The brain's capabilities rely on both the molecular properties of individual cells and their interactions across brain-wide networks. However, relating gene expression to activity in individual neurons across the entire brain remains elusive. Here we developed an experimental-computational platform, WARP, for whole-brain imaging of neuronal activity during behavior, expansion-assisted spatial transcriptomics, and cellular-level registration of these two modalities. Through joint analysis of whole-brain neuronal activity during multiple behaviors, cellular gene expression, and anatomy, we identified functions of molecularly defined populations — including luminance coding in a cckb-pou4f2 midbrain population and task-structured activity in pvalb7-eomesa hippocampal-like neurons — and defined over 2,000 other function-gene-anatomy subpopulations. Analysis of this unprecedented multimodal dataset also revealed that most gene-matched neurons showed stronger activity correlations, highlighting a brain-wide role for gene expression in functional organization. WARP establishes a foundational platform and open-access dataset for cross-experiment discovery, high-throughput function-to-gene mapping, unification of cell biology and systems neuroscience, and scalable circuit modeling at the whole-brain scale. ### Competing Interest Statement The authors have declared no competing interest. Howard Hughes Medical Institute, https://ror.org/006w34k90

An animal’s ability to survive and thrive—whether fleeing from danger, eating a meal, or fighting an infection—arises from the collective moment-to-moment activity of many interacting cell types throughout the body. Physiology seeks to elucidate these cellular interactions that span organs, cell types and timescales, but has been limited by the inability to record this time-varying cellular activity simultaneously throughout the entire body. Here we develop WHOLISTIC (WHole-Organism Live-Imaging System for recording Tissue and IntraCellular activity), a method to image second-timescale activity of cells across the entire vertebrate body at cellular resolution. WHOLISTIC advances and integrates volumetric fluorescence microscopy, machine learning, and pancellular transgenic expression of calcium sensors1, demonstrated in larval zebrafish, with proof of concept in adult Danionella cerebrum. To access information about the molecular and ultrastructural substrates for the measured dynamics, we advanced whole-body expansion microscopy2. At the cellular scale, body-wide screening revealed unexpected responses, including chondrocyte reactions to cold and meningeal responses to ketamine. At the organ scale, WHOLISTIC identified rhythmic travelling waves along the renal nephron. At the multi-organ scale, it revealed unknown muscle synergies and muscle–organ interactions. At the whole-organism scale, the method captured brainstem-controlled redistribution of body-wide blood flow. Combining optogenetics with WHOLISTIC enabled all-optical causal dissection of brain–body interactions. These advances establish a paradigm for systems biology that bridges cellular and organismal physiology, enabling comprehensive discovery across scales—from fundamental mechanisms to therapeutic targets. An imaging system developed to record cellular activity throughout the whole body of zebrafish captures cellular organ dynamics and identifies multiple distributed circuits.

The central complex (CX) plays a key role in many higher-order functions of the insect brain including navigation and activity regulation. Genetic tools for manipulating individual cell types, and knowledge of what neurotransmitters and neuromodulators they express, will be required to gain mechanistic understanding of how these functions are implemented. We generated and characterized split-GAL4 driver lines that express in individual or small subsets of about half of CX cell types. We surveyed neuropeptide and neuropeptide receptor expression in the central brain using fluorescent in situ hybridization. About half of the neuropeptides we examined were expressed in only a few cells, while the rest were expressed in dozens to hundreds of cells. Neuropeptide receptors were expressed more broadly and at lower levels. Using our GAL4 drivers to mark individual cell types, we found that 51 of the 85 CX cell types we examined expressed at least one neuropeptide and 21 expressed multiple neuropeptides. Surprisingly, all co-expressed a small molecule neurotransmitter. Finally, we used our driver lines to identify CX cell types whose activation affects sleep, and identified other central brain cell types that link the circadian clock to the CX. The well-characterized genetic tools and information on neuropeptide and neurotransmitter expression we provide should enhance studies of the CX.

All cells in an animal collectively ensure, moment-to-moment, the survival of the whole organism in the face of environmental stressors1,2. Physiology seeks to elucidate the intricate network of interactions that sustain life, which often span multiple organs, cell types, and timescales, but a major challenge lies in the inability to simultaneously record time-varying cellular activity throughout the entire body. We developed WHOLISTIC (WHole Organism Live Imaging System for recording Tissue and IntraCellular activity), a method to image second-timescale, time-varying intracellular dynamics across cell-types of the vertebrate body. By advancing and integrating volumetric fluorescence microscopy, machine learning, and pancellular transgenic expression of calcium sensors in transparent young Danio rerio (zebrafish) and with proof of concept in adult Danionella, the method enables real-time recording of cellular dynamics across the organism. Calcium is a universal intracellular messenger, with a large array of cellular processes depending on changes in calcium concentration across varying time-scales, making it an ideal proxy of cellular activity3. Using this platform to screen the dynamics of most cells in the body, we discovered unexpected responses of specific cell types to stimuli, such as chondrocyte reactions to cold, meningeal responses to ketamine, and state-dependent activity, such as oscillatory ependymal-cell activity during periods of extended motor quiescence. At the organ scale, the method uncovered pulsating traveling waves along the kidney nephron. At the multi-organ scale, we uncovered muscle synergies and independencies, as well as muscle-organ interactions. Integration with optogenetics allowed us to all-optically determine the causal direction of brain-body interactions. At the whole-organism scale, the method captured the rapid brainstem-controlled redistribution of blood flow across the body. Finally, we advanced Whole-Body Expansion Microscopy4 to provide ground-truth molecular and ultrastructural anatomical context, explaining the spatiotemporal structure of activity captured by WHOLISTIC. Together, these innovations establish a new paradigm for systems biology, bridging cellular and organismal physiology, with broad implications for both fundamental research and drug discovery.

Both neurons and glia communicate through diffusible neuromodulators; however, how neuron-glial interactions in such neuromodulatory networks influence circuit computation and behavior is unclear. During futility-induced behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine (NE) drives fast excitation and delayed inhibition of behavior and circuit activity. We found that astroglial purinergic signaling implements the inhibitory arm of this motif. In larval zebrafish, NE triggers astroglial release of adenosine triphosphate (ATP), extracellular conversion of ATP into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. Our results suggest a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in NE-mediated behavioral and brain state transitions and position astroglia as important effectors in neuromodulatory signaling.

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Unveiling the genetic profiles of spatially distinguished cells is an important aspect in many areas of brain research, as the genetic identity contains information about a cell's physiological properties and internal state. On top of this, knowledge of the genetic details of each cell can reveal structural organization within tissue. As image-based spatial transcriptomics moves toward applications in tissues with dense cellular packing, accurate assignment of detected mRNA transcripts ("spots") to correct segmented cells becomes increasingly difficult, rendering simple methods insufficient with many incorrect assignments to neighboring cells. Here we introduce SpotDMix, a statistical model for assigning spots to cells by modeling spots as coming from a mixture model of distributions matching segmented cell shapes, with assignment probabilities and shape parameters optimized using the Expectation Maximization algorithm. Performance is assessed and compared against several simple methods in various scenarios on both surrogate data and larval zebrafish data. In all tested scenarios SpotDMix outperforms the simple methods on all evaluated metrics, including individual transcript assignment accuracy, total assigned number of spots per cell error and cell type classification. Further, SpotDMix produces a higher degree of exclusivity between genes which are known to not or rarely co-express. ### Competing Interest Statement The authors have declared no competing interest. Dutch Research Council, https://ror.org/04jsz6e67, 016.Vidi.189.052

Many plasticity rules rely on adjusting the strength of synapses between pairs of cells based on their coincident activity. We uncovered a new mechanism for coincidence detection in the Drosophila head direction network. To maintain an accurate sense of direction, head direction neurons that signal orientation during navigation must learn to anchor to relevant external sensory cues in novel environments. Yet the synaptic mechanism for this form of unsupervised learning is unknown in any organism. In Drosophila, GABAergic visual inputs converge onto head direction neurons, and these inhibitory synapses change strength with experience to learn the relationship between visual landmarks and head direction. However, how coincident pre- and postsynaptic activity is detected across this inhibitory synapse is not understood. We discovered that neurons which release the monoamine octopamine close a feedback loop that conveys postsynaptic head direction activity onto presynaptic terminals of visual inputs. This octopamine pathway is required for anchoring the head direction network to visual cues. Furthermore, pairing structured activation of octopamine neurons with a visual cue is sufficient to drive rapid plasticity, even without postsynaptic head direction cell activity. Previous work has extensively characterized coincidence detection mechanisms at excitatory synapses; our work defines a novel mechanism for coincidence detection at an inhibitory synapse, in which postsynaptic activity is relayed via a neuromodulatory neuron onto presynaptic terminals.

The interpretation of Whole Body Imaging (WBI) data necessitates comprehensive anatomical knowledge to accurately determine cell-type identity; however, resources pertaining to the internal anatomy of larval zebrafish are limited. To mitigate this gap, we established an advanced Whole Body Expansion-Microscopy (WB-ExM) protocol, facilitating the acquisition of molecular and cell-type information at subcellular resolution throughout the entire organism. While high-quality resources are available for the larval zebrafish brain1,2,3 there remains a deficiency in materials concerning its visceral anatomy. In order to supplement existing histological, X-ray, and Expansion Microscopy methods aimed at mapping body-wide anatomy4,5,6,7 and to develop a technique that yields high-fidelity molecular and anatomical data with enhanced processing speeds and compatibility with older samples, we devised an enzyme-free, rapid, and robust whole-body expansion microscopy method8,9, effective on larvae up to at least 14 days post-fertilization. This method involves the use of high-temperature (100°C) chemical hydrolysis to uniformly soften tissues, embedding within a medium-density gel with reduced protein-gel anchoring, and repeated embedding post-digestion with moderate ~1.5x expansion factors in each cycle, culminating in a robust and uniform expansion even of challenging structures such as cartilage embedded in soft tissue. This protocol results in excellent optical clearing, retains high levels of antibody signals. A demo dataset can be viewed here. Dorsal view of a double transgenic zebrafish animal labeling the ventricular and vascular systems (Tg(foxj1a:eGFP) x Tg(flk1:dsRed-CAAX)), stained against eGFP (magenta) and dsRed (green) (10 days post-fertilization, expanded ~2×). 1. Kunst, M. et al. A Cellular-Resolution Atlas of the Larval Zebrafish Brain. Neuron 103, 21-38.e5 (2019). 2. Randlett, O. et al. Whole-brain activity mapping onto a zebrafish brain atlas. Nat Methods 12, 1039–1046 (2015). 3. Tabor, K. M. et al. Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping. 4. Copper, J. E. et al. Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 208, 38–46 (2018).5. Ding, Y. et al. Computational 3D histological phenotyping of whole zebrafish by X-ray histotomography. eLife 8, e44898 (2019). 6. Steib, E. et al. TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy. Cell Reports Methods 2, 100311 (2022). 7. Sim, J. et al. Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy. Preprint at https://doi.org/10.1101/2021.05.18.443629 (2021). 8. Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy.9. Wang, Y. et al. EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell 184, 6361-6377.e24 (2021).

Effective classification of neuronal cell types requires both molecular and morphological descriptors to be collected in situ at single cell resolution. However, current spatial transcriptomics techniques are not compatible with imaging workflows that successfully reconstruct the morphology of complete axonal projections. Here, we introduce a new methodology that combines tissue clearing, submicron whole-brain two photon imaging, and Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to assign molecular identities to fully reconstructed neurons in the mouse brain, which we call morphoFISH. We used morphoFISH to molecularly identify a previously unknown population of cingulate neurons projecting ipsilaterally to the dorsal striatum and contralaterally to higher-order thalamus. By pairing whole-brain morphometry, improved techniques for nucleic acid preservation and spatial gene expression, morphoFISH offers a quantitative solution for discovery of multimodal cell types and complements existing techniques for characterization of increasingly fine-grained cellular heterogeneity in brain circuits. ### Competing Interest Statement The authors have declared no competing interest.

A straightforward, robust, and reliable protocol (EASI-FISH) that utilizes expansion microscopy and the hybridization chain reaction for multiplexed in situ hybridization in thick slices of the mouse brain has recently been described (Wang et al., 2021). Below details a modified version of the EASI-FISH protocol for adult Drosophila CNS, which includes antibody detection of fluorescent reporters. The protocol also works well for larval CNS and is expected to be applicable to other tissue types.

Dopamine is a key chemical neuromodulator that plays vital roles in various brain functions. Traditionally, neuromodulators like dopamine are believed to be released in a diffuse manner and are not commonly associated with synaptic structures where pre- and postsynaptic processes are closely aligned. Our findings challenge this conventional view. Using single-bouton optical measurements of dopamine release, we discovered that dopamine is predominantly released from varicosities that are juxtaposed against the processes of their target neurons. Dopamine axons specifically target neurons expressing dopamine receptors, forming synapses to release dopamine. Interestingly, varicosities that were not directly apposed to dopamine receptor-expressing processes or associated with neurons lacking dopamine receptors did not release dopamine, regardless of their vesicle content. The ultrastructure of dopamine release sites share common features of classical synapses. We further show that the dopamine released at these contact sites induces a precise, dopamine-gated biochemical response in the target processes. Our results indicate that dopamine release sites share key characteristics of conventional synapses that enable relatively precise and efficient neuromodulation of their targets. ### Competing Interest Statement The authors have declared no competing interest.

The central nucleus of the amygdala (CEA) is a brain region that integrates external and internal sensory information and executes innate and adaptive behaviors through distinct output pathways. Despite its complex functions, the diversity of molecularly defined neuronal types in the CEA and their contributions to major axonal projection targets have not been examined systematically. Here, we performed single-cell RNA-sequencing (scRNA-seq) to classify molecularly defined cell types in the CEA and identified marker genes to map the location of these neuronal types using expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH). We developed new methods to integrate EASI-FISH with 5-plex retrograde axonal labeling to determine the spatial, morphological, and connectivity properties of ~30,000 molecularly defined CEA neurons. Our study revealed spatiomolecular organization of the CEA, with medial and lateral CEA associated with distinct molecularly defined cell families. We also found a long-range axon projection network from the CEA, where target regions receive inputs from multiple molecularly defined cell types. Axon collateralization was found primarily among projections to hindbrain targets, which are distinct from forebrain projections. This resource reports marker gene combinations for molecularly defined cell types and axon-projection types, which will be useful for selective interrogation of these neuronal populations to study their contributions to the diverse functions of the CEA.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.

Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. The maximum resolution increase is limited by the expansion factor of the gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures. We demonstrate that TREx gels expand 10-fold, can be handled easily, and can be applied to both thick mouse brain tissue sections and cultured human cells enabling high-resolution subcellular imaging with a single expansion step. Furthermore, we show that TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small-molecule stains for both total protein and membranes.

SNT is an end-to-end framework for neuronal morphometry and whole-brain connectomics that supports tracing, proof-editing, visualization, quantification and modeling of neuroanatomy. With an open architecture, a large user base, community-based documentation, support for complex imagery and several model organisms, SNT is a flexible resource for the broad neuroscience community. SNT is both a desktop application and multi-language scripting library, and it is available through the Fiji distribution of ImageJ.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed E xpansion- As sisted Iterative F luorescence In S itu H ybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine novel spatially and molecularly defined subregions. EASI-FISH also facilitates iterative re-analysis of scRNA-Seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization. Highlights - EASI-FISH enables robust gene expression profiling in thick brain slices - A turnkey analysis pipeline for facile analysis of large EASI-FISH image datasets - EASI-FISH reveals novel subregions of the lateral hypothalamus - Identification of rare cell types based on morphological and spatial heterogeneity

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 mm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Prolonged periods of forced social isolation is detrimental to well-being, yet we know little about which genes regulate susceptibility to its effects. In the fruit fly, Drosophila melanogaster, social isolation induces stark changes in behavior including increased aggression, locomotor activity, and resistance to ethanol sedation. To identify genes regulating sensitivity to isolation, I screened a collection of sixteen hundred P-element insertion lines for mutants with abnormal levels of all three isolation-induced behaviors. The screen identified three mutants whose affected genes are likely central to regulating the effects of isolation in flies. One mutant, sex pistol (sxp), became extremely aggressive and resistant to ethanol sedation when socially isolated. sxp also had a high level of male–male courtship. The mutation in sxp reduced the expression of two minor isoforms of the actin regulator hts (adducin), as well as mildly reducing expression of CalpA, a calcium-dependent protease. As a consequence, sxp also had increased expression of the insulin-like peptide, dILP5. Analysis of the social behavior of sxp suggests that these minor hts isoforms function to limit isolation-induced aggression, while chronically high levels of dILP5 increase male–male courtship.

Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, we identified and characterized the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation. We show that hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. Our analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, our data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser.