Three-dimensional visualization of multiple synapses in thick sections using high-voltage electron microscopy in the...

Area of Science:

• Neuroscience
• Microscopy
• Immunohistochemistry

Background:

• Understanding neural circuits requires visualizing synaptic connections at the ultrastructural level.
• Three-dimensional (3-D) analysis using electron microscopy is crucial for this visualization.
• Investigating itch-mediating neurons in the spinal cord necessitates advanced imaging techniques.

Purpose of the Study:

• To present a novel immunohistochemical methodology for effective synaptome analysis.
• To apply high-voltage electron microscopy (HVEM) tomography for 3-D ultrastructural examination of the rat spinal cord.
• To visualize itch-mediating neural networks in the spinal cord.

Main Methods:

• A new sample preparation method involving high-contrast en bloc staining (NCMIR protocol) was employed.
• High-voltage electron microscopy (HVEM) tomography was utilized for 3-D analysis.
• Pre-embedding immunoelectron microscopy was performed on rat spinal cord samples.

Main Results:

• The study successfully visualized numerous itch-mediating synaptic connections and neural networks.
• HVEM tomography allowed direct visualization of ultrastructures in semi-thin sections (~5 μm).
• The applied methodology proved effective for detailed 3-D chemical neuroanatomy.

Conclusions:

• The developed methodology offers a simple and versatile approach for ultrastructural investigations.
• This technique significantly contributes to the study of the central nervous system's neural circuits.
• The findings advance the understanding of spinal cord circuitry involved in itch mediation.