Experiments were conducted on juvenile C57BL/6 mice (Charles River Laboratories, Wilmington, MA, USA) of either sex, post-natal day 14–21. All animal experiments and procedures were approved by the University Health Network Animal Care Committee (protocol AUP 750.50) and carried out in accordance with guidelines outlined by the Canadian Council of Animal Care (CCAC). A total of 63 mice were used for all experimentation and an average of 2 cortical slices were used from each animal. Mice were chosen as their brains are small in size and thus, it is more likely that connectivity between neurons remain intact during slice preparation [50]. Additionally, juvenile mice were chosen as they have shown to be more susceptible to developing seizures in comparison to adults [51,52], and this age correlates with an age range in children wherein cannabinoids have been most studied to date. It has been shown that CBD has beneficial effects in pediatric epilepsies and focal cortical dysplasia is the most common cause of drug-resistant pediatric epilepsy [14,53,54]. Thus, cortical slices from juvenile mice were used to best model pediatric epilepsy.

Mice were first anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital. The pedal reflex was used to ensure that mice were deeply anaesthetized before quickly decapitating the mice. The whole brain was rapidly removed and transferred to a sucrose dissection solution (in mM: 248 sucrose, 26 NaHCO₂, 10 D-glucose, 2 KCl, 3 MgSO₄, 1.25 H₂NaPO₄, 1 CaCl₂) at 4 °C. The cerebellum and forebrain were removed, and a cyanoacrylate adhesive gel was used to fix the brain to a block. Using a Leica 1200 V vibratome, 500 μm thick cortical slices were prepared and hemisected. Slices were incubated in artificial cerebrospinal fluid (ACSF, in mM: 123 NaCl, 25 NaHCO₂, 10 D-glucose, 3.5 KCl, 1.3 MgSO₄, 1.2 HNaPO₄, 1.5 CaCl₂; pH adjusted to 7.4 with 95% O₂, 5% CO₂) for 30 min at 37 °C, followed by 1 h at room temperature in the perfusion chamber prior to recordings.

During recordings, each slice was placed in a submerged recording chamber and was perfused at 10 mL/min with ACSF at 35 °C and aerated with 95% O₂, 5% CO_2. A borosilicate capillary glass electrode (1.5 mm, World Precision Instruments, Sarasota, FL, USA) was filled with ACSF and used to record local field potentials (LFP). This LFP electrode (~2 MΩ resistance) was positioned in cortical layers 2/3. Using a Multiclamp 700B amplifier (Molecular devices), a Digidata 1322A digitizer (Axon Instruments, Burlingame, CA, USA), and the PClamp software (version 10.2) (Axon Instruments/Molecular Devices Corporation, San Jose, CA, USA), signal acquisition was conducted.

Seizures were induced using 4-aminopyridine (4-AP), a blocker of K⁺ channels, which causes prolonged glutamate release with excitation and consequent epileptiform activity. 4-AP is a well-founded model of epilepsy in mice, as is demonstrated by the induction of prolonged recurrent seizures without ‘exhaustion’ of seizure activity overtime in recordings. Thus, the ability of CBD to suppress 4-AP-induced seizures is useful for examining CBD’s efficacy as an anticonvulsant. The 4-AP was dissolved in double-distilled water to create a 100 mM stock solution, was aliquoted in 1 mL tubes and stored at −20 °C. On experimental days, the 4-AP was thawed and diluted in ACSF to a final concentration of 100 µM.

For aim 1, CBD was provided by Avicanna in a powdered form. Working solutions of CBD were freshly prepared daily prior to experiments. CBD was dissolved in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) to create the stock solution before being diluted in ACSF to create the final concentrations of 10, 30, or 100 µM. The concentrations that were seen to be effective were higher than therapeutic concentrations previously shown in the literature [5,55]; however, 4-AP is a very robust model of status epilepticus [56,57] and is more difficult to treat, justifying the anti-seizure benefits potentially observed at higher concentrations.

For aim 2, a CBD isolate and delta-9-tetrahydrocannabidiol (Δ⁹-THC) distillate were donated from Avicanna (Toronto, Canada) in a self-emulsifying drug delivery system comprised of TWEEN80. To prepare the stock solutions, the CBD and Δ⁹-THC solutions were sonicated and final concentrations of 10 mM CBD and 0.5, 1.5, or 2.5 mM Δ⁹-THC were made, aliquoted into 1 mL amber tubes, and stored at −4 °C. On experimental days, the stock solutions were diluted in ACSF to create a final solution of 10 µM CBD:0.5 µM Δ⁹-THC (20:1 ratio), 10 µM CBD:1.5 µM Δ⁹-THC (20:3 ratio), or 10 µM CBD:2.5 µM Δ⁹-THC (20:5 ratio).

For aim 3, The 5-HT1A receptor antagonist and agonist used were N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY100635) and (±)-8-Hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT), respectively (Tocris Bioscience, Ellisville, MO, USA). Stock solutions of 10 µM WAY100635 and 10 mM 8-OH-DPAT were created by dissolving the compounds in sterile water before storing at −20 °C. On experimental days, these solutions were thawed and diluted in ACSF to a final concentration of 10 nM WAY100635 and 10 µM 8-OH-DPAT.

Figure 1 illustrates the protocol of the electrophysiological experimentation. For aims 1 and 2, we assessed the anticonvulsant effects of CBD alone and in combination with low dose Δ⁹-THC, after seizure induction with 4-AP, respectively. An initial 10-min baseline recording was taken. Afterwards, 100 μM 4-AP dissolved in ACSF was added for ~45 min to reliably induce seizure activity, before a 15-min recording was taken. Then, depending on the treatment condition, various concentrations of CBD, Δ⁹-THC, or combinations of CBD and Δ⁹-THC dissolved in ACSF with 4-AP were added for ~45 min, after which another 15-min recording was taken. For aim 1, the drug in the treatment conditions was either 10 (n = 7), 30 (n = 10), or 100 (n = 11) µM CBD (Figure 1A). For aim 2, the drug(s) in the treatment condition was either a combination of 20:1 (n = 5), 20:3 (n = 10), or 20:5 (n = 9) CBD:Δ⁹-THC, or 0.5 (n = 5), 1.5 (n = 7), or 2.5 (n = 10) µM Δ⁹-THC alone (Figure 1B).

In aim 3, we assessed the role that the 5-HT1A receptor plays in CBD’s mechanism of action (Figure 1C). A positive control experiment was first conducted in which an initial 10-min baseline recording was taken. An amount of 100 μM 4-AP dissolved in ACSF was added for ~45 min to reliably induce seizure activity, before a 15-min recording was taken. Then, 10 µM 8-OH-DPAT, a 5-HT1A receptor agonist, dissolved in ACSF with 4-AP was added for ~45 min, after which another 15-min recording was taken (n = 8). In another condition, to confirm that the anticonvulsant effects of 8-OH-DPAT were exerted through the 5-HT1A receptor, slices were pre-treated with 10 nM WAY100635, a 5-HT1A receptor antagonist, for 20 min before adding 100 μM 4-AP dissolved in ACSF with WAY100635 for ~45 min and taking a 15-min recording. Then, 10 µM 8-OH-DPAT dissolved in ACSF with WAY100635 and 4-AP was added for ~45 min, after which another 15-min recording was taken (n = 8) to see if pre-treatment with the 5-HT1A receptor antagonist would abolish the effects of the 5-HT1A receptor agonist. For the treatment condition, an initial 10-min baseline recording was taken before pre-treating the cells with 10 nM WAY100635. Then, 100 μM 4-AP dissolved in ACSF with the WAY100635 was added for ~45 min to reliably induce seizure activity, before a 15-min recording was taken. Afterwards, 30 µM CBD dissolved in ACSF with WAY100635 and 4-AP was added for ~45 min, and a 15-min recording was taken (n = 6).

For all studies, a vehicle control condition was conducted to ensure that DMSO or TWEEN80 as the vehicle did not impact the results of this study. Analysis of acute experiments with these vehicles showed that the addition of DMSO or TWEEN80 after seizure induction did not impact any of the features of interest; thus, neither vehicle impacted the results observed in this study.

Data analyses were initially performed using pClamp 10.2 and MATLAB software, version R2020b. LFP recordings were filtered using a low-pass filter at 1250 Hz and reduced by a factor of 10. A 60 Hz notch filter with three harmonics was applied to eliminate noise before applying a high-pass filter at 0.25 Hz. The features of interest measured included duration (in seconds), amplitude (in mV), coastline length/second, and frequency of seizure-like events as well as duration (in seconds) and frequency of inter-ictal bursting events. Seizure-like events were defined as excitable activity lasting longer than five seconds, typical for 4-AP-induced seizures. Amplitude was defined as the difference between the highest and lowest points within the seizure-like event. Coastline length is the sum of the distance (absolute change in voltage of the signal) for a seizure-like event. Coastline length was divided by duration of that seizure-like event to provide a measure of burst intensity/second. Coastline length/second is indicative of seizure intensity with higher values representing more intense seizures. Frequency was defined as the average number of events in the last 10 min of each recording. Inter-ictal bursting events were defined as any excitable activity that was less than five seconds in duration and occurred after the end of one seizure-like event but before the start of another. Features of interest were measured for the last 10 min of each recording and averaged. Statistical analysis was performed using Graphpad (Version 9). Each sample size (n) equated to a single brain slice and sample sizes subjected to statistical analysis had at least five samples per group. For each experiment, a Wilcoxon’s matched paired two-tailed t-test was used to compare the mean features of interest of the drug condition in each slice to the pre-drug control condition in the same slice. For aim 3, a Mann–Whitney unpaired two-tailed t-test was also used for analyses across conditions to compare pre-treated with antagonist conditions to not pre-treated conditions. Results were considered significant when p < 0.05. For graphing purposes, percent change from pre-drug conditions were calculated using this formula:

The results of this study showed that CBD alone did display anticonvulsant effects, especially at concentrations greater than 30 µM. Interestingly, although the reductions in SLE duration after 30 µM and 100 µM CBD application were both significant, the results displayed a larger reduction in SLE duration after 30 µM CBD (39.7% reduction) compared to 100 µM CBD (31.6% reduction) addition. However, only 100 µM CBD significantly reduced burst amplitude. These findings are in line with the previous literature stating CBD’s potential biphasic effect depending on the feature of interest [7,59].

Overall, after the addition of CBD, we observed more frequent seizures of less intensity. Patients with epilepsy are often prescribed a combination of medications; thus, to reduce adverse side effects, lower doses are preferred. Although 10 µM CBD did not display any significant effects on SLEs, a general anticonvulsant trend was observed through decreases in duration and amplitude of SLEs. It was particularly interesting that 10 µM CBD only caused significant reductions in the duration of inter-ictal bursting events. A dose of 10 µM CBD could exert seemingly nonsignificant anticonvulsant effects through both ictal and inter-ictal characteristics. This compares to the literature showing that certain compounds are more largely involved in the generation of inter-ictal events than ictal events [60]. Further studies are required to fully elucidate the potential anticonvulsant effect of CBD.

4-AP is a well-founded model of epilepsy in mice, as is demonstrated by the induction of prolonged recurrent seizures without ‘exhaustion’ of seizure activity over time in recordings. Thus, the ability of CBD to suppress 4-AP-induced seizures, after the fact, is useful for examining CBD’s efficacy as an anticonvulsant. However, patients more often use medications to prevent, rather than treat, seizures. It is important to note this limitation of our study. Although we investigated the potential of CBD as a treatment of SLEs, our study did not investigate the ability of CBD to prevent SLEs induced by 4-AP. The efficacy of CBD pre-treatment as an anticonvulsant would strengthen the argument for translation into clinical settings, where CBD could potentially prevent prolonged seizures. Further experimentation on the anticonvulsant effect of CBD pre-treatment is needed to elucidate this neuroprotective capacity.

Currently, Epidiolex is a medication approved by the United States Food and Drug Administration (FDA) for patients with Dravet syndrome and Lennox–Gastaut syndrome. Epidiolex is a pure CBD product and although it has been shown to be effective, anecdotal reports have shown that adding other phytocannabinoids is more efficacious in seizure control than CBD alone [24,27]. Considering that the first experiments revealed the anticonvulsant potential of 10 µM CBD through nonsignificant reductions SLEs and inter-ictal bursts, we then examined the potential anticonvulsant interactions between CBD and Δ⁹-THC after seizure induction with 4-AP.

Two important conclusions were drawn from these results. Firstly, the addition of low dose Δ⁹-THC to a low concentration of CBD potentiates the effect of CBD to a therapeutic level. Secondly, when comparing the various ratios tested, only a 20:3 ratio of CBD:Δ⁹-THC demonstrated significant anticonvulsant effect on SLEs. These results suggest that in all features of interest, 10 µM CBD and 1.5 µM Δ⁹-THC display greater anticonvulsant effects in combination than either compound does alone.

To our knowledge, there are a limited number of studies that have looked at the interactions of CBD and Δ⁹-THC in in vitro brain slices made epileptic. One in vitro study using a muscarinic agonist-induced epilepsy model within the piriform cortex of rats showed that CBD and Δ⁹-THC combined did not have greater anticonvulsant effects than Δ⁹-THC alone [61]. These disparities could be due to differences in brain area, the model of epilepsy, or cannabinoid concentrations. Our work parallels in vivo studies which found that the effects of low-dose Δ⁹-THC are enhanced when combined with a subtherapeutic CBD dose [28,29,58]. However, it was also found that chronic use of this combination had proconvulsant effects and increased premature mortality rates [58]. Our study used acute slices and thus, we were unable to examine the long-term effects of a 20:3 CBD: Δ⁹-THC combination. Another study showed a synergistic effect between CBD and Δ⁹-THC only in a 1:1 ratio while the 5:1 ratio displayed a partial non-synergistic effect [62]. Thus, lower doses of Δ⁹-THC relative to CBD did not produce the same potentiating effects that our results displayed. This could be due to differences in species, possibly indicating a species-specific effect when combining CBD and Δ⁹-THC.

Although the literature shows no consensus regarding the therapeutic use of CBD and Δ⁹-THC combined, many drug-resistant patients often turn to CBD-enriched cannabis products. Surveys and studies on these patients show a great reduction in seizure frequency and severity [25,26,63]. However, due to Δ⁹-THC’s psychoactive effects, the compound is clinically unattractive as an independent therapeutic. The safety profile is an important consideration when investigating novel drugs. Unlike Δ9-THC, which has been associated with states of psychosis [64,65], many studies have shown that CBD is well tolerated across many doses. In both short- and long-term administration, no concerning adverse effects were seen on the central nervous system or vital signs when using CBD, including high doses [18]. A study carried out on patients with drug-resistant epilepsy displayed CBD’s long-term safety and tolerability [66]. Thus, CBD also displays high tolerability and low toxicity through its independent use. We showed that lower doses of Δ⁹-THC can potentiate low CBD doses in vitro. Not only are lower doses associated with less adverse side effects, but CBD has also been shown to ameliorate the psychoactive effects of Δ⁹-THC through various mechanisms [67,68,69,70,71,72]. Through these mechanisms, CBD and Δ⁹-THC combined at low doses have great therapeutic potential as we can harness their individual anticonvulsant effects without the potential adverse effects, providing a solution for the reluctance to use Δ⁹-THC in the clinical setting. Our results are important as they parallel what is observed clinically, further justifying more research into the interactions of CBD and Δ⁹-THC.

There has been much research shedding light on the role that the serotonergic system plays in seizures [73,74]. Fenfluramine is a recently approved drug that has high efficacy for the treatment of seizures in Dravet syndrome and Lennox–Gastaut syndrome [75]. Studies have shown that fenfluramine exerts its anticonvulsant effects by enhancing serotonergic transmission through various receptors, including the 5-HT1A receptor [44,76]. Here, we investigated whether CBD exerts its anticonvulsant effects through the 5-HT1A receptor.

After the addition of 8-OH-DPAT, a selective 5-HT1A receptor agonist, we observed significant reductions in the features of interest, leading to the conclusion that 5-HT1A receptor stimulation can exert anticonvulsant effects. The literature supports our results as many studies have observed similar anticonvulsant effects after 5-HT1A receptor stimulation [41,42,43,77]. The anticonvulsant effect of 8-OH-DPAT was very similar to the effect observed after 30 µM CBD. Binding analyses experiments have shown that CBD is a known 5-HT1A receptor agonist, and a multitude of evidence has shown that the 5-HT1A receptor is a pharmacological target for CBD’s other therapeutic effects [45,78,79,80]. Thus, it is imaginable that the 5-HT1A receptor could be a target for CBD’s anticonvulsant effects. Our results provide evidence for this as pre-treatment with a 5-HT1A receptor antagonist abolished the anticonvulsant effects of CBD. In vivo studies have similarly shown that CBD’s anticonvulsant effects are abolished by a 5-HT1A receptor antagonist [46,47]. Conversely, a pentylenetetrazol seizure rat model displayed that CBD’s anticonvulsant effects are independent of the 5-HT1A receptor, but these differences could be due to the dosing and animal model that was used [49]. Knowledge on CBD’s mechanism of action is important to determine the patient population in which this new potential therapeutic is most effective as well as optimal dosing by monitoring the effect on the target pathway in the patient.

Additionally, 5-HT1A receptor stimulation as a potential mechanism by which CBD exerts its anticonvulsant effects is a very promising result, especially for individuals with drug-resistant epilepsy, as these patients are at a higher risk for sudden unexpected death in epilepsy (SUDEP) [2]. Studies using SUDEP animal models have shown decreased firing from serotonergic neurons during and after seizures, implicating the serotonergic system in SUDEP [81,82]. Other studies using a SUDEP animal model showed that stimulation of the serotonergic system inhibited seizure-induced respiratory arrest [83]. Thus, not only does CBD have potential to be a novel anticonvulsant drug that improves seizure control but could also reduce the risk of SUDEP. However, many more studies are required to fully comprehend the role that the serotonergic system plays in SUDEP and how to harness its therapeutic potential.

The data that support the findings of this study are available from the corresponding author upon reasonable request.