Dataset Information

Two-Photon Holographic Stimulation of ReaChR.

ABSTRACT: Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue.

SUBMITTER: Chaigneau E

PROVIDER: S-EPMC5067533 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Publications

Two-Photon Holographic Stimulation of ReaChR.

Chaigneau Emmanuelle E Ronzitti Emiliano E Gajowa Marta A MA Soler-Llavina Gilberto J GJ Tanese Dimitrii D Brureau Anthony Y B AY Papagiakoumou Eirini E Zeng Hongkui H Emiliani Valentina V

Frontiers in cellular neuroscience 20161018

Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of indivi ...[more]

PMID: 27803649

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Project description:Patterned two-photon (2P) photolysis via holographic illumination is a powerful method to investigate neuronal function because of its capability to emulate multiple synaptic inputs in three dimensions (3D) simultaneously. However, like any optical system, holographic projectors have a finite space-bandwidth product that restricts the spatial range of patterned illumination or field-of-view (FOV) for a desired resolution. Such trade-off between holographic FOV and resolution restricts the coverage within a limited domain of the neuron's dendritic tree to perform highly resolved patterned 2P photolysis on individual spines. Here, we integrate a holographic projector into a commercial 2P galvanometer-based 2D scanning microscope with an uncaging unit and extend the accessible holographic FOV by using the galvanometer scanning mirrors to reposition the holographic FOV arbitrarily across the imaging FOV. The projector system utilizes the microscope's built-in imaging functions. Stimulation positions can be selected from within an acquired 3D image stack (the volume-of-interest, VOI) and the holographic projector then generates 3D illumination patterns with multiple uncaging foci. The imaging FOV of our system is 800×800 ?m2 within which a holographic VOI of 70×70×70 ?m3 can be chosen at arbitrary positions and also moved during experiments without moving the sample. We describe the design and alignment protocol as well as the custom software plugin that controls the 3D positioning of stimulation sites. We demonstrate the neurobiological application of the system by simultaneously uncaging glutamate at multiple spines within dendritic domains and consequently observing summation of postsynaptic potentials at the soma, eventually resulting in action potentials. At the same time, it is possible to perform two-photon Ca2+ imaging in 2D in the dendrite and thus to monitor synaptic Ca2+ entry in selected spines and also local regenerative events such as dendritic action potentials.

Dataset Information

Two-Photon Holographic Stimulation of ReaChR.

Publications

Two-Photon Holographic Stimulation of ReaChR.

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