An anti‐epileptiform effect of 100 μM CBDV on the amplitude of 4‐AP‐induced epileptiform LFPs was observed in the CA1 region alone (Figure 1 F; P ≤ 0.05), whereas LFP duration was significantly lowered in all hippocampal regions by ≥10 μM CBDV (Figure 1 G) and, by contrast to the Mg 2+ ‐free model, 4‐AP‐induced LFP frequency was significantly decreased by all CBDV concentrations tested (Figure 1 H; P ≤ 0.05). Thus, CBDV attenuated the duration of amplitude of LFPs in both models, and had differential effects on frequency.

The effects of CBDV (1–100 μM) on epileptiform activity, induced by Mg 2+ ‐free aCSF ( Figure 1 A) or 100 μM 4‐AP (Figure 1 B), in rat acute hippocampal slices were examined. CBDV significantly decreased the amplitude and duration of epileptiform LFPs induced by Mg 2+ ‐free aCSF (Figure 1 C and D; P ≤ 0.05); significant effects were seen at ≥10 μM, and the CA3 region was more resistant to the effects of CBDV than the dentate gyrus (DG) or CA1 (Figure 1 C and D). Conversely, CBDV significantly increased Mg 2+ ‐free‐induced LFP frequency (≥10 μM; Figure 1 E; P ≤ 0.05).

Effects of CBDV on mES and audiogenic seizures in mice. (A) The effect of CBDV on the percentage of animals that exhibited tonic hindlimb extension in response to mES. (B–D) The effect of CBDV (50–200 mg·kg −1 ) on the percentage of animals that displayed tonic convulsions (B), remained seizure‐free (C) or suffered mortality (D) as a result of audiogenic seizure induction. n = 10 in all cases, *** P ≤ 0.001.

The effects of CBDV (50–200 mg·kg −1 ) on mES convulsions and audiogenic seizures in mice were investigated. CBDV had a significant anticonvulsant effect on animals displaying tonic hindlimb extension after mES [χ 2 (3) = 15.000; P ≤ 0.001; Figure 2 A]; significantly fewer animals that received 100 or 200 mg·kg −1 CBDV exhibited hindlimb extension (both groups 30%) than those that received vehicle (90%, Figure 2 A; P ≤ 0.001 vs. vehicle‐treated group). Audiogenic seizures were also significantly attenuated by CBDV (Figure 2 B–D). The incidence of tonic convulsions was significantly lower after CBDV administration [χ 2 (3) = 19.436, P ≤ 0.001; Figure 2 B]; 80% of vehicle‐treated animals developed tonic convulsions compared with only 20% (50 mg·kg −1 CBDV), 10% (100 mg·kg −1 CBDV) and 0% (200 mg·kg −1 CBDV) after drug treatment (each P ≤ 0.001 vs vehicle). The percentage of animals that remained seizure‐free was significantly higher after administration of 200 mg·kg −1 CBDV (90%) than vehicle [0%; χ 2 (3) = 27.461, P ≤ 0.001; Figure 2 C]. Finally, a statistical trend was observed for the mortality rate [χ 2 (3) = 6.667, P ≤ 0.1], with lower mortality after 100 and 200 mg·kg −1 CBDV treatment than vehicle (0% vs 30%, respectively; Figure 2 D). Thus, CBDV exhibits strong and significant anticonvulsant effects in two broad‐screen mouse seizure models. Next, we investigated the anticonvulsant potential of CBDV in two further models of seizure in rat that emulate more specific seizure types.

We extended our studies to investigate the effects of CBDV (50–200 mg·kg −1 ) on the convulsions associated with pilocarpine‐induced status epilepticus (380 mg·kg −1 ). CBDV (50–200 mg·kg −1 ) had no significant effect on the severity ( F 3,59 = 0.049, P > 0.1; Figure 3 E) or resultant mortality of pilocarpine convulsions [χ 2 (3) = 1.779, P > 0.1; Figure 3 F]. Similarly, CBDV did not significantly affect the percentage of animals that remained seizure‐free [χ 2 (3) = 0.110, P > 0.1; Figure 3 G] or the latency to the onset of convulsions ( F 3,53 = 0.404, P > 0.1; Figure 3 H).

Effects of CBDV on PTZ‐ and pilocarpine‐induced seizures in rats. (A–D) The effect of CBDV on PTZ‐induced seizures: seizure severity (A), mortality (B), the proportion of animals remaining seizure‐free (C) and the onset latency (D). (E–H) The effect of CBDV on pilocarpine‐induced convulsions: severity (E), mortality (F), the percentage of animals remaining seizure‐free (G) and the onset latency (H). In (D and H), onset latency is presented as mean ± SEM In (A and E), median severity is represented by a thick horizontal line, the 25th and the 75th percentiles by the box and maxima and minima are represented by ‘whiskers’. n = 15 in all cases. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001.

CBDV significantly decreased PTZ seizure severity ( F 3,58 = 4.423, P ≤ 0.05; Figure 3 A); the median seizure severity after vehicle administration was tonic–clonic convulsion score 5, but after 200 mg·kg −1 CBDV administration seizure severity was significantly lowered to a median severity of bilateral clonic convulsion score 3 ( P ≤ 0.05). CBDV also significantly reduced mortality (χ 2 (3) = 10.356, P ≤ 0.05; Figure 3 B) at 100 and 200 mg·kg −1 CBDV ( P ≤ 0.01). The percentage of animals that remained seizure‐free was significantly increased by CBDV administration [χ 2 (3) = 7.809, P ≤ 0.05; Figure 3 C]; 33.3% of animals that received 200 mg·kg −1 CBDV exhibited no signs of seizure compared with only 6.7% of animals that received vehicle ( P ≤ 0.01). Furthermore, seizure onset was significantly delayed by CBDV treatment ( F 3,50 = 2.971, P ≤ 0.05; Figure 3 D); mean onset latency was significantly longer after administration of 200 mg·kg −1 CBDV than vehicle (65 ± 11 s and 40 ± 4 s, respectively; P ≤ 0.05). Thus, CBDV, administered alone, exhibited strong and significant anticonvulsant effects on PTZ seizures at 200 mg·kg −1 (Figure 3 A–D) with more limited, but significant, effects at 100 mg·kg −1 (Figure 3 B).

Effect of co‐administration of CBDV and AEDs on PTZ‐ and pilocarpine‐induced seizures in rats

We investigated the effects of CBDV when co‐administered with AEDs before PTZ or pilocarpine treatment. The effects of combined drug treatment (CBDV + AED) on seizure parameters are illustrated in Figures 4 and 5, as is the contribution of CBDV to these effects. The contribution of AEDs is illustrated in Figures 4 and 5 while statistical analyses of AED effects and any interaction between CBDV and AEDs are shown in Supporting Information Tables S1 and S2.

Figure 4 Open in figure viewer PowerPoint Effects of co‐administration of CBDV and AEDs on PTZ‐induced seizures in rats. The effects of CBDV co‐administration with VPA (A–D) or ESM (E–I) on PTZ‐induced seizures: severity (A and E), mortality (B and F), the incidence of tonic–clonic seizures (C and G), onset latency (D and H) and (for CBDV + ESM only) the percentage of animals that remained seizure‐free. In (D and H), onset latency is presented as mean ± SEM. In (A and E), median severity is represented by a thick horizontal line, the 25th and 75th percentiles by the box and maxima and minima are represented by ‘whiskers’. Significance of CBDV treatment is given in text. n = 15 in all cases. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 for AED effects.

Figure 5 Open in figure viewer PowerPoint Effects of co‐administration of CBDV and AEDs on pilocarpine‐induced convulsions in rats. The effects of CBDV co‐administration with VPA (A–D) or phenobarbital (E–I) on pilocarpine‐induced convulsions: severity (A and E), mortality (B and F), the incidence of tonic–clonic convulsions (C and G), onset latency (D and H) and (for CBDV + phenobarbital only) the percentage of animals that remained seizure‐free. In (D and H), onset latency is presented as mean ± SEM In (A and E), median severity is represented by a thick horizontal line, the 25th and 75th percentiles by the box and maxima and minima are represented by ‘whiskers’. Significance of CBDV treatment is given in text. n = 15 in all cases. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 for AED effects.

CBDV 200 mg·kg−1 was co‐administered with VPA (50–250 mg·kg−1) or ESM (60–175 mg·kg−1). In the CBDV + VPA experiments, drug co‐administration had significant anticonvulsant effects on all seizure parameters except the percentage of animals remaining seizure‐free. CBDV and VPA co‐administration significantly decreased seizure severity (F 7,112 = 10.449, P ≤ 0.001; Figure 4A). When modelled by log‐linear analyses, our data indicated that drug co‐administration decreased mortality (Figure 4B) and the incidence of the most severe (tonic–clonic) seizures (Figure 4C). Seizure onset was significantly delayed by drug co‐administration (F 7,109 = 13.285, P ≤ 0.001; Figure 4D) and the mean duration of seizures was increased (F 7,103 = 5.250, P ≤ 0.001). VPA contributed significantly to all these effects (Figure 4A–D, Supporting Information Table S1). CBDV significantly contributed to the overall decrease in severity (F 1,112 = 5.748, P ≤ 0.05; Figure 4A) and mortality [χ2(1) = 6.639, P ≤ 0.01; Figure 4B] and the increase in onset latency (F 1,109 = 7.393, P ≤ 0.01; Figure 4C). CBDV did not significantly affect tonic–clonic seizure incidence (Figure 4D) or seizure duration (P > 0.1). No effect of drug treatment on the number of seizure‐free animals was observed [X2(14) = 8.930, P > 0.1] and no significant positive or negative interactions between the effects of 200 mg·kg−1 CBDV and VPA were observed (Supporting Information Tables S1, P > 0.1).

Co‐administration of 200 mg·kg−1 CBDV and ESM (60–175 mg·kg−1) had significant anticonvulsant effects on all parameters of PTZ‐induced seizures: CBDV and ESM co‐administration significantly decreased seizure severity (F 7,110 = 12.556, P ≤ 0.001; Figure 4E), when modelled with log‐linear analysis, our data indicated that co‐administration also decreased mortality (Figure 4F) and the incidence of the most severe seizures (Figure 4G). Seizure onset latency was significantly increased (F 7,76 = 7.885, P ≤ 0.001; Figure 4H), as was the percentage of animals that remained seizure‐free (log‐linear model; Figure 4I); seizure duration was also significantly decreased (F 7,102 = 6.934, P ≤ 0.001). ESM significantly contributed to all anticonvulsant effects (Figure 4E‐I; Supporting Information Table S1). CBDV contributed significantly to the overall decreases in seizure severity (F 1,112 = 7.474, P ≤ 0.01; Figure 4E) and mortality [χ2(1) = 5.174, P ≤ 0.05; Figure 4F]; the contribution of CBDV to the increase in onset latency showed a statistical trend (F 1,76 = 2.791, P ≤ 0.1; Figure 4H). CBDV did not significantly contribute to the effects on seizure duration, the proportion of animals that remained seizure‐free (both P > 0.1) or the incidence of the most severe seizures (P > 0.1; Figure 4G). No significant positive or negative interactions between the effects of 200 mg·kg−1 CBDV and ESM were observed (Supporting Information Tables S1, P > 0.1).

We next investigated whether 200 mg·kg−1 CBDV affected the anticonvulsant actions of VPA or phenobarbital on pilocarpine‐induced convulsions. Interestingly, these co‐administration experiments highlighted significant anticonvulsant effects of 200 mg·kg−1 CBDV not previously observed when CBDV was administered alone. Co‐administration of VPA (50–250 mg·kg−1) with 200 mg·kg−1 CBDV had significant anticonvulsant effects on all the parameters except the percentage of animals that remained convulsion‐free: CBDV and VPA co‐administration significantly decreased severity (F 7,100 = 16.477, P ≤ 0.001; Figure 5A); when modelled by log‐linear analysis, our data indicated that mortality (Figure 5B) and the incidence of the most severe (tonic–clonic) convulsions (Figure 5C) were also decreased by drug co‐administration; onset latency was significantly increased (F 7,105 = 8.649, P ≤ 0.001; Figure 5D). VPA contributed significantly to all anticonvulsant effects (Figure 5A‐D, Supporting Information Table S2) with the interesting exception of mortality. Mortality was higher (but not significantly so) when 62.5 and 125 mg·kg−1 VPA were co‐administered with vehicle (Figure 5B); however, CBDV had an anticonvulsant effect, significantly decreasing mortality compared with administration of its vehicle [χ2(1) = 4.010, P ≤ 0.05; Figure 5D]. CBDV also significantly contributed to the overall anticonvulsant effects of treatment on severity (F 1,110 = 22.711, P ≤ 0.001; Figure 5A) and the incidence of tonic–clonic convulsions [χ2(1) = 4.010, P ≤ 0.01; Figure 5C], although it had no significant effect on onset latency (P > 0.1; Figure 5D). The percentage of animals that remained convulsion‐free [χ2(6) = 1.564, P > 0.1] was unaffected by treatment. No significant interactions between CBDV and VPA effects were observed (Supporting Information Tables S2, P > 0.1).

Co‐administration of 200 mg·kg−1CBDV and phenobarbital (10–40 mg·kg−1) had significant anticonvulsant effects on the severity of pilocarpine‐induced convulsions (F 7,108 = 19.352, P ≤ 0.001; Figure 5E). When modelled with log‐linear analysis, our data indicated that there was no effect of treatment on mortality (Figure 5F), whereas the percentage of animals that developed tonic–clonic convulsions was significantly decreased (Figure 5G). No effect of drug treatment was observed on seizure onset latency (P > 0.1; Figure 5H); however, when modelled with log‐linear analysis, our data indicated that the percentage of animals that remained convulsion‐free was significantly increased (Figure 5I). Phenobarbital significantly contributed to all anticonvulsant effects (Figure 5E–I; Supporting Information Table S2). CBDV significantly contributed to the overall decrease seen in severity (F 1,108 = 4.480, P ≤ 0.05), and the effects of CBDV and phenobarbital interacted significantly due to a convergence of the severity observed in the absence and presence of CBDV (Figure 5F, Supporting Information Table S2; F 3,108 = 3.105, P ≤ 0.05), no further significant interactions between the effects of CBDV and phenobarbital were observed (P > 0.1; Supporting Information Table S2).

Data from the co‐administration experiments demonstrate that the AEDs strongly suppress PTZ‐induced seizures and pilocarpine‐induced convulsions in a dose‐dependent manner (Figures 4 and 5). From several, but not all, of the parameters examined, 200 mg·kg−1 CBDV significantly contributed to the anticonvulsant effects observed in these experiments. To more precisely assess the effect of CBDV on AED actions in these studies, we performed pairwise comparisons at each dose of AED between groups that received CBDV vehicle and groups that received 200 mg·kg−1 CBDV; these analyses were only performed if two‐way ANOVA or log‐linear analysis results indicated an overall effect of CBDV upon a given parameter. Based on these analyses and Figure 5F–I, the effect of CBDV on the actions of phenobarbital in the pilocarpine model appears limited and is not significant. Similarly, the effect of CBDV on the actions of VPA in the PTZ model was limited (Figure 4A–D); the primary effect of CBDV is on delaying seizure onset, as 200 mg·kg−1 CBDV significantly improved the effect of 50 mg·kg−1 VPA (P ≤ 0.05; Figure 4D) and showed a statistical trend towards the same effect with 100 mg·kg−1 VPA (P < 0.1). More notably, CBDV significantly improved the effect of 60 mg·kg−1 ESM on PTZ‐induced seizure severity and onset latency (P ≤ 0.05; Figure 4E and H) and also showed a statistical trend to improvement of the 120 mg·kg−1 ESM effect for both these measures (P < 0.1). Furthermore, when 200 mg·kg−1 CBDV was administered together with VPA before pilocarpine administration, it significantly improved the effects of VPA on severity (62.5 and 250 mg·kg−1; P ≤ 0.05), mortality (62.5 and 125 mg·kg−1; P ≤ 0.05) and the percentage of animals that experienced the most severe seizures (all doses, P ≤ 0.01; Figure 5A–C).

Thus, CBDV is well‐tolerated when co‐administered with AEDs and does not interact antagonistically with any of the AEDs studied in either seizure model. Furthermore, CBDV has significant anticonvulsant effects when co‐administered with ESM in the PTZ model and even greater effects when co‐administered with VPA in the pilocarpine model, where beneficial effects were generally observed at low and medium AED doses. CBDV did not affect the effects of phenobarbital in the pilocarpine model and had only very limited effects on the onset of seizures when co‐administered with VPA before PTZ treatment.