Zebrafish as an Animal Model in Cannabinoid Research
Abstract
:1. Introduction
Type of Compounds | Description |
---|---|
Endocannabinoids | Compounds naturally occurring in living organisms, arachidonic acid derivatives Examples: nandamide (AEA), 2-arachidonoylglycerol (2-AG) Significance: influence on emotions, metabolism, appetite, memory and learning processes, and psychomotor activity [28] |
Phytocannabinoids | Compounds of herbal origin Examples: Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD) Significance: psychoactive effects (Δ9-THC), therapeutic potential (CBD) Side effects (particularly after high doses of Δ9-THC): palpitations, paranoia, anxiety, nausea, vomiting, confusion, coordination disturbances, and seizures [29] |
Synthetic cannabinoids | Synthetic compounds Examples: nabilone, dronabinol Significance: used in experimental pharmacology, and used in the treatment of drug-resistant epilepsy, vomiting and nausea during chemotherapy, anorexia in AIDS patients, used as an adjunctive treatment in multiple sclerosis Side effects: similar to side effects observed after high doses of THC, mentioned above [30] |
Allosteric modulators | Small molecules that allosterically modulate the CB1 receptor Examples: Org27569, PSNCBAM-1, ZCZ011, pepcan-12, lipoxin A4, pregnenolone Significance: better receptor subtype selectivity than cannabinoids, selectively towards tissues where the endogenous ligands are present and function, “ceiling” action preventing drug overdose [14] |
Molecules degrading the endogenous cannabinoid ligands | Inhibitors of fatty acid amide hydrolase (FAAH) Examples: substrate-derived inhibitors (trifluoromethyl ketone analog of oleamide, MAFP), α-ketoheterocycle inhibitors, carbamate inhibitors, urea inhibitors, aryl boronic acids Significance: potential use in the treatment of pain, inflammation, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, insomnia, anxiety, depression, hypertension, cancer, and inflammatory bowel disease [12] Inhibitors of monoacylglycerol lipase (MAGL) Examples: maleimide derivatives, disulfides (e.g., disulfiram), carbamates (URB602, JZL184, KML29, ABX-1431), ureas, arylthoamides, tetrahydrolipstatin-based inhibitors, isothiazolines (e.g., octhilinone), natural terpenoids (e.g., pristimerin, euphol), amide-based derivatives Significance: potential use in the treatment of pain, anxiety, inflammation, cancer, and metabolic disorders [13] |
1.1. Potential Use of Cannabinoids in Medicine
1.2. Preclinical Research of Cannabinoids
2. The Endocannabinoid System in Danio rerio
3. Use of Zebrafish in Cannabinoid Research
3.1. Research on the Impact of Early Life Exposure to Cannabinoids
3.2. Behavioural Research
3.3. Drug Metabolism Studies in Zebrafish
3.4. Disease Modeling and Other Studies in Danio rerio
Tested Substances | Tested Doses | Modeled Disease | Main Findings | Ref. |
---|---|---|---|---|
370 synthetic cannabinoid compounds | 10 and 250 µM, and additionally, 1, 10, and 100 µM | Dravet Syndrome (scn1lab homozygous mutants, 5 dpf) | The used model successfully identified five synthetic cannabinoids with the anti-seizure potential, i.e., indole-based cannabinoids JWH 018 N-(5-chloropentyl) analog, JWH 018 N-(2-methylbutyl) isomer, 5-fluoro PB-22 5-hydroxyisoquinoline isomer, 5-fluoro ADBICA, and AB-FUBINACA 3-fluorobenzyl isomer. | [40] |
CBD, cannabichromene (CBC), cannabidivarin (CBDV), cannabigerol (CBG), cannabinol (CBN) | CBD, CBN, and CBDV: 0.25–4 µM CBC: 0.1–3 µM CBG: 0.25–3 µM | Parkinson’s disease (6-hydroxydopamine (OHDA)-induced model, studies carried out 120 hpf) | The tested cannabinoids, when used separately, did not have any influence on the OHDA-induced hypoactivity, whereas when used as three-component equimolar mixtures (containing CBD + CBDV + CBC or CBD + CBN + CBC or CBD + CBDV + CBG) cannabinoids significantly reduced OHDA-related motor symptoms. | [107] |
Crude CBD extract | 0.1, 0.25, 0.5, 1.25 mg/L | Zebrafish embryo caudal fin amputation model (caudal fin was amputated 3 dpf, and observations were made 48- and 72-h postamputation) | CBD accelerated zebrafish fin regeneration in a dose-dependent manner, partially due to the regulation of the inflammatory response. It also reduced apoptosis after amputation. | [108] |
CBD | 1.25 µM | Tuberous Sclerosis Complex (tsc2 nonsense mutation, 6 dpf) | CBD exerted an anxiolytic effect in behavioral studies (anxiety closely associated with TSC). A decrease in the level of phosphorylated rps6 was observed, which may be due to a reduction in activation of TOR activation (TSC experimental models have increased mTOR activity). | [109] |
THC, CBD | CBD: 1, 1.5, 2 µM THC: 1.5, 2, 3 µM | Neurohyperactivity (pentylenetetrazole-induced model and the genetic model induced by loss-of-function mutations in the GABA receptor subunit alpha1 (GABRA1−/−); 5 dpf) | Both THC and CBD opposed the behavioral hyperactivity induced by pentylenetetrazole and genetic mutation. Higher doses were needed in the case of the genetic model. THC and CBD acted synergistically when used concurrently. | [110] |
Abnormal cannabidiol and O-1602 | 0.1–10 µM | Zebrafish xenograft model (taxol-resistant MDA-MB-231 cells of human breast cancer; 48 hpf | Both tested compounds induced apoptosis of breast cancer cells and they reduced cell migration at a concentration of 2 µM. | [111] |
CBD, THC, CBVD, CBN, linalool (LN) | CBD: 0.3, 0.6, 1.0 µM THC: 1.0, 4.0 µM CBVD: 0.3, 0.6, 1.0 µM CBN: 0.3, 0.6, 1.0 µM LN: 0.3, 0.6, 1.0, 4.0 µM | Dravet Syndrome (scn1lab homozygous mutants) pentylenetetrazole-induced seizures; 6 dpf following 24 h of exposure | In the Dravet Syndrome model, CBD (0.6 µM), THC (1 µM), CBN (0.6 and 1 µM), and LN (4 µM) significantly reduced the number of seizures, with CBN being the most effective. In the pentylenetetrazole-induced model, only CBD and THC were effective. | [112] |
4. Conclusions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Drug | Indication |
---|---|
Epidiolex (oral solution containing 100 mg/mL of cannabidiol) |
|
Marinol (capsules containing 2.5, 5, or 10 mg of dronabinol) |
|
Syndros (oral solution containing 5 mg/mL of dronabinol) | |
Sativex (oromucosal spray containing 2.7 mg/mL of Δ9-tetrahydrocannabinol and 2.5 mg/mL of cannabidiol from Cannabis sativa L.) |
|
Cesamet (capsules containing 1 mg of nabilone) |
|
Tested Substances | Tested Doses | Main Findings | Ref. |
---|---|---|---|
THC, CBD | THC: 2, 4, 6, 8, 10 mg/L CBD: 1, 2, 3, 4 mg/L | Acute experiments. A 5-h exposure during the gastrulation phase led to significant alterations in the heart rate, the morphology of motor neurons, synaptic activity at the neuromuscular junction, as well as changes in the locomotor responses to sound, as well as changes in the expression of postsynaptic nicotinic acetylcholine receptors. | [60] |
THC | 6 mg/L | Acute experiments. Embryos exposed to THC during the gastrulation phase presented minor changes in the axon diameter of Mauthner cells and minor changes in the escape response dynamic to touch. Furthermore, when developed until 5 dpf, animals presented reduced locomotor activity in comparison to the control group. | [61] |
THC, CBD | THC: 1, 2, 4, 8, 16 µM CBD: 0.25, 0.5, 1, 2, 4 µM | Chronic exposure. A 96-h exposure to THC and CBD led to the development of oedemas, body curvature, and disorders in the formation of ears, eyes, and jaws. Animals presented behavioral changes. The LC50 for CBD and THC was 0.53 mg/L and 3.65 mg/L, respectively. | [62] |
Cannabis sativa extract | 2, 20, 200 µL | Chronic experiments. No negative effects on embryo development, embryo hatching, or survival rate were detected after exposure to the Cannabis extract. Larvae exposed to the highest dose of the extract (i.e., containing 1.73 nM and 22.3 nM of THC and cannabidiol CBD, respectively) showed increased locomotion compared to control groups. Moreover, qRT-PCR analysis showed that the highest dose of the extract induced an overexpression of CB1 and CB2 receptors. | [22] |
CBN | 0.25, 0.75, 1, 1.125, 1.2 mg/L | Chronic experiments. A 96-h exposure led to the development of malformations in zebrafish larvae, which increased in a dose-dependent manner. CBN concentrations > 0.75 mg/L induced the development of pericardial edema, anomalies in the yolk sac, tail bending, elongated hearts and shorter in their width, and separation of the ventricle from the atrium. The LD50 value was estimated at 1.12 mg/L. CBN acted as a stimulant and sedative agent, causing both altered velocity and bradycardia in animals. | [63] |
THC | 1, 1.25, 1.5, 2 mg/L | Chronic experiments (a 96-h exposure). The LC50 value was determined as 1.54 mg/L. Tail bending, pericardial edema, etc., were observed even in the LC50. The heart rate, cardiac contractility, and heart rhythm were also significantly affected. | [64] |
5F-APINAC | 0.001, 0.01, 0.1, 1, 10 µM | Acute and chronic experiments. An exposure to 5F-APINAC for 4 (acute treatment) and 96 h (chronic treatment) resulted in changes in GABA-, tryptamine-, acetylcholine-, xanthurenic acid-, picolinic acid-dependent neurotransmission. A 96-h exposure to the 5F-APINAC dose of 10 µM induced embryotoxicity (i.e., disturbed hatching process, tail bending, hyperpigmentation). | [65] |
CBN | 0.0.1, 0.1, 0.5, 1, 2, 3, 4 mg/L | Acute experiments (exposure from 5.25 to 10.75 hpf). Higher mortality rates, reduced mobility, and impaired development of motor neurons were detected with the increase in the tested doses of CBN. Even the lowest tested concentration induced disturbances in the development of hair cells responsible for the auditory function in zebrafish. | [66] |
THC, CBD | THC: 0.024, 0.12, 0.6 mg/L; 0.08, 0.4, 2 µM CBD: 0.006, 0.03, 0.15 mg/L; 0.02, 0.1, 0.5 µM | Multigenerational experiments. After 96-h exposure of the F0 generation to THC and CBD no significant morphological abnormalities were detected in either F0 or F1 generations; however, signs of the developmental neurotoxicity were observed. Furthermore, the fecundity of the F0 generation was reduced, and after exposure to the highest tested dose of THC, animals from the F1 generation spent a significantly shorter time in the periphery. | [67] |
THC, CBD | THC: 1, 2 µg/mL CBD: 1, 2 µg/mL THC + CBD: 1 + 1, 2 + 2 µg/mL | Acute experiments. Early exposure to CBD and THC during the first 10 h of embryonic development led to reduced neural activity in a dose-dependent manner when measured 4 days later. | [26] |
CBD | 0.02, 0.1, 0.5 µM | Multigenerational experiments. A 96-h exposure to CBD of the F0 generation resulted in increased survival and reduced body size in females. A dose of 0.5 µM reduced fertility in males, whereas a dose of 0.1 µM increased the production of eggs in females. These effects were not observed in the F1 generation. | [68] |
CP-55940 | 1, 2.5, 3.8, 5.0 mg/L | Chronic experiments. A 24- or 48-h exposure to CP-55940 resulted in a dose-dependent increase in microphthalmia and midbrain/hindbrain boundary defects. | [58] |
ACEA | 1, 3, 6 mg/L | Chronic experiments. A 48-h exposure to ACEA at a dose of 6 mg/L induced microphthalmia in developing embryos. | [59] |
Tested Substances | Tested Doses | Behavioral Test | Tested Subjects | Main Findings | Ref. |
---|---|---|---|---|---|
CBD | 40 mg/L | Novel tank diving test | Adult fish (6 months) | A 30-min exposure to CDB decreased swimming speed and traveled distance. | [80] |
CBD, WIN55,212-2 | For both agents: 0.5, 1, 5, 10 µg/mL | Light/dark test | Larvae (4–5 dpf) | CBD at a dose of 10 µg/mL reduced the traveled distance and velocity of movement in the darkness. Both lower tested doses (0.5 and 1 µg/mL) of WIN55,212-2 reduced the traveled distance and moving duration, whereas higher doses of the agent (5 and 10 µg/mL) turned out to be lethal to zebrafish larvae within less than 24 h. | [81] |
JWH-018 | THC: 2 µM, JWH-018: 3 µM | Forced light/dark test, startle stimuli test, novel tank diving test | Larvae (5 dpf in the forced light/dark test; 6 dpf in the startle response), adult (4 months in the novel tank diving) | After the developmental exposure to THC and JWH-018, impaired locomotion was detected during baseline and dark periods in the forced light/dark test. In the startle stimuli test, no significant differences in the traveled distance before and during the stimuli were recorded for JWH-018, whereas reduced activity before the stimuli was detected for THC. In the novel tank diving test, adult zebrafish who had been subjected to developmental exposure to JWH-018 spent less time on the bottom of the tank, whereas developmental exposure to THC had no impact on animals’ behavior. | [82] |
Whole-plant Cannabis extracts | 0.25, 0.5, 1, 2 µg/mL | Light/dark test | Larvae (120 hpf) | Zebrafish larvae can be used to assess the bioactivity of Cannabis extracts. Cannabis extracts with various chemical profiles have a distinct impact on baseline larval activity and stress responses. | [83] |
ACEA (CB1 receptor agonist) | 1 mg/kg | Acute restraint stress, novel tank diving test | Adult | Treatment with ACEA prevented both the acute restraint stress-induced anxiety-like behavior and oxidative stress in the zebrafish brain. | [84] |
THC | 40 nM, 1, 2 µM | Swimming pattern | Adult | Animals exposed to THC presented abnormal swimming patterns, i.e., circular swimming (behavioral stereotypy). | [85] |
THC, CBD, THC + CBD | THC: 0.05, 0.1, 0.5, 1.5, 2.0 µM CBD: 0.75, 1.0, 1.75, 2.5, 3.75 µM THC + CBD: 0.5 + 0.5, 1.5 + 0.5 µM | Locomotor activity in Zebrabox and the light/dark test | Larvae (120 hpf) | Exposure to THC (at all tested concentrations) decreased the locomotor activity of larvae, whereas exposure to CBD at concentrations above 1.75 µM increased locomotor activity. | [86] |
9,10-dihydro-5-hydroxy-2, 3,6-trimethoxyphenanthrene-1,4-dione isolated from a commercial cannabis product | 1, 2.5, 3.5 µM | Locomotor activity | Larvae (5 dpf) | Concentrations above 2.5 µM increased the locomotor activity of larvae | [87] |
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Lachowicz, J.; Szopa, A.; Ignatiuk, K.; Świąder, K.; Serefko, A. Zebrafish as an Animal Model in Cannabinoid Research. Int. J. Mol. Sci. 2023, 24, 10455. https://doi.org/10.3390/ijms241310455
Lachowicz J, Szopa A, Ignatiuk K, Świąder K, Serefko A. Zebrafish as an Animal Model in Cannabinoid Research. International Journal of Molecular Sciences. 2023; 24(13):10455. https://doi.org/10.3390/ijms241310455
Chicago/Turabian StyleLachowicz, Joanna, Aleksandra Szopa, Katarzyna Ignatiuk, Katarzyna Świąder, and Anna Serefko. 2023. "Zebrafish as an Animal Model in Cannabinoid Research" International Journal of Molecular Sciences 24, no. 13: 10455. https://doi.org/10.3390/ijms241310455