Open AccessCharacterizing Synaptic Vesicle Proteins Using Synaptosomal Fractions and Cultured Hippocampal Neurons
Author(s) -
DiGiovanni Jerome,
Sun Tao,
Sheng ZuHang
Publication year - 2012
Publication title -
current protocols in neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.307
H-Index - 40
eISSN - 1934-8576
pISSN - 1934-8584
DOI - 10.1002/0471142301.ns0207s59
Subject(s) - synaptic vesicle , hippocampal formation , vesicle , immunogold labelling , biology , neuron , microbiology and biotechnology , synaptic vesicle recycling , neurotransmitter , synaptosome , immunocytochemistry , neuroscience , chemistry , biochemistry , ultrastructure , membrane , central nervous system , anatomy , endocrinology
Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome‐enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre‐embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures. Curr. Protoc. Neurosci . 59:2.7.1‐2.7.22. © 2012 by John Wiley & Sons, Inc.