Search

Animals chain movements into long-lived motor strategies, exhibiting variability across scales that reflects the interplay between internal states and environmental cues. To reveal structure in such variability, we build Markov models of movement sequences that bridge across timescales and enable a quantitative comparison of behavioral phenotypes among individuals. Applied to larval zebrafish responding to diverse sensory cues, we uncover a hierarchy of long-lived motor strategies, dominated by changes in orientation distinguishing cruising versus wandering strategies. Environmental cues induce preferences along these modes at the population level: while fish cruise in the light, they wander in response to aversive stimuli, or in search for appetitive prey. As our method encodes the behavioral dynamics of each individual fish in the transitions among coarse-grained motor strategies, we use it to uncover a hierarchical structure in the phenotypic variability that reflects exploration-exploitation trade-offs. Across a wide range of sensory cues, a major source of variation among fish is driven by prior and/or immediate exposure to prey that induces exploitation phenotypes. A large degree of variability that is not explained by environmental cues unravels hidden states that override the sensory context to induce contrasting exploration-exploitation phenotypes. Altogether, by extracting the timescales of motor strategies deployed during navigation, our approach exposes structure among individuals and reveals internal states tuned by prior experience., Competing Interests: Competing interests statement:The authors declare no competing interest.

Animals constantly integrate sensory information with prior experience to select behavioral responses appropriate to the current situation. Genetic factors supporting this behavioral flexibility are often disrupted in neuropsychiatric conditions, such as the autism-linked ap2s1 gene which supports acoustically evoked habituation learning. ap2s1 encodes an AP2 endocytosis adaptor complex subunit, although its behavioral mechanisms and importance have been unclear. Here, we show that multiple AP2 subunits regulate acoustically evoked behavior selection and habituation learning in zebrafish. Furthermore, ap2s1 biases escape behavior choice in sensory modality-specific manners, and broadly regulates action selection across sensory contexts. We demonstrate that the AP2 complex functions acutely in the nervous system to modulate acoustically evoked habituation, suggesting several spatially and/or temporally distinct mechanisms through which AP2 regulates escape behavior selection and performance. Altogether, we show the AP2 complex coordinates action selection across diverse contexts, providing a vertebrate model for ap2s1 's role in human conditions including autism spectrum disorder., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. Here, we investigate sensory representations in the inferior olive (IO) of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons, we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using two-photon imaging. We found that most IO neurons showed direction-selective and binocular responses to visual stimuli and that the functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between functional properties, soma location, and axonal projection patterns of IO neurons. Taken together, our results suggest that anatomically defined classes of IO neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Félix et al.)

In many brain areas, neuronal activity is associated with a variety of behavioral and environmental variables. In particular, neuronal responses in the zebrafish hindbrain relate to oculomotor and swimming variables as well as sensory information. However, the precise functional organization of the neurons has been difficult to unravel because neuronal responses are heterogeneous. Here, we used dimensionality reduction methods on neuronal population data to reveal the role of the hindbrain in visually driven oculomotor behavior and swimming. We imaged neuronal activity in zebrafish expressing GCaMP6s in the nucleus of almost all neurons while monitoring the behavioral response to gratings that rotated with different speeds. We then used reduced-rank regression, a method that condenses the sensory and motor variables into a smaller number of "features," to predict the fluorescence traces of all ROIs (regions of interest). Despite the potential complexity of the visuo-motor transformation, our analysis revealed that a large fraction of the population activity can be explained by only two features. Based on the contribution of these features to each ROI's activity, ROIs formed three clusters. One cluster was related to vergent movements and swimming, whereas the other two clusters related to leftward and rightward rotation. Voxels corresponding to these clusters were segregated anatomically, with leftward and rightward rotation clusters located selectively to the left and right hemispheres, respectively. Just as described in many cortical areas, our analysis revealed that single-neuron complexity co-exists with a simpler population-level description, thereby providing insights into the organization of visuo-motor transformations in the hindbrain., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recently, we introduced a powerful approach that leverages differences in swimming behaviors of two closely related fish species to identify previously unreported locomotion-related neuronal correlates. Here, we present this analysis approach applicable for any species of fish to compare their short and long timescale swimming kinematics. We describe steps for data collection and cleaning, followed by the calculation of short timescale kinematics using half tail beats and the analysis of long timescale kinematics using mean square displacement and heading decorrelation. For complete details on the use and execution of this protocol, please refer to Rajan et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Social experiences greatly define subsequent social behavior. Lack of such experiences, especially during critical phases of development, can severely impede the ability to behave adequately in social contexts. To date, it is not well characterized how early-life social isolation leads to social deficits and impacts development. In many model species, it is challenging to fully control social experiences, because they depend on parental care. Moreover, complex social behaviors involve multiple sensory modalities, contexts, and actions. Hence, when studying social isolation effects, it is important to parse apart social deficits from general developmental effects, such as abnormal motor learning. Here, we characterized how social experiences during early development of zebrafish larvae modulate their social behavior at 1 week of age, when social avoidance reactions can be measured as discrete swim events. We show that raising larvae in social isolation leads to enhanced social avoidance, in terms of the distance at which larvae react to one another and the strength of swim movement they use. Specifically, larvae raised in isolation use a high-acceleration escape swim, the short latency C-start, more frequently during social interactions. These behavioral differences are absent in non-social contexts. By ablating the lateral line and presenting the fish with local water vibrations, we show that lateral line inputs are both necessary and sufficient to drive enhanced social avoidance reactions. Taken together, our results show that social experience during development is a critical factor in shaping mechanosensory avoidance reactions in larval zebrafish., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Motivation: How to partition a dataset into a set of distinct clusters is a ubiquitous and challenging problem. The fact that data vary widely in features such as cluster shape, cluster number, density distribution, background noise, outliers and degree of overlap, makes it difficult to find a single algorithm that can be broadly applied. One recent method, clusterdp, based on search of density peaks, can be applied successfully to cluster many kinds of data, but it is not fully automatic, and fails on some simple data distributions., Results: We propose an alternative approach, clusterdv, which estimates density dips between points, and allows robust determination of cluster number and distribution across a wide range of data, without any manual parameter adjustment. We show that this method is able to solve a range of synthetic and experimental datasets, where the underlying structure is known, and identifies consistent and meaningful clusters in new behavioral data., Availability and Implementation: The clusterdv is implemented in Matlab. Its source code, together with example datasets are available on: https://github.com/jcbmarques/clusterdv., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart. Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca 2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development. Results: Isoproterenol slightly increased heart rate, diastolic Ca 2+ levels and Ca 2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca 2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ik r blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. I Ca,L blocker nifedipine caused total blockade of Ca 2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile animals demonstrated the possibility of employing an older zebrafish model for in vivo cardiac electrophysiology studies. We observed differences in atrial and ventricular Ca 2+ recovery dynamics between 3 dpf and 14 dpf casper fish, but not in Ca 2+ upstroke dynamics. Conclusion: By introducing the optogenetic sensors chimeric VSFP-butterfly CY and GCaMP6f into the zebrafish we successfully generated an in vivo cellular electrophysiological readout tool for the zebrafish heart. Complementary use of both sensor lines demonstrated the ability to study heart rate, cardiac action potential configuration, spatiotemporal patterning of electrical activation and intracellular Ca 2+ homeostasis in embryonic zebrafish. In addition, we demonstrated the first successful use of an optogenetic sensor to study cardiac function in older zebrafish. These models present a promising new research tool to study the underlying mechanisms of cardiac arrhythmogenesis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

A new study on the zebrafish has discovered a population of forebrain neurons necessary for social orienting, providing a foundation for dissecting social brain networks in this powerful vertebrate model., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

An important concept in ethology is that complex behaviors can be constructed from a set of basic motor patterns. Identifying the set of patterns available to an animal is key to making quantitative descriptions of behavior that reflect the underlying motor system organization. We addressed these questions in zebrafish larvae, which swim in bouts that are naturally segmented in time. We developed a robust and general purpose clustering method (clusterdv) to ensure accurate identification of movement clusters and applied it to a dataset consisting of millions of swim bouts, captured at high temporal resolution from a comprehensive set of behavioral contexts. We identified a set of thirteen basic swimming patterns that are used flexibly in various combinations across different behavioral contexts and show that this classification can be used to dissect the sensorimotor transformations underlying larval social behavior and hunting. Furthermore, using the same approach at different levels in the behavioral hierarchy, we show that the set of swim bouts are themselves constructed from a basic set of tail movements and that bouts are executed in sequences specific to different behaviors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A great challenge in neuroscience is understanding how activity in the brain gives rise to behavior. The zebrafish is an ideal vertebrate model to address this challenge, thanks to the capacity, at the larval stage, for precise behavioral measurements, genetic manipulations, and recording and manipulation of neural activity noninvasively and at single-neuron resolution throughout the whole brain. These techniques are being further developed for application in freely moving animals and juvenile stages to study more complex behaviors including learning, decision making, and social interactions. We review some of the approaches that have been used to study the behavior of zebrafish and point to opportunities and challenges that lie ahead.

Neurons and neural networks often extend hundreds of micrometers in three dimensions. Capturing the calcium transients associated with their activity requires volume imaging methods with subsecond temporal resolution. Such speed is a challenge for conventional two-photon laser-scanning microscopy, because it depends on serial focal scanning in 3D and indicators with limited brightness. Here we present an optical module that is easily integrated into standard two-photon laser-scanning microscopes to generate an axially elongated Bessel focus, which when scanned in 2D turns frame rate into volume rate. We demonstrated the power of this approach in enabling discoveries for neurobiology by imaging the calcium dynamics of volumes of neurons and synapses in fruit flies, zebrafish larvae, mice and ferrets in vivo. Calcium signals in objects as small as dendritic spines could be resolved at video rates, provided that the samples were sparsely labeled to limit overlap in their axially projected images.

A simple neural circuit motif in the zebrafish brain enables robust and reliable behavioral choices.

Week-old zebrafish execute many complex, visually guided actions using a brain that displays the canonical vertebrate organization, but is less than a cubic millimeter in size. Recent studies have made considerable progress towards understanding the underlying neural circuit basis of these innate responses, at two fundamental levels. Large-scale population recordings have revealed how functional properties and activity dynamics are organized within and across brain regions. Meanwhile, single neurons have been specifically labeled to systematically characterize cell types with distinct morphologies and projection patterns. Combining these approaches, in order to investigate the role of individual, identified neurons in generating neural activity dynamics and behavior, has traditionally been possible only for simple invertebrate systems, but is now becoming feasible in a vertebrate model., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We present a protocol to combine behavioral recording and imaging using 2-photon laser-scanning microscopy in head-fixed larval zebrafish that express a genetically encoded calcium indicator. The steps involve restraining the larva in agarose, setting up optics that allow projection of a visual stimulus and infrared illumination to monitor behavior, and analysis of the neuronal and behavioral data.

In recent years, the zebrafish has emerged as an appealing model system to tackle questions relating to the neural circuit basis of behavior. This can be attributed not just to the growing use of genetically tractable model organisms, but also in large part to the rapid advances in optical techniques for neuroscience, which are ideally suited for application to the small, transparent brain of the larval fish. Many characteristic features of vertebrate brains, from gross anatomy down to particular circuit motifs and cell-types, as well as conserved behaviors, can be found in zebrafish even just a few days post fertilization, and, at this early stage, the physical size of the brain makes it possible to analyze neural activity in a comprehensive fashion. In a recent study, we used a systematic and unbiased imaging method to record the pattern of activity dynamics throughout the whole brain of larval zebrafish during a simple visual behavior, the optokinetic response (OKR). This approach revealed the broadly distributed network of neurons that were active during the behavior and provided insights into the fine-scale functional architecture in the brain, inter-individual variability, and the spatial distribution of behaviorally relevant signals. Combined with mapping anatomical and functional connectivity, targeted electrophysiological recordings, and genetic labeling of specific populations, this comprehensive approach in zebrafish provides an unparalleled opportunity to study complete circuits in a behaving vertebrate animal., (Copyright © 2014. Published by Elsevier Ltd.)

Vertebrate locomotion at different speeds is driven by descending excitatory connections to central pattern generators in the spinal cord. To investigate how these inputs determine locomotor kinematics, we used whole-field visual motion to drive zebrafish to swim at different speeds. Larvae match the stimulus speed by utilizing more locomotor events, or modifying kinematic parameters such as the duration and speed of swimming bouts, the tail-beat frequency, and the choice of gait. We used laser ablations, electrical stimulation, and activity recordings in descending neurons of the nucleus of the medial longitudinal fasciculus (nMLF) to dissect their contribution to controlling forward movement. We found that the activity of single identified neurons within the nMLF is correlated with locomotor kinematics, and modulates both the duration and oscillation frequency of tail movements. By identifying the contribution of individual supraspinal circuit elements to locomotion kinematics, we build a better understanding of how the brain controls movement., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Most behaviors, even simple innate reflexes, are mediated by circuits of neurons spanning areas throughout the brain. However, in most cases, the distribution and dynamics of firing patterns of these neurons during behavior are not known. We imaged activity, with cellular resolution, throughout the whole brains of zebrafish performing the optokinetic response. We found a sparse, broadly distributed network that has an elaborate but ordered pattern, with a bilaterally symmetrical organization. Activity patterns fell into distinct clusters reflecting sensory and motor processing. By correlating neuronal responses with an array of sensory and motor variables, we find that the network can be clearly divided into distinct functional modules. Comparing aligned data from multiple fish, we find that the spatiotemporal activity dynamics and functional organization are highly stereotyped across individuals. These experiments systematically reveal the functional architecture of neural circuits underlying a sensorimotor behavior in a vertebrate brain., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rapidly developing imaging technologies including two-photon microscopy and genetically encoded calcium indicators have opened up new possibilities for recording neural population activity in awake, behaving animals. In the small, transparent zebrafish, it is even becoming possible to image the entire brain of a behaving animal with single-cell resolution, creating brain-wide functional maps. In this chapter, we comprehensively review past functional imaging studies in zebrafish, and the insights that they provide into the functional organization of neural circuits. We further offer a basic primer on state-of-the-art methods for in vivo calcium imaging in the zebrafish, including building a low-cost two-photon microscope and highlight possible challenges and technical considerations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.

Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(2+)-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca(2+)]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca(2+) affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

We describe an intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) with signal-to-noise ratio and kinetics appropriate for in vivo imaging. We engineered iGluSnFR in vitro to maximize its fluorescence change, and we validated its utility for visualizing glutamate release by neurons and astrocytes in increasingly intact neurological systems. In hippocampal culture, iGluSnFR detected single field stimulus-evoked glutamate release events. In pyramidal neurons in acute brain slices, glutamate uncaging at single spines showed that iGluSnFR responds robustly and specifically to glutamate in situ, and responses correlate with voltage changes. In mouse retina, iGluSnFR-expressing neurons showed intact light-evoked excitatory currents, and the sensor revealed tonic glutamate signaling in response to light stimuli. In worms, glutamate signals preceded and predicted postsynaptic calcium transients. In zebrafish, iGluSnFR revealed spatial organization of direction-selective synaptic activity in the optic tectum. Finally, in mouse forelimb motor cortex, iGluSnFR expression in layer V pyramidal neurons revealed task-dependent single-spine activity during running.

Background: Although adult vertebrates sense changes in head position by using two classes of accelerometer, at larval stages zebrafish lack functional semicircular canals and rely exclusively on their otolithic organs to transduce vestibular information., Results: Despite this limitation, we find that larval zebrafish perform an effective vestibulo-ocular reflex (VOR) that serves to stabilize gaze in response to pitch and roll tilts. By using single-cell electroporations and targeted laser ablations, we identified a specific class of central vestibular neurons, located in the tangential nucleus, that are essential for the utricle-dependent VOR. Tangential nucleus neurons project contralaterally to extraocular motoneurons and in addition to multiple sites within the reticulospinal complex., Conclusions: We propose that tangential neurons function as a broadband inertial accelerometer, processing utricular acceleration signals to control the activity of extraocular and postural neurons, thus completing a fundamental three-neuron circuit responsible for gaze stabilization., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.

A basic question in the field of motor control is how different actions are represented by activity in spinal projection neurons. We used a new behavioral assay to identify visual stimuli that specifically drive basic motor patterns in zebrafish. These stimuli evoked consistent patterns of neural activity in the neurons projecting to the spinal cord, which we could map throughout the entire population using in vivo two-photon calcium imaging. We found that stimuli that drive distinct behaviors activated distinct subsets of projection neurons, consisting, in some cases, of just a few cells. This stands in contrast to the distributed activation seen for more complex behaviors. Furthermore, targeted cell by cell ablations of the neurons associated with evoked turns abolished the corresponding behavioral response. This description of the functional organization of the zebrafish motor system provides a framework for identifying the complete circuit underlying a vertebrate behavior.

The neural circuitry that constrains visual acuity in the CNS has not been experimentally identified. We show here that zebrafish blumenkohl (blu) mutants are impaired in resolving rapid movements and fine spatial detail. The blu gene encodes a vesicular glutamate transporter expressed by retinal ganglion cells. Mutant retinotectal synapses release less glutamate, per vesicle and per terminal, and fatigue more quickly than wild-type in response to high-frequency stimulation. In addition, mutant axons arborize more extensively, thus increasing the number of synaptic terminals and effectively normalizing the combined input to postsynaptic cells in the tectum. This presumably homeostatic response results in larger receptive fields of tectal cells and a degradation of the retinotopic map. As predicted, mutants have a selective deficit in the capture of small prey objects, a behavior dependent on the tectum. Our studies successfully link the disruption of a synaptic protein to complex changes in neural circuitry and behavior.

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Visual systems break scenes down into individual features, processed in distinct channels, and then selectively recombine those features according to the demands of particular behavioral tasks. In primates, for example, there are distinct pathways for motion and form processing. While form vision utilizes color information, motion pathways receive input from only a subset of cone photoreceptors and are generally colorblind. To explore the link between early channeling of visual information and behavioral output across vertebrate species, we measured the chromatic inputs to the optomotor response of larval zebrafish. Using cone-isolating gratings, we found that there is a strong input from both red and green cones but not short-wavelength cones, which nevertheless do contribute to another behavior, phototaxis. Using a motion-nulling method, we measured precisely the input strength of gratings that stimulated cones in combination. The fish do not respond to gratings that stimulate different cone types out of phase, but have an enhanced response when the cones are stimulated together. This shows that red and green cone signals are pooled at a stage before motion detection. Since the two cone inputs are combined into a single 'luminance' channel, the response to sinusoidal gratings is colorblind. However, we also find that the relative contributions of the two cones at isoluminance varies with spatial frequency. Therefore, natural stimuli, which contain a mixture of spatial frequencies, are likely to be visible regardless of their chromatic composition.

A moving grating elicits innate optomotor behavior in zebrafish larvae; they swim in the direction of perceived motion. We took advantage of this behavior, using computer-animated displays, to determine what attributes of motion are extracted by the fish visual system. As in humans, first-order (luminance-defined or Fourier) signals dominated motion perception in fish; edges or other features had little or no effect when presented with these signals. Humans can see complex movements that lack first-order cues, an ability that is usually ascribed to higher-level processing in the visual cortex. Here we show that second-order (non-Fourier) motion displays induced optomotor behavior in zebrafish larvae, which do not have a cortex. We suggest that second-order motion is extracted early in the lower vertebrate visual pathway.