Enantioselective Toxicity Effects of 2,2',3,5',6-Pentachloro Biphenyl (PCB-95) on Developing Brains in Zebrafish Larvae.

2,2',3,5',6-Pentachlorobiphenyl (PCB-95) is an environmentally relevant, chiral PCB congener that has been shown to act as a developmental neurotoxicant (DNT), targeting the developing brain. However, understanding enantioselective toxic effects for PCB-95 is in its infancy. To investigate these toxic effects, zebrafish embryos were exposed to racemates and enantiomers of PCB-95. Brain areas and pathology were studied. Results indicated dose dependent reduction of brain sizes with increased brain cell death in racemic and Ra (-)-PCB-95 treated groups. To provide a mechanistic basis for the observed neurotoxicity, gene expressions of antioxidant proteins such as Cu/Zn-SOD, Mn-SOD, and GPx were analysed. Antioxidant genes were up regulated with the PCB-95 exposure and racemic PCB-95 showed higher toxicity. These results suggest that the exposure to PCB-95 contributed to developmental neurotoxicity in early developing zebrafish larvae and may confer risks associated with enantioselective enrichment of PCB-95 in the environment.

RNA structure replaces the need for U2AF2 in splicing.

RNA secondary structure plays an integral role in catalytic, ribosomal, small nuclear, micro, and transfer RNAs. Discovering a prevalent role for secondary structure in pre-mRNAs has proven more elusive. By utilizing a variety of computational and biochemical approaches, we present evidence for a class of nuclear introns that relies upon secondary structure for correct splicing. These introns are defined by simple repeat expansions of complementary AC and GT dimers that co-occur at opposite boundaries of an intron to form a bridging structure that enforces correct splice site pairing. Remarkably, this class of introns does not require U2AF2, a core component of the spliceosome, for its processing. Phylogenetic analysis suggests that this mechanism was present in the ancestral vertebrate lineage prior to the divergence of tetrapods from teleosts. While largely lost from land dwelling vertebrates, this class of introns is found in 10% of all zebrafish genes.

Single stimulus learning in zebrafish larvae.

Learning about a moving visual stimulus was examined in zebrafish larvae using an automated imaging system and a t1-t2 design. In three experiments, zebrafish larvae were exposed to one of two inputs at t1 (either a gray bouncing disk or an identical but stationary disk) followed by a common test at t2 (the gray bouncing disk). Using 7days post-fertilization (dpf) larvae and 12 stimulus exposures, Experiment 1 established that these different treatments produced differential responding to the moving disk during testing. Larvae familiar with the moving test stimulus were significantly less likely to be still in its presence than larvae that had been exposed to the identical but stationary stimulus. Experiment 2 confirmed this result in 7dpf larvae and extended the finding to 5 and 6dpf larvae. Experiment 3 found differential responding to the moving test stimulus with 4 or 8 stimulus exposures but not with just one exposure in 7dpf larvae. These results provide evidence for learning in very young zebrafish larvae. The merits and challenges of the t1-t2 framework to study learning are discussed.

Behavioral effects of visual stimuli in adult zebrafish using a novel eight-tank imaging system.

Introduction: Animals respond to various environmental cues. Animal behavior is complex, and behavior analysis can greatly help to understand brain function. Most of the available behavioral imaging setups are expensive, provide limited options for customization, and allow for behavioral imaging of one animal at a time. Methods: The current study takes advantage of adult zebrafish as a model organism to study behavior in a novel behavioral setup allowing one to concurrently image 8 adult zebrafish. Results: Our results indicate that adult zebrafish show a unique behavioral profile in response to visual stimuli such as moving lines. In the presence of moving lines, the fish spent more time exploring the tank and spent more time toward the edges of the tanks. In addition, the fish moved and oriented themselves against the direction of the moving lines, indicating a negative optomotor response (OMR). With repeated exposure to moving lines, we observed a reduced optomotor response in adult zebrafish. Discussion: Our behavioral setup is relatively inexpensive, provides flexibility in the presentation of various animated visual stimuli, and offers improved throughput for analyzing behavior in adult zebrafish. This behavioral setup shows promising potential to quantify various behavioral measures and opens new avenues to understand complex behaviors.

Drug repurposing for neurodegenerative diseases using Zebrafish behavioral profiles.

Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer's disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders.

Combining supervised and unsupervised analyses to quantify behavioral phenotypes and validate therapeutic efficacy in a triple transgenic mouse model of Alzheimer's disease.

Behavioral testing is an essential tool for evaluating cognitive function and dysfunction in preclinical research models. This is of special importance in the study of neurological disorders such as Alzheimer's disease. However, the reproducibility of classic behavioral assays is frequently compromised by interstudy variation, leading to ambiguous conclusions about the behavioral markers characterizing the disease. Here, we identify age- and genotype-driven differences between 3xTg-AD and non-transgenic control mice using a low-cost, highly customizable behavioral assay that requires little human intervention. Through behavioral phenotyping combining both supervised and unsupervised behavioral classification methods, we are able to validate the preventative effects of the immunosuppressant cyclosporine A in a rodent model of Alzheimer's disease, as well as the partially ameliorating effects of candidate drugs nebivolol and cabozantinib.

Finding Drug Repurposing Candidates for Neurodegenerative Diseases using Zebrafish Behavioral Profiles.

Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer's disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders.

Zebrafish Larvae Position Tracker (Z-LaP Tracker): a high-throughput deep-learning behavioral approach for the identification of calcineurin pathway-modulating drugs using zebrafish larvae.

Brain function studies greatly depend on quantification and analysis of behavior. While behavior can be imaged efficiently, the quantification of specific aspects of behavior is labor-intensive and may introduce individual biases. Recent advances in deep learning and artificial intelligence-based tools have made it possible to precisely track individual features of freely moving animals in diverse environments without any markers. In the current study, we developed Zebrafish Larvae Position Tracker (Z-LaP Tracker), a modification of the markerless position estimation software DeepLabCut, to quantify zebrafish larval behavior in a high-throughput 384-well setting. We utilized the high-contrast features of our model animal, zebrafish larvae, including the eyes and the yolk for our behavioral analysis. Using this experimental setup, we quantified relevant behaviors with similar accuracy to the analysis performed by humans. The changes in behavior were organized in behavioral profiles, which were examined by K-means and hierarchical cluster analysis. Calcineurin inhibitors exhibited a distinct behavioral profile characterized by increased activity, acoustic hyperexcitability, reduced visually guided behaviors, and reduced habituation to acoustic stimuli. The developed methodologies were used to identify 'CsA-type' drugs that might be promising candidates for the prevention and treatment of neurological disorders.

An 8-cage imaging system for automated analyses of mouse behavior.

The analysis of mouse behavior is used in biomedical research to study brain function in health and disease. Well-established rapid assays allow for high-throughput analyses of behavior but have several drawbacks, including measurements of daytime behaviors in nocturnal animals, effects of animal handling, and the lack of an acclimation period in the testing apparatus. We developed a novel 8-cage imaging system, with animated visual stimuli, for automated analyses of mouse behavior in 22-h overnight recordings. Software for image analysis was developed in two open-source programs, ImageJ and DeepLabCut. The imaging system was tested using 4-5 month-old female wild-type mice and 3xTg-AD mice, a widely-used model to study Alzheimer's disease (AD). The overnight recordings provided measurements of multiple behaviors including acclimation to the novel cage environment, day and nighttime activity, stretch-attend postures, location in various cage areas, and habituation to animated visual stimuli. The behavioral profiles were different in wild-type and 3xTg-AD mice. AD-model mice displayed reduced acclimation to the novel cage environment, were hyperactive during the first hour of darkness, and spent less time at home in comparison to wild-type mice. We propose that the imaging system may be used to study various neurological and neurodegenerative disorders, including Alzheimer's disease.

Modulation of calcineurin signaling during development.

Calcineurin signaling pathways are suppressed in Down syndrome (trisomy 21), by overexpression of genes that are located on chromosome 21. Two key genes are the regulator of calcineurin 1 (RCAN1), also called the Down syndrome critical region 1 (DSCR1), and the dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). The suppressed calcineurin pathway may potentially be restored using small-molecule DYRK inhibitors, which have been proposed as therapeutics in Down syndrome. However, little is known about the benefits and risks of such treatments during various stages of embryonic development, fetal development, and childhood. We examined the modulation of calcineurin signaling during development, using zebrafish as a model system. To mimic suppressed calcineurin signaling in Down syndrome, zebrafish were exposed to the calcineurin inhibitors cyclosporine and tacrolimus during development. We found that suppression of calcineurin signaling changed specific larval behaviors, including activity and responses to acoustic and visual stimuli, depending on the period of exposure. Cotreatment with the DYRK inhibitor proINDY restored a few of these behaviors but also induced a range of adverse side effects including decreased activity and reduced optomotor responses to visual stimuli. Based on these results, we conclude that proINDY has limited benefits and substantial risks when used during development. We propose that zebrafish is an efficient model system for preliminary safety and efficacy tests of other DYRK inhibitors that aim to restore calcineurin signaling during neural development.

A zebrafish model for calcineurin-dependent brain function.

Small-molecule modulators of calcineurin signaling have been proposed as potential therapeutics in Down syndrome and Alzheimer's disease. Models predict that in Down syndrome, suppressed calcineurin-NFAT signaling may be mitigated by proINDY, which activates NFAT, the nuclear factor of activated T-cells. Conversely, elevated calcineurin signaling in Alzheimer's disease may be suppressed with the calcineurin inhibitors cyclosporine and tacrolimus. Such small-molecule treatments may have both beneficial and adverse effects. The current study examines the effects of proINDY, cyclosporine and tacrolimus on behavior, using zebrafish larvae as a model system. To suppress calcineurin signaling, larvae were treated with cyclosporine and tacrolimus. We found that these calcineurin inhibitors induced hyperactivity, suppressed visually-guided behaviors, acoustic hyperexcitability and reduced habituation to acoustic stimuli. To activate calcineurin-NFAT signaling, larvae were treated with proINDY. ProINDY treatment reduced activity and stimulated visually-guided behaviors, opposite to the behavioral changes induced by calcineurin inhibitors. The opposing effects suggest that activity and visually-guided behaviors are regulated by the calcineurin-NFAT signaling pathway. A central role of calcineurin-NFAT signaling is further supported by co-treatments of calcineurin inhibitors and proINDY, which had therapeutic effects on activity and visually-guided behaviors. However, these co-treatments adversely increased excitability, suggesting that some behaviors are regulated by other calcineurin signaling pathways. Overall, the developed methodologies provide an efficient high-throughput platform for the evaluation of modulators of calcineurin signaling that restore neural function, while avoiding adverse side effects, in a complex neural system.

Novel use of FDA-approved drugs identified by cluster analysis of behavioral profiles.

Repurposing FDA-approved drugs is an efficient and cost-effective approach in the development of therapeutics for a broad range of diseases. However, prediction of function can be challenging, especially in the brain. We screened a small-molecule library with FDA-approved drugs for effects on behavior. The studies were carried out using zebrafish larvae, imaged in a 384-well format. We found that various drugs affect activity, habituation, startle responses, excitability, and optomotor responses. The changes in behavior were organized in behavioral profiles, which were examined by hierarchical cluster analysis. One of the identified clusters includes the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506), which are immunosuppressants and potential therapeutics in the prevention of Alzheimer's disease. The calcineurin inhibitors form a functional cluster with seemingly unrelated drugs, including bromocriptine, tetrabenazine, rosiglitazone, nebivolol, sorafenib, cabozantinib, tamoxifen, meclizine, and salmeterol. We propose that drugs with 'CsA-type' behavioral profiles are promising candidates for the prevention and treatment of Alzheimer's disease.

Imaging escape and avoidance behavior in zebrafish larvae.

This review provides an overview of the assays that are used for measuring escape and avoidance behavior in zebrafish, with a specific focus on zebrafish larvae during the first week of development. Zebrafish larvae display a startle response when exposed to tactile, acoustic, or visual stimuli and will avoid dark areas, moving objects, conspecifics, and open spaces. Emotional states such as fear and anxiety might be induced when larvae are exposed to stimuli that they would normally escape from or avoid. Although these emotional states probably differ between species and change during development, much can be learned about human fear and anxiety using zebrafish as a model system. The molecular mechanisms of fear and anxiety are highly conserved in vertebrates and are present during early zebrafish development. Larvae during the first week of development display elevated cortisol levels in response to stress and are sensitive to the same anxiolytics that are used for the management of anxiety in humans. Zebrafish larvae are well suited for high-throughput analyses of behavior, and automated systems have been developed for imaging and analyzing the behavior of zebrafish larvae in multiwell plates. These high-throughput analyses will not only provide a wealth of information on the genes and environmental factors that influence escape and avoidance behaviors and the emotional states that might accompany them but will also facilitate the discovery of novel pharmaceuticals that could be used in the management of anxiety disorders in humans.

Embryonic Exposure to 2,2',3,5',6-pentachlorobiphenyl (PCB-95) Causes Developmental Malformations in Zebrafish.

2,2',3,5',6-Pentachlorobiphenyl (PCB-95) is an environmental neurotoxicant. There is accumulated evidence that some neurotoxic effects of PCB-95 are caused by increased spontaneous Ca2+ oscillations in neurons resulting from modifying ryanodine receptors (RyR) in calcium-releasing channels. However, there are large gaps in explaining brain and other developmental malformations on embryonic PCB-95 exposure. In the present study, we address those deficiencies by studying the toxic effects of PCB-95 using zebrafish as an ontogenetic model. To characterize these effects, zebrafish embryos with intact chorions were exposed to 4 different concentrations of PCB-95 (0.25, 0.5, 0.75, and 1 ppm) for 3 consecutive days. The controls were maintained in 0.5 × E2 medium or egg water and in 0.1% (v/v) dimethyl sulfoxide (DMSO)/0.5 × E2 medium or egg water. PCB-95-treated groups showed dose-dependent decreases in survival and hatching rates, with increased rates of developmental malformations when compared to controls. These include morphological malformations, brain cell necrosis, and smaller eye sizes at 5 d post fertilization. These data suggest potential mechanisms underlying the abnormal behavior observed in a visual stimulus assay. The present study provides insight into PCB-95-induced developmental toxicity and supports the use of the zebrafish model in understanding the effects of PCB-95 exposure. Environ Toxicol Chem 2019;39:162-170. © 2019 SETAC.

The oxidative burst at fertilization is dependent upon activation of the dual oxidase Udx1.

The sea urchin egg is a quiescent cell...until fertilization, when the egg is activated. The classic respiratory burst at fertilization is the result of prodigious hydrogen peroxide production, but the mechanism for this synthesis is not known. Here we quantitate the kinetics of hydrogen peroxide synthesis at a single-cell level using an imaging photon detector, showing that 60 nM hydrogen peroxide accumulates within the perivitelline space of each zygote. We find that the NADPH oxidation activity is enriched at the cell surface and is sensitive to a pharmacological inhibitor of NADPH oxidase enzymes. Finally, we show that a sea urchin dual oxidase homolog, Udx1, is responsible for generating the hydrogen peroxide necessary for the physical block to polyspermy. Phylogenetic analysis of the enzymatic modules in Udx1 suggests a potentially conserved role for the dual oxidase family in hydrogen peroxide production and regulation during fertilization.

Analysis of vertebrate vision in a 384-well imaging system.

Visual impairment affects 253 million people worldwide and new approaches for prevention and treatment are urgently needed. While small molecules with potential beneficial effects can be examined in various model systems, the in vivo evaluation of visual function remains a challenge. The current study introduces a novel imaging system for measuring visually-guided behaviors in larval zebrafish. The imaging system is the first to image four 96-well plates with a single camera for automated measurements of activity in a 384-well format. In addition, it is the first system to project moving visual stimuli and analyze the optomotor response in the wells of a 96-well plate. We found that activity is affected by tricaine, diazepam and flumazenil. Surprisingly, diazepam treatments induce a loss of visual responses, at concentrations that do not affect activity or induce hyperactivity. Overall, our studies show that the developed imaging system is suitable for automated measurements of vertebrate vision in a high-throughput format.

Cluster analysis profiling of behaviors in zebrafish larvae treated with antidepressants and pesticides.

Antidepressants are used by a substantial number of women in their childbearing years. Treatment may continue during pregnancy, since untreated depression poses a risk to the mother and child. However, many antidepressants readily pass through the placental barrier to reach the fetus or may be ingested by the newborn via breastmilk. Little is known about the effects of antidepressants on brain development and subsequent behavior in young children. In the current study, we used zebrafish as a model system to examine the neurodevelopmental effects of three commonly prescribed antidepressants, sertraline, duloxetine and bupropion. Zebrafish were exposed to these antidepressants during development and were examined for changes in larval avoidance behavior, activity, social behaviors, and anxiety-related behaviors. The results show that antidepressants commonly affect larval swim speeds and resting, and differentially affect other behaviors depending upon the exposure period. Using cluster analysis profiling, we compared the obtained results to previous reports on behavioral defects induced by organophosphate pesticides. We found that the behavioral profiles induced by antidepressants and pesticides overlap, indicating a common mechanism of action. We conclude that developmental antidepressant exposures lead to specific behavioral changes in zebrafish larvae. At present, it is not known if antidepressants have similar effects in human development.

The loss and recovery of vertebrate vision examined in microplates.

Regenerative medicine offers potentially ground-breaking treatments of blindness and low vision. However, as new methodologies are developed, a critical question will need to be addressed: how do we monitor in vivo for functional success? In the present study, we developed novel behavioral assays to examine vision in a vertebrate model system. In the assays, zebrafish larvae are imaged in multiwell or multilane plates while various red, green, blue, yellow or cyan objects are presented to the larvae on a computer screen. The assays were used to examine a loss of vision at 4 or 5 days post-fertilization and a gradual recovery of vision in subsequent days. The developed assays are the first to measure the loss and recovery of vertebrate vision in microplates and provide an efficient platform to evaluate novel treatments of visual impairment.

Effects of embryonic exposure to polychlorinated biphenyls (PCBs) on larval zebrafish behavior.

Developmental disorders such as anxiety, autism, and attention deficit hyperactivity disorders have been linked to exposure to polychlorinated biphenyls (PCBs), a ubiquitous anthropogenic pollutant. The zebrafish is widely recognized as an excellent model system for assessing the effects of toxicant exposure on behavior and neurodevelopment. In the present study, we examined the effect of sub-chronic embryonic exposure to the PCB mixture, Aroclor (A) 1254 on anxiety-related behaviors in zebrafish larvae at 7 days post-fertilization (dpf). We found that exposure to low concentrations of A1254, from 2 to 26 h post-fertilization (hpf) induced specific behavioral defects in two assays. In one assay with intermittent presentations of a moving visual stimulus, 5 ppm and 10 ppm PCB-exposed larvae displayed decreased avoidance behavior but no significant differences in thigmotaxis or freezing relative to controls. In the other assay with intermittent presentations of a moving visual stimulus and a stationary visual stimulus, 5 ppm and 10 ppm PCB-exposed larvae had elevated baseline levels of thigmotaxis but no significant differences in avoidance behavior relative to controls. The 5 ppm larvae also displayed higher terminal levels of freezing relative to controls. Collectively, our results show that exposure to ecologically valid PCB concentrations during embryonic development can induce functional deficits and alter behavioral responses to a visual threat.

Effects of embryonic exposure to polychlorinated biphenyls (PCBs) on anxiety-related behaviors in larval zebrafish.

The zebrafish (Danio rerio) is an excellent model system for assessing the effects of toxicant exposure on behavior and neurodevelopment. In the present study, we examined the effects of sub-chronic embryonic exposure to polychlorinated biphenyls (PCBs), a ubiquitous anthropogenic pollutant, on anxiety-related behaviors. We found that exposure to the PCB mixture, Aroclor (A) 1254, from 2 to 26h post-fertilization (hpf) induced two statistically significant behavioral defects in larvae at 7 days post-fertilization (dpf). First, during 135min of free swimming, larvae that had been exposed to 2ppm, 5ppm or 10ppm A1254 exhibited enhanced thigmotaxis (edge preference) relative to control larvae. Second, during the immediately ensuing 15-min visual startle assay, the 5ppm and 10ppm PCB-exposed larvae reacted differently to a visual threat, a red 'bouncing' disk, relative to control larvae. These results are consistent with the anxiogenic and attention-disrupting effects of PCB exposure documented in children, monkeys and rodents and merit further investigation.