Role Of Endocannabinoid Signaling Research Articles

TRPV1 modulation of contextual fear memory depends on stimulus intensity and endocannabinoid signalling in the dorsal hippocampus

The transient receptor potential vanilloid type-1 (TRPV1) channels have been implicated in the modulation of aversive responses. The endocannabinoid anandamide acts as an endogenous TRPV1 agonist, exerting opposite functions at TRPV1 and type-1 cannabinoid receptors (CB1R). Here we tested the hypothesis that hippocampal TRPV1 modulates contextual fear memory retrieval and investigated the influence of the aversive stimulus intensity as well as the role of endocannabinoid signaling. Male C57BL/6J mice were tested for contextual fear memory after low-, moderate-, or high-intensity shock protocols. The selective TRPV1 blockers SB366791 (1-10 nmol) and 6-I-NC (2 nmol) were infused via intra-dorsal hippocampus before the retrieval test session. The local levels of endocannabinoids and Arc and Zif268 mRNAs, involved in synaptic plasticity and memory, were quantified. First, both TRPV1 blockers reduced memory retrieval in animals exposed to moderate or high (but not low) intensity training protocols. In the second series of results, the magnitude of the freezing responses positively correlated with the hippocampal anandamide levels; TRPV1 and CB1R were found co-localized in this brain region; and the CB1R antagonist, AM251, prevented the effects of SB366791. Thus, endocannabinoid signaling possibly mediates the effects of TRPV1 blockers. Finally, inhibition of memory retrieval by TRPV1 blockers increased Arc and Zif268 mRNAs and impaired fear memory reinstatement. In conclusion, the modulation of fear memories by dorsal hippocampal TRPV1 channels may depend on the aversive stimulus intensity and occur via anandamide/CB1 signaling. Moreover, TRPV1 blockers promote Arc and Zif268 transcription, with subsequent attenuation of aversive memory reinstatement.

Expression of type one cannabinoid receptor in different subpopulation of kisspeptin neurons and kisspeptin afferents to GnRH neurons in female mice

The endocannabinoids have been shown to target the afferents of hypothalamic neurons via cannabinoid 1 receptor (CB1) and thereby to influence their excitability at various physiological and/or pathological processes. Kisspeptin (KP) neurons form afferents of multiple neuroendocrine cells and influence their activity via signaling through a variation of co-expressed classical neurotransmitters and neuropeptides. The differential potency of endocannabinoids to influence the release of classical transmitters or neuropeptides, and the ovarian cycle-dependent functioning of the endocannabinoid signaling in the gonadotropin-releasing hormone (GnRH) neurons initiated us to study whether (a) the different subpopulations of KP neurons express CB1 mRNAs, (b) the expression is influenced by estrogen, and (c) CB1-immunoreactivity is present in the KP afferents to GnRH neurons. The aim of the study was to investigate the site- and cell-specific expression of CB1 in female mice using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. The results support that CB1 mRNAs are expressed by both the GABAergic and glutamatergic subpopulations of KP neurons, the receptor protein is detectable in two-thirds of the KP afferents to GnRH neurons, and the expression of CB1 mRNA shows an estrogen-dependency. The applied estrogen-treatment, known to induce proestrus, reduced the level of CB1 transcripts in the rostral periventricular area of the third ventricle and arcuate nucleus, and differently influenced its co-localization with vesicular GABA transporter or vesicular glutamate transporter-2 in KP neurons. This indicates a gonadal cycle-dependent role of endocannabinoid signaling in the neuronal circuits involving KP neurons.

Expression of Cannabinoid Receptors in Mouse and Human Intrauterine Tissues: Insights into the Role of Endocannabinoid Signaling in Late Pregnancy

Abstract Introduction: Endocannabinoid signaling (ECS), mediated primarily by cannabinoid receptors (CNR) 1 and 2, is implicated in embryo implantation, decidualization, and placentation, but less is known about its role in late pregnancy or labour. Reports of elevated serum endocannabinoid concentrations during labour suggest that ECS may modulate uterine function leading up to and during parturition. Effects on uterine function likely vary depending on: the type of cannabinoid present, the intrauterine tissue, and CNR1/2 expression. Study of ECS gene expression in late pregnancy is therefore important to determine the contribution of this pathway to the normal and pathologic physiology of labour, for instance in preterm labour. Examining ECS gene expression in pregnancy may also contribute insight into the effects of exogenous cannabinoid consumption (reported in ~5% of pregnancies) on the late-pregnant uterus. To understand when and where cannabinoid signaling may impact uterine functions, we conducted an observational study on pregnant human and mouse intrauterine tissues. Methods: Human amnion, chorion, decidua, placenta, and upper/lower myometrium (n=6 participants) were biopsied at cesarean delivery (term, non-labour) to determine CNR expression by qPCR. Additional decidua (n=80) and upper/lower myometrium (n=82) samples were similarly obtained with and without labour, at term and preterm, for CNR expression analysis by qPCR. Mouse uteri for CNR expression analysis by qPCR were obtained from timed-mated C57BL6 mice at days 15-20 of pregnancy, in active labour, or post-partum. Human and mouse expression data were analyzed by Student’s t-test, one-way ANOVA (Bonferroni post-hoc), and Pearson’s correlation, as appropriate. Results: Term non-labour chorion, placenta, myometrium and decidua express CNRs, with 2-fold higher expression of CNR1 versus CNR2. In myometrium and decidua, neither CNR1 or CNR2 expression differed with labour status. CNR1 expression correlated positively with gestational age in decidua (r=0.346, p=0.0266) and lower segment myometrium (r=0.3667, p=0.0270) with labour. In mouse uteri, Cnr1 and Cnr2 expression significantly increased post-partum compared to mid/late pregnancy (vs day 19, p<0.0001 Cnr1; vs days 15-18, p<0.05 Cnr2). Conclusion: ECS may occur in the human decidua and myometrium throughout pregnancy and labour as CNR1 and CNR2 expression is maintained throughout. In our mouse model, higher Cnr1 and Cnr2 expression post-partum suggests that ECS may play a role in uterine resolution following delivery. Additional RNAseq analysis of the mouse tissues studied is underway to address whether expression of genes involved in production, transport or metabolism of cannabinoids are altered in late pregnancy or with the initiation of parturition.

Role of endocannabinoid signaling in a septohabenular pathway in the regulation of anxiety- and depressive-like behavior.

Enhancing endocannabinoid signaling produces anxiolytic- and antidepressant-like effects, but the neural circuits involved remain poorly understood. The medial habenula (MHb) is a phylogenetically-conserved epithalamic structure that is a powerful modulator of anxiety- and depressive-like behavior. Here, we show that a robust endocannabinoid signaling system modulates synaptic transmission between the MHb and its sole identified GABA input, the medial septum and nucleus of the diagonal band (MSDB). With RNAscope in situ hybridization, we demonstrate that key enzymes that synthesize or degrade the endocannabinoids 2-arachidonylglycerol (2-AG) or anandamide are expressed in the MHb and MSDB, and that cannabinoid receptor 1 (CB1) is expressed in the MSDB. Electrophysiological recordings in MHb neurons revealed that endogenously-released 2-AG retrogradely depresses GABA input from the MSDB. This endocannabinoid-mediated depolarization-induced suppression of inhibition (DSI) was limited by monoacylglycerol lipase (MAGL) but not by fatty acid amide hydrolase. Anatomic and optogenetic circuit mapping indicated that MSDB GABA neurons monosynaptically project to cholinergic neurons of the ventral MHb. To test the behavioral significance of this MSDB-MHb endocannabinoid signaling, we induced MSDB-specific knockout of CB1 or MAGL via injection of virally-delivered Cre recombinase into the MSDB of Cnr1loxP/loxP or MgllloxP/loxP mice. Relative to control mice, MSDB-specific knockout of CB1 or MAGL bidirectionally modulated 2-AG signaling in the ventral MHb and led to opposing effects on anxiety- and depressive-like behavior. Thus, depression of synaptic GABA release in the MSDB-ventral MHb pathway may represent a potential mechanism whereby endocannabinoids exert anxiolytic and antidepressant-like effects.

Distinctive Evidence Involved in the Role of Endocannabinoid Signalling in Parkinson's Disease: A Perspective on Associated Therapeutic Interventions.

Current pharmacotherapy of Parkinson’s disease (PD) is symptomatic and palliative, with levodopa/carbidopa therapy remaining the prime treatment, and nevertheless, being unable to modulate the progression of the neurodegeneration. No available treatment for PD can enhance the patient’s life-quality by regressing this diseased state. Various studies have encouraged the enrichment of treatment possibilities by discovering the association of the effects of the endocannabinoid system (ECS) in PD. These reviews delineate the reported evidence from the literature on the neuromodulatory role of the endocannabinoid system and expression of cannabinoid receptors in symptomatology, cause, and treatment of PD progression, wherein cannabinoid (CB) signalling experiences alterations of biphasic pattern during PD progression. Published papers to date were searched via MEDLINE, PubMed, etc., using specific key words in the topic of our manuscript. Endocannabinoids regulate the basal ganglia neuronal circuit pathways, synaptic plasticity, and motor functions via communication with dopaminergic, glutamatergic, and GABAergic signalling systems bidirectionally in PD. Further, gripping preclinical and clinical studies demonstrate the context regarding the cannabinoid compounds, which is supported by various evidence (neuroprotection, suppression of excitotoxicity, oxidative stress, glial activation, and additional benefits) provided by cannabinoid-like compounds (much research addresses the direct regulation of cannabinoids with dopamine transmission and other signalling pathways in PD). More data related to endocannabinoids efficacy, safety, and pharmacokinetic profiles need to be explored, providing better insights into their potential to ameliorate or even regress PD.

Recipient Signaling through the Cannabinoid Type 2 Receptor Regulates IL-6 Independent Neuroinflammation during Graft Versus Host Disease

Neurotoxicity is an increasingly recognized complication of cancer immunotherapy occurring after allogeneic hematopoietic stem cell transplantation (HSCT), immune checkpoint inhibitor administration, and chimeric antigen receptor T cell therapy. While inflammatory cytokines are thought to play a pivotal role, the immunological and biochemical pathways by which neuroinflammation occurs in these settings are still incompletely understood. Using well-defined murine models of graft versus host disease (GVHD) [C57BL/6 (H-2b)→Balb/c (H-2d) and B10.BR(H-2k)→B6(H-2b)] to examine T cell-mediated inflammation in the central nervous system (CNS) after allogeneic HSCT, we previously identified host IL-6 production as a pivotal cytokine responsible for inducing neuroinflammation. Notably, whereas blockade of this signaling pathway reduced donor T cell infiltration, inflammatory cytokine production, and microglial cell expansion in the brain, it did not reverse dysregulation in the tryptophan metabolic pathway and the subsequent production of neurotoxic metabolites, indicating the presence of an IL-6 independent pathway that promoted CNS inflammation. To understand the pathophysiology of this process, we examined the role of endocannabinoid signaling through the type 2 cannabinoid receptor (CB2R) since this receptor is expressed on all immune cells and the natural ligands for this receptor modulate cognitive and behavioral function. Using genetic approaches to define the role of CB2R signaling, we observed that absence of the CB2R on donor cells had no effect on T cell infiltration or expression of inflammatory cytokines (IFN-γ, GM-CSF, IL-6 or TNF-α) when compared to animals transplanted with wild type (WT) grafts. There was also no difference in the production of neurotoxic tryptophan metabolites when assessed by mass spectrometry. Conversely, absence of CB2R expression on host tissues significantly reduced donor T cell infiltration and inflammatory cytokine gene expression, indicating that host CB2R expression potentiated CNS inflammation. In addition, there was a significant decrease in gene expression of indole 2,3 dioxygenase and kynureninase which are key enzymes involved in the pathway by which tryptophan is metabolized to neurotoxic metabolites under inflammatory conditions, as well as a decreased ratio of neurotoxic (i.e. 3-hydroxykynurenine) to neuroprotective (i.e. kynurenic acid) tryptophan metabolites. Pharmacological inhibition of CB2R signaling with a peripherally restricted CB2R-selective antagonist had no effect on reducing GVHD-induced inflammation indicating that peripheral regulation of CB2R did not alter CNS inflammation and that a CB2R-expressing population resident in the brain was critical for regulating disease. To identify CB2R+ cells in the brain, we employed a novel CB2R-EGFP reporter mouse and examined regions with diverse functional roles (i.e. amygdala, brainstem, and cerebellum). These studies revealed that CB2R expression was detected only on host microglial cells. To determine whether microglial cells promoted CNS inflammation, we employed IL-34-/- mice, which specifically lack microglial cells, as recipients and demonstrated a significant reduction in donor T cells and inflammatory cytokines in the brain compared to WT controls. Thus, CB2R expression on microglial cells appeared to be critical for the regulation of inflammation in the CNS. Since we had shown that blockade of IL-6 failed to prevent tryptophan metabolite dysregulation, we examined whether this treatment had any effect on microglial CB2R expression. Treatment with an anti-IL-6 receptor antibody did not alter CB2R expression, indicating that the proinflammatory effects mediated by host CB2R+ microglial cells were IL-6 independent. Collectively, these results indicate that signaling through the CB2R expressed on microglial cells appears to constitute an IL-6 independent pathway by which neuroinflammation and concurrent tryptophan metabolite dysregulation is propagated. These data also provide an explanation for the failure of anti-IL-6R antibody administration with tocilizumab to prevent and/or treat neurotoxicity occurring after T cell-directed immunotherapy, and suggests that strategies targeting key enzymes in the tryptophan metabolic pathway or the CB2R directly may be novel therapeutic approaches to ameliorate this complication. Disclosures No relevant conflicts of interest to declare.

Microglia Sculpt Sex Differences in Social Behavior

Microglia are increasingly recognized as developmental sculptors of neural circuits. In this issue of Neuron, VanRyzin etal. (2019) demonstrate a novel mechanism by which endocannabinoids drive microglia to phagocytose newborn astrocytes in the medial amygdala of male rats, promoting sex differences in social play behavior.

The Lateral Habenula Directs Coping Styles Under Conditions of Stress via Recruitment of the Endocannabinoid System

BackgroundThe ability to effectively cope with stress is a critical determinant of disease susceptibility. The lateral habenula (LHb) and the endocannabinoid (ECB) system have independently been shown to be involved in the selection of stress coping strategies, yet the role of ECB signaling in the LHb remains unknown. MethodsUsing a battery of complementary techniques in rats, we examined the localization of type-1 cannabinoid receptors (CB1Rs) and assessed the behavioral and neuroendocrine effects of intra-LHb CB1R manipulations. We further tested the extent to which the ECB system in the LHb is impacted following chronic unpredictable stress or social defeat stress, and whether manipulation of LHb CB1Rs can bias coping strategies in rats with a history of chronic stress. ResultsElectron microscopy studies revealed CB1R expression on presynaptic axon terminals, postsynaptic membranes, mitochondria, and glial processes in the rat LHb. In vivo microdialysis experiments indicated that acute stress increased the amount of 2-arachidonoylglycerol in the LHb, while intra-LHb CB1R blockade increased basal corticosterone, augmented proactive coping strategies, and reduced anxiety-like behavior. Basal LHb 2-arachidonoylglycerol content was similarly elevated in rats that were subjected to chronic unpredictable stress or social defeat stress and positively correlated with adrenal weight. Finally, intra-LHb CB1R blockade increased proactive behaviors in response to a novel conspecific, increasing approach behaviors irrespective of stress history and decreasing the latency to be attacked during an agonistic encounter. ConclusionsAlterations in LHb ECB signaling may be relevant for development of stress-related pathologies in which LHb dysfunction and stress-coping impairments are hallmark symptoms.

Drinking during pregnancy: Potential role of endocannabinoid signaling in fetal alcohol effects

Drinking during pregnancy: Potential role of endocannabinoid signaling in fetal alcohol effects

Positron Emission Tomography Studies on Cannabinoid Receptor Type 1 in Schizophrenia

A recent issue of Biological Psychiatry titled “Cannabinoids and Psychotic Disorders” offered insights into a rapidly expanding interdisciplinary research field. One of the many important discoveries highlighted in that issue is the involvement of cannabinoid receptor type 1 (CB1R) in a synaptic retrograde signaling mechanism that controls gamma-aminobutyric acid (GABA) and glutamate release, regulates brain oscillatory activity, and contributes to the maturation of neural networks ( 1 Skosnik P.D. Cortes-Briones J.A. Hajos M. It’s all in the rhythm: The role of cannabinoids in neural oscillations and psychosis. Biol Psychiatry. 2016; 79: 568-577 Abstract Full Text Full Text PDF Scopus (43) Google Scholar , 2 Volk D.W. Lewis D.A. The role of endocannabinoid signaling in cortical inhibitory neuron dysfunction in schizophrenia. Biol Psychiatry. 2016; 79: 595-603 Abstract Full Text Full Text PDF Scopus (41) Google Scholar ). Evidence of impairment in these processes in schizophrenia suggests a role for CB1R. Postmortem investigations have yielded a complex pattern of findings, with increased CB1R binding and decreased CB1R messenger RNA and protein levels in the dorsolateral prefrontal cortex in schizophrenia ( 2 Volk D.W. Lewis D.A. The role of endocannabinoid signaling in cortical inhibitory neuron dysfunction in schizophrenia. Biol Psychiatry. 2016; 79: 595-603 Abstract Full Text Full Text PDF Scopus (41) Google Scholar ). To investigate CB1R in vivo, three positron emission tomography (PET) studies so far have compared subjects with schizophrenia with healthy control subjects ( 3 Ranganathan M. Cortes-Briones J. Radhakrishnan R. Thurnauer H. Planeta B. Skosnik P. et al. Reduced brain cannabinoid receptor availability in schizophrenia. Biol Psychiatry. 2016; 79: 997-1005 Google Scholar , 4 Wong D.F. Kuwabara H. Horti A.G. Raymont V. Brasic J. Guevara M. et al. Quantification of cerebral cannabinoid receptors subtype 1 (CB1) in healthy subjects and schizophrenia by the novel PET radioligand [11C]OMAR. Neuroimage. 2010; 52: 1505-1513 Crossref PubMed Scopus (163) Google Scholar , 5 Ceccarini J. De Hert M. Van Winkel R. Peuskens J. Bormans G. Kranaster L. et al. Increased ventral striatal CB1 receptor binding is related to negative symptoms in drug-free patients with schizophrenia. Neuroimage. 2013; 79: 304-312 Crossref PubMed Scopus (84) Google Scholar ), with the most recent one published in the current issue of Biological Psychiatry ( 3 Ranganathan M. Cortes-Briones J. Radhakrishnan R. Thurnauer H. Planeta B. Skosnik P. et al. Reduced brain cannabinoid receptor availability in schizophrenia. Biol Psychiatry. 2016; 79: 997-1005 Google Scholar ). A prior report on the feasibility of CB1R imaging with the radiotracer [124I]AM281 is not discussed here because it included only one participant, a patient with schizophrenia [cited in Ceccarini et al. ( 5 Ceccarini J. De Hert M. Van Winkel R. Peuskens J. Bormans G. Kranaster L. et al. Increased ventral striatal CB1 receptor binding is related to negative symptoms in drug-free patients with schizophrenia. Neuroimage. 2013; 79: 304-312 Crossref PubMed Scopus (84) Google Scholar )]. Reduced Brain Cannabinoid Receptor Availability in SchizophreniaBiological PsychiatryVol. 79Issue 12PreviewSeveral lines of evidence suggest the presence of abnormalities in the endocannabinoid (eCB) system in schizophrenia (SCZ). However, there are limited in vivo measures of the eCB system in SCZ. Full-Text PDF

Role of endocannabinoid signalling in the dorsolateral periaqueductal grey in the modulation of distinct panic-like responses.

Panic attacks, a major feature of panic disorder, can be modelled in rats by exposing animals to stimuli that induce escape reactions, such as the elevated T-maze or the activation of the dorsolateral periaqueductal grey. Since the cannabinoid CB1 receptor modulates various types of aversive responses, this study tested the hypothesis that enhancement of endocannabinoid signalling in the dorsolateral periaqueductal grey inhibits panic-like reactions in rats. Local injection of the CB1 agonist, arachidonoyl 2-Chloroethylamide (0.005-0.5 pmol), attenuated the escape response from the open arm of the elevated T-maze, a panicolytic effect. The anandamide hydrolysis inhibitor, URB597 (0.3-3 nmol), did not induce consistent results. In the test of dorsolateral periaqueductal grey stimulation with d,l-homocysteic acid, arachidonoyl 2-Chloroethylamide, at the lowest dose, attenuated the escape reaction. The highest dose of URB597 also inhibited this response, contrary to the result obtained in the elevated T-maze. This effect was reversed by the CB1 antagonist, AM251 (100 pmol). The present results confirm the anti-aversive property of direct CB1 receptor activation in the dorsolateral periaqueductal grey. The effect of the anandamide hydrolysis inhibitor, however, could be detected only in a model employing direct stimulation of this structure. Altogether, these results suggest that anandamide signalling is recruited only under certain types of aversive stimuli.

P.4.a.002 Role of endocannabinoid signaling in the dorsolateral periaqueductal gray on the modulation of distinct panic-like responses

P.4.a.002 Role of endocannabinoid signaling in the dorsolateral periaqueductal gray on the modulation of distinct panic-like responses

The Endocannabinoid System: From Basic Research to Translational Opportunity

This special issue of ACS Chemical Neuroscience, which was guest-edited by Rao Rapaka, Joni Rutter, and David Shurtleff and relates to a NIDA-ICRS co-sponsored workshop in July of 2011 entitled “Endocannabinoid Metabolic Enzymes and Drug Development”, focuses on the molecular composition, pharmacologic targeting, and physiological significance of the endocannabinoid system, a lipid signaling network that is the target of the psychoactive component of marijuana, Δ9-tetrahydrocannabinol (THC). In the early 1990s, the cannabinoid receptors CB1 and CB2 were identified as the molecular targets of THC and soon thereafter the endogenous cannabinoid ligands (“endocannabinoids”) N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) were discovered.1,2 Endocannabinoid signaling has been implicated in numerous physiological processes, including pain, inflammation, appetite, reproduction, and neurodegeneration. Preparations of the Cannabis plant have been used for medicinal purposes for thousands of years; however, the characteristic “high” elicited by direct cannabinoid agonists such as THC and their concomitant detrimental effects on cognition and motor control limit their widespread clinical use. Alternative strategies to harness the therapeutic potential of the endocannabinoid system include amplifying the actions of the body’s own endocannabinoids by disrupting their metabolism and utilizing bioactive, nonpsychoactive phytocannabinoids. Several of the articles featured in this issue describe the characterization of endocannabinoid metabolic enzymes, which are considered to be promising drug targets for the treatment of conditions such as chronic pain and neuropsychiatric disorders. Piscitelli and Di Marzo present a review highlighting how the complexity of endocannabinoid inactivation may impact endocannabinoid hydrolase-based analgesics.3 Exemplifying this complexity, anandamide and related N-acylethanolamines can be hydrolyzed by multiple enzymes including fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA).4 Kaczocha and colleagues describe FAAH-mediated anandamide hydrolysis in lipid vesicles,5 while Tai and colleagues report on endogenous molecules that stimulate NAAA activity.6 2-AG degradation in the brain and many peripheral tissues is catalyzed primarily by monoacylglycerol lipase (MAGL).4 To investigate the mechanisms of pharmacological inactivation of MAGL, Karageorgos and colleagues describe the generation and characterization of engineered human MAGL mutants.7 In addition to hydrolysis, polyunsaturated endocannabinoids are prone to enzymatic oxidation, which Dainese and colleagues demonstrate affects membrane properties.8 Selective pharmacological agents that disrupt endocannabinoid metabolism have proven to be powerful tools to investigate the effects of enhanced endocannabinoid signaling in vivo. In this issue, two articles describe the development of advanced small-molecule inhibitors of endocannabinoid hydrolases. Otrubova and Boger have contributed a review summarizing the history of α-ketoheterocycle-based FAAH inhibitors.9 Niphakis and colleagues describe a class of O-hydroxyacetamide carbamates, which potently and selectively block endocannabinoid hydrolases.10 This issue also contains two basic science research articles describing the metabolomic and behavioral consequences of blocking endocannabinoid metabolism in mammals. Wiskerke and colleagues employed in vivo microdialysis techniques to directly monitor interstitial endocannabinoid levels in the brains of rats and mice following inhibition of endocannabinoid clearance.11 Wise and colleagues describe the effects of simultaneously inhibiting FAAH and MAGL on short-term memory in mice.12 Two additional papers focus on physiological processes affected by endo- and exocannabinoids. In a review contributed by Sun and Dey, the role of endocannabinoid signaling in female fertility is discussed.13 Valdeolivas and colleagues present new research describing the neuroprotective effects of a combination of phytocannabinoids in an inflammatory model of Huntington’s disease.14 Together, the papers presented in this special issue of ACS Chemical Neuroscience nicely capture the many exciting advances that have been made in our understanding of the endocannabinoid system. This knowledge, combined with the emergence of selective and in vivo active chemical probes to perturb specific protein components of the endocannabinobid system, promises to enable a full exploration of its physiological functions and translational potential for treating a range of human diseases.

Requirement of cannabinoid CB(1) receptors in cortical pyramidal neurons for appropriate development of corticothalamic and thalamocortical projections.

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.

Abstract LB-394: Marijuana use and testicular germ cell tumors

Abstract Introduction: Since the early 1970's the incidence of testicular germ cell tumors (TGCT) in the U.S. has been increasing, however, environmental exposures accounting for this rise have not been identified. Trends in marijuana use have also increased during this period. Chronic marijuana use has been shown to produce adverse effects on the human endocrine and reproductive systems. A prior study of marijuana use and TGCT reported a significant association among frequent and long-term current users. We aimed to evaluate the relationship of marijuana use and TGCT in an existing hospital-based case-control study conducted at the University of Texas M. D. Anderson Cancer Center. Methods: TGCT cases diagnosed between January 1990 and October 1996 (n=187) and male friend controls (n=148) matched on age, race and state of residence were enrolled in the study. Associations of marijuana use and TGCT were estimated using unconditional logistic regression adjusting for age, race, prior cryptorchidism, cigarette smoking and alcohol intake. Associations stratified on histology (seminoma, nonseminoma) were also examined. Results: Overall, TGCT cases were more likely to be frequent marijuana users (daily or >1 day per week) than were controls [OR: 2.2, 95% CI: 1.0, 5.2]. In the analysis limited to nonseminomas, cases were significantly more likely than controls to be frequent users [OR: 3.3, 95% CI: 1.3, 8.6] and to be long-term users (10+ years) [OR: 2.3, 95% CI: 1.0, 5.8]. As the use of friend controls can bias the results of retrospective studies, we evaluated the extent to which reporting of marijuana use among the controls was consistent with data from the 1996 population-based National Survey on Drug Use and Health (NSDUH). We compared the observed number of controls who reported ever using marijuana with the expected number based on age- and race-specific proportions in the NSDUH and found no significant difference [O/E ratio: 0.96; 95% CI: 0.74, 1.22], suggesting selection or reporting bias was not an explanation for the observed associations. Discussion: Our finding of an association between frequent marijuana use and TGCT, particularly among men with nonseminoma is consistent with the findings of a previous report. Marijuana use may be related to nonseminoma due to activation of the cannabinoid receptors during a critical time period in pubertal development; however it is also plausible that biologically active components of marijuana may function through pathways other than the endocannabinoid system. Additional studies of marijuana use and TGCT are warranted, especially studies evaluating the role of endocannabinoid signaling and cannabinoid receptors in TGCT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-394.

Substantially altered expression pattern of cannabinoid receptor 2 and activated endocannabinoid system in patients with severe heart failure

Animal studies suggest that the endocannabinoid system (ECS) plays a role in the regulation of myocardial contractility and in the pathogenesis of heart failure. The current study aimed to proof the existence of endocannabinoid receptors on human ventricular myocardium and to determine whether human chronic heart failure (CHF) is associated with changes in endocannabinoid receptor expression and distribution. Expression of cannabinoid receptor 1 (CB1) and cannabinoid receptor (CB2) on human heart was assessed by means of real-time PCR and immunohistochemistry. On healthy human left ventricular myocardium, mRNA transcripts of CB1 and CB2 receptors were expressed in an almost equal proportion. In patients with CHF, mRNA expression of CB1 receptors was shown to be downregulated 0.7-fold (0.7.±0.15, n=12, p<0.01), whereas expression of CB2 receptors was upregulated more than 11-fold (11.6±4.5; n=12; p<0.005). Corresponding results were obtained by immunohistochemistry. Blood levels of endocannabinoids were significantly elevated (anandamide 3.5-fold (p<0.001); 2-AG 7-fold (p=0.02)) in patients with CHF, as compared to healthy volunteers. Both CB1 and CB2 receptors are present on healthy human left ventricular myocardium in a balanced distribution. Patients suffering from CHF exhibit a shift of the CB1–CB2 receptor ratio towards expression of CB2 receptors combined with significantly elevated peripheral blood levels of endocannabinoids indicating an activation of the ECS. These results might open up new perspectives regarding the role of endocannabinoid signalling in CHF and its potential as a target for pharmacological modulation.

Hot Topic: [Role of Endocannabinoid Signaling in Alcohol and Drug Addiction (Guest Editor: Basalingappa L. Hungund)

Hot Topic: [Role of Endocannabinoid Signaling in Alcohol and Drug Addiction (Guest Editor: Basalingappa L. Hungund)

Editorial: [Role of Endocannabinoid Signaling in Alcohol and Drug Addiction (Guest Editor: Basalingappa L. Hungund)

Editorial: [Role of Endocannabinoid Signaling in Alcohol and Drug Addiction (Guest Editor: Basalingappa L. Hungund)

Differential Effects of Endocannabinoids on Glutamatergic and GABAergic Inputs to Layer 5 Pyramidal Neurons

Endocannabinoids are emerging as potent modulators of neuronal activity throughout the brain, and activation of the type-1 cannabinoid receptor (CB1R) reduces sensory-evoked cortical responses in vivo, presumably by decreasing excitatory transmission. In the neocortex, CB1R is differentially expressed across neocortical laminae, with highest levels of expression in layers 2/3 and 5. Although we have shown that cannabinoid signaling in layer 2/3 of somatosensory cortex targets both gamma-aminobutyric acid (GABA) and glutamate release, the predominant effect is a net increase in pyramidal neuron (PN) activity due to disinhibition. The role of endocannabinoid signaling in layer 5, the main output layer of the neocortex, remains unknown. We found that inducing activity in layer 5 PNs resulted in endocannabinoid-mediated depolarization-induced suppression of excitation (DSE), whereas the majority of inhibitory inputs were cannabinoid insensitive. Furthermore, in contrast to layer 2/3, the net effect of elevations in action potential firing of layer 5 PNs was an endocannabinoid-mediated decrease in PN spike probability. Interestingly, excitatory synaptic currents in layer 5 evoked by intralaminar stimulation were cannabinoid sensitive, whereas inputs evoked from layer 2/3 were insensitive, suggesting specificity of cannabinoid signaling across glutamatergic inputs. Thus, cannabinoids have differential effects on excitation and inhibition across cortical layers, and endocannabinoid signaling in layer 5 may serve to selectively decrease the efficacy of a subset of excitatory inputs.

Synergistic interactions between cannabinoids and environmental stress in the activation of the central amygdala.

Anxiety and panic are the most common adverse effects of cannabis intoxication; reactions potentiated by stress. Data suggest that cannabinoid (CB1) receptor modulation of amygdalar activity contributes to these phenomena. Using Fos as a marker, we tested the hypothesis that environmental stress and CB1 cannabinoid receptor activity interact in the regulation of amygdalar activation in male mice. Both 30 min of restraint and CB1 receptor agonist treatment (Delta9-tetrahydrocannabinol (2.5 mg/kg) or CP55940 (0.3 mg/kg); by i.p. injection) produced barely detectable increases in Fos expression within the central amygdala (CeA). However, the combination of restraint and CB1 agonist administration produced robust Fos induction within the CeA, indicating a synergistic interaction between environmental stress and CB1 receptor activation. An inhibitor of endocannabinoid transport, AM404 (10 mg/kg), produced an additive interaction with restraint within the CeA. In contrast, fatty acid amide hydrolase (FAAH) inhibitor-treated mice (URB597, 1 mg/kg) and FAAH-/- mice did not exhibit any differences in amygdalar activation in response to restraint compared to control mice. In the basolateral (BLA) and medial amygdala, restraint stress produced a low level of Fos induction, which was unaffected by cannabinoid treatment. Interestingly, the CB1 receptor antagonist SR141716 dose-dependently increased Fos expression in the BLA and CeA. These data suggest the CeA is an important neural substrate subserving the interactions between cannabinoids and environmental stress, and could be relevant to understanding the context-dependent emotional and affective changes induced by marijuana intoxication and the role of endocannabinoid signaling in the modulation of amygdalar activity.