780 Corrections Proc. Natl. Acad. Sci. USA 90 (1993) Neurobiology. In the article "Switch in glutamate receptor Sunghee Cho, and William A. Pulsinelli, which appeared in subunit gene expression in CA1 subfield of hippocampus number 21, November 1, 1992, ofProc. Natl. Acad. Sci. USA following global ischemia in rats" by Domenico E. Pellegrini- (89, 10499-10503), the reproduction of Fig. 1 was unsatis- Giampietro, R. Suzanne Zukin, Michael V. L. Bennett, factory. Fig. 1 and its legend are shown below. FIG. 1. Pseudo-color display of density of autoradiograms of GluRi, GluR2, and GluR3 mRNAs in coronal sections of control and postischemic rat brain at the level ofthe hippocampus. (A) GluRl expression in control (sham-operated) brain. (B) GluRl expression in ischemic rats 24 hr after 10 min of global ischemia. (C) GluR2 expression in control. (D) GluR2 expression 24 hr postischemia, showing dramatic and selective reduction in CA1 labeling. (E) GluR3 expression in control brain. (F) GluR3 expression 24 hr postischemia, showing reduction in CA1 that is not as marked as in D. Other experiments did not show as great a decline in CA3 as seen in F. DG, dentate gyrus; Cx, neocortex. (x7.) Downloaded by guest on September 30, 2021 Proc. NatI. Acad. Sci. USA Vol. 89, pp. 10499-10503, November 1992 Neurobiology Switch in glutamate receptor subunit gene expression in CAl subfield of hippocampus following global ischemia in rats caina-at-mino-3-hydroxy-5-umethyl-4-lsoxazole bproplonic add/excdtotoixdty) DOMENICO E. PELLEGRINI-GIAMPIETRO*t, R. SUZANNE ZUKIN**, MICHAEL V. L. BENNETT*, SUNGHEE CHO§, AND WILLIAM A. PULSINELLI§¶ *Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461; and kCerebrovascular Disease Research Center, Department of Neurology and Neuroscience, Cornell University Medical Center, 1300 York Avenue, New York, NY 10021 Contributed by Michael V. L. Bennett, July 20, 1992 ABSTRACT Severe, transient global ischemla ofthe brain resulting from a number of pathological states, including induces delayed damage to specific neuronal populations. ischemia, epilepsy, Huntington chorea, and Alzheimer dis- Sustained Ca2+ influx through glutamate receptor channels Is ease (10, 11). Until recently, the NMDA receptor was con- thought to play a critical role in postischemic cell death. sidered to be unique among glutamate receptors in that it is Although most kainate-type glutamate receptors are Ca2+- Ca2+-permeable (12). It is now established that kainate Impermeable, Ca2+-permeable kainate receptors have been receptors in specific types ofneurons (13, 14) and glia (15, 16) reported in specific kinds of neurons and glia. Recombinant are Ca2+-permeable. Furthermore, receptors formed from receptors assembled from GluRl and/or GluR3 subunits in GluRl and/or GluR3 subunits expressed exogenously are exogenous expression systems are permeable to Ca2+; hetero- also permeable to Ca2+ (17, 18). Coexpression ofGluR2 with meric channels containing GluR2 subunits are Ca2+- GluRi and/or GluR3 results in Ca2+-impermeable channels; impermeable. Thus, altered expression of GluR2 in develop- the GluR2 subunit governs divalent permeability of the ment or following a neurological insult or injury to the brain expressed receptors. We examined the possibility that a can act as a switch to modify Ca2+ permeability. To Investigate modification in expression of kainate/AMPA receptor sub- the molecular mech underlying delayed postischemic cell units leading to increased Ca2+ influx might be a mechanism death, GluRl, GluR2, and GluR3 gene expression was exam- underlying delayed necrosis of CAI neurons caused by ined by in situ hybridization in postischemic rats. Following transient global ischemia. severe, transient forebrain Ischemia GluR2 gene expression was preferentially reduced in CAl hippocampal neurons at a MATERIALS AND METHODS time point that preceded their degeneration. The switch in expression ofkainate/AMPA receptor subunits coincided with Induction of Ischemia in Rats. Transient, severe forebrain the previously reported increase in Ca2+ influx into CAl cells. ischemia was induced in male Wistar rats (175-250 g) by a Timing of the switch indicates that it may play a causal role in modified four-vessel occlusion method (6); cerebral circula- postischemic cell death. tion was restored after 10 min. The animals' body tempera- ture was maintained by means of a heat lamp and rectal thermistor at 37.50C + 0.5OC throughout the ischemic period Transient but severe global or forebrain ischemia, observed and for 6-8 hr after cerebral reperfusion. At the end of10 min in patients successfully resuscitated from cardiac arrest or ofischemia the animals remained unresponsive to stimuli and induced experimentally in animals, damages specific popu- had dilated pupils. Ischemic rats were sacrificed at 1, 6, 12, lations of neurons. Pyramidal cells in the CAl subfield ofthe 18, and 24 hr after reperfusion, times preceding histologically hippocampus are particularly vulnerable and become irre- detectable cell death in CAl. Control rats were sham- versibly damaged after a few minutes ofischemia. However, operated and sacrificed 24 hr later. histological signs of degeneration in these neurons are not In Situ Hybridization. In situ hybridization was performed apparent until 48-72 hr after circulation has been restored (1, in coronal brain sections with uridine 5'-[a-[35S]thio]triphos- 2). Glutamate and aspartate are released in excess during phate (UTP[5S])-labeled antisense RNA probes complemen- cerebral ischemia (3) and are thought to play a role in the tary to GluR1, GluR2, and GluR3 mRNAs (19) by one of us pathological mechanism responsible for this damage. Gluta- (D.E.P.-G.), who was blind to the experimental treatment. mate receptors are enriched in brain regions susceptible to Sections were apposed to Kodak XAR-5 film for 24 hr and ischemic injury (4), and their antagonists are neuroprotective then dipped in Kodak NTB-2 nuclear emulsion, developed in ischemic models. Specific blockers of the N-methyl-D- after 8 days, and counterstained with hematoxylin/eosin. aspartate (NMDA) receptor are protective in moderate (5) Sense RNA probes did not label, and pretreatment with but not severe (6, 7) ischemia, whereas systemic administra- RNase A (100 ,ug/ml) prior to hybridization prevented label- tion of the relatively selective kainate/a-amino-3-hydroxy- ing. Specificity of GluRi, GluR2, and GluR3 probes was 5-methyl-4-isoxazole propionic acid (AMPA) receptor assessed by competition experiments using a 10-fold excess blocker, 2,3-dihydro-6-nitro-7-sulfamoylbenzoquinoxaline of unlabeled cRNAs (19); hybridization to any given probe (NBQX), is effective in preventing delayed CA1 cell death was blocked nearly completely by preincubation of sections following severe global ischemia in gerbils (8) and in rats (9), with an excess ofthe corresponding unlabeled cRNA but was even when given 12 hr after the ischemic challenge (W.A.P., unpublished data). Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4isoxazole Sustained Ca2+ influx through glutamate receptors is propionic acid; NBQX, 2,3-dihydro-6nitro-7-sulfamoylbenzoqui- thought to play an important role in neuronal degeneration noxaline; NMDA, N-methyl-D-aspartate. tPresent address: Department of Pharmacology, University of Flor- ence, Florence, Italy. The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" Present address: Department of Neurology, University of Tennes- in accordance with 18 U.S.C. §1734 solely to indicate this fact. see Health Science Center, Memphis, TN 38163. 10499 10500 Neurobiology: Pellegrini-Giampietro et al. Proc. Natl. Acad. Sci. USA 89 (1992) not blocked by preincubation with the other cRNAs. Con- mRNA in each region. Percent optical densities of control ditions were assumed to be of sufficiently high stringency as were used to compute the ratio (GluRl + GluR3)/GluR2 for to rule out cross-hybridization to GluR4, which is virtually each animal. In control rats labeling of GluRl, GluR2, and absent from regions of rat brain other than cerebellum (20). GluR3 mRNAs in each of the regions examined (CA1, CA3, Other glutamate receptor-related subunits (GluR5, GluR6, and dentate gyrus) was at comparable levels, as determined GluR7, and KA-1) exhibit <40%o homology with GluR1 by in situ hybridization (19) or Northern blotting (24). Since through GluR4 (21) and should not have been labeled. The the control mRNA level for each region examined was set at "flip" and "flop" splice variants do not differ in their 100%o, the control ratio (GluRi + GluR3)/GluR2 was 2. Ca2+-permeability properties (22); we used "pan" probes (23) to label both splice variants. Quantitative Analysis. The rationale of the quantitative RESULTS analysis was based on the constant relationship between Autoradiograms ofcoronal sections ofcontrol rat brain at the densities in film autoradiograms and number of grains over- level ofthe hippocampus showed characteristic distributions lying single neurons in emulsion-dipped sections. Optical of GluR1, GluR2, and GluR3 mRNAs in confirmation of densities and number of grains per cell in each region were earlier studies (19, 20). In hippocampus, all three transcripts essentially constant among sections from the same animal. were expressed at high levels in the granule cell layer, in The concentration of RNA probe used (106 cpm/80 Al per pyramidal cell layers of the CA1, CA3, and CA4 subfields, section) was saturating and afforded the maximal signal-to- and in subiculum (Fig. 1 A, C, and E). noise ratio for GluR1, GluR2, and GluR3. Preexposure of Forebrain ischemia caused a striking change in kainate/ films and analysis of UTP[35S]-labeled brain paste standards AMPA receptor mRNA expression in CA1, the hippocampal indicated that exposure times were in the linear response area most sensitive to ischemic damage (Fig. 1). At 24 hr after range of the film. Corresponding brain sections of different ischemia, GluR2 expression was dramatically reduced in postischemic times (but always including control brain) were CA1 (Fig.