Whole-brain serial-section electron microscopy in larval zebrafish

Abstract

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 neurons 2 -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 techniques 1 , 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.Pioneering studies in invertebrates established that synaptic-resolution wiring diagrams of complete neuronal circuits are valuable tools for relating a nervous system's structure to its function 3-6 . Such resources can be combined with perturbations, activity maps, or behavioural assays to examine how signalling through neuronal networks transforms information from the environment into relevant motor outputs 5-10 . These studies benefited from the small size of the model organisms and stereotypy across individuals, which allow for complete ssEM of an entire individual or mosaicking of data from multiple individuals.Vertebrate model nervous systems, on the other hand, are considerably larger and more variable. Consequently, ssEM of whole vertebrate neuronal circuits requires rapid computerbased technologies for acquiring, storing, and analysing many images from one animal. In many cases, anatomical data must be combined with other experiments on the same individual 11-13 to define the relationship between structure, function, and behaviour. Because verteb...

Zebrafish Differentially Process Color across Visual Space to Match Natural Scenes

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Instructions for Assembling the Early Mammalian Embryo

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Real time large scale in vivo observations reveal intrinsic synchrony, plasticity and growth cone dynamics of midline crossing axons at the ventral floor plate of the zebrafish spinal cord

“…However, current understanding remains to a significant extent based on in vitro data, and it is well known that in vitro experiments may not reflect in vivo processes, only being a projection of what could, and actually often does, occur in vivo. (Andersen, 2005;Denk et al, 2012;Goodhill, 2016;Hildebrand et al, 2017;Mai et al, 2009;Nicol et al, 2011;Rosoff et al, 2004;Yuan et al, 2003).…”