Modulation of pilocarpine-induced seizures by cannabinoid receptor 1

Abstract

Administration of the muscarinic agonist pilocarpine is commonly used to induce seizures in rodents for the study of epilepsy. Activation of muscarinic receptors has been previously shown to increase the production of endocannabinoids in the brain. Endocannabinoids act at the cannabinoid CB1 receptors to reduce neurotransmitter release and the severity of seizures in several models of epilepsy. In this study, we determined the effect of CB1 receptor activity on the induction in mice of seizures by pilocarpine. We found that decreased activation of the CB1 receptor, either through genetic deletion of the receptor or treatment with a CB1 antagonist, increased pilocarpine seizure severity without modifying seizure-induced cell proliferation and cell death. These results indicate that endocannabinoids act at the CB1 receptor to modulate the severity of pilocarpine-induced seizures. Administration of a CB1 agonist produced characteristic CB1-dependent behavioral responses, but did not affect pilocarpine seizure severity. A possible explanation for the lack of effect of CB1 agonist administration on pilocarpine seizures, despite the effects of CB1 antagonist administration and CB1 gene deletion, is that muscarinic receptor-stimulated endocannabinoid production is acting maximally at CB1 receptors to modulate sensitivity to pilocarpine seizures.

Figure 1. CB₁ KO mice are more sensitive to pilocarpine.

Seizure severity scores and the proportion of mice having at least one clonic-tonic seizure after injection with 250 mg/kg pilocarpine were compared in male CB₁ KO (n = 7) and WT (n = 7) littermates. * p<0.05; ** p<0.005. Data are presented as medians ± upper and lower quartiles.

Figure 2. CB₁ receptor antagonist pretreatment increases pilocarpine seizure sensitivity.

SR141716 (SR1, 10 mg/kg) or the corresponding vehicle were given 2 hours prior to injection of 250 or 275 mg/kg pilocarpine. Seizure severity scores and the proportion of mice exhibiting at least one tonic-clonic seizure were compared between SR1 (250 mg/kg, n = 18; 275 mg/kg, n = 13) and vehicle-treated mice (250 mg/kg, n = 15; 275 mg/kg, n = 11). * p<0.05; ** p<0.005; *** p<0.0001. Data are presented as medians ± upper and lower quartiles.

Figure 3. Cell death and cell proliferation after pilocarpine-induced SE are unchanged by SR1 pretreatment.

Mice were pretreated for 2 hours with SR141716 (10 mg/kg) or vehicle before injection with 225–250 mg/kg pilocarpine. Brains were harvested for immunohistochemistry analysis 4 days after pilocarpine treatment. A. Representative images of proliferating cell nuclear antigen (PCNA) immunofluorescence and Fluoro-jade B (FJ) staining from (top to bottom) vehicle-treated mice without SE (V−), SR1-treated mice without SE (SR1−), vehicle-treated mice with SE (V+), and SR1-treated mice with SE (SR1+). Scale bar = 100 µm. B. PCNA and FJ quantifications in the hilus of vehicle-treated mice without SE (V−, n = 6), vehicle-treated mice with SE (V+, n = 3), and SR1-treated mice with SE (SR1+, n = 5). The number of positive cells was normalized to the area of hilus measured per animal. * p<0.05 when compared to vehicle-treated mice without SE (V−). Data are presented as medians ± upper and lower quartiles.

Figure 4. Administration of CP55940 results in CB1-dependent cannabinoid response.

Cannabinoid tetrad behaviors (hypolocomotion, catalepsy, analgesia, and hypothermia) were measured after i.p. administration with 0.3 mg/kg CP in WT (n = 5) and CB₁-KO mice (n = 5). A. Mice were treated with either vehicle or CP, and placed in an open-field chamber 30 minutes after treatment. B. Catalepsy was measured by the bar test either 5 minutes before, or 30 minutes after CP treatment in WT and CB₁ KO mice. C. Analgesia was measured by tail flick both before and after CP treatment. D. Hypothermia was measured by comparing core body temperature before and 30 minutes after treatment with CP. Data are presented as means ± SEM. ***p<0.001.

Figure 5. CB₁ receptor agonist pretreatment does not reduce pilocarpine seizure severity.

Mice were pretreated with CP 55940 (CP, 0.3 mg/kg) or vehicle 30 minutes prior to pilocarpine (325 mg/kg). Seizure severity scores and the proportion of mice having at least one clonic-tonic seizure were compared between CP-treated (n = 8) and vehicle-treated mice (n = 8). Data are presented as medians ± upper and lower quartiles.