Localization and quantification of 5-hydroxytryptophan and serotonin in the central nervous systems of Tritonia and Aplysia

Serotonin (5-hydroxytryptamine, 5-HT) plays a central role in several behaviors in marine molluscs and other species. In an effort to better understand the regulation of 5-HT synthesis, we used high performance liquid chromatography (HPLC) with electrochemical detection and immunohistochemistry to measure and map the distribution of the immediate precursor of 5-HT, 5-hydroxytryptophan (5-HTP), in two model opisthobranch molluscs, the nudibranch Tritonia diomedea and the anaspid Aplysia californica. HPLC measurements showed that 5-HTP is present at approximately the same level as the 5-HT metabolite, 5-hydroxyindolacetic acid (5-HIAA) but is more than 100 times lower in concentration than either 5-HT or dopamine in the same tissue. Specific 5-HTP immunoreactivity was colocalized with serotonin in both species. The overall intensity of 5-HTP immunoreactivity in individual ganglia agreed with HPLC measurements for those ganglia. The intensity of 5-HTP immunolabeling varied between cell types and was correlated with the intensity of 5-HT immunolabeling. In particular, differences in staining intensity were consistently seen among the three dorsal swim interneurons of the Tritonia swim central pattern generator circuit. Some nonserotonergic neurons also displayed low levels of 5-HTP immunolabeling that were above background levels. Together, these results support the notion that production of 5-HTP is a rate-limiting step in serotonin synthesis and suggest that there may be additional regulation that allows 5-HTP to accumulate to varying levels.