Abstract
The actin cytoskeleton of dendritic spines plays a key role in morphological aspects of synaptic plasticity. The detailed analysis of the spine structure and dynamics in live neurons, however, has been hampered by the diffraction-limited resolution of conventional fluorescence microscopy. The advent of nanoscopic imaging techniques thus holds great promise for the study of these processes. We implemented a strategy for the visualization of morphological changes of dendritic spines over tens of minutes at a lateral resolution of 25 to 65 nm. We have generated a low-affinity photoconvertible probe, capable of reversibly binding to actin and thus allowing long-term photoactivated localization microscopy of the spine cytoskeleton. Using this approach, we resolve structural parameters of spines and record their long-term dynamics at a temporal resolution below one minute. Furthermore, we have determined changes in the spine morphology in response to pharmacologically induced synaptic activity and quantified the actin redistribution underlying these changes. By combining PALM imaging with quantum dot tracking, we could also simultaneously visualize the cytoskeleton and the spine membrane, allowing us to record complementary information on the morphological changes of the spines at super-resolution.
Highlights
Fluorescence microscopy using genetically encoded fluorescent proteins has greatly advanced our understanding of many functional biological systems over the last decade
The structure of dendritic spines is defined by the F-actin cytoskeleton and can undergo fast dynamic morphological changes that are believed to contribute to the plasticity of synaptic transmission [9]
photoactivated localization microscopy (PALM) imaging of the actin cytoskeleton using a low affinity photoconvertible probe To study the organization of the actin cytoskeleton we designed an actin probe that combines an actin-binding peptide (ABP) sequence [20] and tandem Eos fluorescent protein
Summary
Fluorescence microscopy using genetically encoded fluorescent proteins has greatly advanced our understanding of many functional biological systems over the last decade. Novel super-resolution imaging methods using photoactivatable proteins or photoswitchable fluorophores bypass this limitation and have the potential to revolutionize the experimental scope of light microscopy [1]. While physiological and morphological aspects of synaptic plasticity are under certain conditions independent from one another [10], the enhancement of synaptic transmission by long-term potentiation (LTP) appears to be generally associated with an increase in spine volume [4,11] In line with these observations, the polymerization state and the dynamic properties of the actin cytoskeleton in dendritic spines have been shown to change during synaptic plasticity [12,13]. The goal of our study was to develop a strategy that would enable us to visualize the dynamic changes of the spine morphology for long periods
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