bioRxiv Subject Collection: Neuroscience's Journal

Hemodynamic and electrophysiological progression of the Rose Bengal photothrombotic stroke model in mice: vasoactive properties of Rose Bengal, tissue heating, wavelength optimization, and sex differences in lesion volume
The photothrombotic stroke model is gaining popularity due to its relative simplicity, minimal invasiveness, and clinical relevance. Photothrombosis involves the delivery of an intravascular photosensitizer (Rose Bengal) followed by its photoactivation, resulting in vessel occlusion and ischemia. Using a combination of complementary optical and non-optical techniques, we characterized the physiological changes in mice undergoing photothrombosis. We report that Rose Bengal acts as a rapid vasoconstrictor, inducing hypoemia both in the brain and periphery even in the absence of its photoactivation. Conversely, we find that light, when used at photothrombosis-appropriate intensities and durations, induces large amounts of tissue heating and hyperemia even in the distal non-illuminated hemisphere. Furthermore, we show that use of the optimal photothrombotic wavelength based on the Rose Bengal absorption spectrum (yellow-561nm) produces a more consistent and pronounced drop in blood flow, and a shorter latency to the initial spreading depolarization (SD), ultimately resulting in a larger stroke. Similarly, when yellow light is used to induce a stroke in ChR2-expressing mice, the electrophysiological and hemodynamic confounds from green light cross activation of ChR2 are eliminated. Finally, we observe across cohorts that male mice have larger strokes than females. Altogether, we extensively describe important caveats and confounds concerning photothrombosis and provide a detailed characterization of its early ischemic events.

Computer models predict differential dendritic vulnerability with ischemia and spreading depression
Ischemia, whether abrupt or chronic, limits ATP production and disrupts ATP-dependent homeostatic mechanisms, leading to alterations in both intracellular and extracellular ion concentrations. Inadequate neuronal ATP triggers K+ release and increased extracellular K+ depolarizes neurons, leading to additional K+ release; this positive feedback phenomenon is known as spreading depolarization (SD). When the depolarizing effects are strong enough, the cells undergo depolarization blockade, known as spreading depression. Excess extracellular K+ increases energy demand from the Na+-K+ pump, producing a pathological confluence of increased demand with reduced delivery of energy. The resulting changes have profound effects at subcellular, cellular, and network scales of brain function. We hypothesized that consequences of ischemic or SD homeostatic failure would differ on the subcellular scale, with differences between disjunct dendritic regions of a hippocampal CA1 pyramidal neuron. To evaluate the interplay between morphology and ion concentrations, we used a mechanistic simulation incorporating neuronal morphology, pumps, exchangers, voltage-, and Ca2+-sensitive ion channels. In both cases, calcium accumulation was greatest in the basilar dendrites, suggesting these dendrites would show the greatest effects of excitotoxicity. By contrast, ischemia, but not SD showed that distal apical dendrites were exposed to greater intracellular chloride concentrations, which may lead to dendritic beading.

Functional independence of entorhinal grid cell modules enables remapping in hippocampal place cells
A systems-level understanding of cortical computation requires insight into how neural codes are transformed across distinct brain circuits. In the mammalian cortex, one of the few systems where such transformations are tractable is the spatial mapping circuit. This circuit comprises interconnected regions of medial entorhinal cortex (MEC) and hippocampus, which encode location using fundamentally different neural codes. A key distinction is that neural activity in MEC, including that of directionally tuned cells and grid cells, evolves along low-dimensional manifolds, preserving stable phase relationships across different environments and behaviors. In contrast, hippocampal place cells frequently undergo global remapping: their collective firing patterns reorganize randomly across different environments, revealing an apparently limitless repertoire of orthogonal spatial representations. The mechanisms by which spatial maps are transformed between the two coding schemes remain unresolved. Here, we used large-scale multi-area Neuropixels recordings to show that when rats were transferred from one familiar environment to another, each module of grid cells underwent a unique change in phase on its low-dimensional manifold, at the same time as simultaneously recorded place cells exhibited global remapping. In contrast, training conditions that produced smaller differences in the phase shifts of simultaneously recorded grid modules resulted in incomplete place cell remapping, mirroring previous reports of 'partial remapping'. Hippocampal remapping was not associated with rotational differences between grid modules under any condition. Taken together, these findings suggest that differential phase shifts across grid cell modules form the basis for the orthogonalization of downstream hippocampal spatial codes during remapping. The transformation from low-dimensional spatial representations in the MEC to high-dimensional codes in the hippocampus may underlie the hippocampus' ability to support high-capacity memory storage.

RAVEN: Robust, generalizable, multi-resolution structural MRI upsampling using Autoencoders
Due to their high inter-tissue contrast, Magnetic resonance images (MRIs) can reflect neuroanatomical changes related to healthy aging and pathological processes. However, standard brain MRI acquisition resolutions hinder the ability to measure the more subtle changes that occur in early disease stages. Increasing the resolution during acquisition poses multiple challenges, including increased noise, higher acquisition times and cost, and discomfort of the scanned individual. In this work, we propose a robust, generalizable single-image super-resolution network for brain MRIs named Resolution Augmentation with Variational auto-Encoder Networks (RAVEN) with generative adversarial networks (GANs). We show RAVEN is capable of upsampling in-vivo and ex-vivo MRIs of diverse modalities (e.g. T1-weighted , T2-weighted, and T2*) and varying field strengths (3T to 7T) to target voxel sizes as small as 0.5mm isotropic using arbitrary upsampling factors. RAVEN achieved state-of-the-art performance against deep learning and non-deep learning methods, best preserving true anatomical information. We have also made RAVEN open access, with the source code as well as training and evaluation scripts available and ready to use at: https://github.com/waadgo/raven.

Calpain-2 inhibition or deletion enhances levels of the transcription Factor, MEIS2, and stimulates neurogenesis
Neurogenesis takes place in the subventricular zone (SVZ) and in the dentate gyrus (DG) of the hippocampus of many adult mammalian species. Recent findings indicate that calpain-2 could participate in neurogenesis regulation through the truncation of the transcription factor, Myeloid Ecotropic Viral Integration Site 2 (MEIS2). The present study aimed to test the effects of calpain-2 inhibition/deletion on MEIS2 levels and neurogenesis in adult mice. Two-to-three month-old mice were injected with a selective calpain-2 inhibitor, NA-184, and sacrificed 24 h later. In addition, two-to-three month-old conditional calpain-2 knock-out (C2KO) and calpain-1 knock-out (C1KO) mice were used. Levels of MEIS2 and of cell markers for neurogenesis were analyzed using immunohistochemistry and western blots. Dendritic spines in hippocampal neurons were also analyzed by Golgi staining. Acute treatment of wild-type (WT) mice with NA-184 increased levels of MEIS2 in various brain structures. It also increased numbers of neurons immunopositive for Ki67 and DCX, two markers for neurogenesis, in both the SVZ and DG. MEIS2 levels were elevated in C2KO mouse brain, while they were decreased in C1KO mouse brain. Compared to those in WT mice, neurons from C2KO mice exhibited a decrease in the number of filipodia spines and an increase in the number of mushroom spines, while those from C1KO exhibited opposite changes. These findings further emphasize the critical and opposite roles of calpain-1 and calpain-2 in brain functions in general, and in neurogenesis in particular with MEIS2 as a major downstream mediator. These findings also underline previous conclusions that calpain-1 promotes spine maturation and synaptic plasticity while calpain-2 hinders spine maturation and synaptic plasticity. These results indicate that calpain-2 inhibition/deletion results in increased neurogenesis, as well as in increased maturation of dendritic spines, potentially due to increased levels and activation of MEIS2.

Neural flexibility in metabolic demand dynamics reveals sex-specific differences and supports cognition in late childhood
Dynamic coordination of metabolic demand across brain networks supports emerging cognitive abilities and may drive overall cognitive development, yet how these dynamics vary by sex and relate to cognition in late childhood remains unclear. Using resting-state fMRI from 2,000 healthy 9- to 11-year-olds in the ABCD study, we applied time-resolved dynamic time warping to quantify amplitude mismatches, a proxy of relative energy demand across brain intrinsic networks. Clustering revealed three recurring states: convergent (globally balanced), divergent (imbalanced), and mixed (intermediate). Females spent engaged more with the flexible mixed state, whereas males lingered longer in convergent and divergent states. Across the cohort, better performance on cognitive flexibility, processing speed, and long-term memory tasks correlated with greater overall time in the mixed state and with higher transition rates, but with shorter dwell in any single state. These findings indicate that neural flexibility, rather than prolonged stability, supports cognition during late childhood and that sex differences in dynamic energy coordination emerge well before adolescence.

Discrete interneuron subsets participate in GluN1/GluN3A excitatory glycine receptor (eGlyR)-mediated regulation of hippocampal network activity throughout development and evolution.
Decades of studies implicating GluN3A N-methyl-D-aspartate receptor (NMDAR) subunits in physiological and pathological function have largely been interpreted through direct regulation of conventional glutamatergic NMDARs. However, emerging evidence indicates that GluN3A frequently assembles with GluN1 forming unconventional glutamate-insensitive NMDARs that operate as native excitatory glycine receptors (eGlyRs). Here we demonstrate that hippocampal somatostatin and neurogliaform interneurons (Sst-INs and NGFCs) express functional eGlyRs from early postnatal through adult ages. In the developing hippocampus eGlyR-mediated excitation of NGFCs with ambient glycine dramatically increases GABAergic tone, with consequences for the generation of giant depolarizing potentials (GDPs). In the mature hippocampus, eGlyR-mediated excitation of Sst-INs regulates sharp wave ripples (SWRs). Finally, we reveal evolutionary conservation of hippocampal Sst-IN eGlyRs and eGlyR-mediated SWR regulation in non-human primates confirming functional eGlyR availability for therapeutic potential in higher species. Our findings underscore that eGlyR mediated regulation of cell and circuit excitability through both cell autonomous and cell non-autonomous mechanisms must be considered to understand GluN3A roles in brain development, plasticity, and disease.

Targeting Amygdala-Brainstem Synapses to Reverse Prepulse Inhibition Deficits
Sensorimotor gating, a fundamental pre-attentive process, can be assessed using the acoustic pre-pulse inhibition (PPI) assay. PPI deficits are a hallmark endophenotype of schizophrenia and are observed across various neuropsychiatric disorders, often predicting symptoms such as attention impairments, psychosis, and other cognitive/motor dysfunctions. Reversal of PPI deficits is routinely tested in disease models as a preclinical trial for antipsychotic drug screening. However, the cellular and circuit-level mechanisms underlying PPI deficits remain unclear, limiting therapeutic progress. We recently identified an uncharted pathway in mice, by which glutamatergic neurons in the central nucleus of the amygdala (CeA) activate glycinergic neurons in the caudal pontine reticular nucleus (PnC), contributing to PPI regulation. Given the prevalence of amygdala dysfunction in disorders associated with PPI deficits, CeA-PnC glutamatergic synapses represent a novel therapeutic target. Here, using "Cal-Light," an in vivo Ca2+-dependent photo-tagging approach, we precisely identified CeA and PnC neurons active during acoustic startle and PPI with high spatiotemporal resolution. Furthermore, we used mice carrying homozygous deficiency in PRODH, a schizophrenia-relevant gene encoding proline dehydrogenase and modulating PPI. While Prodh-/- mice showed aberrant CeA neuronal properties and reduced PPI levels, we re-stored PPI by photo-activating CeA-PnC glutamatergic synapses, underscoring their involvement in pathological states. These findings provide new mechanistic insights into the amygdala-brainstem circuitry that underlies PPI deficits, offering new potential for therapeutic interventions.

Cooperative behavior guided by peer coordination is impaired in a Fragile-X rat model of autism
Cooperative behavior, the ability of individuals to coordinate their actions toward shared goals, is fundamental to survival and social success across species. However, the behavioral mechanisms that support cooperation, and how their disruption leads to social deficits in neurodevelopmental disorders such as autism spectrum disorder (ASD), remain poorly understood. To address these questions, we developed a cooperation task in paired spatial mazes in dyads of wild-type (WT) and Fmr1 knockout (Fmr1) rats, a model of Fragile X syndrome, under deterministic (100%) and probabilistic (50%) reward contingencies. Both WT and Fmr1 rat pairs exhibited mixed leader-follower strategies to coordinate their actions for cooperation, however WT pairs achieved significantly greater cooperation success than Fmr1 pairs. WT and Fmr1 pairs both displayed a follower-tracking-leader strategy during transitions between reward wells, which required partner-directed visual attention, followed by a reactive action to match leader position, with Fmr1 pairs reliant on this strategy to a greater degree. In WT rats, more efficient cooperation was based on a flexible predictive strategy leading to coordination of optimal choice patterns between rat pairs and sensitivity to recent partner choices, whereas Fmr1 rats were significantly weaker in these strategies leading to deficits in adaptive behavior. These findings identify key behavioral strategies for cooperation, reveal their disruption in a rat model of ASD, and provide a framework for linking social cue usage to flexible and strategic cooperative decision-making with relevance to neurodevelopmental disorders.

Activity-dependent control of axonal amphisome trafficking governs norepinephrine release in vivo
The postmitotic nature, exceptional longevity, and elaborate cytoarchitecture of neurons exert extraordinary demands on proteostasis and autophagy regulation. Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. Previous work suggests that neuronal amphisomes also serve as signalling platforms, though their physiological relevance in vivo remains largely unexplored. Here, we demonstrate dynamic trafficking of amphisomes within the long-range, highly branched axons of locus coeruleus norepinephrine (LC-NE) neurons. Using in vivo photoconversion, we show that amphisomes originating in distal axons can traverse the entire axonal length to reach the soma. Two-photon imaging of LC-NE projections to the prefrontal cortex revealed that velocity and directionality of trafficking are tightly regulated by LC activity states, behavioural context, and autocrine norepinephrine signalling. Activation of Gi-coupled receptor signalling unifies directionality of transport, enhances somatic cargo delivery, whereas prolonged distal immobilization correlates with increased norepinephrine release, consistent with a signalling function. Together, these findings establish LC amphisomes as dual-function organelles that integrate degradative transport with activity-dependent signalling in vivo.

Quantifying Rhythmic and Arrhythmic Components of Brain Activity
Brain activity comprises both rhythmic (periodic) and arrhythmic (aperiodic) components. These signal elements vary across healthy aging, and disease, and may make distinct contributions to conscious perception. Despite pioneering techniques to parameterize rhythmic and arrhythmic neural components based on power spectra, the methodology for quantifying rhythmic activity remains in its infancy. Variation in analytical choices for isolating brain rhythms from background arrhythmic activity makes interpreting findings across studies difficult. Whether current approaches can accurately recover the independent contribution of these neural signal elements remains to be established. Here, using simulation and parameter recovery approaches, we show that standard analytic methods for quantifying rhythmic activity conflate these two neurophysiological components, yielding spurious correlations between spectral model parameters. We propose an alternative approach to overcome these limitations and demonstrate effective separation of rhythmic and arrhythmic components in simulated neural time series. We validate these methods using resting-state recordings from a large cohort. Our recommendations for spectral parameterization enable the robust independent quantification of rhythmic and arrhythmic signal components for cognitive neuroscience.

Progenitor Timing Shapes NG2-Glia Fate and Oligodendrocyte Differentiation
The developmental identity and fate of NG2-glia remain debated: are they transient oligodendrocyte precursors or a distinct, self-renewing glial population? Here, we examined how the temporal origin of progenitors influences NG2-glia and oligodendrocyte lineages in the dorsal cortex. Using in utero and postnatal StarTrack electroporation at E12, E14, E16, and P0, we traced their progeny to P30 and P90, performing a clonal analysis in adult mouse brains. Progenitors labeled at E16 generated significantly larger and more widely dispersed NG2-glia clones, whose contribution increased from P30 to P90, suggesting enhanced proliferative capacity. In contrast, P0-derived progenitors showed reduced NG2-glia maintenance and a strong bias toward oligodendrocyte differentiation, forming larger OL clones. Clonal heterogeneity, including mixed NG2-glia/OL clones, was observed across all stages but peaked at E16. These results identify E16 as a critical window for NG2-glia expansion and self-renewal, while P0 marks a transition toward oligodendrocyte lineage restriction, establishing a developmental framework for adult NG2-glia heterogeneity and maybe a regenerative potential.

Octopamine signaling from clock neurons plays dual roles in Drosophila long-term memory
Circadian clock genes are best known for regulating circadian rhythms, but they also play crucial roles in memory processes. This suggests that memory is modulated by neural networks containing clock neurons, although the underlying mechanisms remain unclear. In Drosophila melanogaster, approximately 240 clock neurons are grouped into at least eight distinct clusters. Among them, the dorsal--lateral neurons (LNds) are required for maintaining long-term memory (LTM). In contrast, the neuropeptide Pigment-dispersing factor (Pdf), expressed in both small and large ventral--lateral neurons (s-LNvs and l-LNvs, respectively), functions as a circadian output signal and is also essential for maintaining LTM. In addition, Pdf-expressing neurons (hereafter, Pdf neurons) release neurotransmitters other than Pdf, which are involved in LTM consolidation. However, the specific transmitters used by LNds and Pdf neurons in LTM processing have remained unknown. Here, we show that octopamine signaling from LNds is essential for LTM maintenance, whereas octopamine in Pdf neurons is essential for LTM consolidation. Temporally restricted knockdown of Tyramine {beta} hydroxylase (Tbh), the gene encoding the enzyme required for octopamine synthesis, disrupted LTM maintenance when targeted in LNds, whereas it impaired LTM consolidation when targeted in Pdf neurons. Notably, Tbh knockdown in LNds or Pdf neurons had minimal effects on circadian behavioral rhythms or sleep. These findings reveal that octopamine released from specific subtypes of clock neurons independently regulates distinct phases of LTM in Drosophila.

Fast segmentation with the NextBrain histological atlas
Structural brain analysis at the subregion level offers critical insights into healthy aging and neurodegenerative diseases. The NextBrain histological atlas was recently introduced to support such fine-grained investigations, but its existing Bayesian segmentation framework remains computationally prohibitive, particularly for large-scale studies. We present a new, open-source tool that dramatically accelerates segmentation using a hybrid approach combining: machine learning, contrast-adaptive segmentation; target-specific image synthesis; and fast diffeomorphic registration (all three with GPU support). Our method enables highly granular segmentation of brain MRI scans of any resolution and contrast (in vivo or ex vivo) at a fraction of the computational cost of the original method (<5 minutes on a GPU). We validate our tool on four different modalities (in vivo MRI, ex vivo MRI, HiP-CT, and photography) across a total of approximately 4,000 brain scans. Our results demonstrate that the accelerated approach achieves comparable accuracy to the original method in terms of Dice scores, while reducing runtime by over an order of magnitude. This work enables high-resolution anatomical analysis at unprecedented scale and flexibility, providing a practical solution for large neuroimaging studies. Our tool is publicly available in FreeSurfer (https://surfer.nmr.mgh.harvard.edu/fswiki/HistoAtlasSegmentation).

Rare bioactive tau oligomers from Alzheimer brain support both templated misfolding and fibril formation
The accumulation of hyperphosphorylated tau aggregates is a hallmark of Alzheimer's disease (AD). In addition to long-recognized tau deposits in neurofibrillary tangles, recent studies suggest that diffusible, aqueous soluble (High Molecular Weight, or HMW) species are also bioactive, i.e., able to seed templated misfolding. The characteristics of the diffusible misfolded proteins are largely unknown, and their relationship to classical fibrillar structures is unclear. Using sequential size exclusion and anion exchange chromatography, we fractionated the HMW tau population and identified multiple subspecies varying in retention properties. The subspecies that elute early from the size column, and are retained on the anion exchange column are seed competent, whereas the other soluble fractions are not. Biophysical analyses using super resolution, atomic force, and immunogold electron microscopy confirmed that the size and conformation of both bioactive and non-bioactive tau oligomers are similar, with dimers, trimers, and tetramers predominating. The presence of surface phosphorylations, as detected by recently developed single molecule array (SIMOA) analyses, correlates with seeding capacity. Single bioactive tau oligomers at fMol concentrations can induce seeding and templated misfolding in a reporter cell. The bioactive species can alternatively support aggregation of a truncated repeat domain tau construct into thioflavin T positive fibrils in a real-time quacking-induced conversion (RT-QuIC) assay, whereas full length recombinant tau yields oligomers. These findings provide structural insights into bioactive oligomeric tau species, emphasize the small concentrations necessary for bioactivity, and highlight the possibility that, under different conditions, they can seed either oligomeric or fibrillar structures.

Combining brainwide activity imaging and electron microscopy reveals novel nociceptive circuits
To understand how brains work, it is necessary to connect neural activity to synaptic-resolution circuit architecture. Recent advances in light-sheet microscopy (LSM) enable whole-brain, cellular-resolution imaging of activity of all neuronal cell bodies, however most neurons from such datasets cannot be identified. In most organisms, neurons are identifiable based on their projections (and not based on their cell body position) which cannot be resolved in using LSM. Here, we present a novel methodology to overcome this by combining in vivo whole-brain activity imaging, with subsequent electron microscopy imaging of the same brain to visualise neuronal projections and identify neurons with interesting activity. We used this approach to identify brain neurons involved in nociception in Drosophila larva. After whole-brain imaging of activity during nociceptive stimulation, we imaged the same brain with an enhanced focused ion-beam electron microscope (eFIB-SEM). We registered the functional and anatomical volumes and reconstructed (in the eFIB-SEM volume) the projections of neurons that responded to nociceptive stimulation to determine their developmental lineage and identity. This revealed a distributed nociceptive network spanning 25 distinct lineages and many distinct brain areas, and included direct brain targets of nociceptive projection neurons that integrate nociceptive information with other sensory modalities, as well as brain output neurons (descending neurons [DN]) that likely contribute to action-selection. Surprisingly, we also found neurons previously previously associated with olfaction and learning, such as Kenyon cells. Our workflow provides a powerful framework for mapping neuronal activity onto structure across an entire brain, yielding novel insights into the central processing of noxious stimuli.

An inspiration-off attractor supports the robust and flexible control of breathing
Breathing is a fundamental motor rhythm necessary to sustain life. The rhythm and pattern of breathing arises from the coordination of a bilaterally symmetric, rostro-caudally extended column of heterogeneous neural populations in the medulla called the Ventral Respiratory Column (VRC). By recording from the extent of the VRC using Neuropixels during optogenetic and physiological manipulations, and projecting the population activity into a dynamical latent space, we find that GABAergic lung-stretch feedback circuits promote rhythmic population activity by creating a temporary stable fixed point in the latent state that terminates diaphragmatic contraction. Stimulation of GABAergic circuits either intrinsic to the VRC (VRCGABA) or via afferent pathways from GABAergic neurons of the Nucleus of the Solitary Tract (NTSGABA) advance or delay breathing when activated with specific respiratory phase through temporary stabilization of this fixed point. Lastly, we show that activation of glutamatergic signaling opposes the effects of inhibitory signaling by destabilizing this expiratory fixed point to promote rapid inspiration. Together, we decompose the functional impact of different respiratory control subpopulations to reveal an integrated network level mechanism for respiratory control in vivo.

Encoding and combining naturalistic motion cues in the ferret higher visual cortex
Natural visual scenes contain rich flows of pattern motion that vary not only in orientation but also in spatial sizes and temporal rhythms. To properly interpret the motion, the brain must extract individual features and integrate them into coherent parts, and sometimes also segregate these parts from each other. Here, we performed single-neuron recordings in the motion-sensitive higher-order visual cortex (PMLS) of awake ferrets to investigate how complex motion signals are encoded and combined. We presented motion clouds---naturalistic stimuli with parametrically controlled spatiotemporal frequency content---and found that motion features were encoded in a temporally ordered sequence: orientation and spatial frequency emerged within 120 ms after stimulus onset, while temporal frequency and direction followed at later latencies. Time-resolved decoding revealed that this selectivity evolved dynamically within neurons and was distributed across the population. To probe motion integration, we introduced compound motion clouds composed of two or three localized frequency components. Neuronal responses were well explained by a linear pooling model, suggesting a simple summation mechanism of the individual components. However, a distinct subset of neurons exhibited late responses sensitive to changes in speed content despite matched marginals, consistent with receptive fields differentiating along the speed gradient. Together, we have uncovered a structured and distributed code for motion in high-level visual cortex, and provide mechanistic insights into how the brain parses complex motion in natural scenes.

Mapping the nervous system of the Idiosepius hallami pygmy squid: insights from whole-animal X-ray nanotomography imaging.
The study of a nervous system as big as the cephalopod requires multimodal imaging approaches capable of capturing neural architecture across scales. Here, we present a whole-animal volume of the pygmy squid hatchling Idiosepius hallami, acquired using X-ray holographic nanotomography at the beamline ID16A of the European Synchrotron. The reconstructed 3D volume comprises 40 tiled scans acquired at a voxel size of 125 nm. While individual neurons are not resolved at this resolution, we segmented major body regions and mapped the large-scale connectivity by tracing afferent and efferent nerve bundles, including projections from the olfactory organs, chromatophore lobes, and arm ganglia to the brain. The acquisition of this dataset represents a significant milestone for X-ray nanotomography, being the largest whole animal volume imaged at this spatial resolution. The volume serves as a resource for comparative neuroscience and cephalopod biology.