Visual recognition of social signals by a tectothalamic neural circuit.

Social affiliation emerges from individual-level behavioural rules that are driven by conspecific signals1-5. Long-distance attraction and short-distance repulsion, for example, are rules that jointly set a preferred interanimal distance in swarms6-8. However, little is known about their perceptual mechanisms and executive neural circuits3. Here we trace the neuronal response to self-like biological motion9,10, a visual trigger for affiliation in developing zebrafish2,11. Unbiased activity mapping and targeted volumetric two-photon calcium imaging revealed 21 activity hotspots distributed throughout the brain as well as clustered biological-motion-tuned neurons in a multimodal, socially activated nucleus of the dorsal thalamus. Individual dorsal thalamus neurons encode local acceleration of visual stimuli mimicking typical fish kinetics but are insensitive to global or continuous motion. Electron microscopic reconstruction of dorsal thalamus neurons revealed synaptic input from the optic tectum and projections into hypothalamic areas with conserved social function12-14. Ablation of the optic tectum or dorsal thalamus selectively disrupted social attraction without affecting short-distance repulsion. This tectothalamic pathway thus serves visual recognition of conspecifics, and dissociates neuronal control of attraction from repulsion during social affiliation, revealing a circuit underpinning collective behaviour.

Neural circuitry for stimulus selection in the zebrafish visual system.

When navigating the environment, animals need to prioritize responses to the most relevant stimuli. Although a theoretical framework for selective visual attention exists, its circuit implementation has remained obscure. Here we investigated how larval zebrafish select between simultaneously presented visual stimuli. We found that a mix of winner-take-all (WTA) and averaging strategies best simulates behavioral responses. We identified two circuits whose activity patterns predict the relative saliencies of competing visual objects. Stimuli presented to only one eye are selected by WTA computation in the inner retina. Binocularly presented stimuli, on the other hand, are processed by reciprocal, bilateral connections between the nucleus isthmi (NI) and the tectum. This interhemispheric computation leads to WTA or averaging responses. Optogenetic stimulation and laser ablation of NI neurons disrupt stimulus selection and behavioral action selection. Thus, depending on the relative locations of competing stimuli, a combination of retinotectal and isthmotectal circuits enables selective visual attention.

Deconstructing Hunting Behavior Reveals a Tightly Coupled Stimulus-Response Loop.

Animal behavior often forms sequences, built from simple stereotyped actions and shaped by environmental cues. A comprehensive characterization of the interplay between an animal's movements and its environment is necessary to understand the sensorimotor transformations performed by the brain. Here, we use unsupervised methods to study behavioral sequences in zebrafish larvae. We generate a map of swim bouts, revealing that fish modulate their tail movements along a continuum. During prey capture, larvae produce stereotyped sequences using a subset of bouts from a broader behavioral repertoire. These sequences exhibit low-order transition dynamics and immediately respond to changes in visual cues. Chaining of prey capture bouts is disrupted in visually impaired (lakritz and blumenkohl) mutants, and removing the prey stimulus during ongoing behavior in closed-loop virtual reality causes larvae to immediately abort the hunting sequence. These results suggest that the continuous integration of sensory information is necessary to structure the behavior. This stimulus-response loop serves to bring prey into the anterior dorsal visual field of the larvae. Fish then release a capture strike maneuver comprising a stereotyped jaw movement and tail movements fine-tuned to the distance of the prey. Fish with only one intact eye fail to correctly position the prey in the strike zone, but are able to produce the strike itself. Our analysis shows that short-term integration of binocular visual cues shapes the behavioral dynamics of hunting, thus uncovering the temporal organization of a goal-directed behavior in a vertebrate.

Topography of a Visuomotor Transformation.

The brain converts perceptual information into appropriate patterns of muscle activity depending on the categorization and localization of sensory cues. Sensorimotor information might either be encoded by distributed networks or by "labeled lines" connecting sensory channels to dedicated behavioral pathways. Here we investigate, in the context of natural behavior, how the tectum of larval zebrafish can inform downstream premotor areas. Optogenetic mapping revealed a tectal motor map underlying locomotor maneuvers for escape and approach. Single-cell reconstructions and high-resolution functional imaging showed that two spatially segregated and uncrossed descending axon tracts selectively transmit approach and escape signals to the hindbrain. Moreover, the approach pathway conveys information about retinotopic target coordinates to specific premotor ensembles via spatially ordered axonal projections. This topographic organization supports a tectum-generated space code sufficient to steer orienting movements. We conclude that specific labeled lines guide object-directed behavior in the larval zebrafish brain.

Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging.

We introduce a flexible method for high-resolution interrogation of circuit function, which combines simultaneous 3D two-photon stimulation of multiple targeted neurons, volumetric functional imaging, and quantitative behavioral tracking. This integrated approach was applied to dissect how an ensemble of premotor neurons in the larval zebrafish brain drives a basic motor program, the bending of the tail. We developed an iterative photostimulation strategy to identify minimal subsets of channelrhodopsin (ChR2)-expressing neurons that are sufficient to initiate tail movements. At the same time, the induced network activity was recorded by multiplane GCaMP6 imaging across the brain. From this dataset, we computationally identified activity patterns associated with distinct components of the elicited behavior and characterized the contributions of individual neurons. Using photoactivatable GFP (paGFP), we extended our protocol to visualize single functionally identified neurons and reconstruct their morphologies. Together, this toolkit enables linking behavior to circuit activity with unprecedented resolution.

A Visual Pathway for Looming-Evoked Escape in Larval Zebrafish.

Avoiding the strike of an approaching predator requires rapid visual detection of a looming object, followed by a directed escape maneuver. While looming-sensitive neurons have been discovered in various animal species, the relative importance of stimulus features that are extracted by the visual system is still unclear. Furthermore, the neural mechanisms that compute object approach are largely unknown. We found that a virtual looming stimulus, i.e., a dark expanding disk on a bright background, reliably evoked rapid escape movements. Related stimuli, such as dimming, receding, or bright looming objects, were substantially less effective, and angular size was a critical determinant of escape initiation. Two-photon calcium imaging in retinal ganglion cell (RGC) axons revealed three retinorecipient areas that responded robustly to looming stimuli. One of these areas, the optic tectum, is innervated by a subset of RGC axons that respond selectively to looming stimuli. Laser-induced lesions of the tectal neuropil impaired the behavior. Our findings demonstrate a visually mediated escape behavior in zebrafish larvae exposed to objects approaching on a collision course. This response is sensitive to spatiotemporal parameters of the looming stimulus. Our data indicate that a subset of RGC axons within the tectum responds selectively to features of looming stimuli and that this input is necessary for visually evoked escape.

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.