Journal of NeurochemistryRaven Press, Ltd., New York© 1994 International Society for Neurochemistry

Changes in Metabotropic Glutamate Receptor rnRNA LevelsFollowing Global Ischemia : Increase of a Putative Presynaptic

Subtype (mG1uR4) in Highly Vulnerable Rat Brain Areas

Lars Iversen, tEileen Mulvihill, tBetty Haldeman, *Nils Henrik Diemer, *Frank Kaiser,*Malcolm Sheardown, and Peter Kristensen

Department of Pathology and Histochemistry and *Department ofNeuropharmacology, Pharmaceuticals Division, NovoNordisk A/S, M61ov, Denmark; tZymoGenetics Inc ., Seattle, Washington, U.S.A . ; and $Institute of Neuropathology,

University of Copenhagen, Copenhagen, Denmark

Abstract : Metabotropic glutamate receptors mediatetheir intracellular response by coupling to G proteins andmay be divided into three subfamilies : mGIuR1 andmGIuR5, which stimulate phosphatidylinositol hydrolysis ;mGIuR2 and mGIuR3, which are negatively coupled tocyclic AMP formation ; and mGIuR4 and mGIuR6, whichalso inhibit forskolin-stimulated cyclic AMP formation .The mGIuR4 subtypes may represent L-2-amino-4-phos-phonobutyrate-sensitive presynaptic autoreceptors, andtwo alternatively spliced variants of the mGIuR4 codingfor two receptors with different C termini have been iden-tified . Using in situ hybridization, we measured the levelsof mGIuR1-mGIuR5 mRNA in regions of the rat brain 24h after transient global ischemia, a time point when noneuronal damage can yet be observed morphologically .In the hippocampus, the mRNA levels for mGIuR1,mGIuR2, and mGIuR5 were decreased, mGIuR3 mRNAlevels were unchanged, and the mGIuR4 mRNA levelswere strongly increased . The strongest increase ap-peared to be in the mRNA encoding mGIuR4b . ThemGIuR4 mRNA was also increased in the parietal cortex,whereas the ventral posteromedial thalamic nucleusshowed a small decrease in its mRNA content . Theseresults suggest that vulnerable neurons react to an in-creased extracellular glutamate concentration by differ-ential regulation of the mRNA for pre- and postsynapti-cally located metabotropic glutamate receptors . KeyWords: Metabotropic-Glutamate-Presynaptic-Insitu hybridization-mRNA-Ischemia-Hippocampus .J. Neurochem . 63, 625-633 (1994) .

The amino acid L-glutamate is the principal excit-atory neurotransmitter in the CNS, and excessive re-lease of L-glutamate, followed by stimulation of gluta-mate receptors, is believed to play a major role in thedelayed neuronal death that follows cerebral ischemia(Choi, 1992) . Pharmacological studies and molecularcloning have divided glutamate receptors into two dis-tinct families, ionotropic (ligand-gated ion channels)

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and metabotropic (G protein-coupled) receptors (Na-kanishi, 1992 ; Schoepp and Conn, 1993) . The iono-tropic receptor family includes two receptor subfamil-ies, the N-methyl-D-aspartate (NMDA) receptors andthe non-NMDA receptors . The latter can be dividedfurther into categories of a-amino-3-hydroxy-5-meth-ylisoxazole-4-propionate (AMPA)-preferring and kai-nate-preferring receptors (Monaghan et al ., 1989) .

Metabotropic glutamate receptors are involved inthe relay of sensory information in the thalamus(McCormick and von Krosigk, 1992 ; Eaton et al .,1993) and may play an important role in neuronal plas-ticity and the regulation of synaptic strength, throughthe processes of long-term potentiation and long-termdepression (Anwyl, 1991 ; Baskys, 1992 ; Zheng andGallagher, 1992 ; Bashir et al ., 1993 ; Behnisch andReymann, 1993 ; Schoepp and Conn, 1993) . At least sixmetabotropic receptors have been cloned (mG1uRl-mGluR6) (Houamed et al ., 1991 ; Masu et al ., 1991 ;Abe et al., 1992 ; Tanabe et al ., 1992) . Also, threealternatively spliced versions of mGluR1 (a, ß, and c)have been reported (Pin et al ., 1992 ; Tanabe et al .,1992) .The mRNAs encoding the metabotropic glutamate

receptors differ in their distribution in the brain, and thereceptors are coupled to multiple intracellular signal-transduction pathways . Based on sequence homology,pharmacology, and second-messenger coupling in

Received August 17, 1993 ; revised manuscript received January5, 1994 ; accepted January 5, 1994 .

Address correspondence and reprint requests to Dr . P . Kristensenat Novo Nordisk A/S, Biopharmaceuticals Division, Hagedomsvej1, blgd . HAB 1 .36, DK-2820 Gentofte, Denmark.

Abbreviations used: ACPD, 1-aminocyclopentane-1,3-dicarboxy-late ; AMPA, a-amino-3-hydroxy-5- methyl isoxazole-4-propionate ;t .-AP4, t .-2-amino-4-phosphonobutyrate ; cAMP, cyclic AMP ;mGluR, metabotropic glutamate receptor ; NC, neck-cuff occlusionmodel; NMDA, N-methyl-D-aspartate ; 4VO, four-vessel occlusionmodel ; VPM, ventral posteromedial thalamic nucleus .

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transfected cell lines, these receptors have been dividedinto three groups . The first group consists of themGluRI (a, 6, and c) and mG1uR5 receptors . Theseare coupled to phosphoinositide hydrolysis, and therank order of potency for known agonists is quisqualate> L-glutamate > ibotenate > trans-l-aminocyclopen-tane-1,3-dicarboxylate (t-ACPD) (Masu et al ., 1991 ;Abe et al ., 1992 ; Pin et al., 1992 ; Tanabe et al ., 1992 ;Thomsen et al., 1993) . In addition, the mGluRla isassociated with two additional pathways stimulatingcyclic AMP (CAMP) and arachidonic acid formation(Aramori and Nakanishi, 1992), whereas this is not thecase for the mG1uR5 receptor (at least when expressedin transfected cells) (Abe et al ., 1992) .

The second group includes the mGluR2 and mG1uR3receptors, which inhibit forskolin-induced CAMP for-mation and, thus, are negatively coupled to adenylatecyclase . Injection of these mRNAs into oocytes, fol-lowed by stimulation with L-glutamate, has no effect onchloride conductance, and Chinese hamster ovary cellstransfected with mGluR2 cDNA show only a very smallstimulation of phosphoinositide hydrolysis . The agonistrank order of potency for these receptors is L-glutamate

t-ACPD > ibotenate > quisqualate (Tanabe et al .,1992, 1993) . In addition, other metabotropic receptorslinked to inhibition of cAMP formation, but showing adifferent pharmacological response, have been de-scribed (Prezeau et al ., 1992) .The receptors of the third group, mG1uR4 and

mG1uR6, are also coupled to the inhibition of CAMPformation . The L-2-amino-4-phosphonobutyrate (L-AP4) receptor has been shown recently to be a G pro-tein-coupled receptor (Trombley and Westbrook,1992), and synaptic transmission in the lateral perfor-ant path to the hippocampus has been shown to besensitive to inhibition by L-AP4 (Koerner and Cotman,1981) . In the normal hippocampus, mGluR4 mRNA isfound only in CA2 pyramidal cells, whereas the cellbodies of neurons in the entorhinal cortex forming theperforant path show prominent expression of mGluR4mRNA (Thomsen et al ., 1992) . Furthermore, in vitroL-AP4 potently inhibits forskolin-stimulated cAMPproduction in transfected cells expressing themG1uR4a receptor, and the rank order of potency forthis receptor is L-AP4 > L-glutamate ~! t-ACPD> quisqualate > ibotenate. Thus, the mGluR4 is acandidate for the presynaptic L-AP4-sensitive autore-ceptor (Thomsen et al ., 1992 ; Kristensen et al ., 1993 ;Tanabe et al ., 1993) . The mGluR6 subtype has a high-affinity site for L-AP4 and L-serine 0-phosphate, butit is only expressed in the ON-bipolar cells of the retina(Nakajima et al ., 1993) .

Analysis of the isolated mGluR4 cDNA clones hasshown that, in addition to the mG1uR4 cDNA reportedby others (Tanabe et al ., 1992), an alternatively splicedvariant exists (Simoncini et al ., 1993 ; B . Haldemanet al ., manuscript in preparation) . This variant, in thefollowing designated mG1uR4b, is similar to the pre-viously reported cDNA mGluR4 (Genbank accession

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L. IVERSEN ET AL.

no . M90518), except that base pairs 3,398-4,017 aredeleted . This leads to a different and longer C-terminalregion in the predicted protein sequence; the overalllength of mG1uR4b is 983 amino acids, whereas thetotal length of mG1uR4a is 912 amino acids . The siteof divergence between the two forms of mGluR4 isjust following the proposed seventh transmembraneregion . This results in the replacement of the final 64amino acids of mGluR4a with 135 unique amino acidsin mGluR4b. Both forms of the mRNA can be demon-strated in all brain regions analyzed by cDNA synthe-sis, followed by PCR amplification using primers lo-cated on both sides of the insertion point (P . Kris-tensen, unpublished observations) . Comparison of thein situ hybridization pattern in normal rat brain foundwith a probe detecting both mRNAs (p19) and theprobe specifically detecting the mG1uR4a (p20)showed a similar distribution pattern (data not shown) .Analysis of cells transfected with the mG1uR4b cDNAdemonstrates changes in intracellular calcium follow-ing glutamate stimulation (B . Haldeman et al ., manu-script in preparation) .To increase our knowledge of the biological func-

tions of the metabotropic glutamate receptor subtypes,we have used two different rat models to follow thechanges in metabotropic glutamate receptor mRNAlevels following transient global ischemia .

MATERIALS AND METHODSAll animal experiments were carried out in accordance

with guidelines set by the European Communities Councildirective of 24 November 1986 (86/609/EEC) . All protocolswere approved by the Danish State Animal Inspectorate .Four-vessel occlusion model (4VO) of globalischemiaTwo groups of three male Wistar rats weighing 250-

350 g were used. The day prior to the ischemic insult/shamoperation, the animals had both vertebral arteries occludedby electrocauterization in methohexital anesthesia (50 mg/kg i .p .) . Animals were fasted overnight with free access towater. Immediately prior to ischemia, animals were sub-jected to 1% halothane anesthesia, and both femoral arteriesand one femoral vein were cannulated . Ischemia was inducedby exposing both carotids and clamping them for 12 minwhile keeping the mean arterial blood pressure at 60 mmHg by withdrawal of 6-8 ml of blood from one femoralartery catheter . The temperatures of the body and head werekept at 37.5 °C and 36.5 °C, respectively . In sham-operatedanimals, all procedures were exactly as for ischemic animals,with the exception that no blood was withdrawn and thecarotids were dissected free, but not clamped . After 24 hof recirculation, animals were decapitated and brains wereremoved, frozen in isopentane (-80°C), and sectioned on acryostat . Sections from all animals were cut in the sameweek and stored at -80°C (for a maximum of 4 months)until used for in situ hybridization .Neck-cuff occlusion model (NC) of globalischemia

Experiments were conducted on male Wistar ratsweighing 350-400 g . Two groups of three animals were

METABOTROPIC mRNA FOLLOWING GLOBAL ISCHEMIA

used . Animals were fasted overnight with free access towater. Anesthesia was induced with 3.5% halothane in N20/OZ (70:30) in a closed jar and, after intubation, continuedby ventilation with 1 .2% halothane in N20/OZ (70:30) . Thetail artery was catheterized for recording of blood pressureand for determination of blood gasses, pH, and blood glu-cose . The right jugular vein was catheterized to allow with-drawal and reinfusion ofblood . The temperatures of the bodyand brain were maintained at 37 .5°C and 36.5°C, respec-tively . Blood was withdrawn from the jugular vein to main-tain a mean arterial blood pressure of 50 mm Hg, and a 15-min ischemia was induced by inflating a neck cuff to apressure of 350 mm Hg. Halothane was discontinued at theonset of ischemia . At the end of the 15-min ischemic period,the neck cuff was deflated and the remaining blood rapidlyinfused . All procedures were identical in sham-operated ani-mals, except that blood was not withdrawn and the neck cuffwas not inflated. After 24 h of recirculation, animals weredecapitated, and brains were removed, frozen in dry ice, andsectioned on a cryostat. Sections from all animals were cutin the same week and stored at -80°C (for a maximum of4 months) until used for in situ hybridization .

Molecular cloning and in situ hybridizationThe cDNAs for the different metabotropic glutamate re-

ceptor subtypes were cloned by low stringency hybridizationscreening of a rat brain cDNA library using homology tothe previously isolated mGluR1 subtype (Houamed et al .,1991) or by PCR amplification based on the published se-quence (mGluR5) (Abe et al ., 1992) . Fragments of themGluRla, mGluR2, mGluR3, mGluR4a, and mGluR5cDNAs were isolated by restriction enzyme digestion or PCRamplification and subcloned into pBluescript vectors . Allsubclones were verified by DNA sequencing . The followingparts of the cDNAs (database accession numbers in parenthe-ses) were used for the results presented in this article :mG1uR1 (X57569) : by no . 321-1,650 ; mGluR2 (M92075) :by no . 1,193-2,742 ; mGluR3 (M92076) : by no . 1,812-3,215 ; mGluR4 (M90518) : plasmid p18, a Pstl fragment,by 2,983-4,311 ; plasmid p19, a Pstl fragment, by 2,415-2,983 ; plasmid p20, specific for the 4a subtype, by 3,413-4,009 ; mGluR5 (1310891) : by no . 393-930 . ,5S-UTP-la-belled sense and antisense probes were prepared from CsCl-banded and linearized DNA using T3 or T7 polymerases aspreviously described (Kristensen et al ., 1991) . After repeatedprecipitation, the two corresponding RNA probes transcribedfrom the opposite strands of the same plasmid template wereadjusted to the same concentration of radioactivity . All ex-periments included sections hybridized using the sense RNAprobes . The final hybridization solution contained RNAprobe (50,000 cpm/ptl), deionized formamide (50%), dextransulfate (10%), tRNA (1 wg/pl), Ficoll 400 (0.02%, vol/wt),polyvinylpyrrolidone (0.02%, vol/wt), bovine serum albuminfraction V (0.2%, vol/wt), 10 mM dithiothreitol, 0.3 M NaCl,0 .5 mM EDTA, 10 mM Tris-Cl, and 10 mM sodium phos-phate (pH 6.8) . Hybridization was performed overnight at52°C as described (Kristensen et al., 1991), except that theproteinase K step was omitted and the washing of sectionsin 50% formamide was performed at 62 °C and 67°C . At theexposure lengths used for image analysis, no signal wasobserved on sections hybridized with the sense RNA probes .Hybridization of all sections with one probe was alwaysdone in the same experiment (e.g ., all control and ischemiasections from the 4VO experiment with the mGluRI probe)to assure that minor differences in hybridization and washing

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conditions or other unknown variables did not disturb thecomparison of sham-operated and ischemic animals .

Autoradiography and image analysisAfter hybridization, the sections were exposed to Amer-

sham ßmax film in a closed cassette with a set of ' 4C stan-dards (Amersham microscales) . Exposure times were 4-8days, and sections from both ischemic and sham-operatedanimals hybridized with the same probe were always ex-posed on the same film . Densitometric measurements wereperformed using the NIH-IMAGE program after scanningof the film images at 2,000 dpi resolution in a Howtek Scan-master 1135+ slide scanner, creating images of each frontalsection consisting of -1 .3 x 106 pixels . Average densitiesof individual brain regions were measured after manual de-marcation . The number of pixels included in this averagevaried between regions . In the CAI region, the mean wastypically the average of ^1,500 pixels . Regions measuredwere the hippocampal areas CAI, CA2, CA3, and dentategyrus, the parietal cerebral cortex, and the ventral posterome-dial thalamic nucleus (VPM) . A series of 14C standards in-cluded on each film exposure were also scanned, and a stan-dard curve was prepared using fitting to a third-degree poly-nomial . The average densities measured were always withinthe range of the standards used . After fitting to the standardcurve prepared for each film, the densities were convertedto activity . Each group (ischemic or sham-operated) con-sisted of three animals ; three sections from each animal werehybridized and individual brain regions on both sides of eachsection analyzed . Thus, for each treatment group, a totalof 18 average activities was generated . Plotting of data forindividual regions demonstrated that the variability in aver-age activity of areas could be attributed to differences be-tween both individual sections (variation in thickness, hy-bridization, etc .) and individual animals . The mean ± SEMfor each group was calculated, and comparison betweengroups was done using the Student's t test .

RESULTS

Change in mRNA levels in the hippocampusThe mRNA levels for the metabotropic glutamate

receptors 24 h after transient global ischemia werefound to vary differently depending on the individualsubtype . The mRNA content for the majority of thesubtypes (mG1uRl, mG1uR2, and mGluR5) was lowerin the hippocampus 24 h after the ischemia, whereasthe mGluR3 mRNA level appeared unchanged (Fig .1) . In contrast, the mGluR4 mRNA was increased fol-lowing ischemia, showing a clear signal in all partsof the hippocampal pyramidal cell layer (Fig . 2) . Tovalidate the finding of an increase in mG1uR4 mRNA,hybridization was performed using three different sub-clones, prepared from the mGluR4a cDNA. The over-all structures of the two mG1uR4 subtype cDNAs areoutlined in Fig . 3, where the approximate positions ofthe mG1uR4 subclones used for probe preparation inthis study are also shown . The in situ hybridizationwith the different subclones all confirmed the increasein mG1uR4 mRNA (Fig . 2) .To obtain quantitative evidence for these results, the

average densities of selected regions were measuredafter scanning and digitalization of the autoradio-

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FIG . 1 . Frontal sections from rat forebrain demonstrating the result of in situ hybridization for four metabotropic glutamate receptorsubtypes in sham-operated (a, c, e, g) and ischemic (b, d, f, h) (4VO) animals . Sections were hybridized with 35S-labelled antisenseRNA probes for mGIuR1 (a, b), mGIuR2 (c, d), mGIuR3 (e, f), or mGIuR5 (g, h) . Positive prints were prepared from,ßmax films exposedfor 4 days .

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FIG . 2 . Frontal sections from rat forebrain demonstrating the result of in situ hybridization for the metabotropic glutamate receptormGIuR4 subtype, in sham-operated (a, c, e, g) and ischemic (b, d, f, h) (NC) animals . Sections were hybridized with 35 S-labelledantisense RNA probes from plasmid pl8 (a, b), plasmid p19 (c, d), plasmid p20 (e, f), or a similar amount of 35 S-labelled sense probefrom plasmid p20 (g, h) . Plasmid p20 contains only cDNA sequence specific for the 4a form, whereas probes from p18 and p19 detectboth a and b forms of mGIuR4 (see text). Positive prints were prepared from /8max films exposed for 8 days .

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graphic images (Fig . 4) . In the hippocampus, the levelsof mG1uRl, mG1uR2, and mG1uR5 mRNA were sig-nificantly lower in all parts where expression was mea-surable, except for the dentate gyrus, where the levelof mGluR5 mRNA was unchanged . The quantitationconfirmed that the mGluR4 mRNA was significantlyincreased in all parts of the hippocampus, except forthe CA2 region (the only region with notable expres-sion in the normal brain) . Analysis of the hybridizationwith RNA from the different mG1uR4a cDNA sub-clones showed that the largest increase was found withprobes detecting both the mGluR4a and the mGluR4bforms, whereas the increase was only modest with themGluR4a-specific probe (Fig . 5) .

FIG. 4. Ischemia-induced changes in levels ofmRNA for five metabotropic glutamate recep-tors in different parts of hippocampus in twodifferent experimental models . Average activityin the normal area (sham-operated rats) was setto 100% . Data are expressed as means -- SEM .Three sections from each of the three rats form-ing a group were hybridized . Activity was mea-sured on both sides in each section (see Materi-als and Methods), and the values shown are,therefore, the averages of n = 18.

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FIG. 3. Schematic representation ofthe RNA structure of two forms ofmGIuR4 . The locations of subclonesused for preparation of probes for insitu hybridization are indicated.

Change in mRNA levels in other brain regionsFrom the micrographs of the hybridizations, an in-

crease in cerebral cortical layer IV of the mGluR4mRNA was clearly seen, and was most prominent withthe probes detecting both forms of mG1uR4 mRNA(Fig . 2) . However, in order to make a reliable demarca-tion of the area to be measured on the digitized images,the signal was measured in the entire parietal cortex,and for this reason the changes in percentage (Table1) are smaller than would be expected. In the cerebralcortex, we observed an increase in the apparentmG1uR4b mRNA, whereas the mG1uR4a mRNA wasslightly decreased . In the VPM, a decrease in themG1uR4 mRNA was seen after the transient ischemia,

METABOTROPIC mRNA FOLLOWING GLOBAL ISCHEMIA

FIG. 5. Ischemia-induced changes in levels of mRNA formGIuR4 metabotropic glutamate receptors in hippocampus fromthe NC model. Average activity in the normal area (sham-oper-ated rats) was set to 100% . Data are expressed as meansSEM. p1ß is the probe detecting the mGIuR4a and mGIuR4b

mRNAs, p19 the probe detecting the mGIuR4a and mGIuR4bmRNAs, and p20 the probe detecting only mGIuR4a mRNA .Solid bars, NC controls ; hatched bars, NC ischemia . Three sec-tions were hybridized from each of the three rats forming eachgroup. Activity wasmeasured on both sides in each section (seeMaterials and Methods), and the values shown are, therefore,the averages of n = 18.

with the major apparent change in the levels ofmG1uR4a mRNA (Table 1) . The mRNA for themGluR3 appeared to be slightly increased in whitematter tracts when judged from the micrographs (Fig .1). However, measurement of areas in ischemic andcontrol animals showed that there was no significantchange in the level of this mRNA in corpus callosum(data not shown) .

DISCUSSION

The CAI region of the hippocampus is highly vul-nerable to neuronal damage in the 4VO and NC models

TABLE 1 . Percent change in mRNA levels ofmetabotropic receptor subtypes in ratparietal cerebral cortex and VPM

The changes (in percent) ofthe hybridization signal for mG1uR1-mG1uR5 mRNA in regions of rat parietal cortex andVPM 24 h aftertransient global ischemia are shown. Values are the mean activity inischemia animals as a percentage of the mean activity in sham-operated animals. The probes from p20 detect only mGluR4amRNA, whereas pl8 and p19 detect both mGluR4a and mGluR4bmRNA (see text). Adash (-) indicates that no expression was seenin the area. ND, no experiment was performed.

p G 0.05, using Student's t test .

631

used in this investigation . Twenty-four hours after tran-sient global ischemia, no morphological damage canbe seen, but under the conditions used here, a largepercentage of the CAI pyramidal cells appear severelydamaged if the tissue is analyzed later than 48-66 hafter the ischemic insult. Increasing the period of isch-emia extends the neuronal damage to other parts ofthe hippocampus and to the cerebral cortex (Jorgensenand Diemer, 1982; Pulsinelli et al ., 1982) .

In the hippocampus, the result of hybridization usingRNA probes for five different metabotropic glutamatereceptor cDNAs showed that the mRNA levels of mostreceptor subtypes are either decreased or unaffected24 h after the transient global ischemia . In contrast,the content of mGIuR4 mRNA is increased. A moredetailed analysis showed that the majority of this in-crease is the result of an increase in the expression ofan alternatively spliced version of the mGluR4 recep-tor, most likely the mG1uR4b mRNA. Currently, wedo not know how functionally similar the two mG1uR4forms are, but the mGluR4b, like the mGIuR4a, doesproduce a transient increase in intracellular calcium(B . Haldeman, manuscript in preparation) and the dis-tributions in the normal rat brain of the two mRNAsseem to be identical . It is possible, however, that addi-tional alternatively spliced variants of mG1uR4 existand could account for the observed increase .

Transient global ischemia causes delayed neuronaldeath in the CAI region of the rat hippocampus withan 80% loss of pyramidal neurons and is mediatedby an excitotoxic (glutamatergic) input in the CAIpyramidal neurons by way of the Schaffer collateralsynapse (Benveniste et al ., 1984 ; Wieloch et al ., 1985) .The excitotoxic effect of transient global ischemia onthe pyramidal cells of the hippocampus is believed tobe mediated by the ionotropic glutamate receptors,most notably the AMPA receptor family, as AMPAantagonists, but not those of NMDA, are effective inmodels of transient global ischemia (Buchan et al .,1991 ; Diemer et al ., 1991 ; Nellgârd and Wieloch,1992 ; Sheardown and Nordholm, 1993) . However, anumber of investigations have shown the existence ofan interaction between metabotropic and ionotropicAMPA or NMDA receptors . In a spinal cord slicepreparation, (1S,3R)-ACPD potentiated the inward cur-rents evoked by application of AMPA, NMDA, andkainate, an effect that was rapidly reversible (Bleak-man et al ., 1992) . (IS,3R)-ACPD also potentiatedNMDA-mediated, but not AMPA-mediated, currentsin hippocampal pyramidal cells, an effect that couldbe inhibited by protein kinase C inhibitors (Aniksztejnet al ., 1992) . Protein kinase C was also shown to medi-ate an enhancement of NMDA currents by metabo-tropic glutamate receptors in Xenopus oocytes (Kelsoet al ., 1992) . Protein kinase C inhibitor (staurosporine)prevents postischemic neuronal damage (Hara et al .,1990) . Receptor interactions may also work in the otherdirection, as it has been reported that the activationof AMPA receptors may have an inhibitory effect on

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Parietal

4VO

cortex

NC

VPM

4VO NC

mGIuRI 108 66' 106 60°mG1uR2 57" 70' -mG1uR3 88 102 -mGIuR4, p18 ND 130' ND 93mG1uR4, p19 111° 106 75° 82°mG1uR4, p20 ND 80 . ND 64°mGIuR5 96 97 -

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inositol 1,4,5-trisphosphate formation (Lonart andJohnson, 1993).

Injection of the metabotropic agonist (1 S,3R)-ACPDinto the hippocampus leads to convulsions and damageto the granule cells and pyramidal cells of CAI andCA4, an effect that may be inhibited by simultaneousadministration of an NMDA antagonist (Sacaan andSchoepp, 1992). In contrast to this, others have re-ported a neuroprotective effect of (1S,3R)-ACPD inrat focal ischemia (Chiamulera et al ., 1992) or afterintraocular injection of NMDA (Siliprandi et al .,1992). Blockade of metabotropic receptors by the non-specific antagonist L-2-amino-3-phosphonopropionatewas reported to inhibit hypoxic damage in hippocam-pal slices in vitro (Opitz and Reymann, 1991). Theobservation that mG1uR4a is likely to be a presynapticautoreceptor (Thomsen et al ., 1992) may explain whycontradictory results on the modulation/induction ofexcitoxic damage have been obtained using agonistsaffecting all metabotropic subtypes . The balance be-tween inhibition of glutamate release and increase inpostsynaptic stimulation would thus vary, dependingon which of the receptor subtypes are present. It ishoped that future experiments with subtype-specificcompounds will clarify the influence of the individualmetabotropic receptor subtypes on the excitotoxic pro-cess .

It has been reported previously that stimulation ofcultured cerebellar granule cells with glutamate orquisqualate led to a decrease in mG1uR1 a mRNA (Bes-sho et al., 1993). Our present findings may be interpre-ted as part of the physiological response of vulnerableneurons reacting to the increase in extracellular gluta-mate concentrations . It has been observed that mRNAencoding ionotropic glutamate receptors are down-reg-ulated following ischemia (Pellegrini-Giampietro etal., 1992 ; L. Frank et al ., manuscript submitted) . Like-wise, we have observed a down-regulation of mRNAencoding metabotropic receptors mediating postsynap-tic transmission . The increase in the mRNA for themG1uR4 could increase inhibition of glutamate releasethrough an increase in the number of presynaptic auto-receptors.

Acknowledgment: We are grateful to Winnie Listow-Saabye for excellent technical assistance and to Wayne Ras-band for providing the scientific community with the NIH-IMAGE program (available by ftp from zippy.nimh.nih.gov) .

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