Interplay between group I mGlu and the endocannabinoid system in the hippocampus: studies in models of cerebral ischemia

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. We then investigated the cross-talk between mGlu1 and CB1 receptors in modulating the hippocampal output of GABA in a gerbil model of global ischemia. Using transverse microdialysis, we observed that the CB1 receptor agonist WIN 55212-2 reduced the output of GABA evoked by the mGlu1 receptor antagonist LY367385 in control and ischemic gerbils. When we examined the effects of CB1 agents on mGlu1-mediated CA1 damage in organotypic hippocampal slices exposed to OGD, we observed that when present in the incubation medium, WIN 55212-2 and CP-55940 (1-100 µM) dose-dependently exacerbated CA1 injury induced by a sublethal period of OGD (20 min). Conversely, incubation with the CB1 receptor antagonist AM251 (0.1-1 µM) significantly attenuated CA1 damage induced by 30 min OGD. WIN 55212-2, but not AM251, significantly reverted the neuroprotective effects of LY367385 and 3-MATIDA in the more severe model (30 min OGD). The mGlu1/5 agonist DHPG (100 µM) exacerbated CA1 injury induced by 20 min OGD, but this effect was not synergic with that of WIN 55212-2. AM251, but not WIN 55212-2, was able to revert the neurotoxic effects of the mGlu1/5 agonist DHPG. Immunocytochemistry experiments with specific anti-mGlu1α and anti-CB1 antibodies revealed that OGD did not alter the distribution of mGlu1α receptors in hippocampal slices, but downregulated the expression of CB1 receptors in most interneurons, damaging the network of CB1-positive processes; this effect was prevented by the addition of neuroprotective concentration of 3-MATIDA. We then determined by LC-MS/MS the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in control and OGD-treated hippocampal slices. AEA, and to a lesser extent 2-AG, increased significantly immediately after OGD and returned to basal levels 3 h later; LY367385 was able to prevent the increase induced by OGD. 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. Moreover, the release of GABA appears to contribute to the attenuation of OGD injury induced by mGlu1 receptor antagonists and endocannabinoid receptors appear to be involved in mediating the GABAergic effects of mGlu1 receptors.