| Autor: |
Carroll EC; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;, Berlin S; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;, Levitz J; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;, Kienzler MA; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;, Yuan Z; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;, Madsen D; Department of Chemistry, University of California, Davis, CA 95616;, Larsen DS; Department of Chemistry, University of California, Davis, CA 95616;, Isacoff EY; 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. |
| Abstrakt: |
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. |
