Interview with Mark Fishman, who uses the zebrafish to study body-brain interactions and the genetic structure of social behavior at Harvard University.

Complex collective behaviors such as schooling are believed to emerge from simple, individual-level computations that translate incoming information from conspecifics into actions.1,2,3,4,5,6,7,8,9,10,11,12,13,14 Recently, it has been proposed that discrete behavioral modes, or internal states, may modulate these computations, affecting the resulting collective behaviors.15,16,17,18 Direct evidence for such hierarchical control remains limited due to challenges in inferring hidden perception-action computations and uncovering discrete behavioral modes from continuous behaviors.18,19,20,21,22 To address this, we analyzed swimming behaviors of medaka fish (Oryzias latipes) throughout development.23,24,25 At the group level, medaka exhibit synchronized swimming formations that develop early, emerging around 2 weeks of age and stabilizing within 1 month. Unlike many teleost species that use burst-and-coast swim patterns,19,26,27,28,29,30,31,32,33,34,35 medaka exhibit continuous tail and body undulations.31 We show that this continuous behavior can be segmented into three distinct kinematic states: acceleration, deceleration, and prolonged constant-speed swimming. Using state-dependent computational models,15,18 we tested how medaka translate social information from neighbors into actions across these kinematic states. The models revealed distinct computations governing social information processing and movement responses in each state. Moreover, social responsiveness varied significantly between states: it was strongest during constant-speed epochs, intermediate during accelerations, and lowest during decelerations. These findings highlight discrete behavioral modes as key modulators of social interaction computations underlying collective behavior.

Complex group behavior can emerge from simple inter-individual interactions, yet how prior social experience shapes these interactions remains unclear. Using naturalistic and virtual-reality experiments, we varied prior social experience by exposing zebrafish larvae to high or low group densities, then measured its effects on future social interactions and group structure. We find that inter-individual distances decrease after exposure to high population densities and increase after exposure to low densities. These adaptations develop gradually over tens of minutes and remain stable for hours. Mechanistically, larvae estimate group density from the frequency of neighbor-evoked retinal looming events and couple interaction strength to that estimate. A time-varying computational model incorporating previous visual-social experiences accurately describes our observations. Our findings show that inter-individual interactions are not static, but continuously evolve with social experience and environmental demands, establishing larval zebrafish as a model for studying the neurobiological mechanisms of experience-dependent modulation of collective behavior.

To select behaviors appropriate to their circumstances and needs, animals integrate information about the external environment with information about the state of their bodies, derived from sensing and processing internal signals (interoception). However, the brain-wide circuit activity underlying interoception and its integration with sensorimotor processing remains unclear, partly because of technical barriers to accessing whole-brain activity at the cellular level during physiological perturbations. We developed an all-optical system for whole-brain neuronal imaging in behaving larval zebrafish during optical uncaging of gut- or bloodstream-targeted nutrients and visuo-motor stimulation. Widespread neural activity throughout the brain encoded nutrient delivery, unfolding on multiple timescales across many peripheral and central regions. Evoked activity depended on delivery location and occurred in response to both amino acids and D-glucose, but not L-glucose. Many gut responsive neurons also responded to swimming and visual stimuli, with brainstem areas primarily integrating gut and motor signals and midbrain regions integrating jointly gut, visual, and motor signals. This platform links body-brain communication studies to brain-wide neural computation in awake, behaving vertebrates. Understanding how the brain processes signals from other organs has been limited by a lack of tools. Here the authors introduce an all-optical system for gut or vascular nutrient release during whole-brain imaging in zebrafish.

Animals reprioritize behavioral goals in response to internal physiological states. Using larval zebrafish, we investigated whether engagement with a visuomotor task, the optomotor response (OMR), is coupled to cardiac dynamics. We discovered that threats lead to tachycardia that is synchronized with behavioral suppression. The change in heart rate is represented in the activity of specific neuronal populations. Severing the input to the sympathetic ganglia or ablating the vagus nerve revealed that the threat-related changes to behavioral state do not require interoceptive pathways. Direct tachycardic optopacing of the heart similarly suppressed the OMR response, but by reducing cardiac filling during diastole, thereby impacting oxygen delivery to the CNS. Optopacing also changed the activity of specific brain regions but in neurons distinct from those associated with threat-induced tachycardia. These cardiac function-associated central changes may have relevance to autonomic imbalances in anxiety, stress, and orthostatic disorders.

Organisms must regulate metabolic resources such as oxygen (O2) and nutrients despite environmental variability and the energetic costs of their own actions1-3. Such regulation can occur reactively, through homeostatic corrections of recent imbalances, or predictively, through allostatic adjustments that anticipate future demand4,5. Predictive regulation is particularly important because metabolic resources often continue to be consumed for seconds to minutes after motor actions cease as tissues repay incurred costs, making it advantageous to prevent depletion before it occurs6. However, the cellular and circuit mechanisms for allostatic control remain largely unknown5,7,8. Using whole-brain neuronal and astroglial imaging and O2 measurements in behaving zebrafish, we identified a noradrenergic-astroglial circuit that detects, anticipates, and prevents internal O2 depletion. We found that swimming exacerbated internal hypoxia with a multi-second delay, but behavioral adaptations occurred before such selfgenerated hypoxia manifested, suggesting predictive control, confirmed using computational modeling. Noradrenergic neurons in the nucleus of the solitary tract directly detected brain hypoxia and received efference copies of swimming actions; these inputs summed at the level of membrane voltage to increase spiking and norepinephrine release when actions and resource scarcity co-occurred. Astroglia integrated noradrenergic input into prolonged Ca2+ elevation that tracked the O2 cost of recent actions and thereby predicted O2 debt relative to O2 availability, rising ~8 s before O2 fell. This astroglial prediction reorganized brain-wide activity to suppress locomotion and promote respiration, preempting O2 depletion. Silencing noradrenergic neurons or astroglial signaling abolished these hypoxia coping behaviors, whereas selective activation evoked them. This neuronal-astroglial mechanism constitutes a predictive control system that integrates physiological state with behavioral intent to avert metabolic crisis, revealing a cellular substrate for proactive energy management.

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.

Internal signals from the body and external signals from the environment are processed by brain-wide circuits to guide behavior. However, the complete brain-wide circuit activity underlying interoception - the perception of bodily signals - and its interactions with sensorimotor circuits remain unclear due to technical barriers to accessing whole-brain activity at the cellular level during organ physiology perturbations. We developed an all-optical system for whole-brain neuronal imaging in behaving larval zebrafish during optical uncaging of gut-targeted nutrients and visuo-motor stimulation. Widespread neural activity throughout the brain encoded nutrient delivery, unfolding on multiple timescales across many specific peripheral and central regions. Evoked activity depended on delivery location and occurred with amino acids and D-glucose, but not L-glucose. Many gut-sensitive neurons also responded to swimming and visual stimuli, with brainstem areas primarily integrating gut and motor signals and midbrain regions integrating gut and visual signals. This platform links body-brain communication studies to brain-wide neural computation in awake, behaving vertebrates. ### Competing Interest Statement The authors have declared no competing interest.

Transient exposure to ketamine can trigger lasting changes in behavior and mood. We found that brief ketamine exposure causes long-term suppression of futility-induced passivity in larval zebrafish, reversing the “giving-up” response that normally occurs when swimming fails to cause forward movement. Whole-brain imaging revealed that ketamine hyperactivates the norepinephrine-astroglia circuit responsible for passivity. After ketamine washout, this circuit exhibits hyposensitivity to futility, leading to long-term increased perseverance. Pharmacological, chemogenetic, and optogenetic manipulations show that norepinephrine and astrocytes are necessary and sufficient for ketamine’s long-term perseverance-enhancing aftereffects. In vivo calcium imaging revealed that astrocytes in adult mouse cortex are similarly activated during futility in the tail suspension test and that acute ketamine exposure also induces astrocyte hyperactivation. The cross-species conservation of ketamine’s modulation of noradrenergic-astroglial circuits and evidence that plasticity in this pathway can alter the behavioral response to futility hold promise for identifying new strategies to treat affective disorders.

Studying neural mechanisms in complementary model organisms from different ecological niches in the same animal class can leverage the comparative brain analysis at the cellular level. To advance such a direction, we developed a unified brain atlas platform and specialized tools that allowed us to quantitatively compare neural structures in two teleost larvae, medaka ( Oryzias latipes ) and zebrafish ( Danio rerio ). Leveraging this quantitative approach we found that most brain regions are similar but some subpopulations are unique in each species. Specifically, we confirmed the existence of a clear dorsal pallial region in the telencephalon in medaka lacking in zebrafish. Further, our approach allows for extraction of differentially expressed genes in both species, and for quantitative comparison of neural activity at cellular resolution. The web-based and interactive nature of this atlas platform will facilitate the teleost community’s research and its easy extensibility will encourage contributions to its continuous expansion.### Competing Interest StatementThe authors have declared no competing interest.

Abstract Decision making strategies in the face of conflicting or uncertain sensory input have been successfully described by a drift diffusion to bound model (DDM) in many different species. Here we analyze large behavioral datasets of larval zebrafish engaged in a ‘coherent dot’ optomotor assay and compare DDM performance with trial-to-trial variation in our experimental results. We find a clear discrepancy in the low-level structure of modeled and experimental data, which compels us to implement a critical extension to the classic DDM. To correct for the discrepancy, we propose to add an extra variable that explicitly controls the transition between an engaged and a disengaged state. This addition not only accurately captures the lower-level structure in our data, but also provides a framework to explain general behavioral switches between different tasks and contextual priorities. We use these insights to define two behavioral performance metrics for the zebrafish, labeled ‘focus’ and ‘competence’, which describe two orthogonal aspects of performance in a task. We further show that the extent of ‘focus’ is largely inherited from the parents, while ‘competence’ is influenced mainly by environmental context. This quantitative framework for analyzing decision making can be used to screen genetic perturbations for their impact on these two aspects of performance, and eventually help identify a genetic basis, and a neural mechanism for attention, that extends across organisms.

Mood-altering compounds hold promise for the treatment of many psychiatric disorders, such as depression, but connecting their molecular, circuit, and behavioral effects has been challenging. Here we find that, analogous to effects in rodent learned helplessness models, ketamine pre-exposure persistently suppresses futility-induced passivity in larval zebrafish. While antidepressants are thought to primarily act on neurons, brain-wide imaging in behaving zebrafish showed that ketamine elevates intracellular calcium in astroglia for many minutes, followed by persistent calcium downregulation post-washout. Calcium elevation depends on astroglial α1-adrenergic receptors and is required for suppression of passivity. Chemo-/optogenetic perturbations of noradrenergic neurons and astroglia demonstrate that the aftereffects of glial calcium elevation are sufficient to suppress passivity by inhibiting neuronal-astroglial integration of behavioral futility. Imaging in mouse cortex reveals that ketamine elevates astroglial calcium through conserved pathways, suggesting that ketamine exerts its behavioral effects by persistently modulating evolutionarily ancient neuromodulatory systems spanning neurons and astroglia.### Competing Interest StatementDEO is a co-founder of Delix Therapeutics, Inc., serves as the Chief Innovation Officer and Head of the Scientific Advisory Board, and has sponsored research agreements with Delix Therapeutics. Delix Therapeutics has licensed technology from the University of California, Davis. AEC is a co-founder of Q-State Biosciences. All other authors declare that they have no competing interests.

Mood-altering compounds hold promise for the treatment of many psychiatric disorders, such as depression, but connecting their molecular, circuit, and behavioral effects has been challenging. Here we find that, analogous to effects in rodent learned helplessness models, ketamine pre-exposure persistently suppresses futility-induced passivity in larval zebrafish. While antidepressants are thought to primarily act on neurons, brain-wide imaging in behaving zebrafish showed that ketamine elevates intracellular calcium in astroglia for many minutes, followed by persistent calcium downregulation post-washout. Calcium elevation depends on astroglial α1-adrenergic receptors and is required for suppression of passivity. Chemo-/optogenetic perturbations of noradrenergic neurons and astroglia demonstrate that the aftereffects of glial calcium elevation are sufficient to suppress passivity by inhibiting neuronal-astroglial integration of behavioral futility. Imaging in mouse cortex reveals that ketamine elevates astroglial calcium through conserved pathways, suggesting that ketamine exerts its behavioral effects by persistently modulating evolutionarily ancient neuromodulatory systems spanning neurons and astroglia.

Since Darwin, coordinated movement of animal groups has been believed to be essential to species survival, but it is not understood how changes in the genetic makeup of individuals might alter behavior of the collective. Here we find that even at the early larval stage, zebrafish regulate their proximity and alignment with each other. Two simple visual responses, one that measures relative visual field occupancy and the other global visual motion, suffice to account for the group behavior that emerges. We analyze how mutations in genes known to affect social behavior of humans perturb these simple reflexes in larval zebrafish and thereby affect their collective behaviors. We use model simulations to show that changes in reflexive responses of individual mutant animals predict well the distinctive collective patterns that emerge in a group. Hence group behaviors reflect in part genetically defined primitive sensorimotor “motifs”, which are evident even in young larvae. ### Competing Interest Statement The authors have declared no competing interest.

It is not understood how changes in the genetic makeup of individuals alter the behavior of groups of animals. Here, we find that, even at early larval stages, zebrafish regulate their proximity and alignment with each other. Two simple visual responses, one that measures relative visual field occupancy and one that accounts for global visual motion, suffice to account for the group behavior that emerges. Mutations in genes known to affect social behavior in humans perturb these simple reflexes in individual larval zebrafish and change their emergent collective behaviors in the predicted fashion. Model simulations show that changes in these two responses in individual mutant animals predict well the distinctive collective patterns that emerge in a group. Hence, group behaviors reflect in part genetically defined primitive sensorimotor "motifs," which are evident even in young larvae.

Many animals, including humans, have evolved to live and move in groups. In humans, disrupted social interactions are a fundamental feature of many psychiatric disorders. However, we know little about how genes regulate social behavior. Zebrafish may serve as a powerful model to explore this question. By comparing the behavior of wild-type fish with 90 mutant lines, we show that mutations of genes associated with human psychiatric disorders can alter the collective behavior of adult zebrafish. We identify three categories of behavioral variation across mutants: "scattered," in which fish show reduced cohesion; "coordinated," in which fish swim more in aligned schools; and "huddled," in which fish form dense but disordered groups. Changes in individual interaction rules can explain these differences. This work demonstrates how emergent patterns in animal groups can be altered by genetic changes in individuals and establishes a framework for understanding the fundamentals of social information processing.

Dopamine transporter (SLC6A3) deficiency causes infantile Parkinson disease, for which there is no effective therapy. We have explored the effects of genetically deleting SLC6A3 in zebrafish. Unlike the wild-type, slc6a3-/- fish hover near the tank bottom, with a repetitive digging-like behavior. slc6a3-/- fish manifest pruning and cellular loss of particular tyrosine hydroxylase-immunoreactive neurons in the midbrain. Clozapine, an effective therapeutic for treatment-resistant schizophrenia, rescues the abnormal behavior of slc6a3-/- fish. Clozapine also reverses the abnormalities in the A8 region of the mutant midbrain. By RNA sequencing analysis, clozapine increases the expression of erythropoietin pathway genes. Transgenic over-expression of erythropoietin in neurons of slc6a3-/- fish partially rescues the mutant behavior, suggesting a potential mechanistic basis for clozapine's efficacy.

The hormone αKlotho regulates lifespan in mice, as knockouts die early of what appears to be accelerated aging due to hyperphosphatemia and soft tissue calcification. In contrast, the overexpression of αKlotho increases lifespan. Given the severe mouse phenotype, we generated zebrafish mutants for αklotho as well as its binding partner fibroblast growth factor-23 (fgf23). Both mutations cause shortened lifespan in zebrafish, with abrupt onset of behavioral and degenerative physical changes at around 5 months of age. There is a calcification of vessels throughout the body, most dramatically in the outflow tract of the heart, the bulbus arteriosus (BA). This calcification is associated with an ectopic activation of osteoclast differentiation pathways. These findings suggest that the gradual loss of αKlotho found in normal aging might give rise to ectopic calcification.

α-Klotho is an anti-aging hormone regulating mineral homeostasis together with Fibroblast Growth Factor-23 (FGF23) in mammals. Cellular and molecular mechanisms through which these signals regulate age-related conditions, such as vascular mineralization remain intriguing. It is not known if aging mechanisms controlled by α-Klotho/FGF23 signaling are evolutionarily conserved. To this end, we generated knockouts carrying homozygous mutations in α-klotho and fgf23 in zebrafish. Both knockouts display an adult-onset decline in body condition and behavior, and an early-onset morbidity. Bulbus arteriosus, the outflow tract of zebrafish heart shows extensive mineralization in the mutants. RNA-seq analysis of kidney, heart and gills shows an ectopic activation of bone-remodeling pathway, extracellular matrix remodeling factors osteoclast differentiation and inflammation in the vasculature. Taken together, these findings indicate a conserved role of α-Klotho/FGF23 signaling in regulating vertebrate life span and identify molecular mechanisms underlying vascular mineralization.