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

Abstract

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.

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

“…Preparation and imaging of this sample have been described previously (Wanner et al 2016a, Wanner et al 2016b). Briefly, tissue was stained en bloc with osmium, uranyl acetate and lead aspartate using an established protocol (Deerinck et al, 2010; Tapia et al, 2012) with minor modifications and embedded in Epon resin with silver particles to minimize charging (Wanner et al, 2016a; Wanner et al, 2016b). Multi-tile images were acquired in high vacuum using a scanning electron microscope (QuantaFEG 200; FEI) equipped with an automated ultramicrotome inside the vacuum chamber (3View; Gatan).…”

“…Skeletons of all neurons in the OB were reconstructed previously as described (Wanner et al, 2016a; Wanner et al, 2016b). Briefly, three independent skeletons of each neuron were generated manually from seed points at somata.…”

“…We previously acquired an SBEM image stack of the OB in a zebrafish larva and reconstructed 98% of the neurons in the OB (Wanner et al, 2016a; Wanner et al, 2016b). We now annotated the synaptic connections of all OB neurons to reconstruct the full wiring diagram.…”

“…To achieve this goal we took advantage of the small size of the larval zebrafish brain. We first measured odor responses of neurons in the OB by multiphoton calcium imaging and subsequently reconstructed the synaptic connectivity among all neurons by serial block-face scanning electron microscopy (SBEM) (Denk et al, 2012; Denk and Horstmann, 2004; Wanner et al, 2016a; Wanner et al, 2016b). We found that higher-order features of multisynaptic connectivity specifically suppress the activity of correlated mitral cell ensembles in a stimulus-dependent manner, resulting in a decorrelation and variance normalization.…”

High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation

“…Another approach to mitigate charging is to surround the specimen with conductive silver impregnated epoxy (Wanner et al ., 2016). While this can be effective, it is not applicable to all tissues or with cultured cell monolayers, and it renders the specimen opaque to light, making correlated light and SBEM more difficult.…”