Background and purpose:  The present study evaluated the role of CB ₂ receptors in the regulation of depressive‐like behaviours. Transgenic mice overexpressing the CB ₂ receptor (CB2xP) were challenged with different types of acute and chronic experimental paradigms to evaluate their response in terms of depressive‐like behaviours. Experimental approach:  Tail suspension test (TST), novelty‐suppressed feeding test (NSFT) and unpredictable chronic mild stress tests (CMS) were carried out in CB2xP mice. Furthermore, acute and chronic antidepressant‐like effects of the CB ₂ receptor‐antagonist AM630 were evaluated by means of the forced swimming test (FST) and CMS, respectively, in wild‐type (WT) and CB2xP mice. CB ₂ gene expression, brain‐derived neurotrophic factor (BDNF) gene and protein expressions were studied in mice exposed to CMS by real‐time PCR and immunohistochemistry, respectively. Key results:  Overexpression of CB ₂ receptors resulted in decreased depressive‐like behaviours in the TST and NSFT. CMS failed to alter the TST and sucrose consumption in CB2xP mice. In addition, no changes in BDNF gene and protein expression were observed in stressed CB2xP mice. Interestingly, acute administration of AM630 (1 and 3 mg·kg ⁻¹ , i.p.) exerted antidepressant‐like effects on the FST in WT, but not in CB2xP mice. Chronic administration of AM630 for 4 weeks (1 mg·kg ⁻¹ ; twice daily, i.p.) blocked the effects of CMS on TST, sucrose intake, CB ₂ receptor gene, BDNF gene and protein expression in WT mice. Conclusion and implications:  Taken together, these results suggest that increased CB ₂ receptor expression significantly reduced depressive‐related behaviours and that the CB ₂ receptor could be a new potential therapeutic target for depressive‐related disorders.

Background and Purpose: Heteromerization ofGPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer‐selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositolGPCR55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB₂receptor) in human neutrophils, we investigated the possible heteromerization ofCB₂receptorswith GPR55.Experimental Approach: The direct interaction of humanGPR55 andCB₂receptors heterologously expressed inHEK293 cells was assessed by co‐immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross‐talk on signalling was investigated at downstream levels by label‐free real‐time methods (Epic dynamic mass redistribution andCellKey impedance assays),ERK1/2‐MAPKactivation and gene reporter assays.Key Results: GPR55 andCB₂receptors co‐localized on the surface ofHEK293 cells, co‐precipitated in membrane extracts and formed heteromers in livingHEK293 cells. Whereas heteromerization led to a reduction inGPR55‐mediated activation of transcription factors (nuclear factor of activatedT‐cells,NF‐κBandcAMPresponse element),ERK1/2‐MAPKactivation was potentiated in the presence ofCB₂receptors.CB₂receptor‐mediated signalling was also affected by co‐expression withGPR55. Label‐free assays confirmed cross‐talk between the two receptors.Conclusions and Implications: Heteromers, unique signalling units, form inHEK293 cells expressingGPR55 andCB₂receptors. The signalling by agonists of either receptor was governed (i) by the presence or absence of the partner receptors (with the consequent formation of heteromers) and (ii) by the activation state of the partner receptor.

The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5-20 microM) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 microM was toxic for both cell types. Ciglitazone at 10 microM was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARgamma and production of reactive oxygen species (ROS). PPARgamma does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARgamma. In addition, GW9662, an inhibitor of PPARgamma, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.

Neurons are highly dependent on astrocyte survival during brain damage. To identify genes involved in astrocyte function during ischemia, we performed mRNA differential display in astrocytes after oxygen and glucose deprivation (OGD). We detected a robust down-regulation of S6 kinase 1 (S6K1) mRNA that was accompanied by a sharp decrease in protein levels and activity. OGD-induced apoptosis was increased by the combined deletion of S6K1 and S6K2 genes, as well as by treatment with rapamycin that inhibits S6K1 activity by acting on the upstream regulator mTOR (mammalian target of rapamycin). Astrocytes lacking S6K1 and S6K2 (S6K1;S6K2-/-) displayed a defect in BAD phosphorylation and in the expression of the anti-apoptotic factors Bcl-2 and Bcl-xL. Furthermore reactive oxygen species were increased while translation recovery was impaired in S6K-deficient astrocytes following OGD. Rescue of either S6K1 or S6K2 expression by adenoviral infection revealed that protective functions were specifically mediated by S6K1, because this isoform selectively promoted resistance to OGD and reduction of ROS levels. Finally, "in vivo" effects of S6K suppression were analyzed in the permanent middle cerebral artery occlusion model of ischemia, in which absence of S6K expression increased mortality and infarct volume. In summary, this article uncovers a protective role for astrocyte S6K1 against brain ischemia, indicating a functional pathway that senses nutrient and oxygen levels and may be beneficial for neuronal survival.

BACKGROUND AND PURPOSE This study evaluated gene expression differences between two mouse strains, characterized by opposite impulsivity‐like traits and the involvement of the cannabinoid CB₂ receptor in the modulation of impulsivity.EXPERIMENTAL APPROACH Behavioural tests were conducted to compare motor activity, exploration and novelty seeking, attention and cognitive and motor impulsivity (delayed reinforcement task: session duration 30 min; timeout 30 s) between A/J and DBA/2 mice. Expression of genes for dopamine D₂ receptors, CB₁ and CB₂ receptors were measured in the cingulate cortex (CgCtx), caudate‐putamen (CPu), accumbens (Acc), amygdala (Amy) and hippocampus (Hipp). Involvement of CB₂ receptors in impulsivity was evaluated in DBA/2 mice with a CB₂ receptor agonist (JWH133) and an antagonist (AM630).KEY RESULTS DBA/2 mice presented higher motor and exploratory activity, pre‐pulse inhibition impairment and higher cognitive and motor impulsivity level than A/J mice. In addition, DBA/2 mice showed lower (CgCtx, Acc, CPu) D₂ receptor, lower (Amy) and higher (CgCtx, Acc, CPu, Hipp) CB₁ receptor and higher (CgCtx, Acc, Amy) and similar (CPu, Hipp) CB₂ receptor gene expressions. Treatment with JWH133 (0.5, 1, 3 mg·kg⁻¹, i.p.) reduced cognitive and motor impulsivity level, accompanied by CB₂ receptor down‐regulation (CgCtx, Acc, Amy) but did not modify other behaviours. In contrast, AM630 (1, 2, 3 mg·kg⁻¹, i.p.) improved pre‐pulse inhibition and reduced novelty seeking behaviour in DBA/2 mice.CONCLUSIONS AND IMPLICATIONS CB₂ receptors might play an important role in regulating impulsive behaviours and should be considered a promising therapeutic target in the treatment of impulsivity‐related disorders.