Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

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 -MAG0 ₄₆₀, 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 -MAG0 ₄₆₀. 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. Significance MAGs (maleimide-azobenzene-glutamate) are photoswitches that covalently bind to genetically engineered glutamate receptors (GluRs) and, under the control of light, mimic or block the action of the excitatory neurotransmitter glutamate. However the blue and near-UV light that optimally photoswitch MAGs do not penetrate well into the brain. In this paper, we show how MAGs can instead be photoswitched by two-photon (2P) absorption of near-infrared light, which penetrates deeper into tissue. We demonstrate 2P control of MAG-dependent ionic currents in neurons, and synthesize a new MAG photoswitch to enable 2P activation of a G protein coupled receptor signaling cascade through a metabotropic GluR. These optogenetic tools bring exceptional spatiotemporal resolution and pharmacological specificity to the study of synaptic transmission and plasticity in intact neural circuits.