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The endocannabinoid system is a complex neuronal system involved in a number of biological functions, like attention, anxiety, mood, memory, appetite, reward, and immune responses. It is at the centre of scientific interest, which is driven by therapeutic promise of certain cannabinoid ligands and the changing legalization of herbal cannabis in many countries. The endocannabinoid system is a modulatory system, with endocannabinoids as retrograde neurotransmitters rather than direct neurotransmitters. Neuropharmacology of cannabinoid ligands in the brain can therefore be understood in terms of their modulatory actions through other neurotransmitter systems. The CB 1 receptor is chiefly responsible for effects of endocannabinoids and analogous ligands in the brain. An overview of the neuropharmacology of several cannabinoid receptor ligands, including endocannabinoids, herbal cannabis and synthetic cannabinoid receptor ligands is given in this review. Their mechanism of action at the endocannabinoid system is described, mainly in the brain. In addition, effects of cannabinoid ligands on other neurotransmitter systems will also be described, such as dopamine, serotonin, glutamate, noradrenaline, opioid, and GABA. In light of this, therapeutic potential and adverse effects of cannabinoid receptor ligands will also be discussed., (© 2020 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: Endogenous cannabinoids such as anandamide and 2-arachidonoylglycerol (2-AG) exert important regulatory influences on neuronal signaling, participate in short- and long-term forms of neuroplasticity, and modulate stress responses and affective behavior in part through the modulation of neurotransmission in the amygdala. Alcohol consumption alters brain endocannabinoid levels, and alcohol dependence is associated with dysregulated amygdalar function, stress responsivity, and affective control., Methods: The consequence of long-term alcohol consumption on the expression of genes related to endocannabinoid signaling was investigated using quantitative RT-PCR analyses of amygdala tissue. Two groups of ethanol (EtOH)-exposed rats were generated by maintenance on an EtOH liquid diet (10%): the first group received continuous access to EtOH for 15 days, whereas the second group was given intermittent access to the EtOH diet (5 d/wk for 3 weeks). Control subjects were maintained on an isocaloric EtOH-free liquid diet. To provide an initial profile of acute withdrawal, amygdala tissue was harvested following either 6 or 24 hours of EtOH withdrawal., Results: Acute EtOH withdrawal was associated with significant changes in mRNA expression for various components of the endogenous cannabinoid system in the amygdala. Specifically, reductions in mRNA expression for the primary clearance routes for anandamide and 2-AG (fatty acid amide hydrolase [FAAH] and monoacylglycerol lipase [MAGL], respectively) were evident, as were reductions in mRNA expression for CB(1) , CB(2) , and GPR55 receptors. Although similar alterations in FAAH mRNA were evident following either continuous or intermittent EtOH exposure, alterations in MAGL and cannabinoid receptor-related mRNA (e.g., CB(1) , CB(2) , GPR55) were more pronounced following intermittent exposure. In general, greater withdrawal-associated deficits in mRNA expression were evident following 24 versus 6 hours of withdrawal. No significant changes in mRNA expression for enzymes involved in 2-AG biosynthesis (e.g., diacylglicerol lipase-α/β) were found in any condition., Conclusions: These findings suggest that EtOH dependence and withdrawal are associated with dysregulated endocannabinoid signaling in the amygdala. These alterations may contribute to withdrawal-related dysregulation of amygdalar neurotransmission., (Copyright © 2011 by the Research Society on Alcoholism.)

The endocannabinoid 2-arachidonoylglycerol (2-AG) is a lipid mediator involved in various physiological processes. In response to neural activity, 2-AG is synthesized post-synaptically, then activates pre-synaptic cannabinoid CB1 receptors (CB1Rs) in a retrograde manner, resulting in transient and long-lasting reduction of neurotransmitter release. The signalling competence of 2-AG is tightly regulated by the balanced action between 'on demand' biosynthesis and degradation. We review recent research on monoacylglycerol lipase (MAGL), ABHD6 and ABHD12, three serine hydrolases that together account for approx. 99% of brain 2-AG hydrolase activity. MAGL is responsible for approx. 85% of 2-AG hydrolysis and colocalizes with CB1R in axon terminals. It is therefore ideally positioned to terminate 2-AG-CB1R signalling regardless of the source of this endocannabinoid. Its acute pharmacological inhibition leads to 2-AG accumulation and CB1R-mediated behavioural responses. Chronic MAGL inactivation results in 2-AG overload, desensitization of CB1R signalling and behavioural tolerance. ABHD6 accounts for approx. 4% of brain 2-AG hydrolase activity but in neurones it rivals MAGL in efficacy. Neuronal ABHD6 resides post-synaptically, often juxtaposed with CB1Rs, and its acute inhibition leads to activity-dependent accumulation of 2-AG. In cortical slices, selective ABHD6 blockade facilitates CB1R-dependent long-term synaptic depression. ABHD6 is therefore positioned to guard intracellular pools of 2-AG at the site of generation. ABHD12 is highly expressed in microglia and accounts for approx. 9% of total brain 2-AG hydrolysis. Mutations in ABHD12 gene are causally linked to a neurodegenerative disease called PHARC. Whether ABHD12 qualifies as a bona fide member to the endocannabinoid system remains to be established., (© 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.)

Prenatal development is highly sensitive to maternal drug use due to the vulnerability for disruption of the fetal brain with its ongoing neurodevelopment, resulting in lifelong consequences that can enhance risk for psychiatric disorders. Cannabis and cigarettes are the most commonly used illicit and licit substances, respectively, among pregnant women. Although the behavioral consequences of prenatal cannabis and cigarette exposure have been well-documented in epidemiological and clinical studies, only recently have investigations into the molecular mechanisms associated with the developmental impact of early drug exposure been addressed. This article reviews the literature relevant to long-term gene expression disturbances in the human fetal brain in relation to maternal cannabis and cigarette use. To provide translational insights, we discuss animal models in which protracted molecular consequences of prenatal cannabis and cigarette exposure can be better explored and which enable future evaluation of epigenetic pathways, such as DNA methylation and histone modification, that could potentially maintain abnormal gene regulation and related behavioral disturbances. Altogether, this information may help to address the current gaps of knowledge regarding the impact of early drug exposure that set in motion lifelong molecular disturbances that underlie vulnerability to psychiatric disorders., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Until recently, endocannabinoid (eCB) signalling was largely studied in the context of synaptic plasticity in the postnatal brain in the absence of detailed knowledge of the nature of the enzyme(s) responsible for the synthesis of the eCBs. However, the identification of two diacylglycerol lipases (DAGLα and DAGLβ) responsible for the synthesis of 2-arachidonoylglycerol (2-AG) has increased the understanding of where this eCB is synthesised in relationship to the expression of cannabinoid receptor (CB)1 and CB2. Furthermore, the generation of knockout animals for each enzyme has allowed for the direct testing of their importance for established and emerging eCB functions. Based on this, we now know that DAGLα is enriched in dendritic spines that appose CB1-positive synaptic terminals, and that 2-AG functions as a retrograde signal controlling synaptic strength throughout the nervous system. Consequently, we have built on the principle that expression of eCB components dictates function to identify additional physiological functions of this signalling cassette. A number of studies have now provided support for DAGL-dependent eCB signalling playing important roles in brain development and in cellular plasticity in the adult nervous system. In this article, we will review evidence based on the localisation of the enzymes, as well as from genetic and pharmacological studies, that show DAGL-dependent eCB signalling to play an important role in axonal growth and guidance during development, in retrograde synaptic signalling at mature synapses, and in the control of adult neurogenesis in the hippocampus and subventricular zone., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Endocannabinoid signalling participates in the control of neurogenesis, especially after brain insults. Obesity may explain alterations in physiology affecting neurogenesis, although it is unclear whether cannabinoid signalling may modulate neural proliferation in obese animals. Here we analyse the impact of obesity by using two approaches, a high-fat diet (HFD, 60% fat) and a standard/low-fat diet (STD, 10% fat), and the response to a subchronic treatment with the cannabinoid receptor type 1 (CB1) inverse agonist AM251 (3 mg/kg) on cell proliferation of two relevant neurogenic regions, namely the subventricular zone in the striatal wall of the lateral ventricle (SVZ) and the subgranular zone of the dentate gyrus (SGZ), and also in the hypothalamus given its role in energy metabolism. We found evidence of an interaction between diet-induced obesity and CB1 signalling in the regulation of cell proliferation. AM251 reduced caloric intake and body weight in obese rats, as well as corrected plasma levels of cholesterol and triglycerides. AM251 is shown, for the first time, to modulate cell proliferation in HFD-obese rats only. We observed an increase in the number of 5-bromo-2-deoxyuridine-labelled (BrdU+) cells in the SGZ, but a decrease in the number of BrdU+ cells in the SVZ and the hypothalamus of AM251-treated HFD rats. These BrdU+ cells expressed the neuron-specific βIII-tubulin. These results suggest that obesity may impact cell proliferation in the brain selectively, and provide support for a role of CB1 signalling regulation of neurogenesis in response to obesity., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Objective: To investigate the impact of an experimental model of osteoarthritis (OA) on spinal nociceptive processing and the role of the inhibitory endocannabinoid system in regulating sensory processing at the spinal level., Methods: Experimental OA was induced in rats by intraarticular injection of sodium mono-iodoacetate (MIA), and the development of pain behavior was assessed. Extracellular single-unit recordings of wide dynamic range (WDR) neurons in the dorsal horn were obtained in MIA-treated rats and saline-treated rats. The levels of endocannabinoids and the protein and messenger RNA levels of the main synthetic enzymes for the endocannabinoids (N-acyl phosphatidylethanolamine phospholipase D [NAPE-PLD] and diacylglycerol lipase α [DAGLα]) in the spinal cord were measured., Results: Low-weight (10 gm) mechanically evoked responses of WDR neurons were significantly (P < 0.05) facilitated 28 days after MIA injection compared with the responses in saline-treated rats, and spinal cord levels of anandamide and 2-arachidonoyl glycerol (2-AG) were increased in MIA-treated rats. Protein levels of NAPE-PLD and DAGLα, which synthesize anandamide and 2-AG, respectively, were elevated in the spinal cords of MIA-treated rats. The functional role of endocannabinoids in the spinal cords of MIA-treated rats was increased via activation of cannabinoid 1 (CB(1) ) and CB(2) receptors, and blockade of the catabolism of anandamide had significantly greater inhibitory effects in MIA-treated rats compared with control rats., Conclusion: Our findings provide new evidence for altered spinal nociceptive processing indicative of central sensitization and for adaptive changes in the spinal cord endocannabinoid system in an experimental model of OA. The novel control of spinal cord neuronal responses by spinal cord CB(2) receptors suggests that this receptor system may be an important target for the modulation of pain in OA., (Copyright © 2010 by the American College of Rheumatology.)

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., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Background/aims: Endocannabinoids include anandamide (AEA) and 2-arachidonoylglycerol (2-AG). Endocannabinoid-related molecules like oleoyl-ethanolamine (OEA) and palmitoyl-ethanolamine (PEA) have also been identified. AEA contributes to the pathogenesis of cardiovascular alterations in experimental cirrhosis, but data on the endocannabinoid system in human cirrhosis are lacking. Thus, we aimed to assess whether circulating and hepatic endocannabinoids are upregulated in cirrhotic patients and whether their levels correlate with systemic haemodynamics and liver function., Methods: The endocannabinoid levels were measured in peripheral and hepatic veins and liver tissue by isotope-dilution liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Systemic haemodynamics were assessed by the transthoracic electrical bioimpedance technique. Portal pressure was evaluated by hepatic venous pressure gradient., Results: Circulating AEA and, to a greater extent, PEA and OEA were significantly higher in cirrhotic patients than in controls. PEA and OEA were also increased in the cirrhotic liver tissue. AEA, OEA and PEA levels were significantly higher in peripheral than in the hepatic veins of cirrhotic patients, while the opposite occurred for 2-AG. Finally, circulating AEA, OEA and PEA correlated with parameters of liver function, such as serum bilirubin and international normalized ratio. No correlations were found with systemic haemodynamics., Conclusions: The endocannabinoid system is upregulated in human cirrhosis. Peripheral AEA is increased in patients with a high model of end-stage liver disease score and may reflect the extent of liver dysfunction. In contrast, the 2-AG levels, the other major endocannabinoid, are not affected by cirrhosis. The upregulation of the endocannabinoid-related molecules, OEA and PEA, is even greater than that of AEA, prompting pharmacological studies on these compounds.

Models of neuropathic pain are associated with elevated spinal levels of endocannabinoids (ECs) and altered expression of cannabinoid receptors on primary sensory afferents and post-synaptic cells in the spinal cord. We investigated the impact of these changes on the spinal processing of sensory inputs in a model of neuropathic pain. Extracellular single-unit recordings of spinal neurones were made in anaesthetized neuropathic and sham-operated rats. The effects of spinal administration of the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and the cannabinoid receptor type 2 (CB(2)) receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicycloheptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) on mechanically-evoked responses of spinal neurones were determined. The effects of spinal administration of (5Z,8Z11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707), which binds to CB(2) receptors and alters transport of ECs, on evoked responses of spinal neurones and spinal levels of ECs were also determined. The cannabinoid CB(1) receptor antagonist AM251, but not the CB(2) receptor antagonist, significantly facilitated 10-g-evoked responses of spinal neurones in neuropathic, but not sham-operated, rats. Spinal administration of UCM707 did not alter spinal levels of ECs but did significantly inhibit mechanically-evoked responses of neurones in neuropathic, but not sham-operated, rats. Pharmacological studies indicated that the selective inhibitory effects of spinal UCM707 in neuropathic rats were mediated by activation of spinal CB(2) receptors, as well as a contribution from transient receptor potential vanilloid 1 (TRPV1) channels. This work demonstrates that changes in the EC receptor system in the spinal cord of neuropathic rats influence the processing of sensory inputs, in particular low-weight inputs that drive allodynia, and indicates novel effects of drugs acting via multiple elements of this receptor system.

Striatal medium-sized spiny neurons (MSSNs) receive glutamatergic inputs modulated presynaptically and postsynaptically by dopamine. Mice expressing the gene for enhanced green fluorescent protein as a reporter gene to identify MSSNs containing D1 or D2 receptor subtypes were used to examine dopamine modulation of spontaneous excitatory postsynaptic currents (sEPSCs) in slices and postsynaptic N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) currents in acutely isolated cells. The results demonstrated dopamine receptor-specific modulation of sEPSCs. Dopamine and D1 agonists increased sEPSC frequency in D1 receptor-expressing MSSNs (D1 cells), whereas dopamine and D2 agonists decreased sEPSC frequency in D2 receptor-expressing MSSNs (D2 cells). These effects were fully (D1 cells) or partially (D2 cells) mediated through retrograde signaling via endocannabinoids. A cannabinoid 1 receptor (CB1R) agonist and a blocker of anandamide transporter prevented the D1 receptor-mediated increase in sEPSC frequency in D1 cells, whereas a CB1R antagonist partially blocked the decrease in sEPSC frequency in D2 cells. At the postsynaptic level, low concentrations of a D1 receptor agonist consistently increased NMDA and AMPA currents in acutely isolated D1 cells, whereas a D2 receptor agonist decreased these currents in acutely isolated D2 cells. These results show that both glutamate release and postsynaptic excitatory currents are regulated in opposite directions by activation of D1 or D2 receptors. The direction of this regulation is also specific to D1 and D2 cells. We suggest that activation of postsynaptic dopamine receptors controls endocannabinoid mobilization, acting on presynaptic CB1Rs, thus modulating glutamate release differently in glutamate terminals projecting to D1 and D2 cells.

Both the endocannabinoid and noradrenergic systems have been implicated in neuropsychiatric disorders. Importantly, low levels of norepinephrine are seen in patients with depression, and antagonism of the cannabinoid receptor type 1 (CB1R) is able to induce depressive symptoms in rodents and humans. Whether the interaction between the two systems is important for the regulation of these behaviors is not known. In the present study, adult male Sprague-Dawley rats were acutely or chronically administered the CB1R synthetic agonist WIN 55,212-2, and alpha2A and beta1 adrenergic receptors (AR) were quantified by Western blot. These AR have been shown to be altered in a number of psychiatric disorders and following antidepressant treatment. CB1R agonist treatment induced a differential decrease in alpha2A- and beta1-ARs in the nucleus accumbens (Acb). Moreover, to assess long-lasting changes induced by CB1R activation, some of the chronically treated rats were killed 7 days following the last injection. This revealed a persistent effect on alpha2A-AR levels. Furthermore, the localization of CB1R with respect to noradrenergic profiles was assessed in the Acb and in the nucleus of the solitary tract (NTS). Our results show a significant topographic distribution of CB1R and dopamine beta hydroxylase immunoreactivities (ir) in the Acb, with higher co-localization observed in the NTS. In the Acb, CB1R-ir was found in terminals forming either symmetric or asymmetric synapses. These results suggest that cannabinoids may modulate noradrenergic signaling in the Acb, directly by acting on noradrenergic neurons in the NTS or indirectly by modulating inhibitory and excitatory input in the Acb.

1. Diabetic rats display increased pain responses following injection of formalin into the paw, suggesting the presence of hyperalgesia. In the present study, we investigated the efficacy of the systemic administration of the endocannabinoid transport inhibitors UCM707 and AM404 (1, 10 and 50 mg/kg, i.p.) on hyperalgesia during the formalin test in streptozocin (STZ)-induced diabetic rats. 2. Nociceptive testing was performed in male adult Wistar rats 4 weeks after the onset of hyperglycaemia. At the end of the experiment, all rats were weighed and then underwent plasma glucose measurements. 3. Diabetes caused significant hyperalgesia during both phases of the formalin test. At 10 and 50 mg/kg, both UCM707 and AM404 reversed chemical hyperalgesia in diabetic rats. UCM707 (10 and 50 mg/kg) caused less intensive nociceptive behaviour during both phases of the test, whereas AM404 (10 and 50 mg/kg) only affected pain scores during Phase 1 of the formalin test. At 1 mg, neither drug had any effect on pain behaviour in control and diabetic groups compared with their respective controls. Neither UCM707 nor AM404 had any effect on bodyweight or plasma glucose levels of treated compared with non-treated rats at any of the doses tested. 4. The results of the present study indicate that systemic administration of UCM707 and AM404 is effective in ameliorating chemical hyperalgesia in STZ-diabetic rats. Thus, endocannabinoid transport inhibitors may have potential in the treatment of painful diabetic neuropathy.

Background: The central nervous system cannabinoid CB1 receptors have been implicated in regulation of alcohol consumption. Less data are available on the role of the endogenous ligands for these receptors, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), in alcohol-related behaviors. The purpose of this study was to assess the effects of voluntary alcohol consumption on the levels of these endocannabinoids in key brain areas mediating alcohol reinforcement., Methods: Female and male alcohol-preferring AA (Alko, Alcohol) rats were trained to drink 10% (v/v) alcohol during 90-min limited access sessions every second day. Following establishment of stable alcohol drinking, half of the subjects were killed immediately before the daily alcohol access ("pre-session" group), while the other half was killed after the drinking session ("post-session" group). A separate control group consisted of water-drinking rats. AEA and 2-AG levels were measured from prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), amygdala, and hippocampus using liquid chromatography-tandem mass spectrometry (LC/MS/MS)., Results: Voluntary alcohol drinking caused widespread alterations in the levels of both AEA and 2-AG. Compared to the water group, increased AEA levels were seen in the pre-session group, but they were decreased immediately following limited access drinking in the female AA rats. Also 2-AG levels were significantly elevated after long alcohol exposure, and an additional increase was found after limited access drinking in PFC. In males, however, the only alterations caused by alcohol drinking were significantly elevated AEA levels in NAc and CPu in the post-session group. No changes were seen in the levels of 2-AG., Conclusions: These results demonstrate that voluntary alcohol drinking modulates the levels of endocannabinoids in several brain areas implicated in alcohol reinforcement. AEA and 2-AG were differentially affected, suggesting that they could have partially separate modulatory roles. Alterations were more widespread in females than males, possibly reflecting their higher alcohol intake. Taken together, alcohol-induced release of endocannabinoids may have an important role in alcohol reinforcement and development of alcohol addiction.

Cannabinoid administration suppresses pain by acting at spinal, supraspinal and peripheral levels. Intrinsic analgesic pathways also exploit endocannabinoids; however, the underlying neurobiological substrates of endocannabinoid-mediated analgesia have remained largely unknown. Compelling evidence shows that, upon exposure to a painful environmental stressor, an endocannabinoid molecule called 2-arachidonoylglycerol (2-AG) is mobilized in the lumbar spinal cord in temporal correlation with stress-induced antinociception. We therefore characterized the precise molecular architecture of 2-AG signaling and its involvement in nociception in the rodent spinal cord. Nonradioactive in situ hybridization revealed that dorsal horn neurons widely expressed the mRNA of diacylglycerol lipase-alpha (DGL-alpha), the synthesizing enzyme of 2-AG. Peroxidase-based immunocytochemistry demonstrated high levels of DGL-alpha protein and CB(1) cannabinoid receptor, a receptor for 2-AG, in the superficial dorsal horn, at the first site of modulation of the ascending pain pathway. High-resolution electron microscopy uncovered postsynaptic localization of DGL-alpha at nociceptive synapses formed by primary afferents, and revealed presynaptic positioning of CB(1) on excitatory axon terminals. Furthermore, DGL-alpha in postsynaptic elements receiving nociceptive input was colocalized with metabotropic glutamate receptor 5 (mGluR(5)), whose activation induces 2-AG biosynthesis. Finally, intrathecal activation of mGluR(5) at the lumbar level evoked endocannabinoid-mediated stress-induced analgesia through the DGL-2-AG-CB(1) pathway. Taken together, these findings suggest a key role for 2-AG-mediated retrograde suppression of nociceptive transmission at the spinal level. The striking positioning of the molecular players of 2-AG synthesis and action at nociceptive excitatory synapses suggests that pharmacological manipulation of spinal 2-AG levels may be an efficacious way to regulate pain sensation.

Long-term depression (LTD) at striatal synapses is mediated by postsynaptic endocannabinoid (eCB) release and presynaptic cannabinoid 1 receptor (CB(1)R) activation. Previous studies have indicated that eCB mobilization at excitatory synapses might be regulated by afferent activation. To further address the role of neuronal activity in synaptic plasticity we examined changes in synaptic strength induced by the L-type calcium channel activator 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176, FPL) at glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses in the striatum. We found that the basic mechanisms for FPL-mediated eCB signaling are the same at glutamatergic and GABAergic synapses. FPL-induced LTD (FPL-LTD) was blocked in slices treated with the CB(1)R antagonist AM251 (2 microm), but established depression was not reversed by AM251. FPL-LTD was temperature dependent, blocked by protein translation inhibitors and prevented by intracellular loading of the anandamide transporter inhibitor VDM11 (10 microm) at both glutamatergic and GABAergic synapses. FPL-LTD at glutamatergic synapses required paired-pulse afferent stimulation, while FPL-LTD at GABAergic synapses could be induced even in the absence of explicit afferent activation. By evaluating tetrodotoxin-insensitive spontaneous inhibitory postsynaptic currents we found that neuronal firing is vital for eCB release and LTD induction at GABAergic synapses, but not for short-term depression induced by CB(1)R agonist. The data presented here suggest that the level of neuronal firing regulates eCB signaling by modulating release from the postsynaptic cell, as well as interacting with presynaptic mechanisms to induce LTD at both glutamatergic and GABAergic synapses in the striatum.

Cannabinoids have recently been approved as a treatment for pain in multiple sclerosis (MS). Increasing evidence from animal studies suggests that this class of compounds could also prove efficient to fight neurodegeneration, demyelination, inflammation and autoimmune processes occurring in this pathology. However, the use of cannabinoids is limited by their psychoactive effects. In this context, potentiation of the endogenous cannabinoid signalling could represent a substitute to the use of exogenously administrated cannabinoid ligands. Here, we studied the expression of different elements of the endocannabinoid system in a chronic model of MS in mice. We first studied the expression of the two cannabinoid receptors, CB(1) and CB(2), as well as the putative intracellular cannabinoid receptor peroxisome proliferator-activated receptor-alpha. We observed an upregulation of CB(2), correlated to the production of proinflammatory cytokines, at 60 days after the onset of the MS model. At this time, the levels of the endocannabinoid, 2-arachidonoylglycerol, and of the anti-inflammatory anandamide congener, palmithoylethanolamide, were enhanced, without changes in the levels of anandamide. These changes were not due to differences in the expression of the degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, or of biosynthetic enzymes, diacylglycerol lipase-alpha and N-acylphosphatidylethanolamine phospholipase-D at this time (60 days). Finally, the exogenous administration of palmitoylethanolamide resulted in a reduction of motor disability in the animals subjected to this model of MS, accompanied by an anti-inflammatory effect. This study overall highlights the potential therapeutic effects of endocannabinoids in MS.

Daily life stressors are a major environmental factor contributing to precipitation and exacerbation of mental illness. Animal models using repeated homotypic stress induce anxious and depressive phenotypes and are used to study the pathophysiology of affective disorders. Here we discuss data demonstrating that repeated homotypic stress produces temporally and anatomically distinct changes in endocannabinoid signaling components within stress-responsive brain regions. We also present evidence describing the neural and behavioral correlates of these adaptations in endocannabinoid signaling. These data support a role for endocannabinoid signaling in the central nervous system response to chronic, homotypic stress, and specifically in the process of stress-response habituation. The clinical implications of these findings for the pathophysiology and treatment of affective disorders are discussed.

During puberty, neuronal maturation of the brain, which began during perinatal development, is completed such that the behavioral potential of the adult organism can be fully achieved. These maturational events and processes of reorganization are needed for the occurrence of adult behavioral performance but simultaneously render the organism highly susceptible to perturbations, such as exposure to psychoactive drugs, during this critical developmental time span. Considering the variety of maturational processes occurring in the endocannabinoid system during this critical period, it is not surprising that the still-developing brain might by highly susceptible to cannabis exposure. Emerging evidence from human studies and animal research demonstrates that an early onset of cannabis consumption might have lasting consequences on cognition, might increase the risk for neuropsychiatric disorders, promote further illegal drug intake and increase the likelihood of cannabis dependence. These findings suggest that young people represent a highly vulnerable cannabis consumer group and that they run a higher risk than adult consumers of suffering from adverse consequences from cannabinoid exposure. The aim of the present review is to provide an overview over the possible deleterious residual cannabinoid effects during critical periods of postnatal maturation and to offer a more precise delineation of the vulnerable time window for cannabinoid exposure.

A major finding--that (-)-trans-Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is largely responsible for the psychotropic effects of cannabis--prompted research in the 1970s and 1980s that led to the discovery that this plant cannabinoid acts through at least two types of cannabinoid receptor, CB(1) and CB(2), and that Delta(9)-THC and other compounds that target either or both of these receptors as agonists or antagonists have important therapeutic applications. It also led to the discovery that mammalian tissues can themselves synthesize and release agonists for cannabinoid receptors, the first of these to be discovered being arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol. These 'endocannabinoids' are released onto their receptors in a manner that appears to maintain homeostasis within the central nervous system and sometimes either to oppose or to mediate or exacerbate the unwanted effects of certain disorders. This review provides an overview of the pharmacology of cannabinoid receptors and their ligands. It also describes actual and potential clinical uses both for cannabinoid receptor agonists and antagonists and for compounds that affect the activation of cannabinoid receptors less directly, for example by inhibiting the enzymatic hydrolysis of endocannabinoids following their release.

Stress-induced analgesia (SIA) is an important endogenous mechanism in response to stressful stimuli. Opioid peptides, as well as endocannabinoids, are known mediators of SIA. We were interested in whether the endocannabinoid tone and the resulting SIA could be influenced by changing the activity of cholecystokinin (CCK) in mice. This hypothesis is related to recent evidence showing a cellular colocalization of neuropeptide CCK and cannabinoid type 1 (CB(1)) receptors in many central nervous system structures. However, the physiological relevance of this colocalization is unknown. Our aim was to test whether abolishing the endogenous CCK tone might influence the cannabinoid-mediated form of SIA. As expected, the CB(1) antagonist rimonabant prevented the development of analgesia in response to footshock-induced stress in wild-type mice. In contrast, CCK type 2 (CCK(2)) receptor-deficient mice developed SIA regardless of rimonabant treatment. Naloxone, an opioid antagonist, antagonized SIA in both wild-type and CCK(2) receptor-deficient homozygous mice. This finding suggests that CCK, through CCK(2) receptors, modulates the action of endocannabinoids. Gene expression analysis revealed an up-regulation of CCK(2) receptor gene in the lumbar spinal cord and mesolimbic area in wild-type mice in response to stress. In addition, wild-type mice displayed up-regulation of genes implicated in endocannabinoid-mediated neurotransmission (elevation of CB(1) receptor, 2-arachidonoylglycerol and the anandamide-synthesizing enzymes sn-1-diacylglycerol lipase alpha and N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D) in response to stress in the lumbar spinal cord and mesolimbic area. We did not find any of these changes in CCK(2) receptor-deficient homozygous mice. Altogether, behavioural and gene expression studies reflect an involvement of CCK(2) receptors in the development of endocannabinoid-mediated SIA.

In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30-300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction-liquid chromatography-tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K(+), with or without addition of Ca(2+), or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.

1. Although cannabinoid receptor agonists have analgesic activity in chronic pain states, they produce a spectrum of central cannabinoid CB(1) receptor-mediated motor and psychotropic side-effects. The actions of endocannabinoids, such as anandamide, are terminated by uptake and subsequent intracellular enzymatic degradation. In the present study, we examined the effect of acute administration of the anandamide transport inhibitor AM404 in rat models of chronic neuropathic and inflammatory pain. 2. Systemic administration of AM404 (10 mg/kg) reduced mechanical allodynia in the partial sciatic nerve ligation (PNL) model of neuropathic pain, but not in the complete Freund's adjuvant (CFA) model of inflammatory pain. 3. The effect of AM404 in the PNL model was abolished by coapplication with the selective cannabinoid CB(1) receptor antagonist AM251 (1 mg/kg). AM404 did not produce a reduction in motor performance in either the PNL or CFA models. 4. These findings suggest that acute administration of AM404 reduces allodynia in a neuropathic pain model via cannabinoid CB(1) receptor activation, without causing the undesirable motor disruption associated with cannabinoid receptor agonists.

The endocannabinoid system mediates analgesia expressed following exposure to conditioned or unconditioned aversive stimuli, and controls the extinction of conditioned aversive behaviour. The present study investigated the effects of administration of the cannabinoid(1) (CB(1)) receptor antagonist SR141716A into the right basolateral amygdala (BLA) on expression of conditioned fear, formalin-evoked nociceptive behaviour, fear-conditioned analgesia and associated alterations in monoamine levels in discrete rat brain areas. Re-exposure to a context previously paired with footshock significantly reduced formalin-evoked nociceptive behaviour. Intra-BLA administration of SR141716A did not attenuate fear-conditioned analgesia, but reduced formalin-evoked nociceptive behaviour and attenuated the formalin-induced decrease in freezing and 22-kHz ultrasonic vocalizations in the early part of the trial. Furthermore, intra-BLA SR141716A significantly prolonged the duration of these fear-related behaviours in fear-conditioned rats not receiving formalin. Fear-conditioned analgesia was accompanied by increased homovanillic acid (HVA) : dopamine (DA) ratio and reduced serotonin (5-HT) in the cerebellum, an effect not altered by SR141716A. SR141716A-induced analgesia was accompanied by reduced DA, increased HVA : DA ratio and reduced 5-HT levels in the cerebellum, increased hippocampal HVA levels and increased 5-hydroxyindole-3-acetic acid (5-HIAA) in the amygdaloid cortex. The SR141716A-induced prolongation of contextually induced aversive behaviour was accompanied by reduced DA and 3,4-dihydroxyphenylacetic acid (DOPAC), levels in the hippocampus, and increased DA and 5-HIAA in the periaqueductal grey. These data suggest an important role for CB(1) receptors in the right BLA in mediating short-term extinction of conditioned aversive behaviour but not fear-conditioned analgesia. The results also enhance our understanding of endocannabinoid-monoamine interactions of relevance to conditioned fear and associated analgesia.

Background/aim: Anandamide can activate potassium (K(+)) channels to induce an endothelium-dependent vasorelaxation in normal rat mesenteric arteries. Cannabinoids contribute partly to the splanchnic vasodilation in cirrhosis. This study investigated the roles of vascular K(+) channels in anandamide-induced mesenteric vasorelaxation in isolated rat cirrhotic vessels., Methods: The effects of the pretreatment of AM251, a specific CB(1) receptor antagonist, were assessed on the vascular reactivity to phenylephrine (PE), potassium chloride (KCl), acetylcholine (ACh) and sodium nitroprusside (SNP). Additionally, cannabinoid (CB(1) and CB(2)) receptors' protein expression and the effects of different K(+) channel blockers on vascular reactivity to anandamide were also studied., Results: Cirrhotic mesenteric arteries showed an overexpression of CB(1) receptor associated with hyporeactivity to PE and KCl, and hyper-response to ACh, SNP and anandamide. Pretreatment with AM251 significantly improved the hyporeactivity to KCl and ameliorated the hyper-response to ACh in cirrhotic vessels. Increased relaxation response to anandamide was suppressed by combinations of vascular Ca(2+)-dependent K(+) channel blockers (including apamin+charybdotoxin+iberiotoxin or apamin+TRAM-34+iberiotoxin) (TRAM-34, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole)., Conclusions: In cirrhotic mesenteric arteries, vascular CB(1) receptor and anandamide contribute to the in vitro hyporeactivity to KCl. In addition, hyper-response to ACh may probably act through the modulation of vascular Ca(2+)-dependent K(+) channels.

In models of early stage Parkinson's disease (PD), motor deficits are accompanied by excessive activation of striatal glutamate receptors. Metabotropic glutamate group I receptors (mGluR I) play an important but not well-understood role in PD progression. In mouse brain slices, bath application of the mGluR I agonist (RS)-DHPG (3,5-dihydroxyphenylglycine, 100 microm for 20 min) caused a long-term depression of corticostriatal transmission (LTD(DHPG)), which was reversed by three mGluR I antagonists: LY 367385, CPCCOEt and MPEP. LTD(DHPG) required nitric oxide (NO) synthesis as it was blocked by the broad-spectrum NO synthase (NOS) inhibitor Nomega-nitro-l-arginine (NL-Arg) and impaired under blockade of neuronal NOS and in endothelial NOS-deficient mice. Release of endocannabinoids (eCB) was critically involved in this form of striatal plasticity givem that the CB1 receptor antagonist AM251 prevented LTD(DHPG), while the CB1 agonist ACEA elicited LTD. The NO synthesis necessary for LTD(DHPG) induction occurred downstream of CB1 activation as ACEA-evoked LTD was also abolished by NL-Arg. These findings are relevant for the pathophysiology of PD, as they link the overactivation of group I mGluRs and striatal NO production.

The phytocannabinoid cannabidiol (CBD) possesses no psychotropic activity amid potentially beneficial therapeutic applications. We here characterized interactions between CBD (1 microM) and the endocannabinoid system in cultured rat hippocampal cells. The CBD-induced Ca2+ rise observed in neurons and glia was markedly reduced in the presence of the endogenous cannabinoid anandamide in neurons, with no alteration seen in glia. Neuronal CBD responses were even more reduced in the presence of the more abundant endocannabinoid 2-arachidonyl glycerol, this action was maintained in the presence of the CB1 receptor antagonist AM281 (100 nM). Neuronal CBD responses were also reduced by pre-exposure to glutamate, expected to increase endocannabinoid levels by increasing in [Ca2+]i. Application of AM281 at 1 microM elevated CBD-induced Ca2+ responses in both cell types, further confirming our finding that endocannabinoid-mediated signalling is negatively coupled to the action of CBD. However, upregulation of endogenous levels of endocannabinoids via inhibition of endocannabinoid hydrolysis (with URB597 and MAFP) could not be achieved under resting conditions. Because delta9-tetrahydrocannabinol did not mimic the endocannabinoid actions, and pertussis toxin treatment had no effect on CBD responses, we propose that the effects of AM281 were mediated via a constitutively active signalling pathway independent of CB1 signalling. Instead, signalling via G(q/11) and phospholipase C appears to be negatively coupled to CBD-induced Ca2+ responses, as the inhibitor U73122 enhanced CBD responses. Our data highlight the interaction between exogenous and endogenous cannabinoid signalling, and provide evidence for the presence of an additional pharmacological target, sensitive to endocannabinoids and to AM281.

Endocannabinoids (eCBs) inhibit neurotransmitter release throughout the central nervous system. Using the Ceratomandibularis muscle from the lizard Anolis carolinensis we asked whether eCBs play a similar role at the vertebrate neuromuscular junction. We report here that the CB(1) cannabinoid receptor is concentrated on motor terminals and that eCBs mediate the inhibition of neurotransmitter release induced by the activation of M(3) muscarinic acetylcholine (ACh) receptors. N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, a CB(1) antagonist, prevents muscarine from inhibiting release and arachidonylcyclopropylamide (ACPA), a CB(1) receptor agonist, mimics M(3) activation and occludes the effect of muscarine. As for its mechanism of action, ACPA reduces the action-potential-evoked calcium transient in the nerve terminal and this decrease is more than sufficient to account for the observed inhibition of neurotransmitter release. Similar to muscarine, the inhibition of synaptic transmission by ACPA requires nitric oxide, acting via the synthesis of cGMP and the activation of cGMP-dependent protein kinase. 2-Arachidonoylglycerol (2-AG) is responsible for the majority of the effects of eCB as inhibitors of phospholipase C and diacylglycerol lipase, two enzymes responsible for synthesis of 2-AG, significantly limit muscarine-induced inhibition of neurotransmitter release. Lastly, the injection of (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (an inhibitor of eCB transport) into the muscle prevents muscarine, but not ACPA, from inhibiting ACh release. These results collectively lead to a model of the vertebrate neuromuscular junction whereby 2-AG mediates the muscarine-induced inhibition of ACh release. To demonstrate the physiological relevance of this model we show that the CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide prevents synaptic inhibition induced by 20 min of 1-Hz stimulation.

The endocannabinoid system is a complex physiologic system. One of the most important discoveries related to the endocannabinoid system is that cannabinoid-1 receptors are present throughout the body and that they are linked to obesity and cardiometabolic risk. Adipose tissue was historically thought to be an inert, passive storage vehicle. However, recent findings prove that adipose tissue is a complex endocrine organ that releases adipokines, which influence cardiometabolic risk factors. Elevated endocannabinoid system activity is associated with obesity and coupled with excessive food intake and fat accumulation. Strong evidence suggests that the endocannabinoid system affects the surrogate cardiometabolic end points that greatly influence morbidity and mortality.

The polycystic ovary syndrome (PCOS) is one of the most common causes of infertility due to anovulation in women. The clinical features of PCOS are heterogeneous and may change throughout the lifespan, starting from adolescence to postmenopausal age. This is largely dependent on the influence of obesity and metabolic alterations, including an insulin-resistant state and the metabolic syndrome, which consistently affect most women with PCOS. Obesity does in fact have profound effects on both the pathophysiology and the clinical manifestation of PCOS, by different mechanisms leading to androgen excess and increased free androgen availability and to alterations of granulosa cell function and follicle development. Notably, simple obesity per se represents a functional hyperandrogenic state. These mechanisms involve early hormonal and metabolic factors during intrauterine life, leptin, insulin and the insulin growth factor system and, potentially, the endocannabinoid system. Compared with normal weight women with PCOS, those with obesity are characterised by a worsened hyperandrogenic and metabolic state, poorer menses and ovulatory performance and, ultimately, poorer pregnancy rates. The importance of obesity in the pathogenesis of PCOS is emphasised by the efficacy of lifestyle intervention and weight loss, not only on metabolic alterations but also on hyperandrogenism, ovulation and fertility. The increasing prevalence of obesity among adolescent and young women with PCOS may partly depend on the increasing worldwide epidemic of obesity, although this hypothesis should be supported by long-term prospective epidemiological trials. This may have great relevance in preventive medicine and offer the opportunity to expand our still limited knowledge of the genetic and environmental background favouring the development of the PCOS.

We used a model of neuropathic pain consisting of rats with chronic constriction injury (CCI) of the sciatic nerve, in order to investigate whether endocannabinoid levels are altered in the dorsal raphe (DR) and to assess the effect of repeated treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate, a synthetic cannabinoid agonist, or N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404), an inhibitor of endocannabinoid reuptake, on DR serotonergic neuronal activity and on behavioural hyperalgesia. CCI resulted in significantly elevated anandamide but not 2-arachidonoylglycerol levels in the DR. Furthermore, as well as thermal and mechanical hyperalgesia, CCI caused serotonergic hyperactivity (as shown by the increase of basal activity of serotonergic neurones, extracellular serotonin levels and expression of 5-HT1A receptor gene). Repeated treatment with either (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate or AM404 reverted the hyperalgesia and enhanced serotonergic activity induced by CCI in a way attenuated by N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide, a selective cannabinoid subtype 1 (CB1) receptor antagonist. Despite the elevated levels of anandamide following CCI, N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methyl-3-pyrazolecarboxamide did not produce hyperalgesia or any other effect on serotonergic neuronal activity when administered alone. Furthermore, the effects of AM404 were not accompanied by an increase in endocannabinoid levels in the DR. In conclusion, following CCI of the sciatic nerve, the endocannabinoid and serotonergic systems are activated in the DR, where repeated stimulation of CB1 receptors with exogenous compounds restores DR serotonergic activity, as well as thermal and mechanical nociceptive thresholds, to pre-surgery levels. However, an elevated level of endogenous anandamide in the DR does not necessarily contribute to the CB1-mediated tonic control of analgesia and serotonergic neuronal activity.

The endocannabinoid system has been shown to regulate both the hypothalamic-pituitary-adrenal (HPA) axis and emotionality. The present experiment was designed to examine whether pharmacological modulation of the endocannabinoid system would affect the suppression of hippocampal cell proliferation and increase in defensive behaviours seen following exposure to predator odour (trimethylthiazoline; TMT) stress. Rats were administered either an endocannabinoid uptake inhibitor (AM404; 2 mg/kg) or a cannabinoid CB1 receptor antagonist (AM251; 5 mg/kg) 30 min prior to exposure to TMT. Exposure to TMT reduced cell proliferation in the dentate gyrus and increased the expression of defensive burying. Administration of AM404 significantly inhibited defensive burying, and attenuated the reduction in cell proliferation in response to TMT exposure. Administration of AM251 alone significantly increased cell proliferation; however, pretreatment with AM251 prevented neither the stress-induced suppression of cell proliferation nor the stress-induced increase in behavioural responses. These results support previous research demonstrating that augmentation of endocannabinoid signalling can suppress stress-responsive systems. They also suggest that endocannabinoids may play a complex role in the regulation of neurogenesis via cell proliferation in the hippocampus.

Septohippocampal cholinergic neurons play key roles in learning and memory processes, and in the generation of hippocampal theta rhythm. The range of receptors for endogenous modulators expressed on these neurons is unclear. Here we describe GABA(B) 1a/b receptor (GABA(B)R) and type 1 cannabinoid receptor (CB(1)R) expression in rat septal cholinergic [i.e. choline acetyltransferase (ChAT)-positive] cells. Using double immunofluorescent staining, we found that almost two-thirds of the cholinergic cells in the rat medial septum were GABA(B)R positive, and that these cells had significantly larger somata than did GABA(B)R-negative cholinergic neurons. We detected CB(1)R labelling in somata after axonal protein transport was blocked by colchicine. In these animals about one-third of the cholinergic cells were CB(1)R positive. These cells again had larger somata than CB(1)R-negative cholinergic neurons. The analyses confirmed that the size of GABA(B)R-positive and CB(1)R-positive cholinergic cells were alike, and all CB(1)R-positive cholinergic cells were GABA(B)R positive as well. CB(1)R-positive cells were invariably ChAT positive. All retrogradely labelled septohippocampal cholinergic cells were positive for GABA(B)R and at least half of them also for CB(1)R. These data shed light on the existence of at least two cholinergic cell types in the medial septum: one expresses GABA(B)R and CB(1)R, has large somata and projects to the hippocampus, whereas the other is negative for GABA(B)R and CB(1)R and has smaller somata. The results also suggest that cholinergic transmission in the hippocampus is fine-tuned by endocannabinoid signalling.

The control of endocannabinoid actions on cortical neurons by a putative carrier-mediated uptake is still poorly understood at the level of synaptic transmission. We investigated the effect of an endocannabinoid, 2-arachidonoyl glycerol (2-AG), on inhibitory postsynaptic currents (IPSCs) in hippocampal slices under physiological conditions, and when uptake was altered by a selective blocker or lower temperature. Bath application of 2-AG (20 micro m) caused a 40% reduction in the amplitude of IPSCs evoked in the perisomatic region of CA1 pyramidal neurons at room temperature; this effect could be blocked by a specific CB(1) receptor antagonist, AM251. By contrast, a smaller (20%) but significant suppression of inhibitory transmission was found by 2-AG at 33-35 degrees C. This reduced blocking effect at physiological temperature could be brought back to 40% by coapplying the endocannabinoid uptake blocker, AM404 (10 or 20 micro m) with 2-AG. In parallel experiments, we measured [(3)H]2-AG uptake at different temperatures in primary cultures prepared from cortical neurons. These data confirmed a striking inhibition of [(3)H]2-AG uptake at room temperature compared with values observed at 37 degrees C. Uptake could be significantly modified by anandamide, 2-AG and AM404, suggesting a common transporter for the two endocannabinoids. These findings together demonstrate the presence of an effective endocannabinoid uptake in cortical neurons, which could considerably alter the spatial and temporal constraints of endocannabinoid signalling at physiological temperature, and which may critically change the interpretation of findings at room temperature.

The majority of Parkinson's disease patients undergoing levodopa therapy develop disabling motor complications (dyskinesias) within 10 years of treatment. Stimulation of cannabinoid receptors, the pharmacological target of Delta 9-tetrahydrocannabinol, is emerging as a promising therapy to alleviate levodopa-associated dyskinesias. However, the mechanisms underlying this beneficial action remain elusive, as do the effects exerted by levodopa therapy on the endocannabinoid system. Although levodopa is known to cause changes in CB1 receptor expression in animal models of Parkinson's disease, we have no information on whether this drug alters the brain concentrations of the endocannabinoids anandamide and 2-arachidonylglycerol. To address this question, we used an isotope dilution assay to measure endocannabinoid levels in the caudate-putamen, globus pallidus and substantia nigra of intact and unilaterally 6-OHDA-lesioned rats undergoing acute or chronic treatment with levodopa (50 mg/kg). In intact animals, systemic administration of levodopa increased anandamide concentrations throughout the basal ganglia via activation of dopamine D1/D2 receptors. In 6-OHDA-lesioned rats, anandamide levels were significantly reduced in the caudate-putamen ipsilateral to the lesion; however, neither acute nor chronic levodopa treatment affected endocannabinoid levels in these animals. In lesioned rats, chronic levodopa produced increasingly severe oro-lingual involuntary movements which were attenuated by the cannabinoid agonist R(+)-WIN55,212-2 (1 mg/kg). This effect was reversed by the CB1 receptor antagonist rimonabant (SR141716A). These results indicate that a deficiency in endocannabinoid transmission may contribute to levodopa-induced dyskinesias and that these complications may be alleviated by activation of CB1 receptors.