FIGURE SUMMARY
Title

Molecular psychiatry of zebrafish

Authors
Stewart, A.M., Ullmann, J.F., Norton, W.H., Parker, M.O., Brennan, C.H., Gerlai, R., Kalueff, A.V.
Source
Full text @ Mol. Psychiatry

A brief summary of zebrafish experimental models in neuroscience research

Panel A shows the evolving nature of zebrafish models in the last 50 years, initially used mainly for basic genetic and neurodevelopmental studies, but more recently applied to developing in-vivo models for complex brain disorders, such as autism, depression and psychoses. Inset: selected zebrafish strains useful in biological psychiatry research (top to bottom: adult wild type zebrafish, casper, spiegeldanio and nacre mutants); photos courtesy of the Kalueff (ZENEREI Institute, USA), the Norton (University of Leicester, UK), the Parichy (University of Washington, USA) laboratories and Carolina Biological Supply Company (Burlington, USA).

Panel B illustrates two research strategies which can both be applied to zebrafish models. As a vertebrate species amenable to in-vivo analyses and with high genetic/physiological homology to humans, zebrafish are ideal for ‘intensive’, mechanistically driven neuroscience research into conserved, core molecular pathways or neural circuits (photo). Due to their small size, ease of maintenance and short generation time, zebrafish also represent an excellent model for ‘extensive’ biomedical research, including low-cost high-throughput screening for small molecules or genetic mutations. Both strategies can lead to the development of novel therapies for major psychiatric disorders. Photo: visualizing zebrafish signaling as an example of intensive, pathway-oriented mechanistic research (the left image shows expression patterns of genetically encoded calcium indicator; zebrafish at 5 dpf show pan-neuronal expression of HuC:Gal4; UAS:GCaMP5, a zoomed-in image on the right shows a single cell resolution: note that some cells are activated, as indicated by the increased fluorescence; scale bar 100 µm).

Panel C illustrates the benefits of including inexpensive zebrafish models into preclinical screening batteries. A substantial fiscal saving (shown as $) can be achieved by narrowing screening to potentially active compounds using fish (rather than mice) as the first vertebrate model organism in the screening pipeline. In addition, the risks of missing promising ‘hits’ are lower because the subsequent screening in mammals will be ‘confirmatory’, and based on more valid data generated from zebrafish (rather than invertebrate or in-vitro) tests.

Panel D summarizes major neurotransmitter systems in the adult zebrafish brain: ACh - acetylcholine (dark blue), NA - noradrenergic (purple), DA - dopamine (green), HA - histamine (orange), 5-HT - serotonin (red); GABA - γ-aminobutyric acid (pink), Glu - glutamate (light blue). The highlighted brain regions include olfactory bulb (OB), dorsal pallium (Dp), ventral pallium (Vp), habenula (Ha), optic nerve (ON), facial lobe (LVII) and vagal lobe (LX). Highlighted areas have been overlaid over a minimum deformation model of the adult zebrafish brain.

A brief summary of zebrafish experimental models in neuroscience research

Panel A shows the evolving nature of zebrafish models in the last 50 years, initially used mainly for basic genetic and neurodevelopmental studies, but more recently applied to developing in-vivo models for complex brain disorders, such as autism, depression and psychoses. Inset: selected zebrafish strains useful in biological psychiatry research (top to bottom: adult wild type zebrafish, casper, spiegeldanio and nacre mutants); photos courtesy of the Kalueff (ZENEREI Institute, USA), the Norton (University of Leicester, UK), the Parichy (University of Washington, USA) laboratories and Carolina Biological Supply Company (Burlington, USA).

Panel B illustrates two research strategies which can both be applied to zebrafish models. As a vertebrate species amenable to in-vivo analyses and with high genetic/physiological homology to humans, zebrafish are ideal for ‘intensive’, mechanistically driven neuroscience research into conserved, core molecular pathways or neural circuits (photo). Due to their small size, ease of maintenance and short generation time, zebrafish also represent an excellent model for ‘extensive’ biomedical research, including low-cost high-throughput screening for small molecules or genetic mutations. Both strategies can lead to the development of novel therapies for major psychiatric disorders. Photo: visualizing zebrafish signaling as an example of intensive, pathway-oriented mechanistic research (the left image shows expression patterns of genetically encoded calcium indicator; zebrafish at 5 dpf show pan-neuronal expression of HuC:Gal4; UAS:GCaMP5, a zoomed-in image on the right shows a single cell resolution: note that some cells are activated, as indicated by the increased fluorescence; scale bar 100 µm).

Panel C illustrates the benefits of including inexpensive zebrafish models into preclinical screening batteries. A substantial fiscal saving (shown as $) can be achieved by narrowing screening to potentially active compounds using fish (rather than mice) as the first vertebrate model organism in the screening pipeline. In addition, the risks of missing promising ‘hits’ are lower because the subsequent screening in mammals will be ‘confirmatory’, and based on more valid data generated from zebrafish (rather than invertebrate or in-vitro) tests.

Panel D summarizes major neurotransmitter systems in the adult zebrafish brain: ACh - acetylcholine (dark blue), NA - noradrenergic (purple), DA - dopamine (green), HA - histamine (orange), 5-HT - serotonin (red); GABA - γ-aminobutyric acid (pink), Glu - glutamate (light blue). The highlighted brain regions include olfactory bulb (OB), dorsal pallium (Dp), ventral pallium (Vp), habenula (Ha), optic nerve (ON), facial lobe (LVII) and vagal lobe (LX). Highlighted areas have been overlaid over a minimum deformation model of the adult zebrafish brain.

Neurobehavioral phenotypes in zebrafish relevant to modeling a diverse group of psychiatric disorders (also see <xref rid='T1' ref-type='table'>Table 1</xref>)

Axis represent five major phenotypic domains of brain disorders, and include motor activity, cognition, affect/emotionality, reward and aggression. A dashed- line pentagon represents ‘normal’ healthy phenotype for each respective axis/domains.

Panel A shows typical zebrafish responses relevant to ADHD (modified from 125, 126). The bar diagram shows total distance swum in 5 min for control larvae and lphn3.1 morphants with and without methylphenidate (MPH) treatment. The right panel shows representative locomotion traces (recorded from the top view camera) in 6-dpf control and lphn3.1 morphant zebrafish (green color indicates swimming with normal speed, red color indicates bursts of ‘impulsive’ swimming with high-acceleration).

Panel B illustrates zebrafish mirror-induced aggression (MIA) responses in the mirror exposure test, and typical patterns of zebrafish aggressive confrontations (modified from 139). Note that MIA is particularly simple to record and quantify161, making it also suitable for high-throughput screening (MIA images in the middle: courtesy of the Robison laboratory161, University of Idaho, USA).

Panel C illustrates zebrafish stress- and PTSD-related aversive behaviors, including neophobia (e.g., acute 5-min exposure to the unfamiliar blue marble in the novel object test) and predator avoidance (acute 5-min exposure to a predator fish). Note that zebrafish avoid a blue marble at the bottom of the tank (swim traces in the open field test recorded by Noldus Ethovision XT8 from the top view), as well as display robust aversion of a live predator (e.g., Leaf fish), clearly spending more time in the opposite side of the tank.

Panel D demonstrates zebrafish reward-related addiction behaviors (in the conditioned place preference model, CPP) and molecular alterations in zebrafish brain following exposure to a substance of abuse. In the CPP, fish are initially tested for their preference for both sides of the apparatus, each of which contains a discriminative stimulus (i.e., spots vs. no spots). They are then conditioned with drug to the least-preferred side over a number of sessions. Finally, their preference is assessed again in a probe trial, where the rewarding value of the drug can be ascertained by calculating the change in preference for the least-preferred side following conditioning (in addition to water immersion, also note the use of i.p. drug injections in some zebrafish CPP studies184, 190). The bottom right bar diagram also shows a classic dose-response curve for nicotine in adult fish (also note that mutant heterozygous for a nonsense mutation for the acetlycholinesterase gene (achesb55/+) do not show place preference for 6 µM nicotine; Brock et al., unpublished observations). Top right bar diagram shows fish that display persistent compulsive drug seeking (i.e., drug seeking despite adverse consequences/punishment) following chronic exposure to ethanol or nicotine, also demonstrating long-lasting drug-specific and conserved changes in gene expression197 (brain genes’ microarray, inset).

Panel E shows the reduction in zebrafish social behavior (e.g., shorter distance between a test fish and moving shoaling images delivered on a computer screen; inset). This reduction in hoaling response is abolished by acute administration of a high concentration of alcohol, which was inactive in fish chronically pre-treated with alcohol. Withdrawal from alcohol chronic exposure also abolishes the shoaling response. Note that alcohol-induced behavioral changes in zebrafish are closely paralleled by alterations in DA levels quantified from whole brain samples using HPLC (modified from 203, 230).

Neurobehavioral phenotypes in zebrafish relevant to modeling a diverse group of psychiatric disorders (also see <xref rid='T1' ref-type='table'>Table 1</xref>)

Axis represent five major phenotypic domains of brain disorders, and include motor activity, cognition, affect/emotionality, reward and aggression. A dashed- line pentagon represents ‘normal’ healthy phenotype for each respective axis/domains.

Panel A shows typical zebrafish responses relevant to ADHD (modified from 125, 126). The bar diagram shows total distance swum in 5 min for control larvae and lphn3.1 morphants with and without methylphenidate (MPH) treatment. The right panel shows representative locomotion traces (recorded from the top view camera) in 6-dpf control and lphn3.1 morphant zebrafish (green color indicates swimming with normal speed, red color indicates bursts of ‘impulsive’ swimming with high-acceleration).

Panel B illustrates zebrafish mirror-induced aggression (MIA) responses in the mirror exposure test, and typical patterns of zebrafish aggressive confrontations (modified from 139). Note that MIA is particularly simple to record and quantify161, making it also suitable for high-throughput screening (MIA images in the middle: courtesy of the Robison laboratory161, University of Idaho, USA).

Panel C illustrates zebrafish stress- and PTSD-related aversive behaviors, including neophobia (e.g., acute 5-min exposure to the unfamiliar blue marble in the novel object test) and predator avoidance (acute 5-min exposure to a predator fish). Note that zebrafish avoid a blue marble at the bottom of the tank (swim traces in the open field test recorded by Noldus Ethovision XT8 from the top view), as well as display robust aversion of a live predator (e.g., Leaf fish), clearly spending more time in the opposite side of the tank.

Panel D demonstrates zebrafish reward-related addiction behaviors (in the conditioned place preference model, CPP) and molecular alterations in zebrafish brain following exposure to a substance of abuse. In the CPP, fish are initially tested for their preference for both sides of the apparatus, each of which contains a discriminative stimulus (i.e., spots vs. no spots). They are then conditioned with drug to the least-preferred side over a number of sessions. Finally, their preference is assessed again in a probe trial, where the rewarding value of the drug can be ascertained by calculating the change in preference for the least-preferred side following conditioning (in addition to water immersion, also note the use of i.p. drug injections in some zebrafish CPP studies184, 190). The bottom right bar diagram also shows a classic dose-response curve for nicotine in adult fish (also note that mutant heterozygous for a nonsense mutation for the acetlycholinesterase gene (achesb55/+) do not show place preference for 6 µM nicotine; Brock et al., unpublished observations). Top right bar diagram shows fish that display persistent compulsive drug seeking (i.e., drug seeking despite adverse consequences/punishment) following chronic exposure to ethanol or nicotine, also demonstrating long-lasting drug-specific and conserved changes in gene expression197 (brain genes’ microarray, inset).

Panel E shows the reduction in zebrafish social behavior (e.g., shorter distance between a test fish and moving shoaling images delivered on a computer screen; inset). This reduction in hoaling response is abolished by acute administration of a high concentration of alcohol, which was inactive in fish chronically pre-treated with alcohol. Withdrawal from alcohol chronic exposure also abolishes the shoaling response. Note that alcohol-induced behavioral changes in zebrafish are closely paralleled by alterations in DA levels quantified from whole brain samples using HPLC (modified from 203, 230).

Acknowledgments
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