In Situ Hybridization Chain Reaction and Immunohistochemical Labeling of the Octopamine Production Pathway in the Central Nervous System of Lymnaea stagnalis

Octopamine (OA), a biogenic amine functionally similar to vertebrate norepinephrine, plays an important role in invertebrate neurophysiology. Previous reports of putative octopaminergic cells in the pond snail, Lymnaea stagnalis , have revealed inconsistencies, prompting our investigation that combined in situ hybridization chain reaction with traditional immunohistochemical methods. We mapped tyramine β‐hydroxylase (TBH) mRNA, the corresponding TBH protein, and the OA neurotransmitter. Approximately 40 neurons were labeled by all three methods and are presumably genuinely octopaminergic, including several neurons with previous electrophysiological evidence that OA is their neurotransmitter. Our results also revealed approximately another 40 cells that only showed evidence of mRNA and enzyme labeling, but no neurotransmitter, and another five to eight cells that only labeled for OA. Some of these results are likely explained by antibody cross‐reactivity, but multiple TBH isoforms (some of which may not produce OA) or regulatory mechanisms that block TBH function also need to be considered. Overall, this study underlines the need for a more nuanced interpretation of neuroanatomical mapping and provides new insights into the organization of molluscan octopaminergic systems.