A scalable assay for chemical preference of small freshwater fish.

Sensing the chemical world is of primary importance for aquatic organisms, and small freshwater fish are increasingly used in toxicology, ethology, and neuroscience by virtue of their ease of manipulation, tissue imaging amenability, and genetic tractability. However, precise behavioral analyses are generally challenging to perform due to the lack of knowledge of what chemical the fish are exposed to at any given moment. Here we developed a behavioral assay and a specific infrared dye to probe the preference of young zebrafish for virtually any compound. We found that the innate aversion of zebrafish to citric acid is not mediated by modulation of the swim but rather by immediate avoidance reactions when the product is sensed and that the preference of juvenile zebrafish for ATP changes from repulsion to attraction during successive exposures. We propose an information-based behavioral model for which an exploration index emerges as a relevant behavioral descriptor, complementary to the standard preference index. Our setup features a high versatility in protocols and is automatic and scalable, which paves the way for high-throughput preference compound screening at different ages.

Evolutionary divergence of locomotion in two related vertebrate species.

Locomotion exists in diverse forms in nature; however, little is known about how closely related species with similar neuronal circuitry can evolve different navigational strategies to explore their environments. Here, we investigate this question by comparing divergent swimming pattern in larval Danionella cerebrum (DC) and zebrafish (ZF). We show that DC displays long continuous swimming events when compared with the short burst-and-glide swimming in ZF. We reveal that mesencephalic locomotion maintenance neurons in the midbrain are sufficient to cause this increased swimming. Moreover, we propose that the availability of dissolved oxygen and timing of swim bladder inflation drive the observed differences in the swim pattern. Our findings uncover the neural substrate underlying the evolutionary divergence of locomotion and its adaptation to their environmental constraints.

Thermal modulation of Zebrafish exploratory statistics reveals constraints on individual behavioral variability.

BACKGROUND: Variability is a hallmark of animal behavior. It contributes to survival by endowing individuals and populations with the capacity to adapt to ever-changing environmental conditions. Intra-individual variability is thought to reflect both endogenous and exogenous modulations of the neural dynamics of the central nervous system. However, how variability is internally regulated and modulated by external cues remains elusive. Here, we address this question by analyzing the statistics of spontaneous exploration of freely swimming zebrafish larvae and by probing how these locomotor patterns are impacted when changing the water temperatures within an ethologically relevant range. RESULTS: We show that, for this simple animal model, five short-term kinematic parameters - interbout interval, turn amplitude, travelled distance, turn probability, and orientational flipping rate - together control the long-term exploratory dynamics. We establish that the bath temperature consistently impacts the means of these parameters, but leave their pairwise covariance unchanged. These results indicate that the temperature merely controls the sampling statistics within a well-defined kinematic space delineated by this robust statistical structure. At a given temperature, individual animals explore the behavioral space over a timescale of tens of minutes, suggestive of a slow internal state modulation that could be externally biased through the bath temperature. By combining these various observations into a minimal stochastic model of navigation, we show that this thermal modulation of locomotor kinematics results in a thermophobic behavior, complementing direct gradient-sensing mechanisms. CONCLUSIONS: This study establishes the existence of a well-defined locomotor space accessible to zebrafish larvae during spontaneous exploration, and quantifies self-generated modulation of locomotor patterns. Intra-individual variability reflects a slow diffusive-like probing of this space by the animal. The bath temperature in turn restricts the sampling statistics to sub-regions, endowing the animal with basic thermophobicity. This study suggests that in zebrafish, as well as in other ectothermic animals, ambient temperature could be used to efficiently manipulate internal states in a simple and ethological way.

Trans-inhibition of axon terminals underlies competition in the habenulo-interpeduncular pathway.

Survival of animals is dependent on the correct selection of an appropriate behavioral response to competing external stimuli. Theoretical models have been proposed and underlying mechanisms are emerging to explain how one circuit is selected among competing neural circuits. The evolutionarily conserved forebrain to midbrain habenulo-interpeduncular nucleus (Hb-IPN) pathway consists of cholinergic and non-cholinergic neurons, which mediate different aversive behaviors. Simultaneous calcium imaging of neuronal cell bodies and of the population dynamics of their axon terminals reveals that signals in the cell bodies are not reflective of terminal activity. We find that axon terminals of cholinergic and non-cholinergic habenular neurons exhibit stereotypic patterns of spontaneous activity that are negatively correlated and localize to discrete subregions of the target IPN. Patch-clamp recordings show that calcium bursts in cholinergic terminals at the ventral IPN trigger excitatory currents in IPN neurons, which precede inhibition of non-cholinergic terminals at the adjacent dorsal IPN. Inhibition is mediated through presynaptic GABAB receptors activated in non-cholinergic habenular neurons upon GABA release from the target IPN. Together, the results reveal a hardwired mode of competition at the terminals of two excitatory neuronal populations, providing a physiological framework to explore the relationship between different aversive responses.

FastTrack: An open-source software for tracking varying numbers of deformable objects.

Analyzing the dynamical properties of mobile objects requires to extract trajectories from recordings, which is often done by tracking movies. We compiled a database of two-dimensional movies for very different biological and physical systems spanning a wide range of length scales and developed a general-purpose, optimized, open-source, cross-platform, easy to install and use, self-updating software called FastTrack. It can handle a changing number of deformable objects in a region of interest, and is particularly suitable for animal and cell tracking in two-dimensions. Furthermore, we introduce the probability of incursions as a new measure of a movie's trackability that doesn't require the knowledge of ground truth trajectories, since it is resilient to small amounts of errors and can be computed on the basis of an ad hoc tracking. We also leveraged the versatility and speed of FastTrack to implement an iterative algorithm determining a set of nearly-optimized tracking parameters-yet further reducing the amount of human intervention-and demonstrate that FastTrack can be used to explore the space of tracking parameters to optimize the number of swaps for a batch of similar movies. A benchmark shows that FastTrack is orders of magnitude faster than state-of-the-art tracking algorithms, with a comparable tracking accuracy. The source code is available under the GNU GPLv3 at https://github.com/FastTrackOrg/FastTrack and pre-compiled binaries for Windows, Mac and Linux are available at http://www.fasttrack.sh.

From behavior to circuit modeling of light-seeking navigation in zebrafish larvae.

Bridging brain-scale circuit dynamics and organism-scale behavior is a central challenge in neuroscience. It requires the concurrent development of minimal behavioral and neural circuit models that can quantitatively capture basic sensorimotor operations. Here, we focus on light-seeking navigation in zebrafish larvae. Using a virtual reality assay, we first characterize how motor and visual stimulation sequences govern the selection of discrete swim-bout events that subserve the fish navigation in the presence of a distant light source. These mechanisms are combined into a comprehensive Markov-chain model of navigation that quantitatively predicts the stationary distribution of the fish's body orientation under any given illumination profile. We then map this behavioral description onto a neuronal model of the ARTR, a small neural circuit involved in the orientation-selection of swim bouts. We demonstrate that this visually-biased decision-making circuit can capture the statistics of both spontaneous and contrast-driven navigation.

A Semi-Automatic Dispenser for Solid and Liquid Food in Aquatic Facilities.

We present a novel, low-footprint and low-cost semi-automatic system for delivering solid and liquid food to zebrafish, and more generally to aquatic animals raised in racks of tanks. It is composed of a portable main module equipped with a contactless reader that adjusts the quantity to deliver for each tank, and either a solid food module or a liquid food module. Solid food comprises virtually any kind of dry powder or grains below 2 mm in diameter, and, for liquid-mediated food, brine shrimps (Artemia salina) and rotifers (Rotifera) have been successfully tested. Real-world testing, feedback, and validation have been performed in a zebrafish facility for several months. In comparison with manual feeding this system mitigates the appearance of musculoskeletal disorders among regularly-feeding staff, and let operators observe the animals' behavior instead of being focused on quantities to deliver. We also tested the accuracy of both humans and our dispenser and found that the semi-automatic system is much more reliable, with respectively 7-fold and 84-fold drops in standard deviation for solid and liquid food.

Whole-Brain Calcium Imaging during Physiological Vestibular Stimulation in Larval Zebrafish.

The vestibular apparatus provides animals with postural and movement-related information that is essential to adequately execute numerous sensorimotor tasks. In order to activate this sensory system in a physiological manner, one needs to macroscopically rotate or translate the animal's head, which in turn renders simultaneous neural recordings highly challenging. Here we report on a novel miniaturized, light-sheet microscope that can be dynamically co-rotated with a head-restrained zebrafish larva, enabling controlled vestibular stimulation. The mechanical rigidity of the microscope allows one to perform whole-brain functional imaging with state-of-the-art resolution and signal-to-noise ratio while imposing up to 25° in angular position and 6,000°/s2 in rotational acceleration. We illustrate the potential of this novel setup by producing the first whole-brain response maps to sinusoidal and stepwise vestibular stimulation. The responsive population spans multiple brain areas and displays bilateral symmetry, and its organization is highly stereotypic across individuals. Using Fourier and regression analysis, we identified three major functional clusters that exhibit well-defined phasic and tonic response patterns to vestibular stimulation. Our rotatable light-sheet microscope provides a unique tool for systematically studying vestibular processing in the vertebrate brain and extends the potential of virtual-reality systems to explore complex multisensory and motor integration during simulated 3D navigation.

Sensorimotor computation underlying phototaxis in zebrafish.

Animals continuously gather sensory cues to move towards favourable environments. Efficient goal-directed navigation requires sensory perception and motor commands to be intertwined in a feedback loop, yet the neural substrate underlying this sensorimotor task in the vertebrate brain remains elusive. Here, we combine virtual-reality behavioural assays, volumetric calcium imaging, optogenetic stimulation and circuit modelling to reveal the neural mechanisms through which a zebrafish performs phototaxis, i.e. actively orients towards a light source. Key to this process is a self-oscillating hindbrain population (HBO) that acts as a pacemaker for ocular saccades and controls the orientation of successive swim-bouts. It further integrates visual stimuli in a state-dependent manner, i.e. its response to visual inputs varies with the motor context, a mechanism that manifests itself in the phase-locked entrainment of the HBO by periodic stimuli. A rate model is developed that reproduces our observations and demonstrates how this sensorimotor processing eventually biases the animal trajectory towards bright regions.Active locomotion requires closed-loop sensorimotor co ordination between perception and action. Here the authors show using behavioural, imaging and modelling approaches that gaze orientation during phototaxis behaviour in larval zebrafish is related to oscillatory dynamics of a neuronal population in the hindbrain.

A 2D virtual reality system for visual goal-driven navigation in zebrafish larvae.

Animals continuously rely on sensory feedback to adjust motor commands. In order to study the role of visual feedback in goal-driven navigation, we developed a 2D visual virtual reality system for zebrafish larvae. The visual feedback can be set to be similar to what the animal experiences in natural conditions. Alternatively, modification of the visual feedback can be used to study how the brain adapts to perturbations. For this purpose, we first generated a library of free-swimming behaviors from which we learned the relationship between the trajectory of the larva and the shape of its tail. Then, we used this technique to infer the intended displacements of head-fixed larvae, and updated the visual environment accordingly. Under these conditions, larvae were capable of aligning and swimming in the direction of a whole-field moving stimulus and produced the fine changes in orientation and position required to capture virtual prey. We demonstrate the sensitivity of larvae to visual feedback by updating the visual world in real-time or only at the end of the discrete swimming episodes. This visual feedback perturbation caused impaired performance of prey-capture behavior, suggesting that larvae rely on continuous visual feedback during swimming.

Rheotaxis of Larval Zebrafish: Behavioral Study of a Multi-Sensory Process.

Awake animals unceasingly perceive sensory inputs with great variability of nature and intensity, and understanding how the nervous system manages this continuous flow of diverse information to get a coherent representation of the environment is arguably a central question in systems neuroscience. Rheotaxis, the ability shared by most aquatic species to orient toward a current and swim to hold position, is an innate and robust multi-sensory behavior that is known to involve the lateral line and visual systems. To facilitate the neuroethological study of rheotaxic behavior in larval zebrafish we developed an assay for freely swimming larvae that allows for high experimental throughtput, large statistic and a fine description of the behavior. We show that there exist a clear transition from exploration to counterflow swim, and by changing the sensory modalities accessible to the fishes (visual only, lateral line only or both) and comparing the swim patterns at different ages we were able to detect and characterize two different mechanisms for position holding, one mediated by the lateral line and one mediated by the visual system. We also found that when both sensory modalities are accessible the visual system overshadows the lateral line, suggesting that at the larval stage the sensory inputs are not merged to finely tune the behavior but that redundant information pathways may be used as functional fallbacks.

A microfluidic device to study neuronal and motor responses to acute chemical stimuli in zebrafish.

Zebrafish larva is a unique model for whole-brain functional imaging and to study sensory-motor integration in the vertebrate brain. To take full advantage of this system, one needs to design sensory environments that can mimic the complex spatiotemporal stimulus patterns experienced by the animal in natural conditions. We report on a novel open-ended microfluidic device that delivers pulses of chemical stimuli to agarose-restrained larvae with near-millisecond switching rate and unprecedented spatial and concentration accuracy and reproducibility. In combination with two-photon calcium imaging and recordings of tail movements, we found that stimuli of opposite hedonic values induced different circuit activity patterns. Moreover, by precisely controlling the duration of the stimulus (50-500 ms), we found that the probability of generating a gustatory-induced behavior is encoded by the number of neurons activated. This device may open new ways to dissect the neural-circuit principles underlying chemosensory perception.

Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy.

The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1-2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5-9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20-60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.

The role of exploratory conditions in bio-inspired tactile sensing of single topogical features.

We investigate the mechanism of tactile transduction during active exploration of finely textured surfaces using a tactile sensor mimicking the human fingertip. We focus in particular on the role of exploratory conditions in shaping the subcutaneous mechanical signals. The sensor has been designed by integrating a linear array of MEMS micro-force sensors in an elastomer layer. We measure the response of the sensors to the passage of elementary topographical features at constant velocity and normal load, such as a small hole on a flat substrate. Each sensor's response is found to strongly depend on its relative location with respect to the substrate/skin contact zone, a result which can be quantitatively understood within the scope of a linear model of tactile transduction. The modification of the response induced by varying other parameters, such as the thickness of the elastic layer and the confining load, are also correctly captured by this model. We further demonstrate that the knowledge of these characteristic responses allows one to dynamically evaluate the position of a small hole within the contact zone, based on the micro-force sensors signals, with a spatial resolution an order of magnitude better than the intrinsic resolution of individual sensors. Consequences of these observations on robotic tactile sensing are briefly discussed.

Journey of an intruder through the fluidization and jamming transitions of a dense granular media.

We study experimentally the motion of an intruder dragged into an amorphous monolayer of horizontally vibrated grains at high packing fractions. This motion exhibits two transitions. The first transition separates a continuous motion regime at comparatively low packing fractions and large dragging force from an intermittent motion one at high packing fraction and low dragging force. Associated to these different motions, we observe a transition from a linear rheology to a stiffer response. We thereby call "fluidization" this first transition. A second transition is observed within the intermittent regime when the intruder's motion is made of intermittent bursts separated by long waiting times. We observe a peak in the relative fluctuations of the intruder's displacements and a critical scaling of the burst amplitudes' distributions. This transition occurs at the jamming point phi(J) defined as the point where the static pressure (i.e., the pressure measured in the absence of vibration) vanishes. Investigating the motion of the surrounding grains, we show that below the fluidization transition, there is a permanent wake of free volume behind the intruder. This transition is marked by the evolution of the reorganization patterns around the intruder, which evolve from compact aggregates in the flowing regime to long-range branched shapes in the intermittent regime, suggesting an increasing role of the stress fluctuations. Remarkably, the distributions of the kinetic energy of these reorganization patterns also exhibit a critical scaling at the jamming transition.