The effects of the hypoxia mimetic protocatechuic acid ethyl ester on cell viability and synaptic plasticity in rat hippocampal slices

S. Lanigan1, M. Pickering2, J. J. O'Connor1. 1School of Biomolecular & Biomedical Science, University College Dublin, Dublin, IRELAND, 2School of Medicine, University College Dublin, Dublin, IRELAND.

Introduction: In order to maintain a healthy brain a constant supply of oxygenated blood is required. During hypoxia multiple signalling pathways are activated within neurons including the stabilisation of hypoxia-inducible factors. The activity of these proteins is regulated by O2, Fe2+, 2-OG & ascorbate-dependant hydroxylases which contain proyl-4-hydroxylase domains (PHDs). Very little research has been carried out on the action of PHD inhibitors in the CNS and especially on synaptic transmission and plasticity1,2. In this study we have investigated the effects of the PHD inhibitor and hypoxia mimetic, protocatechuic acid ethyl ester (EDHB) on cell viability and synaptic plasticity in isolated rat (Wistar) hippocampus slices and organotypic hippocampal slice cultures.

Methods: Cell viability was assessed using propidium iodide in cultures. Excitatory post-synaptic potentials were elicited by stimulation of the medial perforant (mDG) or Schaffer collateral pathway (CA1). Long-term potentiation (LTP) was induced by high frequency stimulation consisting of 3 trains of 1s duration every 20s at 100Hz. Responses were analysed using WCP software (J. Dempster, Straythclyde). All values are means±S.E.M., compared by ANOVA.

Results: 24 h treatment with EDHB (100 µM) had no significant effct on hippocampal cell viability (0.2±0.07% versus controls 2.9±0.7%, n=3). Cultures treated with 2h hypoxia, 1h OGD or 24h excitotoxicity (4mM glutamic acid) showed a significantly higher percentage of cell death compared to EDHB treated cultures (8.1±4.1, 31.9±19.9 & 75.6±10.1%, respectively, n=3). EDHB (10-100µM) gave rise to an acute, inhibitory effect on synaptic transmission which was seen in the mDG (70.5±4.5%; n=9) but not in the CA1 region (98.3±3.2%, n=7). EDHB at higher concentrations (100µM), was found to inhibit LTP in both the mDG and CA1 regions (111.7±5.6%, versus control 152.1±10.4%, and 110.0±4.4% versus 143.2±8.2% control, respectively, n=5, P<0.01 for both). Application of exogenous iron (100μM) did not reverse EDHB’s inhibitory effect on LTP in the mDG, suggesting a HIF-independent mechanism of action.

Conclusion: These results highlight a novel modulatory role for the PHD inhibitor EDHB in hippocampal synaptic transmission and plasticity. The effects are unlikely to be mediated pre-synaptically as is observed in hypoxia, where O2 levels are decreased in brain tissue and adenosine receptors are activated. A novel post-synaptic mechanism of action may be involved possibly involving NMDA and GABA receptor activation.

References: 1. Wall A et al. (2014). Neurobiol Dis 62, 8-17. 2. Corcoran A et al. (2013). Hippocampus 23, 861-872.