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Abstract
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 CB2's presence in neurons and to encourage continued rigorous study.
This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit EDITORIAL - Alexander - 2010 - British Journal of Pharmacology - Wiley Online Library
The therapeutic potential of cannabis as well as its psychoactive effects has been known for thousands of years (Adams and Martin, 1996). However, it was not until the discovery of cannabinoid binding sites in brain (Devane et al., 1988; Herkenham et al., 1990; Herkenham et al., 1991; Matsuda et al., 1993) and the subsequent cloning of the CB1 receptor (Matsuda et al., 1990; Alexander et al., 2008) that cellular mechanisms for these effects began to be elucidated. A second cannabinoid receptor (CB2) was identified and first cloned from HL60 cells by Munro et al. in 1993 (Munro et al., 1993). CB2 was dubbed the ‘peripheral cannabinoid receptor’ as a result of in situ hybridization analysis that revealed high levels of CB2 mRNA in spleen and its absence from brain. CB2 receptors were cloned from mouse and rat in later years (Shire et al., 1996; Griffin et al., 2000; Brown et al., 2002).
CB2 and cellular signalling
Around the time that CB1 and CB2 were cloned, anandamide and 2-arachidonylglycerol (2-AG) were identified as endogenous cannabinoid agonists (Devane et al., 1992; Felder et al., 1993; Sugiura et al., 1995; Hanus et al., 2001). 2-AG is a high efficacy agonist at CB2 (Lynn and Herkenham, 1994; Slipetz et al., 1995; Gonsiorek et al., 2000; Sugiura et al., 2000; Shoemaker et al., 2005b). However, anandamide has a low efficacy at CB2, often functioning as a weak partial agonist (Showalter et al., 1996; Gonsiorek et al., 2000; Sugiura et al., 2000). Similar to CB1, CB2 is a Gi/o coupled G protein coupled receptor and as such inhibits adenylyl cyclase (Bayewitch et al., 1995; Felder et al., 1995; Slipetz et al., 1995; Gonsiorek et al., 2000; Sugiura et al., 2000; Shoemaker et al., 2005b). Furthermore, it can also promote MAPK activation (p38 and p42/44), PI3K, ceramide production and gene transcription (Bouaboula et al., 1996; Bouaboula et al., 1999a; Bouaboula et al., 1999b; Howlett, 2002; Howlett et al., 2002; Herrera et al., 2005; Herrera et al., 2006; Grimaldi et al., 2009; Romero-Sandoval et al., 2009). A key difference, however, is that unlike CB1, CB2 appears to poorly modulate calcium channels or inwardly rectifying potassium channels (Felder et al., 1995). Studies using SR144528, a CB2 antagonist/inverse agonist, have revealed that the receptor possesses a high degree of constitutive activity in expression systems (Bouaboula et al., 1999a; Bouaboula et al., 1999b). Of further interest is that CB2 receptors from different species often have different pharmacological responses to identical drugs, complicating the drug discovery process (Mukherjee et al., 2004; Yao et al., 2006; Bingham et al., 2007). Therefore, despite coupling to the same family of G proteins and sharing some ligands, CB1 and CB2 appear to differ significantly from one another in their signalling.
CB2 as a therapeutic target
CB2 is an attractive therapeutic target. The abundant CB2 expression in immune cells presents a plausible explanation for cannabinoid immunomodulatory activity (Lynn and Herkenham, 1994; Berdyshev, 2000; Howlett, 2002; Costa, 2007). Indeed, CB2 activation affects a myriad of immune responses from inflammation to neuroprotection (Cabral and Griffin-Thomas, 2009). Additionally, numerous reports indicate that CB2 activation is analgesic and CB2 agonists suppress responses in many animal models of pain, from acute to neuropathic (Anand et al., 2009), although these effects may involve CB1 activation as well. CB1 is abundant within the brain, where it appears responsible for mediating the psychoactive effects of cannabis (Mackie, 2005). Thus, the scarcity of central nervous system (CNS) CB2 receptors makes CB2 selective drugs attractive as therapeutics as they would presumably lack psychoactivity. In support of this notion, mice with ‘knockout’ of CB2 had typical behavioural responses to Δ9-tetrahydrocannabinol (THC) but lost their normal immune responsiveness to THC (Buckley et al., 2000). CB2 levels can also be increased under certain conditions and disease states further adding to its attractiveness as a therapeutic target (Zhang et al., 2003; Wotherspoon et al., 2005; Yiangou et al., 2006).
CB2 agonists and antagonists
As CB2 is such an attractive therapeutic target, much effort has been made to synthesize selective CB2 agonists and antagonists. Some of the early cannabinoid agonists such as CP55940, WIN55212-2 and HU210 demonstrate high affinity at CB2 and are considered full agonists, but are not selective for CB2 over CB1[see Miller and Stella (2008) for a summary of the binding data]. JWH015 was one of the first potential CB2 selective agonists (Showalter et al., 1996; Griffin et al., 2000), but has since been shown to also be an agonist at GPR55 (Ryberg et al., 2007; Lauckner et al., 2008). Numerous other compounds have been synthesized with the aim of making CB2 selective agonists. AM1241 (Malan et al., 2001) and JWH133 (Huffman et al., 1999) are two of the most commonly used ‘selective’ CB2 agonists. Other early ones include HU308 (Hanus et al., 1999) and GW405833 (L-768242) (Gallant et al., 1996; Valenzano et al., 2005). More recently synthesized compounds include GW833972A (Belvisi et al., 2008), MDA7 (Naguib et al., 2008), A-796260 (Yao et al., 2008) and A-836339 (Yao et al., 2009). Cannabilactones have also been suggested as potential CB2 selective compounds (Khanolkar et al., 2007). For the interested reader, a review by Whiteside et al. contains detailed analysis of many of these compounds as well as numerous others (Whiteside et al., 2007). SR144528 (Rinaldi-Carmona et al., 1998) and AM630 (Pertwee et al., 1995; Ross et al., 1999) are the two of the most commonly used CB2 selective antagonists and have been frequently used to demonstrate specificity of many of these other CB2 selective agonists. However, a fundamental problem with designing selective agonists and antagonists is possible interactions with other unforeseen targets. These compounds may exhibit a strong preference for CB2 over CB1, but as evidenced by JWH015, other non-CB1/CB2 binding sites may still exist. It is extremely difficult to conclusively establish an agonist (or antagonist) is specific for CB2 and no other receptors. This caveat must be kept in mind when evaluating studies that solely use a pharmacological approach and allege CB2 involvement in a process. Furthermore, as demonstrated for AM1241, CB2 agonists may produce very different effects at CB2 receptors from different species (Bingham et al., 2007). Here, a racemic mixture of AM1241 was an agonist at human CB2 but functioned as an inverse agonist at rodent CB2. R-AM1241 has a higher affinity for CB2 than S-AM1241, but functions similar to the racemate. On the other hand S-AM1241 was an efficacious agonist at both human and rodent CB2. Furthermore naloxone, a µ opioid receptor antagonist, can block the analgesic effects of AM1241, but this appears not to be the case for other CB2 agonists (Ibrahim et al., 2005; Yao et al., 2009). It also important to bear in mind that selective agonists and antagonists for a particular receptor may differentially alter coupling to distinct signalling pathways, a concept known as functional selectivity (Urban et al., 2007). Thus, CB2 agonists that share identical binding characteristics may have different potencies in activating different signalling pathways and evoke substantially different physiological responses. For example, with CB2 expressed in Chinese hamster ovary cells, 2-AG, CP55940 and noladin ether had different rank orders of potency depending on the signalling pathway analysed: inhibition of adenylyl cyclase, MAPK activation and stimulation of calcium transients (Shoemaker et al., 2005a). This concept of functional selectivity mandates caution in comparing pharmacological studies that employ different agonists and antagonists for CB2. This may explain the differences previously mentioned between AM1241 and other CB2 agonists and their ability to produce analgesia and may even extend to other agonist-specific effects obtained with other compounds. Thus, it is important that functional evidence obtained using CB2‘selective’ agonists and antagonists be balanced with careful controls and supported by additional genetic and anatomical analyses to assure that some other unanticipated target is not the true site of action.
CB2: the ‘peripheral’ cannabinoid receptor?
CB2 and evidence for absence from CNS
In addition to the data obtained during the CB2 knockout mouse characterization, earlier reports also arrived at the conclusion that CB2 is absent from the CNS. (Of course, ‘absence’ just means below the level of detection of the particular assay being employed.) When CB2 was first cloned, in situ hybridization demonstrated a lack of CB2 mRNA signal in rat brain (Munro et al., 1993). While characterizing CB1 and CB2 in immune cells, Schatz et al. performed Northern blots on mouse brain and rat cerebellums and could not detect the presence of CB2 in these tissues (Schatz et al., 1997). However, RT-PCR demonstrated the presence of CB2 mRNA, at levels too low to be quantified. Northern blot analysis performed by Galiegue et al. is in agreement with the Schatz et al. data (Galiegue et al., 1995). However, in RT-PCR experiments performed as a part of this study, CB2 was undetectable in human cortex, cerebellum, whole brain and pituitary gland. McCoy et al. also did not detect CB2 mRNA in mouse brain using RT-PCR/Southern blot analysis (McCoy et al., 1999). As part of the characterization of SR144528 as a CB2 antagonist, rat brain radioligand binding and GTPγS binding analyses were performed (Griffin et al., 1999). The authors found little SR144528 binding in whole brain and cerebellum and the results of their GTPγS binding analysis supported this. Furthermore, Northern blotting did not detect CB2 mRNA in cerebellum, cortex or spinal cord. Rat cortex has also been reported to lack CB2 mRNA (Beltramo et al., 2006). Included in the review by Howlett et al. (Howlett et al. 2002), Herkenham and Hohmann replicated the in situ hybridization results of Munro and colleagues. Derbenev and colleagues did not detect CB2 mRNA or protein in rat brainstem (Derbenev et al., 2004). As part of an initial characterization of cannabinoid receptors in dorsal root ganglia (DRG), in situ hybridization revealed the presence of CB1 but not CB2 receptors (Hohmann and Herkenham, 1999a,b; ). Price et al. could also not detect CB2 mRNA in rat trigeminal ganglia (Price et al., 2003).
Based on all these data, CB2 was informally referred to as the peripheral cannabinoid receptor. However, a number of more recent reports have suggested that, in contrast to these previous claims of its absence, CB2 may in fact be expressed in the CNS (see below). This finding has had a significant impact on drug discovery and our understanding of the biology of the endocannabinoid system. This review will focus on reports of CB2 in neurons and in the brain and the implications of that presence.
CB2 and the immune system
CB2 research continues to have a large focus on its role in the immune system. Analysis of the presence and function of CB2 in the brain necessitates a discussion concerning CB2 in immune cells. CB2 mRNA has been identified in many immune tissues (Munro et al., 1993; Lynn and Herkenham, 1994; Galiegue et al., 1995; Schatz et al., 1997; Berdyshev, 2000; Buckley et al., 2000). Of specific immune cell types, the highest levels of CB2 are found in macrophages, CD4+ T cells, CD8+ T cells, B cells, natural killer cells, monocytes and polymorphonuclear neutrophils (Derocq et al., 1995; Galiegue et al., 1995; Schatz et al., 1997; Carayon et al., 1998; McCoy et al., 1999; Buckley et al., 2000; Carlisle et al., 2002; Maresz et al., 2007; Dittel, 2008). Of particular relevance for the role of CB2 in the CNS, CB2 mRNA and protein have been found in microglia (Carlisle et al., 2002; Klegeris et al., 2003; Walter et al., 2003; Beltramo et al., 2006; Maresz et al., 2007). Microglia are derived from macrophages and can be viewed as the resident immune cells of the brain where they monitor the brain for pathological damage. In response to specific signals within the brain they transition between different states of activity (Ashton and Glass, 2007; Hanisch and Kettenmann, 2007). The expression levels of CB2 in microglia vary depending on the activation state of the cell (Carlisle et al., 2002; Walter et al., 2003; Stella, 2004; Maresz et al., 2007; Cabral et al., 2008; Pietr et al., 2009). CB2 modulates microglial migration and infiltration into brain areas with active neuroinflammation and degeneration (Walter et al., 2003; Ashton et al., 2007; Fernandez-Ruiz et al., 2008; Miller and Stella, 2008; Price et al., 2009). In healthy brain microglia do not appear to express CB2 (Stella, 2004). However, in Alzheimer's brain tissue, CB2 can be detected in neuritic plaque-associated microglia (Benito et al., 2003). Similarly, in models of neuropathic pain (but not inflammatory pain) CB2 mRNA increases in association with activated microglia in the spinal cord (Zhang et al., 2003). In addition during amyotrophic lateral sclerosis and multiple sclerosis, CB2 microglial expression increases in the spinal cord (Yiangou et al., 2006). According to this evidence it is clear that under certain conditions brain microglia are capable of expressing CB2.
CB2 and tissue distribution
Despite being initially described as an immune cell cannabinoid receptor, CB2 has been identified molecularly and pharmacologically in numerous other cell types. Evidence for the presence of CB2 receptors has been found in pulmonary endothelial cells (Zoratti et al., 2003). In these cells, CB2 activation by anandamide results in phospholipase C-mediated calcium release from smooth ER with subsequent increases in mitochondrial calcium. CB2 can also be found in bone (in osteocytes, osteoblasts and osteoclasts) where it modulates bone formation and turnover (Ofek et al., 2006). The gastrointestinal system appears to express CB2 receptors as well (Storr et al., 2002; Hillsley et al., 2007; Duncan et al., 2008). 2-AG affects meiosis in spermatogonia via CB2 (Grimaldi et al., 2009) as well as a number of other aspects of reproductive function (Maccarrone, 2008; Grimaldi et al., 2009). Keratinocytes release beta-endorphin in response to CB2 selective agonist stimulation (Ibrahim et al., 2005), although this result is controversial (Whiteside et al., 2007; Anand et al., 2008; Yao et al., 2008; Yao et al., 2009). These cells have also been reported to have CB2 immunoreactivity. In the eye, trabecular meshwork cells have been shown to have functional CB2 receptors (Zhong et al., 2005; He and Song, 2007). Mature and precursor adipocytes express functional CB2 receptors that are negatively coupled to adenylyl cyclase (Roche et al., 2006). In cirrhotic liver, CB2 receptors are expressed in hepatic myofibroblasts, but are absent in normal liver (Julien et al., 2005). THC protects cardiomyocytes from hypoxic damage by acting at CB2 receptors resulting in nitric oxide production (Shmist et al., 2006).
CB2 and nociception
To better understand the possible presence of CB2 in neurons it is helpful to consider the role of CB2 in nociception. Cannabinoids have long been known to possess analgesic activity, but evidence for CB2 having a role in analgesia was not presented until 1998 (Calignano et al., 1998). Shortly thereafter, HU308, a CB2 selective agonist, was shown to have analgesic activity without typical cannabinoid CNS side effects (Hanus et al., 1999). AM1241, another CB2 selective agonist was also shown to promote analgesia when injected peripherally and this did not produce CNS side effects, suggesting that CB2 receptors modulate nociception (Malan et al., 2001; Ibrahim et al., 2003; Malan et al., 2003; Ibrahim et al., 2006). It has since been shown that a number of different CB2 agonists can modulate many types of pain: acute, inflammatory, neuropathic, post-surgical and cancer pain (Khanolkar et al., 2007; Whiteside et al., 2007; Jhaveri et al., 2008; Naguib et al., 2008; Ohta et al., 2008; Yao et al., 2008; Anand et al., 2009; Yao et al., 2009). It is still unclear as to where these CB2 agonists exert their analgesic activity. The site could be microglia, astrocytes, neurons, another cell type or a combination of these. Furthermore, as discussed above, the specificity of these CB2‘selective’ compounds may not be as specific as previously thought. Additional work must be performed to state with confidence that these agonists produce analgesia solely via activation of CB2 receptors.
CB2 and peripheral neurons
The first step to determine if CB2 activation has a direct effect on neural mechanisms is to determine whether CB2 is expressed in neurons. The existence of functional CB2 receptors in peripheral neurons has been suggested by a number of studies. The first evidence for CB2 function in peripheral neurons came in 1997 when CB2 mRNA was identified in mouse vas deferens tissue (Griffin et al., 1997). In support of a functional role for CB2, JWH015 and JWH051 (agonists preferring CB2 over CB1) produced concentration dependent inhibition of evoked contractions presumably via a prejunctional site. However, a submicromolar concentration of AM630, a CB2 antagonist, could not block this effect. Further, JWH015 is also an agonist for GPR55 (Ryberg et al., 2007; Lauckner et al., 2008), so the involvement of CB2 cannot be unequivocally asserted.
CB2 and sensory neurons
Functional studies have hinted at the presence of CB2 receptors on sensory neurons. In these studies, it is necessary to consider the possible involvement of CB2-expressing immune cells as microglia can affect synaptic properties (Cullheim and Thams, 2007; Abbadie et al., 2009). Patel and colleagues provided some of the first functional evidence of CB2 in sensory neurons (Patel et al., 2003). Using isolated guinea pig and human vagus nerve preparations, they demonstrated that the CB2 agonist JWH133 inhibited nerve depolarizations in response to capsaicin, PGE2 and hypertonic saline. These three treatments activate vagal C and/or Aδ fibres. SR144528 blocked the effects of JWH133. A follow-up study with another putative CB2 agonist, GW833972A, produced similar results (Belvisi et al., 2008). Neither study was designed to determine a specific site or mechanism of action. While CB2 does not appear to play a role in myenteric contractions, it does seem to play a role in activation of mesenteric sensory nerves. AM1241 administered intravenously inhibits bradykinin induced activation of isolated mesenteric afferents in mice (Hillsley et al., 2007). This effect was blocked by AM630 and absent in CB2 knockout mice. Interestingly, while CB2 agonists do not affect normal enteric contractility, JWH133 can prevent lipopolysaccharide (LPS) induced increases in evoked contractions (Mathison et al., 2004; Duncan et al., 2008). JWH133 also blocks LPS stimulation of Fos expression in enteric neurons. AM630 antagonizes these effects. CB2 receptors in myenteric neurons were identified as the most likely target of this drug effect (see below). CB2 mRNA has also been identified in rat and mouse retina using RT-PCR as well as within specific layers of the retina (ganglion, inner nuclear and photoreceptor inner layers) using in situ hybridization (Lu et al., 2000). Additionally, Burdyga et al. identified low, barely detectable levels of CB2 mRNA in rat nodose ganglion, but were unable to detect CB2 in the human vagal nerve trunk (Burdyga et al., 2004).
CB2 and nociceptive neurons
Further functional studies point to a role for CB2 in sensory neuron function, particularly nociceptive neurons. A study was performed to address AM1241's ability to prevent windup of wide dynamic range (WDR) neurons in spinal cord (Nackley et al., 2004). Here, AM1241 administered locally or systemically reduced the activity of C-fibres synapsing onto WDR neurons and this was reversed by SR144528, but not SR141716A. Significantly, suppression occurred in the presence and absence of inflammation. This, combined with the time course observed suggests long-term changes in presynaptic facilitation, makes the effects less likely to be due to CB2 targeting immune cells. The authors speculate a direct effect of CB2 activation on C-fibre neurons. Elmes et al. performed a similar study using JWH133 as a CB2 agonist to test WDR spinal neuron responses in models of inflammatory and neuropathic pain as well as in non-inflammatory and sham-operated conditions (Elmes et al., 2004). Like the Nackley study, they also found that peripherally administered CB2 agonist inhibits WDR activity in both naïve and inflammatory conditions as well as following neuropathic injury. Once again, the data are suggestive of a non-immune function of CB2, possibly in peripheral neurons. A follow-up study analysed JWH133's ability to inhibit capsaicin-induced calcium increases in DRG neurons cultured from sham-operated and neuropathic rats (Sagar et al., 2005). JWH133 slightly inhibited calcium increases in DRG cultured from both neuropathic and sham rats in a SR144528-sensitive fashion, consistent with the presence of functional CB2 receptors in peripheral neurons. However, spinally administered JWH133 inhibited mechanically evoked responses of dorsal horn neurons from laminae V and VI only in neuropathic rats, but not in sham-operated animals. This points to an up-regulation of CB2 in intrinsic spinal cord neurons in pain states, although does not provide evidence of the site of up-regulation. AM1241 and L768242 (another CB2 agonist) can also decrease capsaicin-induced calcitonin gene-related peptide release, a pain biomarker, from neurons in spinal cord slices and this can be blocked by SR144528 (Beltramo et al., 2006). These studies are most consistent with CB2 participating in neural mechanisms rather than via immune cells, but do not directly answer the question of whether or not CB2 is expressed in neurons.
The initial support for the presence of CB2 protein in neurons came from Ross and colleagues. Using fluorescence-activated cell sorting analysis, they determined that DRG cultures and F-11 cells (DRG neuron × neuroblastoma hybrid) express both CB1 and CB2, but could not conclude that CB2 was functionally expressed in DRG neurons (Ross et al., 2001). To further address the site of CB2 expression in DRG, Wotherspoon and colleagues used immunhistochemistry on DRG and spinal cord of naïve and nerve damaged rats and mice (Wotherspoon et al., 2005). No CB2 could be detected in normal rat or mouse spinal cord and DRG neurons. However, upon nerve sectioning or ligation, CB2 immunoreactivity could be detected in the ipsilateral dorsal horn. This immunoreactivity was strongly reduced in CB2 knockout mice and was blocked by incubation with the immunizing peptide, suggesting specificity of the primary antibody used. Of great interest, and in contrast to what would be expected based on Zhang et al.'s (2003) study, was that the CB2 signal did not co-localize with markers of astrocytes (GFAP) or microglia (OX-42). Instead it co-localized with markers of damaged sensory neuron terminals (GAP-43 and galanin). CB2 immunoreactivity also accumulated in axons proximal to the ligation site. They could not identify CB2 in cell bodies in tissue sections, but were able to identify CB2 in isolated DRG neurons grown in culture from lesioned mice. They also did not observe CB2 immunoreactivity in skin, in contrast to other studies (Stander et al., 2005; Kress and Kuner, 2009) and Ibrahim et al. who found it in keratinocytes (Ibrahim et al., 2005). A few studies have detected CB2 mRNA in DRG and spinal cord using quantitative RT-PCR. Here levels increased following nerve ligation, but this does not necessarily implicate a neuronal source (Zhang et al., 2003; Beltramo et al., 2006).
A more recent study by Anand and colleagues is consistent with the above findings (Anand et al., 2008). Specifically, they found CB2 positive, small diameter neurons in human DRG and peripheral nerves using three different CB2 antibodies. The specificity of the antibodies was assessed using peptide block. CB2 levels increased following nerve injury. They further extended the analysis by demonstrating CB2 colocalization with neuronal (GAP-43), axonal (neurofilament) and nociceptive neuronal markers (TRPV1). Similar staining was observed in mouse, rat and guinea pig DRG. This study also replicated the functional data reported by Sagar et al. in that a CB2 agonist (GW833972) inhibited capsaicin-induced calcium increases in DRG sensory neurons. They further sought to identify a mechanism for this activity and determined that CB2-mediated cAMP depletion attenuated TRPV1 activation. This presumably decreased PKA-mediated phosphorylation of TRPV1, analogous to the effects of µ opioid receptor activation.
CB2 and the enteric nervous system
Despite earlier findings (Griffin et al., 1997), several studies suggest CB2 is expressed in the enteric nervous system. Duncan et al. and Storr et al. found CB2 mRNA in the enteric nervous system (Storr et al., 2002; Duncan et al., 2008). The site of action of JWH133 in preventing LPS-induced increases in ileum contractility was addressed using RT-PCR and immunohistochemistry (Duncan et al., 2008). CB2 mRNA was detected in the full-wall thickness ileum, ileal muscle, submucosal and mucosal layers in normal rats. LPS treatment had no effect on the levels of expression. A number of different antibodies and knockout tissue were used for controls. CB2 protein was detected in all the same tissues in which CB2 mRNA was found. CB2 colocalized with markers of enteric ganglia, pan-neuronal markers and synaptic terminals suggesting a strong presence in myenteric neurons. CB2 immunoreactivity did not colocalize with glial markers.
CB2: another central cannabinoid receptor?
CB2 in the cerebellum
One of the earliest reports of the presence of CB2 in the CNS came from a study performed by Skaper and colleagues (Skaper et al., 1996). In situ hybridization revealed the presence of CB2 mRNA in cultured granule cells. In addition in situ hybridization localized CB2 to the granule and Purkinje cell layers of mouse cerebellum. Radioligand binding analysis of cerebellar membranes revealed the presence of two WIN55212 binding sites. The affinities of WIN55212 at these sites were reported to be close to those of CB1 and CB2, although the exact identity of the binding sites could not be specifically determined. RT-PCR analysis has identified CB2 mRNA in the rat cerebellum and Western blotting has revealed expressed CB2 protein in rat and ferret cerebellum as well (Van Sickle et al., 2005). Peptide block was used as a control for those Western blots.
Additional studies have also attempted to localize CB2 protein in the cerebellum (Ashton et al., 2006; Baek et al., 2008). Using an antibody directed against the C-terminus of CB2, with peptide block control, they identified CB2 protein expression in the granule, Purkinje and white matter layers of the rat cerebellum. The signal did not overlap with astrocytes markers and the staining pattern in the Purkinje layer and parts of the other layers appeared to be capillary endothelial in nature. There were fine fibres in the white matter and granule cell layers that were CB2 positive but their origin remains to be determined. These could possibly arise from microglia or neurons. Onaivi et al. have also reported CB2 expression in the Purkinje and molecular layers of the cerebellum using Western blot, immunohistochemistry and in situ hybridization techniques (Gong et al., 2006; Onaivi et al., 2008b).
CB2 in the brainstem
CB2 has been identified within the brainstem as well. A thorough analysis was performed that investigated CB2 expression in brain, focusing on mRNA, protein and functional expression within the brainstem (Van Sickle et al., 2005). Quantitative RT-PCR showed that the rat brainstem contains CB2 mRNA at significantly lower levels than spleen (1.5% of spleen levels). Western blotting confirmed this expression for rat as well as for ferret. Immunocytochemistry identified the dorsal motor nucleus of the vagus nerve (DMNX) of the mouse, rat and ferret as a brainstem nucleus containing CB2 protein. The CB2 knockout mouse did not show any immunostaining in the DMNX. The DMNX immunoreactivity colocalized with neuronal markers, but in contrast to what Ashton et al. found in the cerebellum (Ashton et al., 2006), the signal did not overlap with glial or blood vessel markers. The authors acknowledged the differences in results between their study and that of Derbenev et al. that did not find CB2 in similar regions (Derbenev et al., 2004) and state that in the latter study a faint signal could be observed in Western blots consistent with low levels of expression. The authors also demonstrated that AM630 blocked the anti-emetic actions of 2-AG treatment in ferrets, suggesting CB2 receptor involvement. Furthermore a sub-efficacious concentration of anandamide combined with AM1241 treatment produced anti-emetic effects. Another, more superficial study of the brainstem using immunohistochemistry was later performed to look for CB2 in other brainstem nuclei (Baek et al., 2008). CB2 immunoreactivity was found in the medial vestibular nucleus as well as the dorsal and ventral cochlear nuclei, but no attempts were made to identify cell types. Peptide block and secondary antibody controls were used to determine CB2 antibody specificity. Viscomi et al. did not find CB2 protein and only low levels of CB2 mRNA in inferior olive and pontine nuclei using immunohistochemistry and quantitative PCR in normal rats (Viscomi et al., 2009). However, following a hemicerebellectomy, CB2 expression dramatically increased in both mRNA and protein levels in these nuclei. The CB2 immunoreactivity colocalized with neuronal markers but not with microglial or astrocytic ones. Further JWH015 had a neuroprotective effect, preventing cell death due to the hemicerebellectomy. This was likely operating through CB2, although they did not report block of the neuroprotective effect with a CB2 antagonist. Gong et al. reported the presence of CB2 in many nuclei of the brainstem using RT-PCR and immunohistochemistry (Gong et al., 2006).
CB2 and the hippocampal formation
Using several approaches, Onaivi and his collaborators have reported finding CB2 immunoreactivity in many areas of the hippocampal formation (Gong et al., 2006; Onaivi, 2006; Onaivi et al., 2006; 2008a,b; Brusco et al., 2008). They report a predominately postsynaptic expression and an association with rough endoplasmic reticulum and Golgi structures. They have also demonstrated CB2 staining in hippocampal cultures. In contrast to their immunohistochemical results, they have had mixed results in finding CB2 mRNA in the hippocampus (Gong et al., 2006; Onaivi et al., 2008b).
Functional evidence for CB2 expression in the cortex comes from recording spontaneous inhibitory postsynaptic currents (sIPSCs) in layers II and V of the medial entorhinal cortex. Here, 2-AG mediated suppression of sIPSCs was not blocked by LY320135, a CB1 antagonist/inverse agonist, whereas they were blocked by AM630 and JTE907 (a structurally distinct CB2 antagonist) (Morgan et al., 2009). Further JWH133 suppressed sIPSCs in a CB2 antagonist sensitive fashion. The site of CB2 agonist action remains to be conclusively demonstrated.
CB2 and other brain regions
Evidence exists for CB2 expression in other brain regions. While recording from the ventral posterior nucleus of the thalamus, Jhaveri et al. found that after spinal nerve ligation, JWH133 reduced spontaneous and evoked responses in a SR144528-sensitive fashion, but that this effect was absent in sham operated rats (Jhaveri et al., 2008). Gong et al. have also reported CB2 immunoreactivity in many thalamic nuclei, but could not detect CB2 mRNA using RT-PCR (Gong et al., 2006). Furthermore, this group has reported finding CB2 mRNA in striatum and hypothalamus, but not in olfactory bulb, cortex and spinal cord and mixed results in midbrain (Gong et al., 2006; Onaivi et al., 2008b). Additionally they report CB2 immunoreactivity in olfactory bulb, cortex, midbrain as well as the other areas already mentioned (Gong et al., 2006; Onaivi, 2006; Onaivi et al., 2006; 2008b; ).
CB2 and neurogenesis
CB2 also appears to play a role in neurogenesis. Both CB1 and CB2 are expressed in stem cells (Jiang et al., 2007; Molina-Holgado et al., 2007). More specifically, RT-PCR, Western blot and immunohistochemical analyses have all revealed the presence of CB2 in embryonic and adult neural progenitor cells (Palazuelos et al., 2006; Molina-Holgado et al., 2007). CB2 blockade or genetic disruption impairs neurosphere formation and prevents progenitor cell proliferation, whereas CB2 agonists promote these activities via ERK and Akt signalling (Palazuelos et al., 2006; Molina-Holgado et al., 2007). However, CB2 expression seems to diminish as the cells differentiate, being nearly absent by the time neuronal and astrocytic markers appear (Palazuelos et al., 2006). Further, CB1 agonists and antagonists have similar effects in neurosphere formation and in COR-1 neural stem cell cultures (Molina-Holgado et al., 2007; Goncalves et al., 2008) suggesting either functional interactions or redundant signalling. In contrast to these data, CB1 agonists and antagonists had no effect on neurogenesis in the subventricular zone (SVZ) of either young or adult mice (Goncalves et al., 2008). On the other hand, JWH133 and WIN55212 stimulated SVZ neurogenesis, whereas AM630 and JTE907 decreased it (Goncalves et al., 2008).
CB2: where is its real home and why do we care?
We feel careful analysis of the studies reviewed above allows us to reach the following conclusions: CB2 is expressed by microglia, with levels increasing as they are activated, and CB2 is present at detectable and functionally relevant levels in a subset of neurons, with increasing levels following injury. We care where CB2 is expressed primarily for understanding pathology that involves CB2 and to develop therapies that target difficult to treat conditions. To this end it is important to have a rigorous understanding of where and under what conditions CB2 is expressed in the CNS.
Approaches aimed at identifying CB2 receptor expression in the brain can be divided into functional (pharmacological), biochemical and anatomical techniques. All three have their strengths and weaknesses. The most convincing studies will incorporate a combination of these techniques. Table 1 summarizes the studies presented here, detailing the brain region analysed and whether or not CB2 was detected and the techniques(s) used to detect it. Pharmacological studies rely on the specificity of the drugs used. When interpreting these studies it is necessary to recall that specificity is never absolute – at sufficiently high concentrations any drug will interact with additional targets. Thus, it is important to relate the concentration of the drug being used to the binding affinity of the CB2 receptor for that drug. The second consideration is that drugs considered to be ‘specific’ or ‘selective’ based on our current understanding may soon be found to interact with other receptors. Examples of this in the cannabinoid system include AM251, often used as a ‘selective’ CB1 receptor antagonist, but it is also a GPR55 agonist (Henstridge et al., 2009; Kapur et al., 2009) and JWH015, sometimes used as a ‘selective’ CB2 agonist, but it, too, is a GPR55 agonist (Ryberg et al., 2007; Lauckner et al., 2008). Approaches to circumvent this issue include using several structurally diverse agonists and antagonists (presumably decreasing the likelihood of having the same ‘off-target’ actions) and knockout or ‘knockdown’ controls, when appropriate.
Biochemical studies include Western blotting and PCR-based approaches. For Western blotting, the key limitations are the sensitivity and specificity of the antibody used. At a minimum, blots from knockout (assuming the antibody is recognizing an epitope present in mouse CB2) and positive control tissues (e.g. spleen) should be shown. Blindly trusting an antibody to ‘work’ without concurrent controls is unacceptable. Block with the immunizing antibody is desirable, but will not rule out a fortuitous interaction of the antibody with an unintended epitope on another protein. The sensitivity of Western blotting will depend on the abundance of CB2 as well as the affinity of the antibody. The lack of detection of CB2 on the blot can only be interpreted as that the level of CB2 in the brain is below a certain level. (This level, relative to a CB2-expressing tissue like spleen, can be determined by serial dilution.) PCR-approached tissues are the most sensitive. Their high sensitivity makes their interpretation subject to several considerations (Suzuki et al., 2000; Lion, 2001). These include amplification of CB2 mRNA from immune cells trapped in the cerebral vasculature and amplification of CB2 mRNA from a very small subset of activated microglia. In order to rationally interpret results from PCR-based experiments it is necessary that they be performed in a quantitative fashion, preferably calculating copy number, to facilitate comparisons.
Anatomical studies need to be conducted and interpreted with a similarly critical approach. These studies fall into three categories: autoradiography, in situ hybridization and immunocytochemistry. As above the issue of sensitivity needs consideration – it is possible to show CB2 is present, but it is very hard to conclusively demonstrate that it is not present, just that it is present at a level below the limit of detection. However, this information, coupled with a lack of functional response, can be very valuable in sorting out the role of CB2 receptors in a particular physiological response. The caveats of autoradiography include the pharmacological considerations discussed above as well as specific technical issues (Frey and Albin, 2001). As this technique has not been widely applied to directly identifying CB2 receptors in the brain, it will not be further discussed here. In situ hybridization studies can yield useful information on which cells express CB2 and thus can complement PCR-based studies. However, the lower sensitivity of in situ hybridization may make this difficult. A necessary control for in situ hybridization includes lack of hybridization in knockout tissues (when possible).
Immunocytochemistry studies have the powerful potential to identify the precise localization of CB2. However, for meaningful information to be drawn from them it is essential that proper controls are followed [as reviewed by Bussolati and Leonardo (2008); Lorincz and Nusser (2008); Saper and Sawchenko (2003)]. Briefly, some of the controls are: adsorption with the immunizing peptide, parallel, blinded staining of wild-type and knockout tissue, the use of two or more antibodies raised against distinct epitopes, antibody titration, omitting the primary antibody from the staining procedure and supporting these findings with RT-PCR, in situ hybridization and other such detection methods. Using just one control for one experimental setup is usually insufficient proof of specificity. These basic controls must be remembered when interpreting the data presented from any study cited in this review and future studies as well.
In conclusion, despite originally being thought of as the ‘peripheral’ cannabinoid receptor, considerable functional and anatomical evidence suggests that CB2 is expressed in the nervous system – certainly in activated microglia and very likely in some neurons. In addition, this raises the point that any report that identifies CB2 in neurons of the nervous system must incorporate careful controls to ensure that the CB2 signal found originates from neurons and not from microglia or immune cells associated with brain blood vessels. Given the importance of determining the functional role of CB2 in the CNS, under what conditions it is up regulated, and the potential therapeutic applications of CB2 agonists it is vital to understand where in the CNS CB2 receptors are expressed. We encourage those working in the field and those reviewing manuscripts to conduct and review these studies in a careful, thoughtful and rigorous fashion.
Source, Graphs and Figures: 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 CB2's presence in neurons and to encourage continued rigorous study.
This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit EDITORIAL - Alexander - 2010 - British Journal of Pharmacology - Wiley Online Library
The therapeutic potential of cannabis as well as its psychoactive effects has been known for thousands of years (Adams and Martin, 1996). However, it was not until the discovery of cannabinoid binding sites in brain (Devane et al., 1988; Herkenham et al., 1990; Herkenham et al., 1991; Matsuda et al., 1993) and the subsequent cloning of the CB1 receptor (Matsuda et al., 1990; Alexander et al., 2008) that cellular mechanisms for these effects began to be elucidated. A second cannabinoid receptor (CB2) was identified and first cloned from HL60 cells by Munro et al. in 1993 (Munro et al., 1993). CB2 was dubbed the ‘peripheral cannabinoid receptor’ as a result of in situ hybridization analysis that revealed high levels of CB2 mRNA in spleen and its absence from brain. CB2 receptors were cloned from mouse and rat in later years (Shire et al., 1996; Griffin et al., 2000; Brown et al., 2002).
CB2 and cellular signalling
Around the time that CB1 and CB2 were cloned, anandamide and 2-arachidonylglycerol (2-AG) were identified as endogenous cannabinoid agonists (Devane et al., 1992; Felder et al., 1993; Sugiura et al., 1995; Hanus et al., 2001). 2-AG is a high efficacy agonist at CB2 (Lynn and Herkenham, 1994; Slipetz et al., 1995; Gonsiorek et al., 2000; Sugiura et al., 2000; Shoemaker et al., 2005b). However, anandamide has a low efficacy at CB2, often functioning as a weak partial agonist (Showalter et al., 1996; Gonsiorek et al., 2000; Sugiura et al., 2000). Similar to CB1, CB2 is a Gi/o coupled G protein coupled receptor and as such inhibits adenylyl cyclase (Bayewitch et al., 1995; Felder et al., 1995; Slipetz et al., 1995; Gonsiorek et al., 2000; Sugiura et al., 2000; Shoemaker et al., 2005b). Furthermore, it can also promote MAPK activation (p38 and p42/44), PI3K, ceramide production and gene transcription (Bouaboula et al., 1996; Bouaboula et al., 1999a; Bouaboula et al., 1999b; Howlett, 2002; Howlett et al., 2002; Herrera et al., 2005; Herrera et al., 2006; Grimaldi et al., 2009; Romero-Sandoval et al., 2009). A key difference, however, is that unlike CB1, CB2 appears to poorly modulate calcium channels or inwardly rectifying potassium channels (Felder et al., 1995). Studies using SR144528, a CB2 antagonist/inverse agonist, have revealed that the receptor possesses a high degree of constitutive activity in expression systems (Bouaboula et al., 1999a; Bouaboula et al., 1999b). Of further interest is that CB2 receptors from different species often have different pharmacological responses to identical drugs, complicating the drug discovery process (Mukherjee et al., 2004; Yao et al., 2006; Bingham et al., 2007). Therefore, despite coupling to the same family of G proteins and sharing some ligands, CB1 and CB2 appear to differ significantly from one another in their signalling.
CB2 as a therapeutic target
CB2 is an attractive therapeutic target. The abundant CB2 expression in immune cells presents a plausible explanation for cannabinoid immunomodulatory activity (Lynn and Herkenham, 1994; Berdyshev, 2000; Howlett, 2002; Costa, 2007). Indeed, CB2 activation affects a myriad of immune responses from inflammation to neuroprotection (Cabral and Griffin-Thomas, 2009). Additionally, numerous reports indicate that CB2 activation is analgesic and CB2 agonists suppress responses in many animal models of pain, from acute to neuropathic (Anand et al., 2009), although these effects may involve CB1 activation as well. CB1 is abundant within the brain, where it appears responsible for mediating the psychoactive effects of cannabis (Mackie, 2005). Thus, the scarcity of central nervous system (CNS) CB2 receptors makes CB2 selective drugs attractive as therapeutics as they would presumably lack psychoactivity. In support of this notion, mice with ‘knockout’ of CB2 had typical behavioural responses to Δ9-tetrahydrocannabinol (THC) but lost their normal immune responsiveness to THC (Buckley et al., 2000). CB2 levels can also be increased under certain conditions and disease states further adding to its attractiveness as a therapeutic target (Zhang et al., 2003; Wotherspoon et al., 2005; Yiangou et al., 2006).
CB2 agonists and antagonists
As CB2 is such an attractive therapeutic target, much effort has been made to synthesize selective CB2 agonists and antagonists. Some of the early cannabinoid agonists such as CP55940, WIN55212-2 and HU210 demonstrate high affinity at CB2 and are considered full agonists, but are not selective for CB2 over CB1[see Miller and Stella (2008) for a summary of the binding data]. JWH015 was one of the first potential CB2 selective agonists (Showalter et al., 1996; Griffin et al., 2000), but has since been shown to also be an agonist at GPR55 (Ryberg et al., 2007; Lauckner et al., 2008). Numerous other compounds have been synthesized with the aim of making CB2 selective agonists. AM1241 (Malan et al., 2001) and JWH133 (Huffman et al., 1999) are two of the most commonly used ‘selective’ CB2 agonists. Other early ones include HU308 (Hanus et al., 1999) and GW405833 (L-768242) (Gallant et al., 1996; Valenzano et al., 2005). More recently synthesized compounds include GW833972A (Belvisi et al., 2008), MDA7 (Naguib et al., 2008), A-796260 (Yao et al., 2008) and A-836339 (Yao et al., 2009). Cannabilactones have also been suggested as potential CB2 selective compounds (Khanolkar et al., 2007). For the interested reader, a review by Whiteside et al. contains detailed analysis of many of these compounds as well as numerous others (Whiteside et al., 2007). SR144528 (Rinaldi-Carmona et al., 1998) and AM630 (Pertwee et al., 1995; Ross et al., 1999) are the two of the most commonly used CB2 selective antagonists and have been frequently used to demonstrate specificity of many of these other CB2 selective agonists. However, a fundamental problem with designing selective agonists and antagonists is possible interactions with other unforeseen targets. These compounds may exhibit a strong preference for CB2 over CB1, but as evidenced by JWH015, other non-CB1/CB2 binding sites may still exist. It is extremely difficult to conclusively establish an agonist (or antagonist) is specific for CB2 and no other receptors. This caveat must be kept in mind when evaluating studies that solely use a pharmacological approach and allege CB2 involvement in a process. Furthermore, as demonstrated for AM1241, CB2 agonists may produce very different effects at CB2 receptors from different species (Bingham et al., 2007). Here, a racemic mixture of AM1241 was an agonist at human CB2 but functioned as an inverse agonist at rodent CB2. R-AM1241 has a higher affinity for CB2 than S-AM1241, but functions similar to the racemate. On the other hand S-AM1241 was an efficacious agonist at both human and rodent CB2. Furthermore naloxone, a µ opioid receptor antagonist, can block the analgesic effects of AM1241, but this appears not to be the case for other CB2 agonists (Ibrahim et al., 2005; Yao et al., 2009). It also important to bear in mind that selective agonists and antagonists for a particular receptor may differentially alter coupling to distinct signalling pathways, a concept known as functional selectivity (Urban et al., 2007). Thus, CB2 agonists that share identical binding characteristics may have different potencies in activating different signalling pathways and evoke substantially different physiological responses. For example, with CB2 expressed in Chinese hamster ovary cells, 2-AG, CP55940 and noladin ether had different rank orders of potency depending on the signalling pathway analysed: inhibition of adenylyl cyclase, MAPK activation and stimulation of calcium transients (Shoemaker et al., 2005a). This concept of functional selectivity mandates caution in comparing pharmacological studies that employ different agonists and antagonists for CB2. This may explain the differences previously mentioned between AM1241 and other CB2 agonists and their ability to produce analgesia and may even extend to other agonist-specific effects obtained with other compounds. Thus, it is important that functional evidence obtained using CB2‘selective’ agonists and antagonists be balanced with careful controls and supported by additional genetic and anatomical analyses to assure that some other unanticipated target is not the true site of action.
CB2: the ‘peripheral’ cannabinoid receptor?
CB2 and evidence for absence from CNS
In addition to the data obtained during the CB2 knockout mouse characterization, earlier reports also arrived at the conclusion that CB2 is absent from the CNS. (Of course, ‘absence’ just means below the level of detection of the particular assay being employed.) When CB2 was first cloned, in situ hybridization demonstrated a lack of CB2 mRNA signal in rat brain (Munro et al., 1993). While characterizing CB1 and CB2 in immune cells, Schatz et al. performed Northern blots on mouse brain and rat cerebellums and could not detect the presence of CB2 in these tissues (Schatz et al., 1997). However, RT-PCR demonstrated the presence of CB2 mRNA, at levels too low to be quantified. Northern blot analysis performed by Galiegue et al. is in agreement with the Schatz et al. data (Galiegue et al., 1995). However, in RT-PCR experiments performed as a part of this study, CB2 was undetectable in human cortex, cerebellum, whole brain and pituitary gland. McCoy et al. also did not detect CB2 mRNA in mouse brain using RT-PCR/Southern blot analysis (McCoy et al., 1999). As part of the characterization of SR144528 as a CB2 antagonist, rat brain radioligand binding and GTPγS binding analyses were performed (Griffin et al., 1999). The authors found little SR144528 binding in whole brain and cerebellum and the results of their GTPγS binding analysis supported this. Furthermore, Northern blotting did not detect CB2 mRNA in cerebellum, cortex or spinal cord. Rat cortex has also been reported to lack CB2 mRNA (Beltramo et al., 2006). Included in the review by Howlett et al. (Howlett et al. 2002), Herkenham and Hohmann replicated the in situ hybridization results of Munro and colleagues. Derbenev and colleagues did not detect CB2 mRNA or protein in rat brainstem (Derbenev et al., 2004). As part of an initial characterization of cannabinoid receptors in dorsal root ganglia (DRG), in situ hybridization revealed the presence of CB1 but not CB2 receptors (Hohmann and Herkenham, 1999a,b; ). Price et al. could also not detect CB2 mRNA in rat trigeminal ganglia (Price et al., 2003).
Based on all these data, CB2 was informally referred to as the peripheral cannabinoid receptor. However, a number of more recent reports have suggested that, in contrast to these previous claims of its absence, CB2 may in fact be expressed in the CNS (see below). This finding has had a significant impact on drug discovery and our understanding of the biology of the endocannabinoid system. This review will focus on reports of CB2 in neurons and in the brain and the implications of that presence.
CB2 and the immune system
CB2 research continues to have a large focus on its role in the immune system. Analysis of the presence and function of CB2 in the brain necessitates a discussion concerning CB2 in immune cells. CB2 mRNA has been identified in many immune tissues (Munro et al., 1993; Lynn and Herkenham, 1994; Galiegue et al., 1995; Schatz et al., 1997; Berdyshev, 2000; Buckley et al., 2000). Of specific immune cell types, the highest levels of CB2 are found in macrophages, CD4+ T cells, CD8+ T cells, B cells, natural killer cells, monocytes and polymorphonuclear neutrophils (Derocq et al., 1995; Galiegue et al., 1995; Schatz et al., 1997; Carayon et al., 1998; McCoy et al., 1999; Buckley et al., 2000; Carlisle et al., 2002; Maresz et al., 2007; Dittel, 2008). Of particular relevance for the role of CB2 in the CNS, CB2 mRNA and protein have been found in microglia (Carlisle et al., 2002; Klegeris et al., 2003; Walter et al., 2003; Beltramo et al., 2006; Maresz et al., 2007). Microglia are derived from macrophages and can be viewed as the resident immune cells of the brain where they monitor the brain for pathological damage. In response to specific signals within the brain they transition between different states of activity (Ashton and Glass, 2007; Hanisch and Kettenmann, 2007). The expression levels of CB2 in microglia vary depending on the activation state of the cell (Carlisle et al., 2002; Walter et al., 2003; Stella, 2004; Maresz et al., 2007; Cabral et al., 2008; Pietr et al., 2009). CB2 modulates microglial migration and infiltration into brain areas with active neuroinflammation and degeneration (Walter et al., 2003; Ashton et al., 2007; Fernandez-Ruiz et al., 2008; Miller and Stella, 2008; Price et al., 2009). In healthy brain microglia do not appear to express CB2 (Stella, 2004). However, in Alzheimer's brain tissue, CB2 can be detected in neuritic plaque-associated microglia (Benito et al., 2003). Similarly, in models of neuropathic pain (but not inflammatory pain) CB2 mRNA increases in association with activated microglia in the spinal cord (Zhang et al., 2003). In addition during amyotrophic lateral sclerosis and multiple sclerosis, CB2 microglial expression increases in the spinal cord (Yiangou et al., 2006). According to this evidence it is clear that under certain conditions brain microglia are capable of expressing CB2.
CB2 and tissue distribution
Despite being initially described as an immune cell cannabinoid receptor, CB2 has been identified molecularly and pharmacologically in numerous other cell types. Evidence for the presence of CB2 receptors has been found in pulmonary endothelial cells (Zoratti et al., 2003). In these cells, CB2 activation by anandamide results in phospholipase C-mediated calcium release from smooth ER with subsequent increases in mitochondrial calcium. CB2 can also be found in bone (in osteocytes, osteoblasts and osteoclasts) where it modulates bone formation and turnover (Ofek et al., 2006). The gastrointestinal system appears to express CB2 receptors as well (Storr et al., 2002; Hillsley et al., 2007; Duncan et al., 2008). 2-AG affects meiosis in spermatogonia via CB2 (Grimaldi et al., 2009) as well as a number of other aspects of reproductive function (Maccarrone, 2008; Grimaldi et al., 2009). Keratinocytes release beta-endorphin in response to CB2 selective agonist stimulation (Ibrahim et al., 2005), although this result is controversial (Whiteside et al., 2007; Anand et al., 2008; Yao et al., 2008; Yao et al., 2009). These cells have also been reported to have CB2 immunoreactivity. In the eye, trabecular meshwork cells have been shown to have functional CB2 receptors (Zhong et al., 2005; He and Song, 2007). Mature and precursor adipocytes express functional CB2 receptors that are negatively coupled to adenylyl cyclase (Roche et al., 2006). In cirrhotic liver, CB2 receptors are expressed in hepatic myofibroblasts, but are absent in normal liver (Julien et al., 2005). THC protects cardiomyocytes from hypoxic damage by acting at CB2 receptors resulting in nitric oxide production (Shmist et al., 2006).
CB2 and nociception
To better understand the possible presence of CB2 in neurons it is helpful to consider the role of CB2 in nociception. Cannabinoids have long been known to possess analgesic activity, but evidence for CB2 having a role in analgesia was not presented until 1998 (Calignano et al., 1998). Shortly thereafter, HU308, a CB2 selective agonist, was shown to have analgesic activity without typical cannabinoid CNS side effects (Hanus et al., 1999). AM1241, another CB2 selective agonist was also shown to promote analgesia when injected peripherally and this did not produce CNS side effects, suggesting that CB2 receptors modulate nociception (Malan et al., 2001; Ibrahim et al., 2003; Malan et al., 2003; Ibrahim et al., 2006). It has since been shown that a number of different CB2 agonists can modulate many types of pain: acute, inflammatory, neuropathic, post-surgical and cancer pain (Khanolkar et al., 2007; Whiteside et al., 2007; Jhaveri et al., 2008; Naguib et al., 2008; Ohta et al., 2008; Yao et al., 2008; Anand et al., 2009; Yao et al., 2009). It is still unclear as to where these CB2 agonists exert their analgesic activity. The site could be microglia, astrocytes, neurons, another cell type or a combination of these. Furthermore, as discussed above, the specificity of these CB2‘selective’ compounds may not be as specific as previously thought. Additional work must be performed to state with confidence that these agonists produce analgesia solely via activation of CB2 receptors.
CB2 and peripheral neurons
The first step to determine if CB2 activation has a direct effect on neural mechanisms is to determine whether CB2 is expressed in neurons. The existence of functional CB2 receptors in peripheral neurons has been suggested by a number of studies. The first evidence for CB2 function in peripheral neurons came in 1997 when CB2 mRNA was identified in mouse vas deferens tissue (Griffin et al., 1997). In support of a functional role for CB2, JWH015 and JWH051 (agonists preferring CB2 over CB1) produced concentration dependent inhibition of evoked contractions presumably via a prejunctional site. However, a submicromolar concentration of AM630, a CB2 antagonist, could not block this effect. Further, JWH015 is also an agonist for GPR55 (Ryberg et al., 2007; Lauckner et al., 2008), so the involvement of CB2 cannot be unequivocally asserted.
CB2 and sensory neurons
Functional studies have hinted at the presence of CB2 receptors on sensory neurons. In these studies, it is necessary to consider the possible involvement of CB2-expressing immune cells as microglia can affect synaptic properties (Cullheim and Thams, 2007; Abbadie et al., 2009). Patel and colleagues provided some of the first functional evidence of CB2 in sensory neurons (Patel et al., 2003). Using isolated guinea pig and human vagus nerve preparations, they demonstrated that the CB2 agonist JWH133 inhibited nerve depolarizations in response to capsaicin, PGE2 and hypertonic saline. These three treatments activate vagal C and/or Aδ fibres. SR144528 blocked the effects of JWH133. A follow-up study with another putative CB2 agonist, GW833972A, produced similar results (Belvisi et al., 2008). Neither study was designed to determine a specific site or mechanism of action. While CB2 does not appear to play a role in myenteric contractions, it does seem to play a role in activation of mesenteric sensory nerves. AM1241 administered intravenously inhibits bradykinin induced activation of isolated mesenteric afferents in mice (Hillsley et al., 2007). This effect was blocked by AM630 and absent in CB2 knockout mice. Interestingly, while CB2 agonists do not affect normal enteric contractility, JWH133 can prevent lipopolysaccharide (LPS) induced increases in evoked contractions (Mathison et al., 2004; Duncan et al., 2008). JWH133 also blocks LPS stimulation of Fos expression in enteric neurons. AM630 antagonizes these effects. CB2 receptors in myenteric neurons were identified as the most likely target of this drug effect (see below). CB2 mRNA has also been identified in rat and mouse retina using RT-PCR as well as within specific layers of the retina (ganglion, inner nuclear and photoreceptor inner layers) using in situ hybridization (Lu et al., 2000). Additionally, Burdyga et al. identified low, barely detectable levels of CB2 mRNA in rat nodose ganglion, but were unable to detect CB2 in the human vagal nerve trunk (Burdyga et al., 2004).
CB2 and nociceptive neurons
Further functional studies point to a role for CB2 in sensory neuron function, particularly nociceptive neurons. A study was performed to address AM1241's ability to prevent windup of wide dynamic range (WDR) neurons in spinal cord (Nackley et al., 2004). Here, AM1241 administered locally or systemically reduced the activity of C-fibres synapsing onto WDR neurons and this was reversed by SR144528, but not SR141716A. Significantly, suppression occurred in the presence and absence of inflammation. This, combined with the time course observed suggests long-term changes in presynaptic facilitation, makes the effects less likely to be due to CB2 targeting immune cells. The authors speculate a direct effect of CB2 activation on C-fibre neurons. Elmes et al. performed a similar study using JWH133 as a CB2 agonist to test WDR spinal neuron responses in models of inflammatory and neuropathic pain as well as in non-inflammatory and sham-operated conditions (Elmes et al., 2004). Like the Nackley study, they also found that peripherally administered CB2 agonist inhibits WDR activity in both naïve and inflammatory conditions as well as following neuropathic injury. Once again, the data are suggestive of a non-immune function of CB2, possibly in peripheral neurons. A follow-up study analysed JWH133's ability to inhibit capsaicin-induced calcium increases in DRG neurons cultured from sham-operated and neuropathic rats (Sagar et al., 2005). JWH133 slightly inhibited calcium increases in DRG cultured from both neuropathic and sham rats in a SR144528-sensitive fashion, consistent with the presence of functional CB2 receptors in peripheral neurons. However, spinally administered JWH133 inhibited mechanically evoked responses of dorsal horn neurons from laminae V and VI only in neuropathic rats, but not in sham-operated animals. This points to an up-regulation of CB2 in intrinsic spinal cord neurons in pain states, although does not provide evidence of the site of up-regulation. AM1241 and L768242 (another CB2 agonist) can also decrease capsaicin-induced calcitonin gene-related peptide release, a pain biomarker, from neurons in spinal cord slices and this can be blocked by SR144528 (Beltramo et al., 2006). These studies are most consistent with CB2 participating in neural mechanisms rather than via immune cells, but do not directly answer the question of whether or not CB2 is expressed in neurons.
The initial support for the presence of CB2 protein in neurons came from Ross and colleagues. Using fluorescence-activated cell sorting analysis, they determined that DRG cultures and F-11 cells (DRG neuron × neuroblastoma hybrid) express both CB1 and CB2, but could not conclude that CB2 was functionally expressed in DRG neurons (Ross et al., 2001). To further address the site of CB2 expression in DRG, Wotherspoon and colleagues used immunhistochemistry on DRG and spinal cord of naïve and nerve damaged rats and mice (Wotherspoon et al., 2005). No CB2 could be detected in normal rat or mouse spinal cord and DRG neurons. However, upon nerve sectioning or ligation, CB2 immunoreactivity could be detected in the ipsilateral dorsal horn. This immunoreactivity was strongly reduced in CB2 knockout mice and was blocked by incubation with the immunizing peptide, suggesting specificity of the primary antibody used. Of great interest, and in contrast to what would be expected based on Zhang et al.'s (2003) study, was that the CB2 signal did not co-localize with markers of astrocytes (GFAP) or microglia (OX-42). Instead it co-localized with markers of damaged sensory neuron terminals (GAP-43 and galanin). CB2 immunoreactivity also accumulated in axons proximal to the ligation site. They could not identify CB2 in cell bodies in tissue sections, but were able to identify CB2 in isolated DRG neurons grown in culture from lesioned mice. They also did not observe CB2 immunoreactivity in skin, in contrast to other studies (Stander et al., 2005; Kress and Kuner, 2009) and Ibrahim et al. who found it in keratinocytes (Ibrahim et al., 2005). A few studies have detected CB2 mRNA in DRG and spinal cord using quantitative RT-PCR. Here levels increased following nerve ligation, but this does not necessarily implicate a neuronal source (Zhang et al., 2003; Beltramo et al., 2006).
A more recent study by Anand and colleagues is consistent with the above findings (Anand et al., 2008). Specifically, they found CB2 positive, small diameter neurons in human DRG and peripheral nerves using three different CB2 antibodies. The specificity of the antibodies was assessed using peptide block. CB2 levels increased following nerve injury. They further extended the analysis by demonstrating CB2 colocalization with neuronal (GAP-43), axonal (neurofilament) and nociceptive neuronal markers (TRPV1). Similar staining was observed in mouse, rat and guinea pig DRG. This study also replicated the functional data reported by Sagar et al. in that a CB2 agonist (GW833972) inhibited capsaicin-induced calcium increases in DRG sensory neurons. They further sought to identify a mechanism for this activity and determined that CB2-mediated cAMP depletion attenuated TRPV1 activation. This presumably decreased PKA-mediated phosphorylation of TRPV1, analogous to the effects of µ opioid receptor activation.
CB2 and the enteric nervous system
Despite earlier findings (Griffin et al., 1997), several studies suggest CB2 is expressed in the enteric nervous system. Duncan et al. and Storr et al. found CB2 mRNA in the enteric nervous system (Storr et al., 2002; Duncan et al., 2008). The site of action of JWH133 in preventing LPS-induced increases in ileum contractility was addressed using RT-PCR and immunohistochemistry (Duncan et al., 2008). CB2 mRNA was detected in the full-wall thickness ileum, ileal muscle, submucosal and mucosal layers in normal rats. LPS treatment had no effect on the levels of expression. A number of different antibodies and knockout tissue were used for controls. CB2 protein was detected in all the same tissues in which CB2 mRNA was found. CB2 colocalized with markers of enteric ganglia, pan-neuronal markers and synaptic terminals suggesting a strong presence in myenteric neurons. CB2 immunoreactivity did not colocalize with glial markers.
CB2: another central cannabinoid receptor?
CB2 in the cerebellum
One of the earliest reports of the presence of CB2 in the CNS came from a study performed by Skaper and colleagues (Skaper et al., 1996). In situ hybridization revealed the presence of CB2 mRNA in cultured granule cells. In addition in situ hybridization localized CB2 to the granule and Purkinje cell layers of mouse cerebellum. Radioligand binding analysis of cerebellar membranes revealed the presence of two WIN55212 binding sites. The affinities of WIN55212 at these sites were reported to be close to those of CB1 and CB2, although the exact identity of the binding sites could not be specifically determined. RT-PCR analysis has identified CB2 mRNA in the rat cerebellum and Western blotting has revealed expressed CB2 protein in rat and ferret cerebellum as well (Van Sickle et al., 2005). Peptide block was used as a control for those Western blots.
Additional studies have also attempted to localize CB2 protein in the cerebellum (Ashton et al., 2006; Baek et al., 2008). Using an antibody directed against the C-terminus of CB2, with peptide block control, they identified CB2 protein expression in the granule, Purkinje and white matter layers of the rat cerebellum. The signal did not overlap with astrocytes markers and the staining pattern in the Purkinje layer and parts of the other layers appeared to be capillary endothelial in nature. There were fine fibres in the white matter and granule cell layers that were CB2 positive but their origin remains to be determined. These could possibly arise from microglia or neurons. Onaivi et al. have also reported CB2 expression in the Purkinje and molecular layers of the cerebellum using Western blot, immunohistochemistry and in situ hybridization techniques (Gong et al., 2006; Onaivi et al., 2008b).
CB2 in the brainstem
CB2 has been identified within the brainstem as well. A thorough analysis was performed that investigated CB2 expression in brain, focusing on mRNA, protein and functional expression within the brainstem (Van Sickle et al., 2005). Quantitative RT-PCR showed that the rat brainstem contains CB2 mRNA at significantly lower levels than spleen (1.5% of spleen levels). Western blotting confirmed this expression for rat as well as for ferret. Immunocytochemistry identified the dorsal motor nucleus of the vagus nerve (DMNX) of the mouse, rat and ferret as a brainstem nucleus containing CB2 protein. The CB2 knockout mouse did not show any immunostaining in the DMNX. The DMNX immunoreactivity colocalized with neuronal markers, but in contrast to what Ashton et al. found in the cerebellum (Ashton et al., 2006), the signal did not overlap with glial or blood vessel markers. The authors acknowledged the differences in results between their study and that of Derbenev et al. that did not find CB2 in similar regions (Derbenev et al., 2004) and state that in the latter study a faint signal could be observed in Western blots consistent with low levels of expression. The authors also demonstrated that AM630 blocked the anti-emetic actions of 2-AG treatment in ferrets, suggesting CB2 receptor involvement. Furthermore a sub-efficacious concentration of anandamide combined with AM1241 treatment produced anti-emetic effects. Another, more superficial study of the brainstem using immunohistochemistry was later performed to look for CB2 in other brainstem nuclei (Baek et al., 2008). CB2 immunoreactivity was found in the medial vestibular nucleus as well as the dorsal and ventral cochlear nuclei, but no attempts were made to identify cell types. Peptide block and secondary antibody controls were used to determine CB2 antibody specificity. Viscomi et al. did not find CB2 protein and only low levels of CB2 mRNA in inferior olive and pontine nuclei using immunohistochemistry and quantitative PCR in normal rats (Viscomi et al., 2009). However, following a hemicerebellectomy, CB2 expression dramatically increased in both mRNA and protein levels in these nuclei. The CB2 immunoreactivity colocalized with neuronal markers but not with microglial or astrocytic ones. Further JWH015 had a neuroprotective effect, preventing cell death due to the hemicerebellectomy. This was likely operating through CB2, although they did not report block of the neuroprotective effect with a CB2 antagonist. Gong et al. reported the presence of CB2 in many nuclei of the brainstem using RT-PCR and immunohistochemistry (Gong et al., 2006).
CB2 and the hippocampal formation
Using several approaches, Onaivi and his collaborators have reported finding CB2 immunoreactivity in many areas of the hippocampal formation (Gong et al., 2006; Onaivi, 2006; Onaivi et al., 2006; 2008a,b; Brusco et al., 2008). They report a predominately postsynaptic expression and an association with rough endoplasmic reticulum and Golgi structures. They have also demonstrated CB2 staining in hippocampal cultures. In contrast to their immunohistochemical results, they have had mixed results in finding CB2 mRNA in the hippocampus (Gong et al., 2006; Onaivi et al., 2008b).
Functional evidence for CB2 expression in the cortex comes from recording spontaneous inhibitory postsynaptic currents (sIPSCs) in layers II and V of the medial entorhinal cortex. Here, 2-AG mediated suppression of sIPSCs was not blocked by LY320135, a CB1 antagonist/inverse agonist, whereas they were blocked by AM630 and JTE907 (a structurally distinct CB2 antagonist) (Morgan et al., 2009). Further JWH133 suppressed sIPSCs in a CB2 antagonist sensitive fashion. The site of CB2 agonist action remains to be conclusively demonstrated.
CB2 and other brain regions
Evidence exists for CB2 expression in other brain regions. While recording from the ventral posterior nucleus of the thalamus, Jhaveri et al. found that after spinal nerve ligation, JWH133 reduced spontaneous and evoked responses in a SR144528-sensitive fashion, but that this effect was absent in sham operated rats (Jhaveri et al., 2008). Gong et al. have also reported CB2 immunoreactivity in many thalamic nuclei, but could not detect CB2 mRNA using RT-PCR (Gong et al., 2006). Furthermore, this group has reported finding CB2 mRNA in striatum and hypothalamus, but not in olfactory bulb, cortex and spinal cord and mixed results in midbrain (Gong et al., 2006; Onaivi et al., 2008b). Additionally they report CB2 immunoreactivity in olfactory bulb, cortex, midbrain as well as the other areas already mentioned (Gong et al., 2006; Onaivi, 2006; Onaivi et al., 2006; 2008b; ).
CB2 and neurogenesis
CB2 also appears to play a role in neurogenesis. Both CB1 and CB2 are expressed in stem cells (Jiang et al., 2007; Molina-Holgado et al., 2007). More specifically, RT-PCR, Western blot and immunohistochemical analyses have all revealed the presence of CB2 in embryonic and adult neural progenitor cells (Palazuelos et al., 2006; Molina-Holgado et al., 2007). CB2 blockade or genetic disruption impairs neurosphere formation and prevents progenitor cell proliferation, whereas CB2 agonists promote these activities via ERK and Akt signalling (Palazuelos et al., 2006; Molina-Holgado et al., 2007). However, CB2 expression seems to diminish as the cells differentiate, being nearly absent by the time neuronal and astrocytic markers appear (Palazuelos et al., 2006). Further, CB1 agonists and antagonists have similar effects in neurosphere formation and in COR-1 neural stem cell cultures (Molina-Holgado et al., 2007; Goncalves et al., 2008) suggesting either functional interactions or redundant signalling. In contrast to these data, CB1 agonists and antagonists had no effect on neurogenesis in the subventricular zone (SVZ) of either young or adult mice (Goncalves et al., 2008). On the other hand, JWH133 and WIN55212 stimulated SVZ neurogenesis, whereas AM630 and JTE907 decreased it (Goncalves et al., 2008).
CB2: where is its real home and why do we care?
We feel careful analysis of the studies reviewed above allows us to reach the following conclusions: CB2 is expressed by microglia, with levels increasing as they are activated, and CB2 is present at detectable and functionally relevant levels in a subset of neurons, with increasing levels following injury. We care where CB2 is expressed primarily for understanding pathology that involves CB2 and to develop therapies that target difficult to treat conditions. To this end it is important to have a rigorous understanding of where and under what conditions CB2 is expressed in the CNS.
Approaches aimed at identifying CB2 receptor expression in the brain can be divided into functional (pharmacological), biochemical and anatomical techniques. All three have their strengths and weaknesses. The most convincing studies will incorporate a combination of these techniques. Table 1 summarizes the studies presented here, detailing the brain region analysed and whether or not CB2 was detected and the techniques(s) used to detect it. Pharmacological studies rely on the specificity of the drugs used. When interpreting these studies it is necessary to recall that specificity is never absolute – at sufficiently high concentrations any drug will interact with additional targets. Thus, it is important to relate the concentration of the drug being used to the binding affinity of the CB2 receptor for that drug. The second consideration is that drugs considered to be ‘specific’ or ‘selective’ based on our current understanding may soon be found to interact with other receptors. Examples of this in the cannabinoid system include AM251, often used as a ‘selective’ CB1 receptor antagonist, but it is also a GPR55 agonist (Henstridge et al., 2009; Kapur et al., 2009) and JWH015, sometimes used as a ‘selective’ CB2 agonist, but it, too, is a GPR55 agonist (Ryberg et al., 2007; Lauckner et al., 2008). Approaches to circumvent this issue include using several structurally diverse agonists and antagonists (presumably decreasing the likelihood of having the same ‘off-target’ actions) and knockout or ‘knockdown’ controls, when appropriate.
Biochemical studies include Western blotting and PCR-based approaches. For Western blotting, the key limitations are the sensitivity and specificity of the antibody used. At a minimum, blots from knockout (assuming the antibody is recognizing an epitope present in mouse CB2) and positive control tissues (e.g. spleen) should be shown. Blindly trusting an antibody to ‘work’ without concurrent controls is unacceptable. Block with the immunizing antibody is desirable, but will not rule out a fortuitous interaction of the antibody with an unintended epitope on another protein. The sensitivity of Western blotting will depend on the abundance of CB2 as well as the affinity of the antibody. The lack of detection of CB2 on the blot can only be interpreted as that the level of CB2 in the brain is below a certain level. (This level, relative to a CB2-expressing tissue like spleen, can be determined by serial dilution.) PCR-approached tissues are the most sensitive. Their high sensitivity makes their interpretation subject to several considerations (Suzuki et al., 2000; Lion, 2001). These include amplification of CB2 mRNA from immune cells trapped in the cerebral vasculature and amplification of CB2 mRNA from a very small subset of activated microglia. In order to rationally interpret results from PCR-based experiments it is necessary that they be performed in a quantitative fashion, preferably calculating copy number, to facilitate comparisons.
Anatomical studies need to be conducted and interpreted with a similarly critical approach. These studies fall into three categories: autoradiography, in situ hybridization and immunocytochemistry. As above the issue of sensitivity needs consideration – it is possible to show CB2 is present, but it is very hard to conclusively demonstrate that it is not present, just that it is present at a level below the limit of detection. However, this information, coupled with a lack of functional response, can be very valuable in sorting out the role of CB2 receptors in a particular physiological response. The caveats of autoradiography include the pharmacological considerations discussed above as well as specific technical issues (Frey and Albin, 2001). As this technique has not been widely applied to directly identifying CB2 receptors in the brain, it will not be further discussed here. In situ hybridization studies can yield useful information on which cells express CB2 and thus can complement PCR-based studies. However, the lower sensitivity of in situ hybridization may make this difficult. A necessary control for in situ hybridization includes lack of hybridization in knockout tissues (when possible).
Immunocytochemistry studies have the powerful potential to identify the precise localization of CB2. However, for meaningful information to be drawn from them it is essential that proper controls are followed [as reviewed by Bussolati and Leonardo (2008); Lorincz and Nusser (2008); Saper and Sawchenko (2003)]. Briefly, some of the controls are: adsorption with the immunizing peptide, parallel, blinded staining of wild-type and knockout tissue, the use of two or more antibodies raised against distinct epitopes, antibody titration, omitting the primary antibody from the staining procedure and supporting these findings with RT-PCR, in situ hybridization and other such detection methods. Using just one control for one experimental setup is usually insufficient proof of specificity. These basic controls must be remembered when interpreting the data presented from any study cited in this review and future studies as well.
In conclusion, despite originally being thought of as the ‘peripheral’ cannabinoid receptor, considerable functional and anatomical evidence suggests that CB2 is expressed in the nervous system – certainly in activated microglia and very likely in some neurons. In addition, this raises the point that any report that identifies CB2 in neurons of the nervous system must incorporate careful controls to ensure that the CB2 signal found originates from neurons and not from microglia or immune cells associated with brain blood vessels. Given the importance of determining the functional role of CB2 in the CNS, under what conditions it is up regulated, and the potential therapeutic applications of CB2 agonists it is vital to understand where in the CNS CB2 receptors are expressed. We encourage those working in the field and those reviewing manuscripts to conduct and review these studies in a careful, thoughtful and rigorous fashion.
Source, Graphs and Figures: CB2: a cannabinoid receptor with an identity crisis
