Reversible silencing of endogenous receptors in intact brain tissue using 2-photon pharmacology

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 μm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles. © 2019 National Academy of Sciences. All rights reserved.
ACKNOWLEDGMENTS. We thank Jordi Hernando (Autonomous University of Barcelona) for useful discussions on 2-photon excitation; Pere Català (Utrecht University) for help with GCaMP; Francisco Ciruela (University of Barcelona) for mGlu5-eYFP plasmid; Erin Schuman and Stephan Junek (Max Planck Institute for Brain Research, Frankfurt) for preliminary 2-photon excitation experiments; and Ashraf Muhaisen (University of Barcelona) for help with slicing. This research received funding from European Union Research and Innovation Programme Horizon 2020 [Human Brain Project SGA2 Grant Agreement 785907 (WaveScalES)], European Research ERA-Net SynBio programme (Modulightor project), and financial support from Agency for Management of University and Research Grants/Generalitat de Catalunya (CERCA Programme; 2017-SGR-1442 project), Fonds Européen de Développement Économique et Régional (FEDER) funds, Ministry of Economy and Competitiveness (MINECO)/FEDER (Grant CTQ2016-80066-R), and the Fundaluce foundation. S.P. was supported by an FI fellowship from the Agency for Management of University and Research Grants/Generalitat de Catalunya (2014FI_B2 00160). H.L. was supported by an Institute for Bioengineering of Catalonia Severo Ochoa International PhD Programme fellowship from MINECO. M.B. was supported by a H2020-MSCA-IF Reintegration Grant. K.E.P. receives support from NIH/National Institute of Neurological Disorders and Stroke Grant R01NS099254 and NSF Biophotonics Grant 1604544. E.S. receives support from MINECO (Grant SAF2016-7426).