Ligands

Elucidation of the delta-9 double bond in plant-derived cannabinoid (phytocannabinoid), Δ9-tetrahydrocannabinol (THC), in the early 1960s instigated the search for endogenously synthesized cannabinoids (endocannabinoids) and led to the detection of anandamide (AEA) and 2-arachidonoylglycerol (2-AG).^{65, 66} Each cannabinoid class and type is distinct in mechanisms of action, behavioral profiles and action in neural circuits. Phytocannabinoids are produced in secretory cells of glandular trichomes at high concentrations in unfertilized female Cannabis flowers. THC is a low potency partial agonist at CB1R and CB2R.^{8, 65} The non-psychoactive analog of THC, cannabidiol (CBD), is of burgeoning clinical interest for its very low efficacy and partial agonism at CB1R and CB2R. At low molar concentrations, CBD acts as an antagonist or inverse agonist (particularly at CB2R) to limit THC effects and contributes to the upregulation of endogenous cannabinoid signaling.⁶⁷ Clinically, CBD is a neuroprotectant used as an anti-inflammatory, anticonvulsant, antiepileptic and antipsychotic.⁸ CBD’s effects are attributed to the full agonism it exhibits for non-cannabinoid G-protein-coupled receptors (for example, peroxisome proliferator-activated and nuclear receptors) and interactions with serotonergic, adenosinergic and vanilloid systems.^{8, 15} Notably, the most efficacious therapeutics are observed with formulations combining THC and CBD (two to four times greater than single cannabinoid preparations⁶⁸) as CBD enhances the therapeutic action of THC by potentiating its psychotropic effects, augmenting THC tolerability, and widening the therapeutic window.⁶⁷ Enhanced efficacy is attributed to the entourage effect,^{65, 68} the synergistic action of the more than 480 biologically active and inactive compounds in Cannabis. It is likely that the as-yet identified major and minor cannabinoids in plant extracts may further enhance the therapeutic benefit observed with THC and CBD through improved stimulation of the endogenous cannabinoid system.⁶⁵

Endocannabinoids (eCBs) are small (<400 Da) lipophilic activity-dependent retrograde messengers in the brain. Produced post-synaptically ‘on demand’ through de novo synthesis from membrane phospholipids in response to increased intracellular Ca²⁺ via depolarization,⁶⁹ eCBs act presynaptically to inhibit GABA and glutamate release.¹⁷ The physiological endpoints of AEA and 2-AG are comparable to THC,⁶⁹ but are functionally and temporally distinct from one another. AEA has high binding affinity (CB1R: K_i=239 nmol l⁻¹, CB2R: 440 nmol l⁻¹),⁷⁰ but low efficacy for cannabinoid receptors. It is most active during steady-state conditions and regulates basal synaptic neurotransmission. Data from rodent autism spectrum disorder (ASD) models demonstrate that genetic mutations in the neuroligin gene disrupt tonic eCB signaling⁷¹ and intimate the importance of eCB signaling mode (tonic or phasic) in neurodevelopment and neuropsychiatric disorders. 2-AG is the more prevalent eCB (~4 nmol g⁻¹ in brain tissue vs AEA at <100 pmol g⁻¹)⁷⁰ and regulates synaptic plasticity.^{17, 69} It is representative of ‘phasic’ signaling evoked in sustained depolarization. The greater overall efficacy and binding affinity of 2-AG (CB1R: K_i=3424nmol l⁻¹, CB2R: 1194 nmol l⁻¹),⁷⁰ particularly at CB2R,⁷² is likely related to the enhanced native tone and role in adaptive response. Inactivation of AEA and 2-AG is primarily through enzymatic degradation. AEA is transported across the transmembrane proteins^{47, 73} and hydrolyzed via fatty acid amide hydrolase (FAAH),⁴⁷ whereas 2-AG can be hydrolyzed by FAAH or monoacylglycerol lipase.⁷⁴

Chronic stress

Stress is an adaptive response to a specific internal or external stressor in preparation for injury or threat. Definitions of stress emphasize the physiological and emotional consequences to relate acute parallel activation of autonomic and endocrine systems. Chronic stress is a response to persistent stressors, like social threat or defeat, which reduces social motivation and social interactions by intensifying fear towards (an emotional reaction) and avoidance (anxiogenic behavior) of unknown conspecifics.^{32, 79} Across development, protracted exposure to stressors imposes a progressive pattern of dysfunction in social functioning, beginning as asociality and culminating in antisociality.⁷⁹ Persistent activation (>4 weeks, ≤48 weeks) of stress neurocircuitry breaks down homeostatic balance^{32, 33} and creates a ‘hypocannabinergic state’ through downregulation of CB1R expression in the hippocampus, hypothalamus, striatum and dorsal root ganglion.^{33, 34} No significant change in CB2R expression was observed in three separate mouse lines, following exposure to chronic stressors, but without analogous data in humans, few conclusions can be made about CB2R function under chronic stress.²⁵ Alterations in CB1R expression are concurrent with changes in eCB signaling in the hippocampus, striatum, dorsal raphe nucleus, hypothalamus, nucleus accumbens (NAc), PFC (at GABAergic terminals) and the amygdala. In the absence of stressors, AEA tone suppresses activity of the hypothalamic–pituitary–adrenal (HPA) axis (the chief modulator of the stress response^{80, 81}) and disruption of this suppression initiates HPA-axis activation.^{35, 81} Chronic stressor exposure impairs AEA signaling and elevates 2-AG content within the amygdala (and increases glucocorticoid hormone secretion), hippocampus, hypothalamus and PFC,³³ likely due to altered FAAH and monoacylglycerol lipase-mediated hydrolysis, as the synthesis of these eCBs is unimpaired.^{34, 82}Glucocorticoid feedback inhibition of HPA-axis activity is suggested to occur on two temporal scales, rapidly via the PFC and 2-AG tone enhancement, or less rapidly via negative feedback inhibition at the paraventricular nucleus of the hypothalamus.^{34, 82} Glucocorticoids then normalize the amygdalar AEA levels to support basal HPA function.³⁴ The enhancement of 2-AG signaling is perceived as an attempt to habituate to the chronic stress exposure and may be crucial in understanding how to properly engage eCB signaling in the modulation of the HPA axis to guarantee a positive adaptive response.³⁴ However, the presence of a mechanism to examine maladaptive responses to chronic stress exposure highlights novel areas for social deficits in which exposure to a chronic stressor is unavoidable (for example, social anxiety and mood disorders).

Social motivation

Social motivation leads to enhancement of social development by promoting social interactions. Abnormal motivation precludes proficient social interaction and has negative consequences on the acquisition of higher-order social abilities.³⁷ The inability to separate motivation from social interest has contributed to the assumption that deficits in social functioning are a cause of disrupted social interest, as opposed to a consequence of them.³⁷ Motivation is necessary to attend to social information, seek and engage in social interaction, as well as foster and maintain social bonds, whereas interest does not necessitate action. Individual differences in social motivation are associated with alterations in the activity of neurocircuitry that overlap with reward learning; high social motivation is correlated with enhanced activation of the amygdala and orbital frontal cortex, whereas weaker activation is related to lower social motivation. Opioid and dopaminergic neurons in the ventral tegmental area have complementary roles in motivated behaviors and require eCB signaling to fine-tune dopamine release in incentive-related reward learning.^{40, 52}Concomitantly, enhanced motivation is observed following CB1R stimulation and with increased AEA signaling in the amygdala, NAc and dorsal striatum and 2-AG in the NAc.^{38, 41, 42} Diminished motivation is associated with CB1R blockade.^{38, 39} Wei et al.⁸⁷ suggest increased AEA content in the forebrain in animal models of ASD can ameliorate social motivation deficits. Extending previous results in rats in which modulation of CB1R or genetic removal of FAAH negatively impacted social interactions,^{79, 88}researchers examined the influence of AEA on social behaviors via FAAH modulation.¹³ A social approach task and elevated plus maze were used to assess the effects of increased AEA (via FAAH inhibition) on social motivation in BTBR mice (an idiopathic ASD model with known deficits in social approach, reciprocal social interaction, and juvenile play) and Fragile X Syndrome mice (a syndromic ASD model with persistent social approach deficits). Increased AEA signaling in the forebrains of ASD mouse models correlated with increased time in the social chamber and preferential interaction with novel animals compared with untreated and control mice.⁸⁷ Effects were not associated with decreased anxiogenic responses, as no change in behavior on the elevated plus maze was observed. Improved prosocial behaviors were ‘generalizable’⁸⁷ and suggest common neural circuitry of social motivation between idiopathic and syndromic ASD.

Working memory

More immediate information processing involves working memory, which actively maintains and updates relevant information, but is capacity-limited.⁵⁰ In social interactions, working memory tracks social information, like the characteristics of, or relationships among, people necessary to competently socially interact. THC exposure in humans negatively impacts working memory via CB1R activation and inhibition of AEA reuptake.^{48, 51} Correspondingly, rodent models with upregulated CB1R expression in the PFC, as well as CB1R knockout mice, demonstrate changes in cognitive flexibility. Low doses of CB1R antagonists improved task switching (a measure of cognitive flexibility) and inhibitory control via inhibition of PFC glutamatergic activity, whereas CB1R agonists increased impulsive behaviors. A neuroimaging study⁹⁰suggests that THC impacts activity in cerebral inhibition response circuits causing increased hyperactivity in the PFC and anterior cingulate cortices. Acute administration of THC reduces response inhibition^{49, 89}(that is, increases behavioral impulsivity) and causes hyperactivity at dopaminergic synapses in the PFC.⁹⁰Data from animal models suggest that cannabinoid signaling interacts with dopaminergic, GABAergic and glutamatergic pathways to mediate behavioral changes. Data in FAAH knockout mice demonstrate that AEA-biased tone improves acquisition in working memory tasks, but the effects are transient and do not persist into later trials.^{65, 66} This evidence suggests that strategies to improve components of working memory (for disorders such as prosopagnosia, schizophrenia and/or depression) should focus on control of AEA tone and local modulation of 2-AG signaling.

Social communication

In humans, high concentrations of cannabinoid receptors are found in the left hemisphere cortical regions that are associated with verbal language function,^{16, 18} which suggests a role for eCB signaling in social communication. However, current findings only supports a role for eCB signaling in nonverbal motor-related aspects of social communication (that is, eye-gaze or body language). Genetic variants (that is, single-nucleotide polymorphisms) in CB1Rs are correlated with greater gaze duration to happy faces and are considered a putative endophenotype for communication deficits, particularly in ASD research.⁹⁹Animal models of social communication impairments demonstrate a direct connection to cannabinergic function, which interacts with the forkhead box (FOXP) protein family.⁵⁸ Interestingly, FOXP are instrumental in ultrasonic vocalizations (USVs) production and vocal learning.^{58, 100} In rodents, USVs serve a communicatory function to elicit social interaction^{61, 62} or to share socially relevant information with known conspecifics.⁶³ Moreover, USVs are developmental- and context-dependent⁶² relating identity, emotionality,^{61, 62, 63} receptivity to affiliative and sexual behaviors, and changes in the environment.⁵⁶Persistent activation of CB1R, via agonists or inhibitors of enzymatic degradation, decrease emission of USVs in rodent pups. Cannabinoids modulate USVs induced by maternal separation in pups,⁶¹ whereas in adult rodents, cannabinoids increase the emission of USVs when exposed to anxiogenic stimuli.^{61, 63}Moreover, CB1R knockout mice have low USV emission throughout development,⁵⁶ which parallels the communication impairments associated with disorders such as ASD.⁶² For clinical populations with impairments in language acquisition, especially nonverbal patients with social deficits, cannabinergic modulation of USVs offers a novel and viable area of biomedical intervention in support of communication acquisition and development.^{59, 60, 100}