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Immunolabeling of Central Serotonin 5‐HT 1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti‐Peptide Antiserum
Author(s) -
Langlois X.,
Gérard C.,
Darmon M.,
Chauveau J.,
Hamon M.,
El Mestikawy S.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.65062671.x
Subject(s) - immunolabeling , receptor , biology , microbiology and biotechnology , colocalization , keyhole limpet hemocyanin , 5 ht receptor , serotonin , peptide , radioligand assay , radioligand , biochemistry , antibody , immunohistochemistry , immunology
A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5‐HT 1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti‐peptide antibodies were detected by enzyme‐linked immunosorbent assay and characterized by immunoprecipitation of the 5‐HT 1B/1Dβ receptor in CHAPS‐solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin‐ O ‐carboxymethylglycyl[ 125 I]iodotyrosinamide ([ 125 I]GTI; a selective 5‐HT 1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5‐HT 1B receptors. The remaining 40% of [ 125 I]GTI binding sites were shown to be 5‐HT 1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5‐HT 1B/1Dβ , but not the 5‐HT 1D/1Dα , receptor in transiently transfected LLC‐PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5‐HT 1Dβ receptor‐like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [ 125 I]GTI in the brain of these species further confirmed that the anti‐peptide antibodies selectively recognized only the 5‐HT 1Dβ component of [ 125 I]GTI specific receptor binding sites.