μM For Dopamine Research Articles

Consequences of Acute or Chronic Methylphenidate Exposure Using Ex Vivo Neurochemistry and In Vivo Electrophysiology in the Prefrontal Cortex and Striatum of Rats.

Methylphenidate (MPH) is among the main drugs prescribed to treat patients with attention-deficit and hyperactivity disease (ADHD). MPH blocks both the norepinephrine and dopamine reuptake transporters (NET and DAT, respectively). Our study was aimed at further understanding the mechanisms by which MPH could modulate neurotransmitter efflux, using ex vivo radiolabelled neurotransmitter assays isolated from rats. Here, we observed significant dopamine and norepinephrine efflux from the prefrontal cortex (PFC) after MPH (100 µM) exposure. Efflux was mediated by both dopamine and norepinephrine terminals. In the striatum, MPH (100 µM) triggered dopamine efflux through both sodium- and vesicular-dependent mechanisms. Chronic MPH exposure (4 mg/kg/day/animal, voluntary oral intake) for 15 days, followed by a 28-day washout period, increased the firing rate of PFC pyramidal neurons, assessed by in vivo extracellular single-cell electrophysiological recordings, without altering the responses to locally applied NMDA, via micro-iontophoresis. Furthermore, chronic MPH treatment resulted in decreased efficiency of extracellular dopamine to modulate NMDA-induced firing activities of medium spiny neurons in the striatum, together with lower MPH-induced (100 µM) dopamine outflow, suggesting desensitization to both dopamine and MPH in striatal regions. These results indicate that MPH can modulate neurotransmitter efflux in brain regions enriched with dopamine and/or norepinephrine terminals. Further, long-lasting alterations of striatal and prefrontal neurotransmission were observed, even after extensive washout periods. Further studies will be needed to understand the clinical implications of these findings.

The Electrochemical Detection of Dopamine Based on the Cobalt-Modified Nitrogen Doping Carbon Aerogels from Biomass

The sensitive detection of dopamine (DA), especially the accurate monitoring of cell secreted DA, is necessary to the study on pathogenesis of central nervous system disease. In this paper, the cobalt-modified nitrogen doping carbon aerogels (Co-NCA) with three dimensional pores were successfully synthesized through the hydrothermal and freeze-drying method of the biomass. As a natural substance, starch is the carbon source of Co-NCA, which is environmentally friendly. The electrochemically active area of electrode modified by Co-NCA was about 1.53 times that of carbon aerogels. Benefiting from the Co and N element, the Co-NCA modified electrode (Co-NCA/GCE) showed that Co-NCA had a wider linear range (0.2–200 μM and 200–1000 μM) and a lower detection limit (55.6 nM) towards DA. The reproducibility and repeatability of Co-NCA/GCE for detecting 500 μM DA was 2.21% and 0.797%, respectively. After stored at 4 °C for 7 days, the current response of Co-NCA/GCE still maintained 96.5% of the initial current value. Finally, the Co-NCA/GCE was successfully applied to the monitoring of DA, which was released from PC12 cells after stimulated by 50 mM K+.

A WSe2@Poly(3,4-ethylenedioxythiophene) nanocomposite based-electrochemical sensor for simultaneous detection of dopamine and uric acid

In the present work, a nanocomposite of two-dimensional WSe2 nanosheets with poly(3,4‑ethylenedioxythiophene (WSe2@PEDOT) was prepared by facile hydrothermal method and characterized in terms of structural and morphological analyses. This nano­composite was used to modify glassy carbon electrode for the construction of an electrochemical sensing platform for simultaneous determination of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA). It was found that the incorporation of PEDOT into WSe2 nano­sheets exhibited enhanced electrochemical behaviors and electro¬catalytic activity against DA and UA. Using differential pulse voltammetry (DPV) measurements, the WSe2@PEDOT modified electrode displayed wide linear detection ranges of 16 to 466 µM for DA and 20 to 582.5 µM for UA. The electrode also exhibited high selectivity against DA and UA in the presence of major interference of ascorbic acid and other interferent substances.

(Digital Presentation) In Vivo Electrochemical Measurement of Dopamine in Adult Drosophila Mushroom Body

Dopamine is a neuromodulator that is secreted to the synapse to relay chemical signals to target neurons. Abnormal levels of dopamine release leads to various neurodegenerative diseases. Therefore, measuring dopamine is essential to understand how dopamine is regulated under normal and pathological conditions. Drosophila melanogaster, the fruit fly, is an ideal model system for studying fundamental neurological processes and diseases because of the availability of sophisticated genetic tools and well conserved neurological processes between mammals and flies. Majority of neuroscience studies involved in modifying a gene and measure the effect of genetic mutation on output behaviors. However, dopamine release is highly dynamic because of the complex activity of dopamine transporters and autoreceptors. Therefore, to understand how dopamine signaling controls the behavior, a direct measurement of changes in dopamine release is necessary. Fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode is an electrochemical technique that trace concentration changes in dopamine release on the sub-second time scale. Our lab pioneered directly measuring various endogenous neuromodulators in the fly central nervous system with FSCV. Initially, these studies were performed in ex vivo preparations, where brains were isolated from larvae and adult fly, and thus could not monitor neuromodulators during behavior. In this study, we developed in vivo FSCV method to measure phasic dopamine in the mushroom body (MB) during behavior for the first time. The MB in fly has been extensively studied as an associative center for regulating olfactory learning and memory. First, acetylcholine stimulation was applied to the MB heel and medial tip to characterize dopamine signaling and to demonstrate the feasibility of in vivo FSCV in intact fly brain. Application of 0.2 pmol acetylcholine released 0.36 ± 0.06 µM dopamine in the medial tip, which is slightly higher than 0.22 ± 0.06 µM dopamine in the heel. Compartmental differences in evoked release suggest heterogeneity of dopamine regulation in the MB. Nisoxetine, a dopamine transporter inhibitor, and flupentixol, a D2 antagonist, increased stimulated dopamine release. We then applied the in vivo method to monitor changes in behaviorally evoked dopamine release during sugar feeding. Sugar feeding evoked 0.31 ± 0.09 µM dopamine in the medial tip of MB. Flupentixol significantly increased sugar evoked release implying D2 receptor acts as autoreceptor and regulates dopamine signaling during sugar feeding. Therefore, this developed in vivo FSCV method is a great addition to the existing tools to measure endogenous neuromodulators in the fly and valuable for studying real-time dopamine signaling during behavior. This in vivo method also can be further extended to better understand how dopamine and other neuromodulators regulate complex behaviors, such as reward associated learning and memory formation.

Abstract 1856: Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206

Abstract ONC201 (originally discovered as TRAIL-Inducing Compound #10 or TIC10) and analogue ONC206 have been found to induce an integrated stress response with suggested primary targets and mechanisms involving agonist activity against mitochondrial ClpP and antagonism of dopamine receptors D2/3 (DRD2/3). We hypothesized that dopamine, the agonist of DRD2, may compete with ONC201 or ONC206 for DRD2/3 and impair the anti-cancer effect of ONC201 or ONC206. We therefore pre-treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, lung cancer, GBM, and DIPG with dopamine for 12 hours, 24 hours or 48 hours, respectively, followed by treatment of ONC201 or ONC206 for 72hours. Cell viability was determined by the CellTiter Glo assay. Single treatment with dopamine did not increase cell viability. We observed that 48 hours of pre-treatment with dopamine at 50 μM, 100 μM or 200 μM impaired the cell viability suppression effect of ONC201 at 4 μM and 8 μM, while ONC206 at 0.5 μM, 1 μM and 2 μM led to similar protection for loss of cell viability in pancreatic cancer cells. The impairment of the anti-cancer effect by dopamine was also observed in colorectal cancer cells with 48 hours pre-treatment with dopamine at 50 μM, 100 μM, 200 μM or 400 μM and treatment with ONC201 at 4 μM and 8 μM, or ONC206 at 0.5 μM, 1 μM, or 2 μM. In addition, we treated pancreatic cancer and colorectal cancer cells with ONC212 after 48 hours pre-treatment with dopamine and observed a similar impairment of the anti-cancer effect, even though ONC212 is not a DRD2 antagonist. We pre-treated pancreatic cancer and colorectal cancer cells with specific DRD2 agonist, sumanirole, for 48 hours followed by treatment of ONC201, ONC206, or ONC212 for 72 hours, and no impairment was seen. We pre-treated multiple cancer cell lines with 2 μM or 10 μM dopamine for one week followed by ONC201, ONC206, or ONC212 treatment and performed colony assays. Pre-treatment with dopamine at 10 μM impaired the anti-cancer effect of ONC201 or ONC206 in pancreatic cancer. Pre-treatment with dopamine at 2 μM or 10 μM impaired the anti-tumor effect of ONC201 in GBM cells. By contrast to the cell viability result, no impairment of ONC212 effect from dopamine was seen in pancreatic cancer cells. Western blotting was conducted to investigate whether dopamine pre-treatment impacted signaling pathways reported to be affected by ONC201. Dopamine pre-treatment did not impact changes induced by ONC201 in ATF4, CHOP, DR5, ClpX, clpP, pERK, pAkt and pS6. We are pursuing whether the protective effect of dopamine is due to the agonism of DRD2 or alternative pro-survival mechanisms. Citation Format: Yiqun Zhang, Wafik S. El-Deiry. Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1856.

In Situ Synthesis of a Bi2Te3-Nanosheet/Reduced-Graphene-Oxide Nanocomposite for Non-Enzymatic Electrochemical Dopamine Sensing.

Dopamine is a neurotransmitter that helps cells to transmit pulsed chemicals. Therefore, dopamine detection is crucial from the viewpoint of human health. Dopamine determination is typically achieved via chromatography, fluorescence, electrochemiluminescence, colorimetry, and enzyme-linked methods. However, most of these methods employ specific biological enzymes or involve complex detection processes. Therefore, non-enzymatic electrochemical sensors are attracting attention owing to their high sensitivity, speed, and simplicity. In this study, a simple one-step fabrication of a Bi2Te3-nanosheet/reduced-graphene-oxide (BT/rGO) nanocomposite was achieved using a hydrothermal method to modify electrodes for electrochemical dopamine detection. The combination of the BT nanosheets with the rGO surface was investigated by X-ray diffraction, X-ray photoelectron spectroscopy, field-emission scanning electron microscopy, transmission electron microscopy, Raman spectroscopy, and Fourier-transform infrared spectroscopy. Electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry were performed to analyze the electrochemical-dopamine-detection characteristics of the BT/rGO nanocomposite. The BT/rGO-modified electrode exhibited higher catalytic activity for electrocatalytic oxidation of 100 µM dopamine (94.91 µA, 0.24 V) than that of the BT-modified (4.55 µA, 0.26 V), rGO-modified (13.24 µA, 0.23 V), and bare glassy carbon electrode (2.86 µA, 0.35 V); this was attributed to the synergistic effect of the electron transfer promoted by the highly conductive rGO and the large specific surface area/high charge-carrier mobility of the two-dimensional BT nanosheets. The BT/rGO-modified electrode showed a detection limit of 0.06 µM for dopamine in a linear range of 10–1000 µM. Additionally, it exhibited satisfactory reproducibility, stability, selectivity, and acceptable recovery in real samples.

Facial Construction of CePO4-Reduced Graphene Oxide Composite for Enhanced Electrochemical Detection of Dopamine and Acetaminophen

A serial of cerium phosphate/reduced graphene oxide (CePO4-rGO) composites were synthesized for the first time by one-pot hydrothermal method. Structural analyses revealed that flaky CePO4 and few-layer rGO were composited homogeneously. It is found that rGO can greatly enhance the conductivity and the adsorption capacity of the composite. With these advantages, the constructed composites were used for electrochemical detection of dopamine (DA) and acetaminophen (APAP). Further studies show that the loading amount of rGO was also very important. When CePO4−0.6rGO was used, wide linear ranges of 1.2−50 μM for DA and 0.5−30 μM for APAP can be obtained, respectively. The detection limits of DA and APAP were 0.013 μM and 0.025 μM with high sensitivities of 25.58 μA μM−1 cm−2 and 13.30 μA μM−1 cm−2, respectively. Moreover, the modified electrode also has high selectivity, good stability and satisfactory reproducibility. In addition, the modified electrode was also successfully applied to detect DA and APAP in real urine samples. This work provides a simple method for preparing new electrode materials for detecting small biomolecules.

Simultaneous voltammetric determination of dopamine and uric acid based on MOF-235 nanocomposite

A highly selective and facile sensing platform based on MOF-235 is fabricated and explored for the synchronous detection of dopamine and uric acid. The MOF-235 material is prepared via a one-step hydrothermal method. The sensing platform is fabricated via drop casting of MOF-235 dispersion on bare glassy carbon electrode (GCE). Then it is sealed using chitosan solution. The as-prepared MOF-235 material has large amount of unsaturated iron metal ions available for electrochemically redox-driven transformation. The aromatic moieties and the uncoordinated carboxylate groups of the MOF-235 are capable of obtain well-separated signals for dopamine (DA) and uric acid (UA), which is attributed to the π-π interactions, hydrogen bond and electrostatic action. These characteristics endow the excellent permselectivity and sensitivity for the MOF-235 modified electrode. The designed sensing platform exhibits linear response over concentration range from 10 to 90 μM for DA, and 10 to 90 μM for UA with the detect limit of 3.34 μM for DA and 3.46 μM for UA separately. To our knowledge, this is the first attempt that MOF-235 is explored the applications in electrochemical determination of medical small molecules. The implementing of MOF-235 in voltammetric detection offers new opportunities for the development of MOF materials based electroanalytical devices.

RGO/ReO3 nano composite modified electrode for the ultra-sensitive determination of dopamine and uric acid

Development of suitable modified electrode in generating the electrochemical signals from dopamine (DA), uric acid (UA) and ascorbic acid (AA) has remained challenging issue even after decades of research in the related field. Under the present investigation the perovskite type metal oxide nano particle has been used in the form of composite with the reduced graphene oxide (rGO) as the active material for sensing. The superior electrical conductivity of Rhenium oxide (ReO3) combined with the adsorptive and conducting nature of rGO has been utilised in enhancing the electrochemical responses form the molecules. Thin films of Nafion was used over the rGO/ReO3 composite materials for stabilization of the modification. The electron transfer property of the molecules has been evaluated from the cyclic voltammetry and electrochemical impedance measurements. The standard electron transfer rate constants have been obtained from the Laviron formulations of the cyclic voltammetry measurements. Simultaneous electrochemical response has been obtained from the three molecules over the rGO/ReO3 nano composite modified electrode, the analytical method has been developed with the detection limit (S/N = 3) of 0.08 nM, 0.06 μM and 0.89 μM for DA, UA and AA respectively. For simultaneous determination however, the determination of DA and UA has been proposed and the interference test was carried out through simultaneous detection of the signals of the molecules. The analytical method has been applied for the detection of analytical signals of DA and UA in synthetic serum samples through spiked recovery test.

In-situ growth of 3D Cu-MOF on 1D halloysite nanotubes/reduced graphene oxide nanocomposite for simultaneous sensing of dopamine and paracetamol

Three-dimensional (3D) metal–organic frameworks (MOFs) a class of porous materials with tunable structure and surface functionality has arisen as electrode materials especially, for electrochemical sensing of analytes. However, MOFs possess intrinsic drawbacks such as poor conductivity with an agglomeration of particles, which restricted the electrochemical signal response in terms of sensitivity and detection limits. In this regard, the present work aims to develop conducting Cu-MOF on HNTs, a good substate for in-situ growth of MOF nanostructures due to the existence of abundant negatively charged Si-OH that can help the growth of nanosized MOFs. The negatively charged siloxane (Si-O-Si) groups on the surface of HNTs can be attracted by positive charged Cu2+ ions present in the reaction mixture through strong electrostatic attraction. When subjected to hydrothermal treatment, the Cu2+ ions can form nano-sized Cu-MOF particles with assistance from 2-methylimidazole. Moreover, the presence of graphene oxide (GO) can improve the electrical conductivity, large surface area, and thus resulting in the formation of conducting Cu-MOF/HNTs/rGO nanocomposite. Owing to the synergetic desirable properties of active metal sites and high porosity offered by Cu-MOF, the high conductivity of rGO, and the large surface area of HNTs, the resultant Cu-MOF/HNTs/rGO modified GC electrode demonstrates superior electrochemical signal response towards dopamine and paracetamol. Moreover, the developed sensor exhibits wide linear ranges of 0.1 µM–130 µM and 0.5–250 µM, with a low detection limit of 0.03 µM and 0.15 µM for dopamine and paracetamol, respectively.

Novel covalent immobilization of cobalt (II) octa acyl chloride phthalocyanines onto phenylethylamine pre-grafted gold via spontaneous amidation

In this work, a gold electrode surface was pre-grafted with phenylethylamine (PEA) thin film for spontaneous covalent immobilization of cobalt (II) octa acyl chloride (CoOAClPc) via the nucleophilic addition reaction. Pre-grafting of PEA thin film was achieved by electrochemical reduction of 4-(2-aminoethyl) benzene diazonium (AEBD) salt. Cobalt (II) octa acyl chloride phthalocyanine (CoOAClPc) was successfully synthesized and covalently immobilized onto the pre-grafted gold electrode surface to form a thin monolayer of Au-PEA-CoOAClPc. Upon hydrolysis of the thin monolayer, the –COCl terminal functional groups were converted to -COOH to form Au-PEA-CoOCAPc. The presence of PEA and PEA-CoOCAPc was confirmed by electrochemical X-ray photoelectron spectroscopy surface characterization. The pH sensitivity of -COOH terminal groups was studied using a negatively charged [Fe(CN)6]3−/4− and a positively charged [Ru(NH3)6]2+/3+ redox probes. The Au-PEA-CoOCAPc was investigated for electrocatalytic and electroanalytical properties towards the detection of catecholamine neurotransmitters (dopamine, epinephrine, and norepinephrine). The electrocatalytic oxidation of the catecholamine neurotransmitters in PBS solution (pH 7.4) was studied using differential pulse voltammetry (DPV) in a linear range up to 50 μM. The limit of detection (LOD) was determined to be 64 nM, 0.22 μM and 0.17 μM for dopamine (DA), epinephrine (EP) and norepinephrine (NOR) respectively. The limit of quantification (LOQ) was determined to be 0.21 μM, 0.73 μM and 0.56 μM for DA, EP and NOR respectively. Stable and reproducible novel method of electrode fabrication to form Au-PEA-CoOCAPc was achieved. Excellent analytical properties (sensitivity, low detection limits and limit of quantification) were obtained by using the fabricated electrochemical sensors, Au-PEA-CoOCAPc, for the determination of catecholamine neurotransmitters and screening of ascorbic acid (AA).

Small intestine neuromuscular dysfunction in a mouse model of dextran sulfate sodium-induced ileitis: Involvement of dopaminergic neurotransmission

AimsAnomalies in dopaminergic machinery have been shown in inflammatory bowel disease (IBD) patients and preclinical models of IBD. Thus, we aimed to evaluate the impact of dextran sodium sulfate (DSS)-induced ileitis on enteric dopaminergic pathways. Materials and methodsMale C57/Bl6 mice (10 ± 2 weeks old) received 2% DSS in drinking water for 5 days and were then switched to regular drinking water for 3 days. To measure ileitis severity inflammatory cytokines (IL-1β, TNFα, IL-6) levels were assessed. Changes in ileal muscle tension were isometrically recorded following: 1) cumulative addition of dopamine on basal tone (0.1–1000 μM); ii) 4-Hz electric field stimulation (EFS) in the presence of 30 μM dopamine with/without 10 μM SCH-23390 (dopamine D1 receptor (D1R) antagonist) or 10 μM sulpiride (D2R antagonist). Immunofluorescence distribution of the neuronal HuC/D protein, glial S100β marker, D1R, and dopamine transporter (DAT) were determined in longitudinal-muscle-myenteric plexus whole-mounts (LMMPs) by confocal microscopy. D1R and D2R mRNA transcripts were evaluated by qRT-PCR. Key findingsDSS caused an inflammatory process in the small intestine associated to dysmotility and altered barrier permeability, as suggested by decreased fecal output and enhanced stool water content. DSS treatment caused a significant increase of DAT and D1R myenteric immunoreactivity as well as of D1R and D2R mRNA levels, accompanied by a significant reduction of dopamine-mediated relaxation, involving primarily D1-like receptors. SignificanceMouse ileitis affects enteric dopaminergic neurotransmission mainly involving D1R-mediated responses. These findings provide novel information on the participation of dopaminergic pathways in IBD-mediated neuromuscular dysfunction.

Synchronous electrochemical detection of dopamine and uric acid by a PMo12@MIL-100(Fe)@PVP nanocomposite

In this work, a noble-metal-free composite electrode was prepared based on PMo12O403− (PMo12), C9H5FeO7 (MIL-100(Fe), a Fe-based metal organic framework) and polyvinylpyrrolidone (PVP), and served as a high performance electrochemical sensor for synchronous detection of dopamine (DA) and uric acid (UA). The PMo12@MIL-100(Fe)@PVP composite electrode was fabricated by a in-situ hydrothermal method. Thanks to the synergistic effect of three active components (PMo12, MIL-100 and PVP), the electrode possesses large specific surface area and high electrical conductivity and therefore it shows high electrocatalytic oxidation performance of DA and UA with a spacing of 0.146 V between the two peak positions. These benefits of the electrode enable its electrochemical sensor to synchronously detect of DA and UA. Namely, the linear ranges can achieve 1–247 μM for DA and 5–406 μM for UA. Meanwhile, the detection limits are 0.586 μM for DA and 0.372 μM for UA. Moreover, the sensor can be applied to simultaneous determination of UA and DA in human serums with satisfactory recovery values.

Molecularly imprinted Ni-polyacrylamide-based electrochemical sensor for the simultaneous detection of dopamine and adenine

Molecularly imprinted polymer (MIP) membranes prepared in situ present several advantages: they maintain the original morphology, adhere strongly to the collector, and exhibit a controllable structure. In this study, a Ni-polyacrylamide (PAM)-MIP matrix was fabricated in situ on glassy carbon via the one-step electro-polymerization of AM monomers in the presence of Ni and template molecules. Ni2+ ions were introduced as oxidants to promote AM polymerization and bulking agents to fabricate a three-dimensional porous PAM-MIP matrix. The Ni-PAM-based MIP sensor exhibited a quantitative dual response toward dopamine (DA) and adenine (Ade) in the pH range of 5.0–9.0. The linear concentration range changed depending on the pH environment, and the concentrations of DA and Ade ranged from 0.6 to 200 μM and from 0.4 to 300 μM, respectively. The ranges of detection limits (S/N = 3) were 0.12–0.37 μM for DA and 0.15–0.36 μM for Ade. In addition, the dual-MIP sensor exhibited high reliability in the detection of DA and Ade in human serum owing to its excellent anti-interference ability and long-term stability. The technique developed in this study is expected to facilitate the construction of multi-target response electrochemical biosensors and the reliable determination of small molecules with high selectivity and stability.

Sensitive electrochemical sensor based on gold nanoparticles assembled ferrocene-functionalised graphene oxide modified glassy carbon electrode for simultaneous determination of dopamine and acetaminophen

In this article, a novel electron transfer mediator based on the ferrocene-functionalised graphene oxide (Fc-GO) was successfully synthesised by the amide linkage between amino (–NH2) in m-ferrocenylaniline (FcAni) and carboxylic (–COOH) in GO sheet through coupling reagents. This novel electroactive Fc-GO can effectively prevent the ferrocene mediator leaking from the electrode surface. Then, gold nanoparticles assembled ferrocene-functionalised graphene oxide (AuNPs@Fc-GO) was prepared and further confirmed by transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDS), Fourier transform-infrared spectroscopy (FTIR) and X-ray powder diffractometer (XRD). The AuNPs@Fc-GO was modified on the surface of glassy carbon electrode (GCE) to construct an electrochemical sensor for sensitively simultaneous determination of dopamine (DA) and acetaminophen (AC). The characteristics toward DA and AC were investigated by the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. Simultaneous determination of both analytes was accomplished under the optimum condition. The oxidation peak potential of DA and AC was 0.20 and 0.43 V, respectively. The linear current response was obtained in the concentration range of 2–200 μM with the detection limit (S/N = 3) of 0.32 μM for DA and 2–200 μM with the detection limit of 0.21 μM for AC, respectively. Finally, the AuNPs@Fc-GO modified GCE was applied for the determination of DA and AC in human urine samples with satisfactory results. Therefore, the AuNPs@Fc-GO was a novel promising candidate for fabricating an electrochemical sensor for the determination of DA and AC in real biological samples.

High-performance Pt/Ti3C2Tx MXene based graphene electrochemical transistor for selective detection of dopamine

Accurate measurement of dopamine (DA) is of great significance for human health monitoring and disease prevention. Herein, two-dimensional Ti3C2Tx MXene with high performance was prepared by layer liquid exfoliation method and used for the modification of gate electrode. The glassy carbon electrode (GCE) modified with Pt/Ti3C2Tx was used as the gate, and assembled with monolayer graphene channel to form a complete transistor device. The device combines the amplification effect of transistors, good electrical conductivity of Ti3C2Tx MXene and excellent catalytic properties of Pt nanoparticles, showing a low detection limit (50 nM) and a wide detection concentration range (50 nM-9 mM) of DA. Ti3C2Tx MXene not only provides a large number of attachment sites for the deposition of Pt nanoparticles, but also improves the electrical conductivity and hydrophilicity of the electrode material, thus enhancing the sensitivity of the device and facilitating the penetration of the solution to be measured. In addition, Ti3C2Tx MXene is negatively charged, it has a certain electrostatic adsorption effect on protonated DA and electrostatic repulsion effect on negatively charged interfering substances. As a result, the device exhibits good selectivity, which provides a great application prospect for the construction of DA sensing platform.

A sensitive Cu(salophen) modified screen-printed electrode for simultaneous determination of dopamine and uric acid

T
 This research applied a nanostructured electrochemical sensor with a screen-printed electrode (SPE) for examining the dopamine (DA) electrocatalytic oxidation when uric acid (UA) was present. Cu(salophen) nanostructured modified SPE Cu(salophen)/SPE was employed to investigate the electrochemical behavior of DA. At optimal pH (pH 7.0), oxidation of DA at the modified electrode takes place at a potential around 100 mV less positive than at the unmodified SPE. Chronoamperometry was used to determine the diffusion coefficient of DA (D = 1.96×10-5 cm2 s-1). Differential pulse voltammetry (DPV) showed linear response in the range between 0.2-450.0 μM for DA. The limit of detection (LOD) of DA was computed to be 0.05 μM. Moreover, Cu(salophen)/SPE was employed for determining DA in the presence of UA using DPV. The DPV results showed that at the modified electrode, two well-separated oxidation peaks of DA and UA could be obtained at potentials of 180 and 325 mV, respectively. This separation forms the basis for the co-detection of these two materials on the surface of Cu(salophen)/SPE. This sensor was then employed to determine DA and UA in real specimens.

P014 Impact of experimental ileitis and Toll-Like Receptor 4 signaling on enteric inhibitory neurotransmission

Abstract Background Alterations in Toll-like receptor 4 (TLR4) expression and changes in enteric neurotransmissions influence the onset and severity of inflammatory bowel disease (IBD). We aimed to assess the interaction between the enteric inhibitory pathways and TLR4 signaling in a mouse model of dinitrobenzene sulfonic acid (DNBS)-induced small intestine inflammation. Methods Male TLR4-/- mice (9±2 weeks old) were intrarectally treated with 2.5% DNBS. Pro-inflammatory cytokines (IL-6 and TNF-α) were measured in ileal samples. Changes in ileal muscle tension were recorded following exposure to: i) 10-Hz electric field stimulation in non-adrenergic-non-cholinergic (NANC) conditions (1 μM guanethidine + 1 μM atropine) with or without 10 µM 1400W (a inducible nitric oxide synthase (iNOS) inhibitor), or 0.1 µM Nω-nitro-L-arginine methyl ester (L-NAME; a pan-NOS inhibitor); ii) 30 μM dopamine or 30 μM SKF38393 (a dopamine 1 receptor (D1R) agonist) or bromocriptine (a D2R agonist). Immunoreactivity of neuronal (HuC/D) and glial (S100β) markers as well as nitrergic chemical coding (i.e. nNOS and iNOS) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy. Results No changes in IL-6 and TNF-α ileal mRNA transcripts were found after DNBS administration. The development of ileitis did not affect the NANC-inhibitory response, that was partially reduced in presence of 1400W or L-NAME, suggesting that TLR4 signaling influences NO-mediated inhibitory responses. DNBS-treated mice showed a significant increase in dopamine-induced relaxation (+31%) and in D1R- and D2R-mediated inhibitory responses (by +57% and +75%, respectively). A significant reduction of the total number of HuC/D+ neurons (-25%), with a proportional increase in nNOS+ neurons (+11%) was found in LMMPs of DNBS mice, associated with a significant reduction of S100β immunofluorescence (-12%) and no changes in iNOS immunoreactivity, highlighting a potential involvement of TLR4 signaling in the development of neuroplasticity and reactive gliosis. Conclusion The present data suggest that the severity of small intestine ileitis and neuronal dysfunction depend on the interplay occurring between TLR4 signaling and enteric inhibitory pathways. This evidence further strengthens the importance of characterizing neuroimmune interactions to identify potential molecular mechanism(s) representing putative pharmacological targets for the development of efficacious IBD therapies.

P044 Enteric dopaminergic pathways in mouse and human intestinal inflammation

Abstract Background Changes in dopamine beta-hydroxylase (DBH) and anomalies in dopaminergic machinery have been shown in inflammatory bowel disease (IBD) patients and related animal models. Thus, we aimed to evaluate the dopaminergic pathways in IBD patients as well as in a mouse model of dextran sodium sulphate (DSS)-induced ileitis. Methods Colon biopsies (CB) obtained from healthy volunteers (N=3) and matched-IBD patients (N=3), were used to evaluate DBH immunoreactivity by confocal microscopy. Male C57/Bl6 (8±2 weeks old; N=16 mice) received 1.5% DSS in drinking water for 5 days, then switched to regular drinking water for 3 days. Inflammatory cytokines (IL-1β, TNFα, IL-6) were measured to assess ileitis severity. Changes in ileal muscle tension were isometrically recorded following 30 μM dopamine or 30 μM SKF38393 (a dopamine receptor 1 (D1R) agonist) or 30 μM bromocriptine (a D2R agonist). Immunofluorescence distribution of Iba1 (a macrophage specific marker), D1R, DBH and dopamine transporter (DAT) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy. D1R and D2R mRNA transcripts were evaluated by qRT-PCR. Results CB from IBD patients and LMMPs from DSS mice showed a significant increase of DBH immunoreactivity compared to healthy patients and sham mice (+25% [p<0.01], +20% [p<0.01], respectively). DSS treatment caused a significant increase of DAT and D1R immunoreactivity as well as D1R mRNA levels (+27% [p<0.05], +24% [p<0.05], +6-fold [p<0.05], respectively), accompanied by a significant reduction of dopamine-mediated relaxation (-27% [p<0.01]). SKF38393 determined a marked inhibitory response in ileal preparations from DSS mice compared to sham mice (+73% [p<0.01]), suggesting that dopamine responses are mainly mediated through D1R. A 2-fold increase of resident Iba1+ macrophages was observed in the myenteric plexus of DSS mice associated with a 2.9- and 1.5-fold enhancement of IL-6 and IL-1β mRNA levels, respectively. Conclusion Human colitis and mouse ileitis affect dopamine machinery in the enteric nervous system. Experimentally induced ileitis impairs dopaminergic neurotransmission altering D1R-mediated responses. These findings provide novel information on the involvement of dopaminergic pathways in IBD.

Neuroprotective effects of catechins in an experimental Parkinson’s disease model and SK-N-AS cells: evaluation of cell viability, anti-inflammatory and anti-apoptotic effects

ABSTRACT Objectives The aim of the study was to establish an in vitro Parkinson’s disease (PD) model and to investigate the cell viability, anti-inflammatory, anti-apoptotic and neuroprotective effects of catechin and EGCG in SK-N-AS and in vitro PD model cells. Method SK-N-AS human neuroblastoma cells were used. To develop an in vitro PD model, SK-N-AS cells were exposed to 6-hydroxydopamine. Model control was performed after ELISA analysis of dopamine and α-synuclein levels in the culture medium. Catechin and EGCG were administered to SK-N-AS and in vitro PD model cells. Cell viability was measured using MTT assay and trypan blue staining. Anti-inflammatory and anti-apoptotic activities of catechin and EGCG were investigated by indirect immunocytochemistry using anti-TNF-α, anti-IL-1β and anti-caspase-3. Results After 24 hours of 6-hydroxydopamine administration at 50 μM, higher αlfa-synuclein and lower dopamine levels were found in PD model than SK-N-AS cells. Cell viability was similar between SK-N-AS and in vitro PD model cells. Treatment with both bioactive components increased cell viability of in vitro PD model cells. Caspase-3 immunoreactivity was significantly reduced in SK-N-AS and PD model cells after EGCG administration, while it was decreased only in PD model cells after catechin administration. IL-1β staining intensity weakened after catechin administration in PD model cells, after EGCG administration in SK-N-AS cells. TNF-α staining intensity was similar in both cells. Conclusion Catechin and EGCG increased cell viability in PD model neuron cells. Both components showed anti-apoptotic and anti-inflammatory effects. Catechin may be more effective in preventing damage to neurons PD.