New Preprint: Mapping functional circuits in octopus arms with calcium imaging

Live calcium imaging in the octopus arm ©CrookLab

The lab’s newest manuscript is available to view on Biorxiv while it makes its way through peer review. This study, led by postdoctoral fellow Gabrielle Winters-Bostwick, uses calcium imaging in the axial ganglia to record neurons firing in response to application of neurotransmitters and neuromodulators secreted from cells mapped in a previous study from our lab. This manuscript, titled “Neurochemically-evoked activity in slice preparations of the octopus arm nerve cord”, is the first to show populations of cells responding to exogenous neurochemicals in slices. Previously we had demonstrated that cells in the arm nerve cord were not organized or clearly laminated according to molecular identity, in contrast to cells in the optic lobe. Here, we extend that finding to show that there is also no evidence for functional organization; i.e., cells responding to specific neurotransmitters are not localised to clusters or lamina either. This finding supports an emerging body of evidence from our lab and others that the arms of the octopus - the most complex example of distributed network control known - do not conform to accepted rules of organization that help simplify neural computations in other nervous systems.

An except of Fig 2, showing glutamate and dopamine produce robust excitation ©CrookLab

In addition, this new study also looks at how network activity changes after application of putative neuromodulators. Gabby’s work demonstrates that subsequent application of fast neurotransmitters glutamate or dopamine, if applied after an initial pulse of serotonin, produces less activation that when they are applied alone. So we can say that glutamate and dopamine act as excitatory neurotransmitters, and serotonin modulates excitation. We also show strong suppression of activity by acetycholine, suggesting that it acts as an inhibitory signal in axial cord.

This study is a remarkable addition to our ongoing work mapping the peripheral nervous system of cephalopods, revealing previously unknown patterns or activation, modulation and inhibition.

New Publication: No evidence for object play in Sepia bandensis

Sepia bandensis housed in a complex, enriched habitat ©CrookLab

In a new paper published this month in Applied Animal Behavior Science, recent MS graduate Kira Lemke demonstrates that unlike octopuses, cuttlefish don’t engage in play with objects.

Welfare standards for cephalopods are generally poorly supported by rigorous studies. Enclosure enrichments are variable, and whether enrichments actually meet needs of different cephalopod taxa is not widely studied. “Enrichment” also has multiple meanings and different applications, including anything from substrate on the enclosure floor, provision of live prey, puzzle boxes, novel objects, housing rearrangement, human interaction and many others. And while there have been a number of studies demonstrating that octopuses in research laboratories and public aquaria engage in extended play-like behavior with novel objects, it is unknown whether these provide a genuine welfare benefit, or whether other cephalopods also display similar behaviors.

In this study based on her thesis work, Kira demonstrates conclusively that dwarf cuttlefish (Sepia bandensis) do not interact with novel objects placed into their housing enclosures, and that this lack of engagement is consistent whether their housing environment contains static enrichment or not. This paper indicates that species- and taxon-specific enrichments need to be assessed and implemented based on the needs of each different cephalopod clade, and also that high levels of cognitive ability (which are certainly present in cuttlefish) do not necessarily correlate with play-like behaviors.

Congratulations to Kira on the publication of an important and timely study!

Congratulations Kira!

Dr. Robyn Crook (L) and Kira Lemke (R), who successfully defensed her MS thesis on May 1

Masters student Kira Lemke successfully defended her thesis titled “How do cuttlefish interact with their environment while in captivity”. Kira’s thesis project included an examination of potential play-like behavior in Sepia bandensis and a separate study of their preference for natural vs. artificial shelter. Surprisingly, Kira’s data suggest that cuttlefish don’t engage in object-based play at all, which was an unexpected finding given the multiple studies showing play behavior in octopuses. Cuttlefish are growing in importance as comparative models in neuroscience, and there is still very little known about best practices for their housing and husbandry. Kira’s work contributes to the lab’s ongoing efforts to produce peer-reviewed. evidence-based findings that support regulation and oversight of cephalopods in research and educational settings.

Kira is applying for PhD positions in the coming cycle, and a manuscript from her thesis will be submitted soon. Congratulations Kira!!!

Press roundup; Octopus arm organization

Octopus bocki. ©CrookLab

Our recent papers in Current Biology have generated some really great scientific press from a range of different sources, including specialist neuroscience publications. It’s great to see growing interest in comparative. evolutionary neurobiological questions and the ways in which cephalopods can inform our understanding of nervous system diversity.

“Octopus arm anatomy, molecular make-up revealed in new maps”. The Transmitter, Sept 25 2024

“SFSU researchers’ unique 3D maps might help solve the mysteries of octopus arms” SF State News, Oct 22, 2024

“Brains, Cowboys and Octopunch!” Danna Staaf’s Octopost, Oct 18, 2024

“Mysterious secrets of octopus arms revealed in unprecedented detail” ScienceAlert, Oct 30 2024

“Scientists get one step closer to figuring out what makes octopuses tick” Popular Mechanics, Nov 4, 2024

New Publication: Stress and cognitive bias in cuttlefish

Sepia bandensis explores an arena with a visual cue signaling food reward. Stress produces negative judgement bias, leading to pessimistic assessment of intermediate cues. ©CrookLab

Congratulations to first author Sarah Giancola-Detmering, whose paper is out today in the Royal Society journal Biology Letters. Sarah’s paper, “Stress produces negative judgement bias in cuttlefish” modifies a common assay for assessing affective state in vertebrate animals and modified it to be used on a cephalopod for the first time. This project is part of the growing applied welfare focus of our lab.

Sarah’s study demonstrates that cuttefish subject to ongoing stressful husbandry practices (impoverished housing and daily restraint) exhibit a pessimistic judgement bias compared with control animals. By training cuttlefish to associate a striped visual cue with food availability, Sarah shows that intermediate cues are interpreted as likely signaling food in control animals but less likely in stressed animals. Cognitive- and judgement-bias tests are used as standard welfare assessment methods in zoos, aquariums and research labs for vertebrate animals, and here we show that the same methods can reveal welfare state and affect in a cephalopod.

This is the first demonstration that cuttlefish have the capacity for negative affective states, and also shows that husbandry refinements can have a large impact on animal welfare for cephalopods. Our work on cephalopod welfare is shaping standards of care and regulatory oversight around the world, and we expect that this important paper will provide further evidentiary support as these oversight structures are continually refined.

Congratulations Sarah!

Two new papers on octopus arms - out today in Current Biology!

Congratulations to first authors Diana Neacsu and Gabby Winters-Bostwick, whose papers are out today in Current Biology! These paired studies of the neuroanatomy of the octopus arm are funded by our Allen Distinguished Investigator grant, which has the broad aim of revealing organisation of nervous systems of less well-studied animals.

A 3D rendering of the ventral tracts of the octopus arm nerve cord, with connections to sucker ganglia ©CrookLab

Diana’s paper uses a large 3D-EM dataset to show previously uncharacterized organizational symmetry in successive ganglia of the arm. It has always been unclear how strictly organized the neural tissues are in the arm, and here we show that each ganglion is a reflection of the ones next to it, making a series of precise A/B patterned structures along the arm, associated with each sucker. She also shows that the oblique connectives, very small tracts previously only mentioned in a few studies, run around the arm in a spiral and potentially allow signal to travel along and around the arm without involvement of other neural centers. This paper is the first from her MS thesis, which also includes a full connectomic reconstruction of the sucker ganglion (hopefully coming soon!)

A cross section through the axial ganglion with neurons of different types labeled with different probes. ©CrookLab

Gabby’s paper uses the relatively new HCR method to label individual cells in the main axial ganglion according to their molecular identity. Using markers for multiple neuronal subtypes, she reveals stratification of cell types within the cord in the oral/aboral, L-R and distal-proximal axes, and shows highly complex co-expression profiles of cells that suggest many more subtypes than previously appreciated. This new information should greatly accelerate future physiological and functional studies of the arm.

Both papers produced high resolution, 3D maps of the octopus arm which can be viewed as animations (here featured at The Transmitter). The reconstruction of the two tissue volumes was a massive computational effort that provides unprecedented detail and new information about how octopus arms achieve their complex behavioral functions. We expect these datasets will inform future studies in our lab and those of others. These are the first two papers that come from the funding to our lab from the Allen Frontiers Group, and we are immensely grateful for their support of our work.

Congratulations Diana!

Diana Neacsu (second from left) with her thesis committee (L-R); Dr. Robyn Crook, Dr. Annette Chan and Dr. Ivan Anastassov. ©CrookLab.

Masters student Diana Neacsu defended her MS thesis today, titled “A Three-dimensional connectome of the sucker ganglion and other associated structures of the octopus arm”. Diana’s work has revealed some fascinating new insights into this minimally-explored structure, as well as revealing previously unappreciated, large-scale organization of the whole arm nervous system. Diana is also a talented scientific illustrator whose beautiful and informative drawings are featured in her thesis and her upcoming publications.

Diana is continuing her work on cephalopod neurobiology for her PhD, and she’s already on her way to KU Leuven to begin her work there in the Fall. Congratulations Diana! The first of Diana’s papers should be coming out soon.

The inaugural CephNeuro conference!

CephNeuro attendees gather for a group photo outside the historic Lillee building at the MBL.

This week the whole lab has been in Woods Hole, MA, for the inaugural Cephalopod Neuroscience Conference. Gabby and Diana presented talks, with Sarah and Kira presenting posters.

Crook Lab alumni Ryan Howard and Abbi Koenigsmark also presented their ongoing work as posters. The conference brought together over 200 researchers interested in neuroscience of cephalopods, and was a great success overall. We certainly had a fun, tiring and stimulating week interacting with cephalopod researchers new and old.

Diana presenting her work on the sucker ganglion connectome ©CrookLab

Gabby presenting her work on molecular mapping of the octopus arm nerve cord. ©CrookLab

We also attended some fantastic, hands-on workshops in the two days before the main conference began. Gabby spent her time learning about Crispr and other techniques for establishing genetic tools for cephalopods, Diana attended a workshop on calcium imaging and electrophysiology, and Sarah, Kira and Diana all also attended a workshop on analysis tools for behavioral, imaging and electrophysiology. Kira, Sarah and Robyn demonstrated handling and husbandry techniques to new cephalopod researchers in the Care, Handling and Welfare workshop. Lastly, Robyn presented the lab’s latest work on connectomics and molecular mapping in the octopus arms to a small group of PIs all working on the octopus nervous system. These intensive training activities allowed sharing of techniques and knowledge among the field, and helped forge connections among labs that we all hope will lead to greater collaboration and cooperation.

Overall it was a great experience that we look forward to making a regular event. Conference organizers are already mobilizing for a second CephNeuro conference in two years’ time. Stay tuned!

A group selfie of Crook Lab members past and present! From left, Ryan Howard (now a PhD student studying the retinas of deep sea squid at AUT in NZ), Diana Neacsu, current MS student in the lab (shortly off to start her PhD at KU Leuven!), Sarah Detmering, currently our research technician but about to start in the MS program at SFSU, Kira Lemke, a first-year MS student in the lab, PI Robyn Crook, and Abbigale Koenigsmark, currently a PhD student at U Oregon. ©CrookLab

PI Robyn Crook quoted on regulation of cephalopod research in The Transmitter

An O. bimaculoides from our lab featured in The Transmitter’s story on regulation of cephalopod research.

The topics of cephalopod welfare and pending federal regulation of their use in laboratories continue to generate broad interest within the scientific community. This week The Trasnmitter published a detailed and nuanced article looking at the current state of knowledge in the field of welfare, and how this might affect or hinder the effective implementation of federal oversight. The story contains quotes from many cephalopod researchers with an interest in welfare, and quotes PI Robyn Crook extensively on what is needed to establish empirically-supported regulations and oversight. The Crook Lab is strongly in favor of additional oversight of cephalopod research, and it is our work on anesthesia, analgesia and affective state that is heavily informing this current effort.

MS Student Diana Neacsu featured for her scientific illustrations

Illustrations featured in the SFSU newsletter, from the illustrated Crook Lab manual. ©Diana Neacsu

Along with being a great student, Diana is also a talented artist and scientific illustrator. Last summer, she was awarded one of three inaurgural grants from the EOS Center to produce a scientific illustration project that would contribute to knowledge either within or outside academia. Diana produced a stunning illustrated lab manual for our lab, making the learning curve for new students joining us much less steep (and also more enjoyable!). Diana and the other two students in her cohort are featured this week in SFSU’s campus newsletter in a detailed story about the grant and the outcomes from the first cohort of fellows. More recently, Diana has been putting her graphical and illustration skills to good use in the sucker ganglion connectome project.

PI Robyn Crook discusses progress on regulatory oversight of cephalopods

The topic of cephalopod welfare is receiving growing attention. In the past year, efforts to secure regulatory protections for cephalopods in the United States have gained considerable traction. Dr. Robyn Crook provided comments on this important topic recently to Nature News and Wired Magazine, for stories describing the progress of these efforts, limitations of current knowledge and the imperative to consider the value of planned experiments in the context of potential suffering of the cephalopods involved. The Crook Lab’s applied welfare work on cephalopod anaesthesia, analgesia, husbandry and handling is recognized as world-leading, and we expect it be relied upon heavily in the establishment of US regulatory standards - if and when they happen - because few other labs are doing this work.

We are strongly in favor of efforts to provide welfare protections to cephalopods in research, and our work in this area is aimed at providing evidence for or against specific interventions that are hypothesized to improve welfare, such as analgesic drugs that have been used previously only in vertebrate animals. While it is critically important to identify interventions that work, it is equally important to identify those that do not. Cephalopod research is growing rapidly and the need for evidence to support refinement procedures is pressing. Despite this, there are currently no active funding mechanisms available to support these types of studies.

We continue to work on these important issues, and hope to see more studies in future that will allow cephalopod researchers and regulators to develop rigorous, empirically supported standards for protecting cephalopod welfare.

Alumni Update: Meghan Holst featured by Reuters for shark conservation

A screen-grab of Meghan being interviewed on a research vessel tagging sevengill shark pups.

MS graduate Meghan Holst (class of 2021) is currently working on her PhD at UC Davis, studying sevengill shark population ecology. Her research was featured this week by Reuters, in a news clip describing the conservation efforts she is leading to ensure that the SF Bay remains a safe place for sevengill shark pups to grow up. Heavy rains in California this previous winter have reduced the salinity in the SF Bay, leading to concerns about the 2023 cohort of pups in this area. Meghan’s work on physiology and population monitoring will be instrumental to efforts to conserve this unique species.

Meghan is also still working with us on an ongoing project to characterize specific cell types that degenerate in the octopus arm nerve cord during senescence. It is great to see her outstanding research getting the attention it deserves. Go Meghan!

New Publication: Cephalopod research and the Social License to Operate

In an invited review article out this week, Dr. Robyn Crook discusses recent progress in cephalopod welfare and husbandry, and outlines a strategy for engaging the public and other stakeholders in discussions of cephalopod use in research, regulatory oversight, and cephalopod welfare. Social license to operate (SLO) is a conceptual framework for examining how a given activity or industry is perceived and evaluated by external observers, whether they have a material interest in the activity or not. This framework has been applied already to commercial aquaculture, where interest in cephalopods is growing, and recreational animal use, including fishing. The application of this well-validated framework to cephalopod research can help structure joint effort from researchers to work to improve animal welfare and community engagement with our science at the same time. This article is currently paywalled and only available as an accepted proof, but will come out in an upcoming, cephalopod-themed special issue of Integrative and Comparative Biology.

Congratulations Rachel!

Rachel defended her MS thesis today, titled “Neural control of quadrupedal walking in the flamboyant cuttlefish, Metasepia pfefferi”. Rachel’s project used video analysis and Deep Lab Cut to characterize the walking gait of metasepia, which is the only cephalopod genus to perform coordinated stepping using front arms and ventral papilla. Her work sheds new light on the organization of neural circuits for locomotion in cephalopods, and opens several new lines of study in these interesting and relatively under-studied animals.

During her time in the lab Rachel also worked on a project examining anesthesia and analgesia in cephalopods, which was published earlier this year. We are working on a manuscript describing her work on M. pfefferi and hope to see that published soon.

Rachel is applying for PhD programs in the upcoming 23/24 cycle, and we expect her to go on to exciting things! Congratulations Rachel!

Congratulations Abbi!

Abbi Koenigsmark defended her MS thesis this week, titled “Sex Differences in the Agonistic Behavior of the Stumpy-Spined Cuttlefish Sepia bandensis (Mollusca: Cephalopoda). Abbi’s work shows that both male and female cuttlefish engage in agonistic displays to conspecifics, and that both sexes use a color pattern that has previously been considered to be male-only. Her work sheds new light on the complex intraspecific communication that occurs in cephalopods, and is one of the few studies of interactions that attempts to manipulate chromatophore function to examine signal fidelity.

During her MS degree Abbi also worked on another project, looking at baseline sex differences in nociceptive thresholds in Euprymna berryi, adding to her interest in sex-specific behavior. We expect that both of these projects will lead to publications soon.

Abbi is heading off to a PhD in Neuroscience at the University of Oregon, where she hopes to work on the octopus visual system. Congratulations Abbi!

Abbi celebrates her successful thesis defense! L-R Robyn Crook, Abbi Koenigsmark, Rachel Parsons, Gabby Bostwick, Sarah Detmering

Crook Lab at the CoSE Showcase

DIana with her poster, and Rachel and Abbi offering support and encouragement!

Diana Neacsu presented her poster on the connectome of the octopus arm at the annual SFSU College of Science and Engineering Showcase. Diana’s work focuses on a large-scale reconstruction of the axons and glial cells in the octopus arm nerve cord and the auxiliary neural structures that underlie sensation and movement. The CoSE showcase is an excellent opportunity for students to present work in progress and show the larger university community the work we do. Great job Diana!

New Publication: Ecology and evolution in a model of persistent pain

A new Perspective article was published this week in Nature Reviews Neuroscience by PI Robyn Crook, along with Ben Seymour (Oxford University) and Zhe Sage Chen (NYU), titled “Post-injury pain and behavior: a control theory perspective”. In this Perspective, we propose a control-theoretic framework to explain the adaptive processes in the brain that drive physiological post-injury behavior. We set out an evolutionary and ethological view on how animals respond to injury, illustrating how the behavioral state associated with persistent pain and recuperation may be just as important as phasic pain in ensuring survival.

This provocative and original approach to thinking about chronic and persistent pain as processes arising from natural selection rather than as a disease state, is likely to generate new lines of enquiry and ways of thinking about management of pain in humans and other animals.

Announcing the inaugural Cephalopod Neurobiology conference, April 17th-21st 2024, MBL

We are pleased to announce the first ever Cephalopod Neuroscience Conference!

April 17-21, 2024, MBL, Woods Hole, MA USA
https://sites.google.com/view/cephneuro/

Get on the mailing list: https://forms.gle/qWfaUTJL4aS4RazM6

Supported by a conference grant from the Simons Collaboration on the Global Brain, and the Frontiers Group of the Allen Foundation.

The organizing committee consists of Cristopher Niell (U Oregon) Caroline Albertin (MBL), Robyn Crook (SFSU) Mandë Holford (CUNY), Joshua Rosenthal (MBL), Simon Sprecher (U. Fribourg), and Paul Katz (U Mass Amherst.

New publication: analgesia and anesthesia in Euprymna berryi

Skyler Deutsch, first author on Deutsch et al., 2023

The lab’s latest paper is published this week in a Special Issue of the journal Biology, “ Anesthetics and analgesics used in aquatic animals”. The study, titled “Evaluation of Candidates for Systemic Analgesia and General Anesthesia in the Emerging Model Cephalopod, Euprymna berryi” provides the first evidence that analgesic drugs from several different drug classes have effects on nociception and pain-like behavior in cephalopods. This work was led by Skyler Deutsch, who was a post-bac researcher in the lab for year, supported by an NSF REPS supplement to the lab. In addition, two of our MS students, four SFSU undergrads, one of our summer REU students, and both our research technician and postdoc, all contributed data to this important and comprehensive study.

Currently there is significant discussion about the welfare of cephalopods in research labs and aquariums, but one constant in these conversations is the lack of information about what interventions are effective for promoting and enhancing welfare for cephalopods, given their physiological and behavioral differences from vertebrate animals. With this study, we provide the first clear evidence that systemic analgesics can reduce sensitivity and pain-like behavior, and we also show that general anesthesia can be performed safely and reliably in this species.

Euprymna berryi is growing rapidly as a newly-emerging model animal in neuroscience and behavior, and we anticipate that this study will encourage and support further evaluation of welfare-promoting interventions for this and other species of cephalopods.

PI Robyn Crook quoted in the Boston Globe

The Boston Globe quotes Robyn Crook on cephalopod research and welfare

A new story in the Boston Globe by David Abel covers the growing efforts to provide federal-level research protection for cephalopods. This controversial subject rests on determinations of cephalopod sentience, intelligence and capacity for affective state, all of which suggest regulation of research on cephalopods is warranted. This article focuses on the cephalopod breeding and research efforts at the Marine Biological Laboratory in Woods Hole, MA, where our laboratory brood-stock originates. The Crook Lab upholds the highest standards of welfare and husbandry for the cephalopods we use in our studies, and our work on applied welfare-based questions has become a touchstone for regulatory efforts in other nations. Our aim is to support the ongoing discussions of cephalopod regulation and welfare with well designed studies, to improve the lives of cephalopods in research, fisheries and display. It is great to see ongoing public-facing discourse on this important topic.