Activation of hippocampal metabotropic excitatory amino acid receptors leads to seizures and neuronal damage

Ncur,;science Letters. 139 (1992) 77 82 '~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92;'S 05.00

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NSL 08597

Activation of hippocampal metabotropic excitatory amino acid receptors leads to seizures and neuronal damage Aida I. Sacaan and Darryle D. Schoepp Lilly Research l.al~oralori~'x, Eli Lilly and Com/~any Lilh' ('orporotc ('elliot, hutianapolis, 13; 462~'~ ~ ~ X..I ; (Reccived 3 .lanuary 1992: Revised version received 4 February 1992: Accepted 5 Februarx 1992 Aey words.

t{xcitalory amino a c i d Glutamate: Metabotropie: Neurotoxicity: ttippocampus: Seizure; I-Aminoc~ch)pentnne-1,3 dicarboxylic acid: N M I)/\

A role t\3r ionotropic ( N M D A , AMPA, and kainate) excitatory amino acid (EAA) receptors in seizure and seizure-related brain damage is well documented. To study the possible role of metabotropic (G-protein linked} EAA receptors in this regard, a highly selective metabotropic EAA agonist was injected into the hippocampus of halothane-anesthetized rats. This resulted in delayed-onset seizures and sclective hippocampal neuronal damage that was indircctly mediated by N M D A receptors. This provides direct evidence t\w a noxel role of metabotropic EAA receptors in the etiology, of seizures and lleuronal damage.

Studies of excitatory amino acid (EAA) neurotransmission have t'0cused primarily on the receptor subtypes that are directly linked to iota channels. The role that these receptors ~N-methyl-D-aspartate (NMDA), kainate (KA) and AMPA) play in epilepsy, seizure-induced brain damage, and glutamate excitotoxicity is well established [2, 3, 7, 8]. Metabotropic receptors are another major EAA receptor subtype which are G-protein linked to the enhanced hydrolysis of cellular phosphoinositides [16]. However, the physiological and/or pathological role of the metabotropic EAA receptor is not well characterized. (1S,3R) I-aminocyclopentane-1,3-dicarboxylicacid ( 1S,3RACPD) has been recently introduced as a highly selective metabotropic EAA receptor agonist [4, 17], which is a useful agent to probe the consequences of activating metabotropic EAA receptors in vitro and in vivo [15, 16]. The rat hippocampal region has one of the highest levels of metabotropic EAA receptor density and coupling [16]. In this study hippocampal metabotropic EAA receptor activation was investigated in vivo by injecting IS.3R-ACPD in the dorsal rat hippocampus. Male Sprague-Dawley rats (250 300 g, Charles Rivet" Labs., Inc., Wilnfington, MA) were anesthetized with halothane and placed in a small animal stereotaxic apparatus. A 28-gauge cannula was introduced into the left dorsal hippocampus through a bun'-hole in the skull. Injection Co;'resl~omh';ic{' D.D. Schoepp. CNS Research, Eli Lilly and Con> pany. Indianapolis. IN 46285, USA. Fax: (I] (317) 276-5546.

coordinates according to system A in the atlas of Pellegrino et al. [12] were (bregma as reference): 2.8 mm anterior, +2.2 mm lateral: and --2.5 turn ventral. Compounds t'o1"injection were dissolved in normal saline then neutralized with 5 N N a O H to pH 7 ~;. Injection was 2/,,'1 in 2 rain, the cannula was left in placc for an additional 5 rain and then withdrawn slowly. The animals were su-

TABLE 1 INCIDENCE A N D ONSET TIME OF LIMBI(.' SEIZURES F()LLOWING INTRAHIPPOCAMPAL INJECI'ION OF" IS,3R-A('I'D Rats were anesthetized with halothanc and placed in a small ammal stereotaxic apparatus. IS.3R-ACPD (1 ,umol in 2 ,ul) or 0.9c4 NaC1 vehicle (2 ul) were infused in the left dorsal hippocampus over a 2 min period. The N M D A receptor antagonist 1.Y274614 was gixen in two equal doses (21} mg,kg i.p.) at 0.5 h before and 6 13after thc infusion of 1S.3 R-ACPD.

Treatment

Controls Vehicle (2 pl) 1S.3R-ACPD(I//mol) LY274614 1S.3R-A('PD(1;umol)

Number of animals seizing:number of aninlals tested

Seizure onset train: mean ' S,E.M.}

(}/6 7/8*

366 " 72.2

1:6

536

*P<0.05 when compared the vehicle (2 ,ul). Fishers exact probability test.

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Fig. 1. Dose-related neuronal damage induced by the intrahippocampal injection of 1S,3R-ACPD. Halothane anesthetized rats were injected with different doses of IS,3R-ACPD in a 2ul volume into the left dorsal hippocampus. Six to 7 days later, frozen coronal brain sections were prepared and stained with Cresyl violet. A,B: 0.25 ¢tmol 1S,3R-ACPD: C,D:0.5 ,umol tS,3R-ACPD: E,F: I ~mol IS,3R-ACPD. Magnification for A,C,E 6>,: B.D,F: 12×.

tured with w o u n d clips and placed in bedded plastic cages while recovering from anesthesia. Following the injection o f either saline vehicle (2/~1) or 1 S , 3 R - A C P D (1 /~mol/2/11) into the left hippocampus, the rats were ob-

served continuously for 12-h by multiple observers blinded to the treatments. Intrahippocampal lS,3R-ACPD-injection in rats p r o d u c e d a highly reproducible, but delayed seizure-related behavioral syndrome. This effect was never ob-

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Fig. 2. Representative Cresyl violet-stained coronal brain sections of rats that were injected with normal saline vehicle, 1R,3S-ACPD, and N M DA. Halothane anesthetized rats were given unilateral (left hemisphere) intrahippocampal injections in a 2//1 volume, Six to 7 days later, frozen brain sections were prepared and stained with Cresyl violet. A,B: saline vehicle: C,D: I pmol 1R,3S-ACPD: E.F: 60 nmol NMI)A. Magnilication for A,C,E 6×: B.D,F-12x.

served following the i n t r a h i p p o c a m p a l injection o f saline vehicle (Table 1). A t the early times p o s t - i n j e c t i o n o f IS,3R-ACPD, behavioral observations included a k i n e s i a and multiple episodes o f wet d o g shakes that

b e g a n within t w o - h o u r s post injection and lasted t h r o u g h o u t the 12-h o b s e r v a t i o n period. At later times a n i m a l s exhibited h i n d - l i m b rearing, j a w twitching, and m o d e r a t e to sevcre limbic-seizures (see Table I). Limbic

80 5 (0

=.=_ om o m..o u.<

z, -r. 03 0 10 ~M NMDA

3 mM 1R,3S-ACPD

3raM 1S,3R-ACPD

Fig. 3. Effect of NMDA. 1R.3S and IS.3R-ACPD on [3H]norepinephrine (NE) release from hippocampal slices. Cross-chopped hippocampal slices were prelabelledwith [3H]NEin the presence of pargyline (10 ~M) and ascorbic acid (0.6 mM t Tissue was then transferred to chambers (Brandel Superfusion System. series B-18Iin which the tissue was supported on Whatman GF-B filter discs and superfused with oxygenated Krebs buffer containing (m mM): NaC[ 119.5. KC1 3.3. CaCI 3.3. KH_,PO4 1.2. EDTA 0.03. and glucose I1.0). After two baseline fractions has been collected, the superfusion buffer was switched to one containing the givenconcentration of test drug. At the end of the experiment the filter and the tissue were removed and the [~H]NE was estimated after extraction on scintillation fluor. The fractional efflux of tritmm was estimated as the amount of radioactivity in the superfusate fraction relative to the total amount in the tissue at that particular point in time. multiplied by 100. Values are mean±S.E.M. (n-3).

smzures in treated animals were defined by the presence of at least one episode of clonic forelimb contracuons followed by rearing, and in most cases falling. These delayed episodes of limbic seizures that occur following a single intrahippocampal injection of 1S. 3R-ACPD are qualitatively similar to those which can be produced by kindling animals electrically to a stage 4 or stage 5 seizure [13]. To determine if 1S,3R-ACPD-induced seizures were associated with neuronal damage, histological examination of the injected hippocampus was performed 6 7 days following 1S,3R-ACPD injection. Brains were frozen quickly on dry ice, then 30/am coronal sections were obtained by using a freezing microtome ( 15 °C) and were mounted on poly-L-lysine-coated slides. The slides were dried and stained with Cresyl violet according to the method of Kluver and Barrera [5]. In contrast to saline vehicle-injected rats (Fig. 2A,B), I S,3R-ACPD injected rats (1 #tool/2 #1) exhibited severe loss of CA t and CA4 pyramidal neurons and dentate gyrus granule neurons (DG), while area CA3 pyramidal neurons were spared (Fig. I E,F). This effect of I S,3R-ACPD was concentration-dependent. Thinning of the dentate granule

cell layer was observed with as little as 0.25 #tool of 1S,3R-ACPD (Fig. IA,B), and 0.5/,mlol IS,3R-ACPD produced more marked damage (Fig. IC,D). In the 7 out of 8 animals receiving I #tool of IS,3R-ACPD, severe (virtually complete) loss of C A I , CA4, and dentate neurons were tbund. One of 8 animals which did not exhibit a seizure episode following 1S,3R-ACPD (see Table I) had little to no loss of C A I , CA4, and dentate neurons. The neuronal damage produced by I S,3R- A C P D was stereoselective. A 1 #tool dose of 1R,3S- ACPD, which is a weak and less selective partial agonist at the metabotropic EAA receptor [17], produced little damage alter hippocampal injection (Fig. 2C,D). The pattern of damage produced by intrahippocampal injection of 1S,3R-ACPD is very similar t~) that produced by N M D A (see Fig. 2E,F). However, we found no evidence that 1S,3R-ACPD can directly activate N M D A receptors. In N M D A receptor binding experiments in rat brain membranes using [3H]CGS19755 as ligand [9], the 1S,3R-ACPD used in this study (Lot 2, Tocris Neuramin, Essex, U K ) had no measurable affinity for the glutamate recognition site on the N M D A receptor (IC50>1 mM). Moreover, when examined Ior ionotropic EAA receptor agonist activity by measurement of neurotransmitter release in slices of the rat hippocampus [14], up to 3mM of 1S,3R-ACPD failed to significantly increase the release of [3H]norepinephrine (Fig. 3). In contrast, N M D A (10t.tM)and I R,3S-ACPD (3 mM), the A C P D isomer which was less potent in vivo in inducing neuronal damage (Fig. 2('.I)), each can directly activate N M D A receptors as indicated by the significant increase in [3H]norepinephrine efflux above baseline (Fig. 3). In addition, qualitative differences between N M D A - and 1S,3R-ACPD-induced seizures were observed. As others have reported [20], the mtrahippocampal injection of N M D A (60 nmol) produced a lhstonset clonic seizure (15-30 min post-injection) which was preceeded by a state of hyperlocomotion. In contrast, 1S,3R-ACPD produced a delayed-onset limbic seizure (average of 6 h post-injection) which was associated with episodes of wet dog shakes and rearing. These observations all support the conclusion that seizures and neuronal damage following the injection of 1S,3R-ACPD in the hippocampus was initiated by the direct activation of the metabotropic EAA receptor subtype. The direct activation of the muscarinic cholinergic receptor, another G-protein phosphoinositide-linked or 'metabotropic' receptor can also lead to seizures and seizure-related neuronal damage to the hippocampus [10]. Muscarinic agonist-induced seizures and the subsequent damage that ensues can be blocked by N M D A receptor antagonists [11, 19]. To determine if seizures and hippocampal damage induced by 1S.3R-ACPD indirectly

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Fig. 4. Protection of IS,3R-ACPD-induced hippocampal damage by the administration of an N M D A receptor antagonist, LY274614. Halothane anesthetized rats werc injected with I S,3R-ACPD (I ,umol in 2#1) into the left dorsal hippocampus. Animals ~elc given drug vehicle (normal saline, ml/kg) or LY274614 (2×20 mg/kg, i.p.) at 0.5 h before and 6 h after IS.3f~.-A('PD. Six to 7 days later, fi'ozen coronal brain sections were prepared and stained with Crcsyl violet. A,B: control animal: C.D: kY274614-treatcd animal. Magnilication Ik~r A.('::8×: B,D I(~×.

involves N M D A receptors, rats were given systemic administration of either drug vehicle (controls) or the systemically active competitive N M DA receptor antagonist LY274614 [18] in conjunction with infusion of 1 /amole 1S,3R-ACPD into the left hippocampus. LY274614 (20 mg/kg, i.p) was given 30 min prior to and 6 h after the intrahippocampal injection of IS,3RACPD. Treatment with LY274614 prevented limbic scizures during the 12 h observation period in 5 of 6 animals, and produced complete protection of IS,3RACPD-induced hippocampal neuronal damage in 4 out of 6 animals (see Table 1. Fig. 4). In two animals LY274614 produced partial protection flom IS,3RACPD, as indicated by protection of CA I pyramidal neurons. However, in these two animals there was still substantial loss of the dentate granule cells (data not shown). This suggests that subsequent to the acute activation of the metabotropic EAA receptor there is enhanced NM DA receptor function that leads to excitotoxicity, presumably via endogenous ghltamatc. When t.-2-

anlino-3-phosphonopropionic acid (L-AP3, 1 #tool), a partial agonist/antagonist of the metabotropic receptor [16] was coinjecled with 1 umol IS,3R-ACPD, we observed a supression of seizures (data not shown). However, it ,aas not possible to assess I S.3R-ACPD-induced damage since t-AP3 alone produced severe non-selective danlage of the entire hippocampus and adjacent areas, with an intense glial reaction. The delayed nature of the I S,3R-ACPD effect suggests the involvement of neuronal plasticity in this phenomenon. The acute actiwltion of the nletabotropic EAA receptors may establish a neuronal network which leads to the spatial spread o[" seizures and neuronal damage. Recently. 1S,3R-ACPD was shown to selectively enhance N M D A currents in CA1 neurons in a time-dependent manner [1]. The circuitry of the hippocampus, namely glutamatergic synapses on glutamatergic neurons, may also be an important factor in the delayed nlanifestation of excitotoxiciiy by metabotropic EAA receptor activation. In the rat striatum where glutamate

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neurons innervate non-glutamatergic neurons, the in vivo activation of metabotropic EAA receptors does not lead to neuronal damage [15]. Furthermore, in cultured neurons which would not possess in situ neuronal circuitry, metabotropic EAA receptor activation is not associated with neurotoxicity [6]. In summary, the in vivo activation of hippocampal metabotropic EAA receptors with 1S,3R-ACPD leads to delayed limbic seizures and selective neuronal damage that manifests through enhanced N M D A receptor activation. This demonstrates a novel cellular mechanism for the in vivo induction of N M D A receptor- mediated brain damage and possibly brain epileptogenesis. I Aniksztejn, L., Bregestovski, E and Ben-Art. Y., Selective activation of quisqualate metabotropic receptor potentiates NMDA but not AMPA responses, Eur. J. Pharmacot,, 205 (1991) 327-328. 2 Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623-634. 3 Dingledine, R., McBain, C.J. and McNamara, J.O., Excitatory amino acid receptors in epilepsy, Trends Pharmacol. Sci., 11 (1991), 334-340. 4 Irving, A.J., Schofield, J.G., Watkins, J.C., Sunter, D.C. and Collingridge, G.L,, IS,3R-ACPD stimulates and L-AP3 blocks Ca > mobilization in rat cerebellar neurons, Eur J. Pharmacol., 186 (1990) 363-365. 5 Kluver, H. and Barrera, E., Cresyl fast violet stain. In E. Labossiere (Edn.), Histological Processing for the Neural Sciences, Charles A. Thomas, Springfield, 1976, p. 39. 6 Koh, J.-y., Palmer E., Lin, A. and Cotman, C,W., A metabotropic glutamate receptor agonist does not mediate neuronal degeneration in cortical culture, Brain Res., 561 (1991) 338-343. 7 Lambert, J.D.C., Jones, R.S.G., Andreasen, M., Jensen, M.S. and Heinemann, U., The role of excitatory amino acids in synaptic transmission in the hippocampus, Comp. Biochem. Physiol., 93A (1989) 195-201. 8 Meldrum, B. and Garthwaite, J., Excitatory amino acid neurotoxicity and neurodegenerative disease, Trends Pharmacol. Sci., 11 (1990), 379-387.

9 Murphy, D.E., Hutchison, A.J., Hurt, S.D., Williams, M., and Sills, M.A., Characterization of the binding of [~H]CGS-19755: a novel N-methyl-D-aspartate receptor antagonist with nanomolar affinity in rat brain, Br. J. Pharmacol., 95, 932-938. 10 Olney, J.W., de Gubareff, T. and Labruyere, J.. Seizure-related brain damage induced by cholinergic agents. Nature, 30t (19831 520 522. II Ormandy, G.C,, Jope, R.S. and Snead, 0.(2". Ill, Anticonvulsant actions of MK-801 on lithium-pilocarpine model of status epilepticus in rats, Exp. Neurol., 106 (1989) 172--18(I. 12 Pellegrino, L.J., Pellegrino A.S. and Cushman, A.J.. 2nd edn., Plenum, New York, 1979. 13 Racinc, R.J., Modification of seizure activity by electrical stimulation: ll. Motor seizure, Electroencephalogr. Clin. Neurophysiol.. 32 (1972) 281 294. 14 Sacaan. A.I. and Johnson, K.M. , Spermidine reverses arcaine's inhibition of N-methyl-D-asparate-induced hippocampal [~H]norepinephrine release, J. Pharmacol. Exp. Thcr., 255 (1991). 11)601063. 15 Sacaan, A.l,, Monn, J.A. and Schoepp. D.D,. lntrastriatal injection of a selective metabotropic excitatory amino acid receptor agonist induces contralateral turning in the rat, J. Pharmacol. Exp+ Ther., 259 (1991) 1366-1370. 16 Schoepp, D.D., Bockaert, J. and Sladeczek, F., Pharmacological and functional characteristics of metabotropic excitatory amino acid receptors, Trends Pharmacol. Sci., 11 (1990) 508 515. 17 Schoepp. D.D., Johnson, B.G., True, R.A. and Monm J.A., Comparison of (1S,3R)-l-aminocyclopentane-|,3-dicarboxylic acid ( I S,3R-ACPD)- and 1R,3S-ACPD-stimulated brain phosphoinositide hydrolysis, Eur. J. Pharmacol.-Mol. Pharmacol, Section, 207 (1991)351 353. 18 Schoepp. D.D.. Ornstem, P.l_.., Salhoff. C R . and Leander, J.D.. Neuroprotectant effects of LY274614, a structurally novel systemically active competitive NMDA reccplor antagonist..1 Neural Transm, 85(1991) 131 143. 19 Turski, L., Cavalheiro, E.A., Turski, W.A. and Meldrum, B.S., Excitatory neurotransmission within subs/antra nigra pars reticulala regulates threshold t\)r seizures produced by pilocarpine in rats: effects of intranigral 2-amino-7-phosphonoheptanoate and 3,methyl-l>aspartate, Neuroscience, 18 (19861 6! 77, 20 Zaczek, R. and Coyle, J.T.. Excitatory amino acid analogues: neurntoxicity and seizures, Neuropharnracology. 21 ~1~82) ! 5 26.