Molecular organization of Octopus brains: Insight into unique memory center signaling

Cephalopods (Octopus, Squid, Cuttlefish, Nautilus) exhibit behavioral flexibility that rivals that of many mammals. The Vertical Lobe (VL), a cephalopod-specific memory circuit, parallels mammalian analogues (hippocampus) in cell number and function, but evolved independently in molluscs. We integrated Next-gen sequencing technology and bioinformatic analyses, followed by anatomical validation, to identify molecular maps of signaling molecules implemented in cephalopod memory circuits. We produced transcriptomes of various Octopus neural tissues (VL, CNS, SFL, Arms), and individual cells from sub-populations within the VL circuit: Amacrine (Am) Interneurons and Large Efferent (LE) Neurons. We mapped these transcriptomes to a publically available Octopus genome and our gastropod transcriptomes including Aplysia. We identified 16,194 transcripts in the VL and found ~25.5% appear to be cephalopod-specific. Next we used targeted and unbiased computational predictions and manual annotation to identify putative signaling molecules form the VL transcriptomes. We have cloned and localized 11 neuropeptides (NP) to components of the VL circuit. NPX3 is abundantly expressed in the cell bodies of the SFL, where the VL circuit originates and NPX1 & 2 localize to the AM cell bodies of each VL gyrus. Among the 7 NPs that localize to the VL LE neurons, 2 unique cephalopod NPs, NPX4 & 5, are abundant in Octopus, but absent in the ancestrally branching Nautilus (which lacks a VL). This expansion of signaling molecules in the VL circuit may be a key feature of unique memory systems of cephalopods, implying extensive parallel evolution of cephalopod brains and memory circuits.