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Nanoscale Photoactivation and Imaging of Synaptic Spine Dynamics

Final Report Summary - DYNASPINE (Nanoscale Photoactivation and Imaging of Synaptic Spine Dynamics)

Executive summary
This project aimed at increasing our understanding of dendritic spines as specialized signalling compartments that constitute tunable computational units of neurons. It relies on application of established electrophysiological techniques, as well as development and application of complex photonic microscopic techniques to visualize and functionally probe dendritic spines at high resolution.
To undertake this task the fellow, Jan Tønnesen, moved from Denmark to the lab of Valentin Nägerl in Bordeaux, France, where the project was carried out. Jan Tønnesen brought electrophysiological expertise to the project, and acquired solid expertise in microscopic approaches from the host during the project. Additional new and advanced photonic techniques were implemented in the host lab by the fellow.
With minor deviations from the initial milestones the project objectives were met, and novel aspects of the physiology and anatomy of dendritic spines were reported. These findings were published in a high impact peer reviewed journal within the neuroscience field. Additionally, the fellow contributed to reviews and methodological papers during the project, disseminating his knowledge and further advertising the lab and the topic.
The findings brought about during the project have spurred on-going research lines in the host lab, where the fellow is currently still working.

Summary of project context and objectives
The overall aim of this project was to push forward our neurobiological understanding of the relationship between structure and function of individual synaptic spines. Specifically, this project sought to understand the role of the spine neck as a chemical and electrical compartmentalizer of the synapse.
To meet this goal we applied a combination of electrophysiological recordings and superresolution stimulated emission depletion (STED) microscopy. Unlike conventional imaging modalities, such as confocal and 2-photon microscopy techniques, STED microscopy can reliably resolve the dendritic spines and report their structural dynamics in plasticity schemes. To functionally address individual spines 2-photon uncaging of photo-releasable glutamate was applied, allowing stimulation of receptors at single synapse level. In addition, fluorescence recovery after two-photon photobleaching (FRAP) in individual spines was performed. This approach allows a detailed structurefunction analysis of live dendritic spines.
Together, these experimental techniques allow a detailed functional and structural analysis of live spines.

Main results
The experimental data collection proceeded in four phases. First, live dendritic spines were imaged by STED microscopy, and their morphology characterized in detail. Second, spines were imaged by STED microscopy and subjected to two photon FRAP experiments, which allowed morphology to be
related to diffusional properties. Third, spine morphology was observed over time in settings of synaptic plasticity, by potentiating individual spines through two photon glutamate uncaging during STED imaging. Fourth, STED, FRAP and glutamate uncaging were combined in individual spines to provide detailed information about the relation between structural and functional plasticity of live spines.
The results obtained revealed that live spines cover a broad range of sizes and shapes, and that spine shape correlates strongly with diffusional properties of the spine, thereby shaping it as a biochemical compartment. Additionally, the spine neck is a likely modulator of synaptic potentials passing the spine, as estimated from morphological and diffusional data.
A major result was to visualize structural spine neck plasticity in settings of synaptic potentiation, with the neck shortening and widening. These structural changes unexpectedly had different influences on biochemical and electrical signaling, revealing a new layer of complexity in spine physiology.