5‐Hydroxytryptamine Measurements in Molluscan Ganglia and Neurons Using a Modified Radioenzymatic Assay

A radioenzymatic procedure for the determination of sub‐picomole amounts of 5‐hydroxytryptamine (5‐HT) is described. It is a modification of the method originally described by Saavedra et al. (1973), in which 5‐HT was measured as the radiolabelled product [ 3 H]5‐ methoxy‐ N ‐acetyltryptamine, after incubation with [ 3 H] S ‐adenosylmethionine, acetyl‐CoA, and the enzymes hydroxyindole‐ O ‐methyltransferase (EC 2.1.1.4) and N‐ acetyltransferase (EC 2.3.1.5). Ganglia from various gastropod molluscs (Aplysia californica, Tritonia diomedia, Lymnaea stagnalis , and Helisoma trivolvis) , as well as individual neuronal somata isolated from these ganglia, were assayed for 5‐HT. Among the homologous giant cerebral cells in these animals, the 5‐HT concentrations were similar. Statistical analysis of the 5‐HT values in paired 5‐HT‐containing neurons demonstrated that the variability was considerably greater in samples obtained from different animals than in those obtained from the same animal. This suggests that experiments aimed at manipulating amine levels in individual neurons may benefit by using a paired‐cell paradigm. The effects of incubating Aplysia ganglia with 5‐HT or with the 5‐HT precursors tryptophan and 5‐hydroxytryptophan (5‐HTP) were studied. High concentrations of 5‐HTP and 5‐HT (100 μ M ) increased the levels of 5‐HT in ganglia, but only incubation in high concentrations of 5‐HTP resulted in an increase of 5‐HT in the isolated somata of 5‐HT‐containing neurons C1 and P5.