Long-range neural pathways for octopus chemotactile processing revealed from periphery-to-brain by centimeter-field microCT

Reviewer #1 (Public review):

Summary:

Sugarman, Vanselow et al. adapted a microCT instrument to permit imaging of an entire organism, a hatchling octopus. In the resulting 3D dataset, they segmented the major organ systems, including the vascular, respiratory, digestive, and nervous systems. The authors released the dataset through a public web interface, and present some observations about body-wide neuroanatomy.

Strengths:

- The dataset is of good quality and access to a whole-cephalopod anatomical resource will be useful for the scientific community.

- The interactive web interface facilitates exploration of the dataset.

Weaknesses:

- The authors identify a series of bundles of nerve fibers between the suckers and the central brain and propose that these structures together constitute the chemotactile pathway, linking sensation to learning and memory. This is an over-interpretation of the available evidence. The data is not presented in a way that allows the reader to independently assess the proposed anatomical relationships: many images include near-opaque colored overlays on the fibers of interest, making it difficult to determine whether these bundles truly merge or interface. Additionally, the mesoscale resolution achieved here reveals the presence of large nerve bundles but cannot resolve the origin or synaptic relationships of the neurons in the bundles - including those from the chemotactile receptors of the suckers. There are likely multiple synapses between the periphery and the central brain, and the location and connectivity of individual neurons are not visible at this resolution. Consequently, this dataset does not demonstrate structural connectivity. Elucidating a neural circuit would require complementary approaches such as neuronal tracing or electron microscopy connectomics.

- The language used in the manuscript is often overly complex and convoluted, making it difficult to follow the main arguments and to assess the strength of the claims. In addition, some vocabulary in the main text is overly technical (e.g. related to microCT or anatomy), making it difficult for a general biology or cephalopod audience to understand, while some neuroscience vocabulary is used imprecisely or in ways that overstate what can be concluded from anatomical data. A substantial rewrite using clearer, more cautious language is recommended. Additionally, a deeper discussion of the observed octopus arm anatomy, and how this may relate to its semi-autonomous function would make this article of greater interest to a broader audience.

Reviewer #2 (Public review):

Sugarman et al show a major advance in the volumetric imaging of the cephalopod body and nervous system, using wide field high resolution micro-CT imaging. The new detection optics are striking in their performance, and the conclusions made from the images seem well-founded. The technical advance and the conclusions both justify the reader's attention, but the authors should make the figures and the text teach the reader so that the findings are more accessible and convincing.

The paper is now written in a style that will impress those ready to be impressed and fail to impress many of the readers, although it should.

(1) The authors must improve the text so that it cleanly states what was known previously, and how the current results extend this. For example. putting a section in the middle of the results section (page 3) that states: "Long-range connections between sucker and brain were demonstrated by fine chemical and tactile sensing by suckers in behavioral experiments with live O. bimaculoides (Buresch et al., 2022, 2024; Sepela et al., 2025; van Giesen et al., 2020; Wells, 1978a; Wells & Young, 1969) and by loss of chemotactile learning and memory observed after ablation of the "inferior frontal system" (i.e., inferior frontal/subfrontal/buccal lobe complex) (Wells, 1978a)..." seems a bit confusing to me. Similarly, putting in a reference to optical imaging approaches for combining data sets (Preibisch et al., 2009) as only the citation does little to make the work accessible. Please expand the text so that it teaches what the authors are thinking.

(2) The authors must improve the figures so the work is more digestible. The data is a pyramid, and the "google earth" range of magnifications and details is not clear in the figures. In short, the figure will impress those who know to be impressed and fail to impress the majority.

(3) The videos are far more useful in this contribution that in almost any other paper. Use them more so the reader realizes how key they are. Revising them to demonstrate the amazing range of scales in the data would be wise.

(4) The demonstration of the data visualization tool is excellent as far as it goes. Expanding the treatment of the multi-scale rendering would be wise.

With proper expansion of the text and the figures, it will become far more obvious that this is landmark work.

Reviewer #3 (Public review):

Summary:

Sugarman et al. present a microCT scan of a hatchling octopus from the species Octopus bimaculoides. The scan is publicly available and poses as a valuable tool for the field of cephalopod biology. Using this scan, the authors uncover two undescribed neural pathways: the intermediate longitudinal tract (iLTs) in the axial nerve cord linking the suckers to the brain, and the arm-to-arm u-tracts (AAUTs) interconnecting neighboring arms. How the eight sucker-lined octopus arms are coordinated with one another and with the brain have been long standing questions in the octopus motor control field, and the results presented here have promise for addressing these questions. However, major weaknesses addressed below limit the interpretability of the dataset.

Strengths:

The authors have publicly published a scan of an entire hatchling octopus, with major organs and subdivisions of the nervous system already segmented. Accessing the data is straightforward, and the authors provide adequate instructions on how to navigate the dataset.

The authors provide validation of the AAUTs using lucifer yellow and wheat germ agglutinin. To overcome motion artifact in the hatchling dataset, the connections between the iLTs and the suckers are validated with a microCT scan of a distal section of adult arm.

Weaknesses:

Given the resolution of the dataset, neural connectivity is determined by texture differences alone, which can be misleading. As such, any claims of anatomical connectivity will need further validation, ideally with tracing techniques. While the authors investigated the AAUTs with other techniques, no such validation exists for the iLTs. Furthermore, the authors themselves state that as the iLTs converge with the brachial nerve, they become indistinguishable from other fibers. Any connections of the iLTs to the brain are only hypothesized, despite their claim of demonstrating a clear pathway from the suckers to the brain.

The relevant prior research on octopus neurobiology is not well explained, making it challenging to understand the significance of the results in a broader context.