Elsevier

Brain Research

Volume 1168, 7 September 2007, Pages 129-138
Brain Research

Research Report
Cortical spreading depression releases ATP into the extracellular space and purinergic receptor activation contributes to the induction of ischemic tolerance

https://doi.org/10.1016/j.brainres.2007.06.070Get rights and content

Abstract

Cortical Spreading Depression (CSD) is a well-studied model of preconditioning that provides a high degree of tolerance to a subsequent ischemic event in the brain. The present study was undertaken in order to determine whether the release of ATP during CSD could contribute to the induction of ischemic tolerance. Direct measurement of ATP levels during CSD indicates that with each CSD wave ATP is released into the extracellular space at levels exceeding 100 μM. Cultures of rat primary cortical neurons exposed to low levels of extracellular ATP developed tolerance to subsequent oxygen-glucose deprivation (OGD) or metabolic hypoxia. The preconditioning effect requires new protein synthesis and develops with time, suggesting that a complex genomic response is required for the induction of tolerance. Multiple purinergic receptors are involved in mediating tolerance, with P2Y receptor activation having the greatest effect. Although extracellular adenosine or glutamate may make a small contribution, most of the tolerance was found to be induced independently of adenosine or glutamate receptor activation. Multiple signal transduction pathways mediate the response to extracellular ATP with the protein kinase A pathway and activation of phospholipase C contributing the most. The results are consistent with the proposal that CSD releases ATP into the extracellular space and the subsequent activation of P2Y receptors makes a major contribution to the induction of ischemic tolerance in the brain.

Introduction

Cortical spreading depression (CSD) was first described as a slowly propagating wave of suppressed electrical activity in rabbit cortex (Leao, 1944). Since then spreading depression (SD) has been characterized as a wave of depolarization of neurons and glia that may be elicited in virtually any grey matter in the nervous system (Sugaya et al., 1975, Somjen, 2001). Results of SD include a net influx of sodium, chloride and calcium, a large efflux of potassium and cell swelling. SD also imparts a substantial degree of tolerance to a subsequent ischemic insult to the brain. This was first shown in hippocampal CA1 neurons in rat brain by Kawahara et al. (1995) and other studies have confirmed this observation for cortical neurons (Kobayashi et al., 1995, Matsushima et al., 1996, Matsushima et al., 1998, Taga et al., 1997, Yanamoto et al., 1998, Yanamoto et al., 2000, Otori et al., 2003). Ischemic tolerance develops with time (Kobayashi et al., 1995, Yanamoto et al., 1998, Taga et al., 1997) and is transient (Yanamoto et al., 1998). In rat brain CSD may reduce cortical infarct volume following transient focal ischemia by up to 50% (Matsushima et al., 1996, Matsushima et al., 1998, Yanamoto et al., 1998, Otori et al., 2003) and the protection is long lasting (Yanamoto et al., 2004).

ATP has been shown to act as extracellular signaling molecule in the brain (Zimmermann, 1994, Fields and Stevens, 2000). It may be released from nerve terminals and act as a co-transmitter (Burnstock, 2004) or released from cells by way of ruptured cell membranes or pass through a variety of channels (Bao et al., 2004, Cotrina et al., 1998, Okada et al., 2004, Reigada and Mitchell, 2005). Two families of receptors for ATP and ADP have been identified. P2X receptors are ligand-gated cationic channels and P2Y receptors are G protein-coupled receptors, both families having a wide distribution in the brain (Illes and Ribeiro, 2004). Extracellular ATP has been shown to mediate a range of effects including neurotransmission, modulation of the effects other neurotransmitters and growth factors, trophic actions and cytotoxicity (Di Virgilio, 2000, Amadio et al., 2002, Volonte et al., 2003). Extracellular ATP has also been reported to induce the expression of a variety of genes (Tsim et al., 2003, Choi et al., 2003, Hanley et al., 2004, D'ambrosi et al., 2004, Priller et al., 1995, Mckee et al., 2006). Some of these genes are associated with enhanced cell survival and activation of P2Y receptors has been reported to suppress apoptosis and activate survival pathways in some tissues (Chorna et al., 2004, Tan et al., 2004, Arthur et al., 2006).

These considerations led us to explore the potential role of extracellular ATP in mediating the increased resistance to ischemic damage imparted by CSD. Direct measurement of ATP levels during CSD shows that ATP is released during CSD waves. Exposure of cultured primary cortical neurons to low levels of extracellular ATP was found to induce a considerable tolerance to subsequent oxygen/glucose deprivation (OGD) and metabolic hypoxia that was not mediated by adenosine or glutamate receptor activation. These observations indicate that the release of ATP plays an important role in induction of ischemic tolerance in mammalian brain and may provide a therapeutic avenue aimed at neuroprotection.

Section snippets

CSD releases ATP into the extracellular space

The response of the ATP sensor was linear in the range of 1 to 50 μM ATP (Fig. 1A). Simultaneous recording of extracellular ATP and the cortical DC potential in vivo showed a spike in ATP concentration occurring at the same time as the negative extracellular potential of the CSD wave (Fig. 1B). This was observed in all rats tested (N = 9). The greatest concentrations of ATP recorded were in the range of 100 μM and the responses were up to 2 min in duration. When consecutive responses to CSD waves

Discussion

In the present study direct measurement of ATP concentration has demonstrated that ATP is released into the extracellular space during SD in rat cortex. It is likely that both glial cells and neurons release ATP during SD, as ATP has been shown to be released from astrocytes during the intercellular progression of cytoplasmic calcium waves (Guthrie et al., 1999, Stout et al., 2002, Coco et al., 2003) and calcium waves accompany SD (Kunkler and Kraig, 1998). The exocytotic release of ATP from

Experimental procedures.

All surgical procedures followed the guidelines of the Canadian Council for Animal Care and were approved by the Animal Care Committee of the University of Ottawa.

Acknowledgments

This work was supported by the Heart and Stroke Foundation Centre for Stroke Recovery, The Canadian Stroke Network and the Children's Hospital of Eastern Ontario Research Institute.

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