Alterations in locomotor activity of feeding zebrafish larvae as a consequence of exposure to different environmental factors.

Behavioral studies are important tools for understanding the development and pathology of neurological diseases. Zebrafish are an emerging alternative model in behavioral and neurological studies as the behavioral repertoire of zebrafish (Danio rerio) is similar to humans, and nervous system structures and functions are highly conserved. In this study, we investigated alterations in day/night locomotor activity of free swimming, feeding wild-type zebrafish larvae (8-15dpf) due to changes in the rhythm of light/dark cycles or caloric content of food. We furthermore exposed zebrafish larvae to continuous stress by applying alternated minor vibrations. Under altered rhythms of light/dark cycle's zebrafish larvae still expressed a distinct light/dark activity pattern but the total activity was reduced compared to control animals. When the larvae were exposed to continuous light, they still had coordinated resting cycles but maximal activity and excitation rates after feeding were increased, indicating that food became the new zeitgeber. Feeding food of high caloric content induced continuously high activity levels during light cycles and significantly elevated activity levels during the dark. Exposure to continuous vibrations lowered total activity levels. We showed previously that changes in environmental factors like light/dark cycles or changes in caloric content of food can affect adipogenesis, lipid composition, and circadian rhythm of free swimming, feeding larvae but this is the first time showing how theses factor alter behavior.

Exposure to endocrine disrupting chemicals perturbs lipid metabolism and circadian rhythms.

A growing body of evidence indicates that exposure to environmental chemicals can contribute to the etiology of obesity by inappropriately stimulating adipogenesis as well as perturbing lipid metabolism and energy balance. One potential mechanism by which chemical exposure can influence lipid metabolism is through disturbance of circadian rhythms, endogenously-driven cycles of roughly 24hr in length that coordinate biochemical, physiological, and behavioral processes in all organisms. Here we show for the first time that exposure to endocrine disrupting compounds (EDCs), including the pesticide tributyltin, two commercial flame retardants, and a UV-filter chemical found in sunscreens, can perturb both circadian clocks and lipid metabolism in vertebrates. Exposure of developing zebrafish to EDCs affects core clock activity and leads to a remarkable increase in lipid accumulation that is reminiscent of the effects observed for longdaysin, a known disruptor of circadian rhythms. Our data reveal a novel obesogenic mechanism of action for environmental chemicals, an observation which warrants further research. Because circadian clocks regulate a wide variety of physiological processes, identification of environmental chemicals capable of perturbing these systems may provide important insights into the development of environmentally-induced metabolic disease.

Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails.

The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail's stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd(2+), Zn(2+), Cu(+)) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors.

Prolonged hypoxia increases survival even in Zebrafish (Danio rerio) showing cardiac arrhythmia.

Tolerance towards hypoxia is highly pronounced in zebrafish. In this study even beneficial effects of hypoxia, specifically enhanced survival of zebrafish larvae, could be demonstrated. This effect was actually more pronounced in breakdance mutants, which phenotypically show cardiac arrhythmia. Breakdance mutants (bre) are characterized by chronically reduced cardiac output. Despite an about 50% heart rate reduction, they become adults, but survival rate significantly drops to 40%. Normoxic bre animals demonstrate increased hypoxia inducible factor 1 a (Hif-1α) expression, which indicates an activated hypoxic signaling pathway. Consequently, cardiovascular acclimation, like cardiac hypertrophy and increased erythrocyte concentration, occurs. Thus, it was hypothesized, that under hypoxic conditions survival might be even more reduced. When bre mutants were exposed to hypoxic conditions, they surprisingly showed higher survival rates than under normoxic conditions and even reached wildtype values. In hypoxic wildtype zebrafish, survival yet exceeded normoxic control values. To specify physiological acclimation, cardiovascular and metabolic parameters were measured before hypoxia started (3 dpf), when the first differences in survival rate occurred (7 dpf) and when survival rate plateaued (15 dpf). Hypoxic animals expectedly demonstrated Hif-1α accumulation and consequently enhanced convective oxygen carrying capacity. Moreover, bre animals showed a significantly enhanced heart rate under hypoxic conditions, which reached normoxic wildtype values. This improvement in convective oxygen transport ensured a sufficient oxygen and nutrient supply and was also reflected in the significantly higher mitochondrial activity. The highly optimized energy metabolism observed in hypoxic zebrafish larvae might be decisive for periods of higher energy demand due to organ development, growth and increased activity. However, hypoxia increased survival only during a short period of development and starting hypoxia before or after this phase reduced survival, particularly in bre animals. Thus, the physiological plasticity, which enables zebrafish larvae to benefit from a hypoxia, occurs only within a narrow developmental window.

HIF signaling and overall gene expression changes during hypoxia and prolonged exercise differ considerably.

Exercise as well as hypoxia cause an increase in angiogenesis, changes in mitochondrial density and alterations in metabolism, but it is still under debate whether the hypoxia inducible factor (HIF) is active during both situations. In this study gene expression analysis of zebrafish larvae that were raised under normoxic, hypoxic, or training conditions were compared, using microarray analysis, quantitative real-time PCR and protein data. Although HIF expression is posttranslationally regulated, mRNA expression levels of all three isoforms (HIF-1α, HIF-2α, and HIF-3α) differed in each of the experimental groups, but the changes observed in hypoxic animals were much smaller than in trained larvae. Prominent changes were seen for Hif-2α expression, which significantly increased after the first day of exercise and then decreased down to values significantly below control values. HIF-3α mRNA expression in turn increased significantly, and at the end of the training period (9-15 days postfertilization) it was elevated three times. At the protein level a transient increase in HIF-1α was observed in hypoxic larvae, whereas in the exercise group the amount of HIF-1α protein even decreased below the level of control animals. The analyzed transcriptome was more affected in hypoxic zebrafish larvae, and hardly any genes were similarly altered by both treatments. These results clearly showed that HIF proteins played different roles in trained and hypoxic zebrafish larvae and that the exercise-induced transition to a more aerobic phenotype was not achieved by persistent activation of the hypoxic signaling pathway.

Chronic reduction in cardiac output induces hypoxic signaling in larval zebrafish even at a time when convective oxygen transport is not required.

In the present study, the zebrafish breakdance mutant (bre) was used to assess the role of blood flow in development because it has been previously shown that bre larvae have a chronically reduced cardiac output as a result of ventricular contraction following only every second atrial contraction in addition to an atrial bradycardia. We confirmed a 50% reduction compared with control fish and further showed that blood flow in the caudal part of the dorsal aorta decreased by 80%. Associated with these reductions in blood flow were indications of developmental retardation in bre mutants, specifically delayed hatching, reduced cell proliferation, and a transiently decreased growth rate. Surprisingly, an increased red blood cell concentration and an earlier appearance of trunk vessels in bre larvae indicated some compensation to convective oxygen transport, although in previous studies it has been shown that zebrafish larvae at this stage obtain oxygen by bulk diffusion. In bre animals immunohistochemical analyses showed a significant increase in hypoxia inducible factor 1 (HIF)-α protein expression, comparable with wild-type larvae that were raised under hypoxic conditions. Accordingly, the expression of some hif downstream genes was affected. Furthermore, Affymetrix microarray analyses revealed a large number of genes that were differently expressed comparing control and bre larvae, and the number even increased with proceeding development. The results showed that a chronic reduction in blood flow generated hypoxic molecular signals despite partial compensation by increased oxygen carrying capacity and transiently slowed the overall development of zebrafish bre larvae.

Ontogenetic development of erythropoiesis can be studied non-invasively in GATA-1:DsRed transgenic zebrafish.

For the erythroid cell lineage development in vertebrates, GATA-1 transcription factor is essential. In our report, we have demonstrated that the approximate developmental status of erythrocytes and the progression of blood formation can be studied non-invasively in GATA-1:DsRed transgenic zebrafish (Danio rerio) embryo and larva by characterization of fluorescence luminance spectra. The study was carried out for animals maintained under normoxic and hypoxic (152 and 20 torr PO(2) respectively) conditions up to 10 days post-fertilization (dpf) and total blood cell concentrations and fluorescent cells' percentage were determined for this purpose. The erythroids were classified into five intensity stages (IS) on the basis of their fluorescence intensity. The luminescent cells with medium intensities (IS3) in normoxic animals were found throughout 2 to 10 dpf although in lower quantity while in hypoxic group they appeared from 5 dpf to 10 dpf showing a maximum of 15% of the total luminescent cells at 8 dpf. The total blood cell concentration dropped after 8 dpf in contrast to hypoxic group which showed further increasing trend. The fluorescent cells' percentage in normoxic group was generally higher as compared to the hypoxic ones. Our method successfully defined various stages of erythroid development. An effort was also made to correlate our luminance data (GATA-1 expression) and total blood cell concentrations with Epo mRNA production. Quantitative RT-PCR of 2-15 dpf old zebrafish was carried out for this purpose. Normoxic animals showed 1-3 Epo mRNA copies per ng RNA in contrast to the hypoxic larvae that showed remarkable fluctuation of 1 to 12 Epo mRNA copies per ng RNA during development. The blood volume (aortic diameter) and production time scale proved to be important factors to define the relationship of Epo mRNA with total blood cell concentration and GATA-1 protein expression respectively.

How does blood cell concentration modulate cardiovascular parameters in developing zebrafish (Danio rerio)?

Blood flow and shear forces are considered to be important parameters possibly stimulating angiogenesis or cardiovascular remodeling. The main objective of this study was to test the hypothesis that a significant reduction in shear forces as a consequence of a significant isovolemic anemia induced by microsurgical techniques during early larval development of the zebrafish might induce a compensatory stimulation of erythropoiesis and/or induce a modification of cardiac activity or even the formation of the heart and may influence the shaping of the vascular bed. Blood from 2 day old zebrafish larvae was withdrawn and replaced by zebrafish Ringer's solution, so that the blood cell concentration was reduced by at least 75%. At 5 days post fertilization (dpf) a partial recovery in blood cell concentration was observed and reached a value of 814.55+/-85.42 cells/nL, while in control animals blood cell concentration amounted to 1856.00+/-131.59 cells/nL. At 7 dpf the value of blood cell concentration was 1023.89+/-95.75 cells/nL versus 1701.54+/-146.03 cells/nL in control animals. Compared to control animals, heart rate and cardiac output were significantly reduced in anemic animals and alterations in the formation of the vascular bed were also observed. A significant decrease in the end-diastolic volume suggested that ventricular volume was reduced. Thus, within a few days zebrafish larvae were nearly able to compensate for an isovolemic anemia by an enhanced erythropoiesis. However, several changes in cardiovascular system indicated that phenotypic plasticity is established even at an early developmental stage.

Cardiac performance in the zebrafish breakdance mutant.

In the Tubingen screen a breakdance mutant of zebrafish (bre) was described as an arrhythmia, in which the ventricle beats only with every second atrial contraction (2:1 rhythm). Surprisingly, a careful analysis of the effect of the breakdance mutation on cardiac performance of the zebrafish during development between 3 d.p.f. and 14 d.p.f revealed that homozygous bre mutants did not always show the 2:1 rhythm. Cardiac activity was continuously recorded for a period of 20 min in each larva, and during this period we observed that heart rate randomly switched between the 2:1 rhythm and a 1:1 rhythm. Furthermore, at 28 degrees C and at 31 degrees C the expression of the 2:1 rhythm decreased with development. At 31 degrees C this was in part due to a significantly reduced survival rate of mutants beyond 4 d.p.f. Besides development, temperature had a marked effect on the expression of the 2:1 rhythm, and during the first days of development the expression of the 2:1 rhythm was significantly higher at elevated incubation temperatures. By contrast, in the 2:1 beating heart ventricular contraction rate was about 80 beats min(-1) throughout development irrespective of the temperature, and even in the 1:1 rhythm mutants showed a significant bradycardia at all three temperatures (25 degrees C, 28 degrees C or 31 degrees C). Compared to wild-type animals, cardiac output was significantly lower in bre mutants. Pressure traces recorded in the ventricle of mutants revealed a prolonged relaxation phase, indicating that the second pacemaker current could not be conveyed to the ventricle (AV-block). This phenotype is comparable to the human Long QT Syndrome, an arrhythmia caused by a modification of an ion channel involved in cardiac repolarization. The bradycardia and the modified temperature sensitivity of heart rate suggested that the activity of the pacemaker cells was also affected by this mutation.