Cannabis and Cannabinoid Signaling: Research Gaps and Opportunities

Abstract

Cannabis and its products have been used for centuries for both medicinal and recreational purposes. The recent widespread legalization of cannabis has vastly expanded its use in the United States across all demographics except for adolescents. Meanwhile, decades of research have advanced our knowledge of cannabis pharmacology and particularly of the endocannabinoid system with which the components of cannabis interact. This research has revealed multiple targets and approaches for manipulating the system for therapeutic use and to ameliorate cannabis toxicity or cannabis use disorder. Research has also led to new questions that underscore the potential risks of its widespread use, particularly the enduring consequences of exposure during critical windows of brain development or for consumption of large daily doses of cannabis with high content Δ⁹-tetrahydrocannabinol. This article highlights current neuroscience research on cannabis that has shed light on therapeutic opportunities and potential adverse consequences of misuse and points to gaps in knowledge that can guide future research.

SIGNIFICANCE STATEMENT

Cannabis use has escalated with its increased availability. Here, the authors highlight the challenges of cannabis research and the gaps in our knowledge of cannabis pharmacology and of the endocannabinoid system that it targets. Future research that addresses these gaps is needed so that the endocannabinoid system can be leveraged for safe and effective use.

Challenges of Cannabis and Δ⁹-Tetrahydrocannabinol (THC) Research

The cannabis plant has been cultivated and used for centuries for medicinal and psychotropic properties and is among the most widely used drugs today (Crocq, 2020). A challenge in research and clinical use of natural products lies in standardizing a product that is a mixture of bioactive components taken in different formats by varied routes of administration. Cannabis is composed of over 500 constituents including phytocannabinoids, terpenes, flavonoids, and alkaloids (Radwan et al., 2021). The landmark discovery of THC as the primary psychoactive compound in cannabis catalyzed a vast field of pharmacologic and biological research focused on the two major phytocannabinoids, THC and cannabidiol (CBD) (Mechoulam and Gaoni, 1965). Other cannabinoids, including cannabinol, cannabidivarin, cannabigerol, cannabichromene, and tetrahydrocannabidivarin, have reported pharmacological and biological activity, although with the exception of cannabinol, they have relatively low affinity and efficacy at cannabinoid receptors (Husni et al., 2014; Filipiuc et al., 2021). Actions of these cannabinoids at other receptors (Morales et al., 2017) and of noncannabinoid components such as terpenes that have reported biological activity, potentially contribute to the net effect of cannabis (Russo, 2011; Liktor-Busa et al., 2021). Given its complex composition of pharmacologically active molecules that can vary depending on the plant species and cultivation, precise knowledge of the pharmacological effects of ingesting or smoking the natural product remains challenging. Policies that call for the standardization of products, including the use of a standard unit THC dose as recommended by the National Institute on Drug Abuse (Volkow and Weiss, 2020) can help address this challenge.

Predicting the effects of ingesting THC alone is also challenging because of the complexity of its primary target, the endocannabinoid system. Knowledge of the endocannabinoid system emerged from a series of landmark discoveries including cannabinoid receptor localization and cloning and isolation and characterization of endogenous ligands (Devane et al., 1988, 1992; Herkenham et al., 1990; Mechoulam et al., 1995). As a partial agonist, THC can both engage cannabinoid receptor signaling and antagonize the endogenous ligands, 2-arachidonoyl glycerol (2-AG) and anandamide (Pertwee et al., 2010; Lu and Mackie, 2021). Because these are released on demand, THC effects can be context-specific, initiating signaling under static conditions and interfering with endogenous signaling under dynamic conditions. Additionally, cannabinoid 1 receptors (CB1R) form heterodimers with other G protein-coupled receptors and the binding of THC to CB1R in heterodimers can have novel, yet unrealized consequences (Hudson et al., 2010). Finally, although the cannabinoid receptors are the primary target of THC, it also interacts with other receptors including GPR55, 5HT3 receptors, glycine receptors, and TRPA1 and TRPV2 channels, and some of these actions may contribute to therapeutic effects such as analgesia (Pertwee et al., 2010; Xiong et al., 2011; Morales et al., 2017).

The Endocannabinoid System

A primary function of the endocannabinoid system in brain is regulation of neurotransmitter release, particularly GABA and glutamate (Castillo et al., 2012; Hillard, 2015; Volkow et al., 2017). The diverse localization of CB1R provides multiple layers of regulation. Agonist binding to Gi/Go-coupled CB1Rs on nerve terminals inhibits neurotransmitter release. Actions at CB1R on mitochondrial membranes may also contribute to presynaptic inhibition by decreasing cellular respiration (Bénard et al., 2012). Neurotransmitter release can be further refined through regulation by astrocytic Gq-coupled CB1R, which promotes the release of gliotransmitters that regulate presynaptic transmitter release in distinct manners (Noriega-Prieto et al., 2023). Because astrocytes are positioned to influence multiple synapses, activation of astrocytic CB1R can affect the activity of local neurons that are not synaptically linked in a synchronized manner through lateral regulation. Research is needed to delineate when these different modes of regulation are engaged, how they are integrated, and their net impact.

The endocannabinoid system is unique in having multiple ligands for the same receptor. Anandamide and 2-AG differ in their synthetic and metabolic pathways and their interactions with CB1R and TRPV1 (Di Marzo and De Petrocellis, 2012; Volkow et al., 2017). Additionally, there are differences in temporal dynamics, with 2-AG regulating neurotransmitter release in a phasic manner and anandamide regulating tonic neurotransmitter release (Di Marzo and De Petrocellis, 2012; deRoon-Cassini et al., 2020). These distinct features give rise to individual but interacting functions that may be differentially recruited depending on the context. This remains a major gap in our understanding of the endocannabinoid system.

Pain, Inflammation, and Stress

The ability to moderate pain and stress responses likely contributes substantially to nonmedical cannabis use. The high expression of CB1R in pain-related brain circuits has implicated this receptor in regulation of pain processing. In contrast, cannabinoid 2 receptor (CB2R) is implicated in inflammation because it is prominent on immune cells and regulates cytokine release. Notably, CB2R is induced in microglia in response to injury or inflammation suggesting that it plays a protective role (Navarro et al., 2016). The ability to inhibit both inflammation and glutamate release may contribute to neuroprotective effects (Al-Khazaleh et al., 2024). Nonetheless, targeting the endocannabinoid system to treat specific pain or inflammatory conditions requires a better understanding of how the endocannabinoid system regulates these processes and more specific tools to manipulate it.

The endocannabinoid system and stress systems interact in a bidirectional manner (deRoon-Cassini et al., 2020). Glucocorticoids released during stress increase 2-AG synthesis (Harris et al., 2019). This could be protective by moderating the activity of stress-response systems, such as the hypothalamic–pituitary–adrenal axis, the brain norepinephrine system, or certain glutamatergic circuits. However, corticotropin-releasing factor release decreases available anandamide by increasing fatty acid amide hydrolase (FAAH) (Gray et al., 2015). In the amygdala, diminished anandamide regulation of glutamatergic transmission has been implicated in anxiety and impaired fear extinction (deRoon-Cassini et al., 2020). FAAH inhibitors that inhibit anandamide metabolism and increase anandamide levels are proposed to mitigate effects of acute stress and enhance fear extinction. Consistent with this, individuals with a FAAH variant that results in elevated anandamide levels have lower baseline anxiety, decreased amygdalar reactivity to threatening stimuli and increased fear extinction (Mayo et al., 2020). These findings support a role for FAAH inhibitors in treating stress-related disorders such as post-traumatic stress disorder or as adjunctive treatments for substance use disorder (SUD) where stress drives relapse and continued drug use.

Reward and Subtance Use Disorder (SUD)

The endocannabinoid system is integral to motivated behavior through its regulation of dopamine (DA) release (for review, see Peters et al., 2021). These effects are mediated through multiple sites including the midbrain where activation of CB1R on GABAergic terminals disinhibits dopamine neurons (Lupica and Riegel, 2005). DA terminal release in the nucleus accumbens is further refined by CB1R receptors on its afferents (Covey et al., 2017; Mateo et al., 2017). CB1R-dopamine receptor 2 (D2R) and CB1R-adenosine receptor 2 heterodimers can also regulate DA release (Przybyla and Watts, 2010; García et al., 2016; Ferré et al., 2023). The regulation of reward circuitry by endocannabinoids contributes to the abuse potential of cannabis and to the risk for developing SUD to other agents. Consistent with this, individuals with a single nucleotide variant of FAAH that results in increased levels of anandamide have a greater risk for developing SUD (Sipe et al., 2002). Cannabis use disorder (CUD) is most prevalent in young adults and the increased availability of cannabis has contributed to its increase in this population, which rose from 14.7% in 2021 to 16.5% in 2022 (Gorelick, 2023; SAMSA, 2024). The increase in THC concentration in cannabis products along with patterns of more regular use are also likely to contribute to the higher incidence of CUD.

Although cannabis use can contribute to the risk to develop a SUD, its modulation of reward circuits and ability to alleviate pain, stress, and perhaps withdrawal has also suggested cannabis as a potential treatment of SUD and as substitute for opioid analgesics (Bachhuber et al., 2014). Some evidence exists for the use of CBD in treating opioid use disorder (Hurd et al., 2015). CBD has a complex pharmacology with actions on different neurotransmitter systems that might contribute to its potential therapeutic effects (for review, see de Almeida and Devi, 2020). As a negative allosteric modulator of CB1R, it does not share psychoactive effects with THC (Laprairie et al., 2015). CBD is also an allosteric modulator of μ and δ opioid receptors, effects that might underlie its ability to attenuate opiate withdrawal (Kathmann et al., 2006). Actions of CBD on 5HT_1A receptors are thought to account for its anxiolytic effects and to its reported ability to decrease opioid craving (Russo et al., 2005). CBD also increases levels of anandamide by inhibiting FAAH and anandamide uptake and this effect could contribute to its anxiolytic activity (Bisogno et al., 2001). CBD decreased opioid reward, cue-induced reinstatement, and attenuated withdrawal in rodents and decreased cue-induced craving and anxiety in a pilot study of individuals with heroin use disorder (Hurd et al., 2015). The beneficial effects of CBD may be specific to opioids, because other studies found no reduction of craving in individuals with cocaine use disorder (Mongeau-Pérusse et al., 2021). Further human studies are required to test CBD’s potential for treating opioid use disorder.

Brain Development

The endocannabinoid system plays essential roles in developmental programming of the brain at multiple stages (for review, see Bara et al., 2021). A major concern with mainstream use of cannabinoids is the potential for exposure during critical windows that could perturb the precision of neurodevelopmental programming resulting in enduring neurocognitive and behavioral consequences. Prenatal THC exposure can downregulate CB1R, which could result in aberrant connectivity in the brain. Adolescent use of THC is of concern because this is a time of pruning and plasticity within brain regions involved in affect and executive function. The endocannabinoid system promotes pruning in adolescence and there is a temporal dynamic of 2-AG and anandamide levels that is disturbed by THC. In humans, adolescent THC use has been associated with increased risk for developing affective disorders, particularly in females and heavy cannabis use before age 18 has been associated with an increased risk of developing schizophrenia, particularly among males (Hjorthøj et al., 2023). A recent study showed that the association between adolescent cannabis use and adult cognitive function was indirect and the result of its effect on academic and socioeconomic outcomes, underscoring the need for further mechanistic studies to test causality (Schaefer et al., 2021). Notably, there has been a substantial increase in the prevalence of cannabis use in older adults (>65 years of age) and there is a gap in research on the impact of cannabis on affect, motor, or cognitive function in this population (Han and Palamar, 2020). Although the literature on cannabis effects on brain development has primarily focused on THC, there is also a potential for effects of CBD exposure during critical windows of development, given its diverse targets. Further research is necessary to identify the developmental impact of CBD.

Sex Differences

Although cannabis use and CUD are more prevalent in males, cannabis legalization has led to escalating use in females, underscoring the importance of considering sex differences in cannabis effects (for review, see Cooper and Craft, 2018). Additionally, the health impact of cannabis on pregnancy and lactation where it can potentially impact gestation, fetal health, or parental–infant bonding requires consideration (Bara et al., 2021). Systematic studies of sex differences in cannabis effects are few and existing data are inconsistent. Sex differences in the characteristics of cannabis use have been reported with women using cannabis more for medicinal than recreational reasons and escalating to cannabis dependence faster after initial use. There are also sex differences in severity and characteristics of cannabis withdrawal. The increased recreational and therapeutic use of cannabis demands rigorous research in sex differences and effects on women’s health across the lifespan.

Pharmacological Targeting

The endocannabinoid system offers multiple therapeutic opportunities but also a complex of unwanted effects (for review, see Maccarrone et al., 2023). Agents that directly interact with CB1R and CB2R orthosteric sites will be most effective and toxic. For example, synthetic cannabinoids such as K2 or spice are potent full agonists and are psychoactive and too toxic for therapeutic use. Rimonabant, a high affinity CB1 inverse agonist, was removed from the market because it was associated with anxiety and suicidal tendencies (Sayburn, 2008). These adverse effects reveal the critical functions of the endocannabinoid in stabilizing affect. Partial agonists such as THC are less psychoactive and toxic and have greater therapeutic potential, although they can also antagonize 2-AG. Allosteric modulators provide a pharmacological refinement because they tune the ligand–receptor interactions by binding to sites other than the orthosteric site and influence the binding and/or efficacy in a positive (positive allosteric modulator) or negative (negative allosteric modulator) manner. They have an advantage of spatiotemporal specificity because they will only have effects where and when the endogenous agonist binds. Positive allosteric modulators could be used to alleviate withdrawal from THC. CBD is a negative allosteric modulator of CB1R, although its effects could be attributed to its many other actions (see above). A notable endogenous negative allosteric modulator of CB1R is pregnenolone, which binds to a unique allosteric site and specifically interferes with THC signaling at CB1R (Raux and Vallée, 2023). This property of pregnenolone could counteract THC toxicity and has been proposed as a potential treatment of CUD (Raux and Vallée, 2023). Other mechanisms for modulating the endocannabinoid system for therapeutic ends include targeting synthetic or metabolic enzymes or transporters (Piomelli and Mabou Tagne, 2022). These mechanisms offer a more measured response by altering the concentration of endogenous ligand availability. The most notable compounds are those that regulate the metabolic enzymes FAAH and monoacylglycerol lipase (MAGL). FAAH inhibitors increase anandamide levels and have analgesic, anxiolytic, and antidepressant properties but lack the rewarding effects and abuse potential of direct agonists. MAGL inhibitors that increase 2-AG levels have analgesic potential in low doses when given acutely. However, chronic administration of MAGL inhibitors can impair function because elevated 2-AG desensitizes and downregulates CB1. Because the metabolism of 2-AG by MAGL is a substantial source of brain arachidonic acid, which is a precursor of proinflammatory substrates, MAGL inhibitors have central anti-inflammatory and neuroprotective potential (Lu and Mackie, 2021). Finally, the multiple mechanisms for transporting endocannabinoids intracellularly and across the synapse offer an array of approaches to fine tune the spatiotemporal dynamics of endocannabinoids (Maccarrone et al., 2023). Further research is necessary to identify the specific roles of transport proteins and systems for the development of selective molecules to manipulate them.

Summary

Changes in the legal regulation of cannabis have inspired a general acceptance of its use that has outpaced the scientific research necessary to inform the public of its safety and efficacy. Figure 1 depicts the many areas where research should be prioritized to fill gaps in knowledge. Because the endocannabinoid system through which cannabinoids act regulates synaptic communication throughout the brain, cannabinoids are positioned to affect diverse functions with both detrimental and advantageous consequences. Research continues to unveil the unique features of this system and the functions of its multiple components, guiding the development of tools to probe the system and therapeutics that leverage the system to enhance neurobehavioral health.

Fig. 1.

Graphical depiction of general categories of research priorities and more specific areas (lists) in which there are gaps in knowledge.

Abbreviations

2-AG

2-arachidonoyl glycerol

CBD

cannabidiol

CB1R

cannabinoid 1 receptors

CB2R

cannabinoid 2 receptors

CUD

cannabis use disorder

DA

dopamine

FAAH

fatty acid amide hydrolase

MAGL

monoacylglycerol lipase

SUD

substance use disorder

THC

Δ⁹-tetrahydrocannabinol

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Valentino, Volkow.