NMDA and DHPG preconditioning induce ischemic tolerance with differential mechanisms in rat organotypic hippocampal slices

Ischemic tolerance is a neuroprotective mechanism by which neurons exposed to a subtoxic insult become resistant to a subsequent lethal stressor. However, the molecular mechanisms underlying the preconditioning response are not yet understood. Because cerebral ischemia and excitotoxicity involve the activation of glutamate receptors, we investigated whether AMPA toxicity was modified following pharmacological preconditioning. For our study, we used organotypic hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later or to 10 µM AMPA for 24 h, which leads to nonselective degeneration of all neuronal populations. We used two models of preconditioning by exposing the slices to subtoxic bouts of either NMDA (3 mM for 60 min) or DHPG (10 mM for 30 min) and 24 h later, to 30 min OGD or AMPA excitotoxicity. After preconditioning, we observed a significant reduction in CA1 damage following our toxic insults. We first performed whole-cell voltage-clamp recordings on control or preconditioned CA1 pyramidal neurons of organotypic slices. 24 h after exposure to NMDA or DHPG preconditioning, AMPA induced-currents were significantly reduced. We then evaluated whether the expression of the GluR1, NR2A and NR2B subunits and of PSD-95 were modified after our preconditioning stimuli, by using western blot analysis performed in postsynaptic densities. Our results show that following NMDA, but not DHPG preconditioning, the expression of GluR1 was significantly reduced. To assess whether the GluR1 reduction induced by NMDA preconditioning was associated with internalization of AMPA receptors, we treated preconditioned slices with the cross-linker bis(sulfosuccinimidyl) suberate and then evaluated the expression levels of GluR1. Western blotting analysis revealed that NMDA, but not DHPG preconditioning, increased the internalization of GluR1. In conclusion, our results show that NMDA and DHPG preconditioning stimuli result in neuroprotection against toxic insults by means of different mechanisms