The endocannabinoid system (ECS) is involved in regulation of excitatory and inhibitory synaptic transmission in the brain [65,66], and consists of two G protein-coupled receptors, CB₁R and CB₂R, with two known endogenous cannabinoid ligands, namely 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamide (NAN), respectively [10,11]. In the past few years, scientists have drawn attention to using a treatment focused on ECS [67,68]. There is already a comprehensive review indicating the roles of ECS dysfunction-induced neuroinflammation in epilepsy [69]. Anticonvulsant-like effects of cannabinoid receptor agonists are dependent on CB₁R. CB₁ agonists could increase ATP-sensitive K⁺ channel (K_ATP) activation by decreasing mitochondrial ATP levels. CB₁R-mediated regulation in neuronal excitability can exert antiseizure effects [70]. Microglia are evoked by pathogens, products of damaged/inflammatory neurons, and destruction of the blood–brain barrier, as well as diverse chemical menace signals. However, there is also a contradiction about suppressing epilepsy by adjusting the activity of CB₁Rs to affect excitability. Owing to the extensive distribution and high level of CB₁ in the CNS under physiological conditions, a risk of side-effects develops when CB₁Rs are activated and targeted.

Tetrahydrocannabinol (THC) and cannabidiol (CBD) have been the most researched at present, especially in psychoactive pharmacology. THC and CBD have many similarities in structure, and their structure–activity relationship has an impact on mental activity. It has been reported that both have certain anticonvulsant effects. THC mainly activates the GPR55 receptor, and partially activates the CB₁Rs and CB₂Rs. However, THC can cause a series of psychological effects, such as coordination problems, slower reaction times, memory loss, anxiety, and addiction. CBD can reduce these side-effects caused by THC. Studies have reported that CBD treats the frequency of spontaneous seizures in DS mice mainly by improving the excitability of hippocampal interneurons, and the excitability of vertebral neurons in the dentate gyrus to strong depolarization stimulation is also reduced [67]. The pharmacological activity of CBD is mainly focused on blocking the effect of GPR55 receptors to inhibit the effect of neurotransmission and significantly reduce the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR)-mediated amplitude and frequency of induced excitatory postsynaptic currents (EPSCs) and miniature EPSCs (mEPSCs) [71]. CBD is also used clinically to treat epileptic seizures caused by Lennox-Gastaut syndrome [19]. A proportion of patients allocated to CBD treatment in these trials were receiving clobazam (CLB). Bialer and Perucca showed an antiseizure effect of CBD independent of an interaction with CLB. A greater antiseizure effect and greater adverse effects were observed when CBD was combined with CLB [72]. However, the therapeutic effect of CBD on other types of epilepsy and the complete mechanism of action remains unclear, but it may involve a non-CLB mechanism. The peripheral side-effect of CBD is mainly diarrhea. There is only indirect proof implying that CBD could modulate endocannabinoid signaling, but no promising data indicating a direct binding or interaction between CBD and CB receptors. CBD can also partially activate CB₂Rs [73]. Negative allosteric modulation of CB₂R activity by CBD might interpret its action for antiseizure and other neural disorders, which provides us with novel insights to develop its medical application [74]. Regarding CB₂R, CBD-DMH, a modification on different pharmacophoric sties, was considered to promote a conformational change in CB₂R, which favors G protein-dependent signaling rather than β-arrestin-dependent signaling [75]. However, there are very few modulators of CB₁R and CB₂R reported in the literature.

Since CB₂Rs exhibit low expression levels in the brain under normal conditions, but are highly inducible during various disease states (including epilepsy), they appear to be an important substrate for neuroprotection [76]. Targeting CB₂Rs will likely offer a novel therapeutic strategy for treating epileptic seizures without the typical CB₁R-mediated side-effects [77,78]. Emerging evidence has indicated that CB₂Rs are involved in epileptic activity in animal models. In an acute pentylenetetrazol (PTZ) rat seizure model, pretreatment with palmitoyl ethanolamide (PEA) increased the latency of seizure initiation and reduced the duration of seizures, and this antiseizure effect was attenuated by the CB₂R antagonist (AM630), suggesting that CB₂Rs mediate PEA’s effect [79]. In developing rats, Huizenga et al. examined the antiseizure effects of a variety of cannabinoid ligands, and found that either combined CB₁R/CB₂R or selective CB₁R agonists exhibited antiseizure effects in either chemo-convulsing methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate or PTZ seizure models of 10-day postnatal rats [80]. Although the CB₂R selective agonist HU-308 did not show an antiseizure effect, the CB₂R selective antagonist AM630 did increase seizure severity [80]. In addition, a recent report showed that CB₁R knockout (KO) mice did not have an epilepsy phenotype, but co-KO of CB₁R and CB₂R caused animal epilepsy [81], suggesting that CB₂R plays a role in stabilizing the neuronal system. Recent studies have also explored the effects of modulating CB₂R activity on seizure susceptibility (Table 1). The activation of CB₂Rs decreases excitatory synaptic transmission in the CNS. The new roles for CB₂R have been identified in inducing hippocampal pyramidal cell hyperpolarization and inhibiting epileptic seizures [12]. The CB₂Rs expressed on hippocampal CA3 neurons also play a critical role in reduced neuronal excitation and oscillations [82]. WIN 55212-2, a non-selective CB receptor agonist, shows striking antiseizure effects in a rat epileptic model [83], and CB₁R and CB₂R double-knockout mice show spontaneous or manual-evoked seizures [81]. CB₂R knockout mice including both heterozygous and homozygous exhibit enhanced epileptic susceptibility, and a reduction in CB₂R activity is associated with increased susceptibility [84], suggesting that absent CB₂Rs can increase seizure susceptibility. The administration of caryophyllene, a CB₂R agonist, was found to improve seizure activity in a mouse model [85]. Collectively, these lines of evidence support the idea that the activation of CB₂Rs exhibits the antiseizure role. On the other hand, some studies reported different responses by CB₂R agonists. For instance, HU-308 [80] and JHW133 [84] show no significant effect on mice seizure occurrence. Moreover, CB₂R agonist AM1241 increases seizure intensity in a PTZ model [86]. Additionally, CB₂R antagonists AM630 and SR144528 can increase seizure susceptibility [80]. Therefore, it is likely that the altered CB₂R activity can affect seizure susceptibility, although the underlying mechanisms are still unclear. The different effects of CB₂R-mediated modulations may come from the differences in using different types of CB₂R ligands, and be based on different experimental designs, including species, epileptic model types, and dosage. We recently found that the commercially available CB₂R agonists showed different effects on pancreatic acinar cell Ca²⁺ oscillations [87]. Nevertheless, numerous lines of evidence have demonstrated that CB₂R agonists ameliorate a variety of epileptic seizures, suggesting that CB₂R is a potential therapeutic target for treating epilepsy.

As a crucial neuro-modulatory system of the brain, the midbrain dopaminergic system plays an important role in neuronal excitability. Temporal lobe epilepsy, involving pathological erethism of the hippocampus, is associated with VTA dopamine neuron activation [88]. The abnormally high synchronous activity of neuronal firing will cause phased impulse stimulation, causing more dopamine neurons to produce impulse firing, so that the dopaminergic system is in a super-reactive state during seizures’ occurrence [89]. This suggests that the dopaminergic system is vital in epileptic brains, and the evidence for a relationship between epilepsy and the dopaminergic system was previously described by Rezaei [2]. Pilocarpine-induced epileptic rats exhibit a significant enhancement in activity of dopaminergic neurons [88]. In the PTZ kindling model of epilepsy in mice, dopamine neurons within the VTA display hyperactivity when compared to saline-injected controls [90]. Anti-seizure treatments (antiseizure drugs or brain stimulation) are applied to downregulate the neuronal excitability for controlling epileptic seizures.

Stimulation of G_s-linked G protein-coupled receptors (GPCRs) triggers adenylate cyclase (AC) to produce cAMP from ATP. Activation of this signal pathway causes calcium influx through plasma membrane channels in a calmodulin-dependent manner [91]. Recent studies using transcriptomic analysis show that the transcription of a set of genes related to cAMP signaling is changed in patients suffering from drug-resistant temporal lobe epilepsy [92,93]. When seizures occur, levels of cAMP increase in the brain. The appearance of cAMP signaling can be divided into temporary influence on neuronal excitability, including ion channel or receptor phosphorylation [75], and long-term effects on epileptogenesis, such as effects regulated by cAMP response factor binding protein, CREB [94]. By coupling to G_i proteins, the activation of CB₂Rs causes inhibition of AC activity and downregulation of cAMP release [95]. 5-HA_1A-CB₂ heteroreceptors were characterized in cortical primary cultures of neurons, and 5-HT_1AR-CB₂ heteroreceptor complex expression and functionality are significantly enhanced in the brain after cerebral ischemia, especially during the neonatal term, also suggesting that this heteromer is associated with NHIBD pathophysiology [96]. Therefore, CB₂R activation may suppress the occurrence of epilepsy via decreasing excitability of the CNS by reducing the level of cAMP.

Most K⁺ channels are controlled by various physiological mediators, such as transmembrane voltage, and intracellular Ca²⁺ and G proteins. The roles of K⁺ in membrane physiology have been extensively investigated in rodent models, and the basic electrophysiological properties and bursting patterns of primate central neurons are generally similar to those reported for the rodent [97]. K⁺ channels are very important in regulating the intrinsic excitability of neurons, and they are the main contributor to neuronal membrane repolarization [98]. K⁺ channels are a promising target for the development of novel anti-epileptic drugs, and activation of the K⁺ channels can be used for restoring control on neuronal excitability in patients with epilepsy [99,100]. Kv7 (M-) channels are an unusual type of K⁺ channel which is different from those that repolarize an individual’s action potential. The M-channels are partially activated within the resting membrane potential of neurons, and further activated by membrane depolarization. The rate of M-channel open and close is slower than other types of K⁺ channels that contribute to the repolarization of action potentials. Activation of M-channels can inhibit highly synchronized neuronal firing that may reduce hyper-excitation and seizure activity [101]. The K⁺ channel opener retigabine is a compelling and selective opener of M-type K⁺ channels and is approved for therapy of drug-resistant focal and focal-to-bilateral tonic-clonic seizures [102]. In VTA DA neurons, G protein-coupled receptor signals regulate the excitability of neurons through a few ion channels, such as G protein-gated internally rectified K⁺ channels (GIRKs) [50,51,52]. The CB₂R agonist JWH133 has been confirmed to effectively modulate the excitability of neurons by regulating voltage-dependent M-type K⁺ channels [53].

More and more lines of evidence show that CB₂Rs are relevant to both immune cell competence at the peripheral region [103] and brain cells in the CNS. Actually, neurons, microglia, and astrocytes cells express CB₂Rs [32,104,105], which are capable of modulating central neural-immune function and impact the related diseases [106,107]. It is critical to accentuate that CB₂R has inducible expression in several immune cells under activated neuroinflammatory conditions. It means that CB₂R levels may be upregulated in the CNS and increased in inflamed brain parenchyma due to the invasion of peripheral immune cells (such as peripheral T cells) that express CB₂Rs [107]. During activated processes, microglia increase the expression of an array of membrane surfaces of CB₂Rs that may be essential in microglial production and/or degeneration within the brain. Both CB₁ and CB₂ receptors were expressed on microglia using an in vitro assay, including immunoglobulin superfamily receptors, cell component receptors, toll-related receptors, opioid receptors, and cannabinoid receptors [13]. A notable example is that neuropathic pain upregulates CB₂Rs in microglia in rat spinal cord, while chronic inflammatory pain does not [108]. When there is an inflammation response in the body, microglia CB₂R is rapidly upregulated and activated, which effectively inhibits the release of harmful factors, including TNF-α and free radicals [103].

In patients with epilepsy, medial temporal lobe sclerosis, cortical dysplasia, encephalitis, and glioma, the astrocytes show increased expression of CB₂Rs, which may change in K⁺ currents during seizures. It may lead to overexcitation and changes in a series of enzymatic pathways, which means that astrocytes may change the M-type K⁺ current by adjusting the expression of CB₂Rs to change the intensity of epilepsy. We believe that the role of the astrocyte/astrocytic CB₂R–cAMP signaling pathway in controlling epileptogenesis is worthy of further exploration.