Brain Research, 579 (1992) 169-172 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
169
BRES 25156
Alterations in spinal cord excitatory amino acid receptors in amyotrophic lateral sclerosis patients H. Allaoua a, I. Chaudieu a, C. Krieger b, P. Boksa a, A.
Privat c and R. Quirion a
aDouglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University, Quebec (Canada), bDivision of Neurology, Department of Medicine, Faculty of Medicine, The University of British Columbia, University Hospital, Vancouver, (Canada) and *INSERM U336, USTL, Montpellier (France) (Accepted 21 January 1992)
Key words: Excitatory amino acid: N-Methyl-D-aspartate; Amyotrophic lateral sclerosis; Excitotoxin; Receptor
Excitatory amino acids (EAA) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We have analyzed the distribution of the N-methyl-o-aspartate (NMDA) 1-(1-(2-thienyl)-cyclohexyl) piperidine (TCP), kainate and a-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMPA) quisqualate subtypes of EAA receptors using quantitative receptor autoradiography in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. We observed that in control spinal cords [3H]TCP/NMDA binding sites were located both in the ventral and dorsal horns with the highest densities being situated in lamina II. [3H]AMPA and [3H]kainate binding sites were present almost exclusively in the substantia gelatinosa of the dorsal horn. In ALS, the distribution of these 3 types of receptors was unchanged, but [3H]TCP/NMDA binding was decreased both in the dorsal and ventral horns. [3H]kainate binding was possibly decreased in substantia gelatinosa, of ALS cords. However, the limited sample size available for [3H]kainate binding did not permit statistical analysis. [aH]AMPA binding sites were unaltered in ALS. These results indicate that there is a preferential reduction in NMDA receptors in ALS. We suggest that should an excitotoxic mechanism be involved in the pathogenesis of ALS, then NMDA receptors may be the target of this effect. The sporadic form of amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by corticospinal tract and motoneuron degeneration producing paralysis and generally resulting in death within 5 years. The disorder has a prevalence of 4 - 6 per 100,000 population in North America and an annual incidence similar to multiple sclerosis 8. Excitatory neurotoxic compounds have been suggested as a possible cause of sporadic A L S 13'14 as well as for the ALS-Parkinsonism-dementia complex observed in the Mariana Islands ~8. In sporadic ALS, concentrations of glutamic acid have been reported to be elevated in fasting plasma ~2, and concentrations of glutamate, aspartate, N-acetyl aspartate and N-acetyl-aspartyl glutamate have been reported as increased in cerebrospinal fluid 16. Although some of these observations have not been supported by other studies 1L12, it is clear that at least one form of motor neuronopathy in humans can result from the ingestion of domoic acic, an excitatory amino acid ( E A A ) present in contaminated mussels 19. Three independent studies of the amino acid contents of spinal cords from patients who have died with A L S have demonstrated decreased contents of glutamate ~° or glutamate and aspartate 14A6. Contents of glutamate and
aspartate are also reduced in most brain regions in patients dying with ALS 1°'14'16. Plaitakis and colleagues have attributed the reduced spinal cord contents of glutamate and aspartate to increased release of E A A from nerve terminals with consequent neuroexcitotoxic damage to motoneurons 14. If the putative excitotoxin(s) involved in producing A L S was a selective agonist at one of the subtypes of E A A receptors, a specific reduction in a given receptor subtype might be observed as a consequence of neuronal death subsequent to receptor activation. For example, in Huntington's disease, an autosomal dominant neurodegenerative disease characterized by progressive dementia and movement disorders, a preferential depletion of the N-methyl-o-aspartate ( N M D A ) subtype of E A A receptors has been observed in the striata of affected patients 22 and presymptomatic individuals ~. The receptor loss and clinical symptomatology seen in this disorder are potentially a consequence of an unknown excitotoxin acting at the N M D A site. To investigate whether there could be a selective loss of any of the 3 major subtypes of E A A receptors in A L S we determined, using quantitative receptor autoradiography, the distribution of the N M D A , kainate and
Correspondence: R. Quirion, Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Quebec, Canada H4H 1R3. Fax: (1) (514) 766-2503.
170 a-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) subtypes of E A A receptors in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. Some of these data were presented in abstract form 2. Earlier studies have reported on the reductions of muscarinic, benzodiazepine, glycine, thyrotropin stimulating hormone (TRH) and substance P receptors in ALS cords 5'21. Cervical and thoracic spinal cords were obtained at autopsy from 4 patients who had ALS, and from 5 individuals who had no evidence of neurological disease. The ALS patients ranged in age from 43 to 71 years (63.8 + 12.0, mean + S.D.) and all had neuropathological verification of their diagnoses and involvement of spinal motoneurons. The death to freezing intervals ranged from 2.0 to 19.8 h (9.9 + 7.6). Individuals with no clinical or neuropathological evidence of neurological disease ranged from 59 to 76 years of age (67.8 +
A
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6.6) and had died following myocardial infarction, congestive heart failure or bronchopneumonia. Death to freezing intervals for this group ranged from 4.3 to 23 h (13.0 + 7.1). All spinal cords were stored at -70°C until analysis. [3H]l-(1-(2-thienyl)-cyclohexyl) piperidine (TCP) (45 Ci/mmol), [3H]kainate (60 Ci/mmol) and [3H]AMPA (27.6 Ci/mmol) were purchased from New England Nuclear, Dupont Canada, Toronto, Ontario. Hyperfilm-3H and microscales were obtained from Amersham Canada, Oakville, Ontario. Unlabelled excitatory amino acids and all other reagents were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Canada (Montreal, Quebec). All frozen samples were sectioned and tissue slices (20 #m) were cut using a cryostat at -17°C, thaw-mounted onto gelatin/chrome alum coated slides, air dried under vacuum at 4°C overnight and then stored at -70°C. The
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Fig. 1. Photomicrographs of the autoradiographic distribution of [3H]TCP/NMDA (A), [3H]kainate (B) and [3H]AMPA/quisqualate (C) binding sites in human spinal cord at the cervical level. D, E and F represent non-specific binding in adjacent sections, respectively. Note that while [3H]TCP binding is located in substantia gelatinosa/lamina II of the dorsal horn as well as in the ventral horn (most likely motoneurons), [3H]kainate and [3H]AMPA binding are mostly restricted to the superficial laminae of the cord. Bar = 4 mm. DH, dorsal horn; VH, ventral horn; II and X, laminae of the spinal cord.
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.~. Fig. 2. Excitatory amino acid (EAA) binding sites in control and amyotrophiclateral sclerosis (ALS) spinal cords. While the amount of [3H]TCP/NMDA binding is decreased in both lamina II and ventral horn in ALS cords (A), [3H]AMPAJquisqualatebinding is unaltered throughout the cord in ALS patients (B). *P < 0.025; **P < 0.02.
NMDA receptor was investigated using [3H]TCP, a specific ligand of the phencyclidine NMDA associated site. Sections were incubated for 45 min at 0°C in presence of 40 nM [3H]TCP in a buffer containing 5 mM Tris pH 7.44. Non-specific binding was evaluated by incubating adjacent sections with 10/~M phencyclidine. The AMPA binding site of the quisqualate receptor was evaluated using [3H]AMPA (50 nM), sections being incubated for 45 min at 2°C in a buffer containing 50 mM Tris, 100 mM KSCN pH 7.29. Non-specific binding was determined in the presence of 100/~M AMPA. Finally, kainate binding was determined by incubating sections for 90 min at 3°C in a buffer containing 20 nM [3H]kainate, 50 mM Tris acetate pH 7.115. Non-specific binding was determined in the presence of 10 aM unlabelled kainate, Autoradiograms were generated by apposing labelled sections to film for 2-4 weeks and the optical density of film autoradiograms was analyzed by microcomputer image analysis (MCID, St. Catharines, Ontario), using tri-
tium standard microscales for film calibration. Under these experimental conditions, the specific binding of [3H]TCP, [3H]AMPA and [3H]kainate represented 40%, 90% and 50% of total binding, respectively. Values are expressed as mean + S.E.M. Student's t-test was used for statistical analysis, P < 0.05 being considered significant. In normal control spinal cord, NMDA/[aH]TCP, [3H]AMPA and [3H]kainate receptor binding sites were almost exclusively concentrated in the grey matter (Fig. 1). NMDA/[3H]TCP sites were localized both in the ventral and dorsal horns with higher densities observed in lamina II than other laminae including the ventral horn (Fig. 2). [3H]AMPA and [3H]kainate receptor sites were present almost exclusively in the substantia gelatinosa of the dorsal horn (lamina II, Fig. 1). In ALS patients, the distribution of these 3 classes of excitatory amino acid binding sites was unchanged. However, as shown in Fig. 2, [3H]TCP binding was decreased significantly in both dorsal (lamina II; 141 + 21 vs. 78 + 7 fmol/mg tissue wet weight, P < 0.025; control vs. ALS cords) and ventral (118 _+ 11 vs. 70 _+ 11 fmol/mg tissue, wet weight, P < 0.02; in control vs. ALS) horns in ALS cords. Decreases in [3H]TCP binding were not correlated to the age of the subject (not shown). [3H]kainate binding was possibly decreased in the substantia gelatinosa (9 + 2 vs. 5.8 _+ 3 fmol/mg tissue weight, control vs. ALS) but the limited sample size (n = 3) of ALS cords did not allow for a statistical evaluation. [3H]AMPA binding was unaltered (e.g. lamina II, 89 + 6 vs. 87 + 6 fmol/mg tissue wet weight; control vs. ALS) in ALS tissues (Fig. 2) revealing the relative specificity of the EAA-receptor modifications in ALS. The present results on the distribution of NMDA, AMPA and kainate receptors in normal human spinal cord are in agreement with some recent reports 6'17. All 3 types of receptor appear to be found predominantly within the dorsal horn and especially within lamina II. This localization has been attributed to the possible involvement of these receptors in primary afferent neurotransmission6. Foci of high [3H]TCP/NMDA binding were also observed within the ventral horn and some of this binding localizes to regions consistent with motoneuron cell bodies 17. In ALS, we observed a reduction in [3H]TCP/NMDA receptor binding in the dorsal and ventral horns at both cervical and thoracic levels. [3H]kainate binding was also possibly reduced in ALS, but due to the limited number of ALS spinal cords available and low amounts of specific [3H]kainate binding (few fmol/mg tissue, wet weight) in the normal control cord definite conclusions could not be made concerning this point. As mammalian motoneurons possess NMDA recep-
172 tors 7, and since all of the spinal cords from A L S patients had m o t o n e u r o n losses, some of the reductions in [3H]TCP/NMDA r e c e p t o r binding in the ventral horn are likely due to the death of N M D A receptor-bearing motoneurons. Surprisingly, we also observed a significant reduction in [ 3 H ] T C P / N M D A r e c e p t o r binding in the dorsal horn; decreases being nearly as p r o n o u n c e d as those seen in the ventral horn. Since afferent pathways are usually spared in A L S , both clinically and pathologically3, it appears unlikely that the reductions in N M D A r e c e p t o r binding are due to cell loss in the dorsal horn. H o w e v e r , recent observations indicate that N-acetyl-aspartate, and the excitatory dipeptide N-acetylaspartyl glutamate are significantly reduced in both the dorsal and ventral horns of cervical spinal cords from A L S patients 2°. This might indicate that some functional alterations occur both in the dorsal and ventral horns in A L S . Alternatively, a possible explanation is that a down-regulation in N M D A and possibly kainate receptor n u m b e r could occur in A L S , possibly due to the continued presence of increased amounts of a N M D A - (or
1 Albin, R.L., Young, A.B., Penney, J.B., Handelin, B., Balfour, R., Anderson, K., Markel, D.S., Tourtellotte, W.W. and Reiner, A., Abnormalities of striatal projection neurons and N-methyl-~aspartate receptors in presymptomatic Huntington's disease, N. Engl. J. Med., 322 (1990) 1293-1298. 2 Allaoua, H., Chaudieu, I., Boksa, P., Perry, T.L., Krieger, C. and Quirion, R., Excitatory amino acid receptors in human spinal cord. Evaluation in amyotrophic lateral sclerosis patients, Ann. N Y Acad. Sci., in press. 3 Brownell, B., Oppenheimer, D.R. and Hughes, J.T., The central nervous system in motor neurone disease, J. Neurol. Neurosurg. Psychiat., 33 (1970) 338-357. 4 Contreras, P.C., Quirion, R. and O'Donohue, T.L., Autoradiographic distribution of phencyclidine receptors in the rat brain using [3H]l-(1-2-thienyl)cyclohexyl) piperidine, [3H]TCP, Neurosci. Lett., 67 (1986) 101-106. 5 Dietl, M.M., Sanchez, M., Probst, A. and Palacios, J.M., Substance P receptors in the human spinal cord: decrease in amyotrophic lateral sclerosis, Brain Res., 483 (1989) 39-49. 6 Jansen, K.L.R., Faull, R.L.M., Dragumow, M. and Waldvogel, H., Autoradiographic localisation of NMDA, quisqualate and kainate receptors in human spinal cord, Neurosci. Lea., 108 (1990) 53-57. 7 Konnerth, A., Keller, B.U. and Lev-Tov, A., Patch clamp analysis of excitatory synapses in mammalian spinal cord slices, Pflugers Arch., 417 (1990) 285-290. 8 Kurtzke, J.E and Kurland, L.T., The epidemiology of neurologic disease. In R.J. Joynt (Ed.), Clinical Neurology, J.B. Lippincott, Philadelphia, 1989, pp. 1-43. 9 Neilsen, E.O., Cha, J.H.J., Penney, J.B. and Young, A.B., Thiocyanate stabilizes AMPA binding to the quisqualate receptor, Eur. J. Pharrnacol., 157 (1988) 197-203. 10 Perry, T.L., Hansen, S. and Jones, K., Brain glutamate deftciency in amyotrophic lateral sclerosis, Neurology, 37 ( 1 9 8 7 ) 1845-1848. 11 Perry, T.L., Krieger, C., Hansen, S. and Eisen, A., Amyotrophic lateral sclerosis: amino acid levels in plasma and cerebrospinal fluid, Ann. Neurol., 28 (1990) 12-17. 12 Perry, T.L., Krieger, C., Hansen, S. and Tabatabei, A., Amyotrophic lateral sclerosis: fasting levels of cysteine and inorganic
kainate) like agonist/excitotoxin in various spinal cord regions in A L S . The present results a p p e a r to be the first autoradiographic investigation of E A A binding in the spinal cord of A L S patients. Selective decreases in [3H]TCP/NMDA receptor binding are noted in both dorsal and ventral horn regions. [3H]kainate binding may also be altered but only in the dorsal horn while [ 3 H ] A M P A binding sites are most likely spared. The preferential alteration in the N M D A r e c e p t o r binding suggests that if an excitotoxic mechanism is involved in the pathogenesis of A L S , then the N M D A receptor would be a likely candidate as a m e d i a t o r of the m o t o n e u r o n loss.
We thank Dr. Wallace W. Tourtellotte of the National Neurological Research Tissue Bank, the Canadian Brain Tissue Bank, and Drs. K. Berry and W. Moore for spinal cord tissues. We acknowledge the late T. Perry for his advice and support. This work was supported by Fonds de la Recherche en Sant6 du Qu6bec (FRSQ) and the Medical Research Council of Canada (MRC). R.Q. is a Chercheur-Boursier, I.C. a Fellow of the FRSQ and C.K. is a MRC Scholar.
sulfate are normal, as are brain contents of cysteine, Neurology, 41 (1991) 487-490.
13 Plaitakis, A. and Caroscio, J.T., Abnormal glutamate metabolism in amyotrophic lateral sclerosis, Ann. Neurol., 22 (1987) 575-579. 14 Plaitakis, A., Constantakakis, E. and Smith, J., The neuroexcitotoxic amino acids glutamate and aspartate are altered in the spinal cord and brain in amyotrophic lateral sclerosis, Ann. Neurol., 24 (1988) 446-449. 15 Represa, A., Tremblay, E. and Ben Ari, Y., Kainate binding sites in the hippocampal mossy fibers: localisation and plasticity, Neuroscience, 20 (1987) 739-745. 16 Rothstein, J.D., Tsai, G., Kunci, R.W., Clawson, L., Cornblath, D.R., Drachman, D.B., Pestronk, A., Stanch, B.L. and Coyle, J.T., Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis, Ann. Neurol., 28 (1990) 18-25. 17 Shaw, P.J., Ince, P.G., Johnson, M., Perry, E.K. and Candy, J., The quantitative autoradiographic distribution of [3H]MK801 binding sites in the normal human spinal cord, Brain Res., 539 (1991) 164-168. 18 Spencer, P.S., Nunn, P.B., Hugon, J., Ludolph, A.C., Ross, S.M., Roy, D.N. and Robertson, R.C., Guam amyotrophic lateral sclerosis-Parkinsonism-dementia linked to a plant excitant neurotoxin, Science, 237 (1987) 517-522. 19 Teitlebaum, J.S., Zatorre, R.J., Carpenter, S., Gendron, D., Evans, A.C., Gjedde, A. and Cashman, N.R., Neurologic sequelae of domoic acid intoxication due to the ingestion of contaminated mussels, N. Engl. J. Med., 322 (1990) 1781-1787. 20 Tsai, G., Stauch-Slusher, B., Sim, L., Hedreen, J.C., Rothstein, J.D., Kuncl, R. and Coyle, J.T., Reductions in acidic amino acids and N-acetylaspartylglutamatein amyotrophic lateral sclerosis CNS, Brain Res., 556 (1991) 151-156. 21 Whitehouse, P.J., Wamsley, J.K., Zarbin, M.A., Price, D.L., Tourtellotte, W.W. and Kuhar, M.J., Amyotrophic lateral sclerosis: alterations in neurotransmitter receptors, Ann. Neurol., 14 (1983) 8-16. 22 Young, A.B., Greenamyre, J.T., Hollingsworth, Z., Albin, R., D'Amato, C., Shoulson, I. and Penney, J.B., NMDA receptor losses in putamen from patients with Huntington's disease, Science, 241 (1988) 981-983.
169
BRES 25156
Alterations in spinal cord excitatory amino acid receptors in amyotrophic lateral sclerosis patients H. Allaoua a, I. Chaudieu a, C. Krieger b, P. Boksa a, A.
Privat c and R. Quirion a
aDouglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University, Quebec (Canada), bDivision of Neurology, Department of Medicine, Faculty of Medicine, The University of British Columbia, University Hospital, Vancouver, (Canada) and *INSERM U336, USTL, Montpellier (France) (Accepted 21 January 1992)
Key words: Excitatory amino acid: N-Methyl-D-aspartate; Amyotrophic lateral sclerosis; Excitotoxin; Receptor
Excitatory amino acids (EAA) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We have analyzed the distribution of the N-methyl-o-aspartate (NMDA) 1-(1-(2-thienyl)-cyclohexyl) piperidine (TCP), kainate and a-amino-3-hydroxy-5-methyl-4 isoxazole proprionic acid (AMPA) quisqualate subtypes of EAA receptors using quantitative receptor autoradiography in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. We observed that in control spinal cords [3H]TCP/NMDA binding sites were located both in the ventral and dorsal horns with the highest densities being situated in lamina II. [3H]AMPA and [3H]kainate binding sites were present almost exclusively in the substantia gelatinosa of the dorsal horn. In ALS, the distribution of these 3 types of receptors was unchanged, but [3H]TCP/NMDA binding was decreased both in the dorsal and ventral horns. [3H]kainate binding was possibly decreased in substantia gelatinosa, of ALS cords. However, the limited sample size available for [3H]kainate binding did not permit statistical analysis. [aH]AMPA binding sites were unaltered in ALS. These results indicate that there is a preferential reduction in NMDA receptors in ALS. We suggest that should an excitotoxic mechanism be involved in the pathogenesis of ALS, then NMDA receptors may be the target of this effect. The sporadic form of amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by corticospinal tract and motoneuron degeneration producing paralysis and generally resulting in death within 5 years. The disorder has a prevalence of 4 - 6 per 100,000 population in North America and an annual incidence similar to multiple sclerosis 8. Excitatory neurotoxic compounds have been suggested as a possible cause of sporadic A L S 13'14 as well as for the ALS-Parkinsonism-dementia complex observed in the Mariana Islands ~8. In sporadic ALS, concentrations of glutamic acid have been reported to be elevated in fasting plasma ~2, and concentrations of glutamate, aspartate, N-acetyl aspartate and N-acetyl-aspartyl glutamate have been reported as increased in cerebrospinal fluid 16. Although some of these observations have not been supported by other studies 1L12, it is clear that at least one form of motor neuronopathy in humans can result from the ingestion of domoic acic, an excitatory amino acid ( E A A ) present in contaminated mussels 19. Three independent studies of the amino acid contents of spinal cords from patients who have died with A L S have demonstrated decreased contents of glutamate ~° or glutamate and aspartate 14A6. Contents of glutamate and
aspartate are also reduced in most brain regions in patients dying with ALS 1°'14'16. Plaitakis and colleagues have attributed the reduced spinal cord contents of glutamate and aspartate to increased release of E A A from nerve terminals with consequent neuroexcitotoxic damage to motoneurons 14. If the putative excitotoxin(s) involved in producing A L S was a selective agonist at one of the subtypes of E A A receptors, a specific reduction in a given receptor subtype might be observed as a consequence of neuronal death subsequent to receptor activation. For example, in Huntington's disease, an autosomal dominant neurodegenerative disease characterized by progressive dementia and movement disorders, a preferential depletion of the N-methyl-o-aspartate ( N M D A ) subtype of E A A receptors has been observed in the striata of affected patients 22 and presymptomatic individuals ~. The receptor loss and clinical symptomatology seen in this disorder are potentially a consequence of an unknown excitotoxin acting at the N M D A site. To investigate whether there could be a selective loss of any of the 3 major subtypes of E A A receptors in A L S we determined, using quantitative receptor autoradiography, the distribution of the N M D A , kainate and
Correspondence: R. Quirion, Douglas Hospital Research Centre, 6875 LaSalle Blvd., Verdun, Quebec, Canada H4H 1R3. Fax: (1) (514) 766-2503.
170 a-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) subtypes of E A A receptors in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. Some of these data were presented in abstract form 2. Earlier studies have reported on the reductions of muscarinic, benzodiazepine, glycine, thyrotropin stimulating hormone (TRH) and substance P receptors in ALS cords 5'21. Cervical and thoracic spinal cords were obtained at autopsy from 4 patients who had ALS, and from 5 individuals who had no evidence of neurological disease. The ALS patients ranged in age from 43 to 71 years (63.8 + 12.0, mean + S.D.) and all had neuropathological verification of their diagnoses and involvement of spinal motoneurons. The death to freezing intervals ranged from 2.0 to 19.8 h (9.9 + 7.6). Individuals with no clinical or neuropathological evidence of neurological disease ranged from 59 to 76 years of age (67.8 +
A
B
6.6) and had died following myocardial infarction, congestive heart failure or bronchopneumonia. Death to freezing intervals for this group ranged from 4.3 to 23 h (13.0 + 7.1). All spinal cords were stored at -70°C until analysis. [3H]l-(1-(2-thienyl)-cyclohexyl) piperidine (TCP) (45 Ci/mmol), [3H]kainate (60 Ci/mmol) and [3H]AMPA (27.6 Ci/mmol) were purchased from New England Nuclear, Dupont Canada, Toronto, Ontario. Hyperfilm-3H and microscales were obtained from Amersham Canada, Oakville, Ontario. Unlabelled excitatory amino acids and all other reagents were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Canada (Montreal, Quebec). All frozen samples were sectioned and tissue slices (20 #m) were cut using a cryostat at -17°C, thaw-mounted onto gelatin/chrome alum coated slides, air dried under vacuum at 4°C overnight and then stored at -70°C. The
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Fig. 1. Photomicrographs of the autoradiographic distribution of [3H]TCP/NMDA (A), [3H]kainate (B) and [3H]AMPA/quisqualate (C) binding sites in human spinal cord at the cervical level. D, E and F represent non-specific binding in adjacent sections, respectively. Note that while [3H]TCP binding is located in substantia gelatinosa/lamina II of the dorsal horn as well as in the ventral horn (most likely motoneurons), [3H]kainate and [3H]AMPA binding are mostly restricted to the superficial laminae of the cord. Bar = 4 mm. DH, dorsal horn; VH, ventral horn; II and X, laminae of the spinal cord.
171
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.~. Fig. 2. Excitatory amino acid (EAA) binding sites in control and amyotrophiclateral sclerosis (ALS) spinal cords. While the amount of [3H]TCP/NMDA binding is decreased in both lamina II and ventral horn in ALS cords (A), [3H]AMPAJquisqualatebinding is unaltered throughout the cord in ALS patients (B). *P < 0.025; **P < 0.02.
NMDA receptor was investigated using [3H]TCP, a specific ligand of the phencyclidine NMDA associated site. Sections were incubated for 45 min at 0°C in presence of 40 nM [3H]TCP in a buffer containing 5 mM Tris pH 7.44. Non-specific binding was evaluated by incubating adjacent sections with 10/~M phencyclidine. The AMPA binding site of the quisqualate receptor was evaluated using [3H]AMPA (50 nM), sections being incubated for 45 min at 2°C in a buffer containing 50 mM Tris, 100 mM KSCN pH 7.29. Non-specific binding was determined in the presence of 100/~M AMPA. Finally, kainate binding was determined by incubating sections for 90 min at 3°C in a buffer containing 20 nM [3H]kainate, 50 mM Tris acetate pH 7.115. Non-specific binding was determined in the presence of 10 aM unlabelled kainate, Autoradiograms were generated by apposing labelled sections to film for 2-4 weeks and the optical density of film autoradiograms was analyzed by microcomputer image analysis (MCID, St. Catharines, Ontario), using tri-
tium standard microscales for film calibration. Under these experimental conditions, the specific binding of [3H]TCP, [3H]AMPA and [3H]kainate represented 40%, 90% and 50% of total binding, respectively. Values are expressed as mean + S.E.M. Student's t-test was used for statistical analysis, P < 0.05 being considered significant. In normal control spinal cord, NMDA/[aH]TCP, [3H]AMPA and [3H]kainate receptor binding sites were almost exclusively concentrated in the grey matter (Fig. 1). NMDA/[3H]TCP sites were localized both in the ventral and dorsal horns with higher densities observed in lamina II than other laminae including the ventral horn (Fig. 2). [3H]AMPA and [3H]kainate receptor sites were present almost exclusively in the substantia gelatinosa of the dorsal horn (lamina II, Fig. 1). In ALS patients, the distribution of these 3 classes of excitatory amino acid binding sites was unchanged. However, as shown in Fig. 2, [3H]TCP binding was decreased significantly in both dorsal (lamina II; 141 + 21 vs. 78 + 7 fmol/mg tissue wet weight, P < 0.025; control vs. ALS cords) and ventral (118 _+ 11 vs. 70 _+ 11 fmol/mg tissue, wet weight, P < 0.02; in control vs. ALS) horns in ALS cords. Decreases in [3H]TCP binding were not correlated to the age of the subject (not shown). [3H]kainate binding was possibly decreased in the substantia gelatinosa (9 + 2 vs. 5.8 _+ 3 fmol/mg tissue weight, control vs. ALS) but the limited sample size (n = 3) of ALS cords did not allow for a statistical evaluation. [3H]AMPA binding was unaltered (e.g. lamina II, 89 + 6 vs. 87 + 6 fmol/mg tissue wet weight; control vs. ALS) in ALS tissues (Fig. 2) revealing the relative specificity of the EAA-receptor modifications in ALS. The present results on the distribution of NMDA, AMPA and kainate receptors in normal human spinal cord are in agreement with some recent reports 6'17. All 3 types of receptor appear to be found predominantly within the dorsal horn and especially within lamina II. This localization has been attributed to the possible involvement of these receptors in primary afferent neurotransmission6. Foci of high [3H]TCP/NMDA binding were also observed within the ventral horn and some of this binding localizes to regions consistent with motoneuron cell bodies 17. In ALS, we observed a reduction in [3H]TCP/NMDA receptor binding in the dorsal and ventral horns at both cervical and thoracic levels. [3H]kainate binding was also possibly reduced in ALS, but due to the limited number of ALS spinal cords available and low amounts of specific [3H]kainate binding (few fmol/mg tissue, wet weight) in the normal control cord definite conclusions could not be made concerning this point. As mammalian motoneurons possess NMDA recep-
172 tors 7, and since all of the spinal cords from A L S patients had m o t o n e u r o n losses, some of the reductions in [3H]TCP/NMDA r e c e p t o r binding in the ventral horn are likely due to the death of N M D A receptor-bearing motoneurons. Surprisingly, we also observed a significant reduction in [ 3 H ] T C P / N M D A r e c e p t o r binding in the dorsal horn; decreases being nearly as p r o n o u n c e d as those seen in the ventral horn. Since afferent pathways are usually spared in A L S , both clinically and pathologically3, it appears unlikely that the reductions in N M D A r e c e p t o r binding are due to cell loss in the dorsal horn. H o w e v e r , recent observations indicate that N-acetyl-aspartate, and the excitatory dipeptide N-acetylaspartyl glutamate are significantly reduced in both the dorsal and ventral horns of cervical spinal cords from A L S patients 2°. This might indicate that some functional alterations occur both in the dorsal and ventral horns in A L S . Alternatively, a possible explanation is that a down-regulation in N M D A and possibly kainate receptor n u m b e r could occur in A L S , possibly due to the continued presence of increased amounts of a N M D A - (or
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kainate) like agonist/excitotoxin in various spinal cord regions in A L S . The present results a p p e a r to be the first autoradiographic investigation of E A A binding in the spinal cord of A L S patients. Selective decreases in [3H]TCP/NMDA receptor binding are noted in both dorsal and ventral horn regions. [3H]kainate binding may also be altered but only in the dorsal horn while [ 3 H ] A M P A binding sites are most likely spared. The preferential alteration in the N M D A r e c e p t o r binding suggests that if an excitotoxic mechanism is involved in the pathogenesis of A L S , then the N M D A receptor would be a likely candidate as a m e d i a t o r of the m o t o n e u r o n loss.
We thank Dr. Wallace W. Tourtellotte of the National Neurological Research Tissue Bank, the Canadian Brain Tissue Bank, and Drs. K. Berry and W. Moore for spinal cord tissues. We acknowledge the late T. Perry for his advice and support. This work was supported by Fonds de la Recherche en Sant6 du Qu6bec (FRSQ) and the Medical Research Council of Canada (MRC). R.Q. is a Chercheur-Boursier, I.C. a Fellow of the FRSQ and C.K. is a MRC Scholar.
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