Orexins are associated with drug relapse in rodents. Here, we show that acute restraint stress in mice activates lateral hypothalamic (LH) orexin neurons, increases levels of orexin A and 2-arachidonoylglycerol (2-AG) in the ventral tegmental area (VTA), and reinstates extinguished cocaine-conditioned place preference (CPP). This stress-induced reinstatement of cocaine CPP depends on type 1 orexin receptors (OX1Rs), type 1 cannabinoid receptors (CB1Rs) and diacylglycerol lipase (DAGL) in the VTA. In dopaminergic neurons of VTA slices, orexin A presynaptically inhibits GABAergic transmission. This effect is prevented by internal GDP-β-S or inhibiting OX1Rs, CB1Rs, phospholipase C or DAGL, and potentiated by inhibiting 2-AG degradation. These results suggest that restraint stress activates LH orexin neurons, releasing orexins into the VTA to activate postsynaptic OX1Rs of dopaminergic neurons and generate 2-AG through a Gq-protein-phospholipase C-DAGL cascade. 2-AG retrogradely inhibits GABA release through presynaptic CB1Rs, leading to VTA dopaminergic disinhibition and reinstatement of cocaine CPP.

Cannabis continues to be the most accessible and popular illicit recreational drug. Whereas current data link adolescence cannabinoid exposure to increased risk for dependence on other drugs, depression, anxiety disorders and psychosis, the mechanism(s) underlying these adverse effects remains controversial. Here we show in a mouse model of female adolescent cannabinoid exposure deficient endocannabinoid (eCB)-mediated signaling and presynaptic forms of long-term depression at adult central glutamatergic synapses in the prefrontal cortex. Increasing endocannabinoid levels by blockade of monoacylglycerol lipase, the primary enzyme responsible for degrading the endocannabinoid 2-arachidonoylglycerol (2-AG), with the specific inhibitor JZL 184 ameliorates eCB-LTD deficits. The observed deficit in cortical presynaptic signaling may represent a neural maladaptation underlying network instability and abnormal cognitive functioning. Our study suggests that adolescent cannabinoid exposure may permanently impair brain functions, including the brain's intrinsic ability to appropriately adapt to external influences.

Indole-based compounds, such as the alkyl-indole (AI) compound WIN55212-2, activate the cannabinoid receptors, CB1 and CB2, two well-characterized G protein-coupled receptors (GPCR). Reports indicate that several indole-based cannabinoid agonists, including WIN55212-2, lack selectivity and interact with at least two additional targets: AI-sensitive GPCRs and microtubules. Studying how indole-based compounds modulate the activity of these 4 targets has been difficult as selective chemical tools were not available. Here we report the pharmacological characterization of six newly-developed indole-based compounds (ST-11, ST-23, ST-25, ST-29, ST-47 and ST-48) that exhibit distinct binding affinities at AI-sensitive receptors, cannabinoid CB1 and CB2 receptors and the colchicine site of tubulin. Several compounds exhibit some level of selectivity for AI-sensitive receptors, including ST-11 that binds AI-sensitive receptors with a Kd of 52nM and appears to have a weaker affinity for the colchicine site of tubulin (Kd=3.2μM) and does not bind CB1/CB2 receptors. Leveraging these characteristics, we show that activation of AI-sensitive receptors with ST-11 inhibits both the basal and stimulated migration of the Delayed Brain Tumor (DBT) mouse glioma cell line. Our study describes a new series of indole-based compounds that enable the pharmacological and functional differentiation of alkylindole-sensitive receptors from cannabinoid receptors and microtubules.

Introduction: The high prevalence of adolescent cannabis use, the association between this use and later psychiatric disease, and increased access to high-potency cannabis highlight the need for a better understanding of the long-term effects of adolescent cannabis use on cognitive and behavioral outcomes. Furthermore, increasing Δ9-tetrahydrocannabinol (THC) in high-potency cannabis is accompanied by a decrease in cannabidiol (CBD), thus an understanding of the interactions between CBD and THC in the neurodevelopmental effects of THC is also important. The current study examined the immediate and long-term behavioral consequences of THC, CBD, and their combination in a mouse model of adolescent cannabis use. Materials and Methods: Male CD1 mice received daily injections of THC (3 mg/kg), CBD (3 mg/kg), CBD+THC (3 mg/kg each), vehicle, or remained undisturbed in their home cage (no handling/injections), either during adolescence (postnatal day [PND] 28-48) or during early adulthood (PND 69-89). Animals were then evaluated with a battery of behavioral tests 1 day after drug treatment, and again after 42 drug-free days. The tests included the following: open field (day 1), novel object recognition (NOR; day 2), marble burying (day 3), elevated plus maze (EPM; day 4), and Nestlet shredding (day 5). Results: Chronic administration of THC during adolescence led to immediate and long-term impairments in object recognition/working memory, as measured by the NOR task. In contrast, adult administration of THC caused immediate, but not long term, impairment of object/working memory. Adolescent chronic exposure to THC increased repetitive and compulsive-like behaviors, as measured by the Nestlet shredding task. Chronic administration of THC, either during adolescence or during adulthood, led to a delayed increase in anxiety as measured by the EPM. All THC-induced behavioral abnormalities were prevented by the coadministration of CBD+THC, whereas CBD alone did not influence behavioral outcomes. Conclusion: These data suggest that chronic exposure to THC during adolescence leads to some of the behavioral abnormalities common in schizophrenia. Interestingly, CBD appeared to antagonize all THC-induced behavioral abnormalities. These findings support the hypothesis that adolescent THC use can impart long-term behavioral deficits; however, cotreatment with CBD prevents these deficits.

The nucleus accumbens (Acb) shell and caudate-putamen nucleus (CPu) are respectively implicated in the motivational and motor effects of dopamine, which are mediated in part through dopamine D₂-like receptors (D₂Rs) and modulated by activation of the cannabinoid-1 receptor (CB₁R). The dopamine D(₂/D3) receptor agonist, quinpirole elicits internalization of D₂Rs in isolated cells; however, dendritic and axonal targeting of D₂Rs may be highly influenced by circuit-dependent changes in vivo and potentially influenced by endogenous CB₁R activation.

G protein coupled receptors (GPCRs) are one of the most widely studied gene superfamilies. Thousands of GPCR research studies have utilized heterologous expression systems such as human embryonic kidney cells (HEK293). Though often treated as 'blank slates', these cell lines nevertheless endogenously express GPCRs and related signaling proteins. The outcome of a given GPCR study can be profoundly influenced by this largely unknown complement of receptors and/or signaling proteins. Little easily accessible information exists that describes the expression profiles of the GPCRs in cell lines. What is accessible is often limited in scope - of the hundreds of GPCRs and related proteins, one is unlikely to find information on expression of more than a dozen proteins in a given cell line. Microarray technology has allowed rapid analysis of mRNA levels of thousands of candidate genes, but though often publicly available, the results can be difficult to efficiently access or even to interpret.

A role for endocannabinoid signaling in neuronal morphogenesis as the brain develops has recently been suggested. Here we used the developing somatosensory circuit as a model system to examine the role of endocannabinoid signaling in neural circuit formation. We first show that a deficiency in cannabinoid receptor type 1 (CB(1)R), but not G-protein-coupled receptor 55 (GPR55), leads to aberrant fasciculation and pathfinding in both corticothalamic and thalamocortical axons despite normal target recognition. Next, we localized CB(1)R expression to developing corticothalamic projections and found little if any expression in thalamocortical axons, using a newly established reporter mouse expressing GFP in thalamocortical projections. A similar thalamocortical projection phenotype was observed following removal of CB(1)R from cortical principal neurons, clearly demonstrating that CB(1)R in corticothalamic axons was required to instruct their complimentary connections, thalamocortical axons. When reciprocal thalamic and cortical connections meet, CB(1)R-containing corticothalamic axons are intimately associated with elongating thalamocortical projections containing DGLβ, a 2-arachidonoyl glycerol (2-AG) synthesizing enzyme. Thus, 2-AG produced in thalamocortical axons and acting at CB(1)Rs on corticothalamic axons is likely to modulate axonal patterning. The presence of monoglyceride lipase, a 2-AG degrading enzyme, in both thalamocortical and corticothalamic tracts probably serves to restrict 2-AG availability. In summary, our study provides strong evidence that endocannabinoids are a modulator for the proposed 'handshake' interactions between corticothalamic and thalamocortical axons, especially for fasciculation. These findings are important in understanding the long-term consequences of alterations in CB(1)R activity during development, a potential etiology for the mental health disorders linked to prenatal cannabis use.

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 μm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.

Selective activation of the peripheral cannabinoid receptor 1 (CB1R) has been shown to suppress neuropathic pain symptoms in rodents. However, relatively little is known about changes in CB1R and its endogenous ligands during development or maintenance of neuropathic pain. Using immunohistochemistry, Western blot, real-time reverse transcription polymerase chain reaction, as well as liquid chromatography/mass spectrometry, we studied the changes in CB1Rs and endocannabinoids N-arachidonoylethanolamine/anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat lumbar (L4 and L5) dorsal root ganglia (DRG) after neuropathic pain induction (L5 spinal nerve ligation: SNL). Immunohistochemistry revealed that in control rats, CB1R is expressed in the majority (76-83%) of nociceptive neurons as indicated by co-labeling with isolectin B4 (IB4) or antibodies recognizing transient receptor potential vanilloid (TRPV1), calcitonin gene related peptide (CGRP), and the NR2C/2D subunits of the N-methyl-D-aspartate receptor. After L5 SNL, CB1R mRNA and protein increases in the ipsilateral uninjured L4 DRG whereas the percentages of CB1R immunoreactive (CB1R-ir) neurons remain unchanged in L4 and L5 DRG. However, for these CB1R-ir neurons, we observe significant increases in percentage of TRPV1-ir cells in ipsilateral L4 DRG, and decreases in percentage of IB4- and CGRP-co-labeled cells in ipsilateral L5 DRG. Levels of both AEA and 2-AG increase significantly only in the ipsilateral L5 DRG. These results are consistent with the preserved analgesic effects of cannabinoids in neuropathic pain and provide a rational framework for the development of peripherally acting endocannabinoid-based therapeutic interventions for neuropathic pain.

Cannabinoid type 1 receptor (CB1R) is expressed and participates in several aspects of cerebral cortex embryonic development as demonstrated with whole-transcriptome mRNA sequencing and other contemporary methods. However, the cellular location of CB1R, which helps to specify molecular mechanisms, remains to be documented. Using three-dimensional (3D) electron microscopic reconstruction, we examined CB1R immunolabeling in proliferating neural stem cells (NSCs) and migrating neurons in the embryonic mouse (Mus musculus) and rhesus macaque (Macaca mulatta) cerebral cortex. We found that the mitotic and postmitotic ventricular and subventricular zone (VZ and SVZ) cells are immunonegative in both species while radially migrating neurons in the intermediate zone (IZ) and cortical plate (CP) contain CB1R-positive intracellular vesicles. CB1R immunolabeling was more numerous and more extensive in monkeys compared to mice. In CB1R-knock out mice, projection neurons in the IZ show migration abnormalities such as an increased number of lateral processes. Thus, in radially migrating neurons CB1R provides a molecular substrate for the regulation of cell movement. Undetectable level of CB1R in VZ/SVZ cells indicates that previously suggested direct CB1R-transmitted regulation of cellular proliferation and fate determination demands rigorous re-examination. More abundant CB1R expression in monkey compared to mouse suggests that therapeutic or recreational cannabis use may be more distressing for immature primate neurons than inferred from experiments with rodents.

Cannabinoid receptor 1 (CB1R), a G protein-coupled receptor, plays a fundamental role in synaptic plasticity. Abnormal activity and deregulation of CB1R signaling result in a broad spectrum of pathological conditions. CB1R signaling is regulated by receptor desensitization including phosphorylation of residues within the intracellular C terminus by G protein-coupled receptor kinases (GRKs) that may lead to endocytosis. Furthermore, CB1R signaling is regulated by the protein Src homology 3-domain growth factor receptor-bound 2-like (SGIP1) that hinders receptor internalization, while enhancing CB1R association with β-arrestin. It has been postulated that phosphorylation of two clusters of serine/threonine residues, 425 SMGDS429 and 460 TMSVSTDTS468 , within the CB1R C-tail controls dynamics of the association between receptor and its interaction partners involved in desensitization. Several molecular determinants of these events are still not well understood. We hypothesized that the dynamics of these interactions are modulated by SGIP1. Using a panel of CB1Rs mutated in the aforementioned serine and threonine residues, together with an array of Bioluminescence energy transfer-based (BRET) sensors, we discovered that GRK3 forms complexes with Gβγ subunits of G proteins that largely independent of GRK3's interaction with CB1R. Furthermore, CB1R interacts only with activated GRK3. Interestingly, phosphorylation of two specific residues on CB1R triggers GRK3 dissociation from the desensitized receptor. SGIP1 increases the association of GRK3 with Gβγ subunits of G proteins, and with CB1R. Altogether, our data suggest that the CB1R signalosome complex is dynamically controlled by sequential phosphorylation of the receptor C-tail and is also modified by SGIP1.

Long-term cannabis use during adolescence has deleterious effects in brain that are largely ascribed to the activation of cannabinoid-1 receptors (CB1Rs) by delta-9-tetrahydrocannabinol (∆9-THC), the primary psychoactive compound in marijuana. Systemic administration of ∆9-THC inhibits acetylcholine release in the prelimbic-prefrontal cortex (PL-PFC). In turn, PL-PFC acetylcholine plays a role in executive activities regulated by CB1R-targeting endocannabinoids, which are generated by cholinergic stimulation of muscarinic-1 receptors (M1Rs). However, the long-term effects of chronic administration of increasing doses of ∆9-THC in adolescent males on the distribution and function of M1 and/or CB1 receptors in the PL-PFC remains unresolved. We used C57BL\6J male mice pre-treated with vehicle or escalating daily doses of ∆9-THC to begin filling this gap. Electron microscopic immunolabeling showed M1R-immunogold particles on plasma membranes and in association with cytoplasmic membranes in varying sized dendrites and dendritic spines. These dendritic profiles received synaptic inputs from unlabeled, CB1R- and/or M1R-labeled axon terminals in the PL-PFC of both treatment groups. However, there was a size-dependent decrease in total (plasmalemmal and cytoplasmic) M1R gold particles in small dendrites within the PL-PFC of mice receiving ∆9-THC. Whole cell current-clamp recording in PL-PFC slice preparations further revealed that adolescent pretreatment with ∆9-THC attenuates the hyperpolarization and increases the firing rate produced by local muscarinic stimulation. Repeated administration of ∆9-THC during adolescence also reduced spontaneous alternations in a Y-maze paradigm designed for measures of PFC-dependent memory function in adult mice. Our results provide new information implicating M1Rs in cortical dysfunctions resulting from adolescent abuse of marijuana.

In the era of combined antiretroviral therapy, HIV-1 infected individuals are living longer lives; however, longevity is met with an increasing number of HIV-1 associated neurocognitive disorders (HAND) diagnoses. The transactivator of transcription (Tat) is known to mediate the neurotoxic effects in HAND by acting directly on neurons and also indirectly via its actions on glia. The Go/No-Go (GNG) task was used to examine HAND in the Tat transgenic mouse model. The GNG task involves subjects discriminating between two stimuli sets in order to determine whether or not to inhibit a previously trained response. Data reveal inhibitory control deficits in female Tat(+) mice (p = .048) and an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group (p < .05). A significant negative correlation was noted between inhibitory control and IL CB1R expression (r = -.543, p = .045), with CB1R expression predicting 30% of the variance of inhibitory control (R2 = .295, p = .045). Furthermore, there was a significant increase in spontaneous excitatory postsynaptic current (sEPSC) frequencies in Tat(+) compared to Tat(-) mice (p = .008, across sexes). The increase in sEPSC frequency was significantly attenuated by bath application of PF3845, a fatty acid amide hydrolase (FAAH) enzyme inhibitor (p < .001). Overall, the GNG task is a viable measure to assess inhibitory control deficits in Tat transgenic mice and results suggest a potential therapeutic treatment for the observed deficits with drugs which modulate endocannabinoid enzyme activity. Graphical Abstract Results of the Go/No-Go operant conditioning task reveal inhibitory control deficits in female transgenic Tat(+) mice without significantly affecting males. The demonstrated inhibitory control deficits appear to be associated with an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group.

The N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) catalyzes the production of N-acylethanolamines (NAEs), a family of endogenous bioactive lipids, which are involved in various biological processes ranging from neuronal functions to energy homeostasis and feeding behaviors. Reward-dependent behaviors depend on dopamine (DA) transmission between the ventral tegmental area (VTA) and the nucleus accumbens (NAc), which conveys reward-values and scales reinforced behaviors. However, whether and how NAPE-PLD may contribute to the regulation of feeding and reward-dependent behaviors has not yet been investigated. This biological question is of paramount importance since NAEs are altered in obesity and metabolic disorders. Here, we show that transcriptomic meta-analysis highlights a potential role for NAPE-PLD within the VTA→NAc circuit. Using brain-specific invalidation approaches, we report that the integrity of NAPE-PLD is required for the proper homeostasis of NAEs within the midbrain VTA and it affects food-reward behaviors. Moreover, region-specific knock-down of NAPE-PLD in the VTA enhanced food-reward seeking and reinforced behaviors, which were associated with increased in vivo DA release dynamics in response to both food- and non-food-related rewards together with heightened tropism towards food consumption. Furthermore, midbrain knock-down of NAPE-PLD, which increased energy expenditure and adapted nutrient partitioning, elicited a relative protection against high-fat diet-mediated body fat gain and obesity-associated metabolic features. In conclusion, these findings reveal a new key role of VTA NAPE-PLD in shaping DA-dependent events, feeding behaviors and energy homeostasis, thus providing new insights on the regulation of body metabolism.

Stimulation of the postsynaptic metabotropic glutamate receptor mGluR5 triggers retrograde signaling of endocannabinoids that activate presynaptic cannabinoid CB1 receptors on juxtaposing axon terminals. To better understand the synaptic structure that supports mGluR5 mediation of CB1 activation in the prefrontal cortex (PFC) and basolateral amygdala (BLA), we examined electron microscopic dual immunolabeling of these receptors in the prelimbic PFC (prPFC) and BLA of adult male rats. CB1 immunoreactivity was detected in axon terminals that were typically large, complex, and contained dense-core and clear synaptic vesicles. Of terminals forming discernible synaptic specializations, 95% were symmetric inhibitory-type in the prPFC and 90% were inhibitory in the BLA. CB1-immunoreactive terminals frequently contacted dendrites containing mGluR5 adjacent to unlabeled terminals forming excitatory-type synapses. Because most CB1-containing terminals form inhibitory-type synapses, the unlabeled axon terminals forming asymmetric synapses are the likely source of the mGluR5 ligand glutamate. In the prPFC, serial section analysis revealed that GABAergic CB1-containing axon terminals targeted dendrites adjacent to glutamatergic axon terminals, often near dendritic bifurcations. These observations provide ultrastructural evidence that cortical CB1 receptors are strategically positioned for integration of synaptic signaling in response to stimulation of postsynaptic mGluR5 receptors and facilitation of heterosynaptic communication between multiple neurons.

Derived from arachidonic acid (AA), the endogenous cannabinoid (eCB) 2-arachidonoyl glycerol (2-AG) is a substrate for α/β hydrolase domain-12 (ABHD12). Loss-of-function mutations of ABHD12 are associated with the neurodegenerative disorder polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract (PHARC). ABHD12 knockout (KO) mice show PHARC-like behaviors in older adulthood. Here, we test the hypothesis that ABHD12 deletion age-dependently regulates bioactive lipids in the CNS. Lipidomics analysis of the brainstem, cerebellum, cortex, hippocampus, hypothalamus, midbrain, striatum and thalamus from male young (3-4 months) and older (7 months) adult ABHD12 KO and age-matched wild-type (WT) mice was performed on over 80 lipids via HPLC/MS/MS, including eCBs, lipoamines, 2-acyl glycerols, free fatty acids, and prostaglandins (PGs). Aging and ABHD12 deletion drove widespread changes in the CNS lipidome; however, the effects of ABHD12 deletion were similar between old and young mice, meaning that many alterations in the lipidome precede PHARC-like symptoms. AA-derived lipids were particularly sensitive to ABHD12 deletion. 2-AG increased in the striatum, hippocampus, cerebellum, thalamus, midbrain, and brainstem, whereas the eCB N-arachidonoyl ethanolamine (AEA) increased in all 8 brain regions, along with at least 2-PGs. Aging also had a widespread effect on the lipidome and more age-related changes in bioactive lipids were found in ABHD12 KO mice than WT suggesting that ABHD12 deletion exacerbates the effects of age. The most robust effects of aging (independent of genotype) across the CNS were decreases in N-acyl GABAs and N-acyl glycines. In conclusion, levels of bioactive lipids are dynamic throughout adulthood and deleting ABHD12 disrupts the wider lipidome, modulating multiple AA-derived lipids with potential consequences for neuropathology.

Microglia activation is central to the neuroinflammation associated with neurological and neurodegenerative diseases, particularly because activated microglia are often a source of proinflammatory cytokines. Despite decade-long research, the molecular cascade of proinflammatory transformation of microglia in vivo remains largely elusive. Here, we report increased β-catenin expression, a central intracellular component of WNT signaling, in microglia undergoing a proinflammatory morphogenic transformation under pathogenic conditions associated with neuroinflammation such as Alzheimer's disease. We substantiate disease-associated β-catenin signaling in microglia in vivo by showing age-dependent β-catenin accumulation in mice with Alzheimer's-like pathology (APdE9). In cultured mouse microglia expressing the WNT receptors Frizzled FZD(4,5,7,8) and LDL receptor-related protein 5/6 (LRP5/6), we find that WNT-3A can stabilize β-catenin. WNT-3A dose dependently induces LRP6 phosphorylation with downstream activation of disheveled, β-catenin stabilization, and nuclear import. Gene-expression profiling reveals that WNT-3A stimulation specifically increases the expression of proinflammatory immune response genes in microglia and exacerbates the release of de novo IL-6, IL-12, and tumor necrosis factor α. In summary, our data suggest that the WNT family of lipoglycoproteins can instruct proinflammatory microglia transformation and emphasize the pathogenic significance of β-catenin-signaling networks in this cell type.

Relative to Δ9-tetrahydrocannabinol (THC), the synthetic cannabinoid CP 55,940 (CP) is significantly more potent and efficacious at cannabinoid receptors, the primary targets for endogenous cannabinoids (eCBs). eCBs belong to a large, interconnected lipidome of bioactive signaling molecules with a myriad of effects in optimal and pathological function. Recreational use of highly potent and efficacious synthetic cannabinoids is common amongst adolescents, potentially impacting brain development. Knowledge of the molecular outcomes of synthetic cannabinoid use will be important to develop more targeted therapies for synthetic cannabinoid intoxication and to prevent long-term disruption to the CNS. Here, we test the hypothesis that CP has age and region-dependent effects on the brain lipidome. Adolescent [post-natal day (PND) 35 and PND 50] and young adult female mice were given either an acute dose of CP or vehicle and brains were collected 2 h later. Eight brain regions were dissected and levels of ∼80 lipids were screened from each region using HPLC/MS/MS. CP had widespread effects on the brain lipidome in all age groups. Interestingly, more changes were observed in the PND 35 mice and more were reductions in a lipid's concentration, including region-dependent lowering of eCB levels. CP levels were highest in the cortex at PND 35, the hippocampus at PND 50, and in the cerebellum in the adult. These data provide novel insights into how high-potency, synthetic cannabinoids drive different, age-dependent, cellular signaling effects in the brain.

Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling.

We recently characterized S426A/S430A mutant mice expressing a desensitization-resistant form of the CB1 receptor. These mice display an enhanced response to endocannabinoids and ∆9-THC. In this study, S426A/S430A mutants were used as a novel model to test whether ethanol consumption, morphine dependence, and reward for these drugs are potentiated in mice with a "hyper-sensitive" form of CB1. Using an unlimited-access, two-bottle choice, voluntary drinking paradigm, S426A/S430A mutants exhibit modestly increased intake and preference for low (6%) but not higher concentrations of ethanol. S426A/S430A mutants and wild-type mice show similar taste preference for sucrose and quinine, exhibit normal sensitivity to the hypothermic and ataxic effects of ethanol, and have normal blood ethanol concentrations following administration of ethanol. S426A/S430A mutants develop robust conditioned place preference for ethanol (2 g/kg), morphine (10 mg/kg), and cocaine (10 mg/kg), demonstrating that drug reward is not changed in S426A/S430A mutants. Precipitated morphine withdrawal is also unchanged in opioid-dependent S426A/S430A mutant mice. Although ethanol consumption is modestly changed by enhanced CB1 signaling, reward, tolerance, and acute sensitivity to ethanol and morphine are normal in this model.