Phenotypes In Adult Zebrafish Research Articles

Environmentally relevant manganese concentrations evoke anxiety phenotypes in adult zebrafish

Manganese (Mn) is an essential metal for living organisms. However, the excess of Mn can be toxic, especially for the central nervous system. Herein, we used adult zebrafish as model organism to investigate the relationship of an environmentally relevant Mn exposure with the onset of neurobehavioral disturbances and brain biochemical alterations. Fish were exposed to MnCl2 at 0.5, 2.0, 7.5 and 15.0 mg/L for 96 h, and after submitted to trials for examining exploratory, locomotor and anxiety-related behaviors. The neurobehavioral parameters were followed by the analyses of cell viability, Mn accumulation and acetylcholinesterase activity in the brain, and whole-body cortisol levels. By Novel tank, Light dark and Social preference test, we found that the exposure to Mn, along with locomotor deficits induced anxiety-like phenotypes in zebrafish. Most of these behavioral changes were evoked by the highest concentrations, which also caused cell viability loss, higher accumulation of Mn and increased AChE activity in the brain, and an increase in the whole-body cortisol content. Our findings demonstrated that zebrafish are quite sensitive to levels of Mn found in the environment, and that the magnitude of the neurotoxic effects may be associated with the levels of manganese accumulated in the brain. Interestingly, we showed that Mn exposure in addition to motor deficits may also cause psychiatric abnormalities, namely anxiety.

Progressive liver, kidney, and heart degeneration in children and adults affected by TULP3 mutations

SummaryOrgan fibrosis is a shared endpoint of many diseases, yet underlying mechanisms are not well understood. Several pathways governed by the primary cilium, a sensory antenna present on most vertebrate cells, have been linked with fibrosis. Ciliopathies usually start early in life and represent a considerable disease burden. We performed massively parallel sequencing by using cohorts of genetically unsolved individuals with unexplained liver and kidney failure and correlated this with clinical, imaging, and histopathological analyses. Mechanistic studies were conducted with a vertebrate model and primary cells. We detected bi-allelic deleterious variants in TULP3, encoding a critical adaptor protein for ciliary trafficking, in a total of 15 mostly adult individuals, originating from eight unrelated families, with progressive degenerative liver fibrosis, fibrocystic kidney disease, and hypertrophic cardiomyopathy with atypical fibrotic patterns on histopathology. We recapitulated the human phenotype in adult zebrafish and confirmed disruption of critical ciliary cargo composition in several primary cell lines derived from affected individuals. Further, we show interaction between TULP3 and the nuclear deacetylase SIRT1, with roles in DNA damage repair and fibrosis, and report increased DNA damage ex vivo. Transcriptomic studies demonstrated upregulation of profibrotic pathways with gene clusters for hypertrophic cardiomyopathy and WNT and TGF-β signaling. These findings identify variants in TULP3 as a monogenic cause for progressive degenerative disease of major organs in which affected individuals benefit from early detection and improved clinical management. Elucidation of mechanisms crucial for DNA damage repair and tissue maintenance will guide novel therapeutic avenues for this and similar genetic and non-genomic diseases.

Integration of multiple imaging platforms to uncover cardiovascular defects in adult zebrafish.

AimsMammalian models have been instrumental in investigating adult heart function and human disease. However, electrophysiological differences with human hearts and high costs motivate the need for non-mammalian models. The zebrafish is a well-established genetic model to study cardiovascular development and function; however, analysis of cardiovascular phenotypes in adult specimens is particularly challenging as they are opaque.Methods and resultsHere, we optimized and combined multiple imaging techniques including echocardiography, magnetic resonance imaging, and micro-computed tomography to identify and analyse cardiovascular phenotypes in adult zebrafish. Using alk5a/tgfbr1a mutants as a case study, we observed morphological and functional cardiovascular defects that were undetected with conventional approaches. Correlation analysis of multiple parameters revealed an association between haemodynamic defects and structural alterations of the heart, as observed clinically.ConclusionWe report a new, comprehensive, and sensitive platform to identify otherwise indiscernible cardiovascular phenotypes in adult zebrafish.

Proteome profile of telencephalon associates attenuated neurogenesis with chronic stress induced mood disorder phenotypes in zebrafish model

Debilitating mental illness like depression and related mood disorders is due to the disruption in circuitry that controls emotion, motivation, and reward, characterized by disparate phenotypes like decrease in socialization, motivation, threshold for threat apprehension, etc. Chronic stress is a major factor in the etiology of these disorders. Here, using a chronic unpredictable stress (CUS) paradigm the characterization of an array of mood disorder phenotypes in adult zebrafish, in comparison to normal control unstressed fish, was achieved using a battery of behavioral assays including novel ones comprising social interaction test, feed approach test, threat response test and novel tank test. For the predictive validity of the model for mood disorders, the mitigative role of a slow (imipramine) and fast (ketamine) acting antidepressant was assessed. The molecular changes associated with CUS-induced mood disorder phenotype was investigated utilizing a high throughput method called isobaric tag for relative and absolute quantification (iTRAQ) in telencephalon, the region critically associated with the processing of emotional information in the fish brain. Out of 222 proteins identified to be significantly altered, 58 were differentially expressed across the stress and antidepressant-treatment groups at more than one fold (in log2) change. Of these proteins, some were implicated in earlier studies on mood disorders such as CABP1, PER2, mTOR, etc. The enrichment of altered proteins by Ingenuity Pathway Analysis (IPA) led us to mTOR and opioid signaling pathways, the top canonical pathways affected in the fish telencephalon. Interestingly, most of the pathways affected converge at the one controlling cell proliferation thus indicating altered neurogenesis, which was validated using immunohistochemistry for cell proliferation markers BrdU, SOX2, and BLBP. The study concludes that molecules that regulate telencephalon neural progenitor cell proliferation or neurogenesis are crucially involved in chronic stress-induced mood disorders by affecting the circuitry that controls emotion and reward.

Leptin induces muscle wasting in kras-driven hepatocellular carcinoma (HCC) model in zebrafish

ABSTRACTCancer cachexia affects up to 80% of patients with advanced solid cancer and leads to excessive muscle wasting. Here, using an inducible zebrafish hepatocellular carcinoma (HCC) model driven by oncogenic krasG12V, we observed a progressive muscle-wasting phenotype in adult zebrafish, characterized by significant loss of body weight and muscle fibers. By differential feeding, we observed that overfeeding caused fatty liver, accelerated carcinogenesis and muscle wasting. Interestingly, leptin, an obesity hormone, was upregulated in oncogenic hepatocytes and overfeeding groups. We also found that leptin expression progressively increased during human liver disease progression. By using leptin receptor (lepr)-knockout fish, we found that tumor fish in the lepr mutant background had a higher survival rate and significantly lower muscle-wasting level after tumor induction than the tumor fish in the wild-type background. Chemical inhibitors targeting leptin signaling also alleviated the muscle-wasting phenotype, indicating that leptin signaling may be a new therapeutic target for cancer patients with muscle wasting.

Understanding nociception-related phenotypes in adult zebrafish: Behavioral and pharmacological characterization using a new acetic acid model

Pain, a severely debilitating symptom of many human disorders, is a growing, unmet biomedical problem. Although the use of zebrafish (Danio rerio) to investigate both behavioral and physiological nociception-related responses is expanding rapidly, the characterization of behavioral phenotypes that reflect injury location is limited, making the results of such studies difficult to interpret. Here, we characterize putative nociception-related behavioral phenotypes in adult zebrafish following an intraperitoneal (i.p.) administration of acetic acid, a well-established protocol for visceral pain in rodents. Acetic acid (2.5 and 5.0%) induced an abdominal constriction-like response, which was assessed by measuring a body curvature index. Moreover, all doses tested (0.5–5.0%) reduced distance traveled and vertical activity in the novel tank test. Freezing duration increased following 5.0% acetic acid, whereas fish injected with 1.0, 2.5, and 5.0% spent more time in top area of the tank. Both morphine (an opioid analgesic) and diclofenac (a nonsteroidal anti-inflammatory drug, NSAID) prevented the 5.0% acetic acid-induced changes in body curvature index, whereas naloxone blocked these effects of morphine. Overall, zebrafish exposed to a single acetic acid i.p. injection display abnormal body curvature and specific changes in behavioral parameters sensitive to anti-nociceptive pharmacological modulation. We suggest that the abdominal constriction-like response represents a novel specific nociceptive-related phenotype in zebrafish. In general, our findings support the growing utility of zebrafish in translational pain research and antinociceptive drug discovery.

Transient Disruption of Adenosine Signaling During Embryogenesis Triggers a Pro-epileptic Phenotype in Adult Zebrafish.

Adenosinergic signaling has important effects on brain function, anatomy, and physiology in both late and early stages of development. Exposure to caffeine, a non-specific blocker of adenosine receptor, has been indicated as a developmental risk factor. Disruption of adenosinergic signaling during early stages of development can change the normal neural network formation and possibly lead to an increase in susceptibility to seizures. In this work, morpholinos (MO) temporarily blocked the translation of adenosine receptor transcripts, adora1, adora2aa, and adora2ab, during the embryonic phase of zebrafish. It was observed that the block of adora2aa and adora2aa + adora2ab transcripts increased the mortality rate and caused high rate of malformations. To test the susceptibility of MO adora1, MO adora2aa, MO adora2ab, and MO adora2aa + adora2ab animals to seizure, pentylenetetrazole (10mM) was used as a convulsant agent in larval and adult stages of zebrafish development. Although no MO promoted significant differences in latency time to reach the seizures stages in 7-day-old larvae, during the adult stage, all MO animals showed a decrease in the latency time to reach stages III, IV, and V of seizure. These results indicated that transient interventions in the adenosinergic signaling through high affinity adenosine receptors during embryonic development promote strong outcomes on survival and morphology. Additionally, long-term effects on neural development can lead to permanent impairment on neural signaling resulting in increased susceptibility to seizure.

Quantitative Responses of Adult Zebrafish to Changes in Ambient Illumination.

The use of zebrafish models to study central nervous system aging and late-onset neurological diseases will be facilitated by assays allowing rapid evaluation of neurological phenotypes in adult zebrafish. We analyzed groups of 12 adult zebrafish swimming simultaneously in single-animal arenas, and quantified their responses to changes in ambient illumination. Under these conditions, stereotypical locomotor patterns were observed and readily quantified using open source software. Continuous, low-velocity movements were observed during 10-min periods of darkness, whereas intermittent high-velocity movements occurred in bright light. At 80%-90% of abrupt light-to-dark or dark-to-light transitions, adult zebrafish produced a synchronous short-latency (20-22 ms) turn, followed by a propulsive movement with a high transient maximum velocity (400-500 mm/s). Between 5 and 35 months of age, latency increased by ∼10%, and peak velocity decreased by ∼30%, suggesting that the response declines in aged adults. Light transition responses can be measured rapidly and automatically in multiple adult zebrafish simultaneously, providing a convenient quantitative method for evaluating sensorimotor function in adult zebrafish models of neurological disease.

Acute embryonic anoxia exposure favours the development of a dominant and aggressive phenotype in adult zebrafish.

Eutrophication and climate change are increasing the incidence of severe hypoxia in fish nursery habitats, yet the programming effects of hypoxia on stress responsiveness in later life are poorly understood. In this study, to investigate whether early hypoxia alters the developmental trajectory of the stress response, zebrafish embryos were exposed to 4 h of anoxia at 36 h post-fertilization and reared to adults when the responses to secondary stressors were assessed. While embryonic anoxia did not affect basal cortisol levels or the cortisol response to hypoxia in later life, it had a marked effect on the responses to a social stressor. In dyadic social interactions, adults derived from embryonic anoxia initiated more chases, bit more often, entered fewer freezes and had lower cortisol levels. Adults derived from embryonic anoxia also performed more bites towards their mirror image, had lower gonadal aromatase gene expression and had higher testosterone levels. We conclude that acute embryonic anoxia has long-lasting consequences for the hormonal and behavioural responses to social interactions in zebrafish. Specifically, we demonstrate that acute embryonic anoxia favours the development of a dominant and aggressive phenotype, and that a disruption in sex steroid production may contribute to the programming effects of environmental hypoxia.

Germ cells are required to maintain a stable sexual phenotype in adult zebrafish

Sex in zebrafish is not determined by a major chromosomal locus, but instead relies on a mechanism that is influenced by a germ cell-derived signal, as animals that lack germ cells, or specifically oocytes, develop as phenotypic males. These data suggest that during primary sex determination, an oocyte-derived signal acts on the bipotential somatic gonad to promote the female-specific program. However, it is not known if germ cells are required only during the primary sex-determining window, or if they are required throughout adult life to maintain the female sexual phenotype. Here, we show that while wild-type zebrafish do not switch sex as adults, germ cell-depleted adult females readily convert to a male phenotype. Notably, when oocytes are depleted, but germline stem cells remain, adult females sex-revert to sperm-producing males, indicating that a germ cell-derived signal acts on the somatic gonad to promote female development directly or indirectly by repressing male-specific gene expression. These results also confirm that signals from the somatic gonad in turn ensure that the sex appropriate gamete is produced.

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.

Modeling seizure-related behavioral and endocrine phenotypes in adult zebrafish

Larval zebrafish ( Danio rerio) have recently been suggested as a high-throughput experimental model of epilepsy-related pathogenetic states. Here we use adult zebrafish to study behavioral symptoms associated with drug-evoked seizures. Experimental epilepsy-like states were evoked in zebrafish by exposure for 20 min to three chemoconvulsant drugs: caffeine (250 mg/L; 1.3 mM), pentylenetetrazole (1.5 g/L; 11.0 mM) and picrotoxin (100 mg/L; 0.17 mM). Fish behavior was analyzed using manual and video-tracking methods (Noldus Ethovision XT7). Compared to their respective controls, all three drug-treated groups showed robust seizure-like responses (hyperactivity bouts, spasms, circular and corkscrew swimming) accompanied by elevated whole-body cortisol levels (assessed by ELISA). In contrast, control fish did not display seizure-like behaviors and had significantly lower cortisol levels. Paralleling behavioral and endocrine phenotypes observed in clinical and rodent studies, our data implicates adult zebrafish as an emerging experimental model for epilepsy research.