Agonist ACEA Research Articles

Cannabinoid receptor ligands prevent dopaminergic neurons death induced by neurotoxic, inflammatory and oxidative stimuli in vitro

Title: Cannabinoid receptor ligands prevent dopaminergic neurons death induced by neurotoxic, inflammatory and oxidative stimuli in vitro. Background: During the last 25 years increasing efforts have been invested for the comprehension of the cannabinoid system in a wide range of healthy and pathological conditions. Previous investigations have indicated the possible protective role of synthetic cannabinoids like arachidonyl-2’-chloroethylamide, ACEA (CB1 agonist), during neurodegenerative diseases such as Parkinson’s disease. Other synthetic CB1 receptor ligands such as N-(Piperidin-1-yl)-5-(4- iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, AM251, commonly known as an antagonist/inverse agonist, have recently been presented as an allosteric modulator of this receptor. In this study, we show the protective effect of ACEA against oxidative and inflammatory damage in in vitro dopaminergic neurons. Methods and Findings: Neuro2A cells differentiated into dopaminergic-like neurons were challenged with neurotoxic, inflammatory and oxidative treatments, 6-hydroxidopamine (6OHDA), lipopolysaccharide (LPS) and hydrogen peroxide (H2O2). Cannabinoid-dependent cell protection was evaluated by means of cellular viability, reactive oxygen species (ROS) production and the pro apoptotic protein caspase 3 expression after CB1 agonist ACEA and antagonist AM251. The ACEA treatment resulted in an increase of cellular viability after the 6OHDA, LPS and H2O2 challenges. When cells were co-treated with ACEA and AM251, an increase in cell death prevention was observed, with a reduction in reactive oxygen species production and caspase 3 expression. Conclusions: Together, we show an ACEA-mediated neuronal protection by means of ROS expression reduction and pro apoptotic protein caspase 3 expression inhibition.

The type-1 cannabinoid receptor modulates the hydroelectrolytic balance independently of the energy homeostasis during salt load

Hydroelectrolytic imbalances, such as saline load (SL), trigger behavioral and neuroendocrine responses, such as thirst, hypophagia, vasopressin (AVP) and oxytocin (OT) release and hypothalamus–pituitary–adrenal (HPA) axis activation. To investigate the participation of the type-1 cannabinoid receptor (CB1R) in these homeostatic mechanisms, male adult Wistar rats were subjected to SL (0.3M NaCl) for four days. SL induced not only increases in the water intake and plasma levels of AVP, OT and corticosterone, as previously described, but also increases in CB1R expression in the lamina terminalis, which integrates sensory afferents, as well as in the hypothalamus, the main integrative and effector area controlling hydroelectrolytic homeostasis. A more detailed analysis revealed that CB1R-positive terminals are in close apposition with not only axons but also dendrites and secretory granules of magnocellular neurons, particularly vasopressinergic cells. In satiated and euhydrated animals, the intracerebroventricular administration of the CB1R selective agonist ACEA (0.1μg/5μL) promoted hyperphagia, but this treatment did not reverse the hyperosmolality-induced hypophagia in the SL group. Furthermore, ACEA pretreatment potentiated water intake in the SL animals during rehydration as well as enhanced the corticosterone release and prevented the increase in AVP and OT secretion induced by SL. The same parameters were not changed by ACEA in the animals whose daily food intake was matched to that of the SL group (Pair-Fed). These data indicate that CB1Rs modulate the hydroelectrolytic balance independently of the food intake during sustained hyperosmolality and hypovolemia.

Pharmacological activation of CB2 receptors counteracts the deleterious effect of ethanol on cell proliferation in the main neurogenic zones of the adult rat brain.

Chronic alcohol exposure reduces endocannabinoid activity and disrupts adult neurogenesis in rodents, which results in structural and functional alterations. Cannabinoid receptor agonists promote adult neural progenitor cell (NPC) proliferation. We evaluated the protective effects of the selective CB1 receptor agonist ACEA, the selective CB2 receptor agonist JWH133 and the fatty-acid amide-hydrolase (FAAH) inhibitor URB597, which enhances endocannabinoid receptor activity, on NPC proliferation in rats with forced consumption of ethanol (10%) or sucrose liquid diets for 2 weeks. We performed immunohistochemical and stereological analyses of cells expressing the mitotic phosphorylation of histone-3 (phospho-H3+) and the replicating cell DNA marker 5-bromo-2'-deoxyuridine (BrdU+) in the main neurogenic zones of adult brain: subgranular zone of dentate gyrus (SGZ), subventricular zone of lateral ventricles (SVZ) and hypothalamus. Animals were allowed ad libitum ethanol intake (7.3 ± 1.1 g/kg/day) after a controlled isocaloric pair-feeding period of sucrose and alcoholic diets. Alcohol intake reduced the number of BrdU+ cells in SGZ, SVZ, and hypothalamus. The treatments (URB597, ACEA, JWH133) exerted a differential increase in alcohol consumption over time, but JWH133 specifically counteracted the deleterious effect of ethanol on NPC proliferation in the SVZ and SGZ, and ACEA reversed this effect in the SGZ only. JWH133 also induced an increased number of BrdU+ cells expressing neuron-specific β3-tubulin in the SVZ and SGZ. These results indicated that the specific activation of CB2 receptors rescued alcohol-induced impaired NPC proliferation, which is a potential clinical interest for the risk of neural damage in alcohol dependence.

Cannabinoid receptor type 1 agonist ACEA improves motor recovery and protects neurons in ischemic stroke in mice.

Brain ischemia produces neuronal cell death and the recruitment of pro-inflammatory cells. In turn, the search for neuroprotection against this type of insult has rendered results involving a beneficial role of endocannabinoid receptor agonists in the Central Nervous System. In this work, to further elucidate the mechanisms associated to this neuroprotective effect, focal brain ischemia was generated by middle cerebral artery occlusion (MCAo) in C57Bl/6 mice. Three, 24 and 48 h after MCAo, animals received CB1R agonist ACEA (1 mg/kg), CB1R antagonist AM251 (1 mg/kg) or vehicle. To assess motor activity, neural deficit scores and motor tests were performed 1 day before and 3, 7, 14, 21, and 28 days after MCAo. At 7 and 28 days post lesion, cytoskeleton structure, astroglial and microglial reaction, and alterations in synapsis were studied in the cerebral cortex. ACEA treatment reduced astrocytic reaction, neuronal death, and dendritic loss. In contrast, AM251 treatment increased these parameters. Motor tests showed a progressive deterioration in motor activity in ischemic animals, which only ACEA treatment was able to counteract. Our results suggest that CB1R may be involved in neuronal survival and in the regulation of neuroprotection during focal cerebral ischemia in mice.

Interaction between Cannabinoid Compounds and Capsazepine in Protection against Acute Pentylenetetrazole-induced Seizure in Mice.

The pharmacological interaction between cannabinoidergic system and vanilloid type 1 (TRPV1) channels has been investigated in various conditions such as pain and anxiety. In some brain structure including hippocampus, CB1 and TRPV1 receptors coexist and their activation produces opposite effect on excitability of neurons. In this study, we tested the hypothesis that TRPV1 channel is involved in the modulation of cannabinoid effects on pentylenetetrazole (PTZ)-induced seizure threshold. In single therapy, male mice (n = 10 per group) received either TRPV1 receptor antagonist capsazepine, CB1 receptor agonist ACEA or anandamide reuptake inhibitor VDM11. In combination therapy, mice were treated with either capsazepine-ACEA or capsazepine-VDM11 combination prior to seizure test. Thirty min later, mice were submitted to infusion of PTZ (1%, 0.25 mL/min) into tail vein and the dose of PTZ to induce clonic convulsion was considered as seizure threshold. Administration of capsazepine and ACEA per se produced protective effects against PTZ-induced seizure, while administration of VDM11 per se did not produce such a protection effect. The anticonvulsant actions of both capsazepine and ACEA were attenuated after co-administration of these compounds. Moreover, the anticonvulsant action of capsazepine was attenuated after co-administration with VDM11. The results suggest an interaction between cannabinoidergic system and TRPV1 receptors in protection against acute PTZ-induced seizure in mice.

Further Evidence for the Neuroplastic Role of Cannabinoids: A Study in Organotypic Hippocampal Slice Cultures.

Endocannabinoid receptors CB1R and CB2R are present in the CNS and modulate synaptic activity. By using an in vitro model, two concentrations of CB1R agonist ACEA at 0.5 and 5μM doses and CB1R antagonist AM251 at 1 and 10μM doses were administered in organotypic slice cultures of mouse hippocampus, and their effects on neurons and glial cells were analyzed at different time points. Exposure to low concentrations of ACEA (0.5μM) did not seem to affect tissue organization, neuronal morphology, or glial response. In contrast, at a higher concentration of ACEA, many neurons in the dentate gyrus exhibited strong caspase-3 immunoreactivity. After treatment with AM251, we observed an increase in caspase-3 immunoreactivity and a downregulation of CB1R expression. Results show that long-term hippocampal slice cultures respond to both CB1R activation and inactivation by changing neuronal protein expression patterns. In the present study, we demonstrate that CB1R agonist ACEA promotes alterations in the neuronal cytoskeleton as well as changes in CB1R expression in organotypic hippocampal slice cultures, and that CB1R antagonist AM251 promotes neuronal death and astroglial reaction.

Cannabinoid CB1 receptors activation and coactivation with D2 receptors modulate GABAergic neurotransmission in the globus pallidus and increase motor asymmetry.

The cannabinoid CB1 (CB1R) and dopaminergic D2 (D2R) receptors modify GABAergic transmission in the globus pallidus. Although dopaminergic denervation produces changes in the expression and supersensitization of these receptors, the consequences of these changes on GABAergic neurotransmission are unknown. The aim of this study was to show the effects of CB1R and D2R activation and coactivation on the uptake and release of [(3) H]GABA in the globus pallidus of hemiparkinsonian rats as well as their effects on motor behavior. The activation of CB1R blocked GABA uptake and decreased GABA release in the globus pallidus in the dopamine denervated side, whereas the co-activation of CB1R-D2R increased GABA release and had no effect on GABA uptake. A microinjection of the CB1R agonist ACEA into the globus pallidus ipsilaterally to a 6-OHDA lesion potentiated turning behavior that was induced by methamphetamine. However, a microinjection of the D2R agonist quinpirole did not modify this behavior, and a microinjection of a mixture of CB1R and D2R agonists significantly potentiated turning behavior. The behavioral effects produced after the activation of the CB1R and the co-activation of CB1R and D2R can be explained by increased GABAergic neurotransmission produced by a block of GABA uptake and an increase in the release of GABA in the globus pallidus, respectively.

Differential effects of CB1 receptor agonism in behavioural tests of unconditioned and conditioned fear in adult male rats

We investigated the effects of the highly selective CB1 receptor agonist ACEA and the CB1 receptor antagonist/inverse agonist AM251 on two behavioural tests of unconditioned fear, the elevated plus maze (EPM) and open field test (OFT), as well as on the recall and extinction of a conditioned auditory fear. Both ACEA and AM251 increased anxiety-like behaviour in the EPM and OFT. There was no effect of either drug on recall of the conditioned fear, and ACEA enhanced and AM251 impaired fear extinction. Further, though both the low (0.1mg/kg) and high (0.5mg/kg) dose of ACEA facilitated fear extinction, the low dose attenuated, and the high dose potentiated, fear induced corticosterone release suggesting independent effects of the drug on fear and stress responses. Although the extent to which cannabinoids are anxiogenic or anxiolytic has been proposed to be dose-dependent, these results indicate that the same dose has differential effects across tasks, likely based in differences in sensitivities of CB1 receptors to the agonist in the neural regions subserving unconditioned and conditioned fear.

Ultralow doses of cannabinoid drugs protect the mouse brain from inflammation-induced cognitive damage.

In our previous studies, we found that a single ultralow dose of tetrahydrocannabinol (THC; 0.002 mg/kg, three to four orders of magnitude lower than the conventional doses) protects the brain from different insults that cause cognitive deficits. Because various insults may trigger a neuroinflammatory response that leads to secondary damage to the brain, the current study tested whether this extremely low dose of THC could protect the brain from inflammation-induced cognitive deficits. Mice received a single injection of THC (0.002 mg/kg) 48 hr before or 1-7 days after treatment with lipopolysccharide (LPS; 10 mg/kg) and were examined with the object recognition test 3 weeks later. LPS caused long-lasting cognitive deficits, whereas the application of THC before or after LPS protected the mice from this LPS-induced damage. The protective effect of THC was blocked by the cannabinoid (CB) 1 receptor antagonist SR14176A but not by the CB2 receptor antagonist SR141528 and was mimicked by the CB1 agonist ACEA but not by the CB2 agonist HU308. The protective effect of THC was also blocked by pretreatment with GW9662, indicating the involvement of peroxisome proliferator-activated receptor-γ. Biochemical examination of the brain revealed a long-term (at least 7 weeks) elevation of the prostaglandin-producing enzyme cyclooxygenase-2 in the hippocampus and in the frontal cortex following the injection of LPS. Pretreatment with the extremely low dose of THC tended to attenuate this elevation. Our results suggest that an ultralow dose of THC that lacks any psychotrophic activity protects the brain from neuroinflammation-induced cognitive damage and might be used as an effective drug for the treatment of neuroinflammatory conditions, including neurodegenerative diseases.

Cannabinoid effects on β amyloid fibril and aggregate formation, neuronal and microglial-activated neurotoxicity in vitro.

Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ1-42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ(9)-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ1-42. Aβ1-42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.

Suppression of outward K+ currents by WIN55212-2 in rat retinal ganglion cells is independent of CB1/CB2 receptors

Cannabinoid CB1 receptor (CB1R) signaling system is extensively distributed in the vertebrate retina. Activation of CB1Rs regulates a variety of functions of retinal neurons through modulating different ion channels. In the present work we studied effects of this receptor signaling on K+ channels in retinal ganglion cells by patch-clamp techniques. The CB1R agonist WIN55212-2 (WIN) suppressed outward K+ currents in acutely isolated rat retinal ganglion cells in a dose-dependent manner, with an IC50 of 4.7μM. We further showed that WIN mainly suppressed the tetraethylammonium (TEA)-sensitive K+ current component. While CB1Rs were expressed in rat retinal ganglion cells, the WIN effect on K+ currents was not blocked by either AM251/SR141716, specific CB1R antagonists, or AM630, a selective CB2R antagonist. Consistently, cAMP-protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways were unlikely involved in the WIN-induced suppression of the K+ currents because both PKA inhibitors H-89/Rp-cAMP and MAPK/ERK1/2 inhibitor U0126 failed to block the WIN effects. WIN-induced suppression of the K+ currents was not observed when WIN was intracellularly applied. Furthermore, an endogenous ligand of the cannabinoid receptor anandamide, the specific CB1R agonist ACEA and the selective CB2R agonist CB65 also suppressed the K+ currents, and the effects were not blocked by AM251/SR141716 or AM630 respectively. All these results suggest that the WIN-induced suppression of the outward K+ currents in rat retinal ganglion cells, thereby regulating the cell excitability, were not through CB1R/CB2R signaling pathways.

CB1 cannabinoid receptor-mediated aggressive behavior

This study examined the role of cannabinoid CB1 receptors (CB1r) in aggressive behavior. Social encounters took place in grouped and isolated mice lacking CB1r (CB1KO) and in wild-type (WT) littermates. Cognitive impulsivity was evaluated in the delayed reinforcement task (DRT). Gene expression analyses of monoaminooxidase-A (MAO-A), catechol-o-methyl-transferase (COMT), 5-hydroxytriptamine transporter (5-HTT) and 5-HT1B serotonergic receptor (5HT1Br) in the median and dorsal raphe nuclei (MnR and DR, respectively) and in the amygdala (AMY) were performed by real time-PCR. Double immunohistochemistry studies evaluated COMT and CB1r co-localization in the raphe nuclei and in the cortical (ACo), basomedian (BMA) and basolateral (BLA) amygdaloid nuclei. The behavioral effects of the CB1r agonist ACEA (1 and 2 mg/kg) on aggression were also evaluated in isolated OF1 mice. CB1KO mice housed in groups showed higher levels of offensive aggression. Isolation increased aggressive behavior only in WT. In grouped CB1KO mice COMT gene expression was significantly higher in the MnR and DR, while MAO-A gene expression was lower in the MnR. Gene expression of 5HT1Br, COMT and MAO-A was higher in the amygdala of CB1KO mice. CB1r double-immunohistochemistry revealed cytoplasmic-labeled COMT-ir cells in the raphe nuclei and in the ACo, BMA and BLA. CB1r immunolabeling was observed only in ACo, BMA and BLA, where it was localized in axons and buttons. The density of labeled processes increased in BLA. Acute administration of the CB1 agonist ACEA (2 mg/kg) significantly decreased the aggression levels of OF1 mice. These results suggest that CB1r plays an important role in social interaction and aggressive behavior.

CB1 cannabinoid receptor agonist prevents NGF-induced sensitization of TRPV1 in sensory neurons

The transient receptor potential vanilloid type 1 channel (TRPV1) and nerve growth factor (NGF) are important mediators of inflammatory pain. NGF released during inflammation sensitizes TRPV1 in afferent nerve endings of peripheral nociceptors, increasing pain sensation. Cannabinoids, by activating CB1 G protein-coupled receptors, produce analgesia in a variety of pain models, though the exact mechanisms are not known. We tested the hypothesis that activation of the CB1 receptor by cannabinoids attenuates NGF-induced TRPV1 sensitization. TRPV1-mediated currents were measured in acutely isolated primary sensory neurons with the whole-cell patch clamp technique using capsaicin (100nM) as the agonist. After the first capsaicin application, during which the baseline current was measured, cells were exposed to NGF (100ng/mL), and the capsaicin application was repeated after 5min. NGF sensitized TRPV1 in 31.0% of cells (13 of 42), with a mean (±SE) increase in the capsaicin-induced current of 262±47% over the baseline current. When the cannabinoid agonist ACEA (arachidonoyl-2′-chloroethylamide; 10nM) was given before NGF, only 10.8% of cells (4 of 37) were sensitized (p<0.05). Neither this rate, nor the magnitude of the sensitization (198±63% of baseline) were different from that seen in cells not treated with NGF (3 of 25 cells sensitized (12.0%), 253±70% of baseline). Pretreatment with the CB1 antagonist AM-251 (100nM) prevented the effect of ACEA on NGF-induced sensitization. These results support the hypothesis that cannabinoids, acting through CB1 receptors, may produce analgesia in part by preventing NGF-induced sensitization of TRPV1 in afferent nociceptor nerve endings.

The role of α2-adrenoceptors in the anti-convulsant effects of cannabinoids on pentylenetetrazole-induced seizure threshold in mice

Cannabinoid system plays a pivotal role in the seizure threshold modulation which is mainly mediated through activation of the cannabinoid CB1 receptor. There is also several evidence of interaction between cannabinoid system and α2-adrenoceptors in different paradigms. Using model of clonic seizure induced by intravenous pentylenetetrazole (PTZ) in male mice, we investigated whether α2-adrenoceptors is involved in the effects of cannabinoids on the seizure threshold. Injection of the selective cannabinoid CB1 agonist ACEA (2mg/kg) significantly (P<0.01) increased the seizure threshold which was prevented by pretreatment with the selective CB1 antagonist AM251 (1mg/kg, i.p.). The highest doses of clonidine, a α2 receptor agonist, (1 and 5mg/kg) showed anticonvulsant effects while yohimbine, a α2 receptor antagonist, (0.01, 0.1, 1, and 10mg/kg) did not induce any significant effect on PTZ seizure threshold. Pretreatment with clonidine (0.1 and 0.5mg/kg) significantly reversed the anticonvulsant effect of ACEA (2mg/kg). Yohimbine (0.1, 1, and 10mg/kg) pretreatment of mice enhanced the clonic seizure threshold of ACEA (1mg/kg), significantly. Combination of non-effective doses of AM251 (0.1mg/kg) and clonidine (0.01mg/kg) showed additive effect in blocking the anticonvulsant effect of ACEA (2mg/kg). In conclusion, our findings demonstrated that α2-adrenoceptors could be involved in the anticonvulsant properties of the specific cannabinoid CB1 agonist ACEA, suggesting that CB1 cannabinoid and α2 receptors have functional interactions in modulation of clonic seizure threshold.

Characterization of cannabinoid-induced relief of neuropathic pain in a rat model of cisplatin-induced neuropathy

Clinical use of antineoplastic drugs is associated with the development of numerous adverse effects that many patients find intolerable, including peripheral neuropathy. Cannabinoids have relieved neuropathic pain in different animal models. But their therapeutic activities could be affected by their psychoactive properties.The aim of this work was to determine the effect of cannabinoids in cisplatin-evoked neuropathy. For this purpose, the non-selective agonist WIN 55,212-2 (WIN), the CB1-selective agonist ACEA or the CB2-selective agonist JWH133 (or their vehicle) was either systemically administered at a non-psychoactive dose or locally injected in cisplatin-treated rats. Selective CB1 and CB2 cannabinoid antagonists (AM251 and SR144528, respectively) were used to characterize cannabinoid effects.Cisplatin-treated rats showed mechanical allodynia but not thermal hyperalgesia. Cannabinoid agonists alleviated mechanical allodynia. This effect was mediated by both CB1 and CB2 cannabinoid receptors when the cannabinoid was systemically applied. At the dose used, cannabinoid agonists had no psychoactive effect. The local effect of the drug involved the activation of peripheral CB1 receptors whereas involvement of CB2 receptors was less clear.In a rat model of cisplatin-induced neuropathy, cannabinoids have an antinociceptive effect, but the cannabinoid receptors involved could be different depending on the route of administration. Non-psychoactive doses of cannabinoid agonists are capable of alleviating the signs of peripheral neuropathy when systemically applied. Interestingly, local administration of selective CB1 agonists or systemic administration of CB2 agonists, which are non-psychoactive, may serve as new therapeutic alternatives for symptom management in painful neuropathy associated with cisplatin treatment.

Type-1 (CB1) cannabinoid receptor promotes neuronal differentiation and maturation of neural stem cells.

Neural stem cells (NSCs) are self-renewing cells that can differentiate into multiple neural lineages and repopulate regions of the brain after injury. We have investigated the role of endocannabinoids (eCBs), endogenous cues that modulate neuronal functions including neurogenesis, and their receptors CB1 and CB2 in mouse NSCs. Real-time PCR and Western blot analyses indicated that CB1 is present at higher levels than CB2 in NSCs. The eCB anandamide (AEA) or the CB1-specific agonist ACEA enhanced NSC differentiation into neurons, but not astrocytes and oligodendrocytes, whereas the CB2-specific agonist JWH133 was ineffective. Conversely, the effect of AEA was inhibited by CB1, but not CB2, antagonist, corroborating the specificity of the response. CB1 activation also enhanced maturation of neurons, as indicated by morphometric analysis of neurites. CB1 stimulation caused long-term inhibition of the ERK1/2 pathway. Consistently, pharmacological inhibition of the ERK1/2 pathway recapitulated the effects exerted by CB1 activation on neuronal differentiation and maturation. Lastly, gene array profiling showed that CB1 activation augmented the expression of genes involved in neuronal differentiation while decreasing that of stemness genes. These results highlight the role of CB1 in the regulation of NSC fate and suggest that its activation may represent a pro-neuronal differentiation signal.

Chronic activation of cannabinoid receptors in vitro does not compromise mouse islet function

We have demonstrated previously that mouse and human islets express ECS (endocannabinoid system) elements, and that short-term activation of islet cannabinoid CB1r and CB2r (cannabinoid type1 and 2 receptors respectively) stimulates insulin secretion invitro. There is evidence that the ECS is overactive in Type2 diabetes, impairing glucose homoeostasis, but little is known about whether it is implicated in islet dysfunction. Therefore the aim of the present study was to investigate the effect of chronic exposure of isolated mouse islets to cannabinoid receptor agonists on islet gene expression and function. Quantitative RT-PCR (reverse transcription-PCR) indicated that mRNAs encoding synthesis [NAPE-PLD (N-acyl-phosphatidyl ethanolamide-hydrolysing phospholipase D)] and degradation [FAAH (fatty acid amide hydrolase)] of the endocannabinoid AEA (anandamide) were the most abundant ECS elements in mouse islets, with much lower levels of CB1r, CB2r, DAGL (diacylglycerol lipase) and MAGL (monoacylglycerol lipase) mRNAs. Maintenance of islets for up to 7days in the presence of the CB1r agonist ACEA [N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eiscosatetraenamide] or the CB2r agonist JWH015 [(2-methyl-1propyl-1H-indol3-yl)-1-napthalenylmethanone] did not compromise islet viability, as assessed by islet morphology and caspase activities, but there were some changes in mRNAs encoding ECS components. Neither glucose-stimulated insulin secretion nor acute insulin secretory responses to ACEA or JWH015 at 16mM glucose were substantially modified by a 48h or 7day pre-exposure to these cannabinoid receptor agonists, but the stimulation of secretion at 3mM glucose by 100nM ACEA was significantly reduced after prolonged treatment with ACEA. Despite JWH015-induced reductions in islet glucagon content at 48h and 7days, there were no reductions in arginine-induced glucagon secretion from islets pre-exposed to JWH015 or ACEA. These data indicate that treatment of islets with agonists of CB1r and CB2r for up to 7days does not have any major impact on islet function, suggesting that the impairments in glucose homoeostasis observed following overactivation of the ECS should be sought in relation to insulin resistance rather than β-cell dysfunction.

The endocannabinoid and endovanilloid systems interact in the rat prelimbic medial prefrontal cortex to control anxiety-like behavior

Cannabinoid receptor 1 (CB1) agonists usually induce dose-dependent biphasic effects on anxiety-related responses. Low doses induce anxiolytic-like effects, whereas high doses are ineffective or anxiogenic, probably due to activation of Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels. In this study we have investigated this hypothesis by verifying the effects of the CB1/TRPV1 agonist ACEA injected into the prelimbic medial prefrontal cortex (PL) and the participation of endocannabinoids in the anxiolytic-like responses induced by TRPV1 antagonism, using the elevated plus-maze (EPM) and the Vogel conflict test (VCT). Moreover, we verified the expression of these receptors in the PL by double labeling immunofluorescence. ACEA induced anxiolytic-like effect in the intermediate dose, which was attenuated by previous injection of AM251, a CB1 receptor antagonist. The higher and ineffective ACEA dose caused anxiogenic- and anxiolytic-like effects, when injected after AM251 or the TRPV1 antagonist 6-iodonordihydrocapsaicin (6-I-CPS), respectively. Higher dose of 6-I-CPS induced anxiolytic-like effects both in the EPM and the VCT, which were prevented by previous administration of AM251. In addition, immunofluorescence showed that CB1 and TRPV1 receptors are closely located in the PL. These results indicate that the endocannabinoid and endovanilloid systems interact in the PL to control anxiety-like behavior.

Effects of Anandamide on Electrical Activity, Calcium Transients and Contraction in Guinea Pig Cardiac Ventricular Myocytes

Anandamide is an endocannabinoid which reduces cardiac contractility. Although the effects of endocannabinoids are generally attributed to their association with G-protein-coupled cannabinoid CB1 and CB2 receptors, the identity of the receptor(s) or target(s) responsible for the effects of anandamide in the heart are unknown.Anandamide (10 μM) reduced action potential (AP) duration and contraction in guinea pig ventricular myocytes stimulated to fire APs using an intracellular microelectrode. In addition, anandamide reduced peak amplitude of L-type Ca2+ currents over the range −30 to + 60 mV (switched voltage clamp, step depolarisations from −40 mV), with a reduction in peak current at 0 mV of 57 ± 11 % (n = 6, p < 0.01). Ca2+ transients measured in field-stimulated cells using Fluo-5F were also reduced. The reduction in AP duration in response to anandamide was partially inhibited by the CB2 receptor antagonist AM 630 (1 μM), but effects appeared non-competitive, and were smaller than predicted based on a Ki value of 31 nM for CB2 receptors. The CB1 receptor antagonist AM 281 (1 μM), the TRPV1 receptor antagonist capsazepine (1 μM), and O-1918 (an antagonist of a reported non CB1/non CB2 cannabinoid receptor; 1 μM) failed to inhibit responses to 10 μM anandamide. Furthermore, the selective CB1 receptor agonist ACEA, and the selective CB2 receptor agonist HU-308 (both 10 μM) were without significant effect on AP duration.The effects of anandamide can, at least in part, be accounted for by an inhibition of L-type Ca2+ currents. However, additional effects on other ion channels or intracellular targets cannot be ruled out. It appears that if a cannabinoid receptor mediates these effects, the characteristics of this receptor are not those expected of conventional CB1 or CB2 receptors.

CB1 receptors and post-ischemic brain damage: Studies on the toxic and neuroprotective effects of cannabinoids in rat organotypic hippocampal slices

Cannabinoids (CBs) are implicated in a number of physiological and pathological mechanisms in the central nervous system, but their exact role in post-ischemic brain injury is unclear. The toxic and neuroprotective effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen–glucose deprivation (OGD) and in gerbils subjected to bilateral carotid occlusion for 5 min. When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1–30 μM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 μM. AM 251 (at 3 mg/kg, i.p.) also attenuated CA1 pyramidal cell death in gerbils in vivo. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1–1 μM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. Our results suggest that exogenous administration of CB1 agonists and the production of endocannabinoids “on demand” may produce different, if not opposite, effects on the fate of neurons following cerebral ischemia.