Nanoscale organization of synaptic proteins in response to glutamate stimulation

[Speaker] Noriko Koganezawa:1
[Co-author] Tomoaki Shirao:1
1:Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Japan

Dendritic spines are postsynaptic responsive regions of excitatory synapses and play an important role in synaptic transmission and plasticity. Various proteins localize within dendritic spines and some of them are known to regulate spine morphology and function. In particular, the subcellular distribution changes of these proteins are thought to be important factors of synaptic plasticity. In conventional fluorescence microscopic images, however, it was hard to detect at which part of spines these proteins exist, although some studies using electron microscopy indicate distribution of the proteins within a spine. To reveal nanoscale organization of synaptic proteins, we used stochastic optical reconstruction microscopy (STORM) in this study. We first observed localizations of several synaptic proteins such as drebrin, synapsin I and PSD-95 during resting state using primary cultured hippocampal neurons. Drebrin is an actin-binding protein which forms stable F-actin and is highly accumulated within dendritic spines. Synapsin I associates with the synaptic vesicles and presents at presynaptic site. PSD-95 is a scaffold protein and exists at postsynaptic site. In the STORM images, drebrin accumulated in the center of the spine heads and synapsin I localized face to face similar to conventional microscope observation. Both drebrin and PSD-95 localized within the dendritic spines, but it was clear that they had slightly different distribution pattern within a spine. Conventional fluorescence microscopy has shown that NMDA receptor activation induces drebrin exodus from dendritic spines. Thus we further observed localization changes of these proteins after glutamate stimulation by STORM. Drebrin changed its distribution from dendritic spine heads to the dendritic shafts after the glutamate stimulation and tended to be scattered along the dendrite. While synapsin I and PSD-95 did not change their distribution pattern drastically. These results explain the existence of a stable core of F-actin which decorated by drebrin in the central region of the spine head. Drebrin exodus from spine heads after glutamate stimulation indicated loss of stable F-actin and dynamic F-actin is thought to be predominant. Scattered drebrin in dendritic shaft during drebrin exodus confirmed that drebrin exodus occurs not due to drebrin degradation but due to distribution change.
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