The zebrafish is a powerful model organism to study the mechanisms governing transition metal ions within whole brain tissue. Zinc is one of the most abundant metal ions in the brain, playing a critical pathophysiological role in neurodegenerative diseases. The homeostasis of free, ionic zinc (Zn2+) is a key intersection point in many of these diseases, including Alzheimer's disease and Parkinson's disease. A Zn2+ imbalance can eventuate several disturbances that may lead to the development of neurodegenerative changes. Therefore, compact, reliable approaches that allow the optical detection of Zn2+ across the whole brain would contribute to our current understanding of the mechanisms that underlie neurological disease pathology. We developed an engineered fluorescence protein-based nanoprobe that can spatially and temporally resolve Zn2+ in living zebrafish brain tissue. The self-assembled engineered fluorescence protein on gold nanoparticles was shown to be confined to defined locations within the brain tissue, enabling site specific studies, compared to fluorescent protein-based molecular tools, which diffuse throughout the brain tissue. Two-photon excitation microscopy confirmed the physical and photometrical stability of these nanoprobes in living zebrafish (Danio rerio) brain tissue, while the addition of Zn2+ quenched the nanoprobe fluorescence. Combining orthogonal sensing methods with our engineered nanoprobes will enable the study of imbalances in homeostatic Zn2+ regulation. The proposed bionanoprobe system offers a versatile platform to couple metal ion specific linkers and contribute to the understanding of neurological diseases.

• Klíčová slova
• fluorescence, gold, nanoparticles, two-photon excitation imaging, zebrafish, zinc,
• MeSH
• dánio pruhované * metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• ionty metabolismus   MeSH
• kovové nanočástice * MeSH
• mozek metabolismus   MeSH
• zinek metabolismus   MeSH
• zlato metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluorescenční barviva MeSH
• ionty MeSH
• zinek MeSH
• zlato MeSH

In this work, we carried out neurochemical and behavioral analysis of zebrafish (Danio rerio) treated with rotenone, an agent used to chemically induce a syndrome resembling Parkinson's disease (PD). Dopamine release, measured with fast-scan cyclic voltammetry (FSCV) at carbon-fiber electrodes in acutely harvested whole brains, was about 30% of that found in controls. Uptake, represented by the first order rate constant (k) and the half-life (t1/2) determined by nonlinear regression modeling of the stimulated release plots, was also diminished. Behavioral analysis revealed that rotenone treatment increased the time required for zebrafish to reach a reward within a maze by more than 50% and caused fish to select the wrong pathway, suggesting that latent learning was impaired. Additionally, zebrafish treated with rotenone suffered from diminished locomotor activity, swimming shorter distances with lower mean velocity and acceleration. Thus, the neurochemical and behavioral approaches, as applied, were able to resolve rotenone-induced differences in key parameters. This approach may be effective for screening therapies in this and other models of neurodegeneration.

• MeSH
• dánio pruhované metabolismus   MeSH
• dopamin metabolismus   MeSH
• kognice MeSH
• modely nemocí na zvířatech MeSH
• Parkinsonova nemoc * MeSH
• rotenon * farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• dopamin MeSH
• rotenon * MeSH

Alzheimer's disease (AD) is a progressive, fatal, neurodegenerative disorder for which only treatments of limited efficacy are available. Despite early mentions of dementia in the ancient literature and the first patient diagnosed in 1906, the underlying causes of AD are not well understood. This study examined the possible role of dopamine, a neurotransmitter that is involved in cognitive and motor function, in AD. We treated adult zebrafish (Danio rerio) with okadaic acid (OKA) to model AD and assessed the resulting behavioral and neurochemical changes. We then employed a latent learning paradigm to assess cognitive and motor function followed by neurochemical analysis with fast-scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes to measure the electrically stimulated dopamine release. The behavioral assay showed that OKA treatment caused fish to have lower motivation to reach the goal chamber, resulting in impeded learning and decreased locomotor activity compared to controls. Our voltammetric measurements revealed that the peak dopamine overflow in OKA-treated fish was about one-third of that measured in controls. These findings highlight the profound neurochemical changes that may occur in AD. Furthermore, they demonstrate that applying the latent learning paradigm and FSCV to zebrafish is a promising tool for future neurochemical studies and may be useful for screening drugs for the treatment of AD.

• Klíčová slova
• Alzheimer’s disease, behavior, dopamine, fast-scan cyclic voltammetry, okadaic acid, zebrafish,
• MeSH
• Alzheimerova nemoc * MeSH
• dánio pruhované MeSH
• dopamin * MeSH
• karbonové vlákno MeSH
• kyselina okadaová MeSH
• mikroelektrody MeSH
• neurotransmiterové látky MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• dopamin * MeSH
• karbonové vlákno MeSH
• kyselina okadaová MeSH
• neurotransmiterové látky MeSH

Oxytocin is a nonapeptide hormone involved in numerous physiological functions. Real-time electrochemical measurements of oxytocin in living tissue are challenging due to electrode fouling and the large potentials needed to oxidize the tyrosine residue. Here, we used fast-scan cyclic voltammetry at carbon-fiber microelectrodes and flow injection analysis to optimize a waveform for the measurement of oxytocin. This optimized waveform employed an accumulation potential of -0.6 V, multiple scan rates, and a 3 ms holding potential at a positive, oxidizing potential of +1.4 V before linearly scanning the potential back to -0.6 V (versus Ag/AgCl). We obtained a limit of quantitation of 0.34 ± 0.02 μM, and our electrodes did not foul upon multiple injections. Moreover, to demonstrate the utility of our method, we measured the release of oxytocin, evoked by light application and mechanical perturbation, in whole brains from genetically engineered adult zebrafish that express channelrhodopsin-2 selectively on oxytocinergic neurons. Collectively, this work expands the toolkit for the measurement of peptides in living tissue preparations.

• MeSH
• dánio pruhované * MeSH
• karbonové vlákno MeSH
• mikroelektrody MeSH
• neurony MeSH
• oxytocin * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• karbonové vlákno MeSH
• oxytocin * MeSH

Caged compounds are molecules that release a protective substrate to free a biologically active substrate upon treatment with light of sufficient energy and duration. A notable limitation of this approach is difficulty in determining the degree of photoactivation in tissues or opaque solutions because light reaching the desired location is obstructed. Here, we have addressed this issue by developing an in situ electrochemical method in which the amount of caged molecule photorelease is determined by fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Using p-hydroxyphenyl glutamate (pHP-Glu) as our model system, we generated a linear calibration curve for oxidation of 4-hydroxyphenylacetic acid (4HPAA), the group from which the glutamate molecule leaves, up to a concentration of 1000 μM. Moreover, we are able to correct for the presence of residual pHP-Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of pHP-Glu in a 3 μL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed method by in situ measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the in situ electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal function.

• MeSH
• dánio pruhované * MeSH
• elektrochemické techniky * MeSH
• fotochemie MeSH
• karbonové vlákno MeSH
• mikroelektrody MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• karbonové vlákno MeSH

We have reported previously on the existence of charge-induced long-range organization in the room-temperature ionic liquid (RTIL), BMIM+BF4-. The induced organization is in the form of a free charge density gradient (ρf) that exists over ca. 100 μm into the RTIL in contact with a charged surface. The fluorescence anisotropy decay of a trace-level charged chromophore in the RTIL is measured as a function of distance from the indium-doped tin oxide support surface to probe this free charge density gradient. We report here on the characterization of the free charge density gradient in five different imidazolium RTILs and use these data to evaluate the magnitude of the induced free charge density gradient. Both the extent and magnitude of this gradient depend on the chemical structures of the cationic and anionic constituents of the RTIL used. Control over the magnitude of ρf has implications for the utility of RTILs for a host of applications that remain to be explored fully.

• Publikační typ
• časopisecké články MeSH

Polyacrylamide-coated, carbon nanotube (PA/CNT) electrodes were prepared by an inkjet printing process and used to measure pyocyanin and uric acid in a wound fluid simulant at 37 °C. These two molecules are potential indicators of infection, and therefore their detection could prove useful for monitoring wound healing. Pyocyanin is a marker for the common wound bacterium Pseudomonas aeruginosa. Our long-term goal is to use these inexpensive and disposable electrodes to measure biomarkers of wound healing directly. In this proof-of-concept work, studies were performed in a wound fluid simulant to evaluate the stability of the electrodes and their responsiveness for the two bioanalytes. The PA/CNT inkjet-printed electrodes and electrical contacts were stable with unchanging physical and electrochemical properties in the wound fluid simulant over a 7-8-day period at 37 °C. The detection figures of merit for pyocyanin in the simulant at 37 °C were as follows: linear over the physiologically relevant range = 0.10 to 100 μmol L-1 (R2 = 0.9992), limit of detection = 0.10 μmol L-1 (S/N = 3), sensitivity = 35.6 ± 0.8 mA-L mol-1 and response variability ≤4% RSD. The detection figures of merit for uric acid in the simulant at 37 °C were as follows: linear over the physiologically relevant range = 100 to 1000 μmol L-1 (R2 = 0.9997), sensitivity = 2.83 ± 0.01 mA-L mol-1, and response variability ≤4% RSD. The limit of detection was not determined. The PA/CNT electrodes were also used to quantify pyocyanin concentrations in cell-free culture media from different strains of P. aeruginosa. The detected concentrations ranged from 1.00 ± 0.02 to 118 ± 6 μM depending on the strain.

• MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• design vybavení MeSH
• elektrody MeSH
• kultivační média analýza   MeSH
• kyselina močová analýza   MeSH
• nanotrubičky uhlíkové chemie   MeSH
• Pseudomonas aeruginosa izolace a purifikace   MeSH
• pyokyanin analýza   MeSH
• rány a poranění mikrobiologie   patologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• kultivační média MeSH
• kyselina močová MeSH
• nanotrubičky uhlíkové MeSH
• pyokyanin MeSH

We report on the analytical performance of a tetrahedral amorphous carbon (ta-C:N) thin-film electrode in flow injection analysis with amperometric detection. Two model redox analytes were used to evaluate the electrode response because of their positive detection potentials and propensity (i.e., reaction products) to adsorb and foul sp2 carbon electrodes: tyrosine and tryptophan. ta-C:N electrodes are attractive for electroanalytical applications because they possesses many of the excellent properties of boron-doped nanocrystalline diamond (BDD) and they can be deposited at or near room temperature. The results show that the ta-C:N electrode exhibits lower background current and noise than glassy carbon (GC). The electrode was stable microstructurally at the positive potentials used for detection, ∼1.1 V, of these two amino acids and it exhibited superior analytical detection figures of merit as compared to GC and as good or superior to BDD. The linear dynamic range for both analytes at ta-C:N was from 0.1 to 100 μmol L-1, the sensitivity was 8-12 mA L mol-1, the short-term response variability was 1-2%, and the minimum detectable concentration was 89.7 ± 0.9 nM (18.3 μg L-1 or 0.46 ng) for tryptophan and 120 ± 11 nM (21.7 μg L-1 or 0.54 ng) for tyrosine. The analytical detection figures of merit for these amino acids at GC and BDD are also presented for comparison as is characterization data for the chemical composition and microstructure of the ta-C:N film.

• Publikační typ
• časopisecké články MeSH

Correction for 'Evaluation of a nitrogen-incorporated tetrahedral amorphous carbon thin film for the detection of tryptophan and tyrosine using flow injection analysis with amperometric detection' by Romana Jarošová, et al., Analyst, 2016, DOI: .

• Publikační typ
• časopisecké články MeSH
• tisková chyba MeSH