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. 2010 Feb;332(2):569-77.
doi: 10.1124/jpet.109.159145. Epub 2009 Nov 11.

Cannabidiol Displays Antiepileptiform and Antiseizure Properties in Vitro and in Vivo

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Free PMC article

Cannabidiol Displays Antiepileptiform and Antiseizure Properties in Vitro and in Vivo

Nicholas A Jones et al. J Pharmacol Exp Ther. .
Free PMC article

Abstract

Plant-derived cannabinoids (phytocannabinoids) are compounds with emerging therapeutic potential. Early studies suggested that cannabidiol (CBD) has anticonvulsant properties in animal models and reduced seizure frequency in limited human trials. Here, we examine the antiepileptiform and antiseizure potential of CBD using in vitro electrophysiology and an in vivo animal seizure model, respectively. CBD (0.01-100 muM) effects were assessed in vitro using the Mg(2+)-free and 4-aminopyridine (4-AP) models of epileptiform activity in hippocampal brain slices via multielectrode array recordings. In the Mg(2+)-free model, CBD decreased epileptiform local field potential (LFP) burst amplitude [in CA1 and dentate gyrus (DG) regions] and burst duration (in all regions) and increased burst frequency (in all regions). In the 4-AP model, CBD decreased LFP burst amplitude (in CA1 only at 100 muM CBD), burst duration (in CA3 and DG), and burst frequency (in all regions). CBD (1, 10, and 100 mg/kg) effects were also examined in vivo using the pentylenetetrazole model of generalized seizures. CBD (100 mg/kg) exerted clear anticonvulsant effects with significant decreases in incidence of severe seizures and mortality compared with vehicle-treated animals. Finally, CBD acted with only low affinity at cannabinoid CB(1) receptors and displayed no agonist activity in [(35)S]guanosine 5'-O-(3-thio)triphosphate assays in cortical membranes. These findings suggest that CBD acts, potentially in a CB(1) receptor-independent manner, to inhibit epileptiform activity in vitro and seizure severity in vivo. Thus, we demonstrate the potential of CBD as a novel antiepileptic drug in the unmet clinical need associated with generalized seizures.

Figures

Fig. 1.
Fig. 1.
Hippocampal slices are amenable to MEA recording. A, schematic representation of hippocampal slice showing the position of CA1, CA3, and DG regions, together with major pathways: Schaffer collateral (SC), mossy fiber (MF), and perforant pathway (PP). B, micrograph showing a hippocampal brain slice (stained with pontamine blue) mounted onto a substrate-integrated MEA (60 electrodes of 30 μm diameter, spaced 200 μm apart in an ∼8 × 8 arrangement). Scale bar, 400 μm. Representative LFP burst activity was recorded at 60 electrodes across a hippocampal slice in (C) Mg2+-free aCSF and (D) 4-AP aCSF. Traces were high pass-filtered in an MC_rack at 2 Hz.
Fig. 2.
Fig. 2.
CBD attenuates epileptiform activity induced by Mg2+-free aCSF. A, representative traces showing the effects of 100 μM CBD on Mg2+-free aCSF-induced LFP bursts in different regions of hippocampal slices. Dotted lines represent an individual LFP (as shown in B). B, effects of 1 and 100 μM CBD on a representative individual Mg2+-free aCSF-induced LFP burst. C, bar graphs showing the effects of acute treatment of increasing CBD concentrations on normalized burst amplitude (Ci), normalized burst duration (Cii), and normalized burst frequency in Mg2+-free aCSF (Ciii). Note that burst amplitudes have been adjusted for run-down and burst frequencies have been adjusted for run-up as described under Materials and Methods. Values are means ± S.E.M. for the last 10 LFP bursts in each condition. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (two-tailed Mann-Whitney U test).
Fig. 3.
Fig. 3.
CBD attenuates epileptiform activity induced by Mg2+-free and 4-AP aCSF. Representative contour plots illustrating CBD effects upon spatiotemporal epileptiform burst features. A, in the continued presence of Mg2+-free aCSF: quiescent period between epileptiform burst events also showing hippocampal slice orientation (i), peak source in the absence of CBD (ii), and peak source in the presence of CBD (100 μM) (iii). B, in the continued presence of 100 μM 4-AP: quiescent period between epileptiform burst events also showing hippocampal slice orientation (i), peak source in the absence of CBD (ii), and peak source in the presence of CBD (100 μM) (iii).
Fig. 4.
Fig. 4.
CBD attenuates epileptiform activity induced by 4-AP aCSF. A, representative traces showing effects of 100 μM CBD on 4-AP aCSF-induced LFP bursts in different regions of a hippocampal slice. Dotted lines represent an individual LFP (as shown in B). B, effects of 1 and 100 μM CBD on a representative individual 4-AP aCSF-induced LFP burst. C, bar graphs showing the effects of acute treatment of increasing CBD concentrations on normalized burst amplitude (Ci), normalized burst duration (Cii), and normalized burst frequency in the 4-AP aCSF (Ciii). Note that burst amplitudes have been adjusted for run-down and burst frequencies have been adjusted for run-up as described under Materials and Methods. Values are means ± S.E.M. for the last 10 LFP bursts in each condition. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (two-tailed Mann-Whitney U test).
Fig. 5.
Fig. 5.
CBD has no clear effects on seizure latency in vivo. Bar graphs showing lack of effects of CBD (1, 10, and 100 mg/kg) on latency to the first sign of a seizure (A), latency to clonic seizures (B), and latency to tonic-clonic seizures (C). Each data set n = 15 animals. Note that CBD (1 mg/kg) reduced latency to tonic-clonic seizures; this proconvulsant action was not observed at higher CBD doses. **, P ≤ 0.01 (one-way analysis of variance) with a post hoc Tukey test).
Fig. 6.
Fig. 6.
CBD reduces seizure severity and mortality in vivo. Bar graphs showing effects of CBD (1, 10, and 100 mg/kg) on median seizure severity (A), percentage of animals reaching tonic-clonic seizures (B), and percent mortality (C). Each data set n = 15 animals. CBD (100 mg/kg) significantly reduced all of these parameters: ***, P ≤ 0.001 (nonparametric binomial test).
Fig. 7.
Fig. 7.
CBD displaces [3H]SR141716A binding with low affinity and lacks agonist effects in [35S]GTPγS binding assays in cortical membranes. A, representative competition curves for the CB receptor agonist WIN55,212-2, the selective CB1 receptor antagonist AM251, and CBD against 1 nM [3H]SR141716A (a selective CB1 receptor antagonist) binding to cortical membranes. Points are means ± S.E.M. of triplicate points. B, agonist-binding curves for the CB receptor agonist WIN55,212-2, the selective CB1 receptor antagonist AM251, and CBD stimulation of [35S]GTPγS binding to cortical membranes. Points are means ± S.E.M. of triplicate points from three separate experiments.

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