Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography

Area of Science:

• Neuroscience
• Cell Biology
• Microscopy Techniques

Background:

• Conventional electron microscopy uses aldehyde fixatives, which alter synaptic morphology and molecular components.
• Understanding synaptic structure is crucial for studying learning and neurological diseases.

Purpose of the Study:

• To investigate synaptic ultrastructure using high-pressure freezing (HPF) as an alternative to aldehyde fixation.
• To capture a more accurate snapshot of living synapses and their molecular organization.

Main Methods:

• High-pressure freezing (HPF) of rat hippocampal CA1 area slices at -173°C.
• Cryosubstitution and embedding for electron microscopy analysis.
• Immunocytochemistry to localize synaptic proteins and cytoskeletal components.

Main Results:

• HPF preserved larger synaptic terminals with less densely packed vesicles compared to aldehyde fixation.
• Filamentous projections linked postsynaptic densities (PSDs) to the spine cytoskeletal meshwork.
• Specific proteins like ProSAP2/Shank3, cortactin, and Ena/VASP were localized to PSD projections, with actin in the underlying cytoskeleton.

Conclusions:

• HPF provides a more realistic view of synaptic ultrastructure by minimizing fixation artifacts.
• This technique reveals novel details about the connection between PSDs and the actin cytoskeleton.
• HPF is a valuable tool for analyzing synaptic structure under near-native conditions.