The Journal of Neuroscience, August 15, 1997, 17(16):6179–6188 Global Ischemia Induces Downregulation of Glur2 mRNA and Increases AMPA Receptor-Mediated Ca21 Influx in Hippocampal CA1 Neurons of Gerbil Jan A. Gorter,1 Jeffrey J. Petrozzino,2 Eleonora M. Aronica,1 Daniel M. Rosenbaum,1 Thoralf Opitz,1 Michael V. L. Bennett,1 John A. Connor,2 and R. Suzanne Zukin1 1Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, and 2Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 Transient, severe forebrain or global ischemia leads to delayed fura-2 and by intracellular recording. Seventy-two hours after cell death of pyramidal neurons in the hippocampal CA1. The ischemia, CA1 neurons that retained the ability to fire action precise molecular mechanisms underlying neuronal cell death potentials exhibited a greatly enhanced AMPA-elicited rise in 21 21 after global ischemia are as yet unknown. Glutamate receptor- [Ca ]i. Basal [Ca ]i in these neurons was unchanged. These mediated Ca 21 influx is thought to play a critical role in this cell findings provide evidence for Ca 21 entry directly through AMPA death. In situ hybridization revealed that the expression of receptors in pyramidal neurons destined to die. Downregulation mRNA encoding GluR2 (the subunit that limits Ca 21 perme- of GluR2 gene expression and an increase in Ca 21 influx ability of AMPA-type glutamate receptors) was markedly and through AMPA receptors in response to endogenous glutamate specifically reduced in gerbil CA1 pyramidal neurons after are likely to contribute to the delayed neuronal death after global ischemia but before the onset of neurodegeneration. To global ischemia. determine whether the change in GluR2 expression is function- Key words: global ischemia; glutamate receptor regulation; ally significant, we examined the AMPA receptor-mediated rise AMPA receptor; GluR2; GluRB; hippocampus; CA1; intracellu- 21 21 in cytoplasmic free Ca level ([Ca ]i ) in individual CA1 pyra- lar calcium; intracellular recording; neurodegeneration; delayed midal neurons by optical imaging with the Ca 21 indicator dye neurodegeneration; optical imaging; gerbil Transient, severe global ischemia, occurring during cardiorespi- permeability, whereas AMPA receptors lacking GluR2 are much ratory arrest in patients or experimentally in animals, induces more Ca 21 permeable (Hollmann et al., 1991; Hume et al., 1991; selective and delayed neuronal cell death (for review, see Burnashev, 1996). AMPA receptors in most principal neurons of Schmidt-Kastner and Freund, 1991). Pyramidal neurons in the adult hippocampus are heteromeric, contain GluR2, and have a CA1 region of the hippocampus and some types of hilar neurons low permeability to Ca 21 (Bochet et al., 1994; Jonas et al., 1994; are particularly vulnerable and die after global ischemia (Kirino, Geiger et al., 1995). GluR2-lacking, relatively Ca 21-permeable 1982; Pulsinelli et al., 1982; Hsu and Buzsaki, 1993). In rats, cell AMPA receptors have recently been implicated in the pathogen- loss is not prominent until 2–3 d after induction of ischemia esis of neuronal degeneration after global ischemia. In rats, global 21 (Pulsinelli et al., 1982). A rise in [Ca ]i is thought to initiate a ischemia leads to reduced expression of GluR2 mRNA in vulner- cascade of events leading to cell death, including activation of able pyramidal neurons of the hippocampal CA1 before the proteases and endonucleases, generation of free radicals that delayed cell death (Pellegrini-Giampietro et al., 1992a; Pollard et destroy cell membranes by lipid peroxidation, and induction of al., 1993); this reduction would lead to increased formation of apoptosis (for review, see Rothman and Olney, 1986; Choi, 1990, Ca21-permeable AMPA receptors. After ischemia, AMPA 1995; Puttfarcken et al., 1993; Bredesen, 1995; Meldrum, 1995). receptor-mediated EPSCs at the CA1–Schaffer collateral synapse Although AMPA receptors were initially thought to be rela- are enhanced and increased in sensitivity to Joro spider toxin and tively impermeable to Ca 21, it is now clear that there are also 1-acetyl naphthyl spermine (Tsubokawa et al., 1995), channel AMPA receptors exhibiting considerable Ca 21 permeability. blockers selective for GluR2-lacking, Ca 21-permeable AMPA AMPA receptors containing the GluR2 subunit exhibit low Ca 21 receptors (Blaschke et al., 1993; Herlitze et al., 1993). Adminis- tration of the AMPA and kainate receptor antagonist 2,3- Received March 19, 1997; revised April 30, 1997; accepted May 28, 1997. dihydroxy-6-nitro-7-sulfamoylbenzo( f)quinoxaline (NBQX) pro- This work was supported by National Institutes of Health Grants NS 20752 and NS tects CA1 neurons in animal models of global ischemia 31282 (R.S.Z.) and NS 07512 (M.V.L.B.), an Aaron Diamond postdoctoral fellow- (Sheardown et al., 1990; Buchan et al., 1991); protection is ob- ship award (E.M.A.), and a Human Frontiers Science Program award (J.A.G). M.V.L.B. is the Sylvia and Robert S. Olnick Professor of Neuroscience. We thank S. served even when NBQX is administered 16–24 hr after ischemia Rybak for technical assistance and C. Roy for excellent histological preparations. (Sheardown et al., 1993). Neuroprotection by NBQX implicates Correspondence should be addressed to Dr. R. Suzanne Zukin, Department of AMPA and/or kainate receptors in delayed neuronal death but Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, 21 Bronx, NY 10461. does not distinguish between more and less of the Ca - Dr. Gorter’s present address: University of Amsterdam, Department of Experi- permeable receptors. mentele Dierkunde, Kruislaan 320, 1098 SM Amsterdam, The Netherlands. Taken together, the data suggest that the “switching off” of Dr. Connor’s present address: The Lovelace Institute, 2425 Ridgecrest Drive, Albuquerque, NM 87108. GluR2 expression in CA1 after an ischemic insult is translated Copyright © 1997 Society for Neuroscience 0270-6474/97/176179-10$05.00/0 into the formation of new AMPA receptors lacking the GluR2 6180 J. Neurosci., August 15, 1997, 17(16):6179–6188 Gorter et al. • AMPA Receptor-Mediated Ca21 Influx After Ischemia subunit. This change in receptor composition increases AMPA presence of excess (100-fold) levels of the same unlabeled probe. This receptor-mediated Ca 21 entry in response to endogenous gluta- procedure resulted in virtually blank autoradiograms. In separate control studies, labeling by sense RNA probes or by antisense probes to sections mate and enhances glutamate pathogenicity in this region (the pretreated with RNase A (100 mg/ml) showed no detectable labeling. GluR2 hypothesis; Pellegrini-Giampietro et al., 1997). Conditions were of sufficiently high stringency as to rule out cross- The present study was performed in gerbils to test whether the hybridization among GluR1, GluR2, and GluR3 (Pellegrini-Giampietro change in AMPA receptor expression enhances AMPA receptor- et al., 1991), and more distantly related glutamate receptor subunits gated Ca 21 entry into CA1 pyramidal neurons. We first show by (GluR5–GluR7, KA1, KA2). The GluR1 and GluR2 probes are “pan” probes (Sommer et al., 1990) in that they label both “flip” and “flop” in situ hybridization that global ischemia in gerbils, as in rats, splice variants. The NR1 probe is a pan probe in that it labels all eight leads to a reduction in GluR2 mRNA in these neurons. We then splice variants; NR1 shares ,20% sequence identity with NR2A–NR2C show by intracellular recording and optical imaging of fura-2- (Kutsuwada et al., 1992; Monyer et al., 1992) and is therefore unlikely to injected CA1 neurons that Ca 21 influx through AMPA receptors cross-react with mRNAs encoding NR2 subunits. Quantitation and statistical analysis. For quantification of mRNA ex- is increased after global ischemia. The changes in GluR2 mRNA pression levels, autoradiograms were analyzed with a Molecular Dynam- expression and AMPA receptor function precede neuronal ics (Sunnyvale, CA) 300A Computing Densitometer equipped with the death. These findings indicate that Ca 21 influx through AMPA National Institutes of Health IMAGE program. Film images were receptors lacking the GluR2 subunit may be an important factor scanned at 2000 dpi resolution, and images of each section (;1 3 10 6 contributing to delayed neurodegeneration after global ischemia. pixels) were created. Mean optical densities in regions of maximal labeling of individual hippocampal subfields were averaged from a min- imum of two sections from each animal for each probe, and film back- MATERIALS AND METHODS ground was subtracted. Optical density values were expressed as grand Global ischemia in the gerbil means (6SEM) of individual means from three to eight gerbils per Global ischemia in the gerbil was produced by temporary bilateral postischemic group. To enable comparisons between groups for any occlusion of the carotid arteries. Adult male Mongolian gerbils (Tumble- given probe, brain sections were cut from control and postischemic brook Farms), weighing between 60 and 80 gm, were fasted overnight and gerbils in the same experimental session, incubated the sections with the anesthetized with intraperitoneal ketamine (100 mg/kg) and xylazine (6 same solutions of RNA probes on the same day, and apposed the sections mg/kg). The carotid arteries were occluded with nontraumatic aneurism to the same sheet of film. clips and released after 5 min to allow cerebral reperfusion. Body Mean optical density readings were statistically