Serotonin-like immunoreactivity and anti-5-hydroxytryptamine (5-HT) antibodies: ultrastructural application in the...

Serotonin immunoreactivity of rat neurons was studied using antibodies to 5-hydroxytryptamine (5-HT) raised in rabbits and the peroxidase-antiperoxidase (PAP) technique. The brains were fixed by perfusion with 4% paraformaldehyde + 0.25% glutaraldehyde. Vibratome sections were incubated in 1:1,000 to 25,000 antiserum dilutions. Specificity of 5-HT immunoreactivity was investigated following immunochemical and immunocytochemical criteria. Ultrastructural analysis of the raphe neurons allowed intracellular localization of 5-HT immunoreactivity in somata, dendrites, and synaptic terminals. Granular reaction product deposits were found attached to microtubules, endoplasmic reticulum membranes, synaptic vesicles, and outer mitochondrial membranes. In all cases the reaction product was confined within the limits of the plasma membrane. It is proposed that binding of 5-HT molecules to tissue proteins via a Mannich reaction and formation of intramolecular condensation products (fluorophores) result from aldehyde fixation. The immunogen (5-HT-bovine serum albumin (BSA) complex) exhibits spectrofluorometric emission and excitation maxima similar to those previously described for the 5-HT fluorophore studied in protein films and in fixed tissue. It is concluded that our antibody detects 5-HT-like immunoreactivity preserved in the tissue with the configuration of the fluorophore (i.e., 3,4-dihydrocarboline). Close agreement between the results and previous studies of 5-HT neurons with other techniques (formaldehyde-induced fluorescence, biochemical assays) support the potential value of this antibody.