Multiplexed peroxidase-based electron microscopy labeling enables simultaneous visualization of multiple cell types

Abstract

Electron microscopy (EM) is a powerful tool for circuit mapping, but identifying specific cell types in EM datasets remains a major challenge. Here we describe a technique enabling simultaneous visualization of multiple, genetically identified neuronal populations so that synaptic interactions between them can be unequivocally defined. We present 15 AAV constructs and six mouse reporter lines for multiplexed EM labeling in the mammalian nervous system. These reporters feature dAPEX2, which exhibits dramatically improved signal compared to previously described ascorbate peroxidases. By targeting this enhanced peroxidase to different subcellular compartments, multiple orthogonal reporters can be simultaneously visualized and distinguished under EM using a protocol compatible with existing EM pipelines. Proof-of-principle double and triple EM labeling experiments demonstrated synaptic connections between primary afferents, descending cortical inputs, and inhibitory interneurons in the spinal cord dorsal horn. Our multiplexed peroxidase-based EM labeling system should therefore greatly facilitate analysis of connectivity in the nervous system.

Arterial Baroreceptors Sense Blood Pressure through Decorated Aortic Claws

“…End-net terminals in the arterial ligament are beaded and labeled by immunohistochemistry for GFP (Figure S7) in Piezo2-ires-Cre mice; a Piezo2-GFP allele is knocked into the endogenous Piezo2 locus along with the ires-Cre cassette resulting in a functional PIEZO2-GFP fusion protein (Woo et al, 2014). Genetically guided electron microscopy was performed to visualize PIEZO2 fibers labeled with a peroxidase targeted to mitochondria (Zhang et al, 2019). Cre-dependent peroxidase-encoding AAVs were injected into NJP ganglia of Piezo2-ires-Cre mice; the aorta was removed, stained with a peroxidase substrate, and analyzed by electron microscopy.…”

Reconstruction of motor control circuits in adultDrosophilausing automated transmission electron microscopy

“…We fixed and stained the central nervous system of one adult female Drosophila melanogaster (aged 1-2 days post-eclosion, genotype y,w/w[1118]; +; P{VT025718-Gal4}attP2/P{pBI-UASC-3×MYC-sbAPEX2-dlg-S97}18). Following fixation (2% paraformaldehyde/2.5% glutaraldehyde) and dissection (Tobin et al, 2017), the specimen was reacted with diaminobenzadine (DAB) and H2O2 as described previously (Zhang et al, 2019), but an EM-dense label was not observed in this sample. The dissected central nervous system was then post-fixed and stained with 1% osmium tetroxide/1.5% potassium ferrocyanide, followed by 1% thiocarbohydrazide, a subsequent incubation in 2% osmium tetroxide, then 1% uranyl acetate, followed by lead aspartate (Walton, 1979), then dehydrated with a graded ethanol series.…”

Dense neuronal reconstruction through X-ray holographic nano-tomography

“…Based on the similarity between XNH and EM images, we reasoned that cell type-specific labeling strategies previously developed for EM could be adapted for XNH. EM-visible labeling of specific cell types has been achieved using peroxidases that deposit dense precipitates inside the organelles of genetically-defined cell populations (Atasoy et al, 2014;Zhang et al, 2019). We developed a fly reporter line that targets the peroxidase APEX2 (Lam et al, 2015) to the nuclei of GABAergic neurons (see Methods).…”