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 sub compartments 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.