Three-dimensional molecular atlas highlights spatial and neurochemical complexity in the axial nerve cord of octopus arms

Octopus arms, notable for their complex anatomy and remarkable flexibility, have sparked significant interest within the neuroscience community. However, there remains a dearth of knowledge about the neurochemical organization of various cell types in the arm’s nervous system. To address this gap, we used hybridization chain reaction (HCR) to identify distinct neuronal types in the axial nerve cords of the pygmy octopus, Octopus bocki, including putative dopaminergic, octopaminergic, serotonergic, GABAergic, glutamatergic, cholinergic, and peptidergic cells. We obtained high-resolution multiplexed fluorescent images at 0.28 × 0.28 × 1.0 μm voxel size from 10 arm base and arm tip cross sections (each 50 μm thick) and created three-dimensional reconstructions of the axial ganglia, illustrating the spatial distribution of multiple neuronal populations. Our analysis unveiled anatomically distinct and molecularly diverse scattered neurons, while also highlighting multiple populations of dense small neurons that appear uniformly distributed throughout the cortical layer and potential glial cells in the neuropil. Our data provide new insights into how different types of neurons may contribute to an octopus’s ability to interact with its environment and execute complex tasks. In addition, our findings establish a benchmark for future studies, allowing pioneering exploration of octopus arm molecular neuroanatomy and offering exciting new avenues in invertebrate neuroscience research. [Display omitted] •3D octopus arm cord maps reveal stratified monoaminergic and peptidergic neurons•Significant neurochemical differences between the arm tip and base ganglia•Cholinergic and glutamatergic neurons are abundant; GABAergic neurons are scarce•Novel identification of neuronal cotransmission and possible protoplasmic glia Winters-Bostwick et al. reveal complex neurochemical organization within the arm nerve cord of Octopus bocki, using HCR to identify diverse neuronal types. High-resolution 3D imaging and reconstruction demonstrate spatial distribution of cell types, offering new insights into octopus neuroanatomy and enhancing understanding of limb function.