Two-Photon Holographic Stimulation of ReaChR.

Emmanuelle Chaigneau,Hongkui Zeng,Gilberto J Soler-Llavina,Eirini Papagiakoumou,Valentina Emiliani,Anthony Y B Brureau,Marta A Gajowa,Dimitrii Tanese,Emiliano Ronzitti

doi:10.3389/fncel.2016.00234

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.

Highlights

Optogenetics has revolutionized neuroscience by enabling remote activation or inhibition of specific populations of neurons in intact brain preparations through genetically-targeted, light-sensitive channels and pumps (Nagel et al, 2003; Boyden et al, 2005; Adamantidis et al, 2015)
Patterns of increasing light power densities (LPDs) and matching the cell shape (65 ± 13 μm2, n = 4 cells) evoked currents of increasing amplitude (Figure 1B) that saturated [Figure 1C, in Methods see Section Voltage clamp measurements of currents evoked by photo-stimulation of opsins and Equation (7)] at 113 ± 54 pA (n = 4 cells) for a LPD of 0.10 ± 0.03 mW/μm2 (n = 4 cells) after the objective
These results demonstrate that ReaChR is sensitive to 2P stimulation even at low LPDs

Summary

Introduction

Optogenetics has revolutionized neuroscience by enabling remote activation or inhibition of specific populations of neurons in intact brain preparations through genetically-targeted, light-sensitive channels and pumps (Nagel et al, 2003; Boyden et al, 2005; Adamantidis et al, 2015). Recent developments of ad hoc opsins (Mattis et al, 2012; Klapoetke et al, 2014) and innovative illumination approaches (reviewed in Papagiakoumou, 2013; Bovetti and Fellin, 2015) offer the possibility of tackling these challenges. Red-shifted variants of channelrhodopsin, such as C1V1 (Yizhar et al, 2011), ReaChR (Lin et al, 2013; Hooks et al, 2015) or Chrimson (Klapoetke et al, 2014), with spectral peaks near and above 600 nm, enable deeper brain stimulation relative to blue-green shifted opsins. Red-orange light illumination of ReaChR, has permitted in vivo trans cranial optogenetics in deep brain structures (Lin et al, 2013). In depth neuronal stimulation using visible light does not enable cellular resolution

Methods

Results

Conclusion