Automated synapse-level reconstruction of neural circuits in the larval zebrafish brain.

Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm3. We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system.

Whole-body integration of gene expression and single-cell morphology.

Animal bodies are composed of cell types with unique expression programs that implement their distinct locations, shapes, structures, and functions. Based on these properties, cell types assemble into specific tissues and organs. To systematically explore the link between cell-type-specific gene expression and morphology, we registered an expression atlas to a whole-body electron microscopy volume of the nereid Platynereis dumerilii. Automated segmentation of cells and nuclei identifies major cell classes and establishes a link between gene activation, chromatin topography, and nuclear size. Clustering of segmented cells according to gene expression reveals spatially coherent tissues. In the brain, genetically defined groups of neurons match ganglionic nuclei with coherent projections. Besides interneurons, we uncover sensory-neurosecretory cells in the nereid mushroom bodies, which thus qualify as sensory organs. They furthermore resemble the vertebrate telencephalon by molecular anatomy. We provide an integrated browser as a Fiji plugin for remote exploration of all available multimodal datasets.

Dense Circuit Reconstruction to Understand Neuronal Computation: Focus on Zebrafish.

The dense reconstruction of neuronal wiring diagrams from volumetric electron microscopy data has the potential to generate fundamentally new insights into mechanisms of information processing and storage in neuronal circuits. Zebrafish provide unique opportunities for dynamical connectomics approaches that combine reconstructions of wiring diagrams with measurements of neuronal population activity and behavior. Such approaches have the power to reveal higher-order structure in wiring diagrams that cannot be detected by sparse sampling of connectivity and that is essential for neuronal computations. In the brain stem, recurrently connected neuronal modules were identified that can account for slow, low-dimensional dynamics in an integrator circuit. In the spinal cord, connectivity specifies functional differences between premotor interneurons. In the olfactory bulb, tuning-dependent connectivity implements a whitening transformation that is based on the selective suppression of responses to overrepresented stimulus features. These findings illustrate the potential of dynamical connectomics in zebrafish to analyze the circuit mechanisms underlying higher-order neuronal computations.

Whitening of odor representations by the wiring diagram of the olfactory bulb.

Neuronal computations underlying higher brain functions depend on synaptic interactions among specific neurons. A mechanistic understanding of such computations requires wiring diagrams of neuronal networks. In this study, we examined how the olfactory bulb (OB) performs 'whitening', a fundamental computation that decorrelates activity patterns and supports their classification by memory networks. We measured odor-evoked activity in the OB of a zebrafish larva and subsequently reconstructed the complete wiring diagram by volumetric electron microscopy. The resulting functional connectome revealed an over-representation of multisynaptic connectivity motifs that mediate reciprocal inhibition between neurons with similar tuning. This connectivity suppressed redundant responses and was necessary and sufficient to reproduce whitening in simulations. Whitening of odor representations is therefore mediated by higher-order structure in the wiring diagram that is adapted to natural input patterns.

Methods for Mapping Neuronal Activity to Synaptic Connectivity: Lessons From Larval Zebrafish.

For a mechanistic understanding of neuronal circuits in the brain, a detailed description of information flow is necessary. Thereby it is crucial to link neuron function to the underlying circuit structure. Multiphoton calcium imaging is the standard technique to record the activity of hundreds of neurons simultaneously. Similarly, recent advances in high-throughput electron microscopy techniques allow for the reconstruction of synaptic resolution wiring diagrams. These two methods can be combined to study both function and structure in the same specimen. Due to its small size and optical transparency, the larval zebrafish brain is one of the very few vertebrate systems where both, activity and connectivity of all neurons from entire, anatomically defined brain regions, can be analyzed. Here, we describe different methods and the tools required for combining multiphoton microscopy with dense circuit reconstruction from electron microscopy stacks of entire brain regions in the larval zebrafish.

3-dimensional electron microscopic imaging of the zebrafish olfactory bulb and dense reconstruction of neurons.

Large-scale reconstructions of neuronal populations are critical for structural analyses of neuronal cell types and circuits. Dense reconstructions of neurons from image data require ultrastructural resolution throughout large volumes, which can be achieved by automated volumetric electron microscopy (EM) techniques. We used serial block face scanning EM (SBEM) and conductive sample embedding to acquire an image stack from an olfactory bulb (OB) of a zebrafish larva at a voxel resolution of 9.25×9.25×25 nm3. Skeletons of 1,022 neurons, 98% of all neurons in the OB, were reconstructed by manual tracing and efficient error correction procedures. An ergonomic software package, PyKNOSSOS, was created in Python for data browsing, neuron tracing, synapse annotation, and visualization. The reconstructions allow for detailed analyses of morphology, projections and subcellular features of different neuron types. The high density of reconstructions enables geometrical and topological analyses of the OB circuitry. Image data can be accessed and viewed through the neurodata web services (http://www.neurodata.io). Raw data and reconstructions can be visualized in PyKNOSSOS.

Dense EM-based reconstruction of the interglomerular projectome in the zebrafish olfactory bulb.

The dense reconstruction of neuronal circuits from volumetric electron microscopy (EM) data has the potential to uncover fundamental structure-function relationships in the brain. To address bottlenecks in the workflow of this emerging methodology, we developed a procedure for conductive sample embedding and a pipeline for neuron reconstruction. We reconstructed ∼98% of all neurons (>1,000) in the olfactory bulb of a zebrafish larva with high accuracy and annotated all synapses on subsets of neurons representing different types. The organization of the larval olfactory bulb showed marked differences from that of the adult but similarities to that of the insect antennal lobe. Interneurons comprised multiple types but granule cells were rare. Interglomerular projections of interneurons were complex and bidirectional. Projections were not random but biased toward glomerular groups receiving input from common types of sensory neurons. Hence, the interneuron network in the olfactory bulb exhibits a specific topological organization that is governed by glomerular identity.

Analyzing the structure and function of neuronal circuits in zebrafish.

The clever choice of animal models has been instrumental for many breakthrough discoveries in life sciences. One of the outstanding challenges in neuroscience is the in-depth analysis of neuronal circuits to understand how interactions between large numbers of neurons give rise to the computational power of the brain. A promising model organism to address this challenge is the zebrafish, not only because it is cheap, transparent and accessible to sophisticated genetic manipulations but also because it offers unique advantages for quantitative analyses of circuit structure and function. One of the most important advantages of zebrafish is its small brain size, both at larval and adult stages. Small brains enable exhaustive measurements of neuronal activity patterns by optical imaging and facilitate large-scale reconstructions of wiring diagrams by electron microscopic approaches. Such information is important, and probably essential, to obtain mechanistic insights into neuronal computations underlying higher brain functions and dysfunctions. This review provides a brief overview over current methods and motivations for dense reconstructions of neuronal activity and connectivity patterns. It then discusses selective advantages of zebrafish and provides examples how these advantages are exploited to study neuronal computations in the olfactory bulb.

Functional development of the olfactory system in zebrafish.

The olfactory system has become a popular model to study the function of neuronal circuits and the molecular and cellular mechanisms underlying the development of neurons and their connections. An excellent model to combine studies of function and development is the zebrafish because it not only permits sophisticated molecular and genetic analyses of development, but also functional measurements of neuronal activity patterns in the intact brain. This article reviews insights into the functional development of the olfactory system that have been obtained in zebrafish. The focus is on the specification of olfactory sensory neurons (OSNs), the mechanisms controlling odorant receptor expression and OSN identity, the pathfinding of OSN axons towards target glomeruli in the olfactory bulb (OB), the development of glomeruli and functional topographic maps in the OB, and the development of inhibitory interneurons in the OB.