Stress Response In Zebrafish Research Articles

Waterborne aripiprazole blunts the stress response in zebrafish.

Here we provide, at least to our knowledge, the first evidence that aripiprazole (APPZ) in the water blunts the stress response of exposed fish in a concentration ten times lower than the concentration detected in the environment. Although the mechanism of APPZ in the neuroendocrine axis is not yet determined, our results highlight that the presence of APPZ residues in the environment may interfere with the stress responses in fish. Since an adequate stress response is crucial to restore fish homeostasis after stressors, fish with impaired stress response may have trouble to cope with natural and/or imposed stressors with consequences to their welfare and survival.

Behind melanocortin antagonist overexpression in the zebrafish brain: A behavioral and transcriptomic approach

Melanocortin signaling is regulated by the binding of naturally occurring antagonists, agouti-signaling protein (ASIP) and agouti-related protein (AGRP) that compete with melanocortin peptides by binding to melanocortin receptors to regulate energy balance and growth. Using a transgenic model overexpressing ASIP, we studied the involvement of melanocortin system in the feeding behaviour, growth and stress response of zebrafish. Our data demonstrate that ASIP overexpression results in enhanced growth but not obesity. The differential growth is explained by increased food intake and feeding efficiency mediated by a differential sensitivity of the satiety system that seems to involve the cocaine- and amphetamine- related transcript (CART). Stress response was similar in both genotypes. Brain transcriptome of transgenic (ASIP) vs wild type (WT) fish was compared using microarrays. WT females and males exhibited 255 genes differentially expressed (DEG) but this difference was reduced to 31 after ASIP overexpression. Statistical analysis revealed 1122 DEG when considering only fish genotype but 1066 and 981 DEG when comparing ASIP males or females with their WT counterparts, respectively. Interaction between genotype and sex significantly affected the expression of 97 genes. Several neuronal systems involved in the control of food intake were identified which displayed a differential expression according to the genotype of the fish that unravelling the flow of melanocortinergic information through the central pathways that controls the energy balance. The information provided herein will help to elucidate new central systems involved in control of obesity and should be of invaluable use for sustaining fish production systems.

Time-dependent sensitization of stress responses in zebrafish: A putative model for post-traumatic stress disorder

Time-dependent sensitization (TDS)—the delayed increase in neurobehavioral responses to heterotypic stressors after exposure to an intense, inescapable stressor—has been proposed as an animal model for post-traumatic stress disorder (PTSD). Translationally relevant stressors used in TDS are capable of affecting more than one behavioral domain and produce interindividual variability in responsiveness. Here, conspecific alarm substance (CAS) is shown to induce TDS in zebrafish in inter- and intra-population-specific way. Exposure to CAS, an ecologically relevant stimulus which produces fear-like responses acutely, increased anxiety and arousal in zebrafish from the blue shortfin (BSF) phenotype 24h after stimulus delivery. Anxiety-like responses were differently affected immediately and 24h after stimulus delivery. Anxiety-like responses were more sensitized in zebrafish from the longfin (LOF) than in the BSF phenotype, an effect which is reminiscent of “basal” differences in anxiety-like behavior. After application of behavioral cutoff criteria, CAS was shown to produce intense TDS in ∼25% of LOF animals, while ∼20% of exposed animals showed little evidence of TDS. Overall, these results suggest that CAS induces TDS in zebrafish after a 24h “incubation” period, with inter- and intra-population variability that underlines its face and ecological validity.

Waterborne Risperidone Decreases Stress Response in Zebrafish.

The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish.

Dynamic expression pattern of corticotropin-releasing hormone, urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish.

Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uIIα and uIIβ genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uIIα and uIIβ mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uIIα that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uIIα and uIIβ mRNA from levels in control fish except at 6dpf when uIIα and uIIβ were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uIIα and uIIβ apart from at 3dpf. The results indicate that the contribution of crh, uI, uIIα and uIIβ genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uIIα and uIIβ in zebrafish.

Diazepam and fluoxetine decrease the stress response in zebrafish.

The presence of pharmaceutical products in the aquatic environment has been reported in several studies. However, the impact of these drugs on living organisms is still uncharacterized. Here, we investigated the effects of acute exposure to either diazepam or fluoxetine on the stress response in Danio rerio. We showed that diazepam and fluoxetine inhibited the stress axis in zebrafish. Intermediate concentrations of diazepam suppressed the stress response as measured by cortisol levels, whereas fluoxetine inhibited cortisol increase at concentrations similar to those found in the environment. These data suggest that the presence of psychoactive drugs in aquatic ecosystems could cause neuroendocrine dysfunction in fish.

Acute embryonic exposure to nanosilver or silver ion does not disrupt the stress response in zebrafish (Danio rerio) larvae and adults

The antibacterial properties of silver nanoparticles (AgNPs) are widely exploited in a variety of medical and consumer products. AgNPs in these products can be released into the aquatic environment, however, the potential toxicity of AgNPs to organisms, including fish, is yet to be fully understood. The present study aimed to investigate the effects of the early life exposure to AgNPs on the hypothalamic–pituitary–interrenal (HPI) axis-mediated stress response in zebrafish (Danio rerio) larvae and adults. Zebrafish embryos were treated with AgNPs (0.5μg/mL) or Ag+ (0.05μg/mL) starting at 2h post fertilization (hpf). At 96hpf the larvae were either subjected to a swirling stress and euthanized, or raised to adulthood (10months) in silver-free water and then net-stressed, euthanized, and sampled. Whole-body basal or stress-induced cortisol levels in larvae were not affected by either AgNPs or Ag+; however, the transcript levels of corticotropin releasing factor (CRF), CRF-binding protein (CRF-BP), CRF-receptor 2 (CRF-R2), and pro-opiomelanocortin (POMCb) were significantly decreased by Ag+. The ability of the adult fish to release cortisol in response to a stressor was also not affected, although the transcript levels of CRF, CRF-BP, and CRF-R1 in the telencephalon were differentially affected in fish exposed to Ag+ as embryos. This is the first study that investigated the potential endocrine-disrupting effects of AgNPs during the early life stages and although AgNPs or Ag+ did not affect the ability of zebrafish to elevate cortisol levels in response to a stressor, the effects on transcript levels by Ag+ should be investigated further since CRF does not solely regulate the HPI axis but is also implicated in other physiological processes.

Development of catecholamine and cortisol stress responses in zebrafish

Both adrenal catecholamines and steroids are known to be involved in the stress response, immune function, blood pressure and energy homeostasis. The response to stress is characterized by the activation of the hypothalamus–pituitary–adrenal (HPA) axis and the sympathetic-adrenomedullary system, though the correlation with activation and development is not well understood. We evaluated the stress response of both cortisol and catecholamines during development in zebrafish. Zebrafish at two different stages of development were stressed in one of two different ways and cortisol and catecholamine were measured. Cortisol was measured by enzyme immune assay and catecholamine was measured by ELISA. Our results show that stress responses are delayed until after the synthesis of both cortisol and catecholamines. These observations suggest that the development of HPA axis may be required for the acquisition of the stress response for cortisol and catecholamines.

Alcohol Impairs Predation Risk Response and Communication in Zebrafish

The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism.

Exposure to tributyltin induces endoplasmic reticulum stress and the unfolded protein response in zebrafish

Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1μM TBT for 0.5–16h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32h post fertilization (hpf), were exposed to 1μM TBT for 16h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK–eIF2α and IRE1–XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner.

One for All and All for One: The Importance of Shoaling on Behavioral and Stress Responses in Zebrafish

Zebrafish has been increasingly used in behavioral studies, but data can present high variability. Most studies have been performed using isolated zebrafish, despite their interactive nature and shoaling behavior. We compared adult zebrafish behavior and cortisol levels after exposure to novelty as well as sensitivity to Pentylenetetrazole (PTZ)-induced seizures in animals tested individually or in groups of three (triplets). In the exploratory behavior task, data from single fish and triplets were not significantly different, but single fish data were more disperse in latency, to enter and time spent in the tank upper part, and crossings. In the light-dark task, time in the light zone and crossings were not different between groups, but latency to enter the dark zone and data variability were. We also observed that the latency to reach stage III seizures induced by PTZ was higher in triplets, but data dispersion was not different from single fish. Finally, cortisol levels of fish individually exposed to a novel environment were higher and more variable than triplets, while both groups had higher levels than unmanipulated animals. Thus, when tested individually, zebrafish are more stressed and present more variable behavior due to disruption of their natural shoal strategies. These features can be beneficial or detrimental depending on study aims and should be considered when designing, analyzing, and interpreting zebrafish behavioral data.

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL and Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6h and 24h exposure to 0.5g/L and 0.05g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24h at 0.1 and 5mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways.

Localization, developmental regulation and stress induction of p8-L1 and p8-L2 in zebrafish

AbstractThe transcription cofactor, p8, has a basic helix-loop-helix motif and is involved in several metabolic processes. It has previously been reported that p8 plays an important role in stress response in zebrafish. We expanded on these studies to further investigate the roles of two p8-like cDNAs found in zerbrafish, p8-L1 and p8-L2. Zebrafish p8-L1 and p8-L2 cDNAs consist of 742 bp and 813 bp, respectively. Both have the same open reading frame despite different genomic structures and encode a 76 amino acid polypeptide with conserved a Phospho_p8 domain. These two cDNA’s were abundant in different tissues in the zebrafish: p8-L1 was high in intestine and muscle tissue and in low abundance in the backbone, whereas the highest amount of p8-L2 was found in the backbone, similar to p8. During embryogenesis, both p8-L1 and p8-L2 were abundant at the cleavage stage and decreased gradually in abundance from blastula to pharyngula stage. However, p8-L1 abundance increased during hatching as observed in p8. Quantitative real-time PCR assay suggested that p8-L1 and p8-L2 were both up-regulated significantly under osmotic pressure and pH value challenges, suggesting an important role of p8-L1 and p8-L2 genes in stress response.

Oxidative stress response in zebrafish (Danio rerio) gill experimentally exposed to subchronic microcystin-LR

The worldwide occurrence of cyanobacterial blooms makes it necessary to perform environmental risk assessment procedures to monitor the effects of microcytins on fish. Oxidative stress biomarkers are valuable tools in this regard. Considering that zebrafish (Danio rerio) is a common model species in fish toxicology and the zebrafish gill is potentially useful in screening waterborne pollutants, this study investigated the oxidative stress response in zebrafish gill exposed to subchronic microcystin-LR (MCLR) concentrations (2 or 20 μg/l) via measurement of toxin accumulation, protein phosphatase (PP) activity, and the antioxidant parameters (glutathione-S-transferase-GST; glutathione-GSH; superoxide dismutase-SOD; catalase-CAT; glutathione peroxide-GPx; glutathione reductase-GR), as well as levels of hydroxyl radical (OH) and lipid peroxidation (LPO). The results showed that after 30 days exposure, MCLR accumulated in zebrafish gill and MCLR exposure induced PP activity in gill. A linear inhibition of GST activity and GSH content was observed in the gills, revealing that they were involved in the first step of MCLR detoxification. The 2 μg/l MCLR treatment neglectably affected OH content and the antioxidant enzymes (SOD, CAT, GPx, and GR), however oxidative stress was induced under the 20 μg/l MCLR treatment in which an enhanced OH content and alterations of the antioxidant enzymes were observed in the treated gills, although both treatments exerted little effect on LPO level. The principal component analysis results indicated that the most sensitive biomarkers of MCLR exposure were GST and GSH in zebrafish gill. So, D. rerio could be regarded as a suitable bioindicator of MCLR exposure by measuring CAT, GR, GST, and GSH as biomarkers.

Stressing zebrafish for behavioral genetics

The stress response is a normal reaction to a real or perceived threat. However, stress response systems that are overwhelmed or out of balance can increase both the incidence and severity of diseases including addiction and mood and anxiety disorders. Using an animal model with both genetic diversity and large family size can help discover the specific genetic and environmental contributions to these behavioral diseases. The stress response has been studied extensively in teleosts because of their importance in food production. The zebrafish (Danio rerio) is a major model organism with a strong record for use in developmental biology, genetic screening, and genomic studies. More recently, the stress response of larval and adult zebrafish has been documented. High-throughput automated tracking systems make possible behavioral readouts of the stress response in zebrafish. This non-invasive measure of the stress response can be combined with mutagenesis methods to dissect the genes involved in complex stress response behaviors in vertebrates. Understanding the genetic and epigenetic basis for the stress response in vertebrates will help to develop advanced screening and therapies for stress-aggravated diseases such as addiction and mood and anxiety disorders.

Stirring Up New Ideas About the Regulation of the Hypothalamic-Pituitary-Interrenal Axis in Zebrafish (Danio rerio)

The dynamic relationships between the changes in cortisol synthesis during and after a stressor and the expression pattern of the key genes that regulate the different levels of the hypothalamic-pituitary-interrenal (HPI) stress axis are poorly understood. This study established a novel vortex stressor and characterized its impact at all levels of the HPI axis in adult zebrafish. Exposure to a moderate vortex speed for 60 min was associated with a marked 18-fold increase in whole-body cortisol after 10 min followed by a gradual return to basal values 30 min poststress. The changes in whole-body cortisol were paralleled by increases in the expression of preoptic area corticotropin-releasing factor, pituitary prohormone convertase 1, and interrenal melanocortin 2 receptor, steroid acute regulatory protein, 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase 2. The response to the vortex stressor also included delayed increases in preoptic area urotensin I and pituitary pro-opiomelanocortin mRNA levels but no change in the expression of other putative HPI axis regulators. Notably, the expression of several genes was depressed below control values 30 min poststress. These findings suggest that multiple genes at all levels of the HPI axis play an active role in the stimulation and termination of the cortisol stress response in zebrafish.

Differential effects of 17β-estradiol and 11-ketotestosterone on the endocrine stress response in zebrafish ( Danio rerio)

Sexually dimorphic stress responses are present in species across all vertebrate taxa and it has been suggested that these effects are mediated by circulating sex steroids. While a few species of fish have been identified as having a sexually dimorphic stress response, there is conflicting evidence as to the effects of sex steroids on the stress axis. In this study, we tested whether zebrafish exhibit a sexually dimorphic cortisol stress response and whether 17β-estradiol (E2) or 11-ketotestosterone (11KT) modulate the activity of the hypothalamic-pituitary-interrenal (HPI) axis. To accomplish this, we quantified the whole body cortisol response to a physical stressor, cortisol release in vitro, and the expression of key HPI axis regulating genes of control and E2- or 11KT-exposed zebrafish. Under control conditions no dimorphisms in the HPI axis were apparent at rest or in response to a standardized stressor. In contrast, E2-exposure blunted the cortisol response of male fish in vivo and in vitro and as well as corticotropin-releasing factor ( crf) expression in the pre-optic area (POA) of the brain. While the expression of some interrenal genes was suppressed by E2-exposure, these changes occurred in both male and female zebrafish. 11KT-exposure increased whole-body cortisol of males at rest and vortex-exposed females, but had no impact on the rate of cortisol synthesis in vitro or on POA crf expression. Therefore, while we found no evidence that zebrafish exhibit a sexually dimorphic cortisol stress response, both E2 and 11KT can modulate the activity of the HPI axis in this species and do so via different mechanisms.

Measuring behavioral and endocrine responses to novelty stress in adult zebrafish

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.

Differential uptake and oxidative stress response in zebrafish fed a single dose of the principal copper and zinc enriched sub-cellular fractions of Gammarus pulex

The sub-cellular compartmentalisation of trace metals and its effect on trophic transfer and toxicity in the aquatic food chain has been a subject of growing interest. In the present study, the crustacean Gammarus pulex was exposed to either 11 μg Cu l −1, added solely as the enriched stable isotope 65Cu, or 660 μg Zn l −1, radiolabeled with 2MBq 65Zn, for 16 days. Post-exposure the heat stable cytosol containing metallothionein-like proteins (MTLP) and a combined granular and exoskeletal (MRG + exo) fractions were isolated by differential centrifugation, incorporated into gelatin and fed to zebrafish as a single meal. Assimilation efficiency (AE) and intestinal lipid peroxidation, as malondialdehyde (MDA) were measured. There was a significant difference ( p < 0.05) between the retention of the MTLP-Zn (39.0 ± 6.4%) and MRG + exo-Zn (17.2 ± 3.7%) and of this zinc retained by the zebrafish a significantly greater proportion of the MTLP-Zn feed had been transported away from the site of uptake. For 65Cu, although the results pointed towards greater bioavailability of the MTLP fraction compared to MRG + exo during the slow elimination phase (24–72 h) these results were not significant ( p = 0.155). Neither zinc feed provoked a lipid peroxidation response in the intestinal tissue of zebrafish compared to control fish (gelatin fed), but both 65Cu labeled feeds did. The greater effect was exerted by the MRG + exo (2.96 ± 0.29 nmol MDA mg protein −1) feed which three-fold greater than control ( p < 0.01) and almost twice the MDA concentration of the MTLP feed (1.76 ± 0.21 nmol MDA mg protein −1, p < 0.05). The oxidative stress response produced by Zn and Cu is in keeping with their respective redox potentials; Zn being oxidatively inert and Cu being redox active. These results are similar, in terms of bioavailability and stress response of each feed, to those in our previous study in which 109Cd labeled G. pulex fractions were fed to zebrafish. Thus it appears that when a metal (Cu or Cd) has the potential to cause cytotoxicity via lipid peroxidation, a feed consisting of a largely unavailable fraction (MRG + exo) causes a greater intestinal stress response than the more bioavailable (MTLP) feed.

Bioavailability and oxidative stress response in Zebrafish fed 109Cd enriched G. pulex sub-cellular fractions

Bioavailability and oxidative stress response in Zebrafish fed 109Cd enriched G. pulex sub-cellular fractions