title: Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics.
abstract: Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors: Elizabeth C
authors: EC
authors: Carroll
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors: Shai
authors: S
authors: Berlin
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors: Joshua
authors: J
authors: Levitz
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors: Michael A
authors: MA
authors: Kienzler
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors: Zhe
authors: Z
authors: Yuan
authors: Department of Chemistry, University of California, Davis, CA 95616;
authors: Dorte
authors: D
authors: Madsen
authors: Department of Chemistry, University of California, Davis, CA 95616;
authors: Delmar S
authors: DS
authors: Larsen
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720; and Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 ehud@berkeley.edu.
authors: Ehud Y
authors: EY
authors: Isacoff
mesh_terms: D000818:Animals; D001391:Azo Compounds / Q000737:chemistry*; D002478:Cells, Cultured; D007536:Isomerism; D009474:Neurons / Q000378:metabolism; D062308:Optogenetics; D017785:Photons; D051381:Rats; D017207:Rats, Sprague-Dawley; D017470:Receptors, Glutamate / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D013057:Spectrum Analysis / Q000379:methods
chemical_list: D001391:Azo Compounds; D017470:Receptors, Glutamate; C009850:azobenzene
keywords: azobenzene; multiphoton; optogenetics; pharmacology; photoswitch
references: Nat Chem Biol. 2014 Apr;10(4):273-80
references: 24561661
references: Mol Ther. 2011 Jul;19(7):1212-9
references: 21610698
references: Nat Neurosci. 2013 Jul;16(7):816-23
references: 23799474
references: Chem Commun (Camb). 2014 Dec 4;50(93):14613-5
references: 25311049
references: Nat Methods. 2008 Sep;5(9):805-11
references: 19160515
references: Nat Commun. 2012;3:736
references: 22415827
references: Nat Methods. 2005 Dec;2(12):932-40
references: 16299478
references: J Am Chem Soc. 2011 Dec 14;133(49):19684-7
references: 22082305
references: Science. 1990 Apr 6;248(4951):73-6
references: 2321027
references: Proc Natl Acad Sci U S A. 1971 Aug;68(8):1820-3
references: 5288770
references: J Physiol. 2012 Feb 15;590(Pt 4):855-73
references: 22219341
references: Proc Natl Acad Sci U S A. 2007 Jun 26;104(26):10865-70
references: 17578923
references: Nat Neurosci. 2013 Oct;16(10):1499-508
references: 23995068
references: Beilstein J Org Chem. 2012;8:1003-17
references: 23019428
references: Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):11981-6
references: 20543137
references: Biochim Biophys Acta. 2004 Jul 9;1657(2-3):82-104
references: 15238266
references: Biophys J. 2009 May 6;96(9):3611-9
references: 19413966
references: Neuron. 2006 Jun 15;50(6):823-39
references: 16772166
references: ACS Chem Neurosci. 2011 Apr 20;2(4):185-197
references: 21731799
references: Nat Methods. 2005 Dec;2(12):905-9
references: 16299475
references: Nat Methods. 2012 Feb;9(2):159-72
references: 22179551
references: Neuron. 2007 May 24;54(4):535-45
references: 17521567
references: Nat Methods. 2012 Dec;9(12):1171-9
references: 23169303
references: Science. 2011 Sep 30;333(6051):1888-91
references: 21903779
references: Cell Signal. 1997 Dec;9(8):551-73
references: 9429760
references: Annu Rev Cell Dev Biol. 2011;27:731-58
references: 21819234
references: Chem Soc Rev. 2011 Aug;40(8):4422-37
references: 21483974
references: Nat Neurosci. 2013 Apr;16(4):507-16
references: 23455609
references: Front Mol Neurosci. 2013 Mar 21;6:3
references: 23519552
references: Chem Soc Rev. 2012 Mar 7;41(5):1809-25
references: 22008710
references: ACS Chem Neurosci. 2011 Sep 21;2(9):536-43
references: 22860175
references: Neuron. 2012 Jun 21;74(6):1005-14
references: 22726831
references: J Chem Phys. 2009 Dec 28;131(24):244516
references: 20059088
references: J Neurosci. 2006 Jul 19;26(29):7650-8
references: 16855092
references: J Am Chem Soc. 2013 Nov 27;135(47):17683-6
references: 24171511
references: Curr Opin Neurobiol. 2002 Oct;12(5):587-92
references: 12367640
references: J Neurosci Methods. 2009 May 30;180(1):9-21
references: 19427524
references: Proc Natl Acad Sci U S A. 2009 Sep 1;106(35):15025-30
references: 19706471
references: Chemphyschem. 2005 Jun 13;6(6):1121-5
references: 15887191
references: Opt Lett. 1995 Dec 1;20(23):2372
references: 19865223
references: J Neurosci Methods. 2001 Oct 15;111(1):29-37
references: 11574117
references: J Am Chem Soc. 2013 Oct 23;135(42):15948-54
references: 24117060
references: Proc Natl Acad Sci U S A. 2009 Apr 21;106(16):6814-9
references: 19342491
references: Nat Chem Biol. 2006 Jan;2(1):47-52
references: 16408092
references: Neuron. 2001 Sep 27;31(6):903-12
references: 11580892
references: Nat Methods. 2008 Apr;5(4):331-8
references: 18311146
references: Proc Natl Acad Sci U S A. 2013 May 28;110(22):9142-7
references: 23674684
references: Nat Methods. 2010 Oct;7(10):848-54
references: 20852649
references: Nat Methods. 2011 May;8(5):393-9
references: 21527931
references: Biomed Opt Express. 2013 Nov 18;4(12):2869-79
references: 24409387
references: Nature. 1977 Mar 24;266(5600):373-4
references: 859605
references: Prog Brain Res. 2012;196:119-43
references: 22341324