are present all over its body and are mainly associated with glandular cells and epithelial tissues. The highest TTXs concentrations were detected in a total extract from the intestine of the anterior part of the body and also in a total extract from the proboscis. However, many questions as to the TTXs distribution in the organs of the anterior part of the worm's body and the functions of the toxins in these organs are still unanswered. In the present report, we provide additional results of a detailed and comprehensive analysis of TTXs distribution in the nemertean's proboscis, buccal cavity, and cephalic gland using an integrated approach including high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), confocal laser scanning microscopy with anti-TTX antibodies, light and electron microscopies, and observations of feeding behavior. For the proboscis, we have found a TTXs profile different from that characteristic of other organs and tissues. We have also shown for the first time that the major amount of TTXs is localized in the anterior part of the proboscis that is mainly involved in hunting. TTX-containing glandular cells, which can be involved in the prey immobilization, have been found in the buccal cavities of the nemerteans. A significant contribution of the cephalic gland to the toxicity of this animal has been shown for the first time, and the role of the gland is hypothesized to be involved not only in protection against potential enemies but also in immobilizing prey. The data obtained have made it possible to extend the understanding of the role and features of the use of TTXs in the organs of the anterior part of nemertean's body.

5, 6, 11-trideoxy tetrodotoxin attracts tiger puffer Takifugu rubripes.

Tetrodotoxin (TTX) is a potent neurotoxin that binds to voltage-gated sodium channels and blocks the passage of sodium ions. TTX is widely distributed in both terrestrial and marine organisms, and the toxic puffers are believed to accumulate TTX through the food chain. Although pufferfish was previously thought to be attracted by TTX, recent finding from electroolfactogram (EOG) studies have indicated that the olfactory epithelium of T. alboplumbeus responded to 5, 6, 11-trideoxyTTX (TDT), but not to TTX itself. In this study, we examined behavioral experiments for Takifugu rubripes to distinguish between TTX and TDT under static and flow-through conditions. Our data clearly suggested that T. rubripes juveniles were attracted to TDT, not TTX. Moreover, we determined that the minimum effective dose of TDT to attract the puffer was 1-2 nmol of TDT under static conditions and 50-60 nmol of TDT under flow-through conditions. Following the experiments under static conditions, numerous bite marks by the pufferfish were found solely on the agarose gel infused with TDT. Based on these finding, we hypothesize that the pufferfish are attracted to TDT derived from prey, leading them effectively become toxic.

Adiponectin affects ileal contractility of mouse preparations.

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.

Sodium currents in naïve mouse dorsal root ganglion neurons: No major differences between sexes.

Sexual dimorphism has been reported in multiple pre-clinical and clinical studies on pain. Previous investigations have suggested that in at least some states, rodent dorsal root ganglion (DRG) neurons display differential sex-dependent regulation and expression patterns of various proteins involved in the pain pathway. Our goal in this study was to determine whether sexual dimorphism in the biophysical properties of voltage-gated sodium (Nav) currents contributes to these observations in rodents. We recently developed a novel method that enables high-throughput, unbiased, and automated functional analysis of native rodent sensory neurons from naïve WT mice profiled simultaneously under uniform experimental conditions. In our previous study, we performed all experiments in neurons that were obtained from mixed populations of adult males or females, which were combined into single (combined male/female) data sets. Here, we have re-analyzed the same previously published data and segregated the cells based on sex. Although the number of cells in our previously published data sets were uneven for some comparisons, our results do not show sex-dependent differences in the biophysical properties of Nav currents in these native DRG neurons.