Super-resolution imaging for synapse reveals structure-function correlation between sub-synaptic protein localization and synaptic plasticity

2020 Fiscal Year Research-status Report

• Project/Area Number: 20K22685
• Research Institution: Kyoto University
• Principal Investigator: 劉 品吾 京都大学, 医学研究科, 研究員 (60886563)
• Project Period (FY): 2020-09-11 – 2022-03-31
• Keywords: AMPAR / PSD / synaptic plasticity / memory formation / super-resolution imaging / STORM

Outline of Annual Research Achievements

To elucidate the molecular mechanism underlying the memory formation, we need to know the effect of excitatory stimulation for sub-synaptic localization of synaptic proteins by Stochastic Optical Reconstruction Microscopy (STORM), one of super-resolution microscopy, which has better spatial resolution than conventional fluorescent microscopy. Especially, the nano-scale localization of postsynaptic proteins are critical for synaptic plasticity. Recently, we have reported the synaptic activity dependent organization of nano-scale localization of postsynaptic proteins, which is critical for the regulation of synaptic plasticity. To expand this research, we need to set up dual-color super resolution microscopy.
In this annual year, we have set up the preparation of low-density primary hippocampal neuron culture and successfully observed AMPA-type glutamate receptor with Nikon STORM system. Also we have tested a novel fluorescent agonist against postsynaptic neurotransmitter receptor for STORM imaging, which has better resolution in principle. Next step, we're going to extend our system from single-color to dual-color in order to observe two different proteins and their interaction at the same time.
We will use this technique to observe postsynaptic nanodomains in dissociated cultured neurons to identify those component proteins.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

In this project, the major challenge is to observe two synaptic protein once with super-resolution imaging. Currently, the single-color observation under 647 nm wavelength is well developed. Now we are trying to establish two-color STORM. Also, we are trying specific fluorescent agonist against glutamate receptor for STORM imaging. The critical advantage of using the agonist instead of antibody is the distance from target proteins. In principle, the position of the signal from antibody has some error due to the size of antibody, which sometimes it becomes critical error in super-resolution imaging. The fluorescent agonist is much smaller than antibody that enables us to obtain more fine images.

Strategy for Future Research Activity

Recent development of super-resolution microscopy revealed that the postsynaptic proteins such as transmitter receptors form nano-scaled clusters on postsynaptic density, so called nanodomains. Emerging evidence suggests that synaptic activity-dependent re-organization of postsynaptic nanodomains regulates the efficacy of synaptic transmission and there by learning and memory. However, which protein forms which nanodomain remained totally unclear. So far we have established dual-color STORM imaging for dissociated neuronal cultures. We will use this technique to clarify the components of nanodomains of postsynaptic proteins.

Causes of Carryover

会計処理の問題により端数が発生してしまった。