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Review
. 2014 May;37(5):264-78.
doi: 10.1016/j.tins.2014.02.011. Epub 2014 Apr 9.

Zebrafish Models for Translational Neuroscience Research: From Tank to Bedside

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Free PMC article
Review

Zebrafish Models for Translational Neuroscience Research: From Tank to Bedside

Adam Michael Stewart et al. Trends Neurosci. .
Free PMC article

Abstract

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes.

Keywords: anxiety; biomarkers; brain disorders; cancer; epilepsy; zebrafish.

Figures

Figure 1
Figure 1
Adult and larval zebrafish (Danio rerio), and their developing utility in biomedical research (from tank to bedside). Panel (A) outlines the life cycle of zebrafish, from embryonic pre-hatching (0–72 hpf) to post-hatching stages, including larval (3–29 dpf), juvenile (30–89 dpf), adult (90 dpf–2 years), and aged zebrafish (>2 years) [92]. Panel (B) shows the utility of zebrafish in biomedical research in 2004–2013. The number of PubMed publications (pie diagram) was assessed in December 2013 for various model organisms, yielding more than 532 000 publications for mice, 361 000 for rats, 54 000 for dogs, 34 000 for fruit flies, 15 000 for zebrafish, and 13 000 for nematodes (Caenorhabditis elegans). Line diagram shows normalized (expressed as % of total) number of publications per respective species (note that zebrafish publications display the sharpest increase compared with other animal models [13], shown as the phylogenetic tree; bottom left). Bottom right: comparative analyses of zebrafish brain versus other model organisms; note generally similar brain characteristics in zebrafish and mammals, including humans [7]. Panel (C) illustrates the zebrafish neuroendocrine stress (hypothalamo–pituitary–inter-renal, HPI) system, which releases cortisol from the inter-renal gland (similar to the adrenal gland in mammals) in response to adrenocorticotropic hormone (ACTH) following the stress-evoked release of hypothalamic corticotropin-releasing hormone (CRH) [16,24,25,93]. Acting via the negative biofeedback mechanism, cortisol released to the circulation activates glucocorticoid receptors (GRs) to inhibit the release of CRH and ACTH in the brain (thereby protecting zebrafish from pathological overactivation of the HPI axis). For example, acute stress evokes fast and robust cortisol responses with similar time dynamics in both humans and zebrafish (bottom row); also note that zebrafish and humans both use cortisol as their main stress hormone, unlike rodents (which use corticosterone) [16,24,25]. Strong behavioral and physiological stress responses can be evoked in zebrafish by acute exposure to their natural predators (e.g., Indian Leaf Fish, Nandus nandus) or other predator fish, such as African Leaf Fish (Ctenopoma acutirostre) and Oscar Fish (Astronotus ocellatus). Acute exposure of zebrafish to their natural predators (e.g., Leaf Fish, LF) induces overt anxiety-like behavior (tight shoaling and avoidance), accompanied by elevated whole body cortisol levels (**P < 0.01 vs control) [6,94]. Other stressors known to elevate cortisol in zebrafish include crowding stress, alarm pheromone exposure, acute restraint stress, novelty stress, social confrontations, drug withdrawal, or pharmacological treatments with various anxiogenic agents (Table 2). Importantly, recent successful applications of optogenetics in zebrafish have enabled selective manipulations of the HPI axis and cortisol signaling [39], further advancing stress physiology research utilizing this model organism. Abbreviations: hpf, hours post-fertilization; dpf, days post-fertilization.
Figure 2
Figure 2
Visualizing zebrafish brain using various imaging techniques in adult zebrafish. Panel (A) shows a 3D reconstructed zebrafish brain using two different types of magnetic resonance (MR) imaging: the MR histology (right) and the diffusion tensor imaging (left). The resolutions are among the highest achieved in a vertebrate brain, further establishing teleost fish as an excellent model for brain imaging [35]. Panel (B) shows a whole mounted zebrafish brain labeled with anti-synaptic vesicle protein 2 and anti-keyhole limpet hemocyanin antibodies [36]. Marked in green are the input (axons from sensory neurons in the sensory epithelia) and partial output from the zebrafish olfactory bulb (OB), and highlighted in red are synaptic terminals formed by long-range and local pathways. The brain in this image was hemisected along its midline (TEL, telencephalon; dorsal part is up and ventral is down). Panel (C) shows an optical section of an intact zebrafish brain showing transgenically labeled inputs to the optic tectum (HuC:chameleon green) and immunostained cholinergic neurons (anti-Chat) likely to receive and/or modulate the incoming neural signals. This panel reveals the interconnectedness of different neuronal pathways at high spatial resolution, and was obtained from an intact zebrafish brain (O. Braubach, 2014 Zebrafish case study). Panel (D) shows central nervous system (CNS) cancers, such as brain neuroblastoma and eye melanoma, in adult zebrafish. Left panel (top): neuroblastoma (side and top view) in adult zebrafish of KOLN wild type strain treated by bath exposure to a mutagen, ethylnitrosourea (ENU, 2.5 mM), as a 3-week-old fry. Left panel (bottom): histological sections of neuroblastoma in adult zebrafish (treated as embryos with the carcinogen agent methylazoxymethanol acetate). Right panel: enlarged right eye and melanoma in a 4-week-old transgenic zebrafish (top) with activated Smoa1 expressed under control of the krt4 promoter [80]. Bottom image: histological appearance of melanoma of zebrafish eye (note poorly differentiated invasive melanocytes in melanoma; J. Spitsbergen, 2014 Zebrafish case study).
Figure 3
Figure 3
Zebrafish models and phenotypes related to epilepsy. Panel (A) illustrates the place of epilepsy (as a neurological disorder) among other groups of central nervous system (CNS) disorders discussed here. Panel (B) shows a larval zebrafish embedded in agarose and paralyzed using a myorelaxant (e.g., d-tubocurarine), with electroencephalographic (EEG) electrodes inserted into the brain areas, such as tectum (invasive EEG), or placed on the skull (non-invasive ‘surface’ EEG) to record brain activity (e.g., typical tectal field recordings shown above). Panel (C) shows experimental seizures in adult zebrafish that can be evoked chemically (e.g., by exposure to various convulsant drugs, such as 10–15 mM pentylenetetrazole, PTZ, a blocker of the Cl ionophore at inhibitory gamma-aminobutyric acid GABA-A receptors). Note characteristic circling and corkscrew swimming, hyperlocomotion, and elevated c-fos expression following pretreatment with PTZ, *** P < 0.001 versus control [3].
Figure 4
Figure 4
Phenotyping zebrafish anxiety-related and social behavior. Panels (A) and (B) illustrate the utility of modern video tracking techniques for larval and adult zebrafish neurophenotyping. Panel (A) shows a 96-well high-throughput screen (HTS) for larval zebrafish, with the typical set-up (top view) and application of video tracking software to quantify zebrafish locomotor responses. Bottom row illustrates general principles of zebrafish HTS, screening a large number of chemical compounds (e.g., 1–12) from the library, and assessing various drug-induced behaviors (B1–B4) using zebrafish (color denotes decrease or increase of individual behaviors, as it moves from blue to red). Based on clustering these responses, HTS can detect psychotropic properties (e.g., anxiolytic versus sedative). Common anxiety-like behaviors in larval zebrafish HTS include increased immobility (freezing) frequency and duration, whereas anxiolytic-like behavior will often manifest in increased center dwelling [12,19]. In a simplified example, drugs evoking anxiolytic-like behaviors B1/B2 without hyperlocomotion B3 are recognized as potential anxiolytic agents; anxiogenic-like behaviors B4 and reduced B1/B2 without hyperlocomotion B3 can be interpreted as potential anxiogenic agents, whereas agents evoking anxiolytic-like behaviors B1/B2 combined with hypoactivity (reduced B3) may be interpreted as ‘sedative’ compounds. Panel (B) shows swimming patterns in the standard novel test tank, one of the most popular zebrafish behavioral assays [6,94]. Note distinct swimming patterns (top row), generated by video tracking software for untreated control (left) and experimental (right) fish treated with the classical antidepressant/anxiolytic drug fluoxetine (0.1 mg/l) for 2 weeks. The traces reveal marked differences in overall exploration and swimming activity, as control fish dwell mostly at the bottom and fluoxetine has the opposite, anxiolytic effect (see [6,94] and Table 2 for details). Consistent with this anxiolytic profile, experimental fish show significantly lower levels of cortisol [6,94] (* P < 0.05 vs control). Middle row: a diagram showing typical zebrafish anxiety-like responses in the novel tank test, including: (i) diving response, (ii) freezing/immobility, (iii) erratic movement, and (iv) thigmotaxis (staying close to the walls), which all increase during high-anxiety states (see [19,50] for detailed definitions of these behaviors), but can be rescued by anxiolytic treatments. Bottom row illustrates the importance of zebrafish behavioral analyses in 3D, to complement traditional 2D approaches; see [50] for details [note that zebrafish swim in 3D (XYZ) coordinates, unlike rodent tests, where animals typically display horizontal locomotion on 2D surfaces]. Right panel: a two-camera set-up which allows 3D neurophenotyping of zebrafish locomotion in XYZ coordinates (images from Noldus IT, The Netherlands in collaboration with the Kalueff laboratory). The 3D neurophenotyping approach reveals robust phenotypic differences between traces in control versus anxiolytic (5–10 mg/l nicotine-treated) zebrafish cohorts, including increased top exploration with reduced bottom dwelling and freezing. Note that nicotine-exposed fish demonstrate a consistent top dwelling, present for the entire duration of the trial. 3D reconstruction of their traces reveals anxiolytic-like ‘top dwelling’, largely concentrated at the water surface, yet with overt active swimming along the tank periphery. Such lack of anxiety coupled with thigmotactic behavior is consistent with typical psychostimulant/anxiolytic action of nicotine, paralleling its profile in various other model organisms (e.g., rodents). Panel (C) shows the predator exposure paradigm (see Table 2 for details) in which the zebrafish tank is exposed to a nearby tank containing a predator fish (e.g., Leaf Fish). As control fish (separated from the predator tank by a non-transparent plastic divider, denoted by the arrow) display low anxiety and swim in the middle and top areas of the tank in relatively ‘relaxed’ loose shoals, the removal of the divider results in overt ‘aversive’ anxiety-like behavior in the zebrafish group, including bottom dwelling, unusually tight shoaling and avoidance of the predator (by gathering in the farthest opposite corner). Panel (D) shows typical zebrafish ‘group’ (shoaling) behaviors and its potential relevance to human disorders. Zebrafish are highly social animals and spend the majority of time in social groups [e.g., staying within 1–2 body lengths (~2.5–5 cm) from each other]. In addition to reflecting anxiety-like responses (panel C), zebrafish shoaling behavior may be useful for modeling normal and pathological social behaviors, such as autism spectrum disorder [21]. For example, tracking zebrafish body shape (by simultaneous tracing three points – nose N, center of body mass C, and tail T; top view) can be used for automated decoding zebrafish social behaviors (photos by Noldus IT). Such automated tests are an invaluable tool to study zebrafish social behavior and its deficits. Typical computer-generated endpoints may include orientation (angle) towards the object, distance between selected body points, and body curve patterns, which may be specific for various treatments. For instance, two zebrafish (#1 and #2) that show proximity of their nose points N1 and N2, tracked by the computer, are most likely to engage in social interaction (left). Heading in the same direction nose-to-tail (middle image) can be detected as ‘chasing/following’ behavior (see [19] for details of zebrafish ethogram). By contrast, two ‘uninterested’ zebrafish (right image) are detected by the software as heading in different directions without proximity of their nose points; the latter pattern is common in zebrafish with social deficits (see [21] for review). Bottom row: examples of normal human social interaction (left) and overt social deficits (right; typically observed in patients with autism; images: www.lovetoknow.com) which parallel zebrafish social phenotypes detected by IT-based tools, also see similar aberrant social behaviors in mouse models of autism [21,65,67].
Figure 4
Figure 4
Phenotyping zebrafish anxiety-related and social behavior. Panels (A) and (B) illustrate the utility of modern video tracking techniques for larval and adult zebrafish neurophenotyping. Panel (A) shows a 96-well high-throughput screen (HTS) for larval zebrafish, with the typical set-up (top view) and application of video tracking software to quantify zebrafish locomotor responses. Bottom row illustrates general principles of zebrafish HTS, screening a large number of chemical compounds (e.g., 1–12) from the library, and assessing various drug-induced behaviors (B1–B4) using zebrafish (color denotes decrease or increase of individual behaviors, as it moves from blue to red). Based on clustering these responses, HTS can detect psychotropic properties (e.g., anxiolytic versus sedative). Common anxiety-like behaviors in larval zebrafish HTS include increased immobility (freezing) frequency and duration, whereas anxiolytic-like behavior will often manifest in increased center dwelling [12,19]. In a simplified example, drugs evoking anxiolytic-like behaviors B1/B2 without hyperlocomotion B3 are recognized as potential anxiolytic agents; anxiogenic-like behaviors B4 and reduced B1/B2 without hyperlocomotion B3 can be interpreted as potential anxiogenic agents, whereas agents evoking anxiolytic-like behaviors B1/B2 combined with hypoactivity (reduced B3) may be interpreted as ‘sedative’ compounds. Panel (B) shows swimming patterns in the standard novel test tank, one of the most popular zebrafish behavioral assays [6,94]. Note distinct swimming patterns (top row), generated by video tracking software for untreated control (left) and experimental (right) fish treated with the classical antidepressant/anxiolytic drug fluoxetine (0.1 mg/l) for 2 weeks. The traces reveal marked differences in overall exploration and swimming activity, as control fish dwell mostly at the bottom and fluoxetine has the opposite, anxiolytic effect (see [6,94] and Table 2 for details). Consistent with this anxiolytic profile, experimental fish show significantly lower levels of cortisol [6,94] (* P < 0.05 vs control). Middle row: a diagram showing typical zebrafish anxiety-like responses in the novel tank test, including: (i) diving response, (ii) freezing/immobility, (iii) erratic movement, and (iv) thigmotaxis (staying close to the walls), which all increase during high-anxiety states (see [19,50] for detailed definitions of these behaviors), but can be rescued by anxiolytic treatments. Bottom row illustrates the importance of zebrafish behavioral analyses in 3D, to complement traditional 2D approaches; see [50] for details [note that zebrafish swim in 3D (XYZ) coordinates, unlike rodent tests, where animals typically display horizontal locomotion on 2D surfaces]. Right panel: a two-camera set-up which allows 3D neurophenotyping of zebrafish locomotion in XYZ coordinates (images from Noldus IT, The Netherlands in collaboration with the Kalueff laboratory). The 3D neurophenotyping approach reveals robust phenotypic differences between traces in control versus anxiolytic (5–10 mg/l nicotine-treated) zebrafish cohorts, including increased top exploration with reduced bottom dwelling and freezing. Note that nicotine-exposed fish demonstrate a consistent top dwelling, present for the entire duration of the trial. 3D reconstruction of their traces reveals anxiolytic-like ‘top dwelling’, largely concentrated at the water surface, yet with overt active swimming along the tank periphery. Such lack of anxiety coupled with thigmotactic behavior is consistent with typical psychostimulant/anxiolytic action of nicotine, paralleling its profile in various other model organisms (e.g., rodents). Panel (C) shows the predator exposure paradigm (see Table 2 for details) in which the zebrafish tank is exposed to a nearby tank containing a predator fish (e.g., Leaf Fish). As control fish (separated from the predator tank by a non-transparent plastic divider, denoted by the arrow) display low anxiety and swim in the middle and top areas of the tank in relatively ‘relaxed’ loose shoals, the removal of the divider results in overt ‘aversive’ anxiety-like behavior in the zebrafish group, including bottom dwelling, unusually tight shoaling and avoidance of the predator (by gathering in the farthest opposite corner). Panel (D) shows typical zebrafish ‘group’ (shoaling) behaviors and its potential relevance to human disorders. Zebrafish are highly social animals and spend the majority of time in social groups [e.g., staying within 1–2 body lengths (~2.5–5 cm) from each other]. In addition to reflecting anxiety-like responses (panel C), zebrafish shoaling behavior may be useful for modeling normal and pathological social behaviors, such as autism spectrum disorder [21]. For example, tracking zebrafish body shape (by simultaneous tracing three points – nose N, center of body mass C, and tail T; top view) can be used for automated decoding zebrafish social behaviors (photos by Noldus IT). Such automated tests are an invaluable tool to study zebrafish social behavior and its deficits. Typical computer-generated endpoints may include orientation (angle) towards the object, distance between selected body points, and body curve patterns, which may be specific for various treatments. For instance, two zebrafish (#1 and #2) that show proximity of their nose points N1 and N2, tracked by the computer, are most likely to engage in social interaction (left). Heading in the same direction nose-to-tail (middle image) can be detected as ‘chasing/following’ behavior (see [19] for details of zebrafish ethogram). By contrast, two ‘uninterested’ zebrafish (right image) are detected by the software as heading in different directions without proximity of their nose points; the latter pattern is common in zebrafish with social deficits (see [21] for review). Bottom row: examples of normal human social interaction (left) and overt social deficits (right; typically observed in patients with autism; images: www.lovetoknow.com) which parallel zebrafish social phenotypes detected by IT-based tools, also see similar aberrant social behaviors in mouse models of autism [21,65,67].

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