The nature of nervous system function and development is inherently global, since all components eventually influence one another. Networks communicate through dense synaptic, electric, and ...modulatory connections and develop through concurrent growth and interlinking of their neurons, processes, glia, and blood vessels. These factors drive the development of techniques capable of imaging neural signaling, anatomy, and developmental processes at ever-larger scales. Here, we discuss the nature of questions benefitting from large-scale imaging techniques and introduce recent applications. We focus on emerging light-sheet microscopy approaches, which are well suited for live imaging of large systems with high spatiotemporal resolution and over long periods of time. We also discuss computational methods suitable for extracting biological information from the resulting system-level image data sets. Together with new tools for reporting and manipulating neuronal activity and gene expression, these techniques promise new insights into the large-scale function and development of neural systems.
Keller and Ahrens provide an overview of light-sheet microscopy approaches for studying the development and function of neural systems. They introduce the technical background of this method and evaluate its potential to advance neuroscience research.
Escape behaviors deliver organisms away from imminent catastrophe. Here, we characterize behavioral responses of freely swimming larval zebrafish to looming visual stimuli simulating predators. We ...report that the visual system alone can recruit lateralized, rapid escape motor programs, similar to those elicited by mechanosensory modalities. Two-photon calcium imaging of retino-recipient midbrain regions isolated the optic tectum as an important center processing looming stimuli, with ensemble activity encoding the critical image size determining escape latency. Furthermore, we describe activity in retinal ganglion cell terminals and superficial inhibitory interneurons in the tectum during looming and propose a model for how temporal dynamics in tectal periventricular neurons might arise from computations between these two fundamental constituents. Finally, laser ablations of hindbrain circuitry confirmed that visual and mechanosensory modalities share the same premotor output network. We establish a circuit for the processing of aversive stimuli in the context of an innate visual behavior.
•Larval zebrafish escape from looming stimuli after a critical image size is reached•Population activity of neurons in the optic tectum encodes critical image size•Modeling predicts the critical role of characterized cell types in the retina and tectum•Motor output is conveyed via multimodal circuitry in the hindbrain
Dunn et al. characterize the parameters influencing visually evoked escape behavior in larval zebrafish. Via large-scale functional imaging, the authors identify the neural circuits underlying the behavior and provide a mechanistic model that incorporates newly classified neural response types.
Discrete populations of brainstem spinal projection neurons (SPNs) have been shown to exhibit behavior-specific responses during locomotion 1–9, suggesting that separate descending pathways, each ...dedicated to a specific behavior, control locomotion. In an alternative model, a large variety of motor outputs could be generated from different combinations of a small number of basic motor pathways. We examined this possibility by studying the precise role of ventromedially located hindbrain SPNs (vSPNs) in generating turning behaviors. We found that unilateral laser ablation of vSPNs reduces the tail deflection and cycle period specifically during the first undulation cycle of a swim bout, whereas later tail movements are unaffected. This holds true during phototaxic 10, optomotor 11, dark-flash-induced 12, and spontaneous turns 13, suggesting a universal role of these neurons in controlling turning behaviors. Importantly, we found that the ablation not only abolishes turns but also results in a dramatic increase in the number of forward swims, suggesting that these neurons transform forward swims into turns by introducing turning kinematics into a basic motor pattern of symmetric tail undulations. Finally, we show that vSPN activity is direction specific and graded by turning angle. Together, these results provide a clear example of how a specific motor pattern can be transformed into different behavioral events by the graded activation of a small set of SPNs.
•Spinal projection neurons serve as a switch to transform forward swims into turns•Visually induced and spontaneously occurring turns are controlled by the same set of SPNs•Turn-controlling SPN activity is direction specific and graded by turning angle
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.
Brains are notoriously hard to understand, and neuroscientists need all the tools they can get their hands on to have a realistic shot at it. Advances in machine learning are proving instrumental, ...illustrated by their recent use to shed light on navigational strategies implemented by zebrafish brains.
Brains are notoriously hard to understand, and neuroscientists need all the tools they can get their hands on to have a realistic shot at it. Advances in machine learning are proving instrumental, illustrated by their recent use to shed light on navigational strategies implemented by zebrafish brains.
Simultaneous recordings of large populations of neurons in behaving animals allow detailed observation of high-dimensional, complex brain activity. However, experimental approaches often focus on ...singular behavioral paradigms or brain areas. Here, we recorded whole-brain neuronal activity of larval zebrafish presented with a battery of visual stimuli while recording fictive motor output. We identified neurons tuned to each stimulus type and motor output and discovered groups of neurons in the anterior hindbrain that respond to different stimuli eliciting similar behavioral responses. These convergent sensorimotor representations were only weakly correlated to instantaneous motor activity, suggesting that they critically inform, but do not directly generate, behavioral choices. To catalog brain-wide activity beyond explicit sensorimotor processing, we developed an unsupervised clustering technique that organizes neurons into functional groups. These analyses enabled a broad overview of the functional organization of the brain and revealed numerous brain nuclei whose neurons exhibit concerted activity patterns.
•Sensory input drives behavior via distributed circuits in larval zebrafish•Activity from nearly all neurons in the brain was recorded in behaving animals•Convergent representations of diverse visual stimuli inform behavioral choices•Unsupervised clustering reveals patterns of brain-wide functional organization
Chen et al. examine brain-wide functional organization in larval zebrafish under diverse visual stimulus conditions. They systematically characterize neurons related to convergent sensorimotor processing as well as extract concerted brain-wide activity patterns beyond sensorimotor contexts.
Genetically encoded voltage indicators (GEVIs) enable monitoring of neuronal activity at high spatial and temporal resolution. However, the utility of existing GEVIs has been limited by the ...brightness and photostability of fluorescent proteins and rhodopsins. We engineered a GEVI, called Voltron, that uses bright and photostable synthetic dyes instead of protein-based fluorophores, thereby extending the number of neurons imaged simultaneously in vivo by a factor of 10 and enabling imaging for significantly longer durations relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In the mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously over a 15-minute period of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.
Calcium imaging with protein-based indicators
is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than ...electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators
. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.
We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were ...identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.
When a behavior repeatedly fails to achieve its goal, animals often give up and become passive, which can be strategic for preserving energy or regrouping between attempts. It is unknown how the ...brain identifies behavioral failures and mediates this behavioral-state switch. In larval zebrafish swimming in virtual reality, visual feedback can be withheld so that swim attempts fail to trigger expected visual flow. After tens of seconds of such motor futility, animals became passive for similar durations. Whole-brain calcium imaging revealed noradrenergic neurons that responded specifically to failed swim attempts and radial astrocytes whose calcium levels accumulated with increasing numbers of failed attempts. Using cell ablation and optogenetic or chemogenetic activation, we found that noradrenergic neurons progressively activated brainstem radial astrocytes, which then suppressed swimming. Thus, radial astrocytes perform a computation critical for behavior: they accumulate evidence that current actions are ineffective and consequently drive changes in behavioral states.
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•Zebrafish “give up” after swim attempts repeatedly fail to generate movement•Whole-brain imaging reveals noradrenergic neurons that encode swim failures•Radial astrocytes accumulate the noradrenergic failure signal and trigger passivity•Glia accumulate evidence that actions are futile, then switch behavioral state
Whole-brain imaging in virtual-reality-immersed zebrafish reveals that failed swim attempts are detected by noradrenergic neurons, which drive glial cells that accumulate calcium until they trigger the suppression of further futile attempts.
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