3D Analysis of Synaptic Ultrastructure in Organotypic Hippocampal Slice Culture by High-Pressure Freezing and Electron Tomography.

ABSTRACT

Transmission electron microscopy serves as a valuable tool for synaptic structure-function analyses aimed at identifying morphological features or modifications associated with specific developmental stages or dysfunctional synaptic states. By utilizing cryo-preparation techniques to minimize the introduction of structural artifacts during sample preparation, and electron tomography to reconstruct the 3D ultrastructural architecture of a synapse, the spatial organization and morphological properties of synaptic organelles and subcompartments can be quantified with unparalleled precision. In this chapter, we present an experimental approach combining organotypic slice culture, high-pressure freezing, automated freeze-substitution, and electron tomography to investigate spatial relationships between synaptic vesicles and active zone release sites in synapses from lethal mouse mutants.