CB1 Receptor Function Research Articles

Male and Female Rats Differ in Brain Cannabinoid CB1 Receptor Density and Function and in Behavioural Traits Predisposing to Drug Addiction: Effect of Ovarian Hormones

Sex-dependent differences are frequently observed in the biological and behavioural effects of substances of abuse, including cannabis. We recently demonstrated a modulating effect of sex and oestrous cycle on cannabinoid-taking and seeking behaviours. Here, we investigated the influence of sex and oestrogen in the regulation of cannabinoid CB1 receptor density and function, measured by [(3)H]CP55940 and CP55940-stimulated [(35)S]GTPγS binding autoradiography, respectively, in the prefrontal cortex (Cg1 and Cg3), caudate- putamen, nucleus accumbens, amygdala and hippocampus of male and cycling female rats, as well as ovariectomised (OVX) rats and OVX rats primed with oestradiol (10 µg/rat) (OVX+E). CB1 receptor density was significantly lower in the prefrontal cortex and amygdala of cycling females than in males and in OVX females, a difference that appeared to be oestradiol-dependent, because it was no more evident in the OVX+E group. CP55940-stimulated [(35)S]GTPγS binding was significantly higher in the Cg3 of OVX rats relative to cycling and OVX+E rats. No difference was observed in CB1 receptor density or function in any of the other brain areas analysed. Finally, sex and oestradiol were also found to affect motor activity, social behaviour and sensorimotor gating in rats tested in locomotor activity boxes, social interaction and prepulse inhibition tasks, respectively. Our findings provide biochemical evidence for sex- and hormone- dependent differences in the density and function of CB1 receptors in selected brain regions, and in behaviours associated with greater vulnerability to drug addiction, revealing a more vulnerable behavioural phenotype in female than in male rats.

P.1.013 Chronic (−)cannabidiol produces antidepressant-like effects in bulbectomised mice, acting on 5-HT1A and CB1 receptor functionality

P.1.013 Chronic (−)cannabidiol produces antidepressant-like effects in bulbectomised mice, acting on 5-HT1A and CB1 receptor functionality

Changes in cannabinoid CB1 receptor functionality in the female rat prefrontal cortex following a high fat diet

AimsA high fat diet (HFD) has been found to affect neurotransmission in the prefrontal cortex, but the effects of this dietary regime upon the endocannabinoid system has not been studied in this brain region. In consequence, in the present study, we have investigated the effect of HFD for up to 20weeks upon the endocannabinoid system in the prefrontal cortex of female rats. Main methodsCB1 receptor functionality was measured using CP55,940-stimulated [35S]GTPγS autoradiography. Fatty acid amide hydrolase and monoacylglycerol lipase activities were analysed in brain regions by assessing rates of [3H]anandamide and JZL184-sensitive [3H]2-oleoylglycerol hydrolysis, respectively. Key findingsIn the prefrontal cortex, a significantly greater stimulation of [35S]GTPγS binding by CP55,940 was seen following 4–12, but not 16–20weeks of HFD. No significant changes were seen for the caudate–putamen, CA1–CA3 region of the hippocampus or the dentate gyrus. The increased response for the 12week animals was not accompanied by a significant change in the receptor density, measured with [3H]CP55,940 autoradiography. No significant changes in the activity of the endocannabinoid hydrolytic enzymes fatty acid amide or monoacylglycerol lipase were seen in the prefrontal cortex, hippocampus, amygdala or hypothalamus following either 12 or 20weeks of HFD. SignificanceIt is concluded that HFD produces an increased CB1 receptor functionality in the prefrontal cortex of female rats. Given that the endocannabinoid system regulates neurotransmission in the prefrontal cortex, the present data would implicate this system in the disturbed prefrontal cortical activity in this region following a high fat diet.

Repeated Low-Dose Administration of the Monoacylglycerol Lipase Inhibitor JZL184 Retains Cannabinoid Receptor Type 1–Mediated Antinociceptive and Gastroprotective Effects

The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug-induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [(3)H]SR141716A binding and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ(9)-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.

Functional Autoradiography Shows Unaltered Cannabinoid CB1 Receptor Signalling in Hippocampus and Cortex of APP/PS1 Transgenic Mice

The cannabinoid CB1-receptor is among the most abundant G-protein-coupled receptors in the mammalian brain. Whereas post-mortem studies in Alzheimer's disease (AD) brains compared to age-matched controls have reported decreased CB1-receptor binding but no change in their protein levels (immunoreactivity), decreased or increased CB1- receptor protein levels have been reported in APP/PS1 transgenic mice modelling AD. To complete the picture, the present study used functional autoradiography to assess CB1-receptor-dependent G(i) protein activation in the hippocampus, entorhinal cortex and medial frontal cortex of 13- to 14-month-old female APPswe/PS1dE9 transgenic and wild-type littermate control mice. The mouse brains were processed for [³⁵S]GTPλS autoradiography so that brain sections were analysed in pairs of one transgenic and one control mouse brain. The autoradiography protocol was completed for each pair both in the absence and presence of dithiotreitol (DTT) to reveal possible redox-dependent alterations in CB1 receptor function. Five treatments were used: baseline, incubation with 10 μM GTPλS to assess nonspecific binding, and CB1 receptor agonist CP55,940 in three concentrations. By and large we found no statistically significant differences between the APP/PS1 transgenic and control mice in CB1 receptor signalling. The only exception was a modest redox-dependent alteration in entorhinal cortical CB1 receptors between the genotypes. Thus, in accordance with the majority of earlier human AD findings, we did not find evidence for notable changes in the number of functional CB1 receptors in the common APPswe/PS1dE9 mouse model of AD.

Endocannabinoid‐dependent plasticity at spinal nociceptor synapses

Neuroplastic changes at the spinal synapses between primary nociceptors and second order dorsal horn neurons play key roles in pain and analgesia. NMDA receptor-dependent forms of long-term plasticity have been studied extensively at these synapses, but little is known about possible contributions of the endocannabinoid system. Here, we addressed the role of cannabinoid (CB)1 receptors in activity-dependent plasticity at these synapses. We report that conditional low-frequency stimulation of high-threshold primary sensory nerve fibres paired with depolarisation of the postsynaptic neuron evoked robust long-term depression (LTD)of excitatory synaptic transmission by about 40% in the vast majority (90%) of recordings made in wild-type mice. When recordings were made from global or nociceptor-specific CB(1) receptor-deficient mice (CB(1) (−/− ) mice and sns-CB(1)(−/−) mice), the portion of neurons exhibiting LTD was strongly reduced to about 25%. Accordingly, LTD was prevented to a similar extent by the CB1 receptor antagonist AM251 and mimicked by pharmacological activation of CB1 receptors. In a subset of neurons with EPSCs of particularly high stimulation thresholds, we furthermore found that the absence of CB(1) receptors in CB(1)(−/−) and sns-CB(1)(−/−) mice converted the response to the paired conditioning stimulation protocol from LTD to long-term potentiation (LTP). Our results identify CB1 receptor-dependent LTD as a form of synaptic plasticity previously unknown in spinal nociceptors. They furthermore suggest that prevention of LTP may be a second hither to unknown function of CB1 receptors in primary nociceptors. Both findings may have important implications for our understanding of endogenous pain control mechanisms and of analgesia evoked by cannabinoid receptor agonists.

Differential Treatment Regimen-Related Effects of HU210 on CB1 and D2-Like Receptor Functionality in the Rat Basal Ganglia

Background/Aims: Functional linkages between the cannabinoid CB₁ and the dopaminergic systems have been reported although the observations and the mechanisms hypothesizing their interactions at the G protein-coupled receptor (GPCR) functionality level are conflicting. Methods: Administration of a potent cannabinoid agonist, HU210, at various doses (25–100 µg/kg) and treatment regimens (1- to 14-day treatment) in rats was carried out to investigate the effect of HU210 treatment on the CB₁ and D₂-like agonist-mediated GPCR activation. Results: The desensitizations (reduced coupling) of both D₂ agonist- and CB₁ agonist-mediated GPCR activation was found to be treatment duration dependent and region specific, suggesting implication of receptor tolerance and adaptation due to the cannabinoid treatment. The effect of HU210 on the CB₁ agonist-mediated GPCR desensitization in all treatment groups was not dose dependent. Conclusions: The desensitization of D₂-like receptors found after a cannabinoid treatment in this study strengthens the evidence that the two neurotransmitter systems interact at the intercellular level; this interaction might occur via multiple mechanisms, which also vary according to region.

Spinal cannabinoid CB2 receptors as a target for neuropathic pain: an investigation using chronic constriction injury

Agonists for the cannabinoid CB2 receptor are antinociceptive in several rodent models and several reports have suggested that the target for these drugs is CB2 expressed in the spinal cord pain pathway. After confirming the efficacy of a systemically delivered CB2-selective agonist, GW405833, we tested this hypothesis by administering the CB2 agonists GW405833 and JWH-133, via intrathecal cannulation, to the lumbar spinal cord of rats that had undergone chronic constriction injury to induce mechanical allodynia. We found that although the non-selective CB1/CB2 cannabinoid receptor agonist WIN55,212-2 reversed mechanical allodynia in both ipsilateral and contralateral hind paws, neither GW405833 nor JWH-133 reversed mechanical allodynia. In addition, we investigated the expression of CB2 receptors in the neuropathic spinal cord using immunohistochemistry, Western blot and CB2 agonist stimulated [35S]GTPγS binding. Although protein-based analysis of CB2 partially matched the results of earlier studies using the same antibody, we found evidence that this antibody may be insufficiently specific for the detection of CB2 in native tissue. Using [35S]GTPγS binding assays, we found no evidence of functional CB2 in the spinal cord, in sham or surgery-treated tissue. However, WIN55,212-2 stimulated [35S]GTPγS binding showed clear evidence of functional CB1 receptors consistent with the known distribution of elements of the pain pathway, and we concluded that spinal CB2 receptors are not a likely target for cannabinoid-mediated antinociception in this model.

Gender-dependent behavioral and biochemical effects of adolescent delta-9-tetrahydrocannabinol in adult maternally deprived rats

Preclinical data support the long-term adverse effects on cognition, emotionality, and psychotic-like behaviors of adolescent exposure to natural and synthetic cannabinoids. To investigate whether the long-lasting adverse effects induced by cannabinoids in adolescence are influenced by early-life stress, female and male rats were subjected to 24-h maternal deprivation at postnatal day (PND) 9 and treated with tetrahydrocannabinol (THC) during adolescence (PND 35–45) according to our previously reported protocol. At adulthood, rats were tested in the novel object recognition, social interaction, and forced swim tests, to evaluate possible alterations in recognition memory, social behavior, and coping strategy. Moreover, cannabinoid CB1 receptor density and functionality, as well as NMDA and dopamine D1 and D2 receptor densities were measured through autoradiographic binding studies. In female maternally deprived rats, THC failed to impair recognition memory, counteracted aggressiveness induced by maternal deprivation, whereas no interaction was observed in the passive coping behavior. In males, the association of the two events increased passive coping response without affecting other behaviors. This behavioral picture was accompanied by gender-dependent and region-specific alterations in NMDA, D1 and D2 receptors. In conclusion, this study demonstrates that adolescent THC exposure might have different behavioral outcomes in animals previously exposed to early-life stress compared with non-stressed controls. The interaction between the two events is not univocal, and different combinations may arise depending on the sex of the animals and the behavior considered. Alterations in NMDA, D1 and D2 receptors might be involved in the behavioral responses induced by maternal deprivation and in their modulation by THC.This article is part of a Special Issue entitled: Stress, Emotional Behavior and the Endocannabinoid System.

Loss of striatal cannabinoid CB1 receptor function in attention‐deficit / hyperactivity disorder mice with point‐mutation of the dopamine transporter

Abnormal dopamine (DA) transmission in the striatum plays a pivotal role in attention-deficit/hyperactivity disorder (ADHD). As striatal DA signalling modulates the endocannabinoid system (ECS), the present study was aimed at investigating cannabinoid CB1 receptor (CB1R) function in a model of ADHD obtained by triple point-mutation in the dopamine transporter (DAT) gene in mice, making them insensitive to cocaine [DAT cocaine-insensitive (DAT-CI) mice]. DAT-CI mice had a marked hyperactive phenotype, and neurophysiological recordings revealed that the sensitivity of CB1Rs controlling GABA-mediated synaptic currents [CB1Rs((GABA)) ] in the striatum was completely lost. In contrast, CB1Rs modulating glutamate transmission [CB1Rs((Glu)) ], and GABA(B) receptors were not affected in this model of ADHD. In DAT-CI mice, the blockade of CB1R((GABA)) function was complete even after cocaine or environmental manipulations activating the endogenous DA-dependent reward system, which are known to sensitize these receptors in control animals. Conversely, the hedonic property of sucrose was intact in DAT-CI mice, indicating normal sweet perception in these animals. Our results point to CB1Rs as novel molecular players in ADHD, and suggest that therapeutic strategies aimed at interfering with the ECS might prove effective in this disorder.

Δ9‐THC increases endogenous AHA1 expression in rat cerebellum and may modulate CB1 receptor function during chronic use

To characterize the long-term effects of adolescent marijuana abuse, we performed a proteomic analysis of cerebellar extracts from adult female rats with and without ovariectomy that were treated with Δ9-THC for 40 days during adolescence. Six proteins were found to significantly differ among the four treatment groups, with Δ9-THC and ovariectomy (OVX) decreasing the mitochondrial proteins, pyruvate carboxylase and NADH dehydrogenase, whereas the levels of putative cytosolic molecular chaperones NM23B, translationally controlled tumor protein, DJ-1 and activator of heat-shock 90kDa protein ATPase homolog 1 (AHA1) were increased. We further analyzed the effects of AHA1, a HSP90 co-chaperone, on CB1R and CB2R trafficking and signaling in transfected HEK293T and Neuro-2A cells. In HEK293T cells, AHA1 over-expression enhanced plasma membrane levels of CB1R and increased CB1R-mediated effects on cAMP levels and on MAPK phosphorylation. AHA1 over-expression also enhanced cell surface levels of endogenous CB1R and the effects of Δ9-THC on the cAMP levels in Neuro-2A cells. In contrast, over-expression of AHA1 did not affect the subcellular localization and signaling of CB2R. Our data indicate that chronic Δ9-THC administration in adolescence altered the endogenous levels of specialized proteins in the cerebellum, such as AHA1, and that this protein can change CB1R cell surface levels and signaling.

Endocannabinoids: An appetite for fat

It has been known for decades that marijuana causes the “munchies,” i.e., a hunger for palatable food, and for more than 10 y that endocannabinoids (eCBs), in some ways marijuana's counterpart in the organism, are orexigenic mediators (1). When injected in the hypothalamus (HT) or nucleus accumbens (NAc), two key brain areas for the homeostatic and hedonic control of food intake, these compounds stimulate food consumption by acting at the cannabinoid CB1 receptor, one of the two G protein-coupled receptors for marijuana's psychotropic and appetite-inducing component, Δ9-tetrahydrocannabinol (2, 3). Conversely, systemic pharmacological blockade of CB1 receptors causes anorectic effects in rodents exposed to palatable food, or food-deprived for a few hours, and in obese animals (3–6). The two most studied eCBs, anandamide and 2-arachidonoylglycerol (2-AG), are considered local mediators produced by cells only following stimulation, and in this they differ from other signals controlling food intake, which are released into the bloodstream and/or prestored in vesicles (1). What makes the eCBs similar to most orexigenic signals is the fact that their concentrations in the HT and NAc, but also in the proximal intestine, which transmits to the brain the state of “emptiness” or “fullness” of the gut, increase during food deprivation and decrease immediately after food consumption (3, 7, 8). This mechanism is presumably caused by the opposing effects on eCB levels of hormones such as leptin, on the one hand, and ghrelin and corticosterone, on the other hand, the levels of which also vary during food deprivation and refeeding (1, 6). These food intake- and hormone-sensitive changes in eCB signaling are thought to play a key role in the regulation of (i) the release of central neurotransmitters and neuropeptides controlling food intake and (ii) the activity of vagal fibers from the duodenum to the brainstem, which signal gastric distension (1). In this scenario, the results of the elegant study by DiPatrizio and coworkers published in PNAS (9), although in full agreement with the general orexigenic function of eCBs and CB1 receptors, might seem somewhat surprising. In fact, the authors report that a fatty meal elevates eCBs selectively in the rat proximal small intestine, and propose that this effect: (i) is induced by the orosensory properties of the meal; (ii) is mediated by vagal afferent and efferent terminals, and (iii) reinforces fat intake via CB1 receptor activation (9).

Differential developmental trajectories for CB1 cannabinoid receptor expression in limbic/associative and sensorimotor cortical areas

Cannabis use during adolescence is associated with an increased risk for schizophrenia and other disorders. The neuronal basis is unclear, but prefrontal cortical mechanisms have been implicated. Here, we investigated developmental changes in the endocannabinoid system by assessing expression and function of the CB1 cannabinoid receptor in prefrontal and other cortical areas in juvenile (postnatal day 25, P25), adolescent (P40), and adult (P70) rats. Overall, the expression of CB1 receptors in the cortex is highest in juveniles and drops thereafter toward adult levels. However, CB1 receptor expression follows distinct developmental trajectories in different cortical areas. The most pronounced and progressive decrease in CB1 expression was observed in medial prefrontal and other limbic/associative regions. In contrast, major changes in sensorimotor cortices occurred only after P40. We also assessed electrophysiological measures of CB1 receptor function and found that CB1-dependent inhibition of synaptic transmission in the prefrontal cortex follows the same developmental trajectory as observed for receptor expression. Together, these findings indicate that CB1 receptor-mediated signaling decreases during development but is differentially regulated in limbic/associative vs. sensorimotor systems. Therefore, cannabis use during adolescence likely differentially affects limbic/associative and sensorimotor cortical circuits.

Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2–OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2–OT column can be used to determine the binding affinities ( K i values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2–OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.

Cannabinoid CB1 receptor ligand binding and function examined through mutagenesis studies of F200 and S383

The cannabinoid CB1 G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptor's three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB1 receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB1 receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB1 receptor ligands.

Long-lasting recovery of psychotic-like symptoms in isolation-reared rats after chronic but not acute treatment with the cannabinoid antagonist AM251

In this work we investigated the ability of AM251 to reverse schizophrenia-like symptoms produced by a neurodevelopmental animal model based on a social isolation procedure. First, we assessed the validity of our isolation-rearing protocol and, as expected, isolation-reared rats showed hyperlocomotion in a novel environment, cognitive impairment in the novel object recognition (NOR) test and a significant increase in the number of aggressive behaviours in the social interaction test compared to group-housed controls. This behavioural picture was associated with a reduction in CB₁ receptor/G protein coupling in specific brain areas as well as reduced c-Fos immunoreactivity in the prefrontal cortex and caudate putamen. In this model, chronic but not acute treatment with the CB₁ receptor antagonist AM251 counteracted isolation-induced cognitive impairment in the NOR test and aggressive behaviours in the social interaction test. This behavioural recovery was accompanied by the rescue of CB₁ receptor functionality and c-Fos levels in all brain regions altered in isolation-reared rats. Moreover, chronic AM251 also increased c-Fos immunoreactivity in the nucleus accumbens, as previously demonstrated for antipsychotic drugs. Interestingly, the behavioural recovery due to chronic AM251 administration persisted until 10 d after discontinuing the treatment, indicating a long-lasting effect of the cannabinoid antagonist on psychotic-like symptoms.

Chronic URB597 treatment at adulthood reverted most depressive-like symptoms induced by adolescent exposure to THC in female rats

We have recently shown that chronic THC administration in adolescent female rats induces subtle but lasting alterations in the emotional circuit ending in depressive-like behaviour at adulthood. Here we describe other relevant depressive-like symptoms present in these animals. Adult female rats pretreated with THC display passive coping strategy towards acute stressful situations as demonstrated by their behaviours in the first session of the forced swim test, develop a profound anhedonic state as demonstrated by the reduced consumption of palatable food and present a decrease in social functioning. Besides the emotional symptoms, adolescent exposure to THC induced a significant deficit in object recognition memory. Since it has been reported that deficits in adult hippocampal neurogenesis may underlie the cognitive dysfunction seen in depression, we then survey cell proliferation in the dentate gyrus of the hippocampus. Adolescent THC exposure significantly reduced the number of BrdU-positive cells in THC-treated rats as well as hippocampal volume. We suggest that this complex depressive-like phenotype is triggered by a long-lasting decrease in CB1 receptor functionality in specific brain regions. To test whether an increase in the endocannabinoid signalling could ameliorate the depressive phenotype, adult female rats pre-exposed to THC were injected with URB597 (0.3mg/kg ip) and then tested in behavioural assays. URB597 was able to reverse most depressive-like symptoms induced by adolescent THC exposure such as the passive coping strategy observed in THC exposed animals in the forced swim test as well as anhedonia and the reduced social activity.These results support a role for the endocannabinoid system in the neurobiology of depression and suggest the use of URB597 as a new therapeutic tool with antidepressant properties.

Deficiency in Endocannabinoid Signaling in the Nucleus Accumbens Induced by Chronic Unpredictable Stress

The nucleus accumbens (NAc) is a critical component of the reward circuitry, and dysfunction of the NAc may account for anhedonia and other symptoms of depression. Here, we investigated whether alterations in endocannabinoid (eCB) signaling in the NAc contribute to depression-like behaviors induced by chronic unpredictable stress (CUS) in mice. We compared three types of eCB/CB1 receptor-mediated synaptic plasticity in slices prepared from the NAc core of control and stress-exposed mice: depolarization-induced suppression of excitation, long-term depression, and the depression of field excitatory postsynaptic potentials (fEPSPs) induced by group I metabotropic glutamate receptor agonist DHPG. CUS (5-6-week exposure to stressors), but not sub-CUS (1 week exposure to stressors), induces depression-like behaviors and impairs these forms of eCB/CB1 receptor-mediated plasticity examined in the NAc core. Neither sub-CUS nor CUS altered the tissue contents of the eCBs, anandamide and 2-arachidonoylglycerol in the striatum. However, exposure to CUS, but not to sub-CUS, attenuated the depression of fEPSPs induced by the CB1 receptor agonist WIN 55 212-2. CUS exposure reduced the maximal effect without affecting the EC(50) of WIN 55 212-2 to induce fEPSP depression. Thus, impaired CB1 receptor function could account for CUS-induced deficiency in eCB signaling in the NAc. Both CUS-induced deficiency in eCB signaling and depression-like behaviors were reversed by in vivo administration of antidepressant fluoxetine. These results suggest that downregulation of eCB signaling in the NAc occurs after CUS and contributes to the pathophysiology of depression.

Brain-Derived Neurotrophic Factor Controls Cannabinoid CB1 Receptor Function in the Striatum

The role of brain-derived neurotrophic factor (BDNF) in emotional processes suggests an interaction with the endocannabinoid system. Here, we addressed the functional interplay between BDNF and cannabinoid CB(1) receptors (CB(1)Rs) in the striatum, a brain area in which both BDNF and CB(1)s play a role in the emotional consequences of stress and of rewarding experiences. BDNF potently inhibited CB(1)R function in the striatum, through a mechanism mediated by altered cholesterol metabolism and membrane lipid raft function. The effect of BDNF was restricted to CB(1)Rs controlling GABA-mediated IPSCs (CB(1)R(GABA)), whereas CB(1)Rs modulating glutamate transmission and GABA(B) receptors were not affected. The action of BDNF on CB(1)R(GABA) function was tyrosine kinase dependent and was complete even after receptor sensitization with cocaine or environmental manipulations activating the dopamine (DA)-dependent reward system. In mice lacking one copy of the BDNF gene (BDNF(+/-)), CB(1)R(GABA) responses were potentiated and were preserved from the action of haloperidol, a DA D(2) receptor (D(2)R) antagonist able to fully abolish CB(1)R(GABA) function in rewarded animals. Haloperidol also enhanced BDNF levels in the striatum, suggesting that this neurotrophin may act as a downstream effector of D(2)Rs in the modulation of cannabinoid signaling. Accordingly, 5 d cocaine exposure both reduced striatal BDNF levels and increased CB(1)R(GABA) activity, through a mechanism dependent on D(2)Rs. The present study identifies a novel mechanism of CB(1)R regulation mediated by BDNF and cholesterol metabolism and provides some evidence that DA D(2)R-dependent modulation of striatal CB(1)R activity is mediated by this neurotrophin.

Preservation of Striatal Cannabinoid CB1 Receptor Function Correlates with the Antianxiety Effects of Fatty Acid Amide Hydrolase Inhibition

The endocannabinoid anandamide (AEA) plays a crucial role in emotional control, and inhibition of its degradation by the fatty acid amide hydrolase (FAAH) has a potent antianxiety effect. The mechanism by which the magnification of AEA activity reduces anxiety is still largely undetermined. By using FAAH mutant mice and both intraperitoneal and intracerebroventricular administration of the FAAH inhibitor (3'-(aminocarbonyl)[1,1'-biphenyl]-3-yl)-cyclohexylcarbamate (URB597), we found that enhanced AEA signaling reversed, via central cannabinoid CB1 receptors (CB1Rs), the anxious phenotype of mice exposed to social defeat stress. This behavioral effect was associated with preserved activity of CB1Rs regulating GABA transmission in the striatum, whereas these receptors were dramatically down-regulated by stress in control animals. The hypothalamic-pituitary-adrenal (HPA) axis was not involved in the antistress effects of FAAH inhibition, although the HPA axis is a biological target of endogenous AEA. We also provided some physiological indications that striatal CB1Rs regulating GABA synapses are not the receptor targets of FAAH inhibition, which rather resulted in the stimulation of striatal CB1Rs regulating glutamate transmission. Collectively, our findings suggest that preservation of cannabinoid CB1 receptor function within the striatum is a possible synaptic correlate of the antianxiety effects of FAAH inhibition.