EARLY MODIFICATIONS IN N-METHYL-D-ASPARTATE RECEPTOR SUBUNIT mRNA LEVELS IN AN OXYGEN AND GLUCOSE DEPRIVATION MODEL USING RAT HIPPOCAMPAL BRAIN SLICES S. DOS-ANJOS, a B. MARTÍNEZ-VILLAYANDRE, b S. MONTORI, a C. C. PÉREZ-GARCÍA c AND A. FERNÁNDEZ-LÓPEZ a * a Área de Biología Celular, Instituto de Biomedicina, Universidad de León, 24071, León, Spain b Área de Bioquímica y Biología Molecular, Instituto de Biomedicina, Universidad de León, 24071, León, Spain c Área de Medicina y Cirugía, Instituto de Biomedicina, Universidad de León, 24071, León, Spain Abstract—Glutamatergic N-methyl-D-aspartate NMDA recep- tors (NMDAR) are considered to play a key role in ischemia- induced damage. Long-term (hours) changes in their expres- sion upon ischemia have been shown. Here we report short- term changes in the mRNA levels of the major hippocampal NMDAR subunits (NR1, NR2A and NR2B), as well as c-fos, in an ex vivo ischemia model using hippocampal slices. This effect can be observed also in a calcium free incubation solution. Striking early decreases in the NMDAR subunit mRNA levels were observed after 30 min of oxygen and glucose deprivation (OGD) as well as a partial recovery when the tissues were returned to the balanced salt solution (reper- fusion-like period) for 3 h. Since OGD-induced damage has been reported to be a consequence of the increase in OGD- related glutamate release, we also analyzed NMDAR mRNA levels following increased glutamate levels in hippocampal sections in which no significant effects on NMDAR subunit mRNA levels were detected. Furthermore, we describe that the presence of MK-801 (a selective NMDAR antagonist), CNQX (a selective AMPA/kainate receptor antagonist) or their combined action in the incubation solution is able to induce a significant decrease in NMDAR expression but in these conditions the OGD does not induce further decreases in mRNA levels. We suggest that the mechanisms triggered during OGD to downregulate mRNA levels of NMDAR sub- units could be the same than those induced by glutamate receptor antagonists. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hippocampus, glutamate, N-methyl-D-aspartate, OGD, real-time PCR. Isolated brain slice preparations were first developed for biochemical studies in the early 1960s (McIlwain, 1961). Later, Yamamoto (Yamamoto and McIlwain, 1966) em- ployed this technique to obtain the first electrophysiological recordings from brain slices. Since then, brain slice prep- arations have been widely used as a model for ex vivo studies on neurotransmitters and receptors. Since the model provides the advantages of highly controlled in vitro environments and preserves much of the cellular complex- ity and interactions inherent to the in vivo brain (Sick and LaManna, 1995), it should be able to provide relevant insight about gene expression. The main problem of using brain slices is the short temporal availability of the slices for experimental procedures (24 h), which limits ex vivo assays aimed at studying mRNA levels in acute experi- mental assays. This time of analysis is, in turn, limited to a temporal working window where gene transcription is pre- served when brain slices are incubated in a balanced salt solution such as Ringer’s solution (Dos Anjos et al., 2008), particularly if results are to be compared with those ob- tained from electrophysiological recordings, where this type of balanced salt solution is commonly used. The complexity of the response of the brain, mainly the highly vulnerable hippocampus (Kirino et al., 1985), to the ischemia-reperfusion damage reported in in vivo studies, has led to the development of a model in which brain slices are subjected to oxygen and glucose deprivation (OGD) in order to reduce the factors involved in the in vivo ischemia (Moro et al., 1998). Ischemia-reperfusion experiments in vivo have been reported to elicit important modifications in the expression of a number of genes, mainly those related to the glutamate receptors and, in particular, glutamate N-methyl-D-aspartate receptors (NMDARs), which are considered to play a key role in the pathogenesis of isch- emia (Lee et al., 1999; Liu et al., 2007). This subfamily of ionotropic glutamate receptors is overactivated by gluta- mate during ischemia leading to intracellular calcium in- crease and finally to neuronal cell death (excitotoxicity) (Arundine and Tymianski, 2003), and their blockade has been found to be neuroprotective in brain ischemia models (Arias et al., 1999). Thus, in vivo studies have been able to detect changes in NMDAR mRNA levels several hours after the ischemic insult (Hsu et al., 1998; Won et al., 2001) but no early changes or slightly reduced levels in NMDAR mRNA levels have been observed following the challenge (Zhang et al., 1997). To date, there are no data concerning the regulation of NMDAR genes following OGD insult. The increase in glutamate levels elicited by OGD (Fujimoto et al., 2004) has been reported to induce an excitotoxic effect responsible for at least part of the damage due to OGD (Brongholi et al., 2006), but it is not clear how glutamate activity is involved in NMDAR subunits mRNA levels. In this report we study for the first time the major hippocampal NMDAR subunits NR1, NR2A and NR2B (Al-Hallaq et al., 2007) mRNA levels in the model involv- *Corresponding author. Tel: 34-987-291485; fax: 34-987-291917. E-mail address: arsenio.fernandez@unileon.es (A. Fernández-López). Abbreviations: CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; LDH, lactate dehydrogenase; NMDA, N-methyl-D-aspartate; OGD, oxygen– glucose deprivation; RT, reverse transcription. Neuroscience 164 (2009) 1119 –1126 0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.09.019 1119