Abstract 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 nm 3 . 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. 

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

The small engineered luciferase NanoLuc has rapidly become a powerful tool in the fields of biochemistry, chemical biology, and cell biology due to its exceptional brightness and stability. The continuously expanding NanoLuc toolbox has been employed in applications ranging from biosensors to molecular and cellular imaging, and currently includes split complementation variants, engineering techniques for spectral tuning, and bioluminescence resonance energy transfer-based concepts. In this review, we provide an overview of state-of-the-art NanoLuc-based sensors and switches with a focus on the underlying protein engineering approaches. We discuss the advantages and disadvantages of various strategies with respect to sensor sensitivity, modularity, and dynamic range of the sensor and provide a perspective on future strategies and applications.

Engineering with NanoLuc : A playground for the development of bioluminescent protein switches and sensors

Genetically encoded sensors towards imaging cAMP and PKA activity in vivo.

A continued effort in neuroscience aims to understand the way brain circuits consisting of diverse neuronal types generate complex behavior following sensory input. A common feature of vertebrate visual systems is that lower-order and higher-order visual areas are reciprocally connected. Feedforward projections confer visual responsiveness to higher-order visual neurons while feedback projections likely serve to modulate responses of lower-order visual neurons in a context-dependent manner. Optic tectum is the largest first-order visual brain area in zebrafish and is reciprocally connected with the torus longitudinalis (TL), a second-order visual brain area that does not receive retinal input. A functional role for feedback projections from TL to tectum has not been identified. Here we aim to understand how this feedback contributes to visual processing.In this study, we demonstrate that TL feedback projections to tectum drive binocular integration and spatial summation in a defined tectal circuit. We performed genetically targeted, cell type-specific functional imaging in tectal pyramidal neurons (PyrNs) and their two input neuron populations: retinal ganglion cells (RGCs) and neurons in TL. We find that PyrNs encode gradual changes in scene luminance using a complement of three distinct response classes that encode different light intensity ranges. Functional imaging of RGC inputs to tectum suggest that these response classes originate in the retina and RGC input specifies PyrN functional classes. In contrast, TL input serves to endow PyrNs with large, compound receptive fields that span both retinal hemifields.These findings reveal a novel role for the zebrafish TL in driving binocular integration and spatial summation in tectal PyrNs. The neural circuit we describe generates a population of tectal neurons with large receptive fields tailored for detecting changes in the visual scene. 

/pdf/input-from-torus-longitudinalis-drives-binocularity-and-2ys8az07.pdf

Input from torus longitudinalis drives binocularity and spatial summation in zebrafish optic tectum

/pdf/input-from-torus-longitudinalis-drives-binocularity-and-233q0hpr.pdf

The torus longitudinalis (TL) is a midbrain structure unique to ray finned fish. Although previously implicated in orienting behaviors elicited by changes in ambient lighting, the role of TL in visual processing is not well-understood. TL is reciprocally connected to tectum and is the only known source of synaptic input to the stratum marginalis (SM) layer of tectal neuropil. Conversely, tectal pyramidal neurons (PyrNs) are the only identified tectal neuron population that forms a dendrite in SM. In this study we describe a zebrafish gal4 transgenic that labels TL neurons that project to SM. We demonstrate that the axonal TL projection to SM in zebrafish is glutamatergic. Consistent with these axons synapsing directly onto PyrNs, SM-targeted dendrites of PyrNs contain punctate enrichments of the glutamatergic post-synaptic marker protein PSD95. Sparse genetic labeling of individual TL axons and PyrN dendrites enabled quantitative morphometric analysis that revealed (1) large, sparsely branched TL axons in SM and (2) small, densely innervated PyrN dendrites in SM. Together this unique combination of morphologies support a wiring diagram in which TL inputs to PyrNs exhibit a high degree of convergence. We propose that this convergence functions to generate large, compound visual receptive fields in PyrNs. This quantitative anatomical data will instruct future functional studies aimed at identifying the precise contribution of TL-PyrN circuitry to visual behavior.

/pdf/pyramidal-neurons-of-the-zebrafish-tectum-receive-highly-59l3jwi4ts.pdf

Pyramidal Neurons of the Zebrafish Tectum Receive Highly Convergent Input From Torus Longitudinalis.

Genetically encoded fluorescent and luminescent indicators have revolutionized our ability to monitor physiology in real time, but the separate development of new sensors for each of these imaging ...

Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

Brain orexin (hypocretin) neurons are implicated in sleep-wake switching and reward-seeking but their roles in rapid arousal dynamics and reward perception are unclear. Here, cell-specific stimulation, deletion and in vivo recordings revealed strong correlative and causal links between pupil dilation-a quantitative arousal marker-and orexin cell activity. Coding of arousal and reward was distributed across orexin cells, indicating that they specialize in rapid, multiplexed communication of momentary arousal and reward states. 

/pdf/control-and-coding-of-pupil-size-by-hypothalamic-orexin-2z4a6i11.pdf

Alexander L. Tesmer

Papers

Input from torus longitudinalis drives binocularity and spatial summation in zebrafish optic tectum

Pyramidal Neurons of the Zebrafish Tectum Receive Highly Convergent Input From Torus Longitudinalis.

Dual-Mode FRET and BRET Sensors for Detecting cAMP Dynamics.

Input from torus longitudinalis drives binocularity and spatial summation in zebrafish optic tectum

Control and coding of pupil size by hypothalamic orexin neurons