Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes

Jigar Modi; Howard Prentice; Jang-Yen Wu

doi:10.21769/BioProtoc.2664

Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes

Bio Protoc. 2017 Dec 20;7(24):e2664. doi: 10.21769/BioProtoc.2664.

Authors

Jigar Modi^{1

2}, Howard Prentice^{1

2

3}, Jang-Yen Wu^{1

2

3}

Affiliations

¹ Department of Biomedical Sciences, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, FL, USA.
² Center of Complex Systems and Brain Sciences, Florida Atlantic University, Boca Raton, FL, USA.
³ Program in Integrative Biology, Florida Atlantic University, Boca Raton, FL, USA.

Abstract

In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and activation of the GABA synthesizing enzyme GAD65 (L-glutamic acid decarboxylase with molecular weight of 65 kDa). By examining protein complexes on the membrane of synaptic vesicles obtained from synaptosomal preparations, it was possible to characterize the role of GAD65 in the coupled synthesis and vesicular uptake of GABA (γ-aminobutyric acid) culminating in GABA vesicular release, which contributes in an important way to fine-tuning of GABAergic neurotransmission.

Keywords: GABA; GAD65; Synaptic Vesicle (SV); Synaptosomes.