New Insights and Potential Therapeutic Targeting of CB2 Cannabinoid Receptors in CNS Disorders.

The endocannabinoid system (ECS) is ubiquitous in most human tissues, and involved in the regulation of mental health. Consequently, its dysregulation is associated with neuropsychiatric and neurodegenerative disorders. Together, the ECS and the expanded endocannabinoidome (eCBome) are composed of genes coding for CB1 and CB2 cannabinoid receptors (CB1R, CB2R), endocannabinoids (eCBs), and the metabolic enzyme machinery for their synthesis and catabolism. The activation of CB1R is associated with adverse effects on the central nervous system (CNS), which has limited the therapeutic use of drugs that bind this receptor. The discovery of the functional neuronal CB2R raised new possibilities for the potential and safe targeting of the ECS for the treatment of CNS disorders. Previous studies were not able to detect CB2R mRNA transcripts in brain tissue and suggested that CB2Rs were absent in the brain and were considered peripheral receptors. Studies done on the role of CB2Rs as a potential therapeutic target for treating different disorders revealed the important putative role of CB2Rs in certain CNS disorders, which requires further clinical validation. This review addresses recent advances on the role of CB2Rs in neuropsychiatric and neurodegenerative disorders, including, but not limited to, anxiety, depression, schizophrenia, Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) and addiction.

Psychiatric Disorders and Cannabinoid Receptors.

With the increasing global use of medical and adult recreational use of cannabis and cannabinoids, this chapter provides overview of evidence from animal and human studies on psychiatric disorders and cannabinoid receptors. We review and present evaluation of the relationship between changes in the ECS and psychiatric disorders. Evidence suggests the existence of a relationship between changes in components of the ECS, and some of the symptoms present in psychiatric disorders. Both CB1Rs and CB2Rs are components of the endocannabinoid system with different cellular and tissue localization patterns that are differentially expressed in the CNS and PNS and are emerging targets for the treatment of number psychiatric disorders. As cannabis preparations are widely used for recreation globally, it is predictable that cannabis use disorders (CUDs) will increase and there is currently no available treatment for CUDs. Although major advances have been reported from cannabinoid and ECS research, there are gaps in scientific knowledge on long-term consequences of cannabis use. Adolescent and cannabis use during pregnancy presents further challenges, and more research will uncover the signaling pathways that couple the gut microbiota with the host ECS. Development of cannabis and cannabinoid nanomedicine for nanotherapy will certainly overcome some of the shortcomings and challenges in medicinal and recreational use of cannabis and cannabinoids. Thus, nanotechnology will allow targeted delivery of cannabinoid formulations with the potential to elevate their use to scientifically validated nanotherapeutic applications as the field of cannabis nanoscience matures.

Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects.

There are two well-characterized cannabinoid receptors (CB1R and CB2R and other candidates): the central nervous system (CNS) enriched CB1R and peripheral tissue enriched CB2R with a wide dynamic range of expression levels in different cell types of human tissues. Hepatocytes and neurons express low baseline CB1R and CB2R, respectively, and their cell-type-specific functions are not well defined. Here we report inducible expression of CB1R in the liver by high-fat and high sugar diet and CB2R in cortical neurons by methamphetamine. While there is less controversy about hepatocyte CB1R, the presence of functional neuronal CB2R is still debated to date. We found that neuron CB2R basal expression was higher than that of hepatocyte CB1R by measuring mRNA levels of specific isoform CB2A in neurons isolated by fluorescence-activated cell sorting (FACS) and CB1A in hepatocytes isolated by collagenase perfusion of liver. For in vivo studies, we generated hepatocyte, dopaminergic neuron, and microglia-specific conditional knockout mice (Abl-Cnr1Δ, Dat-Cnr2Δ, and Cx3cr1-Cnr2Δ) of CB1R and CB2R by crossing Cnr1f/f and Cnr2f/f strains to Abl-Cre, Dat-Cre, and Cx3cr1-Cre deleter mouse strains, respectively. Our data reveals that neuron and microglia CB2Rs are involved in the "tetrad" effects of the mixed agonist WIN 55212-2, CB1R selective agonist arachidonyl-2'-chloroethylamide (ACEA), and CB2R selective agonist JWH133. Dat-Cnr2Δ and Cx3cr1-Cnr2Δ mice showed genotypic differences in hypomobility, hypothermia, analgesia, and catalepsy induced by the synthetic cannabinoids. Alcohol conditioned place preference was abolished in DAT-Cnr2Δ mice and remained intact in Cx3cr1-Cnr2Δ mice in comparison to WT mice. These Cre-loxP recombinant mouse lines provide unique approaches in cannabinoid research for dissecting the complex endocannabinoid system that is implicated in many chronic disorders.

Developmental and behavioral effects in neonatal and adult mice following prenatal activation of endocannabinoid receptors by capsaicin.

Despite the apparent abundance of ligand-gated transient receptor potential vanilloid type 1 (TRPV1) and possible cross talk between the endocannabinoid and endovanilloid systems in the central nervous system (CNS), it is unclear what role TRPV1 receptor activation in CNS plays in neurobehavioral development. We previously reported that capsaicin or WIN55212-2 induces risk aversion in the plus-maze test, which was dependent on the gender and mouse strain used. In this study, pregnant BALBc mice were administered capsaicin (1.0 or 4.0 mg/kg, i.p.) during the second week of gestation. Developmental effects of prenatal exposure to capsaicin were assessed in neonates, and behavioral effects were assessed in adult offspring. Gender- and dose-specific variations in ultrasonic vocalizations, weight gain, righting reflex, and general activity of the pups were observed. Prenatal exposure to capsaicin altered plus-maze performance, especially with further exogenous capsaicin challenge. Furthermore, dose- and gender-specific effects were evident in the conditioned place preference/aversion paradigm following conditioning with capsaicin in adult animals. The capsaicin-induced aversion in the plus-maze test was enhanced by WIN55212-2 and blocked by pretreatment with vanilloid antagonist capsazepine or the CB1 receptor antagonist rimonabant, demonstrating an interaction between the endocannabinoid and endovanilloid systems in CNS. Taken together, the interaction between the endocannabinoid and endovanilloid signaling systems can be exploited for therapeutic applications in health and disease.

Identification of novel mouse and rat CB1R isoforms and in silico modeling of human CB1R for peripheral cannabinoid therapeutics.

Targeting peripheral CB1R is desirable for the treatment of metabolic syndromes without adverse neuropsychiatric effects. We previously reported a human hCB1b isoform that is selectively enriched in pancreatic beta-cells and hepatocytes, providing a potential peripheral therapeutic hCB1R target. It is unknown whether there are peripherally enriched mouse and rat CB1R (mCB1 and rCB1, respectively) isoforms. In this study, we found no evidence of peripherally enriched rodent CB1 isoforms; however, some mCB1R isoforms are absent in peripheral tissues. We show that the mouse Cnr1 gene contains six exons that are transcribed from a single promoter. We found that mCB1A is a spliced variant of extended exon 1 and protein-coding exon 6; mCB1B is a novel spliced variant containing unspliced exon 1, intron 1, and exon 2, which is then spliced to exon 6; and mCB1C is a spliced variant including all 6 exons. Using RNAscope in situ hybridization, we show that the isoforms mCB1A and mCB1B are expressed at a cellular level and colocalized in GABAergic neurons in the hippocampus and cortex. RT-qPCR reveals that mCB1A and mCB1B are enriched in the brain, while mCB1B is not expressed in the pancreas or the liver. Rat rCB1R isoforms are differentially expressed in primary cultured neurons, astrocytes, and microglia. We also investigated modulation of Cnr1 expression by insulin in vivo and carried out in silico modeling of CB1R with JD5037, a peripherally restricted CB1R inverse agonist, using the published crystal structure of hCB1R. The results provide models for future CB1R peripheral targeting.

Endocannabinoid System Components: Overview and Tissue Distribution.

Marijuana/cannabinoid research has been transformed into mainstream science during the last half-century. Evidence based research and remarkable biotechnological advances demonstrate that phytocannabinoids and endocannabinoid (eCBs) acting on cannabinoid receptors (CBRs) regulate various aspects of human physiological, behavioral, immunological and metabolic functions. The distribution and function of the components of the endocannabinoid system (ECS) in the central nervous system (CNS) and immune processes have garnished significant research focus with major milestones. With these advances in biotechnology, rapid extension of the ECS research in the periphery has gained momentum. In this chapter, we review the components and tissue distribution of this previously unknown but ubiquitous and complex ECS that is involved in almost all aspects of mammalian physiology and pathology.

Involvement of CB2 Receptors in the Neurobehavioral Effects of Catha Edulis (Vahl) Endl. (Khat) in Mice.

There is behavioral evidence for the interaction between crude khat extract and the endocannabinoid system, whereby the endocannabinoid system alters khat extract-mediated behavioral effects through modulation of the monoaminergic system. The objective of this study was to investigate the role of the endocannabinoid system on the neurobehavioral effect of khat extract in mice following concomitant administration of khat extract and the CB2R agonist, JWH133. Locomotor activity test, immunohistochemistry, and reverse transcriptase polymerase chain reaction technique were utilized to assess locomotor activity, tyrosine hydroxylase immunoreactivity, and expression of dopamine transporter mRNA gene. The results show sub-acute administration of khat extract alone increased locomotor activity in mice and co-administration of the CB2R agonist, JWH133, reduced khat extract induced hyperlocomotor activity. The data revealed that cell type specific deletion of CB2Rs on dopaminergic neurons increased the hyperlocomotor behavior of khat extract. Furthermore, the results revealed that khat extract attenuated MPTP induced motor deficits, which is enhanced by JWH133. Khat extract also increased expression of tyrosine hydroxylase positive cells and expression of dopamine transporter mRNA gene in wild type mice. Nevertheless, JWH133 did not alter the effect of khat extract on tyrosine hydroxylase immunoreactivity and dopamine transporter mRNA expression when given together with khat extract. Taken together, the results suggest that the CB2Rs selectively interact with khat extract-mediated locomotor effects and could be utilized as therapeutic target in central nervous system movement disorders associated with dopamine dysregulation.

Cannabinoid CB2 Receptor Gene and Environmental Interaction in the Development of Psychiatric Disorders.

CB2 cannabinoid receptor (CB2R) gene is associated with depression. We investigated the gene-environment interaction between CB2R function and diverse stressors. First, anxiety-like behavior during chronic-mild-stress (CMS) was evaluated in C57BL/6JJmsSlc mice following treatment with CB2R agonist JWH015 or inverse-agonist AM630. Second, locomotor activity and anxiety-like behavior were measured following exposure to an immune poly I:C stressor. Gene expressions of HPA axis related molecules, Fkbp5, Nr3c1 and Crf and pro-inflammatory cytokine Il-1b, as well as Bdnf as a key neurotrophin that supports neuron health, function, and synaptic plasticity, were determined in hippocampus of Cnr2 knockout mice, as indicators of stressful environment. CMS-induced anxiety-like behavior was enhanced by AM630 and reduced by JWH015 and fluvoxamine. Poly I:C reduced locomotor activity and increased anxiety-like behavior, and these effects were pronounced in the heterozygote than in the wild type mice. Fkbp5 and Nr3c1 expression were lower in the Cnr2 heterozygotes than in the wild type mice with Poly I:C treatment. These findings indicate that interaction between CB2R gene and stressors increases the risk of depression-like behaviors that may be linked with neuro-immune crosstalk. Further studies in human subjects are necessary to determine the role of CB2R and environmental interaction in the development of depression.

Reconsolidation and update of morphine-associated contextual memory in mice.

Drug addiction can be viewed as a pathological memory that is constantly retrieved and reconsolidated. Since drug abuse takes place in different contexts, it could be considered that reconsolidation plays a role in memory updating. There is consistent evidence supporting the role of reconsolidation in the strength and maintenance of contextual memories induced by drugs of abuse. However, this role is not well established in memory update. The purpose of the current study was to assess the reconsolidation process over memory update. C57BL6 mice were subjected to a morphine-induced, conditioned place preference (CPP) paradigm. Based on CPP results, animals were divided into distinct experimental groups, according to the contextual characteristics of the re-exposure and a second CPP Test. Re-exposure in the original context was important for memory maintenance and re-exposure under discrete contextual changes resulted in memory updating, although original memory was maintained. Interestingly, cycloheximide, an inhibitor of protein synthesis, had different outcomes in our protocol. When the re-exposure was done under discrete contextual changes, cycloheximide treatment just after re-exposure blocked memory updating, without changes in memory maintenance. When re-exposure was done under the original context, only two subsequent cycloheximide injections (3 and 6h) disrupted later CPP expression. Considering the temporal window of protein synthesis in consolidation and reconsolidation, these findings suggest that re-exposure, according to the contextual characteristics in our protocol, could trigger both phenomena. Furthermore, when new information is present on retrieval, reconsolidation plays a pivotal role in memory updating.

NrCAM-regulating neural systems and addiction-related behaviors.

We have previously shown that a haplotype associated with decreased NrCAM expression in brain is protective against addiction vulnerability for polysubstance abuse in humans and that Nrcam knockout mice do not develop conditioned place preferences for morphine, cocaine or amphetamine. In order to gain insight into NrCAM involvement in addiction vulnerability, which may involve specific neural circuits underlying behavioral characteristics relevant to addiction, we evaluated several behavioral phenotypes in Nrcam knockout mice. Consistent with a potential general reduction in motivational function, Nrcam knockout mice demonstrated less curiosity for novel objects and for an unfamiliar conspecific, showed also less anxiety in the zero maze. Nrcam heterozygote knockout mice reduced alcohol preference and buried fewer marbles in home cage. These observations provide further support for a role of NrCAM in substance abuse including alcoholism vulnerability, possibly through its effects on behavioral traits that may affect addiction vulnerability, including novelty seeking, obsessive compulsion and responses to aversive or anxiety-provoking stimuli. Additionally, in order to prove glutamate homeostasis hypothesis of addiction, we analyzed glutamatergic molecules regulated by NRCAM expression. Glutaminase appears to be involved in NrCAM-related molecular pathway in two different tissues from human and mouse. An inhibitor of the enzyme, prolyl-leucyl-glycinamide, treatment produced, at least, some of the phenotypes of mice shown in alcohol preference and in anxiety-like behavior. Thus, NrCAM could affect addiction-related behaviors via at least partially modulation of some glutamatergic pathways and neural function in brain.

Cannabinoid CB2 receptors and hypersensitivity to methamphetamine: Vulnerability to schizophrenia.

The human cannabinoid receptor 2 (CB2R) gene CNR2 has been associated with schizophrenia development. Inbred mice treated with the CB2R inverse agonist AM630 and challenged with methamphetamine (MAP) showed reduced prepulse inhibition (%PPI) response and locomotor hyperactivity, both behavioral measures in rodents that correlate with psychosis. Mice lacking CB2R on striatal dopaminergic neurons exhibit a hyperdopaminergic tone and a hyperactivity phenotype. Hyperdopaminergia plays a role in the etiology of schizophrenia. This study aimed to determine the direct role of CB2R, heterozygous Cnr2 gene knockout (Het) mice treated with MAP to induce behavioral sensitivity mimicking a schizophrenia-like human phenotype. Additionally, the study aims to explore the unique modulation of dopamine activity by neuronal CB2R. Conditional knockout DAT-Cnr2-/- mice were evaluated in response to MAP treatments for this purpose. Sensorimotor gating deficits in DAT-Cnr2-/- mice were also evaluated. Het mice developed reverse tolerance (RT) to MAP-enhanced locomotor activity, and RT reduced the %PPI compared to wild-type (WT) mice. DAT-Cnr2-/- mice showed an increased sensitivity to stereotypical behavior induced by MAP and developed RT to MAP. DAT-Cnr2-/- mice exhibit a reduction in %PPI and alter social interaction, another core symptom of schizophrenia. These results demonstrate that there is an interaction between neuronal CB2R and MAP treatment, which increases the risk of schizophrenia-like behavior in this mouse model. This finding provides evidence for further studies targeting CB2R as a potential schizophrenia therapy.

Crosstalk between the endocannabinoid and mid-brain dopaminergic systems: Implication in dopamine dysregulation.

Endocannabinoids (eCBs) and the expanded endocannabinoid system (ECS)-"endocannabinoidome", consists of the endogenous ligands, eCBs, their canonical and non-canonical receptor subtypes, and their synthesizing and metabolizing enzymes. This system modulates a wide range of body functions and acts as a retrograde signaling system within the central nervous system (CNS) by inhibition of classical transmitters, and plays a vital modulatory function on dopamine, a major neurotransmitter in the CNS. Dopamine is involved in different behavioral processes and contributes to different brain disorders-including Parkinson's disease, schizophrenia, and drug addiction. After synthesis in the neuronal cytosol, dopamine is packaged into synaptic vesicles until released by extracellular signals. Calcium dependent neuronal activation results in the vesicular release of dopamine and interacts with different neurotransmitter systems. The ECS, among others, is involved in the regulation of dopamine release and the interaction occurs either through direct or indirect mechanisms. The cross-talk between the ECS and the dopaminergic system has important influence in various dopamine-related neurobiological and pathologic conditions and investigating this interaction might help identify therapeutic targets and options in disorders of the CNS associated with dopamine dysregulation.

Cannabinoid CB2 receptors modulate alcohol induced behavior, and neuro-immune dysregulation in mice.

The identification of additional lipid mediators, enzymes, and receptors revealed an expanded endocannabinoid system (ECS) called the endocannabinoidome (eCBome). Furthermore, eCBome research using wild type and genetically modified mice indicate the involvement of this system in modulating alcohol induced neuroinflammatory alterations associated with behavioral impairments and the release of proinflammatory cytokines. We investigated the role of cannabinoid type 2 receptors (CB2Rs) in modulating behavioral and neuro-immune changes induced by alcohol using conditional knockout (cKO) mice with selective deletion of CB2Rs in dopamine neurons (DAT-Cnr2) and in microglia (Cx3Cr1-Cnr2) cKO mice. We used a battery of behavioral tests including locomotor and wheel running activity, rotarod performance test, and alcohol preference tests to evaluate behavioral changes induced by alcohol. ELISA assay was used, to detect alterations in IL-6, IL-1α, and IL-1ß in the prefrontal cortex, striatum, and hippocampal regions of mice to investigate the role of CB2Rs in neuroinflammation induced by alcohol in the brain. The involvement of cannabinoid receptors in alcohol-induced behavior was also evaluated using the non-selective cannabinoid receptor mixed agonist WIN 55,212-2. The results showed that cell-type specific deletion of CB2Rs in dopamine neurons and microglia significantly and differentially altered locomotor activity and rotarod performance activities. The result also revealed that cell-type specific deletion of CB2Rs enhanced alcohol-induced inflammation, and WIN significantly reduced alcohol preference in all genotypes compared to the vehicle controls. These findings suggest that the involvement of CB2Rs in modulating behavioral and neuroinflammatory alterations induced by alcohol may be potential therapeutic targets in the treatment of alcohol use disorder.

Acute cannabidiol treatment enhances social interaction in adult male mice.

Cannabidiol (CBD) is a non-intoxicating phytochemical from Cannabis sativa that is increasingly used to manage pain. The potential for CBD to ameliorate dimensional behavior symptoms occurring in multiple psychiatric disorders was suggested, including social interaction impairments. To test this hypothesis, adult male BTBRT+Itpr3tf/J (BTBR) mice, a model of idiopathic autism exhibiting social preference deficits and restrictive repetitive behaviors, were acutely treated with vehicle or 0.1, 1, or 10 mg/kg CBD. Social interaction preference was assessed 50 min after treatment, followed by social novelty preference at 60 min, marble burying at 75 min and social dominance at 120 min. CBD (10 mg/kg) enhanced BTBR social interaction but not social novelty preference, marble burying or dominance, with serum levels = 29 ± 11 ng/mg at 3 h post-injection. Next, acute 10 mg/kg CBD was compared to vehicle treatment in male serotonin transporter (SERT) knock-out mice, since SERT deficiency is an autism risk factor, and in their wildtype background strain controls C57BL/6J mice. CBD treatment generally enhanced social interaction preference and attenuated social novelty preference, yet neither marble burying nor dominance was affected. These findings show acute treatment with as little as 10 mg/kg purified CBD can enhance social interaction preference in male mice that are otherwise socially deficient.

Alcohol induced behavioral and immune perturbations are attenuated by activation of CB2 cannabinoid receptors.

The endocannabinoidome (eCBome) is the expanded endocannabinoid system (ECS) and studies show that there is a link between this system and how it modulates alcohol induced neuroinflammation. Using conditional knockout (cKO) mice with selective deletion of cannabinoid type 2 receptors (CB2Rs) in dopamine neurons (DAT-Cnr2) and in microglia (Cx3Cr1-Cnr2), we investigated how CB2Rs modulate behavioral and neuroinflammation induced by alcohol. Behavioral tests including locomotor and wheel running activity, rotarod performance test, and alcohol preference tests were used to evaluate behavioral changes induced by alcohol. Using ELISA assay, we investigated the level of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1α (IL-1α), and interleukin-1ß (IL-1ß) in the hippocampus of mice. The findings demonstrated that locomotor activity, wheel running, and rotarod performance activities were significantly affected by cell-type specific deletion of CB2Rs in dopamine neurons and microglia. The non-selective CB2R agonist, WIN 55,212-2, reduced alcohol preference in the wild type and cell-type specific CB2R cKO mice. In addition, the result showed that cell-type specific deletion of CB2Rs per se and administration of alcohol to CB2R cKO mice increased the expression of proinflammatory cytokines in the hippocampus. These findings suggest the involvement of CB2Rs in modulating behavioral and immune alterations induced by alcohol.

Potential Role of Cannabinoid Type 2 Receptors in Neuropsychiatric and Neurodegenerative Disorders.

The endocannabinoid system (ECS) is composed of the two canonical receptor subtypes; type-1 cannabinoid (CB1R) and type 2 receptor (CB2R), endocannabinoids (eCBs) and enzymes responsible for the synthesis and degradation of eCBs. Recently, with the identification of additional lipid mediators, enzymes and receptors, the expanded ECS called the endocannabinoidome (eCBome) has been identified and recognized. Activation of CB1R is associated with a plethora of physiological effects and some central nervous system (CNS) side effects, whereas, CB2R activation is devoid of such effects and hence CB2Rs might be utilized as potential new targets for the treatment of different disorders including neuropsychiatric disorders. Previous studies suggested that CB2Rs were absent in the brain and they were considered as peripheral receptors, however, recent studies confirmed the presence of CB2Rs in different brain regions. Several studies have now focused on the characterization of its physiological and pathological roles. Studies done on the role of CB2Rs as a therapeutic target for treating different disorders revealed important putative role of CB2R in neuropsychiatric disorders that requires further clinical validation. Here we provide current insights and knowledge on the potential role of targeting CB2Rs in neuropsychiatric and neurodegenerative disorders. Its non-psychoactive effect makes the CB2R a potential target for treating CNS disorders; however, a better understanding of the fundamental pharmacology of CB2R activation is essential for the design of novel therapeutic strategies.

Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior.

BTBR mice are potentially useful tools for autism research because their behavior parallels core social interaction impairments and restricted-repetitive behaviors. Altered regulation of central serotonin (5-HT) neurotransmission may underlie such behavioral deficits. To test this, we compared 5-HT transporter (SERT), 5-HT(1A) and 5-HT(2A) receptor densities among BTBR and C57 strains. Autoradiographic [(3) H] cyanoimipramine (1 nM) binding to SERT was 20-30% lower throughout the adult BTBR brain as compared to C57BL/10J mice. In hippocampal membrane homogenates, [(3) H] citalopram maximal binding (B(max) ) to SERT was 95 ± 13 fmol/mg protein in BTBR and 171 ± 20 fmol/mg protein in C57BL/6J mice, and the BTBR dissociation constant (K(D) ) was 2.0 ± 0.3 nM versus 1.1 ± 0.2 in C57BL/6J mice. Hippocampal 5-HT(1A) and 5-HT(2A) receptor binding was similar among strains. However, 8-OH-DPAT-stimulated [(35) S] GTPγS binding in the BTBR hippocampal CA(1) region was 28% higher, indicating elevated 5-HT(1A) capacity to activate G-proteins. In BTBR mice, the SERT blocker, fluoxetine (10 mg/kg) and the 5-HT(1A) receptor partial-agonist, buspirone (2 mg/kg) enhanced social interactions. The D(2) /5-HT(2) receptor antagonist, risperidone (0.1 mg/kg) reduced marble burying, but failed to improve sociability. Overall, altered SERT and/or 5-HT(1A) functionality in hippocampus could contribute to the relatively low sociability of BTBR mice.

Distribution of CB2 cannabinoid receptor in adult rat retina.

Cannabinoid effects are mediated through two receptors, CB1 and CB2. In the retina CB1 has been reported in bipolar cells, gabaergic amacrine cells, horizontal cells, and inner plexiform layer. CB2 receptor mRNA localization was shown in photoreceptors, inner nuclear layer, and ganglion cell layer by using RT-PCR. The aim of this work was to localize CB2 receptor in the rat retina by using immunocytochemistry. Our results showed that CB2 receptor was localized in retinal pigmentary epithelium, inner photoreceptor segments, horizontal and amacrine cells, cells localized in ganglion cell layer, and in fibers of inner plexiform layer. These results are in agreement with studies using RT-PCR and provide some additional information about the distribution of CB2 receptor. Further studies are needed to clarify the role of this cannabinoid receptor in the retina.

Functional polymorphism in the GPR55 gene is associated with anorexia nervosa.

Endocannabinoids, anandamide, and 2-arachidonoyl glycerol are involved in food intake and appetite. Although anandamide is now thought to be a ligand for vanilloid receptor, receptors that are targets of anandamide could play a similar role in eating behaviors and related disorders. This study therefore focused on the receptor, which is called G-protein-coupled receptor 55 (GPR55) that had recently been reported to have binding affinity for endocannabinoids. Functional analysis of the sole missense polymorphism, rs3749073 (Gly195Val) in the GPR55 gene was performed by detecting the phosphorylation level of extracellular signal-regulated kinase (ERK) in Chinese-Hamster-Ovary (CHO) cells engineered to express human GPR55. Val195 type GPR55 appeared to induce less phosphorylated ERK than Gly195 type GPR55 when CHO cells were treated with anandamide and lysophosphatidylinositol (LPI). An association between the functional Gly195Val polymorphism and anorexia nervosa was tested in a female Japanese population comprising 235 patients and 1244 controls. The Val195 allele and homozygote of the Val195 allele were more abundant in the group of patients diagnosed with anorexia nervosa (P = 0.023, Odds ratio = 1.31 (95% Cl = 1.03-1.37), P = 0.0048, OR = 2.41 (95% Cl = 1.34-4.34), respectively). In conclusion, the low-functioning Val195 allele of GPR55 appears to be a risk factor for anorexia nervosa.

Cell-Type Specific Deletion of CB2 Cannabinoid Receptors in Dopamine Neurons Induced Hyperactivity Phenotype: Possible Relevance to Attention-Deficit Hyperactivity Disorder.

DAT-Cnr2 mice are conditional knockout (cKO) animals that do not express cannabinoid CB2 receptors (CB2R), in midbrain dopamine neurons. The hyperactivity phenotype of DAT-Cnr2 cKO mice were paradoxically reduced by low dose of amphetamine. Here, we report on the locomotor activity analysis in male and female adolescent (PND 30 ± 2) mice in basal conditions and in response to different doses of amphetamine, using the Open Field (OF), Elevated Plus-Maze (EPM) tests and the Novel Object Recognition (NOR) task as a putative model of attention deficit hyperactivity disorder (ADHD). Results showed that both male and female adolescent DAT-Cnr2 mice displayed significant increases in distance traveled in the OF test compared with WT mice. However, 2 mg/kg dose of amphetamine reduced the distance traveled by the DAT-Cnr2 but was increased in the WT mice. In the EPM test of anxiety-like behavioral responses, DAT-Cnr2 spent more time in the open arms of the maze than the WT mice, suggesting a reduction in anxiety-like response. DAT-Cnr2 mice showed significant increase in the number of unprotected head dips in the maze test and in the cliff avoidance reaction (CAR) test demonstrating impulsivity and risky behavior. DAT-Cnr2 mice also exhibited deficient response in the delay decision making (DDM), with impulsive choice. Both DAT-Cnr2 and WT were able to recognize the new object in the NOR task, but the exploration by the DAT-Cnr2 was less than that of the WT mice. Following the administration of 2 mg/kg of amphetamine, the similarities and differential performances of the DAT-Cnr2 and WT mice in the EPM test and NOR task was probably due to increase in attention. Microglia activation detected by Cd11b immunolabelling was enhanced in the hippocampus in DAT-Cnr2 cKO than in WT mice, implicating neuro-immune modulatory effects of CB2R. The results demonstrates that DAT-Cnr2 cKO mice with cell-type specific deletion of CB2R in midbrain dopaminergic neurons may represent a possible model for studying the neurobiological basis of ADHD.