Three-Dimensional Molecular Atlas of Octopus Arm Neuroanatomy Highlights Spatial and Functional Complexity

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 molecular and functional identities of various cell types in the arms nervous system. To address this gap, we used hybridization chain reaction (HCR) to identify distinct neuronal types in the arms of the pygmy octopus, Octopus bocki, including putative dopaminergic, octopaminergic, serotonergic, GABAergic, glutamatergic, cholinergic, and peptidergic neurons. We obtained high-resolution multiplexed fluorescent images at 0.28x0.28x1.0 M voxel size from 10 arm base and arm tip cross sections (each 50 M 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 excitatory neurons that appear uniformly distributed throughout the cortical layer. Our data provide new insights into how different types of neurons may contribute to the ability of an octopus 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.