Brain Research, 526 (1990) 165-168
165
Elsevier BRES 24243
The selective ionotropic-type quisqualate receptor agonist AMPA is a potent neurotoxin in immature rat brain John W. McDonald 1, William H. Trescher 1 and Michael V. J o h n s t o n 1'2 Departments of INeurology and 2pediatrics, the Johns Hopkins University School of Medicine and the Kennedy Research Institute, Baltimore, MD (U.S.A.)
(Accepted 8 May 1990) Key words: Excitotoxicity;Neurotoxicity; Glutamate; Excitatory amino acid; Rat; Development; Quisqualate;
a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid
Direct unilateral intrastriatal stereotaxic injections of the selective ionotropic-type quisqualate receptor agonist, AMPA, in postnatal day 7 rats produced tonic-clonic seizure activity. Quantitative analysis of the severity of brain injury was assessed by comparison of the disparities in the weights of injected and contralateral cerebral hemispheres 3 days after the excitotoxin injection. The amount of AMPA that produced half-maximal brain injury was 9.5 nmol as assessed by comparison of disparities in cerebral hemisphere weights. In contrast, quisqualate was 26 times less potent. The marked susceptibility of the developing rat brain to AMPA toxicity may provide a useful model to assess the neuroprotective effectiveness and selectivity of ionotropic quisqualate receptor antagonists.
The excitotoxic actions of excitatory amino acids (EAA) are manifested by activation of subclasses of E A A receptors named for their preferential agonists, N-methyI-D-aspartate (NMDA), quisqualate (QUIS), and kainate 1b'ls'23. The mechanisms of NMDA receptormediated brain injury have been the most extensively studied, whereas non-NMDA receptor-mediated injury is less well characterized. Subtypes of quisqualate receptors have been delineated on the basis of their linkage to an ionophore (ionotropic receptor) or phospholipase C (metabotropic receptor). Agonists of both subtypes of receptors are capable of producing excitotoxic neuronal injury in the immature brain 2'7'24. In this study, we compared the excitotoxic characteristics of quisqualate 22, which acts at both receptor subtypes, and the selective ionotropic quisqualate receptor agonist, AMPA ((RS)a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), in a well-characterized in vivo perinatal rat model of excitotoxic brain injury 15'16A9. Intrastriatal injections of AMPA (5 doses; 2.5, 5, 10, 25, 50 nmol; n = 6-8/dose) and QUIS (4 doses; 50, 100, 150, 200 nmol; n = 7-8/dose) were performed in 7-day-old Sprague-Dawley albino rats briefly anesthetized with ether as previously described in detail 16. With reference to bregma, injection coordinates were AP = 0 mm, L = 2 mm, depth from dura = 4 mm. Drugs were dissolved in 0.01 M Tris base (final pH 7.4) and injection
volumes were 0.5/~1 (AMPA) and 1.0/~1 (QUIS). For quantitative analysis, all animals were sacrificed 3 days after excitotoxin injection and the severity of brain injury (% damage) was assessed by comparison of the dry weights of the injected (I) and contralateral (C) cerebral hemispheres using the formula 100x (C-I)/C = % damage 16. Additional animals were sacrificed 5 days after injection and brains were processed for routine light microscopic histoiogic analysis. We have previously demonstrated that comparisons of cerebral hemisphere weights is an accurate and sensitive measure of excitotoxic brain injury in this model 16. Values presented are means _+ S.E.M. ECso values were calculated by the method of Litchfield and Wilcoxon 9. AMPA and QUIS (synthetic) were obtained from Cambridge Research Biochemicals (Cambridge, England). Unilateral intrastriatal injection of QUIS or AMPA in PND 7 rats produced acute tonic-clonic seizure activity and consistently resulted in extensive, dose-dependent, unilateral brain injury that was most predominant in the corpus striatum, overlying neocortex, dorsal hippocampus, amygdala, thalamus and fornix when examined 5 days later (Fig. 1). Tissue damage was usually confined to the injected hemisphere although doses of AMPA 10 nmol or greater produced microscopic damage in the contralateral hemisphere with a propensity to selectively damage the contralateral fornix. As previously described,
Correspondence: M.V. Johnston, Neuroscience Laboratory, Kennedy Research Institute, Room 506, 707 N. Broadway, Baltimore, MD 21205,
U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166 a similar injection of vehicle in PND 7 rats produced few signs of neurotoxicity is-17. Quantitative analysis of the severity of resulting brain injury by comparison of the weights of the injected and contralateral cerebral hemispheres indicated that the excitotoxic effects of QUIS and AMPA were dosedependent (Table I). Based on the doses required to produce 16% of maximal injury (ED16), A M P A was approximately 26 times more potent as a neurotoxin than QUIS. ED16 values were 4.5 nmol (AMPA) and 118 nmol (QUIS). The EDso value for AMPA was 9.5 nmol with a 95% confidence interval of 5.6-16.1 nmol. Direct determination of the corresponding value for QUIS was not possible because of the limited solubility of QUIS but the estimated EDso value for QUIS was 250.0 nmol with a 95% confidence interval ranging from 146.3 to 427.2
/
nmol. Intrastriatal injections of QUIS did not alter the dry weights of the contralateral hemisphere compared to vehicle-injected controls (dry weight of contralateral cerebral hemisphere (mean + S.E.M.): 50 nmol, 52.1 +_ 0.5 mg; 100 nmol, 52.7 _+ 1.1 mg; 150 nmol, 52.0 _+ 1.4 mg: 200 nmol, 51.7 _+ 0.8 mg; vehicle, 53.1 + 0.7 rag). In contrast, intrastriatal injections of A M P A significantly reduced the size of the contralateral cerebral hemisphere (dry weight of contralateral cerebral hemisphere (mean + S.E.M.); 2.5 nmol, 54.2 _+ 0.6 mg; 5 nmol, 53.9 + 0.5 rag; 10 nmol, 47.9 + 1.6 mg; 25 nmol, 39.6 + 0.9 mg; 50 nmol, 39.7 _+ 1.1 mg; vs vehicle, 53.1 + 0.7 mg: P < 0.01, l-way ANOVA). These results extend the earlier observations that quisqualate and A M P A are neurotoxic in the immature brain 2'7'24, and demonstrate that A M P A , a selective
C
QUIS
50nmol
2ram
25nmol
Fig. 1. Comparison of the histopathologic characteristics of quisqualate (A,B) and AMPA (C,D) induced brain injury in PND 12 rats. Quisqualate (50 nmol/I pi) and AMPA (25 nmol/0.5 ~l) were stereotaxically microinjected into the right anterior striatum on PND 7. Animals were sacrificed 5 days later on PND 12.50 pm frozen sections were cut and stained for Nissl substance. In contrast to quisqualate, intrastriatal injection of half as much AMPA produced extensive brain injury. The AMPA induced lesion is characterized by confluent necrosis involving the entire cerebral hemisphere at the level of the striatum (C). The damage extended several mm caudal to the injection site with marked injury to the dorsal hippocampus and surrounding neocortex. Bar = 2 mm.
167 TABLE I Severity of brain injury produced by intrastriatal injections of AMPA and Q UIS
%Damage reflects the severity of injury in the injected (I) cerebral hemisphere relative to the contralateral (C) hemisphere and was calculated using cerebral hemisphere weights (dry) with the formula; %Damage = 100(C-I)/C. Excitotoxin
Dose (nmol)
n
% Damage (mean + S.E.M.)
Quisqualate
50 100 150 200
8 7 8 7
-0.1 + 0.9 3.1 + 0.6 5.7 + 1.1 7.3 + 0.9
8 8 8 7 6
1.2+0.6 3.6+ 1.1 8.4 + 0.9 20.9 + 2.6 21.5 + 1.3
AMPA
2.5 5 10 25 50
ionotropic quisqualate receptor agonist is a potent excitotoxin in PND 7 rat brain when administered intracerebrally. AMPA is approximately 26 times more potent than QUIS as an excitotoxin in this model. AMPA and QUIS toxicity does not appear to simply result indirectly from seizure activity since unilateral intrastriatal injection of these excitotoxins produces bihemispheric, high voltage, high frequency epileptiform discharges but only unilateral brain injurylL The reduction in the weight of the cerebral hemisphere contralateral to the injected hemisphere observed in animals that received higher doses of AMPA (25 nmol, 50 nmol) reflects, in part, microscopic injury to the contralateral hemisphere and growth retardation of the cerebral hemisphere during the 3-day survival period. Because the method of quantifying the severity of brain injury relates the degree of injury to the contralateral non-injected hemisphere, the reduction in the weight of the contralateral hemisphere may bias the apparent calculated degree of injury such that it will be slightly underestimated at the highest doses of A M P A examined. The regional pattern of susceptibility to AMPA and QUIS toxicity corresponds roughly to the distribution of QUIS receptors in brain 3's'18. The pattern of selective vulnerability is similar, but not identical, to the pattern of 1 Campochiaro, P. and Coyle, J.T., Ontogenic development of kainate neurotoxicity: correlates with glutamatergic innervation, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2025-2029. la Chen, R., Aldridge, J.W., Johnston, M.V. and Silverstein, ES., Quisqualic acid: a potent neurotoxin and convulsant in the developing nervous system, Neurology, 376 (Suppl. 1) (1986) 346. lb Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623-634. 2 Ferriero, D.M., Simon, R.E and Soberano, H.Q., The quis-
injury produced by intrastriatal injections of NMDA or by hypoxia-ischemia5'6'13'15'17'1s. Differences in the distribution of these subtypes of E A A receptors may be related to the differences in vulnerability to AMPA/ QUIS and NMDA toxicity8'1s. At PND 7, in stratum radiatum of rodent hippocampus, the density of [3H]AMPA binding is 30% greater in CA3 subfield compared with CA1, whereas the density of [3H]glutamate binding to NMDA recognition sites is 40% greater in CA1 compared to CA38. However, it is apparent that other intrinsic and extrinsic neuronal factors play a role in the selective vulnerability of neurons to excitotoxic injury12'1s. Because the affinities of AMPA and QUIS for the ionotropic quisqualate receptor are similar in receptor binding studies in vitro 4'2°'22, the greater excitotoxic potency of AMPA in this study may relate to active uptake or elimination processes for QUIS present in brain in vivo 1°'11. The relative excitotoxic potency of AMPA and QUIS observed in brain in this study is similar to their relative potency as neurotoxins in chicken retina (as measured by levels of choline acetyltransferase) where AMPA is approximately 30 times more potent than QUIS 21. NMDA is a potent excitotoxin in PND 7 rat brain s'14. Comparison of the doses that produce halfmaximal brain injury in PND 7 rats indicates that AMPA is 1.5 times more potent than NMDA as an excitotoxin ~6. At this developmental age, the selective E A A agonist kainate is a weak excitotoxin ~8. The sensitivity to kainate toxicity increases with postnatal development and correlates with the development of glutamatergic innervation ~. Thus the relative excitotoxic potency of these selective E A A agonists at PND 7 in rats is AMPA > NMDA > > QUIS > > kainate. The rank order of excitotoxic potency of these agonists differs markedly between PND 7 and adult rats TM. The marked potency of AMPA as an excitotoxin in the developing brain may provide a useful model system to examine the characteristics of ionotropic-type quisqualate receptor-mediated brain injury and to assess the effectiveness of potential neuroprotective compounds. Supported by NIH PO1 NS19613, RO1 NS28208 (Javits Neuroscience Investigators Award) and Basic Research Grant 1145 from the March of Dimes Birth Defects Foundation. qualate analogue a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate is neurotoxic to neonatal rat striatum, Ann. Neurol., 26 (1989) 445. 3 Greenamyre, T., Penney, J.B., Young, A.B., Hudson, C., Siiverstein, F.S. and Johnston, M.V., Evidence for transient perinatal glutamatergic innervation of globus pallidus, J. Neurosci., 7 (1987) 1022-1030. 4 Honore, T., Lauridsen, J. and Krogsgaard-Larsen, P., The binding of [3H]AMPA, a structural analogue of glutamic acid, to rat brain membranes, J. Neurochem., 38 (1982) 173-178.
168 5 Ikonomidou, C., Mosinger, J.L., Shahid Salles, K., Labruyere, J. and Olney, J.W., Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methylo-aspartate neurotoxicity, J. Neurosci., 9 (1989) 2809-2818. 6 Ikonomidou, C., Price, M.T., Mosinger, J.L., Frierdich, G., Labruyere, J., Shahid Salles, K. and Olney, J.W., Hypobaricischemic conditions produce glutamate-like cytopathology in infant rat brain, J. Neurosci., 9 (1989) 1693-1700. 7 Ikonomidou, C., Labruyere, J., Benz. A. and Olney, J.W., Antagonistic pharmacology of glutamate neurotoxicity in infant rat brain, Soc. Neurosci. Abstr., 15 (1989) 306.19. 8 Insel, T.R., Miller, L.P. and Gelhard, R.E., The ontogeny of excitatory amino acid receptors in rat forebrain. I. N-Methylo-aspartate and quisqualate receptors, Neuroscience, 35 (1990) 31-43. 9 Litchfield, J.T. and Wilcoxon, E, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther., 96 (1949) 99-113, 10 Lodge, D., Uptake inhibitors, amino acids and spinal neurones. In EV. DeFeudis and E Mandel (Eds.), Amino Acid Neurotransmitters, Raven, New York, 1982, pp. 327-332. 11 Lodge, E, Curtis, D.R., Johnston, G.A.R. and Bronstein, J.C., In vivo inactivation of quisqualate; studies in the rat spinal cord, Brain Research, 182 (1980) 491-495. 12 Mattson, M.P., Guthrie, P.B. and Kater, S.B., Intrinsic factors in the selective vulnerability of hippocampal pyramidal neurons, Alzheimer's Disease and Related Disorders, (1989) 333-351. 13 McDonald, J.W., Silverstein, ES. and Johnston, M.V., MK-801 protects the neonatal brain from hypoxic-ischemic damage, Eur. J. Pharmacol., 140 (1987) 359-361. 14 McDonald, J.W., Silverstein, ES. and Johnston, M.V., Neurotoxicity of N-methyi-o-aspartate is markedly enhanced in developing rat central nervous system, Brain Research, 459 (1988) 200-203.
15 McDonald, J.W., Silverstein, ES., Cardona, D., Hudson, C., Chen, R. and Johnston, M.V., Neuroprotective effects of MK-801, TCP, PCP and CPP against N-methyl-o-aspartate induced neurotoxicity in an in vivo perinatal rat model, Brain Research, 490 (1989) 33-40. 16 McDonald, J.W., Roeser, N.E, Silverstein, ES. and Johnston, M.V., Quantitative assessment of neuroprotection against NMDA-mediated brain injury, Exp. Neurol., 106 (1989) 289296. 17 McDonald, J.W., Silverstein, ES., Cardona, D., Hudson, C., Chen, R. and Johnston, M.V., Systemic administration of MK-801 protects against N-methyl-t~-aspartate and quisqualate mediated neurotoxicity in perinatal rats, Neuroscience, in press. 18 McDonald, J.W. and Johnston, M.V., Physiological and pathophysiologicai roles of excitatory amino acids during central nervous system development, Brain Res. Rev., 15 (1990) 41-70. 19 McDonald, J.W. and Johnston, M.V., Pharmacology of N-methyl-t~-aspartat e induced brain injury in an in vivo perinatal rat model, Synapse, in press. 20 Monaghan, D.T., Yao, D. and Cotman, C.W., Distribution of [3H]AMPA binding sites in rat brain as determined by quantitative autoradiography, Brain Research, 324 (1984) 160-164. 21 Morgan, I.G., AMPA is a powerful neurotoxin in the chicken retina, Neurosci. Left., 79 (1987) 267-271. 22 Rainbow, T.C., Wieczorek, C.M. and Halpain, S., Quantitative autoradiography of binding sites for [3H]AMPA, a structural analogue of glutamatic acid, Brain Research, 309 (1984) 173177. 23 Rothman, S.M. and Olney, J.W., Excitotoxicity and the NMDA receptor, Trends Neurosci., 10 (1987) 299-302. 24 Silverstein, ES., Chen, R. and Johnston, M.V., The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain, Neurosci. Lett., 71 (1986) 13-18.
165
Elsevier BRES 24243
The selective ionotropic-type quisqualate receptor agonist AMPA is a potent neurotoxin in immature rat brain John W. McDonald 1, William H. Trescher 1 and Michael V. J o h n s t o n 1'2 Departments of INeurology and 2pediatrics, the Johns Hopkins University School of Medicine and the Kennedy Research Institute, Baltimore, MD (U.S.A.)
(Accepted 8 May 1990) Key words: Excitotoxicity;Neurotoxicity; Glutamate; Excitatory amino acid; Rat; Development; Quisqualate;
a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid
Direct unilateral intrastriatal stereotaxic injections of the selective ionotropic-type quisqualate receptor agonist, AMPA, in postnatal day 7 rats produced tonic-clonic seizure activity. Quantitative analysis of the severity of brain injury was assessed by comparison of the disparities in the weights of injected and contralateral cerebral hemispheres 3 days after the excitotoxin injection. The amount of AMPA that produced half-maximal brain injury was 9.5 nmol as assessed by comparison of disparities in cerebral hemisphere weights. In contrast, quisqualate was 26 times less potent. The marked susceptibility of the developing rat brain to AMPA toxicity may provide a useful model to assess the neuroprotective effectiveness and selectivity of ionotropic quisqualate receptor antagonists.
The excitotoxic actions of excitatory amino acids (EAA) are manifested by activation of subclasses of E A A receptors named for their preferential agonists, N-methyI-D-aspartate (NMDA), quisqualate (QUIS), and kainate 1b'ls'23. The mechanisms of NMDA receptormediated brain injury have been the most extensively studied, whereas non-NMDA receptor-mediated injury is less well characterized. Subtypes of quisqualate receptors have been delineated on the basis of their linkage to an ionophore (ionotropic receptor) or phospholipase C (metabotropic receptor). Agonists of both subtypes of receptors are capable of producing excitotoxic neuronal injury in the immature brain 2'7'24. In this study, we compared the excitotoxic characteristics of quisqualate 22, which acts at both receptor subtypes, and the selective ionotropic quisqualate receptor agonist, AMPA ((RS)a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), in a well-characterized in vivo perinatal rat model of excitotoxic brain injury 15'16A9. Intrastriatal injections of AMPA (5 doses; 2.5, 5, 10, 25, 50 nmol; n = 6-8/dose) and QUIS (4 doses; 50, 100, 150, 200 nmol; n = 7-8/dose) were performed in 7-day-old Sprague-Dawley albino rats briefly anesthetized with ether as previously described in detail 16. With reference to bregma, injection coordinates were AP = 0 mm, L = 2 mm, depth from dura = 4 mm. Drugs were dissolved in 0.01 M Tris base (final pH 7.4) and injection
volumes were 0.5/~1 (AMPA) and 1.0/~1 (QUIS). For quantitative analysis, all animals were sacrificed 3 days after excitotoxin injection and the severity of brain injury (% damage) was assessed by comparison of the dry weights of the injected (I) and contralateral (C) cerebral hemispheres using the formula 100x (C-I)/C = % damage 16. Additional animals were sacrificed 5 days after injection and brains were processed for routine light microscopic histoiogic analysis. We have previously demonstrated that comparisons of cerebral hemisphere weights is an accurate and sensitive measure of excitotoxic brain injury in this model 16. Values presented are means _+ S.E.M. ECso values were calculated by the method of Litchfield and Wilcoxon 9. AMPA and QUIS (synthetic) were obtained from Cambridge Research Biochemicals (Cambridge, England). Unilateral intrastriatal injection of QUIS or AMPA in PND 7 rats produced acute tonic-clonic seizure activity and consistently resulted in extensive, dose-dependent, unilateral brain injury that was most predominant in the corpus striatum, overlying neocortex, dorsal hippocampus, amygdala, thalamus and fornix when examined 5 days later (Fig. 1). Tissue damage was usually confined to the injected hemisphere although doses of AMPA 10 nmol or greater produced microscopic damage in the contralateral hemisphere with a propensity to selectively damage the contralateral fornix. As previously described,
Correspondence: M.V. Johnston, Neuroscience Laboratory, Kennedy Research Institute, Room 506, 707 N. Broadway, Baltimore, MD 21205,
U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166 a similar injection of vehicle in PND 7 rats produced few signs of neurotoxicity is-17. Quantitative analysis of the severity of resulting brain injury by comparison of the weights of the injected and contralateral cerebral hemispheres indicated that the excitotoxic effects of QUIS and AMPA were dosedependent (Table I). Based on the doses required to produce 16% of maximal injury (ED16), A M P A was approximately 26 times more potent as a neurotoxin than QUIS. ED16 values were 4.5 nmol (AMPA) and 118 nmol (QUIS). The EDso value for AMPA was 9.5 nmol with a 95% confidence interval of 5.6-16.1 nmol. Direct determination of the corresponding value for QUIS was not possible because of the limited solubility of QUIS but the estimated EDso value for QUIS was 250.0 nmol with a 95% confidence interval ranging from 146.3 to 427.2
/
nmol. Intrastriatal injections of QUIS did not alter the dry weights of the contralateral hemisphere compared to vehicle-injected controls (dry weight of contralateral cerebral hemisphere (mean + S.E.M.): 50 nmol, 52.1 +_ 0.5 mg; 100 nmol, 52.7 _+ 1.1 mg; 150 nmol, 52.0 _+ 1.4 mg: 200 nmol, 51.7 _+ 0.8 mg; vehicle, 53.1 + 0.7 rag). In contrast, intrastriatal injections of A M P A significantly reduced the size of the contralateral cerebral hemisphere (dry weight of contralateral cerebral hemisphere (mean + S.E.M.); 2.5 nmol, 54.2 _+ 0.6 mg; 5 nmol, 53.9 + 0.5 rag; 10 nmol, 47.9 + 1.6 mg; 25 nmol, 39.6 + 0.9 mg; 50 nmol, 39.7 _+ 1.1 mg; vs vehicle, 53.1 + 0.7 mg: P < 0.01, l-way ANOVA). These results extend the earlier observations that quisqualate and A M P A are neurotoxic in the immature brain 2'7'24, and demonstrate that A M P A , a selective
C
QUIS
50nmol
2ram
25nmol
Fig. 1. Comparison of the histopathologic characteristics of quisqualate (A,B) and AMPA (C,D) induced brain injury in PND 12 rats. Quisqualate (50 nmol/I pi) and AMPA (25 nmol/0.5 ~l) were stereotaxically microinjected into the right anterior striatum on PND 7. Animals were sacrificed 5 days later on PND 12.50 pm frozen sections were cut and stained for Nissl substance. In contrast to quisqualate, intrastriatal injection of half as much AMPA produced extensive brain injury. The AMPA induced lesion is characterized by confluent necrosis involving the entire cerebral hemisphere at the level of the striatum (C). The damage extended several mm caudal to the injection site with marked injury to the dorsal hippocampus and surrounding neocortex. Bar = 2 mm.
167 TABLE I Severity of brain injury produced by intrastriatal injections of AMPA and Q UIS
%Damage reflects the severity of injury in the injected (I) cerebral hemisphere relative to the contralateral (C) hemisphere and was calculated using cerebral hemisphere weights (dry) with the formula; %Damage = 100(C-I)/C. Excitotoxin
Dose (nmol)
n
% Damage (mean + S.E.M.)
Quisqualate
50 100 150 200
8 7 8 7
-0.1 + 0.9 3.1 + 0.6 5.7 + 1.1 7.3 + 0.9
8 8 8 7 6
1.2+0.6 3.6+ 1.1 8.4 + 0.9 20.9 + 2.6 21.5 + 1.3
AMPA
2.5 5 10 25 50
ionotropic quisqualate receptor agonist is a potent excitotoxin in PND 7 rat brain when administered intracerebrally. AMPA is approximately 26 times more potent than QUIS as an excitotoxin in this model. AMPA and QUIS toxicity does not appear to simply result indirectly from seizure activity since unilateral intrastriatal injection of these excitotoxins produces bihemispheric, high voltage, high frequency epileptiform discharges but only unilateral brain injurylL The reduction in the weight of the cerebral hemisphere contralateral to the injected hemisphere observed in animals that received higher doses of AMPA (25 nmol, 50 nmol) reflects, in part, microscopic injury to the contralateral hemisphere and growth retardation of the cerebral hemisphere during the 3-day survival period. Because the method of quantifying the severity of brain injury relates the degree of injury to the contralateral non-injected hemisphere, the reduction in the weight of the contralateral hemisphere may bias the apparent calculated degree of injury such that it will be slightly underestimated at the highest doses of A M P A examined. The regional pattern of susceptibility to AMPA and QUIS toxicity corresponds roughly to the distribution of QUIS receptors in brain 3's'18. The pattern of selective vulnerability is similar, but not identical, to the pattern of 1 Campochiaro, P. and Coyle, J.T., Ontogenic development of kainate neurotoxicity: correlates with glutamatergic innervation, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2025-2029. la Chen, R., Aldridge, J.W., Johnston, M.V. and Silverstein, ES., Quisqualic acid: a potent neurotoxin and convulsant in the developing nervous system, Neurology, 376 (Suppl. 1) (1986) 346. lb Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623-634. 2 Ferriero, D.M., Simon, R.E and Soberano, H.Q., The quis-
injury produced by intrastriatal injections of NMDA or by hypoxia-ischemia5'6'13'15'17'1s. Differences in the distribution of these subtypes of E A A receptors may be related to the differences in vulnerability to AMPA/ QUIS and NMDA toxicity8'1s. At PND 7, in stratum radiatum of rodent hippocampus, the density of [3H]AMPA binding is 30% greater in CA3 subfield compared with CA1, whereas the density of [3H]glutamate binding to NMDA recognition sites is 40% greater in CA1 compared to CA38. However, it is apparent that other intrinsic and extrinsic neuronal factors play a role in the selective vulnerability of neurons to excitotoxic injury12'1s. Because the affinities of AMPA and QUIS for the ionotropic quisqualate receptor are similar in receptor binding studies in vitro 4'2°'22, the greater excitotoxic potency of AMPA in this study may relate to active uptake or elimination processes for QUIS present in brain in vivo 1°'11. The relative excitotoxic potency of AMPA and QUIS observed in brain in this study is similar to their relative potency as neurotoxins in chicken retina (as measured by levels of choline acetyltransferase) where AMPA is approximately 30 times more potent than QUIS 21. NMDA is a potent excitotoxin in PND 7 rat brain s'14. Comparison of the doses that produce halfmaximal brain injury in PND 7 rats indicates that AMPA is 1.5 times more potent than NMDA as an excitotoxin ~6. At this developmental age, the selective E A A agonist kainate is a weak excitotoxin ~8. The sensitivity to kainate toxicity increases with postnatal development and correlates with the development of glutamatergic innervation ~. Thus the relative excitotoxic potency of these selective E A A agonists at PND 7 in rats is AMPA > NMDA > > QUIS > > kainate. The rank order of excitotoxic potency of these agonists differs markedly between PND 7 and adult rats TM. The marked potency of AMPA as an excitotoxin in the developing brain may provide a useful model system to examine the characteristics of ionotropic-type quisqualate receptor-mediated brain injury and to assess the effectiveness of potential neuroprotective compounds. Supported by NIH PO1 NS19613, RO1 NS28208 (Javits Neuroscience Investigators Award) and Basic Research Grant 1145 from the March of Dimes Birth Defects Foundation. qualate analogue a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate is neurotoxic to neonatal rat striatum, Ann. Neurol., 26 (1989) 445. 3 Greenamyre, T., Penney, J.B., Young, A.B., Hudson, C., Siiverstein, F.S. and Johnston, M.V., Evidence for transient perinatal glutamatergic innervation of globus pallidus, J. Neurosci., 7 (1987) 1022-1030. 4 Honore, T., Lauridsen, J. and Krogsgaard-Larsen, P., The binding of [3H]AMPA, a structural analogue of glutamic acid, to rat brain membranes, J. Neurochem., 38 (1982) 173-178.
168 5 Ikonomidou, C., Mosinger, J.L., Shahid Salles, K., Labruyere, J. and Olney, J.W., Sensitivity of the developing rat brain to hypobaric/ischemic damage parallels sensitivity to N-methylo-aspartate neurotoxicity, J. Neurosci., 9 (1989) 2809-2818. 6 Ikonomidou, C., Price, M.T., Mosinger, J.L., Frierdich, G., Labruyere, J., Shahid Salles, K. and Olney, J.W., Hypobaricischemic conditions produce glutamate-like cytopathology in infant rat brain, J. Neurosci., 9 (1989) 1693-1700. 7 Ikonomidou, C., Labruyere, J., Benz. A. and Olney, J.W., Antagonistic pharmacology of glutamate neurotoxicity in infant rat brain, Soc. Neurosci. Abstr., 15 (1989) 306.19. 8 Insel, T.R., Miller, L.P. and Gelhard, R.E., The ontogeny of excitatory amino acid receptors in rat forebrain. I. N-Methylo-aspartate and quisqualate receptors, Neuroscience, 35 (1990) 31-43. 9 Litchfield, J.T. and Wilcoxon, E, A simplified method of evaluating dose-effect experiments, J. Pharmacol. Exp. Ther., 96 (1949) 99-113, 10 Lodge, D., Uptake inhibitors, amino acids and spinal neurones. In EV. DeFeudis and E Mandel (Eds.), Amino Acid Neurotransmitters, Raven, New York, 1982, pp. 327-332. 11 Lodge, E, Curtis, D.R., Johnston, G.A.R. and Bronstein, J.C., In vivo inactivation of quisqualate; studies in the rat spinal cord, Brain Research, 182 (1980) 491-495. 12 Mattson, M.P., Guthrie, P.B. and Kater, S.B., Intrinsic factors in the selective vulnerability of hippocampal pyramidal neurons, Alzheimer's Disease and Related Disorders, (1989) 333-351. 13 McDonald, J.W., Silverstein, ES. and Johnston, M.V., MK-801 protects the neonatal brain from hypoxic-ischemic damage, Eur. J. Pharmacol., 140 (1987) 359-361. 14 McDonald, J.W., Silverstein, ES. and Johnston, M.V., Neurotoxicity of N-methyi-o-aspartate is markedly enhanced in developing rat central nervous system, Brain Research, 459 (1988) 200-203.
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