Anxiogenic doses of rapamycin prevent URB597-induced anti-stress effects in socially defeated mice.

Repeated exposure to psychosocial stress modulates the endocannabinoid system, particularly anandamide (AEA) signaling in brain regions associated with emotional distress. The mTOR protein regulates various neuroplastic processes in the brain disrupted by stress, including adult hippocampal neurogenesis. This kinase has been implicated in multiple effects of cannabinoid drugs and the anti-stress behavioral effects of psychoactive drugs. Therefore, our hypothesis is that enhancing AEA signaling via pharmacological inhibition of the fatty acid amide hydrolase (FAAH) enzyme induces an anti-stress behavioral effect through an mTOR-dependent mechanism. To test this hypothesis, male C57Bl6 mice were exposed to social defeat stress (SDS) for 7 days and received daily treatment with either vehicle or different doses of the FAAH inhibitor, URB597 (0.1; 0.3; 1 mg/Kg), alone or combined with rapamycin. The results suggested that URB597 induced an inverted U-shaped dose-response curve in mice subjected to SDS (with the intermediate dose of 0.3 mg/kg being anxiolytic, and the higher tested dose of 1 mg/Kg being anxiogenic). In a second independent experiment, rapamycin treatment induced an anxiogenic-like response in control mice. However, in the presence of rapamycin, the anxiolytic dose of URB597 treatment failed to reduce stress-induced anxiety behaviors in mice. SDS exposure altered the hippocampal expression of the mTOR scaffold protein Raptor. Furthermore, the anxiogenic dose of URB597 decreased the absolute number of migrating doublecortin (DCX)-positive cells in the dentate gyrus, suggesting an anti-anxiety effect independent of newly generated/immature neurons. Therefore, our results indicate that in mice exposed to repeated psychosocial stress, URB597 fails to counteract the anxiogenic-like response induced by the pharmacological dampening of mTOR signaling.

The chronic pharmacological antagonism of the CB1 receptor is not involved in the behavioral effects of antidepressants administered in mice submitted to chronic unpredictable stress.

Several pieces of evidence suggest that the monoaminergic theory of depression cannot fully explain all behavioral and neuroplastic changes observed after antidepressant chronic treatment. Other molecular targets, such as the endocannabinoid system, have been associated with the chronic effects of these drugs. In the present study, we hypothesized that the behavioral and neuroplastic effects observed after repeated treatment with the antidepressants (AD) Escitalopram (ESC) or venlafaxine (VFX) in chronically stressed mice depend on CB1 receptor activation. Male mice submitted to the chronic unpredictable stress (CUS) paradigm for 21 days were treated with Esc (10 mg/kg) or VFX (20 mg/kg) once a day in the presence or not of AM251 (0.3 mg/kg), a CB1 receptor antagonist/inverse agonist. At the end of the CUS paradigm, we conducted behavior tests to evaluate depressive- and anxiety-like behaviors. Our results demonstrated that chronic blockade of the CB1 receptor does not attenuate the antidepressant- or the anxiolytic-like effects of ESC nor VFX. ESC increased the expression of CB1 in the hippocampus, but AM251 did not change the pro-proliferative effects of ESC in the dentate gyrus or the increased expression of synaptophysin induced by this AD in the hippocampus. Our results suggest that CB1 receptors are not involved in behavioral and hippocampal neuroplastic effects observed after repeated antidepressant treatment in mice submitted to CUS.

Spontaneous Activity of CB2 Receptors Attenuates Stress-Induced Behavioral and Neuroplastic Deficits in Male Mice.

The monoaminergic theory of depression/anxiety disorders cannot fully explain the behavioral and neuroplastic changes observed after ADs chronic treatment. Endocannabinoid system, which comprises CB2 receptors, has been associated with the chronic effects of these drugs, especially in stressed mice. CB2-KO mice display more vulnerability to stressful stimuli. In the present study, we hypothesized that the behavioral and neuroplastic effects observed after repeated treatment with the AD escitalopram (Esc) in chronically stressed mice depend on CB2 receptor signaling. Male mice submitted to chronic unpredictable stress (CUS) paradigm (21 days) were treated daily with AM630 (0.01; 0.03 or 0.3 mg/kg, i.p) a CB2 receptor antagonist/inverse agonist. At e 19th day of the CUS protocol, mice were submitted to Open field test and Tail-suspension test to evaluate antidepressant-like behavior. At the end of the stress protocol, mice were submitted to Novel Suppressed Feeding test (day 22nd) to evaluate anxiety-like behavior. In a second series of experiments, male mice treated with Esc (10 mg/kg, daily, 21 days) in the presence or not of AM630 (0.30 mg/kg) were submitted to the same round of behavioral tests in the same conditions as performed in the dose-response curve protocol. Animals were then euthanized under deep anesthesia, and their brains/hippocampi removed for immunohistochemistry (Doublecortin-DCX) or Western Blot assay. Our results demonstrated that chronic treatment with AM630, a CB2 antagonist/inverse agonist, induces anxiolytic-like effects in stressed mice. Moreover, chronic reduction of CB2 receptor endogenous activity by AM630 attenuated the neuroplastic (potentiating stress-induced decreased expression of pro-BDNF, but enhanced pmTOR and DAGL expression in the hippocampus reduced in stressed mice), the antidepressant- but not the anxiolytic-like effects of Esc. AM630 alone or in combination with Esc decreased the expression of DCX + cell in both the subgranular and granular layers of the dentate gyrus (DG), indicating a general reduction of DCX + neuroblasts and a decrease in their migration through the DG layers. We suggest that the antidepressant-like behavior and the pro-neurogenic effect, but not the anxiolytic like behavior, promoted by Esc in stressed mice are, at least in part, mediated by CB2 receptors.