Real time electrochemical investigation of the release, distribution and modulation of nitric oxide in the intestine of individual zebrafish embryos.

Nitric oxide (NO) is an important signaling molecule that has been implicated in a variety of physiological and pathophysiological processes in living organisms. NO plays an important role in embryonic development in vertebrates and has been reported to influence early organ development and plasticity. Quantifying NO in single embryos and their developing organs is challenging because of the small size of the embryos, the low dynamically changing concentration and the short life-time of NO. Here, we measured the distribution of NO in the intestine of live zebrafish (Danio rerio) embryos in physiological conditions and under the influence of therapeutic agents. NO measurements were performed using a miniaturized electrochemical sensor fabricated on a single carbon fiber (CF) which enables quantitative real time in vivo monitoring, and by fluorescence imaging using the 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM-DA) dye. NO production was detected in the middle segment the intestine at a level of 3.78 (±0.64) µM, and at lower levels in the anterior and posterior segments, 1.08 (±0.22) and 1.00 (±0.41) µM respectively. In the presence of resveratrol and rosuvastatin, the intestinal NO concentration decreased by 87% and 84%, demonstrating a downregulating effect. These results indicate the presence of variable micromolar concentrations of NO along the intestine of zebrafish embryos and demonstrate the usefulness of CF microelectrodes to measure quantitatively the NO release at the level of a single organ in individual zebrafish embryos. This work provides a unique approach to study in real time the modulatory role of NO in vivo and contributes to further understanding of the molecular basis of embryonic development for developmental biology and drug screening applications.

Psychopathological and sociodemographic features in treatment-resistant unipolar depression versus bipolar depression: a comparative study.

BACKGROUND: Some authors have hypothesized that Treatment-Resistant Unipolar Depression (TRD-UP) should be considered within the bipolar spectrum disorders and that hidden bipolarity may be a risk factor for TRD-UP. However, there are neither studies comparing clinical and sociodemographic data of patients with TRD-UP versus Bipolar (BP) disorders nor are there any examining differences versus Bipolar type I (BP-I) and Bipolar type II (BP-II). METHODS: Charts analysis was conducted on 194 patients followed at the Mood Disorders Clinic of the McGill University Health Center. Sociodemographic, clinical features and depression scales were collected from patients meeting DSM-IV criteria for TRD-UP (n = 100) and BP (n = 94). Binary logistic regression analysis was conducted to examine clinical predictors independently associated with the two disorders. RESULTS: Compared to BP, TRD-UP patients exhibited greater severity of depression, prevalence of anxiety and panic disorders, melancholic features, Cluster-C personality disorders, later onset of depression and fewer hospitalizations. Binary logistic regression indicated that higher comorbidity with anxiety disorders, higher depression scale scores and lower global assessment of functioning (GAF) scores, and lower number of hospitalizations and psychotherapies differentiated TRD-UP from BP patients. We also found that the rate of unemployment and the number of hospitalizations for depression was higher in BP-I than in BP-II, while the rate of suicide attempts was lower in BP-I than in BP-II depressed patients. CONCLUSIONS: These results suggest that TRD-UP constitutes a distinct psychopathological condition and not necessarily a prodromal state of BP depression.

Parkin Knockdown Modulates Dopamine Release in the Central Complex, but Not the Mushroom Body Heel, of Aging Drosophila.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons leading to reduced locomotion. Mutations of parkin gene in Drosophila produce the same phenotypes as vertebrate models, but the effect of parkin knockdown on dopamine release is not known. Here, we report age-dependent, spatial variation of dopamine release in the brain of parkin-RNAi adult Drosophila. Dopamine was repetitively stimulated by local application of acetylcholine and quantified by fast-scan cyclic voltammetry in the central complex or mushroom body heel. In the central complex, the main area controlling locomotor function, dopamine release is maintained for repeated stimulations in aged control flies, but lower concentrations of dopamine are released in the central complex of aged parkin-RNAi flies. In the mushroom body heel, the dopamine release decrease in older parkin-RNAi flies is similar to controls. There is not significant dopaminergic neuronal loss even in older parkin knockdown flies, which indicates that the changes in stimulated dopamine release are due to alterations of neuronal function. In young parkin-RNAi flies, locomotion is inhibited by 30%, while in older parkin-RNAi flies it is inhibited by 85%. Overall, stimulated dopamine release is modulated by parkin in an age and brain region dependent manner. Correlating the functional state of the dopaminergic system with behavioral phenotypes provides unique insights into the PD mechanism. Drosophila can be used to study dopamine functionality in PD, elucidate how genetics influence dopamine, and test potential therapies to maintain dopamine release.

Time-Dependent Monitoring of Dopamine in the Brain of Live Embryonic Zebrafish Using Electrochemically Pretreated Carbon Fiber Microelectrodes.

Neurotransmitters are involved in functions related to signaling, stress response, and pathological disorder development, and thus, their real-time monitoring at the site of production is important for observing the changes related to these disorders. Here, we demonstrate the first time-dependent quantification of dopamine in the brains of live zebrafish embryos using electrochemically pretreated carbon fiber microelectrodes (CFMEs) utilizing differential pulse voltammetry as the measurement technique. The pretreatment of the CFMEs in 0.1 M NaOH held at a potential of +1.0 V for 600 s improves the sensitivity toward dopamine and allows for reliable measurements in low ionic strength media. We demonstrate the measurement of extracellular dopamine concentrations in the zebrafish brain during late embryogenesis. The extracellular dopamine concentration in the tectum of zebrafish varies between 200 and 400 nM. The conventional pharmacological manipulation of neurotransmitter levels in the brain demonstrates the selective detection of dopamine at the implantation site. Exposure to the dopamine transporter inhibitor nomifensine induces an increase in extracellular dopamine from 201.9 (±34.9) nM to 352.2 (±20.0) nM, while exposure to the norepinephrine transporter inhibitor desipramine does not lead to a significant modulation of the measured signal. Furthermore, we report the quantitative assessment of the catecholamine stress response of embryos to tricaine, an anesthetic frequently used in zebrafish assays. Exposure to tricaine induces a short-lived increase in brain dopamine from 198.6 (±15.7) nM to a maximum of 278.8 (±14.0) nM. Thus, in vivo electrochemistry can detect real-time changes in zebrafish neurochemical physiology resulting from drug exposure.

Nanotoxicity Assessment Using Embryonic Zebrafish.

The emergence of nanomaterials in industrial processing and consumer products has generated an increased presence of nano-enabled products in the environment and now pose an increased risk of exposure to living organisms. However, assessing the risks of nanomaterials is a challenging task because of a large variety and great variability in their properties. Here, we describe a methodology for assessing toxicity and evaluate potential risks posed by nanomaterials using zebrafish embryos as a model organism. Zebrafish are a well-established organism that has a number of advantages over other biological models. These include optical transparency, similar structure and arrangement of organs, and conserved genetic pathways compared to other vertebrates. Their rapid development and high numbers of embryos enables high throughput screening to study toxicity of a large number of nanomaterials. The method described in this chapter can be used as a universal screening approach to assess toxicity of any type of nanomaterials, determine both lethal and sublethal effects, measure LD50 doses, evaluate morphological and organ defects, cell apoptosis, and production of reactive species.

Differential lethal and sublethal effects in embryonic zebrafish exposed to different sizes of silver nanoparticles.

Various parameters can influence the toxic response to silver nanoparticles (Ag NPs), including the size and surface properties, as well as the exposure environment and the biological site of action. Herein, we assess the intestinal toxicity of three different sizes (10, 40, and 100 nm) of Ag NPs in embryonic zebrafish, and describe the relationship between the properties and behavior of Ag NPs in the exposure medium, and induction of lethal and sublethal effects. We find that the composition of the medium and the size contribute to differential NPs agglomeration, release of Ag ions, and subsequent effects during exposure. The exposure medium causes dramatic reduction in silver dissolution due to the presence of salts and divalent cations, which limits the lethal potential of silver ions. Lethality is observed primarily for embryos exposed to medium sized Ag NPs (40 nm), but not to the supernatant originated from particles, which suggests that the exposure to particulate silver is the main cause of mortality. On the other hand, the exposure to 10 nm and 100 nm NPs, as well as Ag ions, only causes sublethal developmental defects in skeletal muscles and intestine, and induces a nitric oxide imbalance.

Interaction, transformation and toxicity assessment of particles and additives used in the semiconducting industry.

Chemical mechanical planarization (CMP) is a widely used technique for the manufacturing of integrated circuit chips in the semiconductor industry. The process generates large amounts of waste containing engineered particles, chemical additives, and chemo-mechanically removed compounds. The environmental and health effects associated with the release of CMP materials are largely unknown and have recently become of significant concern. Using a zebrafish embryo assay, we established toxicity profiles of individual CMP particle abrasives (SiO2 and CeO2), chemical additives (hydrogen peroxide, proline, glycine, nicotinic acid, and benzotriazole), as well as three model representative slurries and their resulting waste. These materials were characterized before and after use in a typical CMP process in order to assess changes that may affect their toxicological profile and alter their surface chemistry due to polishing. Toxicity outcome in zebrafish is discussed in relation with the physicochemical characteristics of the abrasive particles and with the type and concentration profile of the slurry components pre and post-polishing, as well as the interactions between particle abrasives and additives. This work provides toxicological information of realistic CMP slurries and their polishing waste, and can be used as a guideline to predict the impact of these materials in the environment.

Bioapplications of Electrochemical Sensors and Biosensors.

Recent progress in the electrochemical field enabled development of miniaturized sensing devices that can be used in biological settings to obtain fundamental and practical biochemically relevant information on physiology, metabolism, and disease states in living systems. Electrochemical sensors and biosensors have demonstrated potential for rapid, real-time measurements of biologically relevant molecules. This chapter provides an overview of the most recent advances in the development of miniaturized sensors for biological investigations in living systems, with focus on the detection of neurotransmitters and oxidative stress markers. The design of electrochemical (bio)sensors, including their detection mechanism and functionality in biological systems, is described as well as their advantages and limitations. Application of these sensors to studies in live cells, embryonic development, and rodent models is discussed.

Developmental toxicity of glycine-coated silica nanoparticles in embryonic zebrafish.

Nanoparticle (NP) surface coatings are known to influence the toxicity of engineered nanomaterials. This work examines the effect of glycine functionalization on silica NPs and investigates changes in viability and developmental defects in the organs of zebrafish embryos upon exposure. Silica NPs and glycine-functionalized silica NPs are synthesized and characterized. Exposure of zebrafish embryos to glycine-silica NPs affects the mortality percentage in a similar manner to soluble glycine. Developmental defects are observed in embryos exposed to soluble glycine, glycine-silica NPs, or silica NPs in comparison with the unexposed embryos. The damage is localized in the brain, heart, and liver of zebrafish embryos. These observations suggest a complex mechanism of toxicity, with glycine maintaining its toxic activity even when covalently bound on silica surface. Our results illustrate that surface modification of non-lethal particles can create different toxicity outcomes in the organs of exposed zebrafish embryos.