Radial astrocyte synchronization modulates the visual system during behavioral-state transitions.

Glial cells support the function of neurons. Recent evidence shows that astrocytes are also involved in brain computations. To explore whether and how their excitable nature affects brain computations and motor behaviors, we used two-photon Ca2+ imaging of zebrafish larvae expressing GCaMP in both neurons and radial astrocytes (RAs). We found that in the optic tectum, RAs synchronize their Ca2+ transients immediately after the end of an escape behavior. Using optogenetics, ablations, and a genetically encoded norepinephrine sensor, we observed that RA synchronous Ca2+ events are mediated by the locus coeruleus (LC)-norepinephrine circuit. RA synchronization did not induce direct excitation or inhibition of tectal neurons. Nevertheless, it modulated the direction selectivity and the long-distance functional correlations among neurons. This mechanism supports freezing behavior following a switch to an alerted state. These results show that LC-mediated neuro-glial interactions modulate the visual system during transitions between behavioral states.

A protocol for whole-brain Ca2+ imaging in Astyanax mexicanus, a model of comparative evolution.

In this protocol, we describe a comparative approach to study the evolution of brain function in the Mexican tetra, Astyanax mexicanus. We developed surface fish and two independent populations of cavefish with pan-neuronal expression of the Ca2+ sensor GCaMP6s. We describe a methodology to prepare samples and image activity across the optic tectum and olfactory bulb.

Blind cavefish retain functional connectivity in the tectum despite loss of retinal input.

Sensory systems display remarkable plasticity and are under strong evolutionary selection. The Mexican cavefish, Astyanax mexicanus, consists of eyed river-dwelling surface populations and multiple independent cave populations that have converged on eye loss, providing the opportunity to examine the evolution of sensory circuits in response to environmental perturbation. Functional analysis across multiple transgenic populations expressing GCaMP6s showed that functional connectivity of the optic tectum largely did not differ between populations, except for the selective loss of negatively correlated activity within the cavefish tectum, suggesting positively correlated neural activity is resistant to an evolved loss of input from the retina. Furthermore, analysis of surface-cave hybrid fish reveals that changes in the tectum are genetically distinct from those encoding eye loss. Together, these findings uncover the independent evolution of multiple components of the visual system and establish the use of functional imaging in A. mexicanus to study neural circuit evolution.

Fourier Motion Processing in the Optic Tectum and Pretectum of the Zebrafish Larva.

In the presence of moving visual stimuli, the majority of animals follow the Fourier motion energy (luminance), independently of other stimulus features (edges, contrast, etc.). While the behavioral response to Fourier motion has been studied in the past, how Fourier motion is represented and processed by sensory brain areas remains elusive. Here, we investigated how visual moving stimuli with or without the first Fourier component (square-wave signal or missing fundamental signal) are represented in the main visual regions of the zebrafish brain. First, we monitored the larva's optokinetic response (OKR) induced by square-wave and missing fundamental signals. Then, we used two-photon microscopy and GCaMP6f zebrafish larvae to monitor neuronal circuit dynamics in the optic tectum and the pretectum. We observed that both the optic tectum and the pretectum circuits responded to the square-wave gratings. However, only the pretectum responded specifically to the direction of the missing-fundamental signal. In addition, a group of neurons in the pretectum responded to the direction of the behavioral output (OKR), independently of the type of stimulus presented. Our results suggest that the optic tectum responds to the different features of the stimulus (e.g., contrast, spatial frequency, direction, etc.), but does not respond to the direction of motion if the motion information is not coherent (e.g., the luminance and the edges and contrast in the missing-fundamental signal). On the other hand, the pretectum mainly responds to the motion of the stimulus based on the Fourier energy.

Naturalistic Behavior: The Zebrafish Larva Strikes Back.

Two recent studies show that zebrafish larvae alternate between two behavioral modes: exploration and hunting. Both behaviors are structured on multiple time scales, and require the integration of internal and external cues to generate sequences of stereotyped swimming movements.

Sensorimotor Transformations in the Zebrafish Auditory System.

Organisms use their sensory systems to acquire information from their environment and integrate this information to produce relevant behaviors. Nevertheless, how sensory information is converted into adequate motor patterns in the brain remains an open question. Here, we addressed this question using two-photon and light-sheet calcium imaging in intact, behaving zebrafish larvae. We monitored neural activity elicited by auditory stimuli while simultaneously recording tail movements. We observed a spatial organization of neural activity according to four different response profiles (frequency tuning curves), suggesting a low-dimensional representation of frequency information, maintained throughout the development of the larvae. Low frequencies (150-450 Hz) were locally processed in the hindbrain and elicited motor behaviors. In contrast, higher frequencies (900-1,000 Hz) rarely induced motor behaviors and were also represented in the midbrain. Finally, we found that the sensorimotor transformations in the zebrafish auditory system are a continuous and gradual process that involves the temporal integration of the sensory response in order to generate a motor behavior.

Functional Integration of Newborn Neurons in the Zebrafish Optic Tectum.

Neurogenesis persists during adulthood in restricted parts of the vertebrate brain. In the optic tectum (OT) of the zebrafish larva, newborn neurons are continuously added and contribute to visual information processing. Recent studies have started to describe the functional development and fate of newborn neurons in the OT. Like the mammalian brain, newborn neurons in the OT require sensory inputs for their integration into local networks and survival. Recent findings suggest that the functional development of newborn neurons requires both activity-dependent and hard-wired mechanisms for proper circuit integration. Here, we review these findings and argue that the study of neurogenesis in non-mammalian species will help elucidate the general mechanisms of circuit assembly following neurogenesis.

Whole-Brain Neuronal Activity Displays Crackling Noise Dynamics.

Previous studies suggest that the brain operates at a critical point in which phases of order and disorder coexist, producing emergent patterned dynamics at all scales and optimizing several brain functions. Here, we combined light-sheet microscopy with GCaMP zebrafish larvae to study whole-brain dynamics in vivo at near single-cell resolution. We show that spontaneous activity propagates in the brain's three-dimensional space, generating scale-invariant neuronal avalanches with time courses and recurrence times that exhibit statistical self-similarity at different magnitude, temporal, and frequency scales. This suggests that the nervous system operates close to a non-equilibrium phase transition, where a large repertoire of spatial, temporal, and interactive modes can be supported. Finally, we show that gap junctions contribute to the maintenance of criticality and that, during interactions with the environment (sensory inputs and self-generated behaviors), the system is transiently displaced to a more ordered regime, conceivably to limit the potential sensory representations and motor outcomes.

Principles of Functional Circuit Connectivity: Insights From Spontaneous Activity in the Zebrafish Optic Tectum.

The brain is continuously active, even in the absence of external stimulation. In the optic tectum of the zebrafish larva, this spontaneous activity is spatially organized and reflects the circuit's functional connectivity. The structure of the spontaneous activity displayed patterns associated with aspects of the larva's preferences when engaging in complex visuo-motor behaviors, suggesting that the tectal circuit is adapted for the circuit's functional role in detecting visual cues and generating adequate motor behaviors. Further studies in sensory deprived larvae suggest that the basic structure of the functional connectivity patterns emerges even in the absence of retinal inputs, but that its fine structure is affected by visual experience.

An integrated calcium imaging processing toolbox for the analysis of neuronal population dynamics.

The development of new imaging and optogenetics techniques to study the dynamics of large neuronal circuits is generating datasets of unprecedented volume and complexity, demanding the development of appropriate analysis tools. We present a comprehensive computational workflow for the analysis of neuronal population calcium dynamics. The toolbox includes newly developed algorithms and interactive tools for image pre-processing and segmentation, estimation of significant single-neuron single-trial signals, mapping event-related neuronal responses, detection of activity-correlated neuronal clusters, exploration of population dynamics, and analysis of clusters' features against surrogate control datasets. The modules are integrated in a modular and versatile processing pipeline, adaptable to different needs. The clustering module is capable of detecting flexible, dynamically activated neuronal assemblies, consistent with the distributed population coding of the brain. We demonstrate the suitability of the toolbox for a variety of calcium imaging datasets. The toolbox open-source code, a step-by-step tutorial and a case study dataset are available at https://github.com/zebrain-lab/Toolbox-Romano-et-al.

Functional Interactions between Newborn and Mature Neurons Leading to Integration into Established Neuronal Circuits.

From development up to adulthood, the vertebrate brain is continuously supplied with newborn neurons that integrate into established mature circuits. However, how this process is coordinated during development remains unclear. Using two-photon imaging, GCaMP5 transgenic zebrafish larvae, and sparse electroporation in the larva's optic tectum, we monitored spontaneous and induced activity of large neuronal populations containing newborn and functionally mature neurons. We observed that the maturation of newborn neurons is a 4-day process. Initially, newborn neurons showed undeveloped dendritic arbors, no neurotransmitter identity, and were unresponsive to visual stimulation, although they displayed spontaneous calcium transients. Later on, newborn-labeled neurons began to respond to visual stimuli but in a very variable manner. At the end of the maturation period, newborn-labeled neurons exhibited visual tuning curves (spatial receptive fields and direction selectivity) and spontaneous correlated activity with neighboring functionally mature neurons. At this developmental stage, newborn-labeled neurons presented complex dendritic arbors and neurotransmitter identity (excitatory or inhibitory). Removal of retinal inputs significantly perturbed the integration of newborn neurons into the functionally mature tectal network. Our results provide a comprehensive description of the maturation of newborn neurons during development and shed light on potential mechanisms underlying their integration into a functionally mature neuronal circuit.

The Emergence of the Spatial Structure of Tectal Spontaneous Activity Is Independent of Visual Inputs.

The brain is spontaneously active, even in the absence of sensory stimulation. The functionally mature zebrafish optic tectum shows spontaneous activity patterns reflecting a functional connectivity adapted for the circuit's functional role and predictive of behavior. However, neither the emergence of these patterns during development nor the role of retinal inputs in their maturation has been characterized. Using two-photon calcium imaging, we analyzed spontaneous activity in intact and enucleated zebrafish larvae throughout tectum development. At the onset of retinotectal connections, intact larvae showed major changes in the spatiotemporal structure of spontaneous activity. Although the absence of retinal inputs had a significant impact on the development of the temporal structure, the tectum was still capable of developing a spatial structure associated with the circuit's functional roles and predictive of behavior. We conclude that neither visual experience nor intrinsic retinal activity is essential for the emergence of a spatially structured functional circuit.

Sustained Rhythmic Brain Activity Underlies Visual Motion Perception in Zebrafish.

Following moving visual stimuli (conditioning stimuli, CS), many organisms perceive, in the absence of physical stimuli, illusory motion in the opposite direction. This phenomenon is known as the motion aftereffect (MAE). Here, we use MAE as a tool to study the neuronal basis of visual motion perception in zebrafish larvae. Using zebrafish eye movements as an indicator of visual motion perception, we find that larvae perceive MAE. Blocking eye movements using optogenetics during CS presentation did not affect MAE, but tectal ablation significantly weakened it. Using two-photon calcium imaging of behaving GCaMP3 larvae, we find post-stimulation sustained rhythmic activity among direction-selective tectal neurons associated with the perception of MAE. In addition, tectal neurons tuned to the CS direction habituated, but neurons in the retina did not. Finally, a model based on competition between direction-selective neurons reproduced MAE, suggesting a neuronal circuit capable of generating perception of visual motion.

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.

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.

Spontaneous neuronal network dynamics reveal circuit's functional adaptations for behavior.

Spontaneous neuronal activity is spatiotemporally structured, influencing brain computations. Nevertheless, the neuronal interactions underlying these spontaneous activity patterns, and their biological relevance, remain elusive. Here, we addressed these questions using two-photon calcium imaging of intact zebrafish larvae to monitor the neuron-to-neuron spontaneous activity fine structure in the tectum, a region involved in visual spatial detection. Spontaneous activity was organized in topographically compact assemblies, grouping functionally similar neurons rather than merely neighboring ones, reflecting the tectal retinotopic map despite being independent of retinal drive. Assemblies represent all-or-none-like sub-networks shaped by competitive dynamics, mechanisms advantageous for visual detection in noisy natural environments. Notably, assemblies were tuned to the same angular sizes and spatial positions as prey-detection performance in behavioral assays, and their spontaneous activation predicted directional tail movements. Therefore, structured spontaneous activity represents "preferred" network states, tuned to behaviorally relevant features, emerging from the circuit's intrinsic non-linear dynamics, adapted for its functional role.

Monitoring tectal neuronal activities and motor behavior in zebrafish larvae.

To understand how visuomotor behaviors are controlled by the nervous system, it is necessary to monitor the activity of large populations of neurons with single-cell resolution over a large area of the brain in a relatively simple, behaving organism. The zebrafish larva, a small lower vertebrate with transparent skin, serves as an excellent model for this purpose. Immediately after the larva hatches, it needs to catch prey and avoid predators. This strong evolutionary pressure leads to the rapid development of functional sensory systems, particularly vision. By 5 d postfertilization (dpf), tectal cells show distinct visually evoked patterns of activation, and the larvae are able to perform a variety of visuomotor behaviors. During the early larval stage, zebrafish breathe mainly through the skin and can be restrained under the microscope using a drop of low-melting-point agarose, without the use of anesthetics. Moreover, the transparency of the skin, the small diameter of the neurons (4-5 µm), and the high-neuronal density enable the use of in vivo noninvasive imaging techniques to monitor neuronal activities of up to ∼500 cells within the central nervous system, still with single-cell resolution. This article describes a method for simultaneously monitoring spontaneous and visually evoked activities of large populations of neurons in the optic tectum of the zebrafish larva, using a synthetic calcium dye (Oregon Green BAPTA-1 AM) and a conventional confocal or two-photon scanning fluorescence microscope, together with a method for measuring the tail motor behavior of the head-immobilized zebrafish larva.

The first mecp2-null zebrafish model shows altered motor behaviors.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder and one of the most common causes of mental retardation in affected girls. Other symptoms include a rapid regression of motor and cognitive skills after an apparently early normal development. Sporadic mutations in the transcription factor MECP2 has been shown to be present in more than 90% of the patients and several models of MeCP2-deficient mice have been created to understand the role of this gene. These models have pointed toward alterations in the maintenance of the central nervous system rather than its development, in line with the late onset of the disease in humans. However, the exact functions of MeCP2 remain difficult to delineate and the animal models have yielded contradictory results. Here, we present the first mecp2-null allele mutation zebrafish model. Surprisingly and in contrast to MeCP2-null mouse models, mecp2-null zebrafish are viable and fertile. They present nonetheless clear behavioral alterations during their early development, including spontaneous and sensory-evoked motor anomalies, as well as defective thigmotaxis.