How to use Zebrafish for Cannabinoid studies

Cannabinoids are compounds that target the cannabinoid receptors. These types of alkaloid compounds come, mostly, from Cannabis genus plants, even though can be of synthetic or semisynthetic nature. They include psychoactive and non-psychoactive active ingredients. Tetrahydrocannabinol (THC) is the main psychoactive compound, and its psychotropic effects are of clinical concern as it may heighten anxiety and can potentially cause addiction and cravings. On the other hand, cannabidiol (CBD) is a non-psychoactive ingredient, and according to the World Health Organisation (WHO), CBD does not seem to have any major health risks or can potentially become a substance of abuse, while has demonstrated to have huge clinical benefits. Cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), and cannabidivarin (CBDV) are also cannabis-derived alkaloids although less studied, have a strong capability to become clinically relevant and of great interest in the medical field, the cosmetic industry, and the nutraceutical industry among other uses.

Consequently, each day they have gained the acceptance of the population and legalization in a vast number of countries in recent years. The legal commercialization of these substances for medicinal, recreational, and consumer goods purposes have created a necessity to adapt the regulatory guidelines and legislation in order to ensure these products have undergone enough testing such as safety analysis and efficacy screenings to assure they are safe for human consumption.

Therefore, there is an important need to test the properties and safety of cannabinoids to implement their use. Traditionally, these assays were done in animals due to the quality of the biological information they bring, despite their massive costs and time consumption. Nowadays, there are many efforts to minimize the use of traditional laboratory animals such as rats and mice and to replace them with New Alternative Methods (NAMs) in line with animal research care policies, following the 3Rs Principles (Replacement, Reduction, and Refinement). In this sense, cannabinoid studies in alternative animal models, such as zebrafish, are being developed to provide time and cost-efficient results and help in the controlled use of them.

Cannabinoids Efficacy Assays in Zebrafish 

In recent years, various cannabinoids have shown potential as therapeutic agents and are also gaining relevance in the cosmetics industry. They have been proposed as a treatment for a wide variety of medical indications, especially many neurodegenerative and rare diseases, like Amyotrophic Lateral Sclerosis (ALS), Parkinson's Disease (PD), Huntington's Disease (HD), epilepsy, anxiety, or inflammatory diseases. Their use has been regulated in several European countries and in the United States by the Food and Drug Administration (FDA). Although more research must be done in order to analyze and demonstrate the safety, side effects, and therapeutic benefits of cannabinoids in a vast array of conditions. In that regard, the use of zebrafish complies with all the characteristics of a perfect NAM for cannabinoids research as well as offers many scientific and ethical benefits. 

Among their many advantages, zebrafish stand out due to their small size, easier breading and manipulation, fast organogenesis (less than 5 days post-fertilization (dpf), and transparency of larvae stages, which allows for cost and time-effective High Content Screening Assays (HCS). The possibility of using fish embryos or larvae instead of adults, is in line with animal care policies (The 3Rs Principles). Besides its practical benefits, zebrafish is an excellent model because of the quality of the biological information it provides due to its unique characteristics including transparency, for direct organ visualization alone or with the help of fluorescent reporters; easiness of genetic manipulation; and its high genetic and physiological homology with humans (over 75%), especially, in neuronal physiology.

Thus, alterations in the mobility pattern of larvae induced by known drugs have been observed to strongly support Zebrafish as a predictive model of neuroactivity in humans. Behavioral profiling in zebrafish reveals relationships between drugs and their targets and demonstrates conserved vertebrate neuropharmacology.

There has been an increase in the number of studies on the pharmacological effects of cannabinoids using zebrafish as an animal model. In 2018 Achenbach et al. evaluated the acute effects of both THC and CBD on larval behavior and the results showed that both CBD and THC had significant effects on behavioral alterations, providing validation for the zebrafish model and providing a foundation for future work with cannabinoids in zebrafish.

Consequently, cannabinoid efficacy assays have been designed in zebrafish to demonstrate the efficacy of these kinds of compounds through behavioral analysis. The behavior analysis in zebrafish is a rapid and non-invasive alternative assay with high sensitivity and specificity that allows the evaluation of the Central Nervous System (CNS) function and locomotor pattern, leading to an early screening of neuroactive/psychoactive candidates in a cost and time-efficient manner.

A good example of these neurological evaluation assays is the locomotor activity analysis assays developed by Biobide, which can identify primary behavioral alterations including variations in movement through different endpoints (distance moved, mean velocity, maximum and minimum velocity, movement patterns, …), evaluation of anxiety-related behaviors, sleep patterns, or learning and memory processes in response to different stimuli (such as light-dark transitions, touching, or vibrations) in an HCS format. 

Biobide offers several behavior assays: 

• General Locomotion Assay: the assa based on the light/dark transition test analyzing parameters such as distance moved, velocity, or high-speed movements to evaluate locomotor activity. By this assay, hyper- or hypoactivity can be determined in 5-6 dpf zebrafish larvae. 

• Thigmotaxis Assay: a well-validated index of anxiety that measures the reduction of exploratory behavior. A stereotypical behavior related to anxiety disorders is the avoidance and reduction of exploration and it is an endpoint widely employed in animal models for anxiety studies. It has been shown that the zebrafish animal model can be employed for this study, even in their larval stage below 6 dpf. Parameters such as total distance moved, distance moved in the periphery and quiescence (time and frequency) are studied.
  
  
• Sleeping Assay: zebrafish a diurnal specie with a sleeping-awake system regulated by homeostatic and circadian mechanisms highly conserved and with melatonin as the principal hormone involved in it, make a perfect model for studying sleeping disorders. Parameters such as distance moved and quiescence (time and frequency) are studied to evaluate sleep-wake cycles. As a result, alterations in resting bouts can be related to altered sleeping and behavior patterns.  

• Habituation Assay: the assay used to assess an organism's response to repeated stimuli over a period of time, when zebrafish are exposed to repeated, non-threatening stimuli, usually vibration. Initially, the fish exhibit a strong response, but with repeated exposures, they gradually habituate and display a reduced response. Therefore, the rate and extent of habituation can provide insights into the fish's primary learning and memory processes, sensory perception, and behavioral plasticity. This assay is widely used in zebrafish research to study neural circuits, drug effects, and genetic factors impacting behavior. 

Evaluation of behavior can be complemented with neurophysiologic evaluations such as electrophysiology, or activation of specific neuronal circuits or brain regions to discern between specific neurologic effects or toxicity and to understand the neurologic patterns underlying these behaviors.

Cannabinoids neurotoxicity assays in zebrafish

A critical aspect of neuroactive compounds, including cannabinoids, is the evaluation of their toxicity to the nervous system, which can cause a variety of neurological disorders.

Zebrafish are growingly being used as model organisms for neurotoxicity studies. Their nervous system arrangement and the process and specific mechanisms of neurogenesis are similar to other vertebrates, including humans; also, they have a Blood Brain Barrier (BBB) that is of added value when conducting assays in the field of neurotoxicity and, overall, becoming an excellent animal model for neurotoxicology research due to their high fidelity with the human nervous system. Several approaches using zebrafish in neurotoxicity screening have been developed. Neurotoxicity endpoints such as changes in gene expression, neural morphogenesis, and neurobehavioral profiling are used together or combined with efficacy assays to assess the effects of substances on the nervous system.

The Central Nervous System in zebrafish is mostly developed within 3 dpf, making them ideal for rapid neurotoxicity assays. At this stage, they also show spontaneous swimming behavior, allowing the automated assessment of locomotor activity under different conditions. Subsequently, at 5 dpf embryos present a quite developed nervous system being able to respond to several external factors such as light changes, noises, or vibrations.  

Biobide can analyze the effects in locomotion for efficacy but also for neurotoxicity assessment. Furthermore, the assays are performed on 96 well plates, allowing fast, scalable, and cost-effective HCS assays. 

Neurotox Assay evaluates the acute neurotoxicity of compounds after exposition on 5 dpf larvae or development neurotoxicity by the exposition larvae for 5 days, from 3 hpf (hours post-fertilization) to 120 hpf. After 120 hpf, the dark-light transition test is performed, and locomotor activity and morphology are evaluated to determine acute neurotoxicity or developmental neurotoxicity (DNT). 

Neurotoxicity and DNT of cannabinoids can be complemented with Dose Range Finding (DRF and Bioavailability Assays, to determine the tolerable levels of a compound and the dose at which a compound produces those therapeutic or toxic effects on the subjects. 

Additional organ-specific toxicity assays can be performed, including the organ-specific toxicity of the heart, liver, muscle, kidney, immune system, or endocrine system, all of them in HCS format.

Conclusions  

Cannabinoids are gaining attention due to their substantial therapeutic effects that must be well determined and their potential detrimental health effects, mainly, neurotoxicity. To perform these evaluations is essential to use reliable and cost-effective NAMs such as zebrafish, which represent an in vivo model that presents many advantages. Especially the ones allowing automated HCS assays with the advantage of being cost and time-effective, but permitting reliable behavioral and neurological results due to high genetic and neurophysiology similarity with humans and the possibility of direct in vivo movement studies.

 Sources 

• Cannabinoids Efficacy Assays (biobide.com)
• Neurotox Assay | Biobide
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