The Journal of Neuroscience, October 1993, 73(10): 4445-4455

Seizures and Brain Injury in Neonatal Rats Induced by 1 S,3R-ACPD, a Metabotropic

John W. McDonald,’ Andrew S. Fix,* Joseph P. Tizzano:* and Darryle D. Schoepp’ ICNS Research Division and *Toxicology Research Laboratories, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

The role of metabotropic excitatory amino acid receptors in receptors, which are coupled to phosphoinositidehydrolysis or seizures and brain injury was examined using the selective modulation of CAMP metabolism,have beencharacterized (Sla- metabotropic agonist 1 S,3R-ACPD [( 1 S,3R)-1 -aminocyclo- deczek et al., 1985; Nicoletti et al., 1986a; Pearceet al., 1986; pentane-1-3-dicarboxylic acid] in 7-d-old neonatal rats. Sys- Sugiyama et al., 1987; Schoepp and Johnson, 1988; Schoeppet temic administration of 1 S,3R-ACPD produced dose-depen- al., 1992b). Nonselective agonistsof metabotropic receptorsin- dent convulsions (ED,, = 16 mg/kg, i.p.) that were clude glutamate, ibotenate, and quisqualate (Schoepp et al., stereoselective for the active metabotropic ACPD isomer, 1990a). Recently, more selective metabotropic have since lR,BS-ACPD was less potent (ED,, = 93 mg/kg, i.p.). been developed, including (+)truns-ACPD [(f )-truns- 1-ami- 1 S,3R-ACPD-induced seizures were antagonized by sys- nocyclopentane- 1,3-dicarboxylic acid (Palmer et al., 1989; De- temic administration of dantrolene, an inhibitor of intracel- sai and Conn, 1990)]. Truns-ACPD is a racemic mixture of lular calcium mobilization, but not by the ionotropic gluta- lS,3R and lR, 3S stereoisomers. lS,3R-ACPD is the active mate antagonists MK-601 or GYKI-52466. As indexed by isomer of trans-ACPD and is currently the most selective and hemispheric brain weight differences 5 d postinjection, uni- potent metabotropic agonist available (Schoeppet al., 199lb). lateral intrastriatal injection of 1 S,3R-ACPD (0.1-2.0 ccrnoll In contrast, 1R,3S-ACPD is a relatively inactive agonistat met- PI), but not lR,SS-ACPD, produced dose-dependent brain abotropic glutamate receptors that are linked to phosphoino- injury (maximal effect of 3.4 f 0.5% damage). 1 S,3R-ACPD sitide hydrolysis or modulation of adenylate cyclase activity brain injury occurred in the absence of prominent behavioral (Irving et al., 1990; Schoepp et al., 1991b). The secondmes- convulsions. Histologic and ultrastructural examination of sengerresponses to metabotropic agonistsare not antagonized 1 S,3f?-ACPD-injected rat brains revealed swelling and de- by selective ionotropic antagonists (Nicoletti et al., 1986a; generation of select neurons at 4 hr postinjection, but little Schoeppand Johnson, 1988; Schoeppet al., 1992a). evidence of injured neurons 5 d later. 1 S,3R-ACPD-mediated In contrast to ionotropic-type excitatory amino acid receptors, brain injury was not attenuated by systemic administration the physiologic and pathologic roles of metabotropic receptors of the NMDA antagonist MK-601 or the AMPA antagonist are just beginning to emerge becauseof the availability of a GYKI-52466. However, cointrastriatal injection of dantrolene selectiveagonist. Recent reports indicate that lS, 3R-ACPD can reduced the severity of lS,3R-ACPD injury by 66 * 7%. produce brain injury. Intrastriatal injection of subtoxic dosesof These studies indicate that seizures and neuronal injury can 1S, 3R-ACPD potentiates NMDA- but not AMPA-mediated be elicited by the selective activation of metabotropic glu- brain injury in neonatal rats (McDonald and Schoepp, 1992). tamate receptors in perinatal rats, and these effects of 1 S,3R- Stereotaxic intrahippocampal injection of higher dosesof lS,3R- ACPD involve the mobilization of intracellular calcium stores. ACPD produces delayed seizuresand prominent neuronal in- [Key words: lSJR-ACPD (1-amino-cyclopentane- 1,3-h jury in adult rats (Sacaanand Schoepp, 1992). carboxyfk acid), glutamate, brain injury, metabotropic glu- The developing CNS provides a unique model to assessmeta- tamate receptors, dantrolene, excitatory amino acid] botropic excitatory amino acid receptor function and interac- tions with ionotropic receptors, since metabotropic responses Basedon biochemical, electrophysiological,and molecular stud- (i.e., phosphoinositidehydrolysis) are transiently enhanceddur- ies, excitatory amino acid receptorshave beenbroadly classified ing the early postnatal period (McDonald and Johnston, 1990; as ionotropic (ion channel linked) or metabotropic (G-protein Schoepp and Hillman, 1990; Schoepp et al., 1990a). Further- linked). Ionotropic receptors are subclassified into NMDA, more, chemosensitivity and excitotoxicity of NMDA and AMPA a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors are also markedly increasedduring this same period (AMPA), and kainate receptors basedon their preferential ag- (McDonald et al., 1988; Ikonomidou et al., 1989; McDonald onists (Monaghan et al., 1989). More recently, metabotropic or and Johnston, 1990). In this study we further examined the guaninenucleotide binding protein-linked excitatory amino acid cellular mechanismsand pharmacology of seizuresand neuronal injury associatedwith the selective activation of 1S, 3R-ACPD- sensitive metabotropic glutamate receptors in neonatal rats. Received Jan. 22, 1993; revised Apr. 19, 1993; accepted Apr. 29, 1993. Correspondence should be addressed to Darryle D. Schoepp, Ph.D., CNS Re- Materials and Methods search, MC907, DC 08 15, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285. Animals. Postnatalday (PND) 7 maleand femaleSprague-Dawley al- Copyright 0 1993 Society for Neuroscience 0270-6474/93/134445-l 1$05.00/O bino rats were used in all experiments (Charles River Laboratories, Inc., 4446 McDonald et al. * 1 S,3R-ACPD-induced Brain Injury in an in viva Model

1W- Table 1. Effect of excitatory amino acid receptor antagonists on lS,3R-ACPD-induced seizures in neonatal rats EO- # Convulsed/ I Pretreatment Agonist # tested Q Eo- Ei Vehicle Vehicle o/10 z lS,SR-ACPD Vehicle lS,3R-ACPD (40 mg/kg) lO/lO 8 40- l&OS-ACPD MK-801 (1 mg/kg) 1S, 3R-ACPD (40 mg/kg) 9/10 z GYIU-52466 (20 mg/kg) lS,3R-ACPD (40 mg/kg) 9/10 20- Dantrolene (500 mg/kg) lS,3R-ACPD (40 mg/kg) O/l@ Antagonists or sterile water (vehicle) were given by the intraperitoneal route 30 min prior to intraperitoneal injection of lS,3R-ACPD or its water vehicle. Ani- mals were observed for behaviors for 30 min before and an additional 30 min O-001 after lS,3R-ACPD injection. DOSE (mg/kg 1.~4 “Minimally effective protective dose (~50% protection). Figure 1. Comparison of dose-effect curves for convulsions produced by intraperitoneal administration of either lS,3R-ACPD or lR,3S- maldehyde/l.4% glutaraldehyde. Examinations made at 5 d postinjec- ACPD. PND 7 rats were observed for behavioral signs of motor seizures tion were limited to rats that received intrastriatal injections and were over a 30 min period following intraperitoneal injection of ACPD iso- only by light microscopy. Fixed brains were removed immediately after mers (0.05 ml in PBS, pH 7.4). Ten animals were tested at each dose. perfusion and stored in cold fixative until trimming with a rat brain The vertical axis represents the percentage of animals that exhibited matrix (Activational Systems Inc., Warren, MI). For light microscopy, motor convulsions at a particular dose of ACPD. trimmed slices were processed by graded dehydration, embed- ded in paraffin blocks, sectioned at 4 pm, stained with hematoxylin and eosin (HE), and examined by light microscopy. For transmission elec- Wilmington, MA). Litters were culled to 12 pups and animals were tron microscopy, select slices were secondarily fixed in a solution of maintained on a 12: 12 hr light : dark cycle. cold 2.0% naraformaldehvde/2.5% alutaraldehvde. rinsed with 0.1 M Convulsions in neonatal rats. Evaluation of convulsant behavior in sodium cacodylate buffer (pH 7.2) postfixed in aqueous 1% osmium PND 7 rats was carried out as described previously (Schoepp et al., tetroxide/ 1.5% potassium ferrocyanide on ice, serially dehydrated in 1990b). Littermate pairs received one of five doses of either l.S,3R- , and embedded in epoxy resin. Epoxy blocks were sectioned ACPD (7.5-100 mg/kg) or lR,3S-ACPD (25-500 mgkg). Five to 10 at 1 wrn, stained with toluidine blue, and examined by light microscopy. animals were tested at each dose. All compounds were administered After identification of select target areas, ultrathin sections were cut with intraperitoneally in phosphate-buffered saline or sterile water (10 ml a diamond knife, stained with uranyl acetate and Sato’s lead citrate, vol/kg body weight). Following the injection of agonists, animals were and examined with a Philips 4 1OLS transmission electron microscope. placed in warmed (36°C) Plexiglas observation chambers, evaluated for Materials. MK-80 1 I( +)-5-methyl- lO,l l-dihydro-5H-diben- 30 min, and scored for the presence or absence of tonic or clonic motor zo[a,d]cyclohepten-5, IO-imine maleate] was a gift from Dr. P. Anderson movements. In some experiments, antagonists were administered 30 (Merck. Sharn and Dohme. West Point. PA). lX3R-ACPD I(lS.3R)- min prior to lS,3R-ACPD. Data were expressed as percentage of ani- 1 -amino-cyclopentane- 1,3-hicarboxylic acidj was’obtained from To&s mals exhibiting convulsions or as number ofanimals convulsed/number Neuramin (Essex, UK). Dantrolene was purchased from Sigma Chem- of animals tested. The convulsant dose of the agonist in 50% of the ical Company (St. Louis, MO). 1R, 3S-ACPD was prepared as described animals tested (ED,,) was calculated using the median-effect plot of by Schoepp et al. (199 1b). GYKI-5246 was from the Institute for Drug Chou and Talalay (1983). Research (Budapest, Hungary). Brain injury in neonatal rats. The pharmacology of ACPD-induced injury was assessed in a well-characterized in vivo neonatal rat model (McDonald et al., 1989). Briefly, PND 7 rats received unilateral in- Results trastriatal or hippocampal stereotaxic injections of IS, 3R-ACPD (or The convulsive effects of systemic administration of lS,3R- 1R, 3S-ACPD). Injection coordinates relative to bregma were (1) stria- ACPD and lR,3S-ACPD were compared in PND 7 rats. Both turn = AP 0 mm, ML 2.5 mm, V 4.0 mm from the dura matter and (2) hippocampus = AP 3 mm, ML 2 mm, V 2.5 mm from the dura ACPD isomersproduced dose-relatedbehavioral seizures(Fig. matter. Injections were completed over a 2 min period using a 26 gauge 1). 1S, 3R-ACPD wasapproximately six times more potent than Hamilton syringe. Drugs for intracerebral injection were dissolved in lR, 3S-ACPD, with ED,, values of 16 mg/kg and 93 mg/kg, 0.01 M Tris (pH 7.4) and injection volumes were either 0.5 or 1.0 ~1 respectively. The pattern of behavioral seizuresproduced by depending on coinjection of other drugs: the severity of brain injury either isomer could not be dissociated.Both 1S, 3R-ACPD and resulting from injection of 1000 nmol of IS, 3R-ACPD did not differ between 0.5 ~1 or 1.O ~1 injection volumes. Administration ofpotentially 1R, 3S-ACPD produced exaggeration of naturally occurring neuroprotective drugs was performed either by cointrastriatal injection myoclonus, followed by lordosis and rear wagging, and then or intraperitoneal administration (10 ml vol/kg body weight). Eight to repetitive tonic-clonic limb extension. The pharmacology of 10 animals per group were used in the neuroprotective studies. Body 1S, 3R-ACPD-induced seizures was examined by pretreating temperature was maintained at normothermia (36°C surface tempera- ture) using a hovabator incubator. animals with dantrolene, an inhibitor of intracellular calcium Five days after the injection, the severity of brain injury was assessed mobilization, the NMDA receptor antagonist MK-80 1, and the by loss of injected (I) cerebral hemisphere weight relative to the con- AMPA receptor antagonist GYKI-52466. At the doses used, tralateral (C) side using the formula % damage = lOO.(C - I)lC. In eachof theseantagonists produced modestto moderate sedation this model reductions of hemisphere weights are highly correlated with as indexed by ataxic motor movements. However, among these regional reductions in ChAT activity and cross-sectional area measure- ments (McDonald et al., 1989). Statistical analysis consisted of Student’s agents,dantrolene was found to prevent selectively and com- t test for independent values. pletely the lS,3R-ACPD-induced seizures(Table 1). Morphologic examination. Morphologic examinations were made by In contrast to systemic administration, intrastriatal injection light and electron microscopy in PND 7 rats that received injections of of lS,3R-ACPD produced few signs of clonic convulsions; in- vehicle and lS,3R-ACPD into the striatum or hippocampus (as de- scribed above). At 4 hr or 5 d postinjection, rats were deeply anesthetized termittent tonic limb extension was occasionally observed. with and perfused through the left ventricle with a However, these intracerebral dosesof 1S, 3R-ACPD produced heparinized saline flush followed by a fixative containing 4% parafor- brain weight disparities indicative of brain injury. The quan- The Journal of Neuroscience, October 1993, 13(10) 4447 titative dose-effects of 1S, 3R-ACPD and 1R, 3S-ACPD are il- lustrated in Figure 2. When examined 5 d after unilateral in- trastriatal injection on PND 7, lS,3R-ACPD produced a dose-related reduction in the weight of the injected cerebral hemisphere. The lowest significant dose of IS, 3R-ACPD pro- ducing injury was 500 nmol, and maximal effect (3.4 + 0.5% damage) was observed at the 2000 nmol dose. The limit for 1S, 3R-ACPD did not allow injection of higher doses. In contrast to the effects of lS,3R-ACPD, the lR,3S-ACPD isomer did not produce significant brain injury at doses up to 1000 nmol as assessed by hemisphere weight disparities. * ,i-Ji’ fy.,..j ,,,, Light microscopy revealed injury to selective neurons in both striatum and hippocampus at 4 hr postinjection in all rats that 100 1000 10000 received 1S, 3R-ACPD (1000 nmol) injections. When compared DOSE (nmol) to normal control neurons from similar regions, injured neurons had clear, swollen perinuclear cytoplasm containing remnant Figure 2. Dose-effects of unilateral intrastriatal injection of lS,3R- ACPD or lR, 3S-ACPD on brain injury in the neonatal rat. Compounds membranous debris. In addition, nuclei were slightly shrunken were dissolved in 0.5 or 1.O ~1 of 0.0 1 M Tris (pH 7.4) and were injected and pale in affected neurons (Figs. 3, 4). Injury was of similar into the right striatum of ether-anesthetized PND 7 rats. The severity character in neurons from both striatum and hippocampus, was of brain injury was assessed 5 d later, on PND 12, by comparison of not detected in glial and ependymal cells, and was consistent cerebral hemisphere weight disparities. Data are presented as percentage loss brain weight (mean k SEM, n = 5-10 per dose) using the formula from animal to animal. lOO.(C - Z)/C, which represents the reduction in the weight of the In striatum, injured neurons were scattered throughout the injected (I) hemisphere relative to the contralateral (C) hemisphere. caudate-putamen near the injection site and were frequently located adjacent to morphologically normal neurons (Fig. 3). Injured neurons with a similar morphologic appearance were and large blebs at the plasmalemma.Dissolution and clearing also evident in other brain regions ipsilateral to the injection of cytoplasmic particulate material were evident in somecells. site, including layers 3,4, 5, and 6 of cingulate, parietal, insular, Although some nuclei were slightly swollen and pale, conden- and piriform cortex, and lateral septal nucleus. In the septum, sation and margination of chromatin, lossof normal nucleolar neuronal injury was more pronounced than in affected cortical organization, and mild nuclear membraneblebbing were char- areas, where injury was rather scattered. Injured neurons con- acteristic. Slight axonal and dendritic swellingwere evident in tralateral to the injection site were observed only in the cingulate the neuropil. cortex and lateral septal nucleus. Brain sections evaluated by The pharmacologicspecificity of 1S,3R-ACPD-induced brain light microscopy 5 d postinjection contained no morphologic injury was examined by assessingthe neuroprotective phar- abnormalities. macology of selective excitatory amino acid antagonists. Sys- In hippocampus, injured neurons were evident throughout temic administration of either the AMPA antagonist GYKI- the polymorphic, pyramidal, and molecular layers of hippocam- 52466 (20 mg/kg twice, concurrent and 1 hr after intrastriatal pal regions CAl-CA4 (Fig. 4). Based on qualitative light mi- injection) or the NMDA antagonist MK-80 1 (1 mg/kg) did not croscopy, susceptibility to the effects of lS,3R-ACPD appeared attenuate the severity of injury produced by intrastriatal injec- to be greatest in the molecular and polymorphic layers. In these tion of 1000 nmol of lS,3R-ACPD. In contrast, cointrastriatal two layers, a greater percentage of neurons were injured than in injection of dantrolene reduced lS,3R-ACPD injury by 88 & the more dense pyramidal cell layer, where only occasional neu- 7% (Fig. 6). rons were injured. Injured neurons were also evident in the dentate gyrus, but not to the extent as in the hippocampus. Other Discussion brain regions contained similarly appearing injured neurons ip- Little information regardingthe pathophysiologic rolesof meta- silateral to the injection site, including layers 3, 4, 5, and 6 of botropic excitatory amino acid receptorsin brain injury is avail- retrosplenial, frontal, parietal, and perirhinal cortex, dorsal tha- able (McDonald and Johnston, 1990; Schoepp et al., 1990a). lamic regions, and amygdala. Neuronal injury was not detected This largely reflectsthe paucity of selectivecompounds for meta- in any contralateral brain regions examined. Evaluations were botropic receptors. Using neonatal rats, we have characterized not made at 5 d postinjection in rats that received hippocampal the in vivo pharmacology of convulsions and brain injury in- injections. ducedby the selectivemetabotropic agonistIS, 3R-ACPD. When Transmission electron microscopic examination at 4 hr post- administered systemically, 1S, 3R-ACPD was six times more injection of rats that received intrastriatal injections of lS,3R- potent as a convulsant than lR,3S-ACPD. In previous work, ACPD revealed severe disruption of normal cytoplasmic or- intrastriatal injection of a nontoxic dose of lS,3R-ACPD (250 ganelles in injured neurons (Fig. 5). The selectivity of injury nmol) was found to potentiate NMDA toxicity in the neonatal noted by light microscopy was confirmed with electron mi- rat (McDonald and Schoepp, 1992). In this study, higher doses croscopy, as injured neurons were frequently adjacent to neu- of lS,3R-ACPD were examined for direct neurotoxicity. We rons that lacked morphologic alterations. In contrast to normal found that IS,3R-ACPD (2 500 nmol) produced dose-depen- control neurons from the same region, injured neurons had dent brain injury when injected intrastriatally, as indexed by swollen, electron-lucent cytoplasm containing remnant organ- hemisphericbrain weight disparities5 d postinjection. Like con- elles and membranous debris. Injured neurons also had de- vulsions, this effect of lS,3R-ACPD was also stereoselective, granulation of endoplasmic reticulum, scattered ribosomes, dis- since 1R, 3S-ACPD produced no significant brain weight losses rupted Golgi apparatus, swollen mitochondria with cristolysis, at similar doses. 4448 McDonald et al. . 1 S,SR-ACPD-induced Brain Injury in an in viva Model

The ACPD isomer potency differences observed for convul- rat (Schoepp et al., 1992b). Thus, lR,3S-ACPD may negate a sions and neurotoxicity in this study agree with observations in portion of the agonist (toxic) properties of lS,3R-ACPD by neonatal rat brain slice assays measuring receptor transduction acting as a functional antagonist. Furthermore, in our previous events such as stimulation of phosphoinositide hydrolysis study with (&)trans-ACPD significant levels of brain injury were (Schoepp et al., 199 lb). In this context, lS,3R-ACPD is at least achieved with only the highest injectable intrastriatal dose of 30 times more potent in activating metabotropic receptors com- truns-ACPD (1200 nmol) in PND 7 rats (Schoeppet al., 1991 a). pared to its affinity for NMDA receptors, and it has little affinity This degreeof injury reflects only the amount of injury expected for AMPA or kainate receptors (Schoepp et al., 199 1b; Sacaan to be induced by 600 nmol of the active lS,3R-ACPD isomer and Schoepp, 1992). Thus, the stereoselectivity of effects in this that is present in a 1200 nmol doseof truns-ACPD. study suggests that the convulsant and neurotoxic effects of The lack of (*)tvuns-ACPD injury in cultured neurons (Koh 1S, 3R-ACPD are likely due to the selective activation of meta- et al., 1991) may reflect the lack of functional or critical masses botropic glutamate receptors in the neonatal rat brain. of interneuronal connections that are present in situ. Further- Histological examination of 1S, 3R-ACPD-injected rat brains more, a family of metabotropic receptors have been cloned confirmed the neurotoxic effects of this compound. This neu- (Houamed et al., 1991; Masu et al., 1991; Tanabe et al., 1992) ronal injury was characterized by select populations of swollen which are coupled to multiple transduction mechanismsin- neurons with shrunken nuclei at 4 hr postinjection, but no in- cluding phosphoinositide hydrolysis, stimulation of CAMP for- jured neurons could be found 5 d later. These data indicate that, mation, inhibition of CAMP formation, and the releaseof ar- similar to lesions produced by ionotropic glutamate agonists achidonic acid. (+)Truns-ACPD activates the subtypes of (McDonald et al., 1989), the brain weight disparities produced metabotropic glutamate receptors coupled to all of thesetrans- by 1S, 3R-ACPD likely reflect earlier selective loss of neurons duction mechanisms.Therefore, it is possiblethat specificmeta- that manifests as reduced tissue volume 5 d later. However, botropic glutamate-receptor effects, such as the neuronal injury lS,3R-ACPD injury differs qualitatively from the injury pro- demonstratedin our study, are linked to a specificmetabotropic duced by direct injection of ionotropic glutamate receptor ag- glutamate receptor subtype. This heterogeneitymay explain why onists such as NMDA. For example, in the neonatal rat and certain tissuesor in vitro preparations do not appearto express mouse species NMDA produces acute changes in neurons that metabotropic glutamate receptor-mediatedneuronal injury. The characteristically include edematous cell swelling, nuclear pyk- lack of compounds selective for the metabotropic glutamate nosis, dark cell degeneration, and massive distention of den- receptor subtypesmakes it difficult to determine the contribu- drosomal regions that spares axons (Olney, 1978; Ikonomidou tion of eachmetabotropic glutamate receptor subtype to 1S, 3R- et al., 1989). Also evident in excitotoxic damage following ACPD-mediated brain injury in vivo. NMDA are acute changes in glial and ependymal cells (Olney, In addition to the direct toxicity of IS, 3R-ACPD shownhere, 197 1). In the present study, acute changes at 4 hr postinjection a lower subtoxic dose of lS,3R-ACPD (250 nmol) selectively were limited to selective neuronal swelling without evidence of potentiated NMDA-mediated (65% enhancement) but not dark cell degeneration or dendrosomal involvement typical of AMPA-mediated brain injury when injected intrastriatally in excitotoxic damage. In addition, the lack of visible damage to neonatalrats (McDonald and Schoepp,1992). Like direct 1S, 3R- glial or ependymal cells provides further support for the qual- ACPD toxicity, this effect wasstereoselective since 1 R,3S-ACPD itative difference between lS,3R-ACPD injury and excitotoxic did not potentiate NMDA toxicity. Thus, mechanismsalso exist injury typical of ionotropic glutamate receptor agonists. for a synergistic interaction between lS,3R-ACPD-sensitive Two previous studies using trans-ACPD suggested that me- metabotropic receptors and NMDA receptors,and thesemech- tabotropic receptor activation did not produce neuronal injury anisms play a prominent role in neonatal brain injury. It has in neocortical cultures (Koh et al., 199 1) or when injected in- been shown that depolarizing responsesto NMDA, but not trastriatally in rats (Schoepp et al., 199 la). These observations AMPA, are selectively potentiated by 1S, 3R-ACPD in areaCA 1 may be peculiar to the use of (+)truns-ACPD. (&)Truns-ACPD ofrat hippocampus(Aniksztein et al., 1991; Harvey et al., 1991). is a racemic mixture of 1S, 3R-ACPD and 1R, 3S-ACPD. 1S, 3R- Thus, potentiation of NMDA responsesmay have important ACPD is the active agonistwhile 1R, 3S-ACPD is a weak partial implications for synaptic plasticity and neuropathology of acute agonist of metabotropic-stimulated phosphoinositide hydroly- and chronic neurologic disorders. However, in this study rela- sis (Schoeppet al., 1991 b), which can attenuate receptor acti- tively higher dosesof lS,3R-ACPD induced brain injury that vation by the full agonist lS,3R-ACPD (Schoeppet al., 1992b). was not altered by concurrent treatment with the selective ion- This apparentantagonist or partial agonisteffect of 1R,3S-ACPD otropic antagonists MK-801 or GYKI-52466. Direct lS,3R- has beenobserved when measuring1 S, 3R-ACPD-induced con- ACPD injury and convulsions following systemic IS, 3R-ACPD tralateral turning, a behavioral consequenceof activating striatal wereblocked by dantrolene, which hasbeen found to antagonize metabotropic glutamatereceptors with 1S, 3R-ACPD in the adult intracellular calcium mobilization in various neuronaland non-

Figure 3. Histologicneuronal injury inducedby lS,3R-ACPD after injectioninto neonatalrat striatum.A, Vehicle-injectedcontrol rat. Neurons are morphologically normal. B, lS,3R-ACPlXnjected rat. Selectively injured neurons have swollen perinuclear cytoplasm containing membranous debris and slightly shrunken, pale nuclei (arrows). Epoxy embedding, toluidine blue stain. Magnification, 320 x . Figure 4. Histologic neuronal injury induced by lS,3R-ACPD after injection into neonatal rat hippocampus. A, Vehicle-injected control rat. Hippocampus is morphologically normal. B, lS,3R-ACPD-injected rat. Injured neurons with swollen cytoplasm (arrows) appear throughout much of the hippocampus, especially in the polymorphic (PO) and molecular (M) layer. C, Detail of normal control hippocampus. D, Detail of swollen cytoplasmand pale nuclei characteristic of lS,3R-ACPD-inducedneuronal injury (arrows). Note the relatively few injuredcells in the pyramidal cell layer (PY). A and B, paraffin embedding, HE stain; C and D, epoxy embedding, toluidine blue stain. Magnification: A and B, 80 x ; C and D, 320x.

4452 McDonald et al. l 1 S,3R-ACPD-induced Brain Injury in an in viva Model

Figure 5. Transmission electron microscopic neuronal injury induced by lS,3R-ACPD after injection into neonatal rat striatum. A, Vehicle- injected control rat. Neuron is morphologically normal. Note cytoplasm (C), nucleus (N), and nucleolus (MI). B, 1S, 3R-ACPlXnjected rat. Note swollen, electron-lucent cytoplasm containing remnant mitochondria (M), membranous debris (0). plasmalemmal blebs (B), and clear areas (C). The Journal of Neuroscience, October 1993, 13(10) 4453

The nucleus has marginated chromatin (CH) and an abnormal nucleolus (NV). Adjacent neurons are morphologically normal. Magnification, 12,000 x . 4454 McDonald et al. - 1 S,SR-ACPD-induced Brain Injury in an in vivo Model

Desai MA, Corm PJ (1990) Selective activation of phosphoinositide hydrolysis by a rigid analogue of glutamate. Neurosci Lett 109: 157- 162. Frandsen A, Schousboe A (1992) Mobilization of dantrolene-sensitive intracellular calcium pools is involved in the cytotoxicity induced by quisqualate and N-methyl-D-aspartate but not by 2-amino-3-(3-hy- droxy-5-methylisoxazol-4-yl)propionate and kainate in cultured ce- rebral cortical neurons. Proc Nat1 Acad Sci USA 89:2590-2594. Harvey J, Frenguelli BG, Sunter DC, Watkins JC, Collingridge GL (1991) The actions of lS,3R-ACPD, a glutamate metabotropic re- ceptor agonist, in the area CA1 of the rat hippocampus. Br J Phar- macol 104:75. Houamed KM, Kuijper JL, Gilbert TL, Haldeman BA, O’Hara PJ, Mulvihill ER, Almers W, Hagen FS (199 1) Cloning, expression, and gene structure of a G protein

functional characteristics of metabotropic excitatory amino acid re- Schoepp DD, Johnson BG, Sacaan AI, True RA, Monn JA (1992b) ceptors. Trends Pharmacol Sci 11:508-5 15. In vitro and in vivo pharmacology of lS,3R- and lR,SS-ACPD: ev- Schoepp DD, Gamble AY, Salhoff CR, Johnson BG, Omstein PL idence for a role of metabotropic glutamate receptors in striatal motor (1990b) Excitatory amino acid-induced convulsions in neonatal rats function. Mol Neuropharmacol 2:33-37. mediated by distinct receptor subtypes. Eur J Pharmacol 182:421- Sladeczek F, Pin JP, Recasens M, Bockaert J, Weiss S (1985) Gluta- 421. mate stimulates inositol phosphate formation in striatal neurons. Na- Schoepp DD, Johnson BG, Salhoff CR, McDonald JW, Johnston MV ture 317:717-719. (199 la) In vitro and in vivo pharmacology of tram- and cis-ACPD: Sugiyama HI, Ito I, Hirono C (1987) A new type of glutamate receptor dissociation of metabotropic and ionotropic excitatory amino acid linked to inositol phospholipid metabolism. Nature 325:531-533. receptoreffects. J Neurochem 56: 1789-l 796. Tanabe Y, Masu M, Ishii T, Shigemoto R, Nakanishi S (1992) A Schoepp DD, Johnson BG, True RA, Monn JA (199 1b) Comparison family of metabotropic glutamate receptors. Neuron 8: 169-l 79. of (1s. 3R )- 1-aminocyclopentane- 1,3-dicarboxylic acid (1 S, 3R- Ward A, Chaffman MO, Sorkin EM (1986) Dantrolene. A review of ACPD)- and 1R, 3S-ACPD-stimulated brain phosphoinositide hy- its pharmacodynamic and pharmacokinetic properties and therapeu- drolysis. Eur J Pharmacol [Mol Pharmacol] 207:35 l-353. tic use in malignant hyperthermia, the neuroleptic malignant syn- Schoepp DD, Johnson BG, Monn JA (1992a) Inhibition of cyclic AMP drome and an update of its use in muscle spasticity. Drugs 32: 130- formation by a selective metabotropic glutamate antagonist. J Neu- 168. rochem 58:1184-1186.