Neural pathways in the pallial nerve and arm nerve cord revealed by neurobiotin backfilling in the cephalopod mollusk Octopus vulgaris

  • Pamela ImperadoreEmail author
  • Maria Grazia Lepore
  • Giovanna Ponte
  • Hans-Joachim Pflüger
  • Graziano Fiorito
Original Article


Here, we report the findings after application of neurobiotin tracing to pallial and stellar nerves in the mantle of the cephalopod mollusk Octopus vulgaris and to the axial nerve cord in its arm. Neurobiotin backfilling is a known technique in other molluscs, but it is applied to octopus for the first time to be best of our knowledge. Different neural tracing techniques have been carried out in cephalopods to study the intricate neural connectivity of their nervous system, but mapping the nervous connections in this taxon is still incomplete, mainly due to the absence of a reliable tracing method allowing whole-mount imaging. In our experiments, neurobiotin backfilling allowed: (1) imaging of large/thick samples (larger than 2 mm) through optical clearing; (2) additional application of immunohistochemistry on the backfilled tissues, allowing identification of neural structures by coupling of a specific antibody. This work opens a series of future studies aimed to the identification of the neural diagram and connectome of octopus nervous system.


Neural tracing Backfilling Neurobiotin Cephalopods Octopus vulgaris 



Authors are grateful to Dr Astrid Schauss and Dr Christian Jüngst (CECAD Imaging facility, CECAD Research Center, Cologne, Germany) and Dr Michael Dübbert (Institute of Zoology, University of Cologne, Cologne, Germany) for accessing the imaging facility, their assistance and guidance in the use of Leica SP8 multiphoton microscope. Authors are also thankful to Leica Microsystems (P. Romano, K. Orellana) for support and assistance. This work benefited of the networking initiative of the COST Action FA1301—CephsInAction.

Authors’ contributions

PI and MGL carried out all experiments; PI analyzed the data, processed images and drafted the manuscript; GP and HJP contributed to the experimental design, the implementation of the method to cephalopod preparations and manuscript editing; GF supervised the work, designed the experiments and revised the final manuscript. All authors contributed to the final writing of the manuscript and approved the final article.


PI is currently supported by a fellowship for the project “Nociception, pain and suffering in octopuses” (Extra-EU Scientific Research & Cooperation Fund—Stazione Zoologica Anton Dohrn, SZN) and has been previously supported by the Association for Cephalopod Research-CephRes for the time this work has been carried out. MGL has been supported by Progetto MO.DO (Model Organism, POR Campania, FSE 2007/2013—SZN) extra-regional networking initiative. GP and GF have been also supported by RITMARE Flagship Project (MIUR and SZN).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interest.

Ethics approval

Cephalopod mollusks are included since January 2013 in the Directive 2010/63/EU which regulates the use of animals for scientific research purposes. Killing animals solely for tissue removal does not require authorization from the National Competent Authority under Directive 2010/63/EU and its transposition into national legislation. Experiments included in this work have been carried out on samples taken from animals humanely killed under a project license from NCA (Italy; Aut. No 19/2014-PR) or as freshly dead from local fishermen following principles stated in Directive 2010/63/EU.

Supplementary material

10158_2019_225_MOESM1_ESM.tif (1.4 mb)
Supplementary Figure 1. Imaging of whole-mount backfilled axial nerve cord counterstained with phalloidin. (a) Schematic 3D enlargement of the ANC. In (b) a zoom-in of the CBT after backfilling of the nerve cord is shown (sagittal plane). Backfilled fibers (in red) are seen to run straight in the nervous tract. Phalloidin (in green) staining filamentous actin was used to counterstain samples. (c) Large neurons in the CL are also detected using Nb (in red). For list of abbreviations, refer to the legend of Figure 1. Scale bar: (b, c) 100 µm. (TIFF 1423 kb)
10158_2019_225_MOESM2_ESM.avi (207 mb)
Supplementary Video 1. Z-stack imaging of a whole-mount backfilled axial nerve cord through its entire depth in latero-lateral orientation. On the dorsal side (on the left), CBT are traced showing individual axon pathways. Nerve fibers are seen to descend into the BG (on the ventral side, shown on the right) where some of them appear to originate from Mn. Other fibers are seen to form the Np of the BG. For more details, refer to Figure 2 and main text, and for list of abbreviations, refer to the legend of Figure 1. Scale bar: 250 µm (AVI 211972 kb)

Supplementary Video 2. 3D reconstruction of the backfilled whole-mount axial nerve cord (as shown in Supplementary Video 1, in latero-lateral orientation). The rotating animation shows traced CBT on the right. Fibers descend into the BG (on the left) where big neurons and small cells are detected. A corner-cut view is also shown in the video, first on the upper left quadrant (cell and fibers of the BG), and then also on the upper right quadrant (fibers of the CBT). For list of abbreviations, refer to the legend of Figure 1. Scale bar: 200 µm. (AVI 194548 kb)

10158_2019_225_MOESM4_ESM.avi (120 mb)
Supplementary Video 3. Z-stack imaging of a whole-mount backfilled pallial nerve (ventro-dorsal orientation). Fibers of different diameters are detected and traced individually. Majority of them run parallel to each other, but some fiber criss-crossing is also observed. Scale bar: 100 µm. (AVI 122877 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biology and Evolution of Marine OrganismsStazione Zoologica Anton DohrnNaplesItaly
  2. 2.Association for Cephalopod Research—CephResNaplesItaly
  3. 3.Institute for NeurobiologyFree University BerlinBerlinGermany
  4. 4.Instituto de Fisiologıá, Biologıá Molecular y Neurociencias (IFIBYNE)Ciudad UniversitariaBuenos AiresArgentina

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