Ultrastructural Study of Dopaminergic Axon Terminals

Abstract

The way in which dopamine is released in the striatum is critical to its function, and a wide range of disorders are characterized by altered dopamine signaling, from Parkinson’s disease to an array of neuropsychiatric disorders. To fully comprehend the pathogenic pathways of dopamine disorders, it is essential that we investigate the subcellular modifications which underpin them. Research into the genetic variants of Parkinson’s disease has identified common pathways mediated by the mutated genes and their proteins, many of which have a role in vesicular function. A growing body of evidence suggests that neurodegeneration in Parkinson’s disease starts at the axon terminal and propagates backwards, ultimately resulting in the death of neurons in the substantia nigra pars compacta. Indeed, some of the early symptoms of Parkinson’s disease, preceding cell death, can be attributed to impaired synaptic transmission caused by alterations in proteins expressed at the axon terminal. In order to examine this, electron microscopy can be used, as it allows us to perform detailed analyses of the ultrastructure of dopaminergic terminals. The aim of this chapter is to provide a comprehensive protocol for the study of the ultrastructural features of dopaminergic axon terminals in the striatum, including terminal area, the length of the active zone, the number of vesicles, and vesicle distribution. This protocol provides detailed methods covering the preparation of the tissue, immunohistochemistry using gold labeling, preparation of sections for electron microscopy, examination of ultrathin sections using a transmission electron microscope, acquisition of digital electron microscope images, and detailed analyses of ultrastructure using freely available open-source software.