**1. Introduction**

The use of zebrafish as a vertebrate model for biological research began in the late 1960s in the lab of George Streisinger at the University of Oregon. However, it was not until the middle of the 1980s that a community of researchers working on zebrafish began to emerge. Since that time the use of zebrafish as a model organism has continued to increase. Over the past 3 decades the use of zebrafish as a model species has contributed to our understanding of developmental biology, toxicology, drug efficacy and disease.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

As a vertebrate, the zebrafish model provides more information than can be obtained from cell lines and invertebrate studies, while at the same time remaining low-cost and highthroughput compared with mammalian models. It has been estimated that screening 1 drug with rodent models costs approximately 50× more than through zebrafish assays and zebrafish testing can be done in days versus weeks to months for analogous rodent assays [1].

The use of zebrafish in the field of neuroscience continues to increase and a number of recent reviews have highlighted both the strengths and weaknesses of using zebrafish to study neuroactive compounds and brain disorders [9–15]. The zebrafish brain has many analogous regions to those of higher vertebrates and the complexity of both juvenile and adult zebrafish brains has been well documented [16]. In addition to brain morphology, the neurochemistry and endocrine responses linked to zebrafish neuroactivity is highly homologous to other vertebrates including the same neurotransmitters, receptors, synthetic/metabolic enzymes and

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It has been demonstrated that zebrafish are sensitive to a large number of neurotropic drugs including: antipsychotics, mood stabilizers, anxiolytics, antidepressants, ethanol, hypnotics, stimulants, hallucinogens, antiepileptics, analgesics and cognitive enhancers [15, 16]. In addition, both adult and larval zebrafish can be used to model numerous neural disorders including pain/nociception, anxiety, stress, PTSD, ADHD, Autism, epilepsy, learning & memory deficits, psychiatric disorders, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, schizophrenia, bi-polar disorder, addiction and brain cancer [6, 9, 11, 14–16, 20–25]. This provides *in vivo* models with which to test not only the bioactivity of various neuroactive compounds, but also allows for the testing of their potential efficacy against numerous models of disease. Use of these models can provide an indication of the level of compound required to oppose a disease phenotype, which is required for the calculation of a therapeutic window for new drugs. The disease models also provide a platform for the testing and potential re-purposing of neuroactive compounds currently on the market. Finding an effective treatment for the disease models may help to provide clues to the etiology of human

Many of the neuronal disease models developed using zebrafish are centered on the assessment of aberrant behavior in both larvae and adults, which each provide their own distinct advantages [9, 11, 15, 16]. One of the major advantages of using larvae over adults stems from their reproducible patterns of behavior and potential to be screened in a high throughput fashion. Activity patterns can be assessed in multi-well plates allowing for up to 96 larvae to be tested simultaneously using benchtop tracking systems. As mentioned, larvae become free swimming between 3 and 5 dpf and develop stereotypical behavioral and stimulus response patterns. These include their response to startling stimuli such as noise, light–dark transitions and touch. Importantly the behavioral activity patterns are highly quantifiable and can be altered by neuroactive compounds with various targets. The assessment of adult behavior, while much lower throughput, does have some advantages over larval testing as it can often provide more intricate behavioral paradigms than can be obtained with larvae. Specifically, adult behavior can be tracked in 3 dimensions and various models of learning and memory, conspecific interactions and place preference exist that are not found for larvae. Many of these

In addition to models of behavior, as previously mentioned, larval zebrafish are nearly transparent for their first week of development and a number of transgenic lines exist that completely lack pigment. This provides unparalleled access to an intact vertebrate brain. Numerous studies have used *in situ* hybridization and immunohistochemistry to map and

hypothalamo-pituitary hormones [9–11, 15, 17–19].

disease and insights into additional therapeutic targets.

models are analogous to rodent behavioral models [16].

Another major advantage to using zebrafish as a model species is that they show high genetic homology to mammals. The sequencing of the zebrafish genome was begun in 2001 and the reference genome was published in 2013 [2]. This revealed that ~70% of human genes have at least 1 zebrafish ortholog and ~84% of genes known to be associated with human disease have a zebrafish counterpart. This then provides an important platform with which to begin to study genes linked to human disease. The initial studies that made use of zebrafish were largely entrenched in forward genetic screening, which revealed their genetic tractability and helped to lead the way to the generation of clonal lines [3]. While these original studies were begun nearly 40 years ago, since then an ever increasing number of genetic tools have been developed and used to alter the zebrafish genome such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) [4, 5]. These tools along with a fully sequenced genome provide a stage for the creation of any number of informative transgenic and knockdown/ knockout lines. Along with this, zebrafish reach maturity by 90 days post fertilization and produce hundreds of eggs per clutch on a weekly basis. As screening for germline transmission is the general bottle neck in the generation of transgenic lines, the high fecundity of zebrafish allows for more rapid screening and development of transgenic lines compared with mammalian models. The use of transgenic models has a broad applicability and can potentially contribute to all facets of zebrafish research.

One of the major advantages of zebrafish as a model species is that their embryos are fertilized and develop externally providing easy access to embryos and larvae. Importantly, all major organs are formed by 1 day post fertilization (dpf) and larvae hatch from their chorion and become free swimming by 3 dpf. Larvae can live off of the nutrition provided by their yolk sac until 5 dpf, at which time they begin to feed. During this period, larvae are largely transparent making the development of organs and body patterning visible. Their rapid development and transparency provides an ideal setting for testing the effects of various compounds on normal development along with their potential acute toxicity. Standard toxicity testing models exist, including the OECD recognized fish embryo toxicity assay (FET) that tests the effects of compound exposure on normal development from 6 to 72 hpf (OECD guideline 236, adopted July 2013). The general and behavioral toxicity (GBT) assay tests the effects of compound exposure on larvae from 72 to 120 hpf [6]. Additionally, the effect of compounds on the larval heart rate has been shown to be a predictive indicator of potential bradycardia related cardiotoxicity [7]. This is important when screening neuroactive compounds as the blockage of numerous ion channels, often the target of neuroactives, can lead to arrhythmias [8]. The toxicity profiling of potential therapeutics at early stages of development allows for the identification of off-target side effects as well as the potential to calculate a therapeutic window when the toxicity profile is compared with the level of compound required to have a positive effect on disease models.

The use of zebrafish in the field of neuroscience continues to increase and a number of recent reviews have highlighted both the strengths and weaknesses of using zebrafish to study neuroactive compounds and brain disorders [9–15]. The zebrafish brain has many analogous regions to those of higher vertebrates and the complexity of both juvenile and adult zebrafish brains has been well documented [16]. In addition to brain morphology, the neurochemistry and endocrine responses linked to zebrafish neuroactivity is highly homologous to other vertebrates including the same neurotransmitters, receptors, synthetic/metabolic enzymes and hypothalamo-pituitary hormones [9–11, 15, 17–19].

As a vertebrate, the zebrafish model provides more information than can be obtained from cell lines and invertebrate studies, while at the same time remaining low-cost and highthroughput compared with mammalian models. It has been estimated that screening 1 drug with rodent models costs approximately 50× more than through zebrafish assays and zebrafish testing can be done in days versus weeks to months for analogous rodent assays [1].

Another major advantage to using zebrafish as a model species is that they show high genetic homology to mammals. The sequencing of the zebrafish genome was begun in 2001 and the reference genome was published in 2013 [2]. This revealed that ~70% of human genes have at least 1 zebrafish ortholog and ~84% of genes known to be associated with human disease have a zebrafish counterpart. This then provides an important platform with which to begin to study genes linked to human disease. The initial studies that made use of zebrafish were largely entrenched in forward genetic screening, which revealed their genetic tractability and helped to lead the way to the generation of clonal lines [3]. While these original studies were begun nearly 40 years ago, since then an ever increasing number of genetic tools have been developed and used to alter the zebrafish genome such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) [4, 5]. These tools along with a fully sequenced genome provide a stage for the creation of any number of informative transgenic and knockdown/ knockout lines. Along with this, zebrafish reach maturity by 90 days post fertilization and produce hundreds of eggs per clutch on a weekly basis. As screening for germline transmission is the general bottle neck in the generation of transgenic lines, the high fecundity of zebrafish allows for more rapid screening and development of transgenic lines compared with mammalian models. The use of transgenic models has a broad applicability and can

One of the major advantages of zebrafish as a model species is that their embryos are fertilized and develop externally providing easy access to embryos and larvae. Importantly, all major organs are formed by 1 day post fertilization (dpf) and larvae hatch from their chorion and become free swimming by 3 dpf. Larvae can live off of the nutrition provided by their yolk sac until 5 dpf, at which time they begin to feed. During this period, larvae are largely transparent making the development of organs and body patterning visible. Their rapid development and transparency provides an ideal setting for testing the effects of various compounds on normal development along with their potential acute toxicity. Standard toxicity testing models exist, including the OECD recognized fish embryo toxicity assay (FET) that tests the effects of compound exposure on normal development from 6 to 72 hpf (OECD guideline 236, adopted July 2013). The general and behavioral toxicity (GBT) assay tests the effects of compound exposure on larvae from 72 to 120 hpf [6]. Additionally, the effect of compounds on the larval heart rate has been shown to be a predictive indicator of potential bradycardia related cardiotoxicity [7]. This is important when screening neuroactive compounds as the blockage of numerous ion channels, often the target of neuroactives, can lead to arrhythmias [8]. The toxicity profiling of potential therapeutics at early stages of development allows for the identification of off-target side effects as well as the potential to calculate a therapeutic window when the toxicity profile is compared with the level of

potentially contribute to all facets of zebrafish research.

12 Recent Advances in Cannabinoid Research

compound required to have a positive effect on disease models.

It has been demonstrated that zebrafish are sensitive to a large number of neurotropic drugs including: antipsychotics, mood stabilizers, anxiolytics, antidepressants, ethanol, hypnotics, stimulants, hallucinogens, antiepileptics, analgesics and cognitive enhancers [15, 16]. In addition, both adult and larval zebrafish can be used to model numerous neural disorders including pain/nociception, anxiety, stress, PTSD, ADHD, Autism, epilepsy, learning & memory deficits, psychiatric disorders, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, schizophrenia, bi-polar disorder, addiction and brain cancer [6, 9, 11, 14–16, 20–25]. This provides *in vivo* models with which to test not only the bioactivity of various neuroactive compounds, but also allows for the testing of their potential efficacy against numerous models of disease. Use of these models can provide an indication of the level of compound required to oppose a disease phenotype, which is required for the calculation of a therapeutic window for new drugs. The disease models also provide a platform for the testing and potential re-purposing of neuroactive compounds currently on the market. Finding an effective treatment for the disease models may help to provide clues to the etiology of human disease and insights into additional therapeutic targets.

Many of the neuronal disease models developed using zebrafish are centered on the assessment of aberrant behavior in both larvae and adults, which each provide their own distinct advantages [9, 11, 15, 16]. One of the major advantages of using larvae over adults stems from their reproducible patterns of behavior and potential to be screened in a high throughput fashion. Activity patterns can be assessed in multi-well plates allowing for up to 96 larvae to be tested simultaneously using benchtop tracking systems. As mentioned, larvae become free swimming between 3 and 5 dpf and develop stereotypical behavioral and stimulus response patterns. These include their response to startling stimuli such as noise, light–dark transitions and touch. Importantly the behavioral activity patterns are highly quantifiable and can be altered by neuroactive compounds with various targets. The assessment of adult behavior, while much lower throughput, does have some advantages over larval testing as it can often provide more intricate behavioral paradigms than can be obtained with larvae. Specifically, adult behavior can be tracked in 3 dimensions and various models of learning and memory, conspecific interactions and place preference exist that are not found for larvae. Many of these models are analogous to rodent behavioral models [16].

In addition to models of behavior, as previously mentioned, larval zebrafish are nearly transparent for their first week of development and a number of transgenic lines exist that completely lack pigment. This provides unparalleled access to an intact vertebrate brain. Numerous studies have used *in situ* hybridization and immunohistochemistry to map and profile neural activity using indicators such as *c-Fos*. Recent technical advances have allowed for a more in depth assessment of neuronal activity than is possible in mammalian systems. The assessment of neural activity has been accomplished using genetically encoded calcium indicators and whole brain imaging in immobilized larvae [26]. More recently the assessment of neural activity in freely behaving larvae at near cellular resolution has become possible [27]. The development of this new technology now allows for links to be made between localized brain activity and various behaviors and stimulus response patterns that is currently not possible with mammalian models.

become expressed between 1 and 12 hpf and their expression levels continue to increase throughout development. These include the AEA biosynthetic enzymes *N*-acylphosphatidylethanolamineselective phospholipase D (NAPE-pld) and αβ-hydrolase domain containing 4 (Abhd4), the AEA catabolic enzyme fatty acid amide hydrolase (faah), the 2-AG biosynthetic enzyme diacylglycerol lipase α (DAGL α) and the 2-AG catabolic enzymes monoglyceride lipase (mgll) and prostaglandin-endoperoxide synthase 2 (ptgs2b) [31, 33]. Importantly the expression of these biosynthetic enzymes is accompanied by an increase in the protein levels of AEA and 2-AG [33]. The tissue distribution pattern of these enzymes is vast with high levels found in the brain, muscle, heart, intestine, eyes and reproductive organs (ovary and testis) of adults (**Table 1**) [31]. In larvae mgll, dagla and cnr1 are expressed in different regions of the brain and all 3 show some

Zebrafish as a High-Throughput In Vivo Model for Testing the Bioactivity of Cannabinoids

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The CB1 receptor is present at early developmental stages in mammals and it has been suggested that the ECS may contribute to CNS development, such as axonal elongation, myelina-

In zebrafish it has been shown that the developmental expression of the CB1R and Dagl2α occurs at the same time during larval development, suggesting that larvae are able to both synthesize and respond to 2-AG during development [35]. The same study demonstrated that morpholino knockdown of the CB1R expression lead to aberrant patterns of axonal growth. It was subsequently shown that knockdown of Dagl2 alters axon formation in the midbrainhindbrain region and alters different patterns of behavior which suggests that 2-AG plays a role in axon formation which subsequently affects the control of vision and movement in larvae [33]. Additionally, the highest level of CB1R expression in the developing larvae occurs at the time of hatching, which may suggest that the proper expression of the CB1R is neces-

In addition to neuronal development, it has also been shown that endocannabinoid signaling is required for normal embryonic liver development and function [36]. Alteration of this normal development appears to impact the structure and function of the adult liver and may impact metabolic homeostasis. It has also been shown that the CB2R plays a role in the production, expansion and migration of hematopoietic stem cells suggesting that it may play a

The consumption of *cannabis* is well known to stimulate appetite and numerous animal and human studies have detailed the role that the endocannabinoid system plays in appetite regulation, weight gain, energy balance, and lipid metabolism. Rodent models have shown that both the endocannabinoids as well as THC stimulate appetite and can produce hyperphagia [38–41], while CB1R antagonists can suppress appetite [42, 43]. In humans genetic variations in the CB1 receptor and a dysregulation of the endocannabinoid system have been linked to obesity [44, 45]. The first therapeutic targeting this system that was brought to market was the inverse agonist for the CB1 receptor rimonabant. It was shown to lead to weight loss in

sary for the increase in movement that is required for the hatching process [30].

overlap with the expression pattern of CB1R [31, 33].

tion, migration, cell proliferation and synaptogenesis [34].

role hematopoiesis during development [37].

**2.3. Feeding and lipid metabolism**

**2.2. Growth and development**

While the original use of zebrafish as a model species was focused on genetics, they are currently contributing ever evolving models to the fields of developmental biology, neuroscience, molecular biology and pharmacology research.
