Nanoscale molecular imaging of presynaptic active zone proteins in cultured hippocampal neurons

Abstract

Neurotransmitter release occurs at a specialized region of the nerve terminal known as the presynaptic active zone, where a large number of synaptic proteins reside. The composition and nanoscale spatial organization of the active zone proteins are crucial determinants of presynaptic functions, and variety in the composition and organization may account for wide diversity of central synapses. Here, we describe stochastic optical reconstruction microscopy (STORM) imaging, a single-molecule localization-based super-resolution imaging, for analyzing nanoscale distributions of active zone proteins in cultured hippocampal neurons. STORM enables multicolor and three-dimensional analysis with remarkable spatial resolution, and thus resolves detailed nanostructures formed by the active zone proteins, such as Munc13-1 supramolecular nanoassemblies which correspond to the quantal synaptic vesicle release sites. In this chapter, we provide the reader with guidelines on how to stain presynaptic active zone proteins, to prepare specimens suitable for STORM imaging, and to perform STORM imaging in a quantitative manner.