Endocannabinoid signalling in reward and addiction

Abstract

Brain endocannabinoid (eCB) signalling influences the motivation for natural rewards (such as palatable food, sexual activity and social interaction) and modulates the rewarding effects of addictive drugs. Pathological forms of natural and drug-induced reward are associated with dysregulated eCB signalling that may derive from pre-existing genetic factors or from prolonged drug exposure. Impaired eCB signalling contributes to dysregulated synaptic plasticity, increased stress responsivity, negative emotional states and cravings that propel addiction. Understanding the contributions of eCB disruptions to behavioural and physiological traits provides insight into the eCB influence on addiction vulnerability.

Figure 1. Endocannabinoid biosynthesis, signaling and clearance

The most commonly accepted route for AEA synthesis is from catalysis of N-arachidonoyl-phosphatidylethanolamine (NAPE) via a specific phospholipase D (NAPE-PLD). 2-AG derives from the hydrolysis of 1,2-diacylglycerol (DAG) via the sn-1-selective DAG lipases DAGLα and DAGLβ. DAGLα is found on the plasma membranes of both dopaminergic and non-dopaminergic neurons in the VTA, opposite CB₁R-expressing glutamate and GABA axon terminals. Termination of EC signaling is initiated by cellular reuptake followed by enzyme-mediated hydrolytic cleavage. 2-AG hydrolysis is primarily mediated by presynaptic monoacylglycerol lipase (MAGL), though post-synaptic enzymes including ABHD6 also contribute to 2-AG clearance. AEA hydrolysis occurs in postsynaptic cells through fatty acid amide hydrolase (FAAH). Although these mechanisms are depicted here in the VTA, the pre- and post-synaptic organization of EC biosynthetic and hydrolytic enzymes is generally conserved throughout the brain.

Figure 2. Distribution of EC signaling mechanisms within the brain reward circuits

CB₁Rs are expressed throughout the regions implicated in reward and addiction including the basolateral amygdala (BLA), prefrontal cortex (PFC), hippocampus (HIPP), ventral pallidum (VP), globus pallidus (GP), dorsolateral striatum (DSTr), NAc, VTA, bed nucleus of the stria terminalis (BNST) and central nucleus of the amygdala (CeA)^{, ,} . In general, the expression patterns of EC biosynthetic enzymes (e.g. NAPE-PLD and DAGLα) and hydrolytic EC clearance enzymes (e.g. FAAH and MAGL) are similar to that for CB₁Rs across the regions depicted here^, . Within the amygdala, CB₁, DAGLα, MAGL and FAAH expression is highest in the lateral and basolateral nuclei, with substantially lesser expression in the central nucleus^, . In the dorsal striatum there is a comparable medial-lateral gradient of CB₁ and DAGLα expression with greater levels of expression evident in lateral aspects^, . Comparatively weaker CB₁, DAGLα and FAAH expression is observed in the NAc. Although little to no CB₁ expression is found in dopamine cells in the NAc, DAGLα has been found in both dopaminergic and non-dopaminergic cells in this region.

Figure 3. Endocannabinoid influences in the VTA and NAc contributing to approach and avoidance behavior

a. EC influences on VTA synaptic signaling. Endocannabinoids produced by dopaminergic VTA neurons act on CB₁Rs on nearby glutamatergic and GABAergic terminals before being degraded by ABHD6 or MAGL. CB₁Rs mediate robust inhibition of GABA inputs arising from the pallidus, RMTg nucleus and local interneurons onto VTA DA cells and most evidence points to a role for 2-AG but not AEA in these processes^, . CB₁Rs are also localized on glutamatergic terminals synapsing on VTA DA neurons, with relatively greater expression on VGLUT1-positive terminals of cortical origin compared with VGLUT2-expressing terminals of subcortical origin. Extensive evidence demonstrates EC-mediated suppression of glutamate signaling in VTA. Thus, ECs play a prominent role in fine-tuning the activity of the mesolimbic DA projection through modulation of both excitatory and inhibitory signaling in the VTA. b. EC influences on NAc synaptic signaling. The majority of NAc neurons (>90%) are GABAergic medium spiny neurons (MSNs) that comprise the direct and indirect projection pathways. Direct pathway MSNs (dMSNs) project to midbrain regions including the VTA and activation of this pathway increases behavior toward a stimulus (approach or appetitive behavior). Indirect pathway MSNs (iMSNs) project to the ventral pallidum (VP) and activation of this pathway increases the stimulus avoidance. dMSNs express excitatory D₁ DA receptors and iMSNs express inhibitory D₂ DA receptors, and thus reward-related phasic DA release activates the direct pathway and inhibits the indirect pathway, thereby increasing approach behavior and reducing avoidance behavior. NAc MSN activity is also heavily modulated by glutamatergic inputs from the PFC, BLA and ventral hippocampus that express CB₁Rs, CB₁-mediated suppression of excitatory signaling (EC-LTD) is preferentially active at iMSN synapses possibly resulting from D₂ receptor-mediated EC production from iMSN cell bodies. Thus, increased NAc EC formation preferentially reduces excitatory input to iMSNs vs. dMSNs, resulting in decreased avoidance behavior. Through these mechanisms, increased EC signaling in the NAc increases approach behavior while reducing avoidance-related processing thereby enhancing appetitive responding toward a stimulus. CB₁Rs are also expressed on terminals of fast-spiking interneurons (FSIs) in the NAc, the majority of which are electrically and chemically coupled and provide direct innervation to adjacent MSNs. FSIs exert important influence on the synchronization of neural ensemble activity and thus EC signaling may also exert critical influence on NAc output through feed-forward modulation of MSN network activity.

Figure 4. Drug-induced alterations in EC-mediated synaptic plasticity

Simplified summary of the effects of cocaine, Δ9-THC, and possibly other drugs of abuse on EC-mediated long-term depression (LTD). a | Under normal circumstances, EC-mediated long-term depression (EC-LTD) is induced by afferent stimulation with or without postsynaptic depolarization resulting in the 2-AG formation from postsynaptic cells. 2-AG activates CB₁Rs on stimulated or neighboring non-stimulated neurons, which together with other events (e.g. increased [Ca⁺²], NMDAR stimulation, DA D₂R stimulation, etc.) results in persistently decreased neurotransmitter release. The presynaptic signaling mechanisms contributing to EC-LTD are not fully understood. Depending on brain region, EC-LTD of both excitatory (glutamatergic) and inhibitory (GABAergic) afferents has been described. b | Repeated cocaine exposure facilitates the EC-LTD of inhibitory signaling in the VTA^, resulting in diminished inhibitory constraint of VTA DA cell activity and increased excitability. c | In contrast, EC-LTD of excitatory signaling is lost in the NAc medium spiny neurons (MSN) following exposure to either cocaine or Δ9-THC^{, ,} , resulting in diminished constraint of glutamatergic release and increased excitation of NAc cells. Thus, drug exposure results in concurrent loss of EC-mediated plasticity that normally provides inhibitory control over VTA DA cell excitation and loss of EC-mediated plasticity that normally constrains excitatory signaling in the NAc terminal region, conferring an overall enhancement of mesolimbic signaling.

Figure 5. CNR1 and FAAH genes and genetic variants associated with addiction

The EC genes primarily studied to date in relation to genetic associations with addiction are CNR1 and FAAH. The human CNR1 gene, which encodes the CB₁R, maps to chromosome 6 specifically in the cytogenetic band 6q14-q15 and is transcribed from the minus strand (3′ to 5′ orientation) of the DNA. The gene contains four exons with the protein coding region located at the 5′ end of exon 4^–. There are multiple mRNA variants of the CNR1 with the prominent form encoding the canonical 472 aa protein. The FAAH gene is located on human chromosome 1, 1p35-34, and is transcribed from the plus strand (5′ to 3′ orientation). The gene contains 15 exons with functional protein domains encoded across multiple exons. Recently, another FAAH gene, FAAH2, was identified on chromosome X in cytogenetic band Xp11.21 that is composed of a 532 amino acids protein that share about 20% sequence identity with the canonical FAAH1. There is evidence, though not all consistent, that genetic variants (red arrows) associated with addiction and related phenotypes such as reward sensitivity, impulsivity and negative affect are located within exon 4 (rs1049353), introns (rs2023239, rs1535255, rs806380) and the 3′UTR (AATn triplet repeat, rs806368) of CNR1^–. For the FAAH gene the polymorphic variant most associated with substance use disorders is rs324420 (exon 3)^, .