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

Irene P Ayuso-Jimeno1, Paolo Ronchi2, Tianzi Wang1

  • 1Epigenetics & Neurobiology Unit, European Molecular Biology Laboratory (EMBL), Via Ramarini 32, 00015, Monterotondo, RM, Italy.

Scientific Reports
|June 17, 2022
PubMed
Summary
This summary is machine-generated.

Multiplexed labeling using enhanced peroxidase (dAPEX2) and DAB is now compatible with 3D volume electron microscopy. This advance enables mapping genetically defined neural circuits in the mouse brain.

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Microscopy

Background:

  • Enzymatic labeling with peroxidases and 3,3'-diaminobenzidine tetrahydrochloride hydrate (DAB) is crucial for identifying synaptic contacts in electron microscopy (EM).
  • Current methods are limited in multiplexing capabilities and compatibility with large-scale 3D volume EM techniques like Serial Block-Face Electron Microscopy (SBEM) and Focused Ion Beam Electron Microscopy (FIBSEM).

Purpose of the Study:

  • To evaluate the compatibility of multiplexed enhanced peroxidase (dAPEX2)/DAB labeling with 3D volume EM.
  • To demonstrate the application of this technique for mapping long-range neural circuits.

Main Methods:

  • Utilized genetically encoded enhanced peroxidase (dAPEX2) for DAB metabolism.
  • Applied dAPEX2/DAB labeling in conjunction with Focused Ion Beam Electron Microscopy (FIBSEM) and Serial Block-Face Electron Microscopy (SBEM).
  • Mapped synaptic inputs from the anterior cingulate cortex to the periaqueductal gray in mouse brain tissue.

Main Results:

  • Demonstrated successful multiplexed dAPEX2/DAB tagging compatible with both FIBSEM and SBEM.
  • Achieved EM-visible DAB product deposition in thick tissue without detergent treatment.
  • Successfully mapped long-range, genetically identified synaptic inputs in the mouse brain.

Conclusions:

  • Multiplexed dAPEX2/DAB labeling is a viable method for 3D volume EM.
  • This technique facilitates large-scale ultrastructural assessment and circuit mapping of genetically defined cell types.