Molecular Brain Research 99 (2002) 75–81 www.elsevier.com / locate / bres Short communication Cortical spreading depression transiently activates MAP kinases * Ava K. Chow, C.S. Thompson , Matthew J. Hogan, D. Banner, L.A. Sabourin, A.M. Hakim Neuroscience Research Institute, Faculty of Medicine, University of Ottawa, 451 Smyth, Ottawa, Ontario, Canada K1H 8M5 Accepted 10 January 2002 Abstract Cortical spreading depression (CSD) has been shown to have neuroprotective effects when administered in advance of cerebral ischemia. The mechanism by which CSD induces its neuroprotective effect however remains to be elucidated. Since MAP kinases have been shown to impart neuroprotection in ischemic preconditioning paradigms, we attempted to determine the role CSD may have in the activation of MAPK. We show that CSD is capable of increasing the phosphorylation of ERK in a MEK-dependent manner. This phosphorylation is, however, transient, as phosphorylated ERK levels return to control levels 45 min after 2 h of CSD elicitation. Immunohistochemical analysis reveals that the phosphorylated form of ERK is located ubiquitously in cells of the CSD-treated cortex while CSD-elicited MEK phosphorylation resides solely in the nuclei. These data suggest that CSD may act via the MAP kinase pathways to mediate preconditioning.  2002 Elsevier Science B.V. All rights reserved. Keywords: Neuroprotection; Preconditioning; Cortical spreading depression; MAP kinase 1. Introduction logical blockade of ERK phosphorylation during precondi- tioning eliminates neuroprotection in vitro [12]. Like CSD, Cortical spreading depression (CSD) is characterized by the timing of ERK activation appears to be essential in slowly propagating waves of depolarizing neurons and determining its effect: ERK activation during precondition- glia. Though peri-infarct depolarizations in ischemic brains ing is neuroprotective [12,14], while ERK inhibition are detrimental to energetically compromised tissue [29], during ischemia is neuroprotective [1,30]. CSD does not cause neuronal injury in the normal brain We examined the effect of CSD on the time-course and [31]. In vivo preconditioning with sublethal ischemia intensity of ERK1 / 2 phosphorylation in order to define the [11,25] or CSD [21,26,27] reduces infarct volume follow- precise relationship between CSD and this specific neuro- ing a lethal ischemic episode. The mechanisms underlying protective response. Our data indicate that CSD results in CSD-induced neuroprotection remain unknown, although the transient activation of ERK 1 / 2 and to our knowledge, CSD has been found to alter the expression of numerous this is the first time ERK phosphorylation has been genes which may mediate tolerance, including neuronal examined in this system of neuroprotection. nitric oxide synthase [39], calcium-independent protein kinase C [22], c-fos, junB, c-jun and MKP-1 [15]. Several studies have suggested that certain mitogen- 2. Materials and methods activated protein (MAP) kinases may be involved in neuroprotection. Phosphorylation of extracellular signal- 2.1. Reagents regulated kinases 1 and 2 (ERK1 / 2) promotes survival of PC12 cells [34] and ischemic preconditioning increases Polyclonal antibodies to phosphorylated forms of ERK1 / 2 phosphorylation in vivo [38]. Elevated intracellu- ERK1/2 (pERK1/2) and to MEK1/2 (pMEK1/2) were lar calcium [17,36], and elevated extracellular glutamate obtained from New England Biolabs (Beverly, MA, USA). [42], both of which occur during CSD [2,9], are capable of Secondary antibodies conjugated with horseradish per- inducing the phosphorylation of ERK1 / 2 and pharmaco- oxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Enhanced chemiluminescence *Corresponding author. Tel.: 11-613-560-5800x8260; fax: 11-613- reagents were obtained from Amersham (Piscataway, NJ, 562-5403. E-mail address: charliet@uottawa.ca (C.S. Thompson). USA). Western blot reagents and the Biorad DC protein 0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X(02)00106-7