Brady K. Atwood

CB2: a cannabinoid receptor with an identity crisis

CB2 was first considered to be the ‘peripheral cannabinoid receptor’. This title was bestowed based on its abundant expression in the immune system and presumed absence from the central nervous system. However, multiple recent reports question the absence of CB2 from the central nervous system. For example, it is now well accepted that CB2 is expressed in brain microglia during neuroinflammation. However, the extent of CB2 expression in neurons has remained controversial. There have been studies claiming either extreme‐its complete absence to its widespread expression‐as well as everything in between. This review will discuss the reported tissue distribution of CB2 with a focus on CB2 in neurons, particularly those in the central nervous system as well as the implications of that presence. As CB2 is an attractive therapeutic target for pain management and immune system modulation without overt psychoactivity, defining the extent of its presence in neurons will have a significant impact on drug discovery. Our recommendation is to encourage cautious interpretation of data that have been presented for and against CB2s presence in neurons and to encourage continued rigorous study.

JWH018, a common constituent of ‘Spice’ herbal blends, is a potent and efficacious cannabinoid CB1 receptor agonist

Background and purpose:  ‘Spice’ is an herbal blend primarily marketed in Europe as a mild hallucinogen with prominent cannabis‐like effects and as a legal alternative to cannabis. However, a recent report identified a number of synthetic additives in samples of ‘Spice’. One of these, the indole derivative JWH018, is a ligand for the cannabinoid receptor 1 (CB1) cannabinoid receptor and inhibits cAMP production in CB1 receptor‐expressing CHO cells. Other effects of JWH018 on CB1 receptor‐mediated signalling are not known, particularly in neurons. Here we have evaluated the signalling pathways activated by JWH018 at CB1 receptors.

Functional Selectivity in CB2 Cannabinoid Receptor Signaling and Regulation: Implications for the Therapeutic Potential of CB2 Ligands

Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB1 and CB2 cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB2 to different extents. Because CB2 is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB1 and CB2 trafficking. Our most striking finding was that (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB2 receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB2 receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB2 internalization. Despite these differences in internalization, both compounds activated CB2 receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of β-arrestin2 to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB2 receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB2 ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB2 receptors and suggest that different classes of CB2 ligands may produce diverse physiological effects, emphasizing that each class needs to be separately evaluated for therapeutic efficacy.

Endocannabinoid Modulation of Orbitostriatal Circuits Gates Habit Formation

Everyday function demands efficient and flexible decision-making that allows for habitual and goal-directed action control. An inability to shift has been implicated in disorders with impaired decision-making, including obsessive-compulsive disorder and addiction. Despite this, our understanding of the specific molecular mechanisms and circuitry involved in shifting action control remains limited. Here we identify an endogenous molecular mechanism in a specific cortical-striatal pathway that mediates the transition between goal-directed and habitual action strategies. Deletion of cannabinoid type 1 (CB1) receptors from cortical projections originating in the orbital frontal cortex (OFC) prevents mice from shifting from goal-directed to habitual instrumental lever pressing. Activity of OFC neurons projecting to dorsal striatum (OFC-DS) and, specifically, activity of OFC-DS terminals is necessary for goal-directed action control. Lastly, CB1 deletion from OFC-DS neurons prevents the shift from goal-directed to habitual action control. These data suggest that the emergence of habits depends on endocannabinoid-mediated attenuation of a competing circuit controlling goal-directed behaviors.

Opioids induce dissociable forms of long-term depression of excitatory inputs to the dorsal striatum

As prescription opioid analgesic abuse rates rise, so does the need to understand the long-term effects of opioid exposure on brain function. The dorsal striatum is an important site for drug-induced neuronal plasticity. We found that exogenously applied and endogenously released opioids induced long-term depression (OP-LTD) of excitatory inputs to the dorsal striatum in mice and rats. Mu and delta OP-LTD, although both being presynaptically expressed, were dissociable in that they summated, differentially occluded endocannabinoid-LTD and inhibited different striatal inputs. Kappa OP-LTD showed a unique subregional expression in striatum. A single in vivo exposure to the opioid analgesic oxycodone disrupted mu OP-LTD and endocannabinoid-LTD, but not delta or kappa OP-LTD. These data reveal previously unknown opioid-mediated forms of long-term striatal plasticity that are differentially affected by opioid analgesic exposure and are likely important mediators of striatum-dependent learning and behavior.

CB2 cannabinoid receptors inhibit synaptic transmission when expressed in cultured autaptic neurons

The role of CB₂ in the central nervous system, particularly in neurons, has generated much controversy. Fueling the controversy are imperfect tools, which have made conclusive identification of CB₂ expressing neurons problematic. Imprecise localization of CB₂ has made it difficult to determine its function in neurons. Here we avoid the localization controversy and directly address the question if CB₂ can modulate neurotransmission. CB₂ was expressed in excitatory hippocampal autaptic neurons obtained from CB₁ null mice. Whole-cell patch clamp recordings were made from these neurons to determine the effects of CB₂ on short-term synaptic plasticity. CB₂ expression restored depolarization induced suppression of excitation to these neurons, which was lost following genetic ablation of CB₁. The endocannabinoid 2-arachidonylglycerol (2-AG) mimicked the effects of depolarization in CB₂ expressing neurons. Interestingly, ongoing basal production of 2-AG resulted in constitutive activation of CB₂, causing a tonic inhibition of neurotransmission that was relieved by the CB₂ antagonist AM630 or the diacylglycerol lipase inhibitor RHC80267. Through immunocytochemistry and analysis of spontaneous EPSCs, paired pulse ratios and coefficients of variation we determined that CB₂ exerts its function at a presynaptic site of action, likely through inhibition of voltage gated calcium channels. Therefore CB₂ expressed in neurons effectively mimics the actions of CB₁. Thus neuronal CB₂ is well suited to integrate into conventional neuronal endocannabinoid signaling processes, with its specific role determined by its unique and highly inducible expression profile.

Endocannabinoid Actions on Cortical Terminals Orchestrate Local Modulation of Dopamine Release in the Nucleus Accumbens

Dopamine (DA) transmission mediates numerous aspects of behavior. Although DA release is strongly linked to firing of DA neurons, recent developments indicate the importance of presynaptic modulation at striatal dopaminergic terminals. The endocannabinoid (eCB) system regulates DA release and is a canonical gatekeeper of goal-directed behavior. Here we report that extracellular DA increases induced by selective optogenetic activation of cholinergic neurons in the nucleus accumbens (NAc) are inhibited by CB1 agonists and eCBs. This modulation requires CB1 receptors on cortical glutamatergic afferents. Dopamine increases driven by optogenetic activation of prefrontal cortex (PFC) terminals in the NAc are similarly modulated by activation of these CB1 receptors. We further demonstrate that this same population of CB1 receptors modulates optical self-stimulation sustained by activation of PFC afferents in the NAc. These results establish local eCB actions on PFC terminals within the NAc that inhibit mesolimbic DA release and constrain reward-driven behavior.

Endocannabinoid-Dependent Synaptic Plasticity in the Striatum

The striatum plays a critical role in mediating the goal-directed and habitual behaviors involved in the development of drug abuse and addiction. Cannabinoid receptor-dependent synaptic plasticity is a prominent regulator of striatal circuit function. It is increasingly clear that striatal endocannabinoid signaling impacts the development of drug abuse. The expression of endocannabinoid-mediated plasticity in the striatum is affected by disparate types of drugs of abuse, such as opiates and alcohol. Understanding the mechanism of endocannabinoid-mediated plasticity and its effects on striatal circuit function may elucidate how endocannabinoids shape addiction-related behavior and perhaps offer new therapeutic avenues for treating drug abuse.