The effects of temperature on the proxies of visual detection of Danio rerio larvae: observations from the optic tectum.

Numerous studies have indicated that temperature improves the visual capabilities of different ectotherms, including a variety of fish species. However, none of these studies has directly tested whether elevated temperature extends the visual detection distance - the distance from which a visual stimulus is detected. To test this hypothesis, we investigated the effect of temperature on the visual detection distance of zebrafish (Danio rerio) larvae by measuring the largest distance from a moving target that induced a neural response in the optic tectum. We applied advanced methods of functional calcium imaging such as selective plane illumination microscopy in combination with a miniature OLED screen. The screen displayed an artificial, mobile prey, appearing in the visual field of the larvae. We performed experiments in three temperature treatments (18, 23 and 28°C) on transgenic fish expressing a fluorescent probe (GCaMP5G) that changes intensity in response to altered Ca2+ concentrations in the nerves in the optic tectum. Based on the obtained data, we also measured three additional parameters of the neural response in the optic tectum, each being a proxy of sensitivity to changes in the stimulus movement. We did not confirm our hypothesis, since the visual detection distance shortened as the temperature increased. Moreover, all of the three additional parameters indicated a negative effect of the temperature on the speed of the neural response to the stimuli. However, the obtained results could be explained not only by worse visual capabilities at the elevated temperature, but also by the differences in the visual field and in turn, the retinotopic location of the visual stimulus between the temperature treatments, since the stimulus in the experiments moved horizontally rather than forward and backward from the fish's eye.

Targeting mitochondrial calcium pathways as a potential treatment against Parkinson's disease.

Parkinson's disease (PD) is a major health problem worldwide affecting millions of people and is a result of neurodegeneration in a small part of the brain known as substantia nigra pars compacta. Aberration in mitochondrial Ca2+ homeostasis plays, among several other factors, an important role for the neuronal loss in PD. Mitochondria are vital for cellular physiology, e.g. for ATP generation, and mitochondrial Ca2+ is a key player in cell functioning and survival. Mitochondrial Ca2+ homeostasis is maintained by a fine balance between the activities of proteins mediating the influx and efflux of Ca2+ across mitochondrial membranes. Malfunctioning of these proteins leading to Ca2+ overload promotes ROS generation, which induces cell death by triggering the opening of mitochondrial permeability transition pore. Till now PD remains incurable and the "gold standard" drug which can only delays the disease progression is l-Dopa from the 1960s and therefore, the situation warrants the search for novel targets for the treatment of the PD patients. In this review, we summarize the current views that suggest mitochondrial Ca2+ regulatory pathways are good candidates for the treatment of PD.

Restriction of mitochondrial calcium overload by mcu inactivation renders a neuroprotective effect in zebrafish models of Parkinson's disease.

The loss of dopaminergic neurons (DA) is a pathological hallmark of sporadic and familial forms of Parkinson's disease (PD). We have previously shown that inhibiting mitochondrial calcium uniporter (mcu) using morpholinos can rescue DA neurons in the PTEN-induced putative kinase 1 (pink1)-/- zebrafish model of PD. In this article, we show results from our studies in mcu knockout zebrafish, which was generated using the CRISPR/Cas9 system. Functional assays confirmed impaired mitochondrial calcium influx in mcu -/- zebrafish. We also used in vivo calcium imaging and fluorescent assays in purified mitochondria to investigate mitochondrial calcium dynamics in a pink1 -/- zebrafish model of PD. Mitochondrial morphology was evaluated in DA neurons and muscle fibers using immunolabeling and transgenic lines, respectively. We observed diminished mitochondrial area in DA neurons of pink1 -/- zebrafish, while deletion of mcu restored mitochondrial area. In contrast, the mitochondrial volume in muscle fibers was not restored after inactivation of mcu in pink1 -/- zebrafish. Mitochondrial calcium overload coupled with depolarization of mitochondrial membrane potential leads to mitochondrial dysfunction in the pink1 -/- zebrafish model of PD. We used in situ hybridization and immunohistochemical labeling of DA neurons to evaluate the effect of mcu deletion on DA neuronal clusters in the ventral telencephalon of zebrafish brain. We show that DA neurons are rescued after deletion of mcu in pink1 -/- and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) zebrafish model of PD. Thus, inactivation of mcu is protective in both genetic and chemical models of PD. Our data reveal that regulating mcu function could be an effective therapeutic target in PD pathology.

A novel conserved enhancer at zebrafish zic3 and zic6 loci drives neural expression.

BACKGROUND: Identifying enhancers and deciphering their putative roles represent a major step to better understand the mechanism of metazoan gene regulation, development, and the role of regulatory elements in disease. Comparative genomics and transgenic assays have been used with some success to identify critical regions that are involved in regulating the spatiotemporal expression of genes during embryogenesis. RESULTS: We identified two novel tetrapod-teleost conserved noncoding elements within the vicinity of the zic3 and zic6 loci in the zebrafish genome and demonstrated their ability to drive tissue-specific expression in a transgenic zebrafish assay. The syntenic analysis and robust green fluorescent expression in the developing habenula in the stable transgenic line were correlated with known sites of endogenous zic3 and zic6 expression. CONCLUSION: This transgenic line that expresses green fluorescent protein in the habenula is a valuable resource for studying a specific population of cells in the zebrafish central nervous system. Our observations indicate that a genomic sequence that is conserved between humans and zebrafish acts as an enhancer that likely controls zic3 and zic6 expression.

Correction: Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish.

[This corrects the article DOI: 10.1371/journal.pgen.1007743.].

Loss of the Mia40a oxidoreductase leads to hepato-pancreatic insufficiency in zebrafish.

Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.

[Microscopic imaging of calcium ions with genetically encoded calcium indicators]. / Mikroskopowe obrazowanie jonów wapnia za pomoca genetycznie kodowanych sond.

Calcium is a second messenger that plays a key role in various cellular processes. Monitoring calcium levels is a prerequisite to their understanding. The first calcium indicators for microscopy were the luminescent protein aequorin and chemical probes. These indicators, however, have serious drawbacks, limiting their use in many types of experiments. Cloning of cDNA for the A. victoria GFP and creation of its first spectral variants has initiated the development of fluorescent, genetically encoded calcium indicators (GECI). They are composed of a fluorescent protein and a calcium-binding protein, usually calmodulin. The binding of calcium to the sensory domain of the indicator affects the fluorescent properties of the chromophore, which enables recording of calcium signals as fluorescent light. GECIs have many advantages and are essential for conducting long-term experiments in vivo. This article gives an overview of the currently available GECIs, the history of their development, applications and microscopic imaging systems.

Inhibition of the mitochondrial calcium uniporter rescues dopaminergic neurons in pink1-/- zebrafish.

Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of early onset Parkinson's disease (PD). Loss of PINK1 function causes dysregulation of mitochondrial calcium homeostasis, resulting in mitochondrial dysfunction and neuronal cell death. We report that both genetic and pharmacological inactivation of the mitochondrial calcium uniporter (MCU), located in the inner mitochondrial membrane, prevents dopaminergic neuronal cell loss in pink1Y431 * mutant zebrafish (Danio rerio) via rescue of mitochondrial respiratory chain function. In contrast, genetic inactivation of the voltage dependent anion channel 1 (VDAC1), located in the outer mitochondrial membrane, did not rescue dopaminergic neurons in PINK1 deficient D. rerio. Subsequent gene expression studies revealed specific upregulation of the mcu regulator micu1 in pink1Y431 * mutant zebrafish larvae and inactivation of micu1 also results in rescue of dopaminergic neurons. The functional consequences of PINK1 deficiency and modified MCU activity were confirmed using a dynamic in silico model of Ca2+ triggered mitochondrial activity. Our data suggest modulation of MCU-mediated mitochondrial calcium homeostasis as a possible neuroprotective strategy in PINK1 mutant PD.

A study of mechanisms responsible for incorporation of cesium and radiocesium into fruitbodies of king oyster mushroom (Pleurotus eryngii).

Ex vitro cultures of Pleurotus eryngii were carried out under controlled conditions using sterile medium composed of barley seeds. The influence of alkali and alkaline earth element salts (CsCl, KCl, NaCl, RbCl, and CaCl(2)) and tetraethylammonium chloride on incorporation of cesium, potassium, sodium, rubidium and calcium, and their distribution within fruitbodies, was examined. The results show that incorporation of cesium into fruitbodies was not suppressed by Na(+) and Rb(+) or tetraethylammonium chloride. However, it was inhibited by Ca(2+) and stimulated by high concentrations of K(+). The inhibition of cesium incorporation by Ca(2+), lack of influence of tetraethylammonium chloride and stimulation by high K(+) concentrations suggest that there may exist two pathways of passive transport of cesium in mycelium: (i) uptake mediated by a non-specific potassium channel localised in plasmalemma (similar to voltage-insensitive cation channel, VICC) followed by diffusive transport inside hyphae and (ii) extracellular transport from the medium through inter-hyphal cavities into fruitbodies. The results highlight distinctiveness of mechanisms responsible for the uptake and incorporation of cesium in mushrooms and plants.

Transport of radiocesium in mycelium and its translocation to fruitbodies of a saprophytic macromycete.

We present a new protocol to study fluxes of radionuclides and other xenobiotics in saprophytic fungi. This simple method has successfully been used to evaluate transport of radiocesium in hyphae of Pleurotus eryngii and its translocation to fruitbodies.

Pilot study of bioaccumulation and distribution of cesium, potassium, sodium and calcium in king oyster mushroom (Pleurotus eryngii) grown under controlled conditions.

This pilot study presents preliminary results on interrelations between alkali and alkaline earth elements during their transfer to mycelium and fruitbodies of saprophytic fungi. The accumulation and distribution of four elements (cesium, potassium, sodium, and calcium) was evaluated in king oyster mushroom (Pleurotus eryngii) cultivated under controlled conditions. Elemental composition of caps, stipes, and the substrate was analyzed by atomic absorption/emission spectroscopy to evaluate discrimination, concentration, and transfer factors. The transfer factors determined for all the investigated elements were different and can be put in the following order: Cs > K > Na > Ca. There has been a higher accumulation of cesium in caps than in stipes. Distribution of cesium in fruitbodies depended on the presence of other ions in the substrate. The addition of Ca2+ limited the transport of cesium and potassium from stipes to caps. Sodium and calcium were mainly accumulated in the stipes. In a control experiment, without supplementation with K+, Na+, and Ca2+, approximately 62% of the cesium present in the substrate was extracted by mycelium and transported to the fruitbodies. Possible applications of fruiting saprophytic fungi in bioremediation are discussed.