Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling

Authors

  • Akari Hagiwara,

    Corresponding author
    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
    2. Department of Physiological Sciences, School of Life Science, the Graduate University for Advanced Studies, Okazaki 444-8787, Japan
    Current affiliation:
    1. Biochemistry and Cell Biology Unit, Horizontal Medical Research Organization, Kyoto University, Faculty of Medicine, Kyoto 606-8501, Japan
    • Division of Cerebral Structure, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8787, Japan
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  • Yugo Fukazawa,

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
    2. Department of Physiological Sciences, School of Life Science, the Graduate University for Advanced Studies, Okazaki 444-8787, Japan
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  • Maki Deguchi-Tawarada,

    1. KAN Research Institute, Kyoto 600-8815, Japan
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  • Toshihisa Ohtsuka,

    1. KAN Research Institute, Kyoto 600-8815, Japan
    Current affiliation:
    1. Department of Clinical and Molecular Pathology, Toyama Medical and Pharmaceutical University, Toyama 930-0152, Japan
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  • Ryuichi Shigemoto

    1. Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan
    2. Department of Physiological Sciences, School of Life Science, the Graduate University for Advanced Studies, Okazaki 444-8787, Japan
    3. Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan
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Abstract

Synaptic vesicle release occurs at a specialized membrane domain known as the presynaptic active zone (AZ). Several membrane proteins are involved in the vesicle release processes such as docking, priming, and exocytotic fusion. Cytomatrix at the active zone (CAZ) proteins are structural components of the AZ and are highly concentrated in it. Localization of other release-related proteins including target soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (t-SNARE) proteins, however, has not been well demonstrated in the AZ. Here, we used sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) to analyze quantitatively the distribution of CAZ and t-SNARE proteins in the hippocampal CA3 area. The AZ in replicated membrane was identified by immunolabeling for CAZ proteins (CAZ-associated structural protein [CAST] and Bassoon). Clusters of immunogold particles for these proteins were found on the P-face of presynaptic terminals of the mossy fiber and associational/commissural (A/C) fiber. Co-labeling with CAST revealed distribution of the t-SNARE proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) in the AZ as well as in the extrasynaptic membrane surrounding the AZ (SZ). Quantitative analysis demonstrated that the density of immunoparticles for CAST in the AZ was more than 100 times higher than in the SZ, whereas that for syntaxin and SNAP-25 was not significantly different between the AZ and SZ in both the A/C and mossy fiber terminals. These results support the involvement of the t-SNARE proteins in exocytotic fusion in the AZ and the role of CAST in specialization of the membrane domain for the AZ. J. Comp. Neurol. 489:195–216, 2005. © 2005 Wiley-Liss, Inc.

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