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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - IVSTED (In vivo super-resolution imaging of synapses in the hippocampus)

Teaser

Summary

The hippocampus is a deeply embedded region in the mammalian brain that has long been considered the archetypical center for memory formation. Hippocampal neurons process information by integrating a vast number of synaptic inputs via dendritic spines. These postsynaptic structures are highly dynamic and their plasticity is hypothesized to be important structural correlate of the memory trace. However, due to their nanometric size and high density, it is extremely challenging to study the function of dendritic spines under realistic experimental conditions. Indeed, conventional light microscopy fails to properly resolve their fine morphological details, while electron microscopy only provides snapshots from fixed brain sections. Therefore, our view of spine dynamics remains very incomplete, limiting our understanding of the synaptic mechanisms underlying brain physiology and animal behavior.

Leveraging recent developments in optical super-resolution microscopy, adaptive optics and mouse brain surgery techniques, the objective of this project was to establish an innovative approach for nanoscale imaging of hippocampal spines in living mice during memory acquisition and recall. Using this approach, the aim was to perform chronic imaging over several weeks in a cohort of animals to investigate the dynamics of hippocampal spines in vivo with unprecedent spatial resolution, focusing on their turnover and nanoscale morphology during behavioral tests of the capacity of mice to learn and remember things. Specifically, my project focused on tackling the following three main objectives:
(1) Develop adaptive optics-based 3D super-resolution microscopy to improve image resolution and penetration,
(2) Establish chronic in vivo super-resolution imaging to determine spine morphology and turnover over the course of weeks in live animals,
(3) Investigate how spine plasticity correlates with memory performance in the same animal.

Due to my recruitment as a permanent researcher I had to end up this Marie Slodowska Curie action project after only seven months. Therefore, this project is far from being finished. However, in the context of my new position I will largely continue working on the thematic. Indeed, in the context of the IVSTED project I was able to establish the foundation of my project first by implementing the adaptive optics setup necessary to improve image resolution in depth and secondly by successfully establishing the surgery protocol to implant hippocampal cranial window, which is an important milestone for the success of this project.

Work performed

\"During the seven monthsâ€™ duration of this work I laid a firm ground for the project.

First, I learnt a surgery technique called \"\"hippocampal window\"\" recently improved by our collaborators and successfully implemented it in my hosting laboratory. This surgery allows to gain imaging access to the deeply embedded hippocampus, by implanting a metal tube sealed with a coverslip. This represents an important milestone in my project by launching the in vivo part of my work. I then apply our optical super-resolution technique to image the hippocampus in vivo with a unprecedent resolution. Having established this technique, I disseminated it by training PhD students during hands-on training schools (Cajal school program).

Then, I implemented chronic STED microscopy of hippocampal spines in living mice. I obtained in vivo data that clearly illustrate the substantial improvement in image quality using our STED approach compare to regular two photon microscopy, making spine density quantifications much more accurate. Then, I monitored spine turnover over several days. These original data show a substantial rewiring of synapse in the hippocampus over a very short time period, since already 40% of synapse have be replaced after 4 days, raising the question of the existence of a stable population of synapses in this brain region.

In parallel, I implemented an important change in the microscope. Indeed, one of my first objective was to improve the capacity of the device to image in depth in the brain of a living mouse. To that end, I incorporated a special device called Spatial Light Modulator (SLM) which allows to shape the light so as to cancel any distortion induced by the sample when imaging in depth. Using this new device, I calibrated the distortion at a specific depth in brain slice which allows to preserve image quality until 100 microns which represents a 5-fold improvement compare to original configuration. The last step will be to apply this approach to image the brain of an anesthetized mouse.\"

Final results

Due to the short duration of this project, which will however continue during the next years, I did not yet reached all its objective. Nevertheless, I obtained the first in vivo super-resolution images in a deep brain region, which demonstrate the feasibility and the usefulness of such an imaging technique to tackle long standing questions in neuroscience. This open avenue for nanometric investigations of brain structures in physiological conditions and upon a variety of context ranging from behavioral analysis to physio-pathological developments.

Going significantly beyond the state-of-the-art, we expect that in the near future, this project will push the frontier in neuroimaging, shedding new light on the anatomical basis of memory formation and providing a framework to analyze in vivo the cellular mechanisms of memory impairment in neurodegenerative diseases.