Introduction
Cannabidiol (CBD), a phytocannabinoid devoid of psychoactive properties, is currently being investigated in a series of clinical trials to determine its potential for treating diseases such as epilepsy, neuropsychiatric disorders, neurodegeneration and neuropathic allodynia [
1‐
4]. The complete pharmacology of CBD is far from understood, as multiple mechanisms of action and several pharmacological effects have been proposed. Unlike Δ
9-tetrahydrocannabinol, the main psychoactive constituent of the marijuana plant, the effects of CBD do not involve direct binding to the endocannabinoid receptors CB1 and CB2 [
5,
6]; instead, CBD behaves as a non-competitive negative allosteric modulator of CB1 receptors [
7]. Allosteric modulation, in conjunction with effects not mediated by CB1 receptors, may explain the in vivo effects of this compound. Indeed, CBD at nanomolar to micromolar concentrations is reported to interact with several non-endocannabinoid signaling systems to impair the function of orphan G-protein-coupled receptor (GPR) 55 [
8], the transient receptor potential of melastatin type 8 channel and, transient receptor potential of ankyrin type 1 channel [
9] and to facilitate the activity of serotonin 5HT1A receptor [
10] and α3 and α1 glycine receptors [
11,
12].
Within this complex framework, CBD exhibits positive effects in situations in which glutamatergic signaling, particularly that mediated by
N-methyl-D-aspartate acid receptor (NMDAR), plays a critical role. Thus, CBD exhibits antioxidant properties and protects neurons from glutamate-induced death but without cannabinoid receptor activation or NMDAR antagonism [
13]. CBD diminishes the neural damage caused by ischemic stroke [
14] and chronic diseases, including Parkinson’s and Alzheimer’s diseases [
15‐
17]. CBD also shows anticonvulsant activity in many acute animal models of seizures [
18,
19], and in preclinical studies, CBD is comparable to antiepileptic drugs currently used in clinical therapy [
20]. CBD also modulates morphine antinociception in mice [
21] and exhibits anti-allodynia effects in rodent models of neuropathy [
22,
23]. Indeed, CBD prevents the onset of mechanical and thermal sensitivity induced by the taxane chemotherapeutic agent paclitaxel in a female mouse model of chemotherapy-induced peripheral neuropathy [
24]. Clinical evidence suggests that CBD can be used to manage epilepsy in adults and children affected by refractory seizures and exhibits a favorable side effect profile [
25].
Similar to CBD, recent work has revealed that sigma 1 receptor (σ1R) antagonism prevents GPCRs from enhancing the function of NMDARs, thereby reducing the cellular impact of excessive glutamatergic activity [
26,
27]. Thus, in the aforementioned situations, σ1R ligands, particularly antagonists, exhibit potential for treating neurological diseases [
28], substance abuse syndromes [
29], and certain neuropsychiatric disorders [
30] and may serve as adjuvants for opioid analgesia [
31]. Accordingly, σ1R antagonists alleviate neuropathic allodynia and inflammatory hyperalgesia in animal models of pain involving NMDAR activation [
32‐
34]. Additionally, σ1R ligands have been shown to enhance neuroplasticity and functional recovery following experimental stroke [
35], a paradigm in which increased NMDAR activity plays a decisive role. The highly selective σ1R antagonist, S1RA, significantly reduces the cerebral infarct size and neurological deficits caused by permanent middle cerebral artery occlusion (pMCAO) [
36]. Likewise, recent data suggest the involvement of σ1R in rare CNS diseases, such as Dravet syndrome [
37], and the sigma ligand ANAVEX 2–73 shows potential for treating certain CNS disorders [
38], including epilepsy [
https://www.anavex.com/anavex-releases-promising-full-preclinical-epilepsy-data-at-antiepileptic-drug-trials-xiii-conference/].
We thus addressed whether this phytocannabinoid modulates glutamatergic NMDAR transmission via σ1R. Our study suggests that CBD displays antagonist activity toward σ1R to inhibit NMDAR function and effectively reduces its ability to dampen morphine-induced analgesia, promote NMDA-mediated convulsive syndrome and cause neuronal damage after pMCAO.
Discussion
The present study shows that CBD acting as an antagonist of σ1R diminishes the influence of glutamate NMDA receptors in three experimental paradigms in which this activity plays a key role, i.e., the level of morphine-evoked antinociception, the incidence of NMDA-induced convulsive activity and the extent of the neural damage caused by experimental ictus. These positive effects of CBD are also achieved by the direct binding of drugs to the NMDAR ionic pore to block NMDAR calcium influx (antagonists) [
45‐
47] and by antagonists, but not agonists, of σ1R, which regulates NMDAR function [
36,
48,
49]. σ1R antagonists and CBD exhibit no such control over NMDAR activity in mice lacking σ1R protein expression. This and previous observations show that CBD does not bind to the NMDAR ionic pore [
13] and thus suggest that CBD displays antagonist-like activity on σ1R to counteract the negative effects of NMDAR hyperfunction in the aforementioned experimental situations.
The activity of glutamate NMDARs falls under the negative influence of some GPCRs, including CB1R [
50], acetylcholine type 1 muscarinic receptor [
51], serotonin 5HT1AR [
52], adrenergic α1R and α2R [
53], dopamine D3R and D4R [
54,
55], and group III mGluR7R [
56]. Among these GPCRs, 5HT1AR is a suitable candidate for mediating CBD effects [
10], and in fact, the 5HT1AR antagonist WAY100635 diminished the capacity of CBD to alleviate NMDA-induced convulsive syndrome. Nevertheless, WAY100635 alone enhanced the capacity of the agonist NMDA to evoke convulsions, suggesting the endogenous inhibitory control of NMDAR activity by 5HT1AR. Certainly, WAY100635 has been described to display agonist activity toward the dopamine D4 receptor, which is negatively coupled to glutamate activity [
57]. In our experimental model, WAY100635 enhanced wild running and tonic seizures and promoted death in mice treated with a lower dose of 0.3 nmol NMDA. Thus, in our convulsive model, the activity of WAY100635 toward dopamine D4 receptors can be disregarded. If CBD displays activity toward certain GPCRs to alleviate NMDA-induced convulsions, these receptors require the presence of σ1R to promote such an effect because CBD failed to do so in σ1R
−/− mice. The σ1R agonist PPCC produced no significant changes in this model but efficaciously counteracted the anticonvulsant effects of CBD. These observations suggest an essential role of σ1R in the negative control that CBD exerts on NMDAR activity. Indeed, in the presence of σ1R agonists, CBD did not exhibit the aforementioned effects, and the in vitro assays showed that CBD acts as a σ1R antagonist to disrupt σ1R-NR1 complexes.
Although σ1R is a chaperone that regulates a series of signaling proteins in the endoplasmic reticulum in a calcium-dependent manner, this protein was discovered as a type of opioid receptor, and σ1R can thus be found in the cell plasma membrane [
58]. In this context, σ1R is a regulator of NMDAR function and cooperates with histidine triad nucleotide-binding protein 1 (HINT1) to regulate NMDAR function via certain GPCRs [
26]. The calcium-dependent binding of σ1R to NMDAR NR1 subunits that carry the cytosolic C1 segment protects the activity of NMDARs, i.e., calcium influx, from the inhibitory action of calcium-activated calmodulin (Ca
2+-CaM). While agonists promote σ1R binding to the NMDAR NR1 subunit, antagonists such as CBD disrupt these complexes to facilitate the Ca
2+-CaM inhibition of NMDAR function. Thus, CBD acts as a σ1R antagonist to reduce NMDAR activity.
As previously mentioned, CBD is involved in a variety of activities and may act as a sedative, anxiolytic, antipsychotic, anti-inflammatory, antioxidative, neuroprotective, anti-emetic, anticancer, antidepressant and mood-stabilizing drug, as well as have therapeutic effects on movement disorders, ischemia, diabetes, and cannabis withdrawal syndrome [
30,
59‐
63]. Moreover, CBD exhibits anti-neuropathic effects [
22,
23] and modulates morphine antinociception in mice [
21]. Our data suggest that CBD acts on σ1R to alleviate the manifestation of epileptic syndrome, to protect against neural damage caused by vascular ischemia and to enhance the antinociception promoted by morphine. These findings suggest the implication of σ1R in other beneficial activities attributed to this phytocannabinoid. Indeed, σ1R is a potential target for the treatment of neuropathic pain because it interacts with and regulates NMDARs and TRPV1 calcium channels, which are key constituents of the mechanisms that modulate activity-induced sensitization in nociceptive pathways [
32‐
34,
64]. Moreover, σ1R ligands also exhibit antidepressant, anxiolytic, neuroprotective and antioxidative effects [
30,
65].
Alterations in σ1R have been consistently related to schizophrenia [
66,
67]. NMDAR function is lower in this mental illness, and the negative control exerted by GPCRs, such as CB1Rs, on glutamate activity may play an essential role in the etiology of this disease [
68‐
70]. The severity of the negative symptoms of schizophrenic patients correlates with alterations in the plasma levels of anandamide [
71] and with those of neurosteroids, the putative ligands of this ligand-operated chaperone/receptor [
72,
73]. Indeed, adjunct treatment with pregnenolone diminishes the negative symptoms of schizophrenia [
74]. The idea that the CB1R localizes primarily in axon terminals have already been challenged [
75], and a series of immunocytochemical and ultrastructural studies have demonstrated the presence of the CB1R in the somatodendritic compartment (post-synapse), both at the spinal and supraspinal levels where it co-localizes with NMDARs and PSD95 proteins. Thus, an anomalous σ1R-regulated connection between CB1R and NMDAR may contribute to the disproportionate downregulation of NMDAR activity (hypofunction), constituting a serious risk factor for the development of schizophrenia [
68,
76]. In the context of negative NMDAR regulation by CB1Rs, σ1R antagonists such as CBD uncouple NMDAR function from the negative influence of GPCRs, such as CB1Rs [
70,
76].
In the sub-micromolar and micromolar range, CBD affects the function of various signaling pathways. Among other targets, CBD regulates cannabinoid receptors without displaying binding directly to them; CBD also impairs the function of the equilibrative nucleoside transporter, that of the orphan GPR55 receptor and that of the transient receptor potential of melastatin type 8 channel [
8,
9]. Conversely, CBD enhances 5HT1AR, α3 and α1 glycine receptor, and transient receptor potential of ankyrin type 1 channel activity [
9]. In hippocampal cultures, the CBD-mediated regulation of calcium levels is bidirectional and depends on the excitability of the cells [
77]. CBD also activates the nuclear peroxisome proliferator-activated receptor γ and transient receptor potential vanilloid type 1 and 2 channels while inhibiting the cellular uptake and fatty acid amide hydrolase-catalyzed degradation of anandamide [
71,
78]. CBD reduces hydroperoxide-induced oxidative damage, tissue cyclooxygenase activity, the nitric oxide production, T-cell responses, bioactive tumor necrosis factor release, and prostaglandin E2, cytokine interferon c and tumor necrosis factor production and blocks voltage-gated Na + channels [
6,
77]. Whether the effects of CBD on σ1R are relevant to these activities remains to be explored. Because this exogenous cannabinoid may alter the function of a wide variety of cellular activities, the key is to determine the molecular mechanisms that are primarily implicated in a particular effect of CBD.
Our study indicates that CBD regulates the function of σ1R in at least several of the abovementioned behavioral effects. Thus, CBD’s enhancement of opioid analgesia, alleviation of convulsive syndrome, protection against ischemic neural damage and anti-allodynia effects appear to involve an antagonist interaction with σ1R and the subsequent reduction of NMDAR function. This finding may help in us to understand the current pharmacology of CBD and provides new avenues for the treatment of several brain-related disorders.