Serotonin transporter antibodies: Production, characterization, and localization in the brain

Feng Zhou, Yi Xu, Sharon Bledsoe, Renee Lin, Mark Kelley

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Serotonin (5-HT) transporter, the mechanism for 5-HT high affinity uptake, is the essential component for the termination of 5-HT transmission. In order to identify transporter sites on 5-HT neurons or on other 5-HT uptaking cells, three rabbit antisera against cocaine sensitive-serotonin transporter (5-HTT) were produced. Antisera 5-HTT55 (against amino acid sequence 55-68 in cytoplasmic N-terminal) and 5-HTT315 (against amino acid sequence 315-325, a 3rd external loop peptide) were produced against synthetic multiple-antigenic peptides (MAP). Antiserum 5-HTTN was produced against a fusion protein of the first 71 amino acids of N-terminal peptide expressed in recombinant DNA transformed bacteria. SDS-PAGE/Western blots indicate that 5-HTT55 and 5-HTT315 recognized bands of 74 and 64 kDa in rat brains with densities in the order of cortex ≤ hippocampus > cerebellum, but not in liver, or muscle. The 5-HTTN recognized the fusion protein expressed in the bacteria, and the 64 kDa band with a similar density profile in the rat brain regions, and negative staining in liver and muscle. The immunocytochemistry of all three antisera revealed 5-HTT-immunostaining (5-HTT-im) in a pattern similar to 5-HT fiber distribution. 5-HTT55 and 5-HTT315 stainings were punctate in appearance; while 5-HTTN outlined the fibers in the 5-HT fiber areas, and neurons in raphe but not in substantia nigra or locus ceruleus. The preimmune serum and immune serum preabsorbed with 5-HTTN showed negative or diminished staining. Specific neurotoxin, 5,7-dihydroxytryptamine lesion removed all of the 5-HTTN fibers from the injection site, indicating that 5-HTTN-im fibers are 5-HT fibers in nature. Our study indicates that the three antibodies we produced recognize various domains of the 5-HTT. Our 5-HTT antibodies could be used as new markers of 5-HT fibers, and are particularly useful for the study of the plasticity of 5-HT fibers free of the complications involved with 5-HT content.

Original languageEnglish
Pages (from-to)267-278
Number of pages12
JournalMolecular Brain Research
Volume43
Issue number1-2
DOIs
StatePublished - Dec 31 1996

Fingerprint

Serotonin Plasma Membrane Transport Proteins
Antibody Formation
Serotonin
Brain
Immune Sera
Dihydroxytryptamines
Peptides
Amino Acid Sequence
Staining and Labeling
Bacteria
Neurons
Negative Staining
Muscles
Locus Coeruleus
Recombinant DNA
Antibodies
Liver
Substantia Nigra
Cocaine
Cerebellum

Keywords

  • 5,7-dihydroxytryptamine
  • Dorsal raphe
  • Fusion protein
  • Immunocytochemistry
  • Multiple antigenic peptide
  • Polymerase chain reaction
  • Site specific peptide

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Serotonin transporter antibodies : Production, characterization, and localization in the brain. / Zhou, Feng; Xu, Yi; Bledsoe, Sharon; Lin, Renee; Kelley, Mark.

In: Molecular Brain Research, Vol. 43, No. 1-2, 31.12.1996, p. 267-278.

Research output: Contribution to journalArticle

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