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author:("kozel, David")
1.  An In Vivo Zebrafish Screen Identifies Organophosphate Antidotes with Diverse Mechanisms of Action 
Journal of biomolecular screening  2012;18(1):108-115.
Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry–based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.
doi:10.1177/1087057112458153
PMCID: PMC4053346  PMID: 22960781
Xenopus; zebrafish; in vivo screening; mass spectrometry; high-content screening
2.  cables1 Is Required for Embryonic Neural Development: Molecular, Cellular, and Behavioral Evidence From the Zebrafish 
SUMMARY
In vitro studies have suggested that the Cables1 gene regulates epithelial cell proliferation, whereas other studies suggest a role in promoting neural differentiation. In efforts to clarify the functions of Cables1 in vivo, we conducted gain- and loss-of-function studies targeting its ortholog (cables1) in the zebrafish embryo. Similar to rodents, zebrafish cables1 mRNA expression is detected most robustly in embryonic neural tissues. Antisense knockdown of cables1 leads to increased numbers of apoptotic cells, particularly in brain tissue, in addition to a distinct behavioral phenotype, characterized by hyperactivity in response to stimulation. Apoptosis and the behavioral abnormality could be rescued by co-expression of a morpholino-resistant cables1 construct. Suppression of p53 expression in cables1 morphants partially rescued both apoptosis and the behavioral phenotype, suggesting that the phenotype of cables1 morphants is due in part to p53-dependent apoptosis. Alterations in the expression patterns of several neural transcription factors were observed in cables1 morphants during early neurulation, suggesting that cables1 is required for early neural differentiation. Ectopic overexpression of cables1 strongly disrupted embryonic morphogenesis, while overexpression of a cables1 mutant lacking the C-terminal cyclin box had little effect, suggesting functional importance of the cyclin box. Lastly, marked reductions in p35, but not Cdk5, were observed in cables1 morphants. Collectively, these data suggest that cables1 is important for neural differentiation during embryogenesis, in a mechanism that likely involves interactions with the Cdk5/p35 kinase pathway.
doi:10.1002/mrd.21263
PMCID: PMC4016823  PMID: 21268180
3.  Photochemical activation of TRPA1 channels in neurons and animals 
Nature chemical biology  2013;9(4):257-263.
Optogenetics is a powerful research tool because it enables high-resolution optical control of neuronal activity. However, current optogenetic approaches are limited to transgenic systems expressing microbial opsins and other exogenous photoreceptors. Here, we identify optovin, a small molecule that enables repeated photoactivation of motor behaviors in wild type animals. Surprisingly, optovin's behavioral effects are not visually mediated. Rather, photodetection is performed by sensory neurons expressing the cation channel TRPA1. TRPA1 is both necessary and sufficient for the optovin response. Optovin activates human TRPA1 via structure-dependent photochemical reactions with redox-sensitive cysteine residues. In animals with severed spinal cords, optovin treatment enables control of motor activity in the paralyzed extremities by localized illumination. These studies identify a light-based strategy for controlling endogenous TRPA1 receptors in vivo, with potential clinical and research applications in non-transgenic animals, including humans.
doi:10.1038/nchembio.1183
PMCID: PMC3604056  PMID: 23396078
4.  Identification of non-visual photomotor response cells in the vertebrate hindbrain 
Non-visual photosensation enables animals to sense light without sight. However, the cellular and molecular mechanisms of non-visual photobehaviors are poorly understood, especially in vertebrate animals. Here, we describe the photomotor response (PMR), a robust and reproducible series of motor behaviors in zebrafish that is elicited by visual wavelengths of light, but does not require the eyes, pineal gland or other canonical deep-brain photoreceptive organs. Unlike the relatively slow effects of canonical non-visual pathways, motor circuits are strongly and quickly (seconds) recruited during the PMR behavior. We find that the hindbrain is both necessary and sufficient to drive these behaviors. Using in vivo calcium imaging, we identify a discrete set of neurons within the hindbrain whose responses to light mirror the PMR behavior. Pharmacological inhibition of the visual cycle blocks PMR behaviors, suggesting that opsin-based photoreceptors control this behavior. These data represent the first known light-sensing circuit in the vertebrate hindbrain.
doi:10.1523/JNEUROSCI.3689-12.2013
PMCID: PMC3600642  PMID: 23447595
5.  Behavioral barcoding in the cloud: Embracing data-intensive digital phenotyping in neuropharmacology 
Trends in Biotechnology  2012;30(8):421-425.
Summary
For decades, studying the behavioral effects of individual drugs and genetic mutations has been at the heart of efforts to understand and treat nervous system disorders. High-throughput technologies adapted from other disciplines (e.g. high-throughput chemical screening, genomics) are changing the scale of data acquisition in behavioral neuroscience. Massive behavioral datasets are beginning to emerge, particularly from zebrafish labs, where behavioral assays can be performed rapidly and reproducibly in 96-well, high-throughput format. Mining these datasets and making comparisons across different assays are major challenges for the field. Here, we review behavioral barcoding, a process by which complex behavioral assays are reduced to a string of numeric features, facilitating analysis and comparison within and across datasets.
doi:10.1016/j.tibtech.2012.05.001
PMCID: PMC3401323  PMID: 22652049
6.  Using the Zebrafish Photomotor Response for Psychotropic Drug Screening 
Methods in cell biology  2011;105:517-524.
Because psychotropic drugs affect behavior, we can use changes in behavior to discover psychotropic drugs. The original prototypes of most neuroactive medicines were discovered in humans, rodents and other model organisms. Most of these discoveries were made by chance, but the process of behavior based drug discovery can be made more systematic and efficient. Fully automated platforms for analyzing the behavior of embryonic zebrafish capture digital video recordings of animals in each individual well of a 96-well plate before, during, and after a series of stimuli. To analyze systematically the thousands of behavioral recordings obtained from a large-scale chemical screen, we transform these behavioral recordings into numerical barcodes, providing a concise and interpretable summary of the observed phenotypes in each well. Systems-level analysis of these behavioral phenotypes generate testable hypotheses about the molecular mechanisms of poorly understood drugs and behaviors. By combining the in vivo relevance of behavior-based phenotyping with the scale and automation of modern drug screening technologies, systematic behavioral barcoding represents a means of discovering psychotropic drugs and provides a powerful, systematic approach for unraveling the complexities of vertebrate behavior.
doi:10.1016/B978-0-12-381320-6.00022-9
PMCID: PMC3635141  PMID: 21951545
7.  Chemical Informatics and Target Identification in a Zebrafish Phenotypic Screen 
Nature Chemical Biology  2011;8(2):144-146.
Target identification is a core challenge in chemical genetics. Here we use chemical similarity to predict computationally the targets of 586 compounds active in a zebrafish behavioral assay. Of 20 predictions tested, 11 had activities ranging from 1 to 10,000nM on the predicted targets. The role of two of these targets was tested in the original zebrafish phenotype. Prediction of targets from chemotype is rapid and may be generally applicable.
doi:10.1038/nchembio.732
PMCID: PMC3262069  PMID: 22179068
8.  Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation 
Science (New York, N.Y.)  2010;327(5963):348-351.
A major obstacle for the discovery of psychoactive drugs is the inability to predict how small molecules will alter complex behaviors. We report the development and application of a high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found that the multi-dimensional nature of observed phenotypes enabled the hierarchical clustering of molecules according to shared behaviors. Behavioral profiling revealed conserved functions of psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds. In addition, behavioral profiling implicated new factors such as ether-a-go-go-related gene (ERG) potassium channels and immunomodulators in the control of rest and locomotor activity. These results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors.
doi:10.1126/science.1183090
PMCID: PMC2830481  PMID: 20075256
9.  Rapid behavior—based identification of neuroactive small molecules in the zebrafish 
Nature chemical biology  2010;6(3):231-237.
Neuroactive small molecules are indispensable tools for treating mental illnesses and dissecting nervous system function. However, it has been difficult to discover novel neuroactive drugs. Here, we describe a high—throughput (HT) behavior—based approach to neuroactive small molecule discovery in the zebrafish. We use automated screening assays to evaluate thousands of chemical compounds and find that diverse classes of neuroactive molecules cause distinct patterns of behavior. These `behavioral barcodes' can be used to rapidly identify novel psychotropic chemicals and to predict their molecular targets. For example, we identify novel acetylcholinesterase and monoamine oxidase inhibitors using phenotypic comparisons and computational techniques. By combining HT screening technologies with behavioral phenotyping in vivo, behavior—based chemical screens may accelerate the pace of neuroactive drug discovery and provide small—molecule tools for understanding vertebrate behavior.
doi:10.1038/nchembio.307
PMCID: PMC2834185  PMID: 20081854
10.  Chemobehavioural phenomics and behaviour-based psychiatric drug discovery in the zebrafish 
Despite their ubiquity and impact, psychiatric illnesses and other disorders of the central nervous system remain among the most poorly treated diseases. Most psychiatric medicines were discovered due to serendipitous observations of behavioural phenotypes in humans, rodents and other mammals. Extensive behaviour-based chemical screens would likely identify novel psychiatric drugs. However, large-scale chemical screens in mammals are inefficient and impractical. In contrast, zebrafish are very well suited for high-throughput behaviour-based drug discovery. Furthermore, the vast amounts of data generated from large-scale behavioural screens in zebrafish will facilitate a systems-level analysis of how chemicals affect behaviour. Unlike serendipitous discoveries in mammals, a comprehensive and integrative analysis of zebrafish chemobehavioural phenomics may identify functional relationships that would be missed by more reductionist approaches. Thus, behaviour-based chemical screens in the zebrafish may improve our understanding of neurobiology and accelerate the pace of psychiatric drug discovery.
doi:10.1093/bfgp/eln040
PMCID: PMC2722257  PMID: 18784194
phenomics; chemical genetics; zebrafish
11.  Caenorhabditis elegans drp-1 and fis-2 regulate distinct cell death execution pathways downstream of ced-3 and independent of ced-9 
Molecular cell  2008;31(4):586-597.
SUMMARY
The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3, or the profission gene drp-1, are not required for apoptosis activation in C. elegans. However minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9, and downstream of the CED-3 caspase, to promote elimination of mitochondria in dying cells, an event that could facilitate cell death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1’s proapoptotic function but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell death execution downstream of caspase activation.
doi:10.1016/j.molcel.2008.07.015
PMCID: PMC2548325  PMID: 18722182

Results 1-11 (11)