Publications by authors named "Robbert Creton"

29 Publications

  • Page 1 of 1

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

Sci Rep 2019 Sep 27;9(1):13989. Epub 2019 Sep 27.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.

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.
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http://dx.doi.org/10.1038/s41598-019-50372-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764987PMC
September 2019

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

Environ Toxicol Chem 2020 01;39(1):162-170

Environmental Toxicology Program, Clemson University, Clemson, South Carolina, USA.

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 Ca 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.
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http://dx.doi.org/10.1002/etc.4587DOI Listing
January 2020

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

Neurotoxicol Teratol 2018 Sep - Oct;69:54-62. Epub 2017 Oct 31.

Brown University, Department of Molecular and Cellular Biology and Biochemistry, Providence, RI 02912, United States.

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.
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http://dx.doi.org/10.1016/j.ntt.2017.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930167PMC
October 2019

The loss and recovery of vertebrate vision examined in microplates.

PLoS One 2017 17;12(8):e0183414. Epub 2017 Aug 17.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183414PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560659PMC
October 2017

Phenotypic Plasticity Regulates Candida albicans Interactions and Virulence in the Vertebrate Host.

Front Microbiol 2016 26;7:780. Epub 2016 May 26.

Department of Molecular Microbiology and Immunology, Brown University Providence, RI, USA.

Phenotypic diversity is critical to the lifestyles of many microbial species, enabling rapid responses to changes in environmental conditions. In the human fungal pathogen Candida albicans, cells exhibit heritable switching between two phenotypic states, white and opaque, which yield differences in mating, filamentous growth, and interactions with immune cells in vitro. Here, we address the in vivo virulence properties of the two cell states in a zebrafish model of infection. Multiple attributes were compared including the stability of phenotypic states, filamentation, virulence, dissemination, and phagocytosis by immune cells, and phenotypes equated across three different host temperatures. Importantly, we found that both white and opaque cells could establish a lethal systemic infection. The relative virulence of the two cell types was temperature dependent; virulence was similar at 25°C, but at higher temperatures (30 and 33°C) white cells were significantly more virulent than opaque cells. Despite the difference in virulence, fungal burden, and dissemination were similar between cells in the two states. Additionally, both white and opaque cells exhibited robust filamentation during infection and blocking filamentation resulted in decreased virulence, establishing that this program is critical for pathogenesis in both cell states. Interactions between C. albicans cells and immune cells differed between white and opaque states. Macrophages and neutrophils preferentially phagocytosed white cells over opaque cells in vitro, and neutrophils showed preferential phagocytosis of white cells in vivo. Together, these studies distinguish the properties of white and opaque cells in a vertebrate host, and establish that the two cell types demonstrate both important similarities and key differences during infection.
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http://dx.doi.org/10.3389/fmicb.2016.00780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880793PMC
June 2016

An asymptomatic mutation complicating severe chemotherapy-induced peripheral neuropathy (CIPN): a case for personalised medicine and a zebrafish model of CIPN.

NPJ Genom Med 2016 8;1:16016. Epub 2016 Jun 8.

Department of Pediatrics, Division of Pediatric Hematology-Oncology, Hasbro Children's Hospital and The Warren Alpert Medical School at Brown University, Providence, RI, USA.

Targeted next-generation sequencing (NGS) identified a novel loss of function mutation in , a gene linked to Charcot-Marie-Tooth disease (CMT), in a paediatric acute lymphoblastic leukaemia patient with severe chemotherapy-induced peripheral neuropathy (CIPN) due to vincristine. The patient was clinically asymptomatic, and lacked a family history of neuropathy. The effect of the mutation was modelled in a zebrafish knockdown system that recapitulated the symptoms of the patient both prior to and after treatment with vincristine. Confocal microscopy of pre- and post-synaptic markers revealed that the GARS knockdown results in changes to peripheral motor neurons, acetylcholine receptors and their co-localisation in neuromuscular junctions (NMJs), whereas a sensitive and reproducible stimulus-response assay demonstrated that the changes correlating with the GARS mutation in themselves fail to produce peripheral neuropathy symptoms. However, with vincristine treatment the GARS knockdown exacerbates decreased stimulus response and NMJ lesions. We propose that there is substantial benefit in the use of a targeted NGS screen of cancer patients who are to be treated with microtubule targeting agents for deleterious mutations in CMT linked genes, and for the screening in zebrafish of reagents that might inhibit CIPN.
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http://dx.doi.org/10.1038/npjgenmed.2016.16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685301PMC
June 2016

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

Neurotoxicology 2016 Mar 31;53:93-101. Epub 2015 Dec 31.

Department of Cognitive, Linguistic & Psychological Sciences, Brown University, Providence, Rhode Island, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.neuro.2015.12.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808333PMC
March 2016

RNA structure replaces the need for U2AF2 in splicing.

Genome Res 2016 Jan 13;26(1):12-23. Epub 2015 Nov 13.

Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA; Center for Computational Molecular Biology, Brown University, Providence, Rhode Island 02912, USA;

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.
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http://dx.doi.org/10.1101/gr.181008.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691745PMC
January 2016

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

Neurotoxicol Teratol 2016 Jan-Feb;53:1-10. Epub 2015 Nov 10.

Department of Cognitive, Linguistic & Psychological Sciences, Brown University, Providence, Rhode Island, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ntt.2015.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703504PMC
October 2016

Chlorpyrifos and malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity.

Neurotoxicology 2015 Jul 14;49:50-8. Epub 2015 May 14.

Brown University, Department of Molecular and Cellular Biology and Biochemistry, Providence, RI 02912, United States.

Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size.
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http://dx.doi.org/10.1016/j.neuro.2015.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4523399PMC
July 2015

Effects of embryonic cyclosporine exposures on brain development and behavior.

Behav Brain Res 2015 Apr 13;282:117-24. Epub 2015 Jan 13.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Electronic address:

Cyclosporine, a calcineurin inhibitor, is successfully used as an immunosuppressant in transplant medicine. However, the use of this pharmaceutical during pregnancy is concerning since calcineurin is thought to play a role in neural development. The risk for human brain development is difficult to evaluate because of a lack of basic information on the sensitive developmental times and the potentially pleiotropic effects on brain development and behavior. In the present study, we use zebrafish as a model system to examine the effects of embryonic cyclosporine exposures. Early embryonic exposures reduced the size of the eyes and brain. Late embryonic exposures did not affect the size of the eyes or brain, but did lead to substantial behavioral defects at the larval stages. The cyclosporine-exposed larvae displayed a reduced avoidance response to visual stimuli, low swim speeds, increased resting, an increase in thigmotaxis, and changes in the average distance between larvae. Similar results were obtained with the calcineurin inhibitor FK506, suggesting that most, but not all, effects on brain development and behavior are mediated by calcineurin inhibition. Overall, the results show that cyclosporine can induce either structural or functional brain defects, depending on the exposure window. The observed functional brain defects highlight the importance of quantitative behavioral assays when evaluating the risk of developmental exposures.
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http://dx.doi.org/10.1016/j.bbr.2015.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323675PMC
April 2015

High-throughput analysis of behavior in zebrafish larvae: effects of feeding.

Zebrafish 2014 Oct 25;11(5):455-61. Epub 2014 Aug 25.

1 Department of Molecular Biology, Cell Biology and Biochemistry, Brown University , Providence, Rhode Island.

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. Identification of these factors is facilitated by high-throughput analyses of behavior in zebrafish larvae, which can be imaged in multiwell or multilane plates. However, the nutritional needs of zebrafish larvae during the behavioral experiments are not fully understood. Zebrafish larvae begin feeding between 4 and 5 days postfertilization (dpf), but can live solely on nutrients derived from the yolk until at least 7 dpf. To examine whether feeding affects behavior, we measured a broad range of behaviors with and without feeding at 5, 6, and 7 dpf. We found that feeding did not have a significant effect on behavior in 5-day-old larvae. In contrast, fed 6- and 7-day-old larvae displayed increased avoidance responses to visual stimuli, increased swim speeds, and decreased resting in comparison to unfed larvae. In addition, the fed 7-day-old larvae displayed a decrease in thigmotaxis and a decrease in the distance between larvae in the presence of visual stimuli. Thus, feeding affects a range of behaviors in 6- and 7-day-old larvae. We conclude that 5-day-old larvae are well-suited for high-throughput analyses of behavior, since effects of feeding can be avoided at this time. For high-throughput analyses of behavior in older larvae, standard feeding protocols need to be developed.
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http://dx.doi.org/10.1089/zeb.2014.0989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172468PMC
October 2014

Single stimulus learning in zebrafish larvae.

Neurobiol Learn Mem 2014 Feb 6;108:145-54. Epub 2013 Sep 6.

Department of Cognitive, Linguistic, and Psychological Sciences, Brown University, Providence, RI, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.nlm.2013.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946257PMC
February 2014

Automated high-throughput behavioral analyses in zebrafish larvae.

J Vis Exp 2013 Jul 4(77):e50622. Epub 2013 Jul 4.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University.

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.
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http://dx.doi.org/10.3791/50622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731428PMC
July 2013

Developmental sub-chronic exposure to chlorpyrifos reduces anxiety-related behavior in zebrafish larvae.

Neurotoxicol Teratol 2012 Jul 8;34(4):458-65. Epub 2012 May 8.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.

Neurobehavioral disorders such as anxiety, autism, and attention deficit hyperactivity disorders are typically influenced by genetic and environmental factors. Although several genetic risk factors have been identified in recent years, little is known about the environmental factors that either cause neurobehavioral disorders or contribute to their progression in genetically predisposed individuals. One environmental factor that has raised concerns is chlorpyrifos, an organophosphate pesticide that is widely used in agriculture and is found ubiquitously in the environment. In the present study, we examined the effects of sub-chronic chlorpyrifos exposure on anxiety-related behavior during development using zebrafish larvae. We found that sub-chronic exposure to 0.01 or 0.1 μM chlorpyrifos during development induces specific behavioral defects in 7-day-old zebrafish larvae. The larvae displayed decreases in swim speed and thigmotaxis, yet no changes in avoidance behavior were seen. Exposure to 0.001 μM chlorpyrifos did not affect swimming, thigmotaxis, or avoidance behavior and exposure to 1 μM chlorpyrifos induced behavioral defects, but also induced defects in larval morphology. Since thigmotaxis, a preference for the edge, is an anxiety-related behavior in zebrafish larvae, we propose that sub-chronic chlorpyrifos exposure interferes with the development of anxiety-related behaviors. The results of this study provide a good starting point for examination of the molecular, cellular, developmental, and neural mechanisms that are affected by environmentally relevant concentrations of organophosphate pesticides. A more detailed understanding of these mechanisms is important for the development of predictive models and refined health policies to prevent toxicant-induced neurobehavioral disorders.
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http://dx.doi.org/10.1016/j.ntt.2012.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573706PMC
July 2012

Imaging escape and avoidance behavior in zebrafish larvae.

Rev Neurosci 2011 ;22(1):63-73

Department of Psychology, Brown University, Providence, RI 02912, USA.

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.
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http://dx.doi.org/10.1515/RNS.2011.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092434PMC
July 2011

A novel high-throughput imaging system for automated analyses of avoidance behavior in zebrafish larvae.

Behav Brain Res 2011 Sep 28;223(1):135-44. Epub 2011 Apr 28.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA.

Early brain development can be influenced by numerous genetic and environmental factors, with long-lasting effects on brain function and behavior. The identification of these factors is facilitated by recent innovations in high-throughput screening. However, large-scale screening in whole organisms remains challenging, in particular when studying changes in brain function or behavior in vertebrate model systems. In this study, we present a novel imaging system for high-throughput analyses of behavior in zebrafish larvae. The three-camera system can image 12 multiwell plates simultaneously and is unique in its ability to provide local visual stimuli in the wells of a multiwell plate. The acquired images are converted into a series of coordinates, which characterize the location and orientation of the larvae. The developed imaging techniques were tested by measuring avoidance behaviors in seven-day-old zebrafish larvae. The system effectively quantified larval avoidance and revealed an increased edge preference in response to a blue or red 'bouncing ball' stimulus. Larvae also avoid a bouncing ball stimulus when it is counter-balanced with a stationary ball, but do not avoid blinking balls counter-balanced with a stationary ball. These results indicate that the seven-day-old larvae respond specifically to movement, rather than color, size, or local changes in light intensity. The imaging system and assays for measuring avoidance behavior may be used to screen for genetic and environmental factors that cause developmental brain disorders and for novel drugs that could prevent or treat these disorders.
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http://dx.doi.org/10.1016/j.bbr.2011.04.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111907PMC
September 2011

Locomotor behaviors in zebrafish (Danio rerio) larvae.

Behav Processes 2011 Feb 13;86(2):222-9. Epub 2010 Dec 13.

Department of Psychology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA. Ruth

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.
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http://dx.doi.org/10.1016/j.beproc.2010.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063417PMC
February 2011

Automated analysis of behavior in zebrafish larvae.

Authors:
Robbert Creton

Behav Brain Res 2009 Oct 3;203(1):127-36. Epub 2009 May 3.

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Box G-L127, 185 Meeting Street, Providence, RI 02912, USA.

Zebrafish larvae have become a popular model system to examine genetic and environmental factors that affect behavior. However, studying complex behavior in large numbers of fish larvae can be challenging. The present study describes a novel high-resolution imaging system that is unique in its ability to automatically analyze the location and orientation of zebrafish larvae in multiwell plates. The system revealed behaviors in zebrafish larvae that would have been missed by more manual approaches, including a preference to face a threatening stimulus from a distance and a clockwise orientation in a two-fish assay. The clockwise orientation of the larvae correlates with a clockwise orientation of molecular structures during early development. Larvae with reversed embryonic asymmetries display a counter-clockwise orientation in the two-fish assay, suggesting that embryonic asymmetry and chiral behavior are regulated by the same developmental mechanisms. The developed imaging techniques may be used in large-scale screens to identify genes, pharmaceuticals, and environmental toxicants that influence complex behaviors.
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http://dx.doi.org/10.1016/j.bbr.2009.04.030DOI Listing
October 2009

Suppression of the endoplasmic reticulum calcium pump during zebrafish gastrulation affects left-right asymmetry of the heart and brain.

Mech Dev 2008 May-Jun;125(5-6):396-410. Epub 2008 Feb 20.

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Box G-L127, 185 Meeting Street, Providence, RI 02912, USA.

Vertebrate embryos generate striking Ca(2+) patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca(2+) during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca(2+) along the dorsal-ventral axis, with higher Ca(2+) concentrations in the ventral margin and lower Ca(2+) concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca(2+) pump with 0.5 microM thapsigargin elevates cytosolic Ca(2+) in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left-right asymmetry. Brain defects include a left-right reversal of pitx2 expression in the dorsal diencephalon and a left-right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca(2+) pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca(2+) pump during gastrulation inhibits expression of no tail (ntl) and left-right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer's vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca(2+) plays a role in Kupffer's vesicle function, influencing ciliary motility and translating the vesicle's counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca(2+) plays an additional role in the formation of Kupffer's vesicle.
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http://dx.doi.org/10.1016/j.mod.2008.02.004DOI Listing
August 2008

Analysis of Kupffer's vesicle in zebrafish embryos using a cave automated virtual environment.

Dev Dyn 2007 Jul;236(7):1963-9

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.

Previous studies show that cilia in Kupffer's vesicle (KV) generate a counterclockwise flow of fluid and provide convincing evidence that this flow regulates left/right asymmetry. We hypothesized that the distribution of cilia in KV leads to this directional flow. However, there are limitations in determining the localization of structures when viewing a three-dimensional (3-D) image on a 2-D computer screen. We analyzed the distribution of KV cilia in the Cave, an immersive virtual environment that displays stacks of confocal images in 3-D. We found 80% of the cilia are located on the dorsal surface and 20% were located on the ventral surface of the vesicle. We confirmed the ventral location of some cilia by electron microscopy. There is an asymmetrical distribution of cilia on the dorsal surface, with the anterior one third containing 50% and the posterior one third containing 20% of the cilia. This dorsal-anterior patch could explain the directionality of the flow, and could drive local differences in flow rate.
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http://dx.doi.org/10.1002/dvdy.21191DOI Listing
July 2007

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

Dev Cell 2004 Dec;7(6):801-14

Department of Molecular Biology, Cell Biology, and Biochemistry, Box G, Brown University, Providence, RI 02912 USA.

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.
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http://dx.doi.org/10.1016/j.devcel.2004.10.014DOI Listing
December 2004

The calcium pump of the endoplasmic reticulum plays a role in midline signaling during early zebrafish development.

Authors:
Robbert Creton

Brain Res Dev Brain Res 2004 Jul;151(1-2):33-41

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, box G-B187, Providence, RI 02912, USA.

During early vertebrate development, a signaling network is activated along the midline of the embryo. This signaling network induces the neural tube floor plate and ventral brain regions. In turn, induction of the ventral brain region is important for bilateral division of the forebrain and bilateral separation of the eyes. The present study provides direct evidence for a role of the endoplasmic reticulum Ca(2+) pump in zebrafish midline signaling. The endoplasmic reticulum Ca(2+) pump was inhibited in zebrafish embryos using thapsigargin or cyclopiazonic acid. Inhibition of the endoplasmic reticulum Ca(2+) pump during early gastrulation induces cyclopia, mimicking defects observed in cyclops, squint, one-eyed pinhead, and silberblick mutant embryos. In contrast, inhibition of the endoplasmic reticulum Ca(2+) pump during mid-gastrulation does not induce cyclopia, but does induce tail defects, mimicking defects observed in no-tail mutant embryos. This study is the first to relate thapsigargin and cyclopiazonic acid with induction of cyclopia. In addition, obtained results provide new information on the roles of Ca(2+) in embryonic development and may lead to new insights on the mechanisms underlying holoprosencephaly, a relatively common brain defect in human development.
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http://dx.doi.org/10.1016/j.devbrainres.2004.03.016DOI Listing
July 2004

Retinoic acid modulates the pattern of cell division in embryos of Lymnaea stagnalis (Mollusca).

Rouxs Arch Dev Biol 1994 Oct;204(1):70-74

Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

All-trans retinoic acid is well known as a modulator of positional specification in vertebrate development. A similar mechanism may operate in molluscan development. Molluscan development is characterized by an invariant pattern of cell divisions, which allows the study of individual cells in the developing organism. Low concentrations of exogenous retinoic acid applied during gastrulation affect the cell division pattern in the early larval stage of the mollusc Lymnaea stagnalis. A few cells from the apical plate, a larval organ consisting of seven large cleavage-arrested cells, were induced by retinoic acid to resume cell division. They typically formed an area of proliferating small cells that resembles the adjacent areas of precursor cells of adult ectoderm. The identification of individual cells that are transformed by retinoic acid may provide new insights into the mechanisms underlying positional specification within the embryo.
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http://dx.doi.org/10.1007/BF00189070DOI Listing
October 1994

Retinoic acid modulates the pattern of cell division in embryos ofLymnaea stagnalis (Mollusca).

Rouxs Arch Dev Biol 1994 Jan;204(1):70-74

Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

All-trans retinoic acid is well known as a modulator of positional specification in vertebrate development. A similar mechanism may operate in molluscan development. Molluscan development is characterized by an invariant pattern of cell divisions, which allows the study of individual cells in the developing organism. Low concentrations of exogenous retinoic acid applied during gastrulation affect the cell division pattern in the early larval stage of the molluscLymnaea stagnalis. A few cells from the apical plate, a larval organ consisting of seven large cleavage-arrested cells, were induced by retinoic acid to resume cell division. They typically formed an area of proliferating small cells that resembles the adjacent areas of precursor cells of adult ectoderm. The identification of individual cells that are transformed by retinoic acid may provide new insights into the mechanisms underlying positional specification within the embryo.
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http://dx.doi.org/10.1007/BF00744875DOI Listing
January 1994

Manipulation of cytokinesis affects polar ionic current around the egg of Lymnaea stagnalis.

Rouxs Arch Dev Biol 1992 Oct;201(6):346-353

Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

The fertilized egg of the mollusc Lymnaea stagnalis generates a polarized current pattern as measured with the vibrating probe. Here we investigated the basis of these polar ionic currents. Ionic currents were measured around eggs during the second meiotic division after interference with cytokinesis. Cytokinesis was either displaced by centrifugation or inhibited with cytochalasin or nocodazole. Furthermore, ectopic constrictions were induced with lectin treatment. It appeared that the inward current of the animal pole can be displaced by centrifugation and remains associated with the position of the meiotic apparatus. The influence of the meiotic apparatus on the polar current pattern seems to be directly related to membrane constrictions rather than to karyokinesis. This was demonstrated by a change in current density after induction of an ectopic constriction at the vegetal pole and by the abolishment of currents after cytochalasin treatment. Since the location of the outward current was not sensitive to centrifugation, it may be concluded that the vegetal outward current depends upon properties of the vegetal cortex. On the basis of these results, we conclude that the Lymnaea egg generates two types of ionic currents during the second meiotic division. The first is an inward current activated at the site of membrane constrictions. The second is an outward current associated with the vegetal cortex.
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http://dx.doi.org/10.1007/BF00365122DOI Listing
October 1992

Localized activity of Ca-stimulated ATPase and transcellular ionic currents during mesoderm induction in embryos ofLymnaea stagnalis (Mollusca).

Rouxs Arch Dev Biol 1991 Nov;200(6):320-329

Department of Experimental Zoology, University of Utrecht, Padualaan 8, NL-3584, Utrecht, CT, The Netherlands.

InLymnaea stagnalis, mesoderm induction occurs at the 24-cell stage, when the apical tip of the macromere 3D establishes a close contact with a number of micromeres. Via its tip, the macromere 3D is supposed to receive an inductive signal from the micromeres, resulting in the determination of the mesodermal stem cell 4d at the next division. In view of the possibility that transcellular ionic currents might somehow be involved, either in the processes that bring about this particular configuration of blastomeres or in the induction process itself, we mapped the electric field around the embryo during the 24-cell stage, using a vibrating probe. We detected a reversal of the current direction as compared to the uncleaved egg, whilst the polarity of the field along the animal-vegetal axis was maintained. We also mapped the localization of Ca-stimulated AT-Pase, an enzyme that drives the Ca-efflux from the cell. We found that this enzyme is localized exclusively along the cytoplasmic face of the apical plasma membrane of macromere 3D, and that its presence is restricted to the period from 110 to 135 min after the fifth cleavage, when there is close contact between macormere 3D and the micromeres. Since the localization of the Ca-stimulated ATPase coincides both in time and space with the induction of the mesoderm-mother cell, we suggest that localized calcium fluxes may play a role in this induction process.
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http://dx.doi.org/10.1007/BF00665527DOI Listing
November 1991

Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca/Mg ATPase activity during early cleavages of Lymnaea stagnalis embryos.

Rouxs Arch Dev Biol 1991 Aug;200(3):120-131

Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca-stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 μA/cm have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca/Mg ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca-stimulated ATPase. The ionic currents and Ca-stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca-stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca-stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.
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http://dx.doi.org/10.1007/BF00190231DOI Listing
August 1991

Polar localization of plasma membrane Ca/Mg ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca).

Rouxs Arch Dev Biol 1990 Nov;199(3):134-145

Department of Experimental Zoology, University of Utrecht, Padualaan 8, Utrecht, The Netherlands.

During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca /Mg ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca /Mg ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca-stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.
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http://dx.doi.org/10.1007/BF01681486DOI Listing
November 1990