Mapping neuronal gene expression reveals aspects of ganglionic organization in a gastropod mollusc

Gastropod molluscs such as Aplysia, Lymnaea, and Tritonia have been important for determining fundamental rules of motor control, learning, and memory because of their large, individually identifiable neurons. Yet for the vast majority of gastropod neurons, as well as glia, there are no established molecular markers, limiting the ability to establish brain-wide structure-function relations. Here we combine high-throughput, single-cell RNA sequencing (scRNAseq) with in-situ hybridization chain reaction (HCR) in the nudibranch Berghia stephanieae to identify and visualize the expression of markers for cell types. Broad neuronal classes were characterized by genes associated with neurotransmitters, like acetylcholine, glutamate, serotonin, and GABA, as well as neuropeptides. These classes were subdivided by other genes including transcriptional regulators and unannotated genes. Marker genes expressed by neurons and glia formed discrete, previously unrecognized regions within and between ganglia. This study provides the foundation for understanding the fundamental cellular organization of gastropod nervous systems.