Phytocannabinoids Reduce Seizures in Larval Zebrafish and Affect Endocannabinoid Gene Expression

Abstract

Cannabis has demonstrated anticonvulsant properties, and about thirty percent of epileptic patients do not have satisfactory seizure management with standard treatment and could potentially benefit from cannabis-based intervention. Here, we report the use of cannabinoids to treat pentylenetetrazol (PTZ)-induced convulsions in a zebrafish model, their effect on gene expression, and a simple assay for assessing their uptake in zebrafish tissues. Using an optimized behavioral assay, we show that cannabidiol (CBD) and cannabichromene (CBC) and cannabinol (CBN) are effective at reducing seizures at low doses, with little evidence of sedation, and our novel HPLC assay indicates that CBC is effective with the lowest accumulation in larval tissues. All cannabinoids tested were effective at higher concentrations. Pharmacological manipulation of potential receptors demonstrates that Gpr55 partially mediates the anticonvulsant effects of CBD. Treatment of zebrafish larvae with endocannabinoids, such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA), altered larvae movement, and the expression of genes that regulate their metabolism was affected by phytocannabinoid treatment, highlighting the possibility that changes to endocannabinoid levels may represent one facet of the anticonvulsant effect of phytocannabinoids.

Keywords: GPR55; cannabis; endocannabinoid; epilepsy; phytocannabinoid; seizure; zebrafish.

Figure 1

Analysis of movement in 6 dpf larvae exposed to phytocannabinoids and vehicle control (methanol). (A–F) Baseline activity of 6 phytocannabinoids. The “0 μM” indicates incubation in methanol only. Data presented as mean +/− SEM. ** p < 0.01, *** p < 0.001 (compared to 0 μM). n = 67–72 per condition.

Figure 2

Analysis of PTZ-induced hyperactivity after pre-treatment with phytocannabinoids. (A–F) Total activity after PTZ and pretreatment with three doses (1, 2, and 4 μM, green bars) of 6 phytocannabinoids. The solid black line indicates the mass of cannabinoid isolated through HPLC analysis. Data graphed as mean +/− SEM, ** p< 0.01, *** p < 0.001 (movement data compared to 0 μM). n = 67–72 per treatment.

Figure 3

Inhibition of endocannabinoid receptors in zebrafish PTZ hyperactivity assay. Movement tracking after addition of CBD (2 μM) and receptor antagonists demonstrating the effect on baseline movement (A) all data not significant from vehicle controls) and after the addition of PTZ (2.75 mM (B). Final concentrations of receptor antagonists were 2.5 μM (ML-193 and PSB-CB5) and 1 μM AM251. Data graphed as mean +/− SEM. n = 34–36 larvae for each condition. *** statistically significant p < 0.001 when compared to PTZ only. Red X (XXX) indicates statistical significance from the combined PTZ and CBD treatment p < 0.001.

Figure 4

The endocannabinoid 2-AG alters baseline movement and PTZ-induced hyperactivity. Movement assayed before the addition of PTZ demonstrates increased activity at the 1 μM dose for 2-AG and reduced movement at 2 and 4 μM doses (A). The 4 μM dose of AEA significantly increased baseline movement (C). After the addition of PTZ, reduced movement is observed compared to controls at the 4 μM dose of 2-AG (B), with no effect observed for AEA (D). Data presented as mean +/− SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 (compared to 0 μM). n = 40–48 larvae per condition.

Figure 5

qPCR expression analysis of seizure markers and endocannabinoid system genes. (A–G) Methanol was used as the vehicle control, with 4 µM cannabinoid doses of cannabinoids used. Significance is noted via one-factor ANOVA with Tukey HSD, * p < 0.05, ** p < 0.01 (compared to WT). Red (X) indicates significance from PTZ/VC-treated larvae (p < 0.05). Error bars are SEM.