Abstract
Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs.
Highlights
Relating neural circuits to the computations they perform requires mapping patterns of structural and functional connectivity among neurons
Only electron microscopy (EM) provides sufficient resolution to visualize the complete complement of synapses that neurons form and receive
Recombinant horseradish peroxidase (HRP) is enzymatically inactive in the cytosol because it fails to form disulfide bonds or become glycosylated, but this limitation can be overcome by directing the protein to topologically extracellular compartments such as vesicles (Li et al, 2010; Atasoy et al, 2014; Schikorski et al, 2007)
Summary
Relating neural circuits to the computations they perform requires mapping patterns of structural and functional connectivity among neurons. An algorithm pieces together the individual images to produce a reconstruction of the cell This pipeline of steps reduces the time required to map the shapes and connectivity of neurons with electron microscopy by some two orders of magnitude. Our pipeline includes the following series of steps: (a) tagging a specific cell type with a genetically encoded EM tracer, (b) enhancing the electron-density of the stain without compromising ultrastructure of the surrounding tissue, (c) imaging the cell rapidly at relatively low resolution, (d) reimaging selected small volumes at higher resolution to map connectivity and (e) segmenting the cell using a novel algorithm that is reliable, fast and does not require computationally intense pre-training. We call the method ARTEMIS for Assisted Reconstruction Technique for Electron Microscopic Interrogation of Structure
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