Serotonin transporter antibodies: production, characterization, and localization in the brain.
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 > or = 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 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.
Zhou
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Department of Anatomy, Indiana University School of Medicine, Indianapolis 46202, USA. imce100@indyvax.iupui.edu
Xu
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Bledsoe
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Lin
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Kelley
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001921:Brain / Q000276:immunology* / Q000378:metabolism*; D002352:Carrier Proteins / Q000276:immunology*; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000276:immunology*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D011817:Rabbits; D051381:Rats; D050486:Serotonin Plasma Membrane Transport Proteins
D000906:Antibodies; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat