The mesencephalic locomotor region recruits V2a reticulospinal neurons to drive forward locomotion in larval zebrafish.

The mesencephalic locomotor region (MLR) is a brain stem area whose stimulation triggers graded forward locomotion. How MLR neurons recruit downstream vsx2+ (V2a) reticulospinal neurons (RSNs) is poorly understood. Here, to overcome this challenge, we uncovered the locus of MLR in transparent larval zebrafish and show that the MLR locus is distinct from the nucleus of the medial longitudinal fasciculus. MLR stimulations reliably elicit forward locomotion of controlled duration and frequency. MLR neurons recruit V2a RSNs via projections onto somata in pontine and retropontine areas, and onto dendrites in the medulla. High-speed volumetric imaging of neuronal activity reveals that strongly MLR-coupled RSNs are active for steering or forward swimming, whereas weakly MLR-coupled medullary RSNs encode the duration and frequency of the forward component. Our study demonstrates how MLR neurons recruit specific V2a RSNs to control the kinematics of forward locomotion and suggests conservation of the motor functions of V2a RSNs across vertebrates.

Spatiotemporal visual statistics of aquatic environments in the natural habitats of zebrafish.

Animal sensory systems are tightly adapted to the demands of their environment. In the visual domain, research has shown that many species have circuits and systems that exploit statistical regularities in natural visual signals. The zebrafish is a popular model animal in visual neuroscience, but relatively little quantitative data is available about the visual properties of the aquatic habitats where zebrafish reside, as compared to terrestrial environments. Improving our understanding of the visual demands of the aquatic habitats of zebrafish can enhance the insights about sensory neuroscience yielded by this model system. We analyzed a video dataset of zebrafish habitats captured by a stationary camera and compared this dataset to videos of terrestrial scenes in the same geographic area. Our analysis of the spatiotemporal structure in these videos suggests that zebrafish habitats are characterized by low visual contrast and strong motion when compared to terrestrial environments. Similar to terrestrial environments, zebrafish habitats tended to be dominated by dark contrasts, particularly in the lower visual field. We discuss how these properties of the visual environment can inform the study of zebrafish visual behavior and neural processing and, by extension, can inform our understanding of the vertebrate brain.

Optic flow in the natural habitats of zebrafish supports spatial biases in visual self-motion estimation.

Animals benefit from knowing if and how they are moving. Across the animal kingdom, sensory information in the form of optic flow over the visual field is used to estimate self-motion. However, different species exhibit strong spatial biases in how they use optic flow. Here, we show computationally that noisy natural environments favor visual systems that extract spatially biased samples of optic flow when estimating self-motion. The performance associated with these biases, however, depends on interactions between the environment and the animal's brain and behavior. Using the larval zebrafish as a model, we recorded natural optic flow associated with swimming trajectories in the animal's habitat with an omnidirectional camera mounted on a mechanical arm. An analysis of these flow fields suggests that lateral regions of the lower visual field are most informative about swimming speed. This pattern is consistent with the recent findings that zebrafish optomotor responses are preferentially driven by optic flow in the lateral lower visual field, which we extend with behavioral results from a high-resolution spherical arena. Spatial biases in optic-flow sampling are likely pervasive because they are an effective strategy for determining self-motion in noisy natural environments.

Manipulation of the Tyrosinase gene permits improved CRISPR/Cas editing and neural imaging in cichlid fish.

Direct tests of gene function have historically been performed in a limited number of model organisms. The CRISPR/Cas system is species-agnostic, offering the ability to manipulate genes in a range of models, enabling insights into evolution, development, and physiology. Astatotilapia burtoni, a cichlid fish from the rivers and shoreline around Lake Tanganyika, has been extensively studied in the laboratory to understand evolution and the neural control of behavior. Here we develop protocols for the creation of CRISPR-edited cichlids and create a broadly useful mutant line. By manipulating the Tyrosinase gene, which is necessary for eumelanin pigment production, we describe a fast and reliable approach to quantify and optimize gene editing efficiency. Tyrosinase mutants also remove a major obstruction to imaging, enabling visualization of subdermal structures and fluorophores in situ. These protocols will facilitate broad application of CRISPR/Cas9 to studies of cichlids as well as other non-traditional model aquatic species.

BonZeb: open-source, modular software tools for high-resolution zebrafish tracking and analysis.

We present BonZeb-a suite of modular Bonsai packages which allow high-resolution zebrafish tracking with dynamic visual feedback. Bonsai is an increasingly popular software platform that is accelerating the standardization of experimental protocols within the neurosciences due to its speed, flexibility, and minimal programming overhead. BonZeb can be implemented into novel and existing Bonsai workflows for online behavioral tracking and offline tracking with batch processing. We demonstrate that BonZeb can run a variety of experimental configurations used for gaining insights into the neural mechanisms of zebrafish behavior. BonZeb supports head-fixed closed-loop and free-swimming virtual open-loop assays as well as multi-animal tracking, optogenetic stimulation, and calcium imaging during behavior. The combined performance, ease of use and versatility of BonZeb opens new experimental avenues for researchers seeking high-resolution behavioral tracking of larval zebrafish.

Parallel Channels for Motion Feature Extraction in the Pretectum and Tectum of Larval Zebrafish.

Non-cortical visual areas in vertebrate brains extract relevant stimulus features, such as motion, object size, and location, to support diverse behavioral tasks. The optic tectum and pretectum, two primary visual areas in zebrafish, are involved in motion processing, and yet their differential neural representation of behaviorally relevant visual features is unclear. Here, we characterize receptive fields (RFs) of motion-sensitive neurons in the diencephalon and midbrain. We show that RFs of many pretectal neurons are large and sample the lower visual field, whereas RFs of tectal neurons are mostly small-size selective and sample the upper nasal visual field more densely. Furthermore, optomotor swimming can reliably be evoked by presenting forward motion in the lower temporal visual field alone, matching the lower visual field bias of the pretectum. Thus, tectum and pretectum extract different visual features from distinct regions of visual space, which is likely a result of their adaptations to hunting and optomotor behavior, respectively.

Genetic targeting and anatomical registration of neuronal populations in the zebrafish brain with a new set of BAC transgenic tools.

Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression. We used image registration to characterize, compare, and digitally superimpose the labeling patterns from our newly generated transgenic lines. This analysis revealed highly restricted and mutually exclusive tissue distributions, with striking resolution of layered brain regions such as the tectum or the rhombencephalon. We further show that a combination of Gal4 and Cre transgenes allows intersectional expression of a fluorescent reporter in regions where the expression of the two drivers overlaps. Taken together, our study offers new tools for functional studies of specific neural circuits in zebrafish.

A stochastic neuronal model predicts random search behaviors at multiple spatial scales in C. elegans.

Random search is a behavioral strategy used by organisms from bacteria to humans to locate food that is randomly distributed and undetectable at a distance. We investigated this behavior in the nematode Caenorhabditis elegans, an organism with a small, well-described nervous system. Here we formulate a mathematical model of random search abstracted from the C. elegans connectome and fit to a large-scale kinematic analysis of C. elegans behavior at submicron resolution. The model predicts behavioral effects of neuronal ablations and genetic perturbations, as well as unexpected aspects of wild type behavior. The predictive success of the model indicates that random search in C. elegans can be understood in terms of a neuronal flip-flop circuit involving reciprocal inhibition between two populations of stochastic neurons. Our findings establish a unified theoretical framework for understanding C. elegans locomotion and a testable neuronal model of random search that can be applied to other organisms.

A dedicated visual pathway for prey detection in larval zebrafish.

Zebrafish larvae show characteristic prey capture behavior in response to small moving objects. The neural mechanism used to recognize objects as prey remains largely unknown. We devised a machine learning behavior classification system to quantify hunting kinematics in semi-restrained animals exposed to a range of virtual stimuli. Two-photon calcium imaging revealed a small visual area, AF7, that was activated specifically by the optimal prey stimulus. This pretectal region is innervated by two types of retinal ganglion cells, which also send collaterals to the optic tectum. Laser ablation of AF7 markedly reduced prey capture behavior. We identified neurons with arbors in AF7 and found that they projected to multiple sensory and premotor areas: the optic tectum, the nucleus of the medial longitudinal fasciculus (nMLF) and the hindbrain. These findings indicate that computations in the retina give rise to a visual stream which transforms sensory information into a directed prey capture response.

Descending control of swim posture by a midbrain nucleus in zebrafish.

The reticular formation in the brainstem controls motor output via axonal projections to the hindbrain and spinal cord. It remains unclear how individual groups of brainstem neurons contribute to specific motor functions. Here, we investigate the behavioral role of the nucleus of the medial longitudinal fasciculus (nMLF), a small group of reticulospinal neurons in the zebrafish midbrain. Calcium imaging revealed that nMLF activity is correlated with bouts of swimming. Optogenetic stimulation of neurons in the left or right nMLF activates the posterior hypaxial muscle and produces a graded ipsilateral tail deflection. Unilateral ablation of a subset of nMLF cells biases the tail position to the intact side during visually evoked swims, while sparing other locomotor maneuvers. We conclude that activity in the nMLF provides postural control of tail orientation and thus steers the direction of swimming. Our studies provide an example of fine-grained modularity of descending motor control in vertebrates.

An image-free opto-mechanical system for creating virtual environments and imaging neuronal activity in freely moving Caenorhabditis elegans.

Non-invasive recording in untethered animals is arguably the ultimate step in the analysis of neuronal function, but such recordings remain elusive. To address this problem, we devised a system that tracks neuron-sized fluorescent targets in real time. The system can be used to create virtual environments by optogenetic activation of sensory neurons, or to image activity in identified neurons at high magnification. By recording activity in neurons of freely moving C. elegans, we tested the long-standing hypothesis that forward and reverse locomotion are generated by distinct neuronal circuits. Surprisingly, we found motor neurons that are active during both types of locomotion, suggesting a new model of locomotion control in C. elegans. These results emphasize the importance of recording neuronal activity in freely moving animals and significantly expand the potential of imaging techniques by providing a mean to stabilize fluorescent targets.

Optogenetic analysis of synaptic transmission in the central nervous system of the nematode Caenorhabditis elegans.

A reliable method for recording evoked synaptic events in identified neurons in Caenorhabditis elegans would greatly accelerate our understanding of its nervous system at the molecular, cellular and network levels. Here we describe a method for recording synaptic currents and potentials from identified neurons in nearly intact worms. Dissection and exposure of postsynaptic neurons is facilitated by microfabricated agar substrates, and ChannelRhodopsin-2 is used to stimulate presynaptic neurons. We used the method to analyse functional connectivity between a polymodal nociceptor and a command neuron that initiates a stochastic escape behaviour. We find that escape probability mirrors the time course of synaptic current in the command neuron. Moreover, synaptic input increases smoothly as stimulus strength is increased, suggesting that the overall input-output function of the connection is graded. We propose a model in which the energetic cost of escape behaviours in C. elegans is tuned to the intensity of the threat.

The neural network for chemotaxis to tastants in Caenorhabditis elegans is specialized for temporal differentiation.

Chemotaxis in Caenorhabditis elegans depends critically on the rate of change of attractant concentration computed as the worm moves through its environment. This computation depends, in turn, on the neuron class ASE, a left-right pair of pair of chemosensory neurons that is functionally asymmetric such that the left neuron is an on-cell, whereas the right neuron is an off-cell. To determine whether this coding strategy is a general feature of chemosensation in C. elegans, we imaged calcium responses in all chemosensory neurons known or in a position to contribute to chemotaxis to tastants in this organism. This survey revealed one new class of on-cells (ADF) and one new class of off-cells (ASH). Thus, the ASE class is unique in having both an on-cell and an off-cell. We also found that the newly characterized on-cells and off-cells promote runs and turns, respectively, mirroring the pattern reported previously for ASEL and ASER. Our results suggest that the C. elegans chemotaxis network is specialized for the temporal differentiation of chemosensory inputs, as required for chemotaxis.

Functional asymmetry in Caenorhabditis elegans taste neurons and its computational role in chemotaxis.

Chemotaxis in Caenorhabditis elegans, like chemotaxis in bacteria, involves a random walk biased by the time derivative of attractant concentration, but how the derivative is computed is unknown. Laser ablations have shown that the strongest deficits in chemotaxis to salts are obtained when the ASE chemosensory neurons (ASEL and ASER) are ablated, indicating that this pair has a dominant role. Although these neurons are left-right homologues anatomically, they exhibit marked asymmetries in gene expression and ion preference. Here, using optical recordings of calcium concentration in ASE neurons in intact animals, we demonstrate an additional asymmetry: ASEL is an ON-cell, stimulated by increases in NaCl concentration, whereas ASER is an OFF-cell, stimulated by decreases in NaCl concentration. Both responses are reliable yet transient, indicating that ASE neurons report changes in concentration rather than absolute levels. Recordings from synaptic and sensory transduction mutants show that the ON-OFF asymmetry is the result of intrinsic differences between ASE neurons. Unilateral activation experiments indicate that the asymmetry extends to the level of behavioural output: ASEL lengthens bouts of forward locomotion (runs) whereas ASER promotes direction changes (turns). Notably, the input and output asymmetries of ASE neurons are precisely those of a simple yet novel neuronal motif for computing the time derivative of chemosensory information, which is the fundamental computation of C. elegans chemotaxis. Evidence for ON and OFF cells in other chemosensory networks suggests that this motif may be common in animals that navigate by taste and smell.

Step-response analysis of chemotaxis in Caenorhabditis elegans.

The sensorimotor transformation underlying Caenorhabditis elegans chemotaxis has been difficult to measure directly under normal assay conditions. Thus, key features of this transformation remain obscure, such as its time course and dependence on stimulus amplitude. Here, we present a comprehensive characterization of the transformation as obtained by inducing stepwise temporal changes in attractant concentration within the substrate as the worm crawls across it. We found that the step response is complex, with multiple phases and a nonlinear dependence on the sign and amplitude of the stimulus. Nevertheless, the step response could be reduced to a simple kinetic model that predicted the results of chemotaxis assays. Analysis of the model showed that chemotaxis results from the combined effects of approach and avoidance responses to concentration increases and decreases, respectively. Surprisingly, ablation of the ASE chemosensory neurons, known to be necessary for chemotaxis in chemical gradient assays, eliminated avoidance responses but left approach responses intact. These results indicate that the transformation can be dissected into components to which identified neurons can be assigned.

Natural variation in the npr-1 gene modifies ethanol responses of wild strains of C. elegans.

Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Here we show that allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of Caenorhabditis elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations.

A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans.

The activities of many neuronal proteins are modulated by ethanol, but the fundamental mechanisms underlying behavioral effects of ethanol remain unclear. To identify mechanisms responsible for intoxication, we screened for Caenorhabditis elegans mutants with altered behavioral responses to ethanol. We found that slo-1 mutants, which were previously recognized as having slightly uncoordinated movement, are highly resistant to ethanol in two behavioral assays. Numerous loss-of-function slo-1 alleles emerged from our screens, indicating that slo-1 has a central role in ethanol responses. slo-1 encodes the BK potassium channel. Electrophysiological analysis shows that ethanol activates the channel in vivo, which would inhibit neuronal activity. Moreover, behaviors of slo-1 gain-of-function mutants resemble those of ethanol-intoxicated animals. These results demonstrate that selective activation of BK channels is responsible for acute intoxicating effects of ethanol in C. elegans. BK channel activation may explain a variety of behavioral responses to ethanol in invertebrate and vertebrate systems.