**2. Functions of the zebrafish endocannabinoid system**

The initial use of zebrafish for testing the toxicity of THC occurred in 1975 [28]. However, it has only been the last 10–15 years that interest in the study of the zebrafish endocannabinoid system (ECS) has begun to grow. As outlined below, the zebrafish ECS shows genetic homology to mammalian systems and is involved in many of the same physiological processes. Importantly, the route of administration for cannabinoids to zebrafish is relatively straight forward as they can be added to the bath solution with either methanol or dimethyl sulfoxide (DMSO) as a solvent.

#### **2.1. Gene expression patterns**

The initial sequencing and mapping of the expression pattern of the CB1 receptor (CB1R) in both larvae and adults found that the zebrafish CB1R showed a 69% nucleotide identity and a 73.6% amino acid identity with the human CB1R [29]. Larvae begin to express the CB1R by the 3 somite stage of development [30] and, as expected, show a widespread and distinct expression pattern throughout the CNS (preoptic area, dorsal telencephalon, periventricular hypothalamus, tegmentum and anterior hindbrain) by 48 hpf that continues into adulthood [29, 31]. The general pattern of expression for the CB1R in the adult zebrafish brain appears to be homologous to that of mammals.

Shortly after the cloning of the CB1R zebrafish were found to express two CB2 receptor (CB2R) orthologs that showed 98% genetic identity with each other and a 39% amino acid identity with the human CB2R [32]. Importantly, similar to the CB1R, the expression patterns of the CB2R were homologous to those found in mammals with low levels in the brain and higher levels in the intestine, retina, gills, heart, muscle, pituitary and spleen.

Zebrafish also express the transient receptor potential vanilloid type 1 cation channel (TrpV1) and the G-protein coupled receptor 55 (Gpr55) early in development. Both receptors are known to bind endocannabinoids [33]. The cannabinoid receptor interacting protein (CRIP1A) is also expressed early in development [31].

In addition to the cannabinoid receptor genes, the genes responsible for the synthesis and catabolism of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) begin to 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 overlap with the expression pattern of CB1R [31, 33].

#### **2.2. Growth and development**

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

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, neurosci-

The initial use of zebrafish for testing the toxicity of THC occurred in 1975 [28]. However, it has only been the last 10–15 years that interest in the study of the zebrafish endocannabinoid system (ECS) has begun to grow. As outlined below, the zebrafish ECS shows genetic homology to mammalian systems and is involved in many of the same physiological processes. Importantly, the route of administration for cannabinoids to zebrafish is relatively straight forward as they can be added to the bath solution with either methanol or dimethyl sulfoxide (DMSO) as a solvent.

The initial sequencing and mapping of the expression pattern of the CB1 receptor (CB1R) in both larvae and adults found that the zebrafish CB1R showed a 69% nucleotide identity and a 73.6% amino acid identity with the human CB1R [29]. Larvae begin to express the CB1R by the 3 somite stage of development [30] and, as expected, show a widespread and distinct expression pattern throughout the CNS (preoptic area, dorsal telencephalon, periventricular hypothalamus, tegmentum and anterior hindbrain) by 48 hpf that continues into adulthood [29, 31]. The general pattern of expression for the CB1R in the adult zebrafish brain appears to

Shortly after the cloning of the CB1R zebrafish were found to express two CB2 receptor (CB2R) orthologs that showed 98% genetic identity with each other and a 39% amino acid identity with the human CB2R [32]. Importantly, similar to the CB1R, the expression patterns of the CB2R were homologous to those found in mammals with low levels in the brain and higher

Zebrafish also express the transient receptor potential vanilloid type 1 cation channel (TrpV1) and the G-protein coupled receptor 55 (Gpr55) early in development. Both receptors are known to bind endocannabinoids [33]. The cannabinoid receptor interacting protein (CRIP1A)

In addition to the cannabinoid receptor genes, the genes responsible for the synthesis and catabolism of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) begin to

levels in the intestine, retina, gills, heart, muscle, pituitary and spleen.

possible with mammalian models.

14 Recent Advances in Cannabinoid Research

**2.1. Gene expression patterns**

be homologous to that of mammals.

is also expressed early in development [31].

ence, molecular biology and pharmacology research.

**2. Functions of the zebrafish endocannabinoid system**

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

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

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 role hematopoiesis during development [37].

#### **2.3. Feeding and lipid metabolism**

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

#### **Distribution**


for the potential role its dysregulation may play in obesity [44]. Unfortunately, the side effect

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

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17

Zebrafish provide a model with which to study the role of the endocannabinoid system in appetite regulation and lipid bioaccumulation. Similar to what was found for mammals, rimonabant led to the suppression of feeding in juvenile fish [48]. In larvae it was found that rimonabant exposure led to larger yolk sacs during development, suggesting a decrease in the use of fat stores, which may be related to a decreased appetite. Exposure of adult zebrafish to melatonin, a known regulator of energy homeostasis, suppressed appetite through the downregulation of the CB1R gene expression [49]. It then appears that similar to rodents,

Zebrafish are also an established model for the study of lipid biology [50–52]. With respect to the endocannabinoid system, it has been shown that overexpression of the CB1R in liver leads to hepatic lipid accumulation, while suppression leads to a loss of lipid accumulation during hepatogenesis [53]. It has also been found that bisphenol A exposure produces hepatostenosis in adult zebrafish liver by increasing the liver levels of 2-AG and AEA [33]. Stimulation of the endocannabinoid system through CB1 and CB2 receptor activation can influence lipid deposition during embryogenesis through an up-regulation of the lipoprotein lipase gene [54]. Additionally, exposure to two non-psychoactive cannabinoids, namely CBD and tetrahydrocannabivarin (THCV), can lead to a decrease in intracellular lipid levels in zebrafish yolk along with human hepatocytes and adipocytes [55]. This activity does not appear to be linked to CB1R or TRPV1-R activation, but it may suggest a use for both cannabinoids in the

The effects of cannabinoids on learning and memory in mammalian models is complex and often depends on the model employed and the neural pathways that are activated. However, cannabinoid exposure has been shown to lead to memory impairments for numerous rodent

Zebrafish also have a number of different learning paradigms that include habituation learning, conditioned place preference, avoidance learning, associative learning and spatial memory tests. These learning paradigms are largely based on appetitive and/or fear conditioning [57]. Importantly, a number of these training models have been used to test the cognitive effects of various psychoactive drugs [58]. As many of these models involve the activation of different neuronal pathways, only some of which express cannabinoid receptors, the role of cannabinoid exposure on the development, retention and recall of memories can vary. One such example is a model of fear learning where adult fish were taught to associate the presentation of the alarm pheromone known as the Schreckstoff substance [59] with the presentation of a red light [60]. The response to Schreckstoff substance typically resulted in an increase in bottom dwelling and an increase in erratic movements, both of which are linked to stressful stimuli. Following training, the fish then respond to the red light stimulus, a previously inert stimulus, by showing a similar pattern of behavior. Pre-exposure to THC reduced, but did not eliminate the bottom dwelling and had only a minor effect on erratic movements

profile of rimonabant resulted in its withdrawal from the marketplace.

modulation of the zebrafish endocannabinoid system can regulate appetite.

treatment of obesity.

**2.4. Learning and memory**

learning paradigms [56].

**Table 1.** Organ distribution patterns of cannabinoid related proteins in adult zebrafish

overweight subjects and was marketed as a therapeutic for the treatment of obesity [46]. In line with this, it has been shown that CB1 receptor knockout animals are thinner than controls and have less adipose tissue, which is thought to relate to both decreased caloric intake as well as changes in metabolic factors [47]. Less adipose tissue may also be linked to the therapeutic potential of targeting the CB1 receptor in the treatment of obesity, as obesity in humans is linked to hepatic stenosis, which was shown to be reduced by treatment with rimonabant [44]. Since the initial development of rimonabant, the endocannabinoid system has been of interest for the potential role its dysregulation may play in obesity [44]. Unfortunately, the side effect profile of rimonabant resulted in its withdrawal from the marketplace.

Zebrafish provide a model with which to study the role of the endocannabinoid system in appetite regulation and lipid bioaccumulation. Similar to what was found for mammals, rimonabant led to the suppression of feeding in juvenile fish [48]. In larvae it was found that rimonabant exposure led to larger yolk sacs during development, suggesting a decrease in the use of fat stores, which may be related to a decreased appetite. Exposure of adult zebrafish to melatonin, a known regulator of energy homeostasis, suppressed appetite through the downregulation of the CB1R gene expression [49]. It then appears that similar to rodents, modulation of the zebrafish endocannabinoid system can regulate appetite.

Zebrafish are also an established model for the study of lipid biology [50–52]. With respect to the endocannabinoid system, it has been shown that overexpression of the CB1R in liver leads to hepatic lipid accumulation, while suppression leads to a loss of lipid accumulation during hepatogenesis [53]. It has also been found that bisphenol A exposure produces hepatostenosis in adult zebrafish liver by increasing the liver levels of 2-AG and AEA [33]. Stimulation of the endocannabinoid system through CB1 and CB2 receptor activation can influence lipid deposition during embryogenesis through an up-regulation of the lipoprotein lipase gene [54]. Additionally, exposure to two non-psychoactive cannabinoids, namely CBD and tetrahydrocannabivarin (THCV), can lead to a decrease in intracellular lipid levels in zebrafish yolk along with human hepatocytes and adipocytes [55]. This activity does not appear to be linked to CB1R or TRPV1-R activation, but it may suggest a use for both cannabinoids in the treatment of obesity.

#### **2.4. Learning and memory**

overweight subjects and was marketed as a therapeutic for the treatment of obesity [46]. In line with this, it has been shown that CB1 receptor knockout animals are thinner than controls and have less adipose tissue, which is thought to relate to both decreased caloric intake as well as changes in metabolic factors [47]. Less adipose tissue may also be linked to the therapeutic potential of targeting the CB1 receptor in the treatment of obesity, as obesity in humans is linked to hepatic stenosis, which was shown to be reduced by treatment with rimonabant [44]. Since the initial development of rimonabant, the endocannabinoid system has been of interest

**Protein name Abbreviation High levels Low levels** Cannabinoid receptor 1 CB1R Brain Eyes, testis

TrpV1 Sensory neurons

NAPE-pld All organs

testis, spleen

reproductive organs

eyes

αβ-Hydrolase domain containing 4 Abhd4 Spleen, testis All organs Fatty acid amide hydrolase faah Brain Skin, testis Fatty acid amide hydrolase 2a faah2a Brain Intestine, eyes,

Diacylglycerol lipase β DAGL β Brain, muscle, kidney, eyes, testis Spleen

muscle, pituitary, kidney, spleen

Brain, testis

testis

Kidney, heart, intestine

Cannabinoid receptor 2 CB2R Intestine, eyes, gills, heart,

Diacylglycerol lipase α DAGL α Brain, muscle, kidney, eyes,

Monoglyceride lipase mgll Brain, kidney, spleen, eyes

Prostaglandin-endoperoxide synthase 2 ptgs2a Skin, spleen, eyes αβ-Hydrolase domain containing 6b Abhd6b Not detectable αβ-Hydrolase domain containing 6a Abhd6a Intestine, liver, testis αβ-Hydrolase domain containing 12 abhd12 Brain, muscles, eyes,

Glycerophosphodiester phosphodiesterase1 gde1 All organs

N-acylspihingosine amidohydrolase 1a naaa1a Reproductive organs

Peroxisome proliferator-activated receptor γ pparg Muscles, spleen, testis

**Table 1.** Organ distribution patterns of cannabinoid related proteins in adult zebrafish

Peroxisome proliferator-activated receptor αβ pparab Muscles, spleen, brain, heart,

G protein-coupled receptor 55A GPR55A Brain, spleen, testis Cannabinoid receptor interacting protein (CRIP1A) Brain, eyes, testis

**Distribution**

cation channel

phospholipase D

Transient receptor potential vanilloid type 1

16 Recent Advances in Cannabinoid Research

N-acylphosphatidylethanolamine-selective

Adapted from Oltrabella et al. [31].

The effects of cannabinoids on learning and memory in mammalian models is complex and often depends on the model employed and the neural pathways that are activated. However, cannabinoid exposure has been shown to lead to memory impairments for numerous rodent learning paradigms [56].

Zebrafish also have a number of different learning paradigms that include habituation learning, conditioned place preference, avoidance learning, associative learning and spatial memory tests. These learning paradigms are largely based on appetitive and/or fear conditioning [57]. Importantly, a number of these training models have been used to test the cognitive effects of various psychoactive drugs [58]. As many of these models involve the activation of different neuronal pathways, only some of which express cannabinoid receptors, the role of cannabinoid exposure on the development, retention and recall of memories can vary. One such example is a model of fear learning where adult fish were taught to associate the presentation of the alarm pheromone known as the Schreckstoff substance [59] with the presentation of a red light [60]. The response to Schreckstoff substance typically resulted in an increase in bottom dwelling and an increase in erratic movements, both of which are linked to stressful stimuli. Following training, the fish then respond to the red light stimulus, a previously inert stimulus, by showing a similar pattern of behavior. Pre-exposure to THC reduced, but did not eliminate the bottom dwelling and had only a minor effect on erratic movements [60]. A previous study from the same group evaluating spatial memory and found that THC exposure did not affect associative memory but did impair spatial cognition and memory retrieval [60]. In addition to THC, high levels of CBD also appear to reduce memory retention in a spatial memory test [61]. While the number of studies testing the role of cannabinoids on learning and memory using zebrafish is currently limited, it appears the model has great potential in assessing the role of the endocannabinoid system in multiple aspects of learning and how this can be influenced by various cannabinoids.

on the various zebrafish models of nociception, the initial indications are that zebrafish will be

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

http://dx.doi.org/10.5772/intechopen.79321

19

Recent data suggests that approximately 9% of individuals that use cannabis show symptoms associated with addiction, including tolerance and withdrawal [88]. Comparatively the rate of dependence for tobacco is 67.5% and for alcohol is 22.7% [89]. Zebrafish represent an underutilized model with which to study the addictive properties of cannabinoids. While it has been demonstrated that zebrafish can be used to study the pathology of addiction to numerous drugs of abuse, including, alcohol, cocaine, morphine, nicotine, amphetamine, diazepam and salvinorin A [90–95], thus far their use to study the addictive properties of cannabinoids has been minimal. Changes in both larval and adult zebrafish behavior can be linked to numerous phenotypes associated with addiction that include conditioned place preference for drugs of abuse, relapse, changes in social behavior, along with symptoms indicating the development of tolerance and withdrawal [90, 93, 96–99]. It has also been found that the genetic pathways linked to addiction are highly conserved in zebrafish [100]. Currently, with respect to cannabinoids, only one study has shown that zebrafish larvae develop tolerance to the effects of cannabinoids after chronic exposure [101]. As the levels of THC in cannabis plant strains is varied and the refinement and extraction processes allow for other cannabinoids to be used at higher levels both medicinally and recreationally, there is a need to develop models with which to test the addictive properties of both pure cannabinoids on their own, in combination and as part of

valuable for assessing the efficacy of potential therapeutics for pain management.

a complex mixture or extract. Zebrafish have the potential to be such a model.

when cannabinoids are used to treat anxiety related disorders such as PTSD.

One of the known difficulties in using cannabinoids as therapeutics lies in their effect on stress and anxiety. A sought after symptom of cannabis use is the euphoric feeling that often leads to it being considered an anxiolytic. However, it has been broadly shown that as the levels of cannabinoids (specifically THC) are elevated there can be an increase in anxiety-related effects [102]. This is important not only from a side effect perspective, but also becomes an issue

Zebrafish provide numerous models with which to assess stress responses in both larvae and adults. Measurements such as scototaxis (light-dark preference), thigmotaxis (wall hugging), shoaling and the amount of time spent in the bottom of a tank are used as standard measures of stress. Induction of stress can occur by chemical means such as neuro-hyperactive compounds or exposure to the alarm substance. Stress can also be induced physically by touch or following the placement of a fish in a novel setting (novel tank response). Various visible stimuli can also lead to stress responses such as changes in back ground light/dark levels or the appearance of an image of prey. All of these models seem to activate both unique and overlapping neural pathways and thus could provide insight into the mechanism of action of any potential anxiolytic effect [103–105]. An example of the use of zebrafish stress models for testing cannabinoids was outlined in a recent paper that evaluated the acute effects of both THC and CBD on larval behavior [106]. Zebrafish larvae show a preference for light and a transition from a light to a dark setting results in an increase in activity in the form of darting type movements which are

**3.2. Addiction**

**3.3. Stress and anxiety**
