Abstracts

Rationale:
The endocannabinoid system (ECS) consists of lipid signaling molecules (endocannabinoids), cannabinoid receptors and the enzymes that synthesize and degrade endocannabinoids. In the central nervous system, endocannabinoids such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are retrograde neurotransmitters released on-demand to regulate both excitatory and inhibitory synaptic activity. Therefore, it is not surprising that perturbation of the ECS has been shown to contribute to seizures by causing an imbalance in excitatory and inhibitory neurotransmission. We sought to determine whether dysfunction of the ECS contributes to the Dravet syndrome phenotype. Dravet syndrome is a severe, treatment-resistant childhood epilepsy characterized by febrile seizures, afebrile spontaneous seizures, cognitive and behavioral deficits and an increased risk for sudden unexpected death. Heterozygous deletion of Scn1a (Scn1a+/-) in mice mimics the phenotype hallmarks of Dravet syndrome.
Method:
We measured the function of the ECS in Scn1a+/- mice. The Scn1a+/- mouse model exhibits a strain-dependent epilepsy phenotype, with mice on the 129S6/SvEvTac (129) background having no overt phenotype and mice on a mixed F1 (129S6/SvEvTac x C57BL/6J) strain displaying hyperthermia-induced seizures, spontaneous seizures and premature death. We compared hippocampal expression of components within the ECS between strains, using digital droplet RT-PCR and Western blot analysis. Endocannabinoids, 2-AG and AEA, were quantified using LC-MS/MS. Additionally, we used F1.Scn1a+/- mice to examine the anticonvulsant potential of two compounds that target the endocannabinoid system: GAT229, a positive allosteric modulator (PAM) of CB1 receptors, and ABX1431, an inhibitor of monoacylglycerol lipase (MAGL), the predominant 2-AG degrading enzyme.
Results:
A reduced ECS tone was observed in mice on the seizure susceptible F1 background strain. F1 mice had significantly less mRNA expression of Cnr1, the gene encoding CB1 receptors, than mice on the 129 background strain (n=8-11, p = 0.0117), which translated to significantly lower levels of protein expression (n=7-8, p = 0.0208). To examine whether reduced CB1 receptor expression might contribute to the seizure phenotype of F1.Scn1a+/- mice, we treated mice with the CB1 receptor PAM, GAT229. GAT229 (30 and 100 mg/kg) significantly elevated temperature thresholds for thermally-induced seizures compared to vehicle controls (n=18-22, p = 0.0152 and p < 0.0059, respectively). We also found that hippocampal 2-AG concentrations were significantly lower in F1 mice (n=14-16, p = 0.035). F1.Scn1a+/- mice treated with the MAGL inhibitor, ABX1431 (10 mg/kg) to increase levels of 2-AG had a significantly elevated temperature threshold for hyperthermia-induced seizures compared to vehicle-treated mice (n=17, p = 0.0008).