Identifying long-range synaptic inputs using genetically encoded labels and volume electron microscopy

Abstract

Enzymes that facilitate the local deposition of electron dense reaction products have been widely used as labels in electron microscopy (EM). Peroxidases, in particular, can efficiently metabolize 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB) to produce precipitates with high contrast under EM following heavy metal staining, and can be genetically encoded to facilitate the labeling of specific cell-types or organelles. Nevertheless, the peroxidase/DAB method has so far not been reported to work in combination with 3D volume EM techniques (e.g. Serial blockface electron microscopy, SBEM; Focused ion beam electron microscopy, FIBSEM) because the surfactant treatment needed for efficient reagent penetration disrupts tissue ultrastructure and because these methods require the deposition of large amounts of heavy metals that can obscure DAB precipitates. However, a recently described peroxidase with enhanced enzymatic activity (dAPEX2) appears to successfully deposit EM-visible DAB products in thick tissue without surfactant treatment. Here we demonstrate that multiplexed dAPEX2/DAB tagging is compatible with both FIBSEM and SBEM volume EM approaches and use them to map long-range genetically identified synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in the mouse brain.

MoBIE: A Fiji plugin for sharing and exploration of multi-modal cloud-hosted big image data

“…Panel (h) shows a sponge choanocyte chamber imaged in EM 16 and zooms in on the spatial interaction of a neuroid cell with several choanocytes, which is also rendered in the 3d viewer. Finally, (j) shows neural tissue imaged in EM from a part of the mouse brain 17 .…”

“…i immunofluorescence assay for SARS-COV2 antibodies 20 , imaged with high-throughput microscopy and shown in the multi-well plate layout recreaded with two nested grid transformations. j EM volume of neural tissue from a mouse brain 26 .…”