Abstract
Genetically-encoded voltage indicators (GEVIs) of the VSFP (voltage-sensitive fluorescent protein) family based on the Ci-VSP (Ciona intestinalis voltage-sensitive phosphatase) voltage sensor domain were demonstrated to report membrane voltage fluctuations of genetically-targeted cells in a number of in-vitro and in-vivo preparations using single photon (1P) fluorescence excitation and wide field image detection [1, 2]. However, in-vivo 1P epi-fluorescence imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon (2P) excitation imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here, we show GEVI two-photon imaging in mouse brain slices and mouse cortex in-vivo expressing VSFP Butterfly 1.2 in cortical layer2/3 pyramidal cells. We show that 2P excitation at 920-940 nm of the mCitrine/mKate2 FRET reporter in Butterfly 1.2 yields an optical voltage signal from neuron membranes in brain slices in response to field stimulation with a time course identically revealed by 1P excitation and with a 2-3 larger ΔR/R value. Two-photon imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3 of Butterfly 1.2-expressing pyramidal cells. In somatosensory barrel cortex we recorded sensory responses to single whisker deflections in anesthetized mice at 30 frames per second in full frame view. The 2P-excited voltage signal was localized within the functional map established by 1P VSFP imaging in the same preparation. Our results demonstrate the feasibility of GEVI-based functional 2P imaging of sensory-evoked electrical activity in mouse cortex.[1] Akemann W, Mutoh H, Perron A, Rossier J, Knopfel T, Nat Methods 7, 2010[2] Akemann W, Mutoh H, Perron A, Park KY, Iwamoto Y, Knopfel T, J Neurophysiol, 2012 Jul 18
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.