Laprell L , Repak E , Franckevicius V , Hartrampf F , Terhag J , Hollmann M , Sumser M , Rebola N , DiGregorio DA , Trauner D .

ERC_268795 European Research Council, 268795 European Research Council

	Figure 1. Design and synthesis of ATG.(a) Structures of GluAzo, a photochromic agonist of kainate receptors, and ATA, a photochromic agonist of AMPA receptors in their respective trans isoform. (b) Structure and photophysical properties of ATG. The molecule consists of a photoswitchable azobenzene, a triazole and a glutamate moiety. The trans- and cis-configuration of ATG are shown. (c) Synthesis of the azobenzene ATG using click chemistry. (d) Synthesis of the stilbene cis-STG using click chemistry.
	Figure 2. Photopharmacology of ATG.(a) Action spectrum of ATG recorded in layer 2/3 cortical neurons in an acute slice preparation in presence of 200 μM ATG in ACSF. Current amplitude was measured after 5 s light stimulation with the respective wavelength and normalized to the maximal current amplitude at 360 nm. (b) Wavelength screening for τoff kinetics of ATG-mediated currents between 400 and 560 nm light. Best τoff kinetics were achieved at 400–450 nm light. (c) Dose–response relationship of ATG-mediated currents in cortical slice preparations. Concentrations from 1 to 500 μM were tested. The EC50 is 185 μM (black dashed line) and was calculated using the Hill-equation. (d) Current-clamp recording of a layer 2/3 cortical neuron. Irradiation with 370 nm light (purple) induces robust action potential firing that is terminated by irradiation with 420 nm light (blue). (e) Washing in D-AP-5 (40 μM), an NMDA-specific antagonist, blocks the ATG-mediated light-dependent action potential firing. (f) Current–voltage relationships indicative of NMDARs as targets for ATG. Black; current–voltage relationship of puff-applied NMDA (200 μM) currents (n=12 cells). Red; current–voltage relationship of ATG-mediated currents under 370 nm light (n=10 cells). Blue; current–voltage relationship of ATG-mediated currents in the absence of Mg2+ ions (n=10 cells). Error bars indicate s.e.m.
	Figure 3. Dendritic NMDAR currents evoked by rapid laser-mediated photoswitching of ATG.(a) Schematic diagram showing putative sculpting of NMDAR gating by ATG photoswitching. A near-diffraction-limited spot of 375 nm light switches ATG to an active cis-conformation (top) that can activate the NMDAR transiently. When the 375 nm laser light is followed quickly by a brief 405 nm laser pulse focused over a larger volume, ATG is converted to the inactive trans-conformation (bottom), eliminating cis-ATG-mediated current more quickly than via diffusional clearance of cis-ATG. (b) Upper traces show light-evoked NMDAR currents recorded in CA1 pyramidal neurons while bath applying 200 μM ATG, in response to a 500 ms 375 nm laser pulse immediately followed by various durations of 405 nm laser pulses. Lower traces show smaller currents evoked by 405 nm pulses alone. Inset: confocal image of dendrite stimulated in these recordings. Purple dot indicates targeted point of ATG stimulation. Scale bar 3 μm (c) Normalized population averages of NMDAR currents evoked by 375 nm laser pulse (100 ms) alone (red; n=9 cells), or 375nm followed by 405 nm laser pulse (50 ms; magenta; n=9 cells) when locally applying ATG (100 μM) with a patch pipette. Blue trace represents uncaging-evoked NMDAR responses when locally applying MNI-glutamate (100 μM; n=5 cells). Dotted line on the magenta trace in the inset indicates the double exponential decay function. (d) Bar graph shows half-decay of NMDAR currents from cells in c. Error bars indicate s.e.m. *P<0.05 for all three comparisons (Steel Dwass all pairs nonparametric multiple comparison test).
	Figure 4. Comparison of ATG photoswitching responses between wild-type and GluN2A KO animals.(a) Population averages of light-evoked currents from WT CA1 pyramidal cells in response to 375 nm (100 ms) only, 375 nm followed by 405 nm (50 ms), and 405 nm only when locally applying ATG (100 μM) with a patch pipette (n=9 cells). (b) Population averages of photoswitching currents from GluN2A KO animals under same conditions as (a) (n=5 cells). (c) (left) Normalized currents in response to 375–405 nm photoswitching from (a) and (b) and population averages of NMDAR EPSCs in wild-type (n=13 cells) and KO animals (n=10 cells). Traces were aligned on their peaks and electrical artifacts from presynaptic stimulation have been blanked. Right: Bar graph of half-decays. Error bars indicate s.e.m. *P<0.05 and NS indicates comparisons that are not significantly different (Steel Dwass all pairs nonparametric multiple comparison test).
	Figure 5. Localized two-photon activation of ATG.(a) Cis-ATG-mediated current evoked by two-photon illumination (1 ms, 740 nm) in a CA1 pyramidal cell while bath applying 400 μM ATG. (b) 2P-evoked cis-ATG-mediated currents with illumination spot parked at 0.5, 2 and 4 μm away from spine head. Illumination duration was 1 ms, and the wavelength set at 740 nm. (inset) Enlarged view of distance-dependent ATG evoked responses. Box over traces illustrates the time window over which spatial dependence was estimated for isochronal amplitude plots in (c) (Scale bar 2 μm). This was chosen to correspond to the time point at which the largest current reached 75% of its amplitude. (c) Normalized isochronal plots for six cells, with the average in black (half-width half-maximum=2.0 μm, red dotted lines). Error bars indicate s.e.m.
	Figure 6. Calcium imaging using ATG in acute hippocampal slices.(a) Cis-ATG-mediated (200 μM) electrical signals in ACSF (left) and in the presence of 40 μM felodipine (Fel) and 1 μM TTX (right), elicited with 370 nm light and terminated with 420 nm (250 ms light pulse for each wavelength). (b) Calcium transients from responsive cells in the field of view corresponding to cis-ATG-mediated recording presented in (a). Bar graph: quantification of calcium transients (ATG+TTX: n=18 experiments and ATG+TTX+felodipine: n=10 experiments). *P<0.05, Wilcoxon rank-sum test. Error bars indicate s.e.m. (c) Changes in fluorescence (ΔF/F) at different time points of the calcium transient; prior to light stimulation, immediately after illumination and after returning to basal calcium levels.
	Figure 7. Coincidence detection using ATG in layer 2/3 cortical neurons.Coincidence detection of cis-ATG mediated current (200 μM) paired with antidromic stimulation. (a) Antidromic stimulation (black bars) of the postsynaptic cell 10 ms before, during and 10 ms after the light stimulation (purple trace). (b) As in (a), but with 50 ms intervals. (c) As in (a), but with 100 ms intervals. (d) Quantification of coincidence detection. Relative number of spikes compared with condition ZERO, when both stimuli were applied together (n=11 cells). Statistics were calculated using the Wilcoxon rank-sum test (*P<0.05,**P<0.01, ***P<0.001).

Amatrudo, Wavelength-selective one- and two-photon uncaging of GABA. 2014, Pubmed

Amatrudo, Wavelength-selective one- and two-photon uncaging of GABA. 2014, Pubmed
Bagal, Long-term potentiation of exogenous glutamate responses at single dendritic spines. 2005, Pubmed
Banke, Activation of NR1/NR2B NMDA receptors. 2003, Pubmed
Bidoret, Presynaptic NR2A-containing NMDA receptors implement a high-pass filter synaptic plasticity rule. 2009, Pubmed , Xenbase
Bischofberger, Patch-clamp recording from mossy fiber terminals in hippocampal slices. 2006, Pubmed
Busetto, Developmental presence and disappearance of postsynaptically silent synapses on dendritic spines of rat layer 2/3 pyramidal neurons. 2008, Pubmed
Carroll, Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics. 2015, Pubmed
Carter, State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons. 2004, Pubmed
Coan, MK-801 blocks NMDA receptor-mediated synaptic transmission and long term potentiation in rat hippocampal slices. 1987, Pubmed
DiGregorio, Desensitization properties of AMPA receptors at the cerebellar mossy fiber granule cell synapse. 2007, Pubmed
Dingledine, The glutamate receptor ion channels. 1999, Pubmed
Erreger, Subunit-specific gating controls rat NR1/NR2A and NR1/NR2B NMDA channel kinetics and synaptic signalling profiles. 2005, Pubmed
Fehrentz, Optochemical genetics. 2011, Pubmed
Fino, RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines. 2009, Pubmed
Gee, Synthesis and photochemistry of a photolabile precursor of N-methyl-D-aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor. 1999, Pubmed
Gray, Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo. 2011, Pubmed
Hansen, Distinct functional and pharmacological properties of Triheteromeric GluN1/GluN2A/GluN2B NMDA receptors. 2014, Pubmed , Xenbase
Izquierdo-Serra, Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches. 2014, Pubmed
Karakas, Crystal structure of a heterotetrameric NMDA receptor ion channel. 2014, Pubmed
Kramer, New photochemical tools for controlling neuronal activity. 2009, Pubmed
Lee, NMDA receptor structures reveal subunit arrangement and pore architecture. 2014, Pubmed , Xenbase
Lester, Channel kinetics determine the time course of NMDA receptor-mediated synaptic currents. 1990, Pubmed
Maier, Comparative analysis of inhibitory effects of caged ligands for the NMDA receptor. 2005, Pubmed
Makara, Variable dendritic integration in hippocampal CA3 pyramidal neurons. 2013, Pubmed
Matsuzaki, Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. 2001, Pubmed
Nevian, Single spine Ca2+ signals evoked by coincident EPSPs and backpropagating action potentials in spiny stellate cells of layer 4 in the juvenile rat somatosensory barrel cortex. 2004, Pubmed
Nielsen, Modulation of glutamate mobility reveals the mechanism underlying slow-rising AMPAR EPSCs and the diffusion coefficient in the synaptic cleft. 2004, Pubmed
Noguchi, Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites. 2005, Pubmed
Paoletti, NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. 2013, Pubmed
Reiter, A photoswitchable neurotransmitter analogue bound to its receptor. 2013, Pubmed
Rodríguez-Moreno, Presynaptic induction and expression of timing-dependent long-term depression demonstrated by compartment-specific photorelease of a use-dependent NMDA receptor antagonist. 2011, Pubmed
Sobczyk, Activity-dependent plasticity of the NMDA-receptor fractional Ca2+ current. 2007, Pubmed
Stawski, A photochromic agonist of AMPA receptors. 2012, Pubmed
Stroebel, Controlling NMDA receptor subunit composition using ectopic retention signals. 2014, Pubmed , Xenbase
Thompson, Flashy science: controlling neural function with light. 2005, Pubmed
Tovar, Triheteromeric NMDA receptors at hippocampal synapses. 2013, Pubmed
Trigo, Laser photolysis of caged compounds at 405 nm: photochemical advantages, localisation, phototoxicity and methods for calibration. 2009, Pubmed
Volgraf, Reversibly caged glutamate: a photochromic agonist of ionotropic glutamate receptors. 2007, Pubmed