Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues: findings with a novel liquid phase radioimmunoassay

Angela S Guarda, Michael B. Robinson, Laure Ory-Lavollée, Gian Luigi Forloni, Randy D. Blakely, Joseph T. Coyle

Research output: Contribution to journalArticle

Abstract

Antibodies were raised in rabbits against the neuropeptide N-acetyl-l-aspartyl-l-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100% of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons. We have also used this RIA to identify NAAG-LIR in striated muscle and other peripheral tissues.

Original languageEnglish (US)
Pages (from-to)223-231
Number of pages9
JournalMolecular Brain Research
Volume3
Issue number3
DOIs
StatePublished - 1988

Fingerprint

Radioimmunoassay
Glutamic Acid
Brain
N-acetyl-1-aspartylglutamic acid
Tissue Extracts
Immune Sera
Rabbits
Red Nucleus
Serotonergic Neurons
Carbodiimides
Pressure
Adrenergic Neurons
Cholinergic Neurons
Antibody Affinity
Locus Coeruleus
Striated Muscle
Antibodies
Bovine Serum Albumin
Neuropeptides
Aspartic Acid

Keywords

  • Glutamate
  • Immunocytochemistry
  • N-Acetyl-aspartate
  • N-Acetyl-aspartyl-glutamate
  • Optic tract
  • Radioimmunoassay

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues : findings with a novel liquid phase radioimmunoassay. / Guarda, Angela S; Robinson, Michael B.; Ory-Lavollée, Laure; Forloni, Gian Luigi; Blakely, Randy D.; Coyle, Joseph T.

In: Molecular Brain Research, Vol. 3, No. 3, 1988, p. 223-231.

Research output: Contribution to journalArticle

Guarda, Angela S ; Robinson, Michael B. ; Ory-Lavollée, Laure ; Forloni, Gian Luigi ; Blakely, Randy D. ; Coyle, Joseph T. / Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues : findings with a novel liquid phase radioimmunoassay. In: Molecular Brain Research. 1988 ; Vol. 3, No. 3. pp. 223-231.
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abstract = "Antibodies were raised in rabbits against the neuropeptide N-acetyl-l-aspartyl-l-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100{\%} of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons. We have also used this RIA to identify NAAG-LIR in striated muscle and other peripheral tissues.",
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AU - Robinson, Michael B.

AU - Ory-Lavollée, Laure

AU - Forloni, Gian Luigi

AU - Blakely, Randy D.

AU - Coyle, Joseph T.

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