The selective antagonism of P2X7 and P2Y1 receptors prevents synaptic failure and affects cell proliferation induced by oxygen and glucose deprivation in rat dentate gyrus.

Giovanna Maraula,Alessia Melani,Anna Maria Pugliese,Daniele Lana,Maria Grazia Giovannini,Francesca Gentile,Tommaso Mello,Elisabetta Coppi,Felicita Pedata,Andrea Galli,Gilberto Fisone

doi:10.1371/journal.pone.0115273

Abstract

Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X7 and P2Y1 receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y1 receptor) and BBG (selective antagonist of P2X7 receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU+ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU+ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y1 antagonist reduced the number of BrdU+ cells at this time. The data demonstrate that P2X7 and P2Y1 activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y1 receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.

Highlights

The hippocampus comprises two distinct subfields that show different responses to hypoxic-ischemic brain injury
In a first series of experiments we characterized the synaptic response of evoked field excitatory post-synaptic potentials (fEPSPs) in the dentate gyrus (DG) in the absence or following severe (9-min) oxygen and glucose deprivation (OGD), an ischemic insult that under our experimental conditions constantly produces an irreversible loss of synaptic transmission [2]
In the present study we report that the selective P2X7 receptor (P2X7R) antagonist Brilliant Blue G (BBG) and the selective P2Y1 receptor (P2Y1R) antagonist MRS2179, applied before, during and after a severe OGD period, prevented the appearance of anoxic depolarization (AD), an unequivocal sign of neuronal injury during ischemia, and allowed a significant recovery of fEPSP amplitude

Summary

Introduction

The hippocampus comprises two distinct subfields that show different responses to hypoxic-ischemic brain injury. The CA1 region is susceptible to hypoxia, whereas the dentate gyrus (DG), which serves as a gateway to the hippocampus, is usually more resistant [1]. After OGD initiation, the large efflux of K+ ions into the extracellular space, combined with activation of Na+ and Ca2+ channels, triggers sustained depolarization of hippocampal cells that coincides with the appearance of AD. Increased intracellular Ca2+ and/or massive glutamate receptor activation are additional mechanisms that concur to produce AD [4, 5] and that contribute to cell damage during ischemia [3]. A delay in the appearance of AD can be obtained by treating the slices with glutamate receptor antagonists [4, 5]

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