Behavioural analysis of mouse mutants with altered neuronal and glial genes

Im ersten Teil dieser Doktorarbeit beabsichtigte meine Arbeit, die funktionelle Beteiligung des CB1 Rezeptors, einer Hauptkomponente des neuronalen Endocannabinoid-Systems (ECS), an der Ausbildung von verschiedenen Verhaltensphänotypen mit Hilfe von konditionalen Mausmutanten, denen der CB1 Rezeptor auf verschiedenen neuronalen Unterpopulationen fehlt, aufzuschlüsseln und zu untersuchen. Verschiedene Verhaltensmodelle wurden hierzu getestet. Dabei lag der Fokus dieser Arbeit auf der CB1f/f;D1-Cre Mauslinie, welche der CB1 Rezeptor auf den D1 Rezeptor exprimierenden Neuronen des Striatums fehlt. Ich konnte zeigen, dass der Verlust des CB1 Rezeptors auf diesen Neuronen keinen Einfluss auf basale neurologische Funktionen, Gewicht, Bewegung, Exploration, Sozialverhalten, Angst und Stressbewältigung der Tiere hat, jedoch eine Beteiligung an der Entwicklung von Suchtverhalten gegeben ist. Bei Betrachtung des Kokain-induzierten Suchtverhaltens zeigten die konditionalen Mausmutanten eine reduzierte Suchtanfälligkeit sowohl im Vergleich zu Tieren mit einem totalen CB1 Rezeptor Verlust in allen Körperzellen, als auch zu genetisch unveränderten Kontrollmäusen beider Linien.rnDes Weiteren zeigen die Ergebnisse dieser Studie eine große, aber gegensätzliche Beteiligung des ECS bei der Regulation von Exploration in Abhängigkeit des Verlustes des CB1 Rezeptors auf GABAergen Neuronen des Vorderhirns und kortikalen glutamatergen Neuronen, jedoch nicht auf striatalen Neuronen alleine. Zusätzlich war ich in der Lage, die Wichtigkeit des genetischen Hintergrunds von Mauslinien nicht nur auf die Ausbildung von spezifischen Verhaltensphänotypen, sondern auch auf die Genexpression zu zeigen.rnIn dem zweiten Teil dieser Arbeit, in dem ich mich auf die Funktion von Gliazellen konzentrierte, wurden ebenfalls Mausmutanten in verschiedenen Verhaltensmodellen getestet. Ein genetisches Auslöschen des NG2 Glykoproteins in Gliazellen sorgt in den Knock-out Mäusen für ein schlechteres Hörvermögen und ein reduziertes Depressionsverhalten im Vergleich zu ihren Wildtyp-Kontrollmäusen. Interessanterweise zeigten diese Tiere auch eine reduzierte Empfänglichkeit bei chemisch induzierten epileptischen Krämpfen, was eine Rolle des NG2 Glykoproteins bei der Kontrolle der glutamatergen Homöostase vorschlägt, die wahrscheinlich durch Strukturänderungen der Neuron-Glia-Synapse verursacht wird. rn

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'.

A critical review of both the synthesis approach and the receptor profile of the 8-chloro-1-(2′,4′-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide and analogue derivatives

We thank European Commission (project “PET BRAIN: Mapping the brain with PET radiolabeled cannabinoid CB1 ligands”; FP7-People-2009-IAPP; Grant Agreement N.25142).

Role of organic acids and phytonutrients as natural alternatives to antibiotics in supporting intestinal functionality

The gastrointestinal tract (GIT) represents the major portion of the body that interfaces with the external environment, with the double function of food processing and line of defense of the body. Numerous components support and regulate the barrier function of the GIT, such as tight junctions (TJs), cytokines, commensal and pathogenic microorganisms, and other systems of the organism, as the endocannabinoid system (ECS). The ECS can control several gastrointestinal functions, as well as the regulation of intestinal inflammation. Failure of the intestinal barrier function triggers an increase of the concentration of pro-inflammatory cytokines and leads to a reduction in intestinal functionality. This thesis aimed to explore the potential of natural compounds as a new alternative approach to antibiotics not only as antimicrobial, but also supporting intestinal maturation and integrity, and as immune-boosting agents. Different experiments were performed to evaluate the potential of nature-identical compounds (NICs), organic acids (OAs), and essential oils (EOs) to support and fight various stressful stimuli. In vitro, a well characterized blend of NICs and OAs were able to improve TJs and transepithelial electrical resistance (TEER) in an intestinal cell line, exerting an anti-inflammatory potential. EOs enhanced TEER and TJs mRNA levels, with a reduction of paracellular permeability, showing antioxidant and antimicrobial properties. In vivo, thymol modulates the gene expression of ECS and gut chemosensing in the GIT of piglets, where the precise localization of the cannabinoid receptors was immunohistochemically confirmed, suggesting an anti-inflammatory potential. In conclusion, natural alternative molecules represent an effective alternative to support or replace the classical pharmacological prophylaxis. These alternative molecules act not only as antimicrobial agents, but also exerted a crucial role in supporting the intestinal barrier function, preventing oxidative stress, and reducing inflammation. Moreover, thymol seems able to modulate the ECS, representing a novel frontier to support animal health and productivity.

Transgenic mice as a tool for depression research: examples from the endocannabinoid and the corticotropin-releasing hormone system

Major depression belongs to the most serious and widespread psychiatric disorders in today’s society. There is a great need for the delineation of the underlying molecular mechanisms as well as for the identification of novel targets for its treatment. In this thesis, transgenic mice of the endocannabinoid and the corticotropin-releasing hormone (CRH) system were investigated to determine the putative role of these systems for depression-like phenotypes in mice. In the first part of the thesis, we found that the endocannabinoid system was prominently involved in a brain region-specific and temporally controlled manner in acute as well as in chronic stress processing. Genetic deletion in combination with pharmacological intervention revealed the importance of a fully functional endocannabinoid system for efficient neuroendocrine and behavioral stress coping. Accordingly, cannabinoid type 1 (CB1) receptor-deficient mice displayed several depression-like symptoms and molecular alterations, including “behavioral despair”, stress hormone hypersecretion and decreased glucocorticoid receptor and brain-derived neurotrophic factor expression in the hippocampus. However, the endocannabinoid system was dispensable for the efficacy of currently used antidepressant drugs. To facilitate future endocannabinoid research, a transgenic mouse was generated, which overexpressed the CB1 receptor protein fused to a fluorescent protein. In the second part of the thesis, conditional brain region-specific CRH overexpressing mice were evaluated as a model for pathological chronic CRH hyperactivation. Mutant mice showed aberrant neuroendocrine and behavioral stress coping and hyperarousal due to CRH-induced activation of the noradrenergic system in the brain. Mutant mice appeared to share similarities with naturally occurring endogenous CRH activation in wild-type mice and were sensitive to acute pharmacological blockade of CRH receptor type 1 (CRH-R1). Thus, CRH overexpressing mice serve as an ideal in vivo tool to evaluate the efficacy of novel CRH-R1 antagonists. Together, these findings highlight the potential of transgenic mice for the understanding of certain endo-phenotypes (isolated symptoms) of depression and their molecular correlates.

FACILITATION OF ENDOCANNABINOID EFFECTS IN THE VENTRAL HIPPOCAMPUS MODULATES ANXIETY-LIKE BEHAVIORS DEPENDING ON PREVIOUS STRESS EXPERIENCE

Although several pieces of evidence indicate that the endocannabinoid system modulates anxiety-like behaviors and stress adaptation, few studies have investigated the brain sites of these effects. The ventral hippocampus (VHC) has been related to anxiety behaviors and has a high expression of cannabinoid-1 (CBI) receptors. Moreover, endocannabinoid signaling in the hippocampus is proposed to regulate stress adaptation. In the present study we investigated the role of previous stressful experience on the effects of AM404, an anandamide uptake inhibitor, microinjected into the VHC of rats submitted to the elevated plus maze (EPM), a widely used animal model of anxiety. Stressed animals were forced restrained for two h 24 h before the test. AM404 (5-50 pmol) microinjection promoted an anxiogenic-like effect in non-stressed rats but decreased anxiety in stressed animals. AM251 (0.01 to 1000 pmol), a CBI receptor antagonist, failed to change behavior in the EPM over a wide dose range but prevented the effects of AM404. Anxiolytic-like effects of AM404 (5 pmol) intra-VHC injection were also observed in the Vogel conflict test (VCT), another model of anxiety that involves previous exposure to stressful situations (48 h of water deprivation). These results suggest that facilitation of endocannabinoid system neurotransmission in the ventral hippocampus modulates anxiety-like behaviors and that this effect depends on previous stress experience. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Endocannabinoid regulation of acute and protracted nicotine withdrawal: effect of FAAH inhibition.

Evidence shows that the endocannabinoid system modulates the addictive properties of nicotine. In the present study, we hypothesized that spontaneous withdrawal resulting from removal of chronically implanted transdermal nicotine patches is regulated by the endocannabinoid system. A 7-day nicotine dependence procedure (5.2 mg/rat/day) elicited occurrence of reliable nicotine abstinence symptoms in Wistar rats. Somatic and affective withdrawal signs were observed at 16 and 34 hours following removal of nicotine patches, respectively. Further behavioral manifestations including decrease in locomotor activity and increased weight gain also occurred during withdrawal. Expression of spontaneous nicotine withdrawal was accompanied by fluctuation in levels of the endocannabinoid anandamide (AEA) in several brain structures including the amygdala, the hippocampus, the hypothalamus and the prefrontal cortex. Conversely, levels of 2-arachidonoyl-sn-glycerol were not significantly altered. Pharmacological inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for the intracellular degradation of AEA, by URB597 (0.1 and 0.3 mg/kg, i.p.), reduced withdrawal-induced anxiety as assessed by the elevated plus maze test and the shock-probe defensive burying paradigm, but did not prevent the occurrence of somatic signs. Together, the results indicate that pharmacological strategies aimed at enhancing endocannabinoid signaling may offer therapeutic advantages to treat the negative affective state produced by nicotine withdrawal, which is critical for the maintenance of tobacco use.

Role of the cannabinoid system in the effects induced by nicotine on anxiety-like behaviour in mice

Rationale: Acute behavioural effects and motivational responses induced by nicotine can be modulated by the endocannabinoid system supporting the existence of a physiological interaction between these two systems. Objectives: The present study was designed to examine the possible involvement of the cannabinoid system in the anxiolytic- and anxiogenic-like responses induced by nicotine in mice. Methods: Animals were only exposed once to nicotine. The acute administration of low (0.05, sc) or high (0.8 mg/kg, sc) doses of nicotine produced opposite effects in the elevated plus-maze, i.e., anxiolytic- and anxiogenic-like responses, respectively. The effects of the pretreatment with the CB1 cannabinoid receptor antagonist, rimonabant (0.25, 0.5 and 1 mg/kg, ip), and the cannabinoid agonist, 9-tetrahydrocannabinol (0.1 mg/kg, ip), were evaluated on the anxiolytic- and anxiogenic-like responses induced by nicotine. Results: Rimonabant completely abolished nicotine-induced anxiolytic-like effects and increased the anxiogenic-like responses of nicotine, suggesting an involvement of CB1 receptors in these behavioural responses. On the other hand, 9-tetrahydrocannabinol failed to modify nicotine anxiolytic-like responses, but attenuated its anxiogenic-like effects. In addition the association of non-effective doses of 9-tetrahydrocannabinol and nicotine produced clear anxiolytic-like responses. Conclusions: These results demonstrate that the endogenous cannabinoid system is involved in the regulation of nicotine anxiety-like behaviour in mice, and provide new findings to support the use of cannabinoid antagonists in the treatment of tobacco addiction.

Cannabis sativa and the endogenous cannabinoid system: therapeutic potential for appetite regulation

The herb Cannabis sativa (C. sativa) has been used in China and on the Indian subcontinent for thousands of years as a medicine. However, since it was brought to the UK and then the rest of the western world in the late 19th century, its use has been a source of controversy. Indeed, its psychotropic side effects are well reported but only relatively recently has scientific endeavour begun to find valuable uses for either the whole plant or its individual components. Here, we discuss evidence describing the endocannabinoid system, its endogenous and exogenous ligands and their varied effects on feeding cycles and meal patterns. Furthermore we also critically consider the mounting evidence which suggests non‐tetrahydrocannabinol phytocannabinoids play a vital role in C. sativa‐induced feeding pattern changes. Indeed, given the wide range of phytocannabinoids present in C. sativa and their equally wide range of intra‐, inter‐ and extra‐cellular mechanisms of action, we demonstrate that non‐Δ9tetrahydrocannabinol phytocannabinoids retain an important and, as yet, untapped clinical potential.

Phytocannabinoids as novel therapeutic agents in CNS disorders

The Cannabis sativa herb contains over 100 phytocannabinoid (pCB) compounds and has been used for thousands of years for both recreational and medicinal purposes. In the past two decades, characterisation of the body's endogenous cannabinoid (CB) (endocannabinoid, eCB) system (ECS) has highlighted activation of central CB1 receptors by the major pCB, Δ9-tetrahydrocannabinol (Δ9-THC) as the primary mediator of the psychoactive, hyperphagic and some of the potentially therapeutic properties of ingested cannabis. Whilst Δ9-THC is the most prevalent and widely studied pCB, it is also the predominant psychotropic component of cannabis, a property that likely limits its widespread therapeutic use as an isolated agent. In this regard, research focus has recently widened to include other pCBs including cannabidiol (CBD), cannabigerol (CBG), Δ9tetrahydrocannabivarin (Δ9-THCV) and cannabidivarin (CBDV), some of which show potential as therapeutic agents in preclinical models of CNS disease. Moreover, it is becoming evident that these non-Δ9-THC pCBs act at a wide range of pharmacological targets, not solely limited to CB receptors. Disorders that could be targeted include epilepsy, neurodegenerative diseases, affective disorders and the central modulation of feeding behaviour. Here, we review pCB effects in preclinical models of CNS disease and, where available, clinical trial data that support therapeutic effects. Such developments may soon yield the first non-Δ9-THC pCB-based medicines.

Il sistema degli endocannabinoidi nella cirrosi epatica complicata da infezione batterica: studio nell’uomo ed in un modello sperimentale animale

Gli endocannabinoidi (EC) sono una classe di composti che mimano gli effetti del Δ9-tetraidrocannabinolo. Essi comprendono l’anandamide (AEA) ed il 2-arachidonoilglicerolo (2-AG), molecole che interagiscono preferenzialmente con due specifici recettori, il CB1 ed il CB2. Più recente è la scoperta di due molecole EC simili, il palmitoiletanolamide (PEA) e l’oleiletanolamide (OEA), che tuttavia agiscono legando recettori diversi tra cui il PPARα ed il TRVP1. Studi sperimentali dimostrano che il sistema degli EC è attivato in corso di cirrosi epatica ed è coinvolto nel processo fibrogenico e nella patogenesi delle alterazioni emodinamiche tipiche della malattia. Esso partecipa alla patogenesi di alcune delle maggiori complicanze della cirrosi quali ascite, encefalopatia, cardiomiopatia ed infezioni batteriche. Scopo del presente studio è stato quello di studiare il ruolo degli EC nella patogenesi delle infezioni batteriche in corso di cirrosi. A tale scopo sono stati eseguiti un protocollo clinico ed uno sperimentale. Nel protocollo sperimentale la cirrosi è stata indotta mediante somministrazione di CCl4 per via inalatoria a ratti maschi Wistar. In tale protocollo i livelli circolanti di tutti gli EC sono risultati significativamente aumentati a seguito della somministrazione di LPS. La somministrazione dell’antagonista del recettore CB1, Rimonabant, inoltre, è stata efficace nel ridurre del 50% la mortalità a 24 ore dei ratti trattati col farmaco rispetto ai ratti trattati col solo LPS. Parallelamente il Rimonabant ha determinato una riduzione dell’espressione genica di molecole pro-infiammatorie e sostanze vasoattive. Lo studio clinico, condotto su 156 pazienti, ha confermato l’attivazione del sistema degli EC in corso di cirrosi epatica. Inoltre è stata identificata una forte correlazione tra il PEA e l’OEA e l’emodinamica sistemica ed una associazione con alcune delle maggiori complicanze. L’analisi statistica ha inoltre individuato l’OEA quale predittore indipendente di insufficenza renale e di sopravvivenza globale.

Der Einfluss des Endocannabinoidsystems auf die Alzheimer Pathologie

Die Alzheimer Krankheit ist eine der häufigsten neurodegenerativen Erkrankungen, deren Ursache, abgesehen von einem geringen Prozentsatz vererbter Formen, bisher nicht bekannt ist. Ein wichtiges Ziel der Grundlagenforschung liegt derzeit in der Modulation der APP-spaltenden Enzyme. Durch die Modulation dieser Enzyme könnten weniger schädigende Amyloid β-Peptide entstehen. Die Aktivität des ECS ist in vielen neurodegenerativen Krankheiten verändert. Protektive Eigenschaften der Cannabinoidrezeptoren wurden bei der Alzheimer Krankheit beschrieben. Deshalb sollte in dieser Arbeit der Einfluss des ECS auf die Pathogenese der Alzheimer Erkrankung untersucht werden. In Zellkultursystemen wurde der Einfluss von Cannabinoiden auf die Prozessierung des Amyloid-Vorläuferproteins analysiert. Durch Inkubation der Zellen mit CB1-Rezeptor Agonisten konnte die APP-Prozessierung zugunsten von sAPPα moduliert werden. Gleichzeitig führte die Inkubation mit Cannabinoiden zur reduzierten Amyloid β Menge im Medium der Zellen. In dieser Arbeit konnte die APP-Prozessierung durch die Aktivierung des CB1-Rezeptors zugunsten des nicht-amyloiden Wegs moduliert werden.rnIn einem Tiermodell wurde der Einfluss des CB1-Rezeptors in APP23 transgenen Mäusen untersucht. Der Knockout des CB1-Rezeptors führte in APP23 transgenen Tieren zu weitreichenden biochemischen Veränderungen. APP23/CB1-/--Tiere zeigten eine erhöhte Mortalität und ein sehr geringes Durchschnittsgewicht. Im Vergleich zu APP23/CB1+/+-Tieren führte der CB1-Rezeptor Knockout zur Reduktion der APP-Expression und dessen Prozessierungsprodukten. In den histologischen Untersuchungen wurde eine reduzierte Anzahl an amyloiden Plaques, sowie eine reduzierte Neuroinflammation ermittelt. Biochemische Untersuchungen zeigten, dass der CB1-Rezeptor einen möglichen regulatorischen Einfluss auf die Expression und Prozessierung von APP ausübt. Die Tiere mit der geringsten Plaque-Menge (APP23/CB1-/-) und einer reduzierten Prozessierung von sAPPα- und den CTFs zeigten die schlechteste Lernleistung im Morris Water-Maze. Deshalb müssen andere Faktoren (z.B. die Degradation der Myelinschicht) für die schlechte Lernleistung verantwortlich sein. Mit einem zweiten Tiermodell könnte in CB1-Knockout Mäusen durch den viral-vermittelten Gentransfer eine mögliche Toxizität von Aβ Peptiden untersucht werden. Die in dieser Arbeit ermittelten Ergebnisse zeigen, dass der CB1-Rezeptor an der Regulation der APP-Prozessierung beteiligt ist und zu proteinbiochemischen Veränderungen im Zell- und Tiermodell führt.

The major central endocannabinoid directly acts at GABA(A) receptors

GABA(A) receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA(A) receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of β(2) confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB(1)/CB(2) cannabinoid receptor double-KO, whereas β(2)-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA(A) receptor may have far-reaching consequences for the study of locomotion and sedation.

Endocannabinoid transport revisited.

Endocannabinoids are arachidonic acid-derived endogenous lipids that activate the endocannabinoid system which plays a major role in health and disease. The primary endocannabinoids are anandamide (AEA, N-arachidonoylethanolamine) and 2-arachidonoyl glycerol. While their biosynthesis and metabolism have been studied in detail, it remains unclear how endocannabinoids are transported across the cell membrane. In this review, we critically discuss the different models of endocannabinoid trafficking, focusing on AEA cellular uptake which is best studied. The evolution of the current knowledge obtained with different AEA transport inhibitors is reviewed and the confusions caused by the lack of their specificity discussed. A comparative summary of the most important AEA uptake inhibitors and the studies involving their use is provided. Based on a comprehensive literature analysis, we propose a model of facilitated AEA membrane transport followed by intracellular shuttling and sequestration. We conclude that novel and more specific probes will be essential to identify the missing targets involved in endocannabinoid membrane transport.