Brain Research, 529 (1990) 339-344
339
Elsevier
BRES 24312
Antibodies to glutamate and aspartate recognize non-endogenous ligands for excitatory amino acid receptors Peter Petrusz 1, Susan L. Van Eyck 1, Richard J. Weinberg 1 and Aldo Rustioni L2 Departments of 1Cell Biology and Anatomy, and 2Physiology, University of North Carolina, Chapel Hill, NC 27599 (U.S.A.)
(Accepted 19 June 1990) Key words: Glutamate; Aspartate; Antiserum; Receptor; Quisqualate; Kainate; N-Methyi-D-aspartate
Antisera raised against glutaraldehyde conjugates of glutamate (Glu) and aspartate (Asp) with hemocyanin proved highly specific for their respective unconjugated amino acid haptens when tested in immunocytochemicalblocking experiments on sections of the rat spinal cord. In addition, immunocytochemicalstaining by the Glu antiserum was effectivelyblocked by quisqualate but not by kainate or N-methyl-o-aspartate (NMDA); staining with the Asp antiserum was effectivelyblocked by kainate, to a lesser extent by quisqualate, and was not affected by NMDA. These results may be explained by assuming that the specific binding regions of the antibodies tested share certain recognition characteristics with endogenous binding sites or receptors for excitatory amino acids and their agonists.
Antisera raised in our laboratories against glutamate (Glu) and aspartate (Asp) have been shown to be highly specific for their respective amino acids 1°. Nevertheless, the identity of the antigens recognized by these antibodies in neural tissue remains uncertain. For example, in addition to Glu and Asp, they may recognize dipeptides that have the respective amino acid in a C-terminal position. Among these, N-acetyI-Asp-Glu (NAAG) is present in brain tissue and has been proposed as a neurotransmitter 3. At least 3 types of excitatory amino acid (EAA) receptors have been identified pharmacologically: the quisqualate (Quis)-, kainate (KA)-, and the N-methylD-aspartate (NDMA)-preferring receptors 17'2s. However, Quis, KA, and NMDA do not occur in the central nervous system (CNS), and the possibility remains open that endogenous ligands other than Glu and Asp bind to these receptors 7"~3"14. As a step toward the identification of putative endogenous ligands specific for these receptors, we tested whether our antisera recognized the non-endogenous compounds Quis, KA, and NMDA. A brief account of this study has been published previously26. All chemicals were purchased, unless stated otherwise, from Sigma Chemical Co. (St. Louis, MO). Four male Sprague-Dawley rats (200-300 g, Zivic-Miller Laboratories, Zelienople, PA) were perfused at room temperature with a mixture of 2% carbodiimide (Cyanamide),
0.2% picric acid and 0.3% glutaraldehyde dissolved in 0.1 M, pH 7.4 phosphate buffer (PB), followed by 4% paraformaldehyde in PB. Spinal cords were removed and postfixed overnight in 4% paraformaldehyde at 4 °C. Forty-pm thick vibratome sections were cut in a chilled saline bath and stored refrigerated in PB. The two primary antisera used in these studies, anti-Glu 482a and anti-Asp 484a, were produced in rabbits against each amino acid conjugated to keyhole limpet hemocyanin (Calbiochem, San Diego, CA) with glutaraldehyde, as described by Storm-Mathisen et al. 25. A detailed account of the characterization of these antisera has been published 1°. The optimal dilution for the present studies, established in preliminary experiments, was 1:60,000 for both antibodies. Aliquots of the optimally diluted antisera were prepared to contain 0.01, 0.1, 1, and 10 mM (final concentrations) of free (unconjugated) Glu, Asp, Quis, KA, and NMDA. The Quis and KA preparations, tested by thin-layer chromatography, were >99% free of contamination with Glu or other amino acids (Sigma Chemical Co., personal communication). Sections were incubated overnight in these antiserum-agonist combinations, or with antiserum only (as controls). They were subsequently processed for horseradish peroxidase immunohistochemistry with the avidinbiotin complex method (Vector Laboratories, Inc., Burlingame, CA) using 3,3"-diaminobenzidine tetrahydrochloride as chromogen.
Correspondence: P. Petrusz, Department of Cell Biologyand Anatomy, University of North Carolina, Box 7090, 108 Taylor Hall, Chapel Hill, NC 27599, U.S.A.
0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
Quis
KA
NMDA
Asp
10.0
1.0
0.1
0.01
mM
Fig. 1. Vibratome sections of the rat spinal cord stained with the anti-Glu serum (1:60,000) without (single section at the left) and with the addition of increasing concentrations (shown at the right) of the 5 compounds indicated at the top. ×8.
Control
Anti-Glu
Glu
Quis
KA
NMDA
Asp
10.0
1.0
0.1
0.01
mM
Fig. 2. Vibratome sections of the rat spinal cord stained with the anti-Asp serum (1:60,000) without (single section at the left) and with the addition of increasing concentrations (shown at the right) of the 5 compounds indicated at the top. x8.
Control
Anti-Asp
Glu
L~
342 A n t i - G l u Blocking
Anti-Asp Blocking
1,0-
1.0
0.8-
0.8
"0 Glu NMDA
Glu ~ ,.Q
100" > 100" > 100"
> 100" 11 > 100" 3.5 0.03
* Extrapolated values.
In all animals, staining was denser in gray matter than in white matter with both antisera. With the Glu antiserum, staining was relatively uniform within the gray matter, with the exception of dense staining of cells and neuropil in superficial dorsal horn laminae 18"29. Increasing concentrations of Glu progressively blocked the reaction (Fig. 1). Quis blocked the staining in a similar manner and was even more effective than Glu at the lower concentrations; none of the other compounds blocked the staining with the Glu antiserum to any appreciable extent (Fig. 1). Staining with the anti-Asp serum was most prominent in the large motoneurons of the ventral horn. Increasing concentrations of Asp produced progressive blocking of the staining (Fig. 2). A similar, but less pronounced blocking was seen with KA, whereas the other compounds did not seem to have an appreciable effect. Normalized absorbances resulting from densitometric analysis of a complete series of spinal cord sections from one rat confirmed that immunostaining with anti-Glu was blocked by Quis, but not significantly affected by Asp, KA or N M D A (Fig. 3). In contrast, staining with anti-Asp was blocked by Asp and KA only, partially blocked by Quis, but not affected by Glu or NMDA. These results were quantitatively summarized by fitting least-squares lines to the data of Fig. 3, permitting estimation of the blocking concentrations required to give 50% inhibition of the immunostaining (Table I). These ECs0 values summarize the above results: the Glu antiserum is highly specific for Giu and Quis, while the Asp antiserum is highly specific for Asp and KA,
343 recognizes Quis to a lesser degree, and does not recognize Glu or N M D A in the dose ranges tested. These e x p e r i m e n t s confirm that the antibodies to Glu and A s p selectively recognize their respective haptens and do not require an amino acid-glutaraldehyde-protein conjugate for recognition 2"t5'16'2°'27. O u r results also d e m o n s t r a t e , for the first time, that antibodies against Glu and A s p possess certain 'receptorlike' characteristics: they preferentially recognize, and distinguish b e t w e e n , n o n - e n d o g e n o u s ligands which are specific for subclasses of E A A receptors. A l t h o u g h this observation m a y be coincidental for the particular antibodies studied, it is consistent with the general concept that a n t i b o d y binding sites can mimic cell surface receptors 23. F o r e x a m p l e , antibodies raised against h a l o p e r i d o l 4, m o r p h i n e s and benzodiazepines 6 have been shown to possess receptor-like binding characteristics. A n t i - i d i o t y p e s H (antibodies directed against the antigenbinding domains of antibodies raised against natural or
1 Amit, T., Barkey, R.J., Gavish, M. and Youdim, M.B.H., Anti-idiotypic antibodies raised against anti-prolactin (PRL) antibodies recognize the PRL receptor, Endocrinology, 118 (1986) 835-843. 2 Beitz, A.J., Larson, A.A., Monaghan, P., Altschuler R.A., Mullett, M.M. and Madl, J.E., Immunohistochemical localization of glutamate, glutaminase and aspartate aminotransferase in neurons of the pontine nuclei of the rat, Neuroscience, 17 (1986) 741-753. 3 Blakely, R.D. and Coyle, J.T., The neurobiology of Nacetylaspartylglutamate, Int. Rev. Neurobiol., 30 (1988) 39-100. 4 Bolger, M.B., Sherman, M.A. and Linthicum, D.S., Molecular characterization of anti-neuroleptic idiotypes and anti-idiotypes. In D.S. Linthicum and N.R. Farid (Eds.), Anti-ldiotypes, Receptors, and Molecular Mimicry, Springer, New York, 1988, pp. 93-133. 5 Couraud, J.Y., Escher, E., Regoli, D., Imhoff, V., Rossignol, B. and Pradelles, P., Anti-substance P anti-idiotypic antibodies. Characterization and biological activities, J. Biol. Chem., 260 (1985) 9461-9469. 6 De Bias, A.L., Park, D., Victorica, J. and Sangameshwaran, L., Monoclonal antibodies to benzodiazepines. Their application in making anti-idiotypic antibodies to the benzodiazepine receptor and in revealing the existence of benzodiazepines and benzodiazepine-like molecules in the brain. In D.S. Linthicum and N.R. Farid (Eds.), Anti-ldiotypes, Receptors, and Molecular Mimicry, Springer, New York, 1988, pp. 155-177. 7 Do, K.Q., Herding, P.L., Streit, P. and Cu6nod, M., Release of neuroactive substances: homocysteic acid as an endogenous agonist of the NMDA receptor, J. Neural Transm., 72 (1988) 185-190. 8 Glasel, J.A., Opiate receptors and molecular shapes. In D.S. Linthicum and N.R. Farid (Eds.), Anti-ldiotypes, Receptors, and Molecular Mimicry, Springer, New York, 1988, pp. 136-153. 9 Guillet, J.G., Chamat, S., Hoebeke, J. and Strosberg, A.D., Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-fl-adrenergic ligand antibody, J. Immunol. Methods, 74 (1984) 163-171. 10 Hepler, J.R., Toomim, C.S., McCarthy, K.D., Conti, F., Battaglia, G., Rustioni, A. and Petrusz, P., Characterization of antisera to glutamate and aspartate, J. Histochem. Cytochem., 36 (1988) 13-22. 11 Jerne, N.K., Towards a network theory of the immune system,
synthetic r e c e p t o r ligands) have b e e n shown to recognize receptors for prolactin ~, insulin 22, substance pS, acetylcholine 12, d o p a m i n e 21, n o r e p i n e p h r i n e 9 and m o r p h i n e 19. T h e validity of this general concept (i.e. r e c e p t o r mimicry by antibodies) remains to be d e m o n strated for antisera to Glu and A s p . The failure of our antisera to recognize N M D A m a y be i n t e r p r e t e d as an indication of the existence of an additional, as yet unidentified, e n d o g e n o u s E A A receptor agonist, conformationally similar to N M D A . O u r results suggest that such a putative e n d o g e n o u s ligand for the N M D A r e c e p t o r could be d e t e c t e d with the help of antibodies that selectively recognize N M D A , but not Glu and A s p .
We are grateful to K.D. Phend and G. Grossman for excellent technical assistance. This work was supported by USPHS Grants NS12440 and NS16264 (A.R.), NS27679, and by the University of North Carolina Research Council (P.P.).
Ann. lmmunol. (Paris), 125C (1974) 373-389. 12 Leiber, D., Harbon, S., Guillet, J.G., Andre, C. and Strosberg, A.D., Monoclonal antibodies to purified muscarinic receptor display agonist-like activity, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 4331-4334. 13 Lodge, D., Modulating glutamate pharmacology, Trends Pharmacol. Sci., 8 (1987) 243-244. 14 Luini, A., Tal, N., Goldberg, O. and Teichberg, V.I., An evaluation of selected brain constituents as putative excitatory neurotransmitters, Brain Research, 324 (1984) 271-277. 15 Madl, J.E., Beitz, A.J., Johnson, R.L. and Larson, A.A., Monoclonal antibodies specific for fixative-modified aspartate: immunocytochemical localization in the rat CNS, J. Neurosci., 7 (1987) 2639-2650. 16 Madl, J.E., Larson, A.A. and Beitz, A.J., Monoclonal antibody specific for carbodiimide-fixed glutamate: immunocytochemical localization in the rat CNS, J. Histochem. Cytochem., 34 (1986) 317-326. 17 McLennan, H., Receptors for the excitatory amino acids in the mammalian central nervous system, Progr. Neurobiol., 20 (1983) 251-271. 18 Miller, K.E., Clements, J.R., Larson, A.A. and Beitz, A.J., Organization of glutamate-like immunoreactivity in the rat superficial dorsal horn: light and electron microscopic observations, Synapse, 2 (1988) 28-36. 19 Ng, D.S. and Isom, G.E., Binding of antimorphine antiidiotypic antibodies to opiate receptors, Eur. J. Pharmacol., 102 (1984) 187-190. 20 Ottersen, O.P., Storm-Mathisen, J., Madsen, S., Skumlien, S. and Stromhaug, J., Evaluation of the immunocytochemical method for amino acids, Med. Biol., 64 (1986) 147-158. 21 Schreiber, M., Fogelfeld, L., Souroujon, M.C., Kohen, E and Fuchs, S., Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors, Life Sci., 33 (1983) 1519-1526. 22 Schechter, Y., Elias, D., Maron, R. and Cohen, I.R., Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies, J. Biol. Chem., 259 (1984) 6411-6415. 23 Sege, K. and Peterson, P.A., Use of anti-idiotypic antibodies as cell surface probes, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2443-2447. 24 Shiosaka, S., Kiyama, H., Wanaka, A. and Tohyama, M., A
344 new method for producing a specific and high titre antibody against glutamate using colloidal gold as a carrier, Brain Research, 382 (1986) 399-403. 25 Storm-Mathisen, J., Leknes, A.K., Bore, A.T., Vaaland, J.L., Edminson, P., Haug, EM.S. and Ottersen, O.P., First visualization of glutamate and GABA in neurons by immunocytochemistry, Nature (Lond.), 301 (1983) 517-520. 26 Van Eyck, S.L., Weinberg, R.J., Rustioni, A. and Petrusz, P., Antibodies to glutamate and aspartate selectively recognize ligands for subtypes of excitatory amino acid receptors, J. Histochem. Cytochem., 37 (1989) 927. 27 Wanaka, A., Shiotami, Y., Kiyama, H., Matsuyama, T.,
Kamada, T., Shiosaka, S. and Tohyama, M., Glutamate-like immunoreactive structures in primary sensory neurons in the rat detected by a specific antiserum against glutamate, Exp. Brain Res., 65 (1987) 691-694. 28 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Ann. Rev. PharrnacoL Toxicol., 21 (1981) 165-204. 29 Weinberg, R.J., Conti, E, Van Eyck, S.L., Petrusz, P. and Rustioni, A., Glutamate immunoreactivity in superficial laminae of rat dorsal horn and spinal trigeminal nucleus. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York. 1987, pp. 173-176.
339
Elsevier
BRES 24312
Antibodies to glutamate and aspartate recognize non-endogenous ligands for excitatory amino acid receptors Peter Petrusz 1, Susan L. Van Eyck 1, Richard J. Weinberg 1 and Aldo Rustioni L2 Departments of 1Cell Biology and Anatomy, and 2Physiology, University of North Carolina, Chapel Hill, NC 27599 (U.S.A.)
(Accepted 19 June 1990) Key words: Glutamate; Aspartate; Antiserum; Receptor; Quisqualate; Kainate; N-Methyi-D-aspartate
Antisera raised against glutaraldehyde conjugates of glutamate (Glu) and aspartate (Asp) with hemocyanin proved highly specific for their respective unconjugated amino acid haptens when tested in immunocytochemicalblocking experiments on sections of the rat spinal cord. In addition, immunocytochemicalstaining by the Glu antiserum was effectivelyblocked by quisqualate but not by kainate or N-methyl-o-aspartate (NMDA); staining with the Asp antiserum was effectivelyblocked by kainate, to a lesser extent by quisqualate, and was not affected by NMDA. These results may be explained by assuming that the specific binding regions of the antibodies tested share certain recognition characteristics with endogenous binding sites or receptors for excitatory amino acids and their agonists.
Antisera raised in our laboratories against glutamate (Glu) and aspartate (Asp) have been shown to be highly specific for their respective amino acids 1°. Nevertheless, the identity of the antigens recognized by these antibodies in neural tissue remains uncertain. For example, in addition to Glu and Asp, they may recognize dipeptides that have the respective amino acid in a C-terminal position. Among these, N-acetyI-Asp-Glu (NAAG) is present in brain tissue and has been proposed as a neurotransmitter 3. At least 3 types of excitatory amino acid (EAA) receptors have been identified pharmacologically: the quisqualate (Quis)-, kainate (KA)-, and the N-methylD-aspartate (NDMA)-preferring receptors 17'2s. However, Quis, KA, and NMDA do not occur in the central nervous system (CNS), and the possibility remains open that endogenous ligands other than Glu and Asp bind to these receptors 7"~3"14. As a step toward the identification of putative endogenous ligands specific for these receptors, we tested whether our antisera recognized the non-endogenous compounds Quis, KA, and NMDA. A brief account of this study has been published previously26. All chemicals were purchased, unless stated otherwise, from Sigma Chemical Co. (St. Louis, MO). Four male Sprague-Dawley rats (200-300 g, Zivic-Miller Laboratories, Zelienople, PA) were perfused at room temperature with a mixture of 2% carbodiimide (Cyanamide),
0.2% picric acid and 0.3% glutaraldehyde dissolved in 0.1 M, pH 7.4 phosphate buffer (PB), followed by 4% paraformaldehyde in PB. Spinal cords were removed and postfixed overnight in 4% paraformaldehyde at 4 °C. Forty-pm thick vibratome sections were cut in a chilled saline bath and stored refrigerated in PB. The two primary antisera used in these studies, anti-Glu 482a and anti-Asp 484a, were produced in rabbits against each amino acid conjugated to keyhole limpet hemocyanin (Calbiochem, San Diego, CA) with glutaraldehyde, as described by Storm-Mathisen et al. 25. A detailed account of the characterization of these antisera has been published 1°. The optimal dilution for the present studies, established in preliminary experiments, was 1:60,000 for both antibodies. Aliquots of the optimally diluted antisera were prepared to contain 0.01, 0.1, 1, and 10 mM (final concentrations) of free (unconjugated) Glu, Asp, Quis, KA, and NMDA. The Quis and KA preparations, tested by thin-layer chromatography, were >99% free of contamination with Glu or other amino acids (Sigma Chemical Co., personal communication). Sections were incubated overnight in these antiserum-agonist combinations, or with antiserum only (as controls). They were subsequently processed for horseradish peroxidase immunohistochemistry with the avidinbiotin complex method (Vector Laboratories, Inc., Burlingame, CA) using 3,3"-diaminobenzidine tetrahydrochloride as chromogen.
Correspondence: P. Petrusz, Department of Cell Biologyand Anatomy, University of North Carolina, Box 7090, 108 Taylor Hall, Chapel Hill, NC 27599, U.S.A.
0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
Quis
KA
NMDA
Asp
10.0
1.0
0.1
0.01
mM
Fig. 1. Vibratome sections of the rat spinal cord stained with the anti-Glu serum (1:60,000) without (single section at the left) and with the addition of increasing concentrations (shown at the right) of the 5 compounds indicated at the top. ×8.
Control
Anti-Glu
Glu
Quis
KA
NMDA
Asp
10.0
1.0
0.1
0.01
mM
Fig. 2. Vibratome sections of the rat spinal cord stained with the anti-Asp serum (1:60,000) without (single section at the left) and with the addition of increasing concentrations (shown at the right) of the 5 compounds indicated at the top. x8.
Control
Anti-Asp
Glu
L~
342 A n t i - G l u Blocking
Anti-Asp Blocking
1,0-
1.0
0.8-
0.8
"0 Glu NMDA
Glu ~ ,.Q
100" > 100" > 100"
> 100" 11 > 100" 3.5 0.03
* Extrapolated values.
In all animals, staining was denser in gray matter than in white matter with both antisera. With the Glu antiserum, staining was relatively uniform within the gray matter, with the exception of dense staining of cells and neuropil in superficial dorsal horn laminae 18"29. Increasing concentrations of Glu progressively blocked the reaction (Fig. 1). Quis blocked the staining in a similar manner and was even more effective than Glu at the lower concentrations; none of the other compounds blocked the staining with the Glu antiserum to any appreciable extent (Fig. 1). Staining with the anti-Asp serum was most prominent in the large motoneurons of the ventral horn. Increasing concentrations of Asp produced progressive blocking of the staining (Fig. 2). A similar, but less pronounced blocking was seen with KA, whereas the other compounds did not seem to have an appreciable effect. Normalized absorbances resulting from densitometric analysis of a complete series of spinal cord sections from one rat confirmed that immunostaining with anti-Glu was blocked by Quis, but not significantly affected by Asp, KA or N M D A (Fig. 3). In contrast, staining with anti-Asp was blocked by Asp and KA only, partially blocked by Quis, but not affected by Glu or NMDA. These results were quantitatively summarized by fitting least-squares lines to the data of Fig. 3, permitting estimation of the blocking concentrations required to give 50% inhibition of the immunostaining (Table I). These ECs0 values summarize the above results: the Glu antiserum is highly specific for Giu and Quis, while the Asp antiserum is highly specific for Asp and KA,
343 recognizes Quis to a lesser degree, and does not recognize Glu or N M D A in the dose ranges tested. These e x p e r i m e n t s confirm that the antibodies to Glu and A s p selectively recognize their respective haptens and do not require an amino acid-glutaraldehyde-protein conjugate for recognition 2"t5'16'2°'27. O u r results also d e m o n s t r a t e , for the first time, that antibodies against Glu and A s p possess certain 'receptorlike' characteristics: they preferentially recognize, and distinguish b e t w e e n , n o n - e n d o g e n o u s ligands which are specific for subclasses of E A A receptors. A l t h o u g h this observation m a y be coincidental for the particular antibodies studied, it is consistent with the general concept that a n t i b o d y binding sites can mimic cell surface receptors 23. F o r e x a m p l e , antibodies raised against h a l o p e r i d o l 4, m o r p h i n e s and benzodiazepines 6 have been shown to possess receptor-like binding characteristics. A n t i - i d i o t y p e s H (antibodies directed against the antigenbinding domains of antibodies raised against natural or
1 Amit, T., Barkey, R.J., Gavish, M. and Youdim, M.B.H., Anti-idiotypic antibodies raised against anti-prolactin (PRL) antibodies recognize the PRL receptor, Endocrinology, 118 (1986) 835-843. 2 Beitz, A.J., Larson, A.A., Monaghan, P., Altschuler R.A., Mullett, M.M. and Madl, J.E., Immunohistochemical localization of glutamate, glutaminase and aspartate aminotransferase in neurons of the pontine nuclei of the rat, Neuroscience, 17 (1986) 741-753. 3 Blakely, R.D. and Coyle, J.T., The neurobiology of Nacetylaspartylglutamate, Int. Rev. Neurobiol., 30 (1988) 39-100. 4 Bolger, M.B., Sherman, M.A. and Linthicum, D.S., Molecular characterization of anti-neuroleptic idiotypes and anti-idiotypes. In D.S. Linthicum and N.R. Farid (Eds.), Anti-ldiotypes, Receptors, and Molecular Mimicry, Springer, New York, 1988, pp. 93-133. 5 Couraud, J.Y., Escher, E., Regoli, D., Imhoff, V., Rossignol, B. and Pradelles, P., Anti-substance P anti-idiotypic antibodies. Characterization and biological activities, J. Biol. Chem., 260 (1985) 9461-9469. 6 De Bias, A.L., Park, D., Victorica, J. and Sangameshwaran, L., Monoclonal antibodies to benzodiazepines. Their application in making anti-idiotypic antibodies to the benzodiazepine receptor and in revealing the existence of benzodiazepines and benzodiazepine-like molecules in the brain. In D.S. Linthicum and N.R. Farid (Eds.), Anti-ldiotypes, Receptors, and Molecular Mimicry, Springer, New York, 1988, pp. 155-177. 7 Do, K.Q., Herding, P.L., Streit, P. and Cu6nod, M., Release of neuroactive substances: homocysteic acid as an endogenous agonist of the NMDA receptor, J. Neural Transm., 72 (1988) 185-190. 8 Glasel, J.A., Opiate receptors and molecular shapes. In D.S. Linthicum and N.R. Farid (Eds.), Anti-ldiotypes, Receptors, and Molecular Mimicry, Springer, New York, 1988, pp. 136-153. 9 Guillet, J.G., Chamat, S., Hoebeke, J. and Strosberg, A.D., Production and detection of monoclonal anti-idiotype antibodies directed against a monoclonal anti-fl-adrenergic ligand antibody, J. Immunol. Methods, 74 (1984) 163-171. 10 Hepler, J.R., Toomim, C.S., McCarthy, K.D., Conti, F., Battaglia, G., Rustioni, A. and Petrusz, P., Characterization of antisera to glutamate and aspartate, J. Histochem. Cytochem., 36 (1988) 13-22. 11 Jerne, N.K., Towards a network theory of the immune system,
synthetic r e c e p t o r ligands) have b e e n shown to recognize receptors for prolactin ~, insulin 22, substance pS, acetylcholine 12, d o p a m i n e 21, n o r e p i n e p h r i n e 9 and m o r p h i n e 19. T h e validity of this general concept (i.e. r e c e p t o r mimicry by antibodies) remains to be d e m o n strated for antisera to Glu and A s p . The failure of our antisera to recognize N M D A m a y be i n t e r p r e t e d as an indication of the existence of an additional, as yet unidentified, e n d o g e n o u s E A A receptor agonist, conformationally similar to N M D A . O u r results suggest that such a putative e n d o g e n o u s ligand for the N M D A r e c e p t o r could be d e t e c t e d with the help of antibodies that selectively recognize N M D A , but not Glu and A s p .
We are grateful to K.D. Phend and G. Grossman for excellent technical assistance. This work was supported by USPHS Grants NS12440 and NS16264 (A.R.), NS27679, and by the University of North Carolina Research Council (P.P.).
Ann. lmmunol. (Paris), 125C (1974) 373-389. 12 Leiber, D., Harbon, S., Guillet, J.G., Andre, C. and Strosberg, A.D., Monoclonal antibodies to purified muscarinic receptor display agonist-like activity, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 4331-4334. 13 Lodge, D., Modulating glutamate pharmacology, Trends Pharmacol. Sci., 8 (1987) 243-244. 14 Luini, A., Tal, N., Goldberg, O. and Teichberg, V.I., An evaluation of selected brain constituents as putative excitatory neurotransmitters, Brain Research, 324 (1984) 271-277. 15 Madl, J.E., Beitz, A.J., Johnson, R.L. and Larson, A.A., Monoclonal antibodies specific for fixative-modified aspartate: immunocytochemical localization in the rat CNS, J. Neurosci., 7 (1987) 2639-2650. 16 Madl, J.E., Larson, A.A. and Beitz, A.J., Monoclonal antibody specific for carbodiimide-fixed glutamate: immunocytochemical localization in the rat CNS, J. Histochem. Cytochem., 34 (1986) 317-326. 17 McLennan, H., Receptors for the excitatory amino acids in the mammalian central nervous system, Progr. Neurobiol., 20 (1983) 251-271. 18 Miller, K.E., Clements, J.R., Larson, A.A. and Beitz, A.J., Organization of glutamate-like immunoreactivity in the rat superficial dorsal horn: light and electron microscopic observations, Synapse, 2 (1988) 28-36. 19 Ng, D.S. and Isom, G.E., Binding of antimorphine antiidiotypic antibodies to opiate receptors, Eur. J. Pharmacol., 102 (1984) 187-190. 20 Ottersen, O.P., Storm-Mathisen, J., Madsen, S., Skumlien, S. and Stromhaug, J., Evaluation of the immunocytochemical method for amino acids, Med. Biol., 64 (1986) 147-158. 21 Schreiber, M., Fogelfeld, L., Souroujon, M.C., Kohen, E and Fuchs, S., Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors, Life Sci., 33 (1983) 1519-1526. 22 Schechter, Y., Elias, D., Maron, R. and Cohen, I.R., Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies, J. Biol. Chem., 259 (1984) 6411-6415. 23 Sege, K. and Peterson, P.A., Use of anti-idiotypic antibodies as cell surface probes, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2443-2447. 24 Shiosaka, S., Kiyama, H., Wanaka, A. and Tohyama, M., A
344 new method for producing a specific and high titre antibody against glutamate using colloidal gold as a carrier, Brain Research, 382 (1986) 399-403. 25 Storm-Mathisen, J., Leknes, A.K., Bore, A.T., Vaaland, J.L., Edminson, P., Haug, EM.S. and Ottersen, O.P., First visualization of glutamate and GABA in neurons by immunocytochemistry, Nature (Lond.), 301 (1983) 517-520. 26 Van Eyck, S.L., Weinberg, R.J., Rustioni, A. and Petrusz, P., Antibodies to glutamate and aspartate selectively recognize ligands for subtypes of excitatory amino acid receptors, J. Histochem. Cytochem., 37 (1989) 927. 27 Wanaka, A., Shiotami, Y., Kiyama, H., Matsuyama, T.,
Kamada, T., Shiosaka, S. and Tohyama, M., Glutamate-like immunoreactive structures in primary sensory neurons in the rat detected by a specific antiserum against glutamate, Exp. Brain Res., 65 (1987) 691-694. 28 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Ann. Rev. PharrnacoL Toxicol., 21 (1981) 165-204. 29 Weinberg, R.J., Conti, E, Van Eyck, S.L., Petrusz, P. and Rustioni, A., Glutamate immunoreactivity in superficial laminae of rat dorsal horn and spinal trigeminal nucleus. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York. 1987, pp. 173-176.
