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High-throughput behavioral phenotyping is critical to genetic or chemical screening approaches. Zebrafish larvae are amenable to high-throughput behavioral screening because of their rapid development, small size, and conserved vertebrate brain architecture. Existing commercial behavioral phenotyping systems are expensive and not easily modified for new assays. Here, we describe a modular, highly adaptable, and low-cost system. Along with detailed assembly and operation instructions, we provide data acquisition software and a robust, parallel analysis pipeline. We validate our approach by analyzing stimulus response profiles in larval zebrafish, confirming prepulse inhibition phenotypes of two previously isolated mutants, and highlighting best practices for growing larvae prior to behavioral testing. Our new design thus allows rapid construction and streamlined operation of many large-scale behavioral setups with minimal resources and fabrication expertise, with broad applications to other aquatic organisms.

Natural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use continuous brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as each mating unfolds in its own sequence of component behaviors, the brain operates similarly between instances of each component, but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed, but change with behavioral dynamics. From the contribution of individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions within their natural context.

SUMMARYMany animals use coordinated limb movements to interact with and navigate through the environment. To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to map synaptic connectivity within a neuronal network that controls limb movements. We present a synapse-resolution EM dataset containing the ventral nerve cord (VNC) of an adult femaleDrosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we reconstructed 507 motor neurons, including all those that control the legs and wings. We show that a specific class of leg sensory neurons directly synapse onto the largest-caliber motor neuron axons on both sides of the body, representing a unique feedback pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM data acquisition more accessible and affordable to the scientific community.

Investigating the dense meshwork of wires and synapses that form neuronal circuits is possible with the high resolution of serial-section electron microscopy (ssEM)1. However, the imaging scale required to comprehensively reconstruct axons and dendrites is more than 10 orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons2—some of which span nearly the entire brain. The difficulties in generating and handling data for relatively large volumes at nanoscale resolution has thus restricted all studies in vertebrates to neuron fragments, thereby hindering investigations of complete circuits. These efforts were transformed by recent advances in computing, sample handling, and imaging techniques1, but examining entire brains at high resolution remains a challenge. Here we present ssEM data for a complete 5.5 days post-fertilisation larval zebrafish brain. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management. The resulting dataset can be analysed to reconstruct neuronal processes, allowing us to, for example, survey all the myelinated axons (the projectome). Further, our reconstructions enabled us to investigate the precise projections of neurons and their contralateral counterparts. In particular, we observed that myelinated axons of reticulospinal and lateral line afferent neurons exhibit remarkable bilateral symmetry. Additionally, we found that fasciculated reticulospinal axons maintain the same neighbour relations throughout the extent of their projections. Furthermore, we use the dataset to set the stage for whole-brain comparisons of structure and function by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. We provide the complete dataset and reconstructions as an open-access resource for neurobiologists and others interested in the ultrastructure of the larval zebrafish.

To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to acquire a synapse-resolution dataset containing the ventral nerve cord (VNC) of an adult female Drosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we studied neuronal networks that control leg and wing movements by reconstructing all 507 motor neurons that control the limbs. We show that a specific class of leg sensory neurons synapses directly onto motor neurons with the largest-caliber axons on both sides of the body, representing a unique pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM more accessible and affordable to the scientific community.

Transmission electron microscopy (TEM) is an essential tool for studying cells and molecules. We present a tape-based, reel-to-reel pipeline that combines automated serial sectioning with automated high-throughput TEM imaging. This acquisition platform provides nanometer-resolution imaging at fast rates for a fraction of the cost of alternative approaches. We demonstrate the utility of this imaging platform for generating datasets of biological tissues with a focus on examining brain circuits.

Circuits in the cerebral cortex consist of thousands of neurons connected by millions of synapses. A precise understanding of these local networks requires relating circuit activity with the underlying network structure. For pyramidal cells in superficial mouse visual cortex (V1), a consensus is emerging that neurons with similar visual response properties excite each other1–5, but the anatomical basis of this recurrent synaptic network is unknown. We combined physiological imaging and large-scale electron microscopy (EM) to study an excitatory network in V1. We found that layer 2/3 neurons organized into subnetworks defined by anatomical connectivity, with more connections within than between groups. More specifically, we found that pyramidal neurons with similar orientation selectivity preferentially formed synapses with each other, despite the fact that axons and dendrites of all orientation selectivities pass near (< 5 μm) each other with roughly equal probability. Therefore, we predict that mechanisms of functionally specific connectivity take place at the length scale of spines. Neurons with similar orientation tuning formed larger synapses, potentially enhancing the net effect of synaptic specificity. With the ability to study thousands of connections in a single circuit, functional connectomics is proving a powerful method to uncover the organizational logic of cortical networks.