Carroll Elizabeth C, Berlin Shai, Levitz Joshua, Kienzler Michael A, Yuan Zhe, Madsen Dorte, Larsen Delmar S, Isacoff Ehud Y
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
Department of Chemistry, University of California, Davis, CA 95616;
Proc Natl Acad Sci U S A. 2015 Feb 17;112(7):E776-85. doi: 10.1073/pnas.1416942112. Epub 2015 Feb 4.
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.
哺乳动物的神经递质门控受体可与光开关连接配体（PTL）结合，以实现光激活或光拮抗作用，同时在神经元突触处保持正常功能。用于离子型和代谢型谷氨酸受体（GluR）的“MAG” PTL基于一种偶氮苯光开关，该光开关通过蓝光或近紫外光最佳地切换到配体状态，而这些波长的光穿透大脑的能力较差。为了便于用近红外光进行深层组织光激活，我们使用非线性光谱测量了两种MAG分子的双光子（2P）激发效率。基于定量表征，我们发现最近设计的第二代PTL，L-MAG0460，在850nm处有一个良好的2P吸收峰，能够有效地对海人藻酸受体GluK2（LiGluR）进行2P激活。我们还用一种新的代谢型谷氨酸受体特异性PTL，D-MAG0460，实现了代谢型受体LimGluR3的2P光激活。利用数字全息术对培养神经元的单个胞体上的照明进行整形，有效地实现了2P光开关。同时进行的Ca(2+)成像以高时间分辨率报告多个细胞中的2P光开关情况。将电生理学或Ca(2+)成像与通过光波前整形进行的2P激活相结合，应该会使第二代PTL控制的受体适用于完整神经回路的研究。
