Immunocytochemical localization and direct assays of serotonin-containing neurons in Aplysia

Serotonin-containing neurons were localized in the central nervous system of Aplysia californica by the combination of an immunohistochemical procedure for wholemounts of Aplysia ganglia and, in parallel experiments, by the direct measurement of serotonin in individual neurons with a radioenzymatic assay. Paraformaldehyde fixed, desheathed ganglia were incubated in a commercially available antiserum against a conjugate of serotonin and bovine serum albumin. The bound antibody was visualized by an indirect antibody procedure using the horseradish peroxidase catalyzed reduction of the chromogen, diaminobenzidine, and a cobalt-nickel intensification procedure. The specificity of the immunoreaction for serotonin-containing cells was demonstrated by (1) the consistent staining of previously identified serotonin-containing neurons; (2) the absence of staining in identifiable neurons which do not contain measurable serotonin; and (3) the absence of staining in ganglia treated with antiserum which had been absorbed by serotonin and the serotonin conjugate. Previously unidentified serotonin-containing neurons were localized in the cerebral and pedal ganglia by the combination of immunocytochemistry and direct assay for serotonin. Immunoreactive fibers were found surrounding many neuronal somata. In addition, serotonin assays of known cholinergic neurons that were covered by immunoreactive fibers indicated that measurable amounts of serotonin were associated with such neurons, but the concentration of serotonin was an order of magnitude lower than in neurons known to stain with the anti-serotonin serum. These studies have localized more than a hundred neurons that appear to contain serotonin in concentrations (greater than 0.5 mM) in the Aplysia central nervous system. In addition, it appears that the long-held belief that the somata of invertebrate neurons are relatively free of impinging nerve fibers may no longer be tenable. The immunoreactive fibers surrounding many cell bodies may be the source of measurable serotonin associated with neurons known to utilize or to contain transmitter substances other than serotonin. Immunocytochemical techniques applied to wholemounts of molluscan preparations facilitate identification of stained neurons for parallel physiological and chemical experiments.