Increased seizure susceptibility and proconvulsant activity of anandamide in mice lacking fatty acid amide hydrolase

Abstract

A number of recent in vitro studies have described a role for endogenous cannabinoids ("endocannabinoids") as transsynaptic modulators of neuronal activity in the hippocampus and other brain regions. However, the impact that endocannabinoid signals may have on activity-dependent neural events in vivo remains mostly unknown and technically challenging to address because of the short half-life of these chemical messengers in the brain. Mice lacking the enzyme fatty acid amide hydrolase [FAAH (-/-) mice] are severely impaired in their ability to degrade the endocannabinoid anandamide and therefore represent a unique animal model in which to examine the function of this signaling lipid in vivo. Here, we show that the administration of anandamide dramatically augments the severity of chemically induced seizures in FAAH (-/-) mice but not in wild-type mice. Anandamide-enhanced seizures in FAAH (-/-) mice resulted in significant neuronal damage in the CA1 and CA3 regions of the hippocampus for the bicuculline and kainate models, respectively. Notably, in the absence of anandamide treatment, FAAH (-/-) mice exhibited enhanced seizure responses to high doses of kainate that correlated with greatly elevated endogenous levels of anandamide in the hippocampus of these animals. Collectively, these studies suggest that both exogenously administered and endogenously produced anandamide display FAAH-regulated proconvulsant activity and do not support a general neuroprotective role for this endocannabinoid in response to excitotoxic stimuli in vivo. More generally, these findings demonstrate that the disinhibitory actions of endocannabinoids observed in hippocampal slices in vitro may also occur in vivo.

Fig. 1.

The effects of anandamide on bicuculline- and kainate-induced seizures in FAAH (+/+) and (−/−) mice. Treatment with anandamide (6.25–50 mg/kg, i.p.) 1 hr before administration of either bicuculline (A; 4 mg/kg, i.p.) or kainate (C; 15 mg/kg, i.p.) significantly enhanced the severity of seizures in FAAH (−/−) mice (filled bars) but not in FAAH (+/+) mice (open bars); n = 4 mice per group for 6.25 and 12.5 mg/kg anandamide, and n = 6–14 mice per group for 25 and 50 mg/kg anandamide. Pretreatment with a low dose of anandamide (6.25 mg/kg, i.p.) did not affect the seizure responses to high doses of bicuculline (B; 6 mg/kg) or kainate (D; 25 mg/kg) in FAAH (+/+) or (−/−) mice;n = 6–14 mice per group. For A–D, seizure scores are presented as mean ± SEM. All data were compared with the Mann–Whitney U test. ★p < 0.05 for FAAH (+/+) versus FAAH (−/−) mice receiving the same treatment. *p < 0.05 and ***p < 0.001 for FAAH (−/−) mice under different treatment conditions.

Fig. 2.

The effects of CB₁ receptor agonists and antagonists on chemically induced seizures in FAAH (+/+) and (−/−) mice. A, FAAH (+/+) and (−/−) mice were treated with the CB₁ receptor antagonist SR141716A (1 mg/kg, i.p.) or vehicle 10 min before administration of anandamide (25 mg/kg, i.p.) or vehicle. Seizures were induced with (bicuculline, 4 mg/kg, i.p.) 60 min after anandamide injection. SR141716A blocked the effects of anandamide on seizures in FAAH (−/−) mice but did not affect seizure responses in vehicle-treated animals of either genotype (SR group). n = 6–12 mice per group. ★p < 0.05 for FAAH (+/+) versus FAAH (−/−) mice receiving the same treatment. **p < 0.01 and ***p < 0.001 for FAAH (−/−) mice under different treatment conditions (Mann–Whitney U test).B, Treatment with the CB₁ agonist (R)-(+)-WIN 55,212–2, but not its inactive enantiomer (S)-(−)-WIN 55,212–3 (15 mg/kg, i.p.), 1 hr before administration of bicuculline (4 mg/kg) significantly enhanced the severity of seizures in both FAAH (+/+) and (−/−) mice. n = 6–12 mice per group. *p < 0.05 and ***p < 0.001 for WIN 55,212–2-treated versus WIN 55,212–3-treated and vehicle-treated mice of the same genotype, respectively (Mann–WhitneyU test). C, SR141716A augmented kainate-induced seizures in wild-type (WT), FAAH (−/−), and CB₁ (−/−) mice. Subjects from each genotype were treated with either vehicle or SR141716A (1 mg/kg, i.p.) 70 min before kainic acid (15 mg/kg, i.p.). Data are depicted as means ± SEM (n = 8–14 FAAH (−/−) and CB₁ (−/−) mice; 20–22 wild-type mice). ***p < 0.001 for SR141716A-treated versus vehicle-treated animals (ANOVA).

Fig. 3.

The effects of anandamide on hippocampal neurotoxicity associated with bicuculline- and kainate-induced seizures in FAAH (−/−) mice. Brains from FAAH (−/−) mice (n = 5–7 for each treatment group) were removed 3 d after drug treatment, and hematoxylin–eosin-stained slices through the hippocampal region were examined for neuronal cell death. Subthreshold doses of bicuculline (4 mg/kg) and kainate (15 mg/kg) treatment that did not result in clonic–tonic seizures were not found to induce neuronal cell death in the hippocampal region (A, B, left panels). Administration of anandamide (50 mg/kg, i.p.) 60 min before treatment with bicuculline or kainate resulted in severe seizures (Fig. 1) that were accompanied by prominent hippocampal neuronal death. In the bicuculline-treated group, five of the seven surviving animals exhibiting seizure scores of 4–5 displayed pyramidal cell death mainly in the CA1 region of the hippocampus (A, middle panels). Arrows point to examples of injured neurons, which appear as dark, eosinophilic cells with shrunken nuclei. Administration of SR147161A (SR) (3 mg/kg, i.p.) 10 min before treatment with anandamide blocked the neurotoxic (A, right panels) effects of this endocannabinoid. B, In the kainate model, three of the five surviving FAAH (−/−) mice pretreated with anandamide that exhibited seizure scores of 4–5 showed substantial neuronal damage mainly in the CA3 region (B, middle panels). A similar degree of neuronal damage was observed in a separate group of FAAH (−/−) mice that exhibited strong seizure responses (scores of 4–5) to a high dose of kainate (25 mg/kg, i.p.) (B, right panels).

Fig. 4.

Seizure sensitivities of naive FAAH (+/+) and (−/−) mice. In response to high (30 mg/kg, i.p.), but not low (15 and 25 mg/kg), doses of kainate, FAAH (−/−) mice exhibited more intense seizures (A, filled bars) and reduced survival (B, solid line) compared with FAAH (+/+) mice (A, open bars; B, dashed line). No genotype differences were observed in the severity of seizures induced by low (4 mg/kg, i.p.) and high (6 mg/kg, i.p.) doses of bicuculline (C). **p < 0.01 for FAAH (+/+) versus (−/−) mice receiving the same treatment (Mann–Whitney U test). The survival curve (B) showed a significantly higher mortality for FAAH (−/−) mice than for FAAH (+/+) mice (p < 0.01; Fisher's exact test) after treatment with 30 mg/kg kainate. n = 10 mice per group.