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Spatially selective photoactivation and functional fluorescence imaging in freely-behaving mice with a fiberscope

le 28 mai 2015 à 11 h
Cathie Ventalon, Institut de Biologie, ENS, Paris

Despite evident advances made in the past few years, understanding the mechanisms governing information integration in the central nervous system remains a challenge. By providing numerous tools for perturbing and recording neural activity, optogenetics has shown great potential to advance these questions. To establish the links between specific spatiotemporal activity patterns and behaviors, optical methods should ideally provide simultaneous access to a group of neurons with single cell resolution in freely behaving animals. However, so far, optogenetic control of neural activity in freely behaving rodents has been performed with illumination volumes larger than 50µm on a side, yielding significant limitations with respect to spatial resolution. In addition, these approaches have yet been coupled with functional imaging. Here, we demonstrate, for the first time to our knowledge, photoactivation with near-cellular resolution in freely behaving mice [1]. Photoactivation patterns are generated with computer-generated holography, and transmitted to the mouse using a fiber bundle coupled to a micro-objective. The optical system also includes a fluorescence imaging pathway to locate cells and record neuronal activity. Imaging is based on intensity modulation of a laser beam which provides easy switching between epifluorescence, structured illumination, and scanless multi-point confocal microscopy. We tested the system for photoactivation and calcium imaging in freely-behaving mice co-expressing GCaMP5-G and ChR2-tdTomato in cerebellar molecular layer interneurons. We demonstrated that the field of view remained stable during our acquisitions and that we could reliably elicit an increase in GCaMP5-G fluorescence signal by targeting a single soma with a 5µm diameter photoactivation spot. In addition, this somatic calcium signal vanished when the photoactivation spot was moved just 20µm away from the soma, thus demonstrating that cells could be photoactivated with near-cellular resolution in freely behaving mice.

[1] V. Szabo*, C. Ventalon*, V. De Sars, J. Bradley, and Valentina Emiliani, “Spatially selective photoactivation with computer generated holography and functional fluorescence imaging in freely behaving mice with a fiberscope”, Neuron, 84 (6), 1157-1169.

 


Lieu(x) :         Amphithéatre Curie, Ecole Polytechnique


Contact :       Emmanuel Beaurepaire
                      emmanuel.beaurepaire at polytechnique.edu


À télécharger :       Séminaire VENTALON.pdf - PDF