The dual blocker of FAAH/TRPV1 N-arachidonoylserotonin reverses the behavioral despair induced by stress in rats and modulates the HPA-axis.

In recent years, several studies have explored the involvement of the deregulation of the hypothalamus-pituitary-adrenal (HPA) axis in the pathophysiology of stress-related disorders. HPA hyper-activation as a consequence of acute/chronic stress has been found to play a major role in the neurobiological changes that are responsible for the onset of such states. Currently available medications for depression, one of the most relevant stress-related disorders, present several limitations, including a time lag for treatment response and low rates of efficacy. N-Arachidonoylserotonin (AA-5-HT), a dual blocker at fatty acid amide hydrolase (FAAH, the enzyme responsible for the inactivation of the endocannabinoid anandamide) and transient receptor potential vanilloid type-1 channel (TRPV1), produces anxiolytic-like effects in mice. The present study was designed to assess the capability of AA-5-HT to reverse the behavioral despair following exposure to stress in rats and the role of the HPA-axis. Behavioral tasks were performed, and corticosterone and endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were measured in selected brain areas critically involved in the pathophysiology of stress-related disorders (medial PFC and hippocampus) under basal and stress conditions, and in response to treatment with AA-5-HT. Our data show that AA-5-HT reverses the rat behavioral despair in the forced swim test under stress conditions, and this effect is associated with the normalization of the HPA-axis deregulation that follows stress application and only in part with elevation of anandamide levels. Blockade of FAAH and TRPV1 may thus represent a novel target to design novel therapeutic strategies for the treatment of stress-related disorders.

Behavioural and neurochemical changes induced by stress-related conditions are counteracted by the neurokinin-2 receptor antagonist saredutant.

These experiments were undertaken to assess the mechanisms underlying the antidepressant-like effects of the neurokinin-2 (NK(2)) receptor antagonist saredutant (SR48968) in rats tested in the forced swim test (FST), by analysing hippocampal brain-derived neurotrophic factor (BDNF) and plasma corticosterone [as index of hypothalamic-pituitary-adrenal (HPA) axis activity]. Male Wistar rats received three intraperitoneal injections over 24 h of vehicle, saredutant (5 mg/kg), citalopram (15 mg/kg), clomipramine (50 mg/kg). Rats were subjected to restraint stress (4 h) 24 h prior to the FST procedure. This stress procedure increased immobility and decreased swimming behaviour in the FST; furthermore, it lowered hippocampal BDNF protein expression and increased plasma corticosterone levels. Saredutant and clomipramine or citalopram, used here as positive controls, reduced the immobility time in the FST both under basal conditions and after stress exposure. This effect was not attributable to changes in locomotion, because locomotor activity was unchanged when assessed in the open field test. Pretreatment with para-cholorophenylalanine (150 mg/kg, 72 h and 48 h prior to FST) abolished the effect of citalopram and saredutant on immobility time. At neurochemical level, saredutant attenuated activation of HPA axis in stressed animals more than clomipramine or citalopram. The behavioural effects of saredutant support the hypothesis that NK(2) receptor activity is involved in stress-related disorders. These effects of saredutant may be related to normalization of the HPA axis. Moreover, saredutant increases BDNF expression in the hippocampus, confirming the role of NK(2) receptor blockade in BDNF activation following stressor application.

Enhanced cognitive performance of dopamine D3 receptor "knock-out" mice in the step-through passive-avoidance test: assessing the role of the endocannabinoid/endovanilloid systems.

Increasing evidence suggests a pivotal role of the D3 receptor (D3R) in cognitive processes and the involvement of endocannabinoid/endovanilloid signaling in the pathophysiology of neurodegenerative disorders such as Alzheimer's disease. This study was undertaken to investigate both the basal and beta-amyloid peptide 1-42 (BAP 1-42)-impaired cognitive performance of D3R((-/-)) mice, and the role therein of endocannabinoids/endovanilloids. D3R((-/-)) mice were either untreated or injected i.c.v. with 400 pMol BAP 1-42 or vehicle to be tested 14 days later in a step-through passive-avoidance paradigm. The CB(1) receptor antagonist, rimonabant (1mg/kg), or the transient receptor potential vanilloid-type 1 channel (TRPV1) antagonist SB366791, were injected intraperitoneally for 11 or 7 days. The retention test was performed 1, 7 and 14 days after the learning trial. Wild-type (WT) mice were subjected to the same procedures. D3R((-/-)) mice exhibited a better basal cognitive performance as compared to WT mice (p<0.001), which was reversed by TRPV1 antagonism. BAP 1-42 induced a pronounced worsening of the passive-avoidance response in all tests and in both genotypes (p<0.001). Rimonabant treatment never affected the cognitive performance of healthy mice, but fully counteracted BAP 1-42-induced amnesic effects in both D3R((-/-)) and WT mice only when administered for 11 days, whereas, when administered for 7 days, only transiently affected WT mice and caused more prolonged cognitive ameliorations in D3R((-/-)) mice. These results support the involvement of D3R and TRPV1 in cognitive processes and the concept that A beta peptides inhibit memory retention in mice through the involvement of endocannabinoids.

Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: Possible involvement of the endocannabinoid and endovanilloid systems.

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB(1) receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D(3) receptor null (D3R((-/-))) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R((-/-)) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R((-/-)) mice also exhibited different endocannabinoid and TRPV1, but not CB(1), levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB(1) and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R((-/-)) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.

Behavioral effects of saredutant, a tachykinin NK2 receptor antagonist, in experimental models of mood disorders under basal and stress-related conditions.

The present study was made to investigate the role of tachykinin NK2 receptors in the expression of stress-related behaviors in animals. Under basal conditions, intraperitoneal (i.p.) administration of the selective tachykinin NK2 receptor antagonist, saredutant (1 and 3 mg/kg) or diazepam (1 mg/kg) exerted anxiolytic-like effects in rodents, as they reduced grooming score of Wistar male rats tested in the novelty-induced grooming sampling test (NGT) and increased percentage of time and entries in open arms of Swiss male mice tested in the elevated plus maze (EPM) test. After previous exposure to stress-related conditions, as induced by a 2-min forced swim made 5 min prior to the EPM test, saredutant but not diazepam, exhibited anxiolytic-like effects in mice. To study the antidepressant-like activity of tachykinin NK2 receptor antagonist under basal conditions, different groups of rats were injected i.p. with saredutant (2.5, 5 and 10 mg/kg) or the tricyclic antidepressant, clomipramine (50 mg/kg) and tested in the forced swim test (FST), a widely used antidepressant-responsive test. The influence of stress-related conditions was studied in rats subjected to electric foot-shocks (1 mA, 1 s) 24, 5 and 1 h prior to FST, after drugs injection. In the FST, clomipramine decreased the immobility time only under basal conditions, but not after application of acute foot-shocks. To the contrary, saredutant-treated rats also exhibited more active behavior in FST after previous exposure to stressors. These results give further support to the hypothesis that tachykinin NK2 receptors may be a therapeutic target for pharmacological treatment of stress-related diseases, such as anxiety and depression.

Reversible inhibition of vasoconstriction by thiazolidinediones related to PI3K/Akt inhibition in vascular smooth muscle cells.

Thiazolidinediones (also referred to as glitazones), agonists for Peroxisome Proliferator-Activated Receptor gamma (PPARγ), are used for treating type 2 diabetes mellitus, where they decrease insulin resistance and cardiovascular risk. Compounds bearing the thiazolidinedione structure have also been shown to inhibit phosphoinositide 3-kinase (PI3K). Here we tried to elucidate the poorly defined role of PI3K/Akt in the physiology of vascular smooth muscle cell contraction and tested the hypothesis that thiazolidinediones, by affecting the PI3K/Akt pathway, may influence vascular physiology. Isolated rat femoral arteries segments were mounted in a wire myograph and challenged with 100mM KCl or phenylephrine (PE), in the absence or presence of troglitazone, rosiglitazone, pioglitazone, LY294002 (PI3K inhibitor) and 10-DEBC (Akt inhibitor). All these compounds dose-dependently inhibited vasoconstriction to KCl or PE; their effect was reversible (after 60-120 min washout) and not affected by GW9662 (a PPARγ antagonist) or by N(G)-nitro-L-arginine (LNNA, an inhibitor of NO biosynthesis). Analysis of phospho-Akt (ser 473) in lysates from rat arteries (by immunoblot) revealed that thiazolidinediones, LY294002 and 10-DEBC, at the same concentration and kinetics inhibiting vasoconstriction, produced a similar decrease of Akt phosphorylation. PI3K/Akt pathway therefore appears to be an important, fast acting, modulator of contraction of vascular smooth muscle. Thiazolidinediones decrease vasoconstriction of isolated vessels possibly by inhibiting PI3K/Akt pathway. Such an effect of glitazones, if occurring in vivo, may impact cardiovascular syndromes related to vasospasm in diabetic patients.

The selective norepinephrine reuptake inhibitor atomoxetine counteracts behavioral impairments in trimethyltin-intoxicated rats.

This study was carried out to assess the behavioral effects of the non-psychostimulant drug atomoxetine, in rats prenatally-exposed to the organic compound trimethyltin chloride (TMT) and in spontaneously hypertensive rat (SHR), two rodent models of Attention Deficit/Hyperactivity Disorder (ADHD). At birth, neonatal reflexes (righting, cliff aversion, forelimb placing, forelimb grasping, bar holding and startle) had an earlier onset (i.e. percent of appearance) and completion (maximum appearance, i.e. 100% of the brood exhibiting each reflex) in prenatally TMT-exposed and SHR pups as compared to control groups. Two months after birth, TMT-exposed and SHR rats showed impaired cognitive performances in both the step-through passive avoidance test and the shuttle box active avoidance test. Atomoxetine (1, 3 and 6 mg/kg, i.p.), already at the lowest dose tested, improved learning and memory capacity of prenatally TMT-exposed rats and SHR; while methylphenidate (1, 3 and 6 mg/kg, i.p.), used here as positive control, elicited a significant cognitive enhancing effect only at the higher doses. In the open field test, both TMT-exposed rats and SHR displayed enhanced locomotor activity. Methylphenidate further increased locomotor activity in all groups, whereas atomoxetine reduced the enhanced locomotor activity of TMT-exposed rats and SHR down to the level of controls. These results suggest that prenatal TMT-exposure could be considered as a putative experimental model of ADHD and further support the effectiveness of atomoxetine in the ADHD pharmacotherapy. Furthermore, despite the similar effect of the two drugs on cognitive tasks, they exhibit distinct profiles of activity on locomotion, in ADHD models.

The beta3 adrenoceptor agonist, amibegron (SR58611A) counteracts stress-induced behavioral and neurochemical changes.

These experiments were made to study the mechanisms underlying the antidepressant-like effects of the beta(3) adrenoceptor agonist amibegron (SR58611A). To this purpose, the expression levels of the hippocampal cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB), brain-derived neurotrophic factor (BDNF), B-cell lymphoma-2 (Bcl-2) and Bax proteins were assessed, by using western blot analysis, in rats tested in the forced swim test (FST). Under basal conditions (no previous exposure to stressors), different groups of male Wistar rats received acutely or repeatedly (once/day for 7days) intraperitoneal (i.p.) injections of amibegron (1, 5 and 10mg/kg), the tricyclic antidepressant (TCA) clomipramine (50mg/kg), the selective serotonin reuptake inhibitor (SSRI) citalopram (15mg/kg) or their vehicles. The influence of stress-related conditions was studied in rats subjected to acute (4h) or repeated (4h/day for 7days) restraint stress, applied prior to the FST procedure. Compared to the control groups, both stressor procedures increased the immobility time in the FST and reduced hippocampal BDNF and Bcl-2/Bax ratio proteins expression, which were counteracted by amibegron (5 and 10mg/kg) treatment. Opposite effects were found in the CREB expression, since it was lower after acute and higher after repeated stress procedure, respectively. Again, these effects were reversed by amibegron treatment. Different results were obtained in animals treated with clomipramine or citalopram. Hence, it is likely that the observed behavioral effects of amibegron could be due, at least in part, to its action on hippocampal expression of neurotrophic and/or anti-apoptotic factors, supporting the hypothesis that beta(3) adrenoceptors may be a therapeutic target for the treatment of stress-related disorders.

Antidepressant properties of the 5-HT4 receptor partial agonist, SL65.0155: behavioral and neurochemical studies in rats.

This study was undertaken to investigate the potential antidepressant-like properties of SL65.0155, a serotonin 5-HT(4) receptor partial agonist, in male rats of the Wistar strain tested in the forced swim test (FST), an experimental model widely used to assess antidepressant-like activity. The expression of hippocampal neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), the phosphorilated cAMP response element-binding protein (p-CREB), the B cell lymphoma-2 (Bcl-2), the Bax and the vascular endothelium growth factor (VEGF) were also evaluated by Western Blot analysis. Different groups of rats received intraperitoneally (i.p.) injections of SL65.0155 (0.1, 0.5 and 1 mg/kg), clomipramine (50 mg/kg), citalopram (15 mg/kg) or vehicle, respectively, 24, 5 and 1 h prior to the FST. Compared to the control group, SL65.0155 (0.5 and 1 mg/kg), clomipramine or citalopram injected animals showed an increased swimming and climbing behavior and reduced immobility time in the FST. Interestingly, this effect was not due to changes in the locomotor activity since all treated groups failed to show any change in motor ability as assessed in the open field test. Western blot analysis of hippocampal homogenates showed an enhancement of p-CREB, BDNF Bcl-2 and VEGF protein levels in SL65.0155 treated groups, but not in citalopram or clomipramine treated groups, used here as positive control. No change was found in Bax expression in any treated group. These findings give further support to the hypothesis that the stimulation of serotonin 5-HT(4) receptors may be a therapeutic target for depression.

Anxiolytic effects in mice of a dual blocker of fatty acid amide hydrolase and transient receptor potential vanilloid type-1 channels.

The endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH), and the transient receptor potential vanilloid type-1 (TRPV1) channel are new targets for the development of anxiolytic drugs. We studied the effect on anxiety-like behavior in the elevated plus maze of a dual FAAH/TRPV1 blocker, N-arachidonoyl-serotonin (AA-5-HT). In male C57BL/6J mice, acute intraperitoneal administration of AA-5-HT (0.1-2.5 mg/kg) increased both the time spent and the number of entries in the open arm, while being inactive at the highest dose tested (5 mg/kg). AA-5-HT was more potent than selective blockers of FAAH or TRPV1 (URB597 and SB366791, respectively). In male Swiss mice, AA-5-HT had to be administered chronically to observe an anxiolytic effect at an intermediate dose (2.5 mg/kg), the highest dose (5 mg/kg) being anxiogenic, and 1 mg/kg being ineffective. In both strains, the anxiolytic effects of AA-5-HT were paralleled by elevation of brain endocannabinoid levels and were reversed by per se inactive doses of the cannabinoid receptors of type-1 (CB(1)) receptor antagonist AM251, or the TRPV1 agonist, olvanil. Immunohistochemical localization of CB(1) and TRPV1 receptors was observed in mouse prefrontal cortex, nucleus accumbens, amygdala, and hippocampus. Simultaneous 'indirect' activation of CB(1) receptors following FAAH inhibition, and antagonism at TRPV1 receptors might represent a new therapeutic strategy against anxiety.