In vivo fast imaging and optogenetic manipulation using genetically-encoded fluorescent indicators and actuators. Serena Bovetti
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1 In vivo fast imaging and optogenetic manipulation using genetically-encoded fluorescent indicators and actuators Serena Bovetti Istituto Italiano di Tecnologia Genova, Italy Bogliasco, June
2 Analyzing structure and function Ramon y Cajal ( ) 2P microscopy (1990-present) From
3 Analyzing structure and function Living cells Small elements High number millions glutamatergic cells just into the mouse neocortex 3D and the overall complexity of NS Light scattering tissue Fast signalling deflection of a light «ray» from its original direction and depend on refractive index of the structures that interact with the ray µm deep ms Non-stationary 3D From
4 From circuits to behaviour: how we do that? Circuits Behaviour How we do that? From
5 From circuits to behaviour: how we do that? Genetic and molecular tools Fluorescent molecules Cre-lox technology Viral vector based tools etc... specification Cell types Functional optical strategies assembly Readout Genes Circuits Optogenetics evolution physiology Probing Behaviour From
6 Outline 1- Optical functional indicators and actuators of cells activity using GCaMPs to monitor cell activity using opsins to manipulate cell activity 2- In vivo functional two-photon imaging: improving the temporal resolution of functional 2P imaging using scanless approach 3- Combining imaging methods with optical manipulation of cell activity 4- Future perspectives
7 Optical functional indicators of cell activity A first necessary step toward elucidating the basic principles underlying brain function is to precisely map the activity of individual cellular elements in space and time in vivo In the last 20 years, the development of multiphoton microscopy in combination with fluorescent activity reporters has provided a valuable tool to reach this goal Voltage indicators Calcium indicators
8 The Calcium Ion as an Indirect Reporter of Neuronal Activity Both neurons and glia display increase calcium concentration in response to neuronal activity One way to measure free cytosolic calcium variations optically is using molecules that change their fluorescence or absorbance properties upon calcium binding Synthetic dyes: OGB Fluo-2 Fluo-4... No labelling of specific cell population Acute loading Short life time Genetically encoded indicators: Camgoroo Pericams GCaMPs Expressed in cell type specific manner Allow chronic imaging
9 The Calcium Ion as an Indirect Reporter of Neuronal Activity Synthetic dyes: OGB Fluo-2 Fluo-4... No labelling of specific cell population Acute loading Short life time Genetically encoded indicators: Camgoroo Pericams GCaMPs Expressed in cell type specific manner Allow chronic imaging Biophysical properties: Affinity Dynamic range Selectivity Kinetics From Bovetti et al., 2014
10 The Calcium Ion as an Indirect Reporter of Neuronal Activity GCaMP structure M13 domain of the myosin light chain Ca2+ Calmodulin cpgfp Transgenic mouse lines Promoter specific expression Thy1-GCaMP6 CAMKII-GCaMP6 Cre-dependent expression (cre-lox technology) Promoter-lox-STOP-lox-GCaMP6 (i.e. CAG) Cre-mouse line (i.e.pv-cre) X Flex- GCaMP6 Adeno-associated virus (AAVs) Small (25 nm), Single-stranded DNA Viral vector delivery Each virus has characteristic tropism (targeting of cells) and spread from injection sites, in some cases via retrograde or anterograde transport of viral particles, which are important to consider when designing experiments. Different serotypes that influence virus tropism Now mix serotypes are available Small capacity for effective packaging (4.7 kb) Both anterograde and retrograde (depending on serotypes) transport Do not integrate into the host genome, remain as an episome (extragenomic circular DNA)
11 The Calcium Ion as an Indirect Reporter of Neuronal Activity GCaMP structure M13 domain of the myosin light chain Ca2+ Calmodulin cpgfp Transgenic mouse lines Promoter specific expression Thy1-GCaMP6 CAMKII-GCaMP6 Promoter specific expression Viral vector delivery AAV1.Syn.GCaMP6f.WPRE.SV40 serotype promoter indicator Cre-dependent expression (cre-lox technology) Promoter-lox-STOP-lox-GCaMP6 (i.e. CAG) Cre-dependent expression (cre-lox technology) AAV1.Syn.flex.GCaMP6f.WPRE.SV40 X Cre mouse line Flox-GCaMP6 WT or Cre mouse line ~10 13 GC/ml (tirtrate!!!) very low inj rate (20-50 nl/min)
12 The Calcium Ion as an Indirect Reporter of Neuronal Activity GCaMP structure M13 domain of the myosin light chain Ca2+ Calmodulin cpgfp Transgenic mouse lines Viral vector delivery Advantages: More homogeneous expression across brain regions Advantages: Higher expression Disadvantages: Low expression Disadvantages: Less homogeneous expression across brain regions
13 Bovetti et al., 2014 The Calcium Ion as an Indirect Reporter of Neuronal Activity AAV1.Syn.GCaMP6f.WPRE.SV40 AAV1.Syn.flexGCaMP6f.WPRE.SV40 Cre-expressing mouse lines WT
14 Optical manipulation of cell activity Optogenetics: combination of genetic and optical methods to cause or inhibit well defined events in specific cells or living tissue and behaving animals (Deisseroth, 2015) 1) Microbial opsins: proteins that directly elicits electrical current across cellular membranes in response to light (transduce photons into electrical current) Extra Intra Chow et al NphR Hyperpolarizating current Deisseroth 2015 Depolarizating current Arch, earch, earch3.0, ArchT NphR, NphR3.0 ChR2, ChETa, C1V1 Beltramo et al., 2013
15 Optical manipulation of cell activity Extra Intra Deisseroth 2015 Biophysical properties Efficiency of light absorption (cross-section) defined in term of extinction coefficient (ε max : how strongly a substance absorbs light at a given wavelength) and quantum efficiency (Φ: the fraction of absorbed photons that are efficacious in driving the relevant conformational change) Conductance and photocurrent Kinetics defined in term of turnover time of the photocycle Mutation at different residues change the biophysical properties of opsins
16 Optical manipulation of cell activity Methods for targeting sufficiently strong and specific opsin gene expression to well-defined cellular elements in the brain Thy1-ChR2 Thy1-eNphR... Transgenic mouse lines Promoter specific expression Promoter specific expression AAVs delivery AAV1.CAG.ChR2.EYFPWPRE.SV40 serotype promoter actuator reporter Cre-dependent expression (cre-lox technology) Promoter-lox-STOP-lox-ChR2 (i.e. CAG) Cre-dependent expression (cre-lox technology) AAV1.EF1.flex.ChR2.mCherry.WPRE.SV40 X Cre mouse line Flox-GCaMP6 WT or Cre mouse line ~10 13 GC/ml (tirtrate!!!) very low inj rate (20-50 nl/min)
17 Optical manipulation of cell activity Methods for targeting sufficiently strong and specific opsin gene expression to well-defined cellular elements in the brain Thy1-ChR2 Thy1-eNphR... Transgenic mouse lines Promoter specific expression Special attention to: AAVs delivery Membrane trafficking Targeting specific cellular compartments Cre-dependent expression (cre-lox technology) Promoter-lox-STOP-lox-ChR2 (i.e. CAG) X Cre mouse line Flox-GCaMP6 Prakash et al., 2012
18 Multiphoton microscopy A first necessary step toward elucidating the basic principles underlying brain function is to precisely map the activity of individual cellular elements in space and time in vivo In the last 20 years, the development of multiphoton microscopy in combination with fluorescent activity reporters has provided a valuable tool to reach this goal Multiphoton microscopy is a powerful technique based on non-linear interactions between photons and matter. The most commonly used multiphoton imaging procedure is the two-photon excitation microscopy.
19 absorption Multiphoton microscopy Multiphoton microscopy is a powerful technique based on non-linear interactions between photons and matter. The most commonly used multiphoton imaging procedure is the two-photon excitation microscopy. Linear microscopy: one photon is adsorbed by a fluorescence molecule and one single fluorescent photon is emitted Non-Linear microscopy: uses «higher order» lightmatter interactions involving multiple photons Excited state 1P fluorescence 2P 2P: rare event in which 2 photons interact with the same molecule at the same time (interval less then s) Maria Goppert-Mayer ( ) Nobel prize 1963 Ground state
20 Two-photon microscopy 2P fluorescence Longer wavelengths = less energy Longer wavelenghts = less subjected to scattering Longer wavelengths = deeper penetration Near-infrared light penetrates deeper into scattering tissue and is generally less phototoxic nm Visible spectral range
21 A two-photon microscope Raster scanning FOV Detection Excitation Bovetti et al., 2014 Pinhole increase peak intensity -concentration in time (pulsed excitation) 2P = 1P 2P
22 Laser scanning two-photon microscopy Laser scanning imaging of GCaMP6-expressing layer 2/3 neurons in the somatosensory cortex of the awake mouse imaging large field of view attaining high spatial resolution discriminating between different cell types Limited temporal resolution From
23 Cell sampling rate (Hz) How to Laser improve scanning to improve two-photon the acquisition microscopy speed Raster scanning i i + 1 Max acquisition frequency = 1/(t d *N + t m *(N-1)) t d : dwell time t m : time to move from point i to i+1 N: total number of points y N - 2 x Random access scanning N - 1 N N - 1 N - 2 y x N Number of cells
24 Development of a structured light microscope for fast imaging in vivo From Grunwald and Bock, 2010 Scanless or parallel illumination Dal Maschio et al., 2010 Dal Maschio et al., 2011 N-1 N-2 Dwell time = Exposure time y x N Max. acquisition frequency = Camera max. frame rate
25 Acknowledgements Tommaso Fellin, PI Claudio Moretti Andrea Antonini Angelo Forli Stefano Zucca Dania Vecchia Francesca Succol Angela De Stasi Marco Brondi Noemi Binini Stefano Varani Paolo Bonifazi Marco Dal Maschio Pasqualina Farisello Giulia D Urso Thanks for your attention
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