TY - JOUR
T1 - Flash-and-freeze
T2 - Coordinating optogenetic stimulation with rapid freezing to visualize membrane dynamics at synapses with millisecond resolution
AU - Watanabe, Shigeki
N1 - Publisher Copyright:
� 2016 Watanabe.
Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 2016
Y1 - 2016
N2 - Electron microscopy depicts subcellular structures at synapses exquisitely but only captures static images. To visualize membrane dynamics, we have developed a novel technique, called flash-and-freeze, which induces neuronal activity with a flash of light and captures the membrane dynamics by rapid freezing. For characterizing membrane movements during synaptic transmission, a light-sensitive cation channel, channelrhodopsin, is heterologously expressed in mouse hippocampal neurons or in Caenorhabditis elegans motor neurons. A brief pulse of blue light activates channelrhodopsin and induces an action potential, leading to synaptic transmission. Following the light stimulation, neurons are frozen at different time intervals ranging from 10 ms to 20 s. Electron micrographs are then acquired from each time point to visualize the morphological changes. Using this approach, we have characterized a novel form of endocytosis, ultrafast endocytosis, which rapidly removes excess membrane added to the surface during neurotransmission. The flash-and-freeze approach can be adapted to study other cellular phenomena that can be induced by light-sensitive genetic or pharmacological tools.
AB - Electron microscopy depicts subcellular structures at synapses exquisitely but only captures static images. To visualize membrane dynamics, we have developed a novel technique, called flash-and-freeze, which induces neuronal activity with a flash of light and captures the membrane dynamics by rapid freezing. For characterizing membrane movements during synaptic transmission, a light-sensitive cation channel, channelrhodopsin, is heterologously expressed in mouse hippocampal neurons or in Caenorhabditis elegans motor neurons. A brief pulse of blue light activates channelrhodopsin and induces an action potential, leading to synaptic transmission. Following the light stimulation, neurons are frozen at different time intervals ranging from 10 ms to 20 s. Electron micrographs are then acquired from each time point to visualize the morphological changes. Using this approach, we have characterized a novel form of endocytosis, ultrafast endocytosis, which rapidly removes excess membrane added to the surface during neurotransmission. The flash-and-freeze approach can be adapted to study other cellular phenomena that can be induced by light-sensitive genetic or pharmacological tools.
KW - Electron microscopy
KW - Flash-and-freeze
KW - High-pressure freezing
KW - Optogenetics
KW - Synaptic cell biology
KW - Synaptic transmission
KW - Synaptic vesicle exocytosis
KW - Synaptic vesicles
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U2 - 10.3389/fnsyn.2016.00024
DO - 10.3389/fnsyn.2016.00024
M3 - Article
C2 - 27594835
AN - SCOPUS:84993966769
VL - 8
JO - Frontiers in Synaptic Neuroscience
JF - Frontiers in Synaptic Neuroscience
SN - 1663-3563
IS - AUG
M1 - 24
ER -