David Kokel

Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation

Behavioral Profiling The complexity of the brain makes it difficult to predict how a drug will affect behavior without direct testing in live animals. Rihel et al. (p. 348) developed a high-throughput assay to assess the effects of thousands of drugs on sleep/wake behaviors of zebrafish larvae. The data set reveals a broad conservation of zebrafish and mammalian sleep/wake pharmacology and identifies pathways that regulate sleep. Moreover, the biological targets of poorly characterized small molecules can be predicted by matching their behavioral profiles to those of well-known drugs. Thus, behavioral profiling in zebrafish offers a cost-effective way to characterize neuroactive drugs and to predict biological targets of novel compounds. The effects of most neuroactive drugs are conserved and can be detected by behavioral screening. 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 multidimensional 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.

Chemical informatics and target identification in a zebrafish phenotypic screen

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.

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.

Live imaging of apoptotic cells in zebrafish

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium‐dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single‐cell resolution in vivo.—Van Ham, T. J., Mapes, J., Kokel, D., Peterson, R. T. Live imaging of apoptotic cells in zebrafish. FASEB J. 24, 4336–4342 (2010). www.fasebj.org

Photochemical activation of TRPA1 channels in neurons and animals

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, optovins 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.

Identification of Nonvisual Photomotor Response Cells in the Vertebrate Hindbrain

Nonvisual photosensation enables animals to sense light without sight. However, the cellular and molecular mechanisms of nonvisual 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 nonvisual 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.

Discovering novel neuroactive drugs through high-throughput behavior-based chemical screening in the zebrafish

Most neuroactive drugs were discovered through unexpected behavioral observations. Systematic behavioral screening is inefficient in most model organisms. But, automated technologies are enabling a new phase of discovery-based research in central nervous system (CNS) pharmacology. Researchers are using large-scale behavior-based chemical screens in zebrafish to discover compounds with new structures, targets, and functions. These compounds are powerful tools for understanding CNS signaling pathways. Substantial differences between human and zebrafish biology will make it difficult to translate these discoveries to clinical medicine. However, given the molecular genetic similarities between humans and zebrafish, it is likely that some of these compounds will have translational utility. We predict that the greatest new successes in CNS drug discovery will leverage many model systems, including in vitro assays, cells, rodents, and zebrafish.

Using the Zebrafish Photomotor Response for Psychotropic Drug Screening

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.

Apoptotic Cells Are Cleared by Directional Migration and elmo1-Dependent Macrophage Engulfment

Apoptotic cell death is essential for development and tissue homeostasis. Failure to clear apoptotic cells can ultimately cause inflammation and autoimmunity. Apoptosis has primarily been studied by staining of fixed tissue sections, and a clear understanding of the behavior of apoptotic cells in living tissue has been elusive. Here, we use a newly developed technique to track apoptotic cells in real time as they emerge and are cleared from the zebrafish brain. We find that apoptotic cells are remarkably motile, frequently migrating several cell diameters to the periphery of living tissues. F-actin remodeling occurs in surrounding cells, but also within the apoptotic cells themselves, suggesting a cell-autonomous component of motility. During the first 2 days of development, engulfment is rare, and most apoptotic cells lyse at the brain periphery. By 3 days postfertilization, most cell corpses are rapidly engulfed by macrophages. This engulfment requires the guanine nucleotide exchange factor elmo1. In elmo1-deficient macrophages, engulfment is rare and may occur through macropinocytosis rather than directed engulfment. These findings suggest that clearance of apoptotic cells in living vertebrates is accomplished by the combined actions of apoptotic cell migration and elmo1-dependent macrophage engulfment.

Behavioral barcoding in the cloud: embracing data-intensive digital phenotyping in neuropharmacology

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.