Imaging cellular activity simultaneously across all organs of a vertebrate reveals body-wide circuits

Background:

The foundational principle of physiology, articulated by Claude Bernard and later canonized by Walter Cannon, is that complex organisms maintain a stable internal milieu-homeostasis through a symphony of coordinated responses across organs¹. This “wisdom of the body” relies on intricate feedback loops that span neural, endocrine, and immune systems, allowing an animal to adapt to external stressors and internal demands². For decades, our understanding of these networks has been largely inferential, pieced together from studies that examine one organ, one pathway, or one molecule at a time. This approach, while invaluable, is akin to trying to understand an orchestral piece by listening to individual instruments in separate rooms; the score of the whole remains elusive. This gap between conceptual framework and observational capability has been a central challenge in biology and medicine³.

The rise of functional neuroscience offered a new paradigm for observing biological processes at scale. Pioneering work, particularly in transparent model organisms like larval zebrafish, demonstrated the power of using genetically encoded calcium indicators (GECIs) to monitor the activity of thousands of neurons simultaneously during behavior^4,5. This “brain-wide” approach led to fundamental insights into sensorimotor processing⁶ and neural coding⁷, proving that observing a system in its entirety could reveal principles invisible to reductionist methods. However, this powerful lens remained almost exclusively focused on the central nervous system. The rest of the body, the very organs with which the brain continuously communicates to regulate physiology, was rendered a black box. This created a critical knowledge gap: we could decode a brain’s response to a threat, but not the concurrent, real-time cellular events in the immune system, gut, and vasculature that together constitute the full physiological response.

Bridging this gap presented monumental technical hurdles. Extending functional imaging to the entire body was not simply a matter of scaling up the field of view. The viscera exhibit complex, non-rigid motion that confounds standard image registration algorithms developed for the relatively stable brain⁸. Tissues outside the CNS have diverse optical properties that scatter light, degrading resolution, and no transgenic strategy existed for robust, non-toxic expression of sensors across the vast diversity of non-neuronal cell types. While recent studies provided glimpses into specific body-brain connections revealing, for instance, neurons that regulate inflammation⁹ or link gut cues to behavior¹⁰, these were still discovered and examined one circuit at a time. The field lacked a generalizable, unbiased platform to screen the entire vertebrate for coordinated cellular activity, a tool that could move beyond testing specific hypotheses to generating them de novo from a complete functional dataset.

In this groundbreaking preprint, Ruetten and colleagues directly confront this long-standing challenge. They present WHOLISTIC: a unified methodological platform that finally enables the comprehensive observation of cellular calcium dynamics across nearly all tissues of a living vertebrate. By converging key innovations, the authors achieve a systems-level view of physiology that has been a central goal of the field for decades. Furthermore, by integrating their functional imaging with a newly developed Whole-Body Expansion Microscopy (WB-ExM) protocol^11,12, they provide the essential anatomical context, allowing them to trace the subcellular structures, such as the elaborate ventral projections of ependymal cells (Fig. 1), that underlie the observed body-wide dynamics.

Fig. 1 – Propagation of Ca2+ waves and whole-body expansion microscopy of hindbrain ependymal cells. Fig. 1 of the preprint, made available under a CC-BY-NC-ND 4.0 International license.

Key Findings:

A unified platform for whole-body functional imaging (WHOLISTIC):

The authors engineered a Tg(ubi:tTA; TRE:GCaMP7f) zebrafish line for sustained, high-fidelity sensor expression across >15 cell types. Bypassing standard light-sheet microscopy, they identified spinning-disk confocal as uniquely capable for whole-body volumetric imaging due to its superior scattering rejection. Their custom iterative optical flow algorithm solves the critical problem of non-rigid motion in viscera, enabling stable tracking of individual cells over hours. The computational workflow segments individual cells and then uses a new coherence-based spectral clustering method to group them into functionally coupled anatomical units.

Discovery of New Functional Units and Couplings:

Applying WHOLISTIC as an unbiased discovery tool yielded multiple insights: a) Revised Muscle Anatomy: The preprint authors identified a novel functional synergy between the cervical-epaxial and abdominal muscles, linked to dual innervation by the spino-occipital nerve, revising classical anatomical maps. b) Body-Wide Motor Coupling:A regression model revealed that skeletal muscle activity triggers calcium transients in diverse tissues. A key discovery was that ependymal cells, glia-like cells lining the central canal, show prolonged, movement-locked calcium signals. c) State-Dependent Ultraslow Oscillations: During extended motor quiescence, a network of hindbrain/spinal cord ependymal cells exhibited high-amplitude, traveling calcium waves with a ~3-7 minute period, potentially linked to rest-state physiology. d) Causal Vagus-Sphincter Circuit: Combining WHOLISTIC with optogenetics, the research team validated a direct, topographical circuit from motor vagal neurons to the gastropharyngeal sphincter, demonstrating causal control of visceral smooth muscle.

Visualizing a Conserved Hypoxia Response Circuit:

Under hypoxic conditions, WHOLISTIC captured the rapid constriction of the mesenteric artery and shunting of blood flow away from the gut in real-time, a classic stress response never before imaged dynamically at cellular resolution. The researchers proved this is neurally regulated: optogenetic inhibition of the hindbrain instantly restored gut perfusion. WB-ExM then revealed the responsible sympathetic ganglion and its axonal projections along the artery, defining a complete brain-body circuit for oxygen prioritization (Fig. 2).

Anatomical Ground Truth with WB-ExM:

The team developed an enzyme-free, rapid WB-ExM protocol for ~5x uniform expansion of whole zebrafish and Danionella, compatible with immunofluorescence and in situhybridization. This provided the essential ultrastructural context for their functional observations, enabling high-resolution molecular phenotyping across the entire organism.

Fig. 2 – Uncovering of body-wide circuit engaged in response to stress. Fig. 2 of the preprint, made available under a CC-BY-NC-ND 4.0 International license.

Why I highlight this preprint?

This preprint represents the kind of transformative science that redefines what is possible in a field. As someone working in zebrafish systems neuroscience, I have admired the Ahrens lab’s philosophy: to build the tools needed to answer the biggest, most integrative questions. Misha has consistently pushed the boundaries of in vivo imaging, and WHOLISTIC feels like the culmination of this vision: a platform that shifts the very unit of observation from the brain to the entire organism. It’s a breathtaking technical and conceptual achievement that embodies the collaborative, tool-building spirit of Janelia Farm.

My excitement for this work is multifaceted. It solves a fundamental measurement problem. For decades, we’ve studied physiology in fragments. WHOLISTIC provides the first unified lens to see the organism as an integrated circuit, where the state of a kidney tubule, a skin cell, and a neuron can be correlated simultaneously. This is not merely incremental; it’s a paradigm shift from reductionism to holistic systems biology, echoing the foundational principles of homeostasis but now with the tools to visualize it.

The methodology itself is a masterpiece of integration. Each component from the pancellular driver and motion correction to the coherence-based clustering and WB-ExM is a significant innovation. Together, they form a closed loop of discovery: unbiased functional imaging generates hypotheses, optogenetics tests causality, and expansion microscopy provides mechanistic anatomical explanation. This pipeline sets a new standard for in vivo physiology.

Finally, the implications are vast. Beyond the immediate, fascinating discoveries about muscle synergy, ependymal networks, and hypoxia circuits, WHOLISTIC opens entirely new frontiers. For disease modelling and drug discovery, it offers a platform to screen for organism-wide effects and off-target actions, moving beyond single-organ endpoints. For basic research, it enables the study of previously intractable problems like inter-organ communication during sleep, stress, or metabolic regulation. This work doesn’t just advance zebrafish research; it provides a blueprint for holistic biology that could eventually inform a new understanding of human physiology and integrative medicine. It is, in every sense, a foundational study for the next era of systems biology.

Reference:

1. Cannon, W. B. (1939). The wisdom of the body.
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3. Buchman, T. G. (2002). The community of the self. Nature, 420(6912), 246-251.
4. Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M., & Keller, P. J. (2013). Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nature methods, 10(5), 413-420
5. Vladimirov, N., Mu, Y., Kawashima, T., Bennett, D. V., Yang, C. T., Looger, L. L., … & Ahrens, M. B. (2014). Light-sheet functional imaging in fictively behaving zebrafish. Nature methods, 11(9), 883-884.
6. Naumann, E. A., Fitzgerald, J. E., Dunn, T. W., Rihel, J., Sompolinsky, H., & Engert, F. (2016). From whole-brain data to functional circuit models: the zebrafish optomotor response. Cell, 167(4), 947-960.
7. Portugues, R., Feierstein, C. E., Engert, F., & Orger, M. B. (2014). Whole-brain activity maps reveal stereotyped, distributed networks for visuomotor behavior. Neuron, 81(6), 1328-1343.
8. Voleti, V., Patel, K. B., Li, W., Perez Campos, C., Bharadwaj, S., Yu, H., … & Hillman, E. M. (2019). Real-time volumetric microscopy of in vivo dynamics and large-scale samples with SCAPE 2.0. Nature methods, 16(10), 1054-1062.
9. Jin, H., Li, M., Jeong, E., Castro-Martinez, F., & Zuker, C. S. (2024). A body–brain circuit that regulates body inflammatory responses. Nature, 630(8017), 695-703.
10. Tan, H. E., Sisti, A. C., Jin, H., Vignovich, M., Villavicencio, M., Tsang, K. S., … & Zuker, C. S. (2020). The gut–brain axis mediates sugar preference. Nature, 580(7804), 511-516.
11. Chen, F., Tillberg, P. W., & Boyden, E. S. (2015). Expansion microscopy. Science, 347(6221), 543-548.
12. Ruetten, V. M., Hu, A., Eddison, M., Close, K., He, Y., Ahrens, M. B., & Tillberg, P. (2025). WHOLISTIC ExM: Whole-Body Expansion Microscopy with Immunofluorescence and Histological Stains.

Tags: systems neuroscience, zebrafish

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