Pharmacological enhancement of endocannabinoid signaling reduces the cholinergic toxicity of diisopropylfluorophosphate

Diisopropylfluorophosphate (DFP) elicits cholinergic toxicity by inhibiting acetylcholinesterase, leading to accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors throughout the body. Endocannabinoids inhibit the release of neurotransmitters including acetylcholine via a widely distributed retrograde signaling pathway. Endocannabinoid signaling is therefore a potential therapeutic target for the management of OP poisoning. We first evaluated the relative in vitro and in vivo (2.5mg/kg, sc) effects of DFP on cholinesterase, fatty acid amide hydrolase (FAAH, an endocannabinoid degrading enzyme), monoacylglycerol lipase (MAGL, another endocannabinoid degrading enzyme) and cannabinoid receptor (CB1) binding in rat hippocampus. The effects of WIN 55212-2 (cannabinoid receptor agonist, 1.5mg/kg), URB597 (FAAH inhibitor, 3mg/kg), URB602 (MAGL inhibitor, 10mg/kg) or AM404 (endocannabinoid uptake inhibitor, 10mg/kg) on DFP toxicity were then examined. Adult male rats were given either peanut oil or DFP followed immediately by vehicle or one of the four cannabinomimetic drugs. Functional signs of toxicity were evaluated for 24h and then rats were sacrificed for neurochemical measurements. DFP inhibited cholinesterase, FAAH, MAGL and CB1 receptor binding in vitro in a concentration-dependent manner, with highest and lowest potency against cholinesterase and FAAH, respectively. In vivo, DFP inhibited hippocampal cholinesterase (89%) and FAAH (42%), but had no significant effect on MAGL or CB1 binding. Rats treated with DFP alone showed typical signs of cholinergic toxicity including involuntary movements and excessive secretions (SLUD signs). WIN 55212-2, URB597, URB602 and AM404 all significantly reduced involuntary movements following DFP exposure in a time-dependent manner, and most (URB597, URB602 and AM404) also significantly reduced DFP-induced SLUD signs. These results suggest that enhancing endocannabinoid signaling can attenuate the acute toxicity of DFP and provide rationale for further investigations on the role of endocannabinoids in cholinergic toxicity.     

Estrogen recruits the endocannabinoid system to modulate emotionality

Estrogen administration elicits anxiolytic and antidepressant-like effects in female rats; however, the mechanism of this effect is unknown. Fatty acid amide hydrolase (FAAH), the enzyme which degrades the endocannabinoid anandamide, has been shown to be regulated by estrogen. Thus, we examined if the anxiolytic and antidepressant effects of estrogen implicated the endocannabinoid system. In the first experiment, ovariectomized female rats were administered a single injection of 17beta-estradiol (10 microg) or oil, and 48 h later were given an injection of the cannabinoid CB1 receptor antagonist AM251 (1 mg/kg) or vehicle. One hour after AM251 or vehicle administration, subjects were tested in either the open field test (OFT), elevated plus maze (EPM) or the forced swim test (FST). Estradiol treatment resulted in a significant increase in open arm entries in the EPM and time spent in the center quadrant of the OFT, which were reversed by co-treatment with AM251, suggesting that endocannabinoids are integral to the anxiolytic effects of estrogen. No significant effects of estradiol or AM251 were seen in the FST. In the second experiment, administration of the FAAH inhibitor URB597 (0.1 and 0.3 mg/kg) increased open arm entries in the EPM and time spent in the center quadrant in the OFT as well as significantly reduced immobility in the FST. Collectively, these data demonstrate that estrogen may elicit changes in emotional behavior through an endocannabinoid mechanism, and suggest that inhibition of FAAH represents a therapeutic target for anxiety and depression in women.     

Facilitation of CB1 receptor-mediated neurotransmission decreases marble burying behavior in mice

Obsessive-compulsive disorder (OCD) is a common psychiatric disorder characterized by the occurrence of obsessions and compulsions. Glutamatergic abnormalities have been related to the pathophysiology of OCD. Cannabinoids inhibit glutamate release in the central nervous system, but the involvement of drugs targeting the endocannabinoid system has not yet been tested in animal models of repetitive behavior. Thus, the aim of the present study was to verify the effects of the CB1 receptor agonist WIN55,212-2, the inhibitor of anandamide uptake AM404 and the anandamide hydrolysis inhibitor URB597, on compulsive-associate behavior in male C57BL/6J mice submitted to the marble burying test (MBT), an animal model used for anti-compulsive drug screening. WIN55,212-2 (1 and 3 mg/kg), AM404 (1 and 3 mg/kg) and URB597 (0.1, 0.3 and 1 mg/kg) induced a significant decrease in the number of buried marbles compared to controls. Pretreatment with the CB1 receptor antagonist, AM251, prevented both WIN55,212-2 and URB597 effects. These results suggest a potential role for drugs acting on the cannabinoid system in modulating compulsive behavior.     

Cannabinoids prevent the differential long‐term effects of exposure to severe stress on hippocampal‐ and amygdala‐dependent memory and plasticity

Exposure to excessive or uncontrolled stress is a major factor associated with various diseases including posttraumatic stress disorder (PTSD). The consequences of exposure to trauma are affected not only by aspects of the event itself, but also by the frequency and severity of trauma reminders. It was suggested that in PTSD, hippocampal‐dependent memory is compromised while amygdala‐dependent memory is strengthened. Several lines of evidence support the role of the endocannabinoid (eCB) system as a modulator of the stress response. In this study we aimed to examine cannabinoids modulation of the long‐term effects (i.e., 1 month) of exposure to a traumatic event on memory and plasticity in the hippocampus and amygdala. Following exposure to the shock and reminders model of PTSD in an inhibitory avoidance light‐dark apparatus rats demonstrated: (i) enhanced fear retrieval and impaired inhibitory extinction (Ext), (ii) no long‐term potentiation (LTP) in the CA1, (iii) impaired hippocampal‐dependent short‐term memory in the object location task, (iv) enhanced LTP in the amygdala, and (v) enhanced amygdala‐dependent conditioned taste aversion memory. The cannabinoid CB1/2 receptor agonist WIN55‐212,2 (0.5mg/kg, i.p.) and the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.3mg/kg, i.p.), administered 2 hr after shock exposure prevented these opposing effects on hippocampal‐ and amygdala‐dependent processes. Moreover, the effects of WIN55‐212,2 and URB597 on Ext and acoustic startle were prevented by co‐administration of a low dose of the CB1 receptor antagonist AM251 (0.5mg/kg, i.p.), suggesting that the preventing effects of both drugs are mediated by CB1 receptors. Exposure to shock and reminders increased CB1 receptor levels in the CA1 and basolateral amygdala 1 month after shock exposure and this increase was also prevented by administering WIN55‐212,2 or URB597. Taken together, these findings suggest the involvement of the eCB system, and specifically CB1 receptors, in the opposite effects of severe stress on memory and plasticity in the hippocampus and amygdala.     

Evaluation of interactions between cannabinoid compounds and diazepam in electroshock-induced seizure model in mice

Several studies have shown that cannabinoids have anticonvulsant properties that are mediated through activation of the cannabinoid CB1 receptors. In addition, endogenous cannabinoid compounds (endocannabinoids) regulate synaptic transmission and dampen seizure activity via activation of the same receptors. The aim of this study was to evaluate the possible interactions between antiepileptic effects of cannabinoid compounds and diazepam using electroshock-induced model of seizure in mice. Electroconvulsions were produced by means of an alternating current (ear-clip electrodes, fixed current intensity 35 mA, stimulus duration 0.2 s) and tonic hindlimb extension was taken as the endpoint. All experiments were performed on groups of ten mice and the number of animals who did not display seizure reported as percent protection. Intraperitoneal (i.p.) administration of diazepam (0.25-2 mg/kg) and CB1 receptor agonist WIN55212-2 (0.5-4 mg/kg) dose dependently produced an antiepileptic effect evaluated in terms of increased percentage of protection against electroshock-induced seizure. Logistic regression analysis indicated synergistic interactions in anticonvulsant action after co-administration of diazepam and WIN55212-2 in fixed-ratio combination of 3:1 (diazepam:WIN55212-2), while an additive effect was resulted after co-administration of 1:1 and 1:3 fixed-ratio combinations. Administration of various doses of the endocannabinoid reuptake inhibitor, AM404, did not produce any effect on electroshock-induced seizure. Moreover, co-administration of AM404 and diazepam did not produce significant interaction in antiepileptic properties of these compounds. Administration of the fatty acid amide hydrolase inhibitor, URB597, produced significant antiepileptic effect. Co-administration of URB597 and diazepam led to an antagonistic interaction in protection against shock-induced seizure. Co-administration of different doses of the cannabinoid CB1 receptor antagonist, AM251 did not alter the antiepileptic effect of diazepam in the electroshock-induced seizure test. These results demonstrate that endocannabinoid system participates in the modulation of seizure and combination of small doses of exogenous CB1 receptor agonists with diazepam may have effective consequences in seizure control. Furthermore, inhibiting the endocannabinoid degradation could be more efficacious in modulating seizure than preventing their uptake. This study also suggests that the effects of cannabinoids on epilepsy depend on the relative cannabinoid responsiveness of GABAergic and glutamatergic neurotransmission. While, the antiepileptic effects of cannabinoid compounds are likely by affecting excitatory glutamate neurotransmission, the antagonistic interaction between cannabinoid compounds and diazepam to protect seizure is due to the cannabinoid action on inhibitory GABAergic system.     

Interactions between endocannabinoids and stress-induced decreased sensitivity to natural reward

Since endocannabinoids modulate reward processing and the stress response, we tested the hypothesis that endocannabinoids regulate stress-induced decreased sensitivity to natural reward. Restraint was used to produce stress-induced reductions in sucrose consumption and preference in male mice. Central cannabinoid receptor (CB(1)) signaling was modulated pharmacologically prior to the application of stress. The preference for sucrose over water was significantly decreased in mice exposed to restraint. Treatment of mice with a cannabinoid receptor agonist (CP55940) or fatty acid amide hydrolase inhibitor (URB597) attenuated, while the CB(1) receptor antagonist/inverse agonist, rimonabant (SR141716), enhanced, stress-induced decreases in sucrose preference. These data are consistent with a tonically active, stress-inhibitory role for the CB(1) receptor. Mice treated with 10 daily episodes of restraint showed reduced sucrose preference that was unaffected by CP55940 and URB597. However, rimonabant produced a greater reduction in sucrose preference on day 10 compared to day 1. These data suggest that on day 10, endocannabinoid signaling is maximally activated and essential for reward sensitivity. The findings of the present study indicate that the CB(1)/endocannabinoid signaling system is an important allostatic mediator that both modulates the responses of mice to stress and is itself modulated by stress.     

Antidepressant-like activity of the fatty acid amide hydrolase inhibitor URB597 in a rat model of chronic mild stress.

BACKGROUND: The endocannabinoid anandamide may be involved in the regulation of emotional reactivity. In particular, it has been shown that pharmacological inhibition of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of anandamide, elicits anxiolytic-like and antidepressant-like effects in rodents. METHODS: We investigated the impact of chronic treatment with the selective FAAH inhibitor, URB597 (also termed KDS-4103), on the outcomes of the chronic mild stress (CMS) in rats, a behavioral model with high isomorphism to human depression. RESULTS: Daily administration of URB597 (.3 mg kg(-1), intraperitoneal [IP]) for 5 weeks corrected the reduction in body weight gain and sucrose intake induced by CMS. The antidepressant imipramine (20 mg kg(-1), once daily, IP) produced a similar response, whereas lower doses of URB597 were either marginally effective (.1 mg kg(-1)) or ineffective (.03 mg kg(-1)). Treatment with URB597 (.3 mg kg(-1)) resulted in a profound inhibition of brain FAAH activity in both CMS-exposed and control rats. Furthermore, the drug regimen increased anandamide levels in midbrain, striatum, and thalamus. CONCLUSIONS: URB597 exerts antidepressant-like effects in a highly specific and predictive animal model of depression. These effects may depend on the ability of URB597 to enhance anandamide signaling in select regions of the brain.     

Inhibition of anandamide hydrolysis enhances noradrenergic and GABAergic transmission in the prefrontal cortex and basolateral amygdala of rats subjected to acute swim stress

Limbic forebrain endocannabinoid (eCB) signaling is critically involved in stress integration by modulating neurotransmitters release. The purpose of this study was to examine, by brain microdialysis, the effects of fatty acid amide hydrolase (FAAH) inhibition on noradrenergic and γ‐aminobutyric acid (GABA)‐ergic neurotransmission in the prefrontal cortex (PFC) and basolateral amygdala (BLA) of rats subjected to a 20‐min swim stress. Microdialysis started on stress‐ and drug‐naïve rats that were treated with the FAAH inhibitor URB597 (0.1 or 0.3 mg/kg) 30 min before undergoing the stress procedure. Dialysate samples were collected every 20 min from the beginning of the experiment. Concentrations of noradrenaline (NA) and GABA were determined by HPLC coupled to electrochemical and fluorescence detection, respectively. We found that neither URB597 treatment nor 20 min of swim stress exposure per se altered NA and GABA extracellular levels in PFC or BLA. Interestingly, rats treated with 0.1 mg/kg of URB597 followed by 20 min of stress showed significantly higher NA and GABA levels in PFC and BLA. These effects were absent in rats treated with 0.3 mg/kg URB597, indicating a dose‐specific effect. Moreover, we found that the pretreatment with the CB1 receptor antagonist rimonabant blocked the URB597 effects on NA and GABA release in PFC and BLA of animals subjected to forced swimming. The present study might provide an important first step toward understanding the mechanisms through which URB597 modulates stress‐induced neuroendocrine secretion and behavioral coping strategies. © 2015 Wiley Periodicals, Inc.     

'One-trial sensitization' to the anxiolytic-like effects of cannabinoid receptor antagonist SR141716A in the mouse elevated plus-maze

Significant variability in the effects of cannabinoid CB1 receptor ligands on emotional reactivity in animals and humans suggests that the endocannabinoid system may selectively modulate certain types of anxiety. In view of substantial evidence for qualitative differences in the nature of anxiety elicited on initial and subsequent exposures to the elevated plus-maze, the present studies contrasted the behavioural effects of the selective CB1 receptor antagonist SR141716A (0.1-10.0 mg/kg) and the reference benzodiazepine chlordiazepoxide (CDP, 15 mg/kg) both in maze-naive mice (trial 1) and in mice that had been given a single undrugged exposure to the maze 24 h prior to testing (trial 2). Results confirmed the anxioselective effect of CDP on trial 1 but a complete absence of such activity on trial 2 (i.e. one trial tolerance). In marked contrast, SR141716A had no behavioural effects in maze-naive mice but, at doses of 1.0-3.0 mg/kg (effect maximal at 1.0 mg/kg), significantly reduced anxiety-like responses in maze-experienced animals. Like the effect of CDP on trial 1, the antianxiety profile of SR141716A on plus-maze trial 2 was observed in the absence of any change in general activity levels. The apparent experientially induced 'sensitization' to the anxiolytic-like effects of SR141716A in the plus-maze contrasts markedly with the widely reported loss of benzodiazepine efficacy in test-experienced animals. Data are discussed in relation to the recently described phenotypes of CB1 receptor knockout mice and, in particular, to mounting evidence for the existence of a novel SR141716A-sensitive neuronal cannabinoid receptor.     

Spinal anandamide inhibits nociceptive transmission via cannabinoid receptor activation in vivo

The endocannabinoid anandamide has affinity for cannabinoid and vanilloid receptors, which have opposing effects on nociceptive transmission. Effects of spinal administration of anandamide on innocuous and noxious evoked spinal neuronal responses in non-inflamed and carrageenin-inflamed rats were studied. Anandamide (0.1-50 microg/50 microl) had inconsistent effects in non-inflamed rats. Following carrageenin inflammation, anandamide (50 microg/50 microl) significantly reduced evoked neuronal responses, C-fibre mediated non-potentiated and post-discharge responses of neurones reduced to 65 +/- 5% and 57 +/- 10% of control, respectively. Effects of anandamide were blocked by SR141716A, a selective CB1 receptor antagonist. Spinal SR141716A (0.001-1 ng/50 microl) alone did not influence neuronal responses in inflamed rats. Spinal anandamide inhibited nociceptive transmission via CB1 receptors; following inflammation there is evidence for a loss of spinal endogenous cannabinoid tone.     

Effects of inhibition of fatty acid amide hydrolase vs. the anandamide membrane transporter on TRPV1-mediated calcium responses in adult DRG neurons; the role of CB receptors

The aim of the present study was to investigate the relationship between TRPV1 stimulation and endocannabinoid-driven CB(1) receptor-mediated inhibition of activity in adult rat dorsal root ganglion (DRG) neurons, a model of primary afferent nociceptors. Calcium-imaging studies were performed to compare the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 microm) vs. the anandamide (AEA) uptake inhibitor UCM707 (1 microm) on capsaicin (100 nm) and N-arachidonoyl dopamine (NADA; 1 microm)-evoked changes in intracellular calcium [Ca(2+)](i) in DRG neurons. The ability of the CB(1) receptor antagonist AM251 (1 microm) to modulate the effects of URB597 and UCM707 was also determined. Suprafusion of NADA and capsaicin evoked robust increases in [Ca(2+)](i) in DRG neurons (89 +/- 4% and 132 +/- 6% of the depolarizing KCl response, respectively). Co-incubation with URB597 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (39 +/- 3% and 79 +/- 4% of KCl response, respectively). Similarly, co-incubation with UCM707 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (59 +/- 7% and 72 +/- 4% of KCl response, respectively). The CB(1) receptor antagonist AM251 significantly attenuated the effects of URB597 on NADA-evoked increases in [Ca(2+)](i) but not the effects of URB597 on capsaicin-evoked increases in [Ca(2+)](i). By contrast, AM251 significantly attenuated the inhibitory effects of UCM707 on both NADA and capsaicin-evoked increases in [Ca(2+)](i.) These data suggest that transport of both NADA and capsaicin into DRG neurons and the subsequent activation of TRPV1 is partly governed by FAAH-dependent mechanisms as well as via the putative AEA membrane transporter.     

Effects of CB1 cannabinoid receptor modulating compounds on the hyperkinesia induced by high-dose levodopa in the reserpine-treated rat model of Parkinson's disease.

The present study was designed to determine the potential of CB1 cannabinoid receptor modulating compounds in the treatment of L-3,4-dihydroxyphenylalanine (L-dopa)-induced dyskinesia in Parkinson's disease. In the reserpine-treated rat model of parkinsonism, administration of a high dose of L-dopa (150 mg/kg) but not of Cl-APB (0.5 mg/kg) or quinpirole (0.5 mg/kg) produced a hyperkinetic state characterised by an increase in horizontal and vertical activity, which likely represent correlates of antiparkinsonian and dyskinetic activity, respectively. Injection of the CB1 cannabinoid receptor antagonist SR141716 (0.1-3 mg/kg) reduced the increase in vertical activity elicited by L-dopa without affecting the increase in horizontal activity. Injection of the CB1 cannabinoid receptor agonist WIN55,212-2 (0.1-3 mg/kg) reduced the L-dopa-induced increase in vertical activity and, at the highest dose only (3 mg/kg), also reduced horizontal activity elicited by L-dopa. WIN55,212-2 (1 mg/kg) reduced motor activity induced by both the D1 receptor agonist Cl-APB (0.5 mg/kg) and the D2 receptor agonist quinpirole (0.5 mg/kg) in the reserpine-treated rat. SR141716 (1 mg/kg) had no effects on motor activity induced by Cl-APB (0.5 mg/kg) nor quinpirole (0.5 mg/kg) in the reserpine-treated rat. Injection of the inhibitor of endocannabinoid transport AM404 (0.1-1 mg/kg) did not affect the increase in horizontal or vertical activity elicited by L-dopa (150 mg/kg) in the reserpine-treated rat. The data suggest that both CB1 cannabinoid receptor antagonists and agonists can modulate the behavioural effects of L-dopa and may be useful for the treatment of the dyskinesia associated with long-term L-dopa treatment of Parkinson's disease.     

Extinction of avoidance behavior by safety learning depends on endocannabinoid signaling in the hippocampus

The development of exaggerated avoidance behavior is largely responsible for the decreased quality of life in patients suffering from anxiety disorders. Studies using animal models have contributed to the understanding of the neural mechanisms underlying the acquisition of avoidance responses. However, much less is known about its extinction. Here we provide evidence in mice that learning about the safety of an environment (i.e., safety learning) rather than repeated execution of the avoided response in absence of negative consequences (i.e., response extinction) allowed the animals to overcome their avoidance behavior in a step-down avoidance task. This process was context-dependent and could be blocked by pharmacological (3 mg/kg, s.c.; SR141716) or genetic (lack of cannabinoid CB1 receptors in neurons expressing dopamine D1 receptors) inactivation of CB1 receptors. In turn, the endocannabinoid reuptake inhibitor AM404 (3 mg/kg, i.p.) facilitated safety learning in a CB1-dependent manner and attenuated the relapse of avoidance behavior 28 days after conditioning. Safety learning crucially depended on endocannabinoid signaling at level of the hippocampus, since intrahippocampal SR141716 treatment impaired, whereas AM404 facilitated safety learning. Other than AM404, treatment with diazepam (1 mg/kg, i.p.) impaired safety learning. Drug effects on behavior were directly mirrored by drug effects on evoked activity propagation through the hippocampal trisynaptic circuit in brain slices: As revealed by voltage-sensitive dye imaging, diazepam impaired whereas AM404 facilitated activity propagation to CA1 in a CB1-dependent manner. In line with this, systemic AM404 enhanced safety learning-induced expression of Egr1 at level of CA1. Together, our data render it likely that AM404 promotes safety learning by enhancing information flow through the trisynaptic circuit to CA1.     

Cannabinoids modulate hippocampal memory and plasticity

Considerable evidence demonstrates that cannabinoid agonists impair whereas cannabinoid antagonists improve memory and plasticity. However, recent studies suggest that the effects of cannabinoids on learning do not necessarily follow these simple patterns, particularly when emotional memory processes are involved. We investigated the involvement of the cannabinoid system in hippocampal learning and plasticity using the fear‐related inhibitory avoidance (IA) and the non‐fear‐related spatial learning paradigms, and cellular models of learning and memory, i.e., long‐term potentiation (LTP) and long‐term depression (LTD). We found that microinjection into the CA1 of the CB1/CB2 receptor agonist WIN55,212‐2 (5 μg/side) and an inhibitor of endocannabinoid reuptake and breakdown AM404 (200 ng/side) facilitated the extinction of IA, while the CB1 receptor antagonist AM251 (6 ng/side) impaired it. WIN55,212‐2 and AM251 did not affect IA conditioning, while AM404 enhanced it, probably due to a drug‐induced increase in pain sensitivity. However, in the water maze, systemic or local CA1 injections of AM251, WIN55,212‐2, and AM404 all impaired spatial learning. We also found that i.p. administration of WIN55,212‐2 (0.5 mg/kg), AM404 (10 mg/kg), and AM251 (2 mg/kg) impaired LTP in the Schaffer collateral‐CA1 projection, whereas AM404 facilitated LTD. Our findings suggest diverse effects of the cannabinoid system on CA1 memory and plasticity that cannot be categorized simply into an impairing or an enhancing effect of cannabinoid activation and deactivation, respectively. Moreover, they provide preclinical support for the suggestion that targeting the endocannabinoid system may aid in the treatment of disorders associated with impaired extinction‐like processes, such as post‐traumatic stress disorder. © 2009 Wiley‐Liss, Inc.     

Endocannabinoids modulate stress-induced suppression of hippocampal cell proliferation and activation of defensive behaviours

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.     

Differential effects of the sleep-inducing lipid oleamide and cannabinoids on the induction of long-term potentiation in the CA1 neurons of the rat hippocampus in vitro

Cannabinoids have been shown to impair cognition in vivo and block long-term potentiation (LTP), a candidate experimental model of learning and memory in vitro, via cannabinoid receptor (CB1) activation. cis-Oleamide (cOA) is an endogenous sleep-inducing lipid with putative cannabinomimetic properties. We hypothesise that cOA is cannabinomimetic and perform a comparative study with synthetic and endogenous cannabinoids on their effects on synaptic conditioning via two different patterns of stimulation in the hippocampal slice. CB1 agonists, R(+)-WIN55212-2 and anandamide, but not cOA blocked high frequency stimulation (HFS)-LTP. R(+)-WIN55212-2 and cOA (stereoselectively) attenuated responses to theta-burst-LTP, while anandamide did not. The anandamide transport inhibitor, AM404, attenuated HFS-LTP, an effect reversed by the CB1 receptor antagonist SR141716A but not mimicked by the vanilloid receptor agonist capsaicin. TFNO, an inhibitor of fatty acid amide hydrolase (FAAH), the enzyme responsible for degrading anandamide, failed to block HFS-LTP alone or in combination with cOA. On the contrary, this combination was as effective as cOA on its own in attenuating theta-burst-LTP. cOA effects on theta-burst-LTP were prevented in the presence of the GABA(A) receptor blocker picrotoxin, but not by pretreatment with SR141716A. These findings suggest that cOA neither directly activates CB1 receptors nor acts via the proposed "entourage" effect [Nature 389 (1997) 25] to increase titres of anandamide through FAAH inhibition. The selective effects of cOA on theta-burst-conditioning may reflect modulation of GABAergic transmission. Anandamide uptake inhibition, but not blockade of FAAH, effectively increases synaptic concentrations of endocannabinoids.     

Cannabinoids alter spontaneous firing,bursting, and cell synchrony of hippocampal principal cells

Both natural and synthetic cannabinoid receptor (e.g., CB1) agonists such as Δ⁹‐THC, WIN 55,212‐2 (WIN‐2), and HU‐210 disrupt spatial cognition presumably through the inhibition of synchrony of hippocampal ensemble firing to task‐related events. Although the CB1 receptor agonist CP 55,940 also disrupts the synchronous firing of hippocampal neurons, it does not seem to affect the average firing rate. This difference is not readily explained by the chemical structure and pharmacology of the different compounds thus warranting a more detailed examination into (i) how other cannabinoids affect the spontaneous firing, bursting, and cell synchrony of hippocampal principal cells located in CA3 and CA1 subfields, and (ii) whether these effects are indeed mediated through CB1 receptors, which will be explored by the selective antagonist AM‐251. Male Long‐Evans rats surgically implanted with multielectrode arrays to hippocampal CA3 and CA1 were anesthetized and principal cells discharging at 0.25–6.0 Hz were isolated and “tracked” following the systemic administration of Tween‐80, Δ⁹‐THC (1 or 3 mg/kg) or WIN‐2 (1 mg/kg) or HU‐210 (100 μg/kg), and 1.5 mg/kg AM‐281. All cannabinoids except for 1 mg/kg Δ⁹‐THC reliably reduced average firing rates and altered “burst” characteristics, which were reversible with AM‐281 for Δ⁹‐THC and WIN‐2 but not for HU‐210. In addition, all cannabinoids disrupted intrasubfield and intersubfield ensemble synchrony of pyramidal cells, which is an effect insensitive to AM‐281 and thus unlikely to be CB1 mediated. We consider these cannabinoid effects on spike timing and firing/bursting of principal hippocampal neurons carried by CB1 and non‐CB1 receptors to be physiological underpinnings of the cognitive impairments inherent to cannabinoid exposure. © 2010 Wiley‐Liss, Inc.     

Endocannabinoid modulation of jejunal afferent responses to LPS

Background Endocannabinoids influence immune function and nociceptive signaling. This study examines cannabinoid modulation of sensory signaling from the GI tract following an acute inflammatory response triggered by systemic administration of bacterial lipopolysaccharide (LPS). Methods A segment of proximal jejunum was intubated, to measure intraluminal pressure, in anesthetized rats. Afferent impulse traffic was recorded from a single isolated paravascular nerve bundle supplying the jejunal loop. Drugs and LPS were administered intravenously and changes in afferent firing were determined. Key Results The non‐selective cannabinoid agonist, WIN55,212‐2 (1 mg kg^?1 i.v.) and the anandamide transport inhibitor, VDM11 (1 mg kg^?1 i.v.) but not the fatty acid amide hydrolase (FAAH) inhibitor, URB597 (0.3 mg kg^?1) caused a significant increase in afferent activity. The WIN55,212‐2‐induced afferent response was mediated by activation of CB₁ receptors whereas the VDM11 response was mediated by both CB₁ and CB₂ receptor mechanisms. LPS (10 mg kg^?1) evoked an increase in afferent activity which was significantly reduced in the presence of WIN55,212‐2 and VDM11 but not URB597. The inhibitory effect of WIN55,212‐2 was prevented by CB₁ but not CB₂ receptor antagonism. In contrast, the inhibitory effect of VDM11 remained unaltered after CB₁ or CB₂ receptor blockade. Conclusions & Inferences Endocannabinoids play a role in modulating afferent signaling and may represent a target for the treatment of visceral hypersensitivity. In contrast to the effects of blocking endocannabinoid uptake (VDM11), inhibiting breakdown of endocannabinoids (URB597) had no effect on baseline or LPS induced afferent firing. Therefore, uptake of cannabinoids rather than breakdown via FAAH terminates their action in the GI tract.     

CB1 cannabinoid receptor agonist WIN 55,212-2 decreases intravenous cocaine self-administration in rats

The effect of the CB1 cannabinoid receptor agonist WIN 55,212-2 on intravenous cocaine self-administration (IVSA) in rats was evaluated. Male Long Evans rats were implanted with silastic catheters through the external jugular vein. The IVSA was conducted in 3-h daily sessions with a fixed ratio (FR1) schedule: the experimental apparatus had a nose-poking response-like operandum. Intravenous pre-treatment with WIN 55,212-2 (0.25, 0.5 and 1 mg/kg) to rats self-administering cocaine (0.25 or 0.5 mg/kg/inj) at stable baseline, reduces cocaine intake in a dose-dependent manner. The CB1 receptor antagonist SR 141716A (3 mg/kg i.p.) completely reversed the WIN 55,212-2-induced decrease of cocaine intake. However, pre-treatment of SR 141716A alone (up to dose of 9 mg/kg i.p.) was unable to modify cocaine IVSA. These results indicate that stimulation of CB1 cannabinoid receptors activates rewarding mechanisms which produce reinforcing effects additional to those induced by cocaine.     

Endocannabinoid modulation of amphetamine sensitization is disrupted in a rodent model of lesion‐induced dopamine dysregulation

We tested the hypothesis that increased dopaminergic sensitivity induced by olfactory bulbectomy is mediated by dysregulation of endocannabinoid signaling. Bilateral olfactory bulbectomy induces behavioral and neurobiological symptomatology related to increased dopaminergic sensitivity. Rats underwent olfactory bulbectomy or sham operations and were assessed 2 weeks later in two tests of hyperdopaminergic responsivity: locomotor response to novelty and locomotor sensitization to amphetamine. Amphetamine (1 mg/kg i.p.) was administered to rats once daily for 8 consecutive days to induce locomotor sensitization. URB597, an inhibitor of the anandamide hydrolyzing enzyme fatty‐acid amide hydrolase (FAAH), was administered daily (0.3 mg/kg i.p.) to sham and olfactory bulbectomized (OBX) rats to investigate the impact of FAAH inhibition on locomotor sensitization to amphetamine. Pharmacological specificity was evaluated with the CB₁ antagonist/inverse agonist rimonabant (1 mg/kg i.p). OBX rats exhibited heightened locomotor activity in response to exposure either to a novel open field or to amphetamine administration relative to sham‐operated rats. URB597 produced a CB₁‐mediated attenuation of amphetamine‐induced locomotor sensitization in sham‐operated rats. By contrast, URB597 failed to inhibit amphetamine sensitization in OBX rats. The present results demonstrate that enhanced endocannabinoid transmission attenuates development of amphetamine sensitization in intact animals but not in animals with OBX‐induced dopaminergic dysfunction. Our data collectively suggest that the endocannabinoid system is compromised in olfactory bulbectomized rats. Synapse 63:941–950, 2009. © 2009 Wiley‐Liss, Inc.