ARTICLE https://doi.org/10.1038/s41467-019-08796-9 OPEN Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation Gisela Cabré1, Aida Garrido-Charles2, Miquel Moreno1, Miquel Bosch2, Montserrat Porta-de-la-Riva3, Michael Krieg3, Marta Gascón-Moya1, Núria Camarero2, Ricard Gelabert1, José M. Lluch 1, Félix Busqué1, Jordi Hernando1, Pau Gorostiza 2,4,5 & Ramon Alibés 1 1234567890():,; Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photo- isomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on the- oretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These com- pounds provide optimized and sustained two-photon neuronal stimulation both in light- scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions. 1 Departament de Química, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès 08193, Spain. 2 Institut de Bioenginyeria de Catalunya (IBEC), Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain. 3 Institut de Ciències Fotòniques (ICFO), The Barcelona Institute of Science and Technology (BIST), Castelldefels, Barcelona 08860, Spain. 4 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain. 5 Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Zaragoza 50018, Spain. These authors contributed equally: G. Cabré, A. Garrido-Charles. Correspondence and requests for materials should be addressed to P.G. (email: pau@icrea.cat) or to R.A. (email: ramon.alibes@uab.cat) NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 A zobenzene photoswitches1 are at the core of most recently a developed strategies to manipulate biological functions with light2–4, which among other systems, enable remote control of cell receptors and channels5–7. Toward these applica- tions, much effort is being devoted to optimize the light-induced S S S S response of azobenzenes (e.g. long-wavelength operation8–13 or hν1 modulation of cis state thermal stability9,13). A crucial aspect that must ineluctably be addressed to unleash hν2 or Δ the full potential of azobenzene photoswitches is multiphoton excitation with near-infrared radiation (NIR, ~700–1400 nm)14,15, which enables three-dimensional (3D) sub-micrometric resolu- S Thiol-maleimide conjugate Azobenzene Glutamate tion16, deeper penetration into tissue with lower photodamage17, and patterned illumination18,19. However, in contrast to the pro- b H N H N gress made with optogenetics20,21, efficient multiphoton operation of azobenzenes still remains a challenge, mainly due to the low O N N O N N O two-photon (2P) absorption cross sections (σ2) of most of these N N compounds under NIR light excitation22,23. H MAG MAG2P This is the case of MAG, the first-generation azobenzene-based MAG0 MAG460 photoswitchable tethered ligand (PTL) used for the preparation of Large cis thermal stability Electronic asymmetry light-gated glutamate receptors (LiGluR, Fig. 1a and Supple- but electronic symmetry but low cis thermal stability mentary Fig. 1)24,25. Upon conjugation to a cysteine residue + genetically engineered in kainate-type ionotropic glutamate H3N COOH c receptor GluK2, MAG trans-cis photoisomerization allows optical control of ion channel opening and closing in LiGluR24,25, a behavior profusely applied to the study of neurotransmission O O COOH in vitro and in vivo26–30. While effective light-gating of LiGluR is R NH N achieved via regular one-photon (1P) absorption of ultraviolet- O N H visible (UV-vis) radiation24–30, multiphoton operation with NIR O N light is preferred to stimulate selected cells located deep into N N slow tissues at high spatiotemporal resolution31,32. Indeed, 2P O H MAG 2P (R = H) switching at ~750–900 nm was recently demonstrated for LiGluR slow MAG 2P_F (R = F) after functionalization with MAG33 and its analogous ligand Electronic asymmetry and large cis thermal stability MAG0 (Supplementary Fig. 1)34; however, rather limited responses were obtained owing to the very low 2P absorption Fig. 1 Strategy toward optimized azobenzene photoswitches for the two- cross section of the symmetrically substituted azobenzene core of photon (2P) excitation of light-gated glutamate receptors (LiGluR). these compounds (σ2 = 10 GM for trans-MAG0 at 820 nm34, a Operating mode of MAG-type photoswitchable tethered ligands (PTLs) Fig. 1b). on LiGluR, which are composed of three covalently tethered units: a Two different strategies have been explored to enhance the 2P glutamate ligand, an azobenzene core, and a maleimide group that binds to activity of MAG-type PTLs for multiphoton LiGluR operation. a cysteine residue genetically engineered in the receptor. Ultraviolet-visible On one hand, sensitized photoswitching with NIR light was (one-photon; 1P) or near-infrared (2P) light excitation induces glutamate attempted by tethering a 2P-absorbing antenna to the ligand33,35, recognition and channel opening via trans → cis isomerization, which results which however compromised its biological activity due to in ion flow across the membrane. This process is reverted by illumination decreased solubility in water, low conjugation efficiency to with visible light excitation (1P) or thermal back-isomerization of the cis cysteine-tagged GluK2, and/or reduced affinity toward the state of the switch. b Structures of the azobenzene cores of MAG, MAG0, receptor-binding site33,35. On the other hand, electronically MAG2P, and MAG460 PTLs proposed for the photoswitching of LiGluR asymmetric azobenzenes were proposed to intrinsically increase under 1P and 2P excitation conditions. c Structures of PTLs MAGslow 2P and their 2P absorption cross sections22,23,36–38. For MAG-type PTLs MAGslow 2P F and other related compounds, this concept was examined by introducing a strong electron-donating amino group in the fourth thermal stability of its cis state. Importantly, this strategy could be position of their azobenzene core (e.g. in MAG2p and MAG460, expanded to other azobenzene-based photoswitches for tailoring Fig. 1b and Supplementary Fig. 1)33,34,39. A notable increase in 2P their response under multiphoton excitation. absorption was observed in these cases (σ2 = 80 GM for trans- MAG460 at 850 nm34), though at the expense of dramatically decreasing the thermal stability of the cis isomer of the photo- Results switch down to the sub-second timescale. As such, this prevented Design and synthesis of high 2P-responsive MAG-type PTLs. large photoresponses to be obtained for the 2P operation of Although the most efficient manner to increase the 2P absorption LiGluR with MAG2p and MAG460, since rapid thermal relaxation cross section of azobenzenes lies in the push-pull substitution of of the cis state of the switch impeded building up a large popu- their aromatic core22,23, the introduction of strong mesomeric lation of the open state of the ion channel33,34. electron-donating (EDG), and/or electron-withdrawing (EWG) Here we report the computationally based rational design and groups concomitantly accelerates their cis → trans thermal back- preparation of MAG-type photoswitches displaying both high 2P isomerization in the dark (e.g. with EDG = 4-NR2 and EWG = 4- biological activity with NIR light and large cis isomer thermal NO2)1. A compromise must therefore be met to obtain azo lifetime (τcis). Our approach toward this goal relies on the accu- derivatives with both large σ2 and τcis values. In order to rationally rate selection of the substitution pattern of the azoaromatic core devise these compounds, we computed the 2P absorption cross of the system, which should allow for electronic asymmetry (i.e. section and the cis state thermal stability for a series of model enhanced σ2 in the NIR region) without compromising the azoaromatic photochromes using the time-dependent density 2 NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 ARTICLE Table 1 2P absorption cross sections and cis thermal dependence on the electronic asymmetry of their azo core: stabilities of model azo compounds in water selective introduction of o-fluoro and o-nitro EWGs in one of the azobenzene aryl rings of trans-Azo2–Azo4 led to a noteworthy increase in σ2 with respect to o-unsubstituted trans-Azo1. On the Azo compounda σ2,trans (GM)b ΔE zcistrans (kJ mol−1)c contrary, electronic symmetrization of the azobenzene chromo- AzoMAG phore in trans-Azo5 bearing four o-fluoro substituents inhibited R = 4-NHCOMe 0d 91.2 (25.5 min)f R′ = 4′-NHCOMe this effect and even resulted in a slight decrease of the 2P AzoMAG2p absorption cross section with respect to trans-AzoMAG2p. More R = 4-NHCOMe 56e 67.7 (118 ms)f importantly, the enhancement in σ2,trans observed for Azo1–Azo3 R′ = 4′-NMe2 was not found to detrimentally affect the stability of their cis Azo1 isomer and rather large τcis values were predicted for these R = 4-NHCOMe 58 97.2 (4.9 h) compounds in water, in contrast to the behavior observed for R′ = 4′-CONH2 AzoMAG2p in cis-MAG2p/cis-MAG460 and cis-Azo4 bearing a Azo2 nitro group. This clearly demonstrates the advantages of designing R = 4-NHCOMe 69 98.1 (7.0 h) electronically asymmetric azoaromatic photochromes with weak R1′ = 4′-CONH2 mesomeric EDG and EWG as well as strong inductive EWG, R2′ = 2′-F Azo3 which emerge as ideal candidates for the preparation of R = 4-NHCOMe 82 101.0 (22.6 h) azobenzene-based switches with high 2P activity for biological R1′ = 4′-CONH2 applications. R2′ = 2′,4′-F Because of the optimal σ2,trans and τcis values computed for Azo4 Azo1–Azo3, the 2P absorption properties of the cis isomers of R = 4-NHCOMe 112 46.0 (18.6 μs) these compounds were theoretically predicted (Supplementary R1′ = 4′-CONH2 Tables 1 and 2), since they also affect the efficiency of the R2′ = 2′-NO2 photoisomerization process of azobenzenes. On one hand, larger Azo5 σ2,trans/σ2,cis ratios were calculated for Azo1–Azo3 with respect to R = 4-NHCOMe 51 112.0 (80 days) AzoMAG and AzoMAG2p for their 2P-allowed S0 → S2 transition. R1′ = 4′-CONH2 R2 = R2′ = 2′,4′-F On the other hand, different excitation energies were found for both isomers of Azo1–Azo3 as to favor selective 2P excitation of Calculations performed at the CAM-B3LYP/6–31G(d) level and accounting for solvent (water) their trans states, although this behavior was observed to decrease effects with a self-consistent PCM continuum method 2P two-photon, PCM polarizable continuum model with the number of fluorine substituents introduced. This, in aStructures shown in Supplementary Fig. 2 b2P absorption cross section for the S → S transition of the trans isomer combination with their high σ2,trans and τcis values and assuming 0 2 cIn all the cases, the lowest-energy barrier height for the thermal cis → trans isomerization was similar trans-cis and cis-trans photoisomerization quantum found to correspond to an inversion mechanism. In parentheses τcis values estimated at 298 K yields34, should result in a large 2P activity for trans-Azo1– from Eyring equation are shown dσ = 10 GM34 for trans-MAG containing an AzoMAG core 2 0 Azo3 in comparison to the trans-azoaromatic cores of eσ = 80 GM34 for trans-MAG 2 460 containing an Azo MAG2p core fEstimated from the experimental τ values reported for MAG25 and MAG 33 at room MAG/MAG0 and MAG2P/MAG460. temperature using Eyring equation cis 2P Encouraged by our theoretical results on trans-Azo1–Azo3, two MAG-type PTLs were designed (MAGslow 2P and MAGslow2P F , Fig. 1c). To favor synthetic accessibility, selective excitation of the functional theory (TDDFT, Table 1, Supplementary Fig. 2, and trans isomer, as well as structural resemblance with MAG and, as Supplementary Tables 1 and 2). The azobenzene cores of such, replication of its light-gated control of LiGluR, we: (a) took MAG/MAG0 (AzoMAG, R = R′ = 4-NHCOMe) and MAG2P/ the azo chromophores of Azo1 and Azo2 as models for the MAG460 (AzoMAG2p, R = 4-NHCOMe, R′ = 4′-NMe2) were preparation of MAGslow slow taken as reference systems in these calculations, while several 2P and MAG2P F , and (b) employed similar alternative azo compounds (Azo1–Azo3) were explored on the linkers to those used in MAG to tether the maleimide and basis of two main design principles: (a) a push-pull substitution glutamate units to the azobenzene core. The preparation of MAGslow 2P (R = H) and MAG2P F (R = F) slow pattern to enhance σ2 with respect to MAG and MAG0; and (b) the use of weak mesomeric EDG (R = 4-NHCOMe) and EWG was achieved via a linear sequence where glutamate and (R′ = 4′-CONH2) as well as a strong inductive EWG (R′ = 2′-F maleimide functions were sequentially introduced into the and 2′,4′-F) to increase τcis relative to MAG2P and MAG460. For azoaromatic photochrome of choice (Fig. 2)24,25,33,35,41. In this comparison purposes, two other model cases were considered: (a) case, however, the azobenzene cores of MAGslow 2P and MAG2P F slow an azo group alternatively bearing a strong mesomeric EWG were not commercially available and they had to be previously substituent (Azo4, R′ = 2′-NO2); and (b) an azo core with strong synthesized by diazotization of aminobenzoic acids 4a (R = H) inductive EWGs on both aryl rings (Azo5, R = R′ = 2,4-F), a and 4b (R = F) followed by coupling with sodium phenylamino- substitution pattern that enables long-wavelength 1P isomeriza- methanesulfonate 1, which was readily available from aniline and tion of azobenzenes9 and was recently reported to allow for 2P the formaldehyde sodium bisulfite adduct42. Posterior removal of operation with NIR light in biological samples40. the amino protecting group delivered amino acids 5a and 5b in As shown in Table 1 and Supplementary Tables 1 and 2, null 2P moderate yields. Then, the sequence continued by joining the absorption was predicted for both the S0 → S1 and S0 → S2 monoprotected ethyldiamino tether to 5a and 5b using the transitions of trans-AzoMAG, which is in good agreement with the carbodiimide coupling reagent N-ethyl-N′-(3-dimethyldiamino- behavior expected for purely centrosymmetric azobenzenes22,23 propyl)-carbodiimide HCl along with 1-hydroxybenzotriazole and the minimal σ2,trans value experimentally determined for hydrate and diisopropylethylamine as a base. Subsequent acid MAG034. By contrast, a major σ2 value was computed for the removal of the tert-butyl carbamate protection and introduction S0 → S2 band of electronically asymmetric trans-AzoMAG2p, of previously described glutamate derivative 235 using the same which is consistent with that measured for trans-MAG46034. coupling conditions afforded intermediates 6a and 6b in 47 and Interestingly, larger 2P absorption cross sections were calculated 49%, respectively, for the three steps. The next reaction between for model compounds trans-Azo1–Azo4, which showed a clear these intermediates and the freshly prepared acid chloride of NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 O NHCH2SO3–Na+ HO O N O NHBoc O tBuO2C CO2tBu OH 1 2 3 H2N H2N COOH R R (a,b) N N (c, d, e) N N (f) H R N OH NH NH2 O O O 4a (R = H) 5a, 5b 6a, 6b 4b (R = F) NHBoc t BuO2C CO2t Bu O O N O N O O O HN HN R R (g) N N N N H H N N NH NH O O O O slow 7a, 7b MAG2P (R = H) NHBoc NH3+ slow MAG2P_F (R = F) t BuO2C CO2t Bu HO2C CO2H Fig. 2 Synthesis of MAGslow slow 2P and MAG2P F . Reagents and conditions: (a) 5.5 M HCl, NaNO2; (b) (i) 1, 0.86 M NaOAc; (ii) 1 M NaOH; (iii) 5.5 M HCl (38%, over the two steps, for 5a, 47% for 5b); (c) tert-butyl (2-aminoethyl)carbamate, N-ethyl-N′-(3-dimethyldiaminopropyl)-carbodiimide HCl (EDCI), 1- hydroxybenzotriazole hydrate (HOBt), diisopropylethylamine (DIPEA), THF; (d) 37% HCl, MeOH; (e) 2, EDCI, HOBt, DIPEA, THF (47%, over the three steps, for 6a, 49% for 6b); (f) (i) 3, ClCOCOCl, CH2Cl2, DMF; (ii) DIPEA, THF (86% for 7a, 84% for 7b); (g) trifluoroacetic acid (TFA), CH2Cl2 (80% for MAGslow slow 2P F , quantitative yield for MAG2P F ) maleimide derivative 343 furnished compounds 7a and 7b, which trans → cis photoisomerization, as confirmed by 1H nuclear feature all the envisioned functional fragments of the final magnetic resonance (NMR) (Supplementary Figs 3-4). High photoswitches. Finally, acid removal of all the protecting groups trans → cis photoisomerization quantum yields and cis-enriched of the glutamate moiety delivered the target compounds MAGslow 2P photostationary states were determined for both MAGslow 2P and and MAGslow2P F as their monotrifluroacetate salts in good yields. MAGslow2P F at these irradiation conditions (Φ trans → cis ~ 0.15 and % Figure 3a shows the 1P absorption spectra of trans-MAGslow 2P cisPSS ~ 70% in aqueous buffer), which were similar to those and trans-MAGslow 2P F in aqueous buffer, which are compared to measured for their azoaromatic cores and MAG (Supplementary those of trans-MAG and trans-MAG2p. As predicted by TDDFT Table 3). UV-vis absorption spectroscopy and 1H NMR also calculations (Supplementary Table 2), very similar absorption revealed efficient 1P cis → trans photoisomerization of MAGslow 2P signals were registered for the azoaromatic cores of trans-MAG, and MAGslow 2P F upon excitation of their nπ* band at 473 nm trans-MAGslow slow 2P , and trans-MAG2P F , which are typical for (Φcis→trans ~ 0.26 and %transPSS ~ 85% in aqueous buffer), in azobenzene-type switches in the absence of strong mesomeric analogy to the behavior registered for their azobenzene units and EDG and EWG1: they show an intense absorption band at λmax ~ MAG (Supplementary Fig. 5 and Supplementary Table 4). In 360 nm corresponding to the allowed 1P S0 → S2 transition (ππ* addition, thermal back-isomerization of cis-MAGslow 2P and cis- band), and a broad shoulder at λ ~ 400–500 nm arising from the MAGslow was also observed at room temperature, which occurred 2P F forbidden 1P S0 → S1 transition (nπ* band). This is in contrast in the time span of tens of minutes in aqueous media (τcis ~ 10 min with the absorption spectrum of trans-MAG2p bearing an amino- in 99% PBS:1% DMSO, Supplementary Fig. 6 and Supplementary substituted azo core, the ππ* band of which notably red-shifts and Table 5). Importantly, this demonstrates that the azoaromatic cores overlaps with the nπ* band33. selected for both MAGslow and MAGslow 2P F possess long-lived cis 2P and trans-MAG2P F ππ* band Upon excitation of trans-MAGslow slow 2P at 365 nm in either organic (dimethylsulfoxide (DMSO)) or aqueous isomers, as anticipated by our theoretical calculations. Therefore, (99% phosphate-buffered solution (PBS):1% DMSO) media, spectral these PTLs fairly reproduce the large τcis value of MAG and clearly changes were observed in absorption that were consistent with 1P surpass the thermal stability of cis-MAG2p and cis-MAG460 despite 4 NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 ARTICLE a c 1.0 1.0 MAG MAG MAG2sPlow MAG2sPlow Photocurrent (arb. u.) 0.8 0.8 slow slow MAG2P_F MAG2P_F 0.6 0.6 Abs MAG2P MAG2P 0.4 0.4 0.2 0.2 0.0 0.0 300 400 500 600 300 400 500 600 Wavelength (nm) Wavelength (nm) b d 1.2 1000 MAG MAG2sPlow 0.8 100 slow MAG2P_F cis (s) ΔF/F 0.4 10 1 0.0 0.1 0.0 slow MAG MAG2sPlow MAG2P_F MAG2P 300 350 400 450 Wavelength (nm) Fig. 3 Photochemical and physiological characterization of MAGslow slow 2P and MAG2P F under one-photon (1P) stimulation. a Normalized absorption spectra of trans-MAG, trans-MAGslow 2P , trans-MAG slow 2P F , and trans-MAG 2P in 99% phosphate-buffered solution (PBS):1% dimethylsulfoxide (DMSO). b Thermal lifetimes of cis-MAG, cis-MAGslow2P , cis-MAG2P F , and cis-MAG2P at room temperature in 99% PBS:1% DMSO. Errors from the monoexponentials fits to slow obtain τcis are shown. c Normalized 1P action spectra recorded using whole-cell patch-clamp in human embryonic kidney 293 (HEK293) cells expressing GluK2-L439C after conjugation to MAG, MAGslow 2P , MAG2P F , and MAG2P (n = 5, 7, 3, and 8 biologically independent cells, respectively). Errors are slow standard error of the mean (SEM). d Normalized 1P action spectra recorded using calcium imaging in HEK293 cells co-expressing GluK2-L439C and GCaMP6s after conjugation to MAG, MAGslow 2P , and MAG2P F (n = 33, 20, and 25 biologically independent cells, respectively). Errors are SEM. In c and slow d wavelength-dependent photoresponses were normalized to the maximum signal along the spectral range measured for each cell before averaging over different cells. Source data for c and d are provided as a source Data file the push-pull substitution pattern of their azoaromatic photo- similar spectral response was registered for MAGslow slow 2P , MAG2P F , chromes (Fig. 3b). and MAG, which generated maximal electrophysiological signals In view of the optimal photochemical behavior established for when irradiating LiGluR-expressing cells at ~375 nm. By contrast, MAGslow2P and MAGslow 2P F , we next explored their capacity to a broader red-shifted action spectrum was measured for MAG2P photocontrol LiGluR channels in living cells under 1P stimula- peaking at ~425 nm. In addition, further electrophysiological tion. Thus, GluK2 receptors bearing a cysteine residue at position measurements were conducted to demonstrate that the photo- L439C (GluK2-L439C) were expressed in human embryonic switches MAGslow slow 2P and MAG2P F : (a) preserve their long thermal kidney 293 (HEK293) cells and incubated with the azobenzene- cis lifetimes after tethering to GluK2 (~10 min, Supplementary based compound of choice for selective conjugation (MAGslow 2P , Fig. 8) as well as the fast channel opening and closing times MAGslow2P F , MAG, and MAG 2P ). (<0.5 s, Supplementary Fig. 9 and Supplementary Table 6) In a first set of experiments, the photoinduced operation of the previously reported for MAG24,25; and (b) do not inhibit the resulting LiGluRs was evaluated using whole-cell patch-clamp, a native physiological activity of the receptor, which retains their technique that allows measuring the currents elicited across the intrinsic response to free glutamate (Supplementary Fig. 10) and cell membrane when modulating ion fluxes via channel opening rapid desensitization kinetics (Supplementary Fig. 11) after and closing24,25. Large and repetitive electrophysiological signals photoswitch conjugation. were recorded in all the cases upon trans → cis photoisomeriza- Taking advantage of the Ca2+ permeability of LiGluR channels, tion of the PTLs (i.e. LiGluR channel opening) with UV and their light-gated operation under 1P stimulation was further violet light (MAGslow slow 2P , MAG2P F , and MAG) or broadband visible quantified by means of calcium imaging, using GCaMP6s co- light (MAG2P). Cell basal current levels could be next recovered expressed with GluK2-L439C in HEK293 cells. GCaMP6s is a by reverting back this process (i.e. LiGluR channel closing) upon genetically encoded intracellular fluorescent calcium indicator illumination with blue and green radiation (MAGslow slow that undergoes a large increase in emission upon calcium ion 2P , MAG2P F , and MAG) or thermally in the dark (MAG2P; Supplementary complexation44. In this case, we focused on MAGslow 2P -, Fig. 7). By scanning the excitation wavelength used to induce MAGslow2P F -, and MAG-tethered LiGluRs, since the low thermal LiGluR channel opening, the action spectra could be measured stability of cis-MAG2P is reported to lead to poor photoinduced for each of the photoswitchable compounds under analysis calcium imaging signals33. In contrast, the large τcis values of (Fig. 3c). As expected from their photochemical properties, a very MAGslow slow 2P , MAG2P F , and MAG yielded intense, reversible, and NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 reproducible light-triggered fluorescent responses in GCaMP6s- a b ΔF/F 1.0 ΔF/F 0.75 expressing cells upon repetitive illumination with sequential slow pulses of UV-violet and green radiation (Supplementary Fig. 12). MAG MAG2P_F Similar action spectra were again measured for these PTLs using 120 s calcium imaging (Fig. 3d), and the maximal responses obtained 120 s for the three compounds at 360 nm were nearly equivalent (Supplementary Fig. 13). This, together with the whole-cell patch- clamp measurements conducted, proves that the 1P biological activity of MAG in LiGluRs is preserved for MAGslow 2P and MAGslow2P F photoswitchable ligands bearing electronically asym- MAG2sPlow metric azoaromatic cores and slightly different structures. 2P stimulation in cultured cells. NIR-induced 2P operation of LiGluR with PTLs MAGslow slow 2P and MAG2P F was studied by cal- cium imaging, since this enabled all-optical control and mon- itoring of the light-gated ion channels. Experiments were conducted in a confocal fluorescence microscope equipped with slow MAG2P_F both continuous-wave visible lasers and a femtosecond pulsed Ti: Sapphire laser. This allowed for sequential and independent 1P and 2P stimulation of cells by raster-scanning the focused laser beam of choice over the sample while detecting the fluorescence signal of the calcium ion indicator for the whole field of view. To evaluate the 2P physiological activity of MAGslow 2P and 2P F , we first undertook experiments on HEK293 cells MAGslow expressing both GluK2-L439C and R-GECO145, a red genetically Fig. 4 One-photon (1P) and two-photon (2P) stimulation of MAG, encoded fluorescent Ca2+ probe. Figure 4a depicts calcium MAGslow slow 2P , and MAG2P F in cultured cells. a Individual (thin lines) and imaging fluorescence responses measured for these cells after average (thick lines) calcium imaging fluorescence traces recorded for conjugation with MAG, MAGslow slow 2P , and MAG2P F (see also human embryonic kidney 293 (HEK293) cells co-expressing GluK2-L439C Supplementary Movies 1-2). For comparison, all the cells were and R-GECO1 after conjugation to MAG (n = 16 biologically independent subjected to two consecutive cycles of 1P LiGluR photostimula- 2P (n = 14 biologically independent cells), and MAG2P F (n = cells), MAGslow slow tion with violet light (405 nm) followed by two cycles of 2P 34 biologically independent cells). The bands around average traces plot excitation with NIR radiation (780 nm). After every stimulation, the corresponding SEM. Both 1P (violet, 405 nm, power density = 0.37 LiGluR was deactivated by 1P absorption of green light (514 nm). mW μm−2) and 2P excitation scans (red, 780 nm, power density = 2.8 In averaged recordings over n > 10 cells in the field of view, no or mW μm−2) were applied to open LiGluR channels and trigger calcium- very low signals were observed for MAG-tethered LiGluR upon induced R-GECO1 fluorescence enhancement, while 1P excitation scans irradiation with NIR light, which highlights the poor efficiency of (green, 514 nm, power density = 0.35 mW μm−2) were applied to revert 2P trans → cis photoisomerization for this ligand, as already back the process. b Repetitive 2P-induced calcium imaging fluorescence reported33,34. In agreement with our theoretical calculations, a responses recorded in five different HEK293 cells co-expressing GluK2- remarkable increase in 2P responses was observed when replacing L439C and R-GECO1 after conjugation to MAGslow 2P F . Source data for a are MAG with MAGslow 2P and MAGslow 2P F bearing push-pull azoaro- provided as a source Data file matic cores. In fact, 1P- and 2P-induced calcium imaging responses of similar intensities were measured with MAGslow 2P F , stimulation of LiGluR at the spectral maximum (780 nm) and for which the highest σ2 value was anticipated owing to the high averaging over a large number of cells (n > 25), 3.5- and 6-fold electronic asymmetry of its trans-azobenzene photochrome. increases in 2P/1P intensity ratio were measured for MAGslow 2P Notably, such efficient 2P stimulation of LiGluR upon conjuga- and MAGslow 2P F relative to MAG, respectively (Fig. 5b). Even more tion to MAGslow2P F was found to be reproducible, repetitive, and to importantly, 2P stimulation of MAGslow slow 2P - and MAG2P F -tethered occur with minimal photodegradation, since negligible variation LiGluR was observed for all the cells analyzed, while no in calcium imaging response was observed after four consecutive multiphoton response could be measured for ~30% of the light-gating cycles for different individual cells (Fig. 4b). GluK2-L439C-expressing cells conjugated with MAG under the By comparing the 1P- and 2P-induced calcium imaging same illumination conditions (Fig. 5c). Overall, these results responses recorded on the same cells (2P/1P ratio), a detailed indicate that MAGslow 2P and, especially, MAGslow2P F are very assessment of the high multiphoton physiological activity of advantageous photoswitches to efficiently and reliably control MAGslow slow 2P and MAG2P F was performed. First, we determined the LiGluR in neurotransmission studies under 2P excitation with 2P action spectra of these compounds and MAG when NIR radiation. conjugated to HEK293 cells co-expressing GluK2-L439C and R- GECO1, which showed a similar spectral distribution (Fig. 5a). In all the cases, rather broad 2P action spectra were found with 2P stimulation in hippocampal organotypic slices. In view of maxima at ~780 nm, which reasonably agrees with twice the the superior 2P stimulation performance observed for MAGslow 2P F wavelength of the 1P absorption band of the 2P allowed S0 → S2 in GluK2-L439C-expressing HEK cells, we explored the use of transition of the trans-azobenzene core of these compounds this PTL to the multiphoton control with NIR light of neuronal (~360 nm). However, much higher 2P/1P ratios were obtained for cells embedded in their physiological environment. In this way we MAGslow 2P and MAGslow 2P F compared to MAG at equivalent could not only assess the efficiency of MAGslow 2P F in neurons with excitation conditions. In particular, when considering 2P mature synapses naturally containing all endogenous glutamate 6 NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 ARTICLE a As depicted in Fig. 6b, d, slices incubated with MAG showed MAG 1.2 robust photoresponses during 1P stimulation (405 nm), but no or MAGs2Plow minimal photoresponses when applying 2P stimulation (780 nm). (ΔF/F)2P/(ΔF/F)1P@405 MAGs2Plo_wF On the contrary, slices incubated with MAGslow 2P F did not only 0.8 show clear photoresponses upon illumination at 405 nm, but comparable light-induced signals were also recorded for the same cells by excitation at 780 nm, thus indicating very similar 1P 0.4 and 2P photoswitching efficacies (Fig. 6c, e). In all the cases, the photoresponses observed upon stimulation at 405 and/or 780 nm were completely inhibited under green light illumination 0.0 (514 nm), which demonstrated the long τcis of both MAG and 720 760 800 840 MAGslow 2P F switches (see also Supplementary Movies 3-4). Wavelength (nm) To compare the efficacy of LiGluR photocontrol in slices conjugated with MAG or MAGslow 2P F , we determined both the b 1.0 c 100 calcium-induced fluorescence enhancement responses after 1P and 2P stimulation (Fig. 6f) and the 2P/1P response ratio (ΔF/F )2P@780 /(ΔF/F)1P@405 0.8 80 (Fig. 6g). Clearly, MAG could efficiently activate LiGluR and thus induce neuronal activity under 1P excitation at 405 nm, but not % reliability 0.6 60 upon 2P stimulation at 780 nm. In contrast, MAGslow 2P F stimula- tion reliably activated neurons using both 1P and 2P excitation 0.4 40 with violet and NIR light, respectively, and similar 1P and 2P signals were indeed measured at our experimental conditions. In 0.2 20 addition, the 2P-induced control of neuronal activity with MAGslow 2P F was found to be robust and reproducible in many 0.0 0 different cells (n = 6 cells) located at distinct depths of the brain tissue (0–100 μm) from hippocampal slices cultured for different 2P l ow 2P l ow 2P l ow 2P l ow AG AG _F _F s s s s M M AG AG AG AG days (8–15 days in vitro). Importantly, cells expressing RCaMP2 M M M M but not LiGluR-eGFP did not respond to MAG or MAGslow 2P F Fig. 5 Average two-photon (2P) activity of MAG, MAGslow slow 2P , and MAG2P F slow slow photostimulation (Supplementary Fig. 14). in cultured cells. a 2P action spectra of MAG, MAG2P , and MAG2P F after conjugation to GluK2-L439C-expressing human embryonic kidney 293 (HEK293) cells. Fluorescence calcium responses were measured using R- 2P stimulation in vivo in Caenorhabditis elegans. We further GECO1. Before averaging over different cells, the 2P responses of each cell tested the ability of MAGslow 2P F to control neuronal activity in vivo were normalized with respect to the 1P response at 405 nm (MAG: 740, 760, 780, 800, and 820 nm; n = 28, 33, 40, 7, and 12 biologically using 2P excitation. For that purpose, we used Caenorhabditis independent cells, respectively; MAGslow elegans as a model of choice to analyze neuronal circuits. The 2P : 740, 760, 780, 800, and 820 nm; n = 20, 9, 12, 16, and 17 biologically independent cells, respectively; and morphology and function of its 302 neurons are characterized in detail, and allow scrutinizing sensory and motor circuits, among 2P F : 720, 740, 760, 780, 800, 820, and 840 nm; n = 14, 17, 18, 86, MAGslow 15, 23, and 17 biologically independent cells, respectively). Errors are SEM. others. Figure 7 summarizes the results of all-optical experiments b Ratio between the 2P and 1P responses of MAG, MAGslow slow with GluK2-L439C-mCherry and GCaMP6s fluorescent calcium 2P , and MAG2P F for the same cells excited at 780 and 405 nm, respectively. Errors are SEM. reporter co-expressed in touch receptor neurons (TRNs). From c Reliability of the 2P calcium imaging response elicited in GluK2-L439C- the six TRNs, which tile the receptive field of the animal in expressing HEK293 cells after conjugation to MAG, MAGslow anterior/posterior and left/right, we specifically focused on the 2P , and single pair PLML/R, which is located near the tail of the nema- 2P F (n = 72, 25, and 86 biologically independent cells, respectively). MAGslow Reliability is expressed as the percentage of transfected cells showing tode (Fig. 7a). Like in other neurons within the nervous system, measurable 2P stimulation signals. Source data for a and b are provided as pairs of TRNs can be selectively stimulated in the anterior and a source Data file posterior part of the animal using spatial light patterning, but differential unilateral activation is difficult, which poses a hurdle to photomanipulate their activity at high resolution using opto- receptors but also test the ultimate advantages of 2P stimulation. genetics47. This is especially relevant when neurons are over- With this aim, we prepared organotypic slice cultures from lapping along the optical axis, as shown in Fig. 7b (compare neonatal rat hippocampi and biolistically transfected them with confocal projection top view, and side view of the confocal sec- GluK2-L439C-eGFP and RCaMP246. This allowed the cells tions) or are closely packed as in the head. In such cases, 2P expressing LiGluR within the slices to be localized by the green excitation provides a unique advantage over 1P excitation to fluorescence of enhanced green fluorescent protein (eGFP), while activate cells with 10 μm axial plane selectivity48. simultaneously recording their activity after 1P or 2P stimulation Upon expression of GluK2-L439C-mCherry in TRNs, we by monitoring the red fluorescence of the calcium ion probe observed localization of mCherry on the plasma membrane and RCaMP2 (Fig. 6a). Transfected hippocampal slices were finally in vesicles that are transported along the sensory neurite incubated with MAG or MAGslow 2P F and their photoresponses after (Supplementary Movie 5). After delivery of MAGslow 2P F to living consecutive 1P and 2P stimulation were measured and compared. animals (see details in the Methods section), calcium activity was Neither Concanavalin A nor toxins were used in these experi- monitored in the posterior TRNs (PLM) expressing GluK2- ments to inhibit GluK2 desensitization upon prolonged binding L439C-mCherry and GCaMP6s using a confocal fluorescence to the glutamate unit of trans-MAG and trans-MAGslow 2P F . Thus, microscope. Clear photoresponses to 2P excitation were observed our measurements on brain slices truly reported on intact neu- in every animal responding to 1P excitation (n = 5 neurons from ronal gating and connectivity. four different individuals, a 2P/1P excitation efficacy of 100%, NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 a Fig. 6 Two-photon (2P) Ca2+ photoresponses in rat hippocampal RCaMP2 LiGluR-eGFP Merge organotypic slices expressing GluK2-L439C-eGFP and RCaMP2. a Microphotograph of a neuron expressing both RCaMP2 (red) and GluK2- L439C-eGFP (green) (scale bar = 20 μm). b, c Real time traces of a single- cell neuronal activity of slices incubated with b MAG or c MAGslow 2P F . d, e Average one-photon (1P) and 2P responses of neurons incubated with d MAG (n = 3 biologically independent cells) or e MAGslow 2P F (n = 6 biologically independent cells). In b–e 1P stimulation was performed at 405 nm (purple bar, 0.81 mW μm−2) and 514 nm (green bar, 0.35 mW μm−2), and 2P stimulation at 780 nm (red bar, 2.8 mW μm−2). f, g Quantification of photoresponses in slices incubated with MAG (black bars, n = 5 b biologically independent cells) and MAGslow 2P F (red bars, n = 6 biologically ΔF/F 0.2 independent cells): f fluorescence enhancement; g ratio between the 2P and 1P responses of MAG and MAGslow 2P F for the same cells. Error bars are 60 s SEM. Source data for d–g are provided as a source Data file activity of single, individual neurons with high efficacy and selectivity. c Discussion Genetically targeted and pharmacologically selective49 synthetic ΔF/F 0.2 photoswitches offer great potential to dissect neuronal circuits based respectively on specific promoters and endogenous recep- 60 s tors. However, the efficiency of azobenzene-based photoswitch- able ligands is ultimately limited by the cis isomer population that can be achieved upon irradiation, which is determined by dif- ferent experimental (excitation intensity and wavelength) and photochemical parameters (trans and cis absorptivities and iso- merization quantum yields, cis state thermal lifetime). Among them, the 2P activity of long τcis azobenzene switches is mainly d ΔF/F 0.2 controlled by the σ2 values for both isomers at the excitation conditions, since they normally show relatively similar iso- 30 s merization quantum yields (e.g. Φtrans→cis = 0.18 and Φcis→trans = 0.30 for MAG in 99% PBS:1% DMSO, Supplementary Table 3). In the case of MAG and MAG0 under NIR illumination, σ2,trans is low and, worse still, even smaller than σ2,cis, which strongly dis- e favors 2P-induced trans → cis isomerization34. This leads to ΔF/F 0.2 photostationary states with very low cis content and, as such, poor 2P LiGluR responses with NIR light even at high excitation 30 s intensities (~20 mW μm−2)33,34. As discussed above, compounds MAGslow slow 2P and, especially, MAG2P F overcome this drawback by introducing rationally designed azobenzene cores with both large σ2,trans and τcis values, which allow for robust and reliable 2P g signals at milder, more cell-compatible excitation conditions (2.8 f 1.2 mW μm−2) that are comparable to 1P responses. (ΔF/F)2P@780/(ΔF/F))1P@405 1P 2P 1.0 1.0 As for MAG2P and MAG460 biological photoactivity, it is mainly governed by a different factor: the millisecond cis state 0.8 0.8 lifetime of both switches in physiological media33,34. Although this enables single-wavelength, fast neuronal stimulation, it dra- ΔF/F 0.6 1P 0.6 matically limits the extent of the photostationary state reached 0.4 0.4 even when using high excitation intensities. As a result, MAG2P 0.2 0.2 and MAG460 also lead to smaller 2P responses with NIR 2P light than under 1P stimulation (~10–40%) despite their 0 0 enhanced σ2,trans values with respect to MAG and MAG033,34. MAG MAGs2lPo_wF MAG MAGs2lPo_wF This, together with their low cis state lifetimes, is a severe con- straint for the 2P stimulation of calcium-evoked photoresponses with MAG2P and MAG460. On the contrary, the higher stability Figs. 7c, e, Supplementary Movies 6–7), whereas control-treated of MAGslow slow 2P and MAG2P F cis isomers enables sustained receptor animals (vehicle) only showed a partial fluorescence reduction activation, which combined with their optimized 2P excitability due to bleaching of mCherry and GCaMP6s (n = 5 neurons from results in larger photoresponse amplitudes and allows the four different individuals, Fig. 7d). No signs of toxicity were manipulation of calcium-regulated processes with NIR light. observed after compound injection, during recovery and imaging. Thus, while very small R-GECO1 calcium responses were Thus, the rationally designed 2P excitation properties of described for LiGluR-expressing HeLa cells under 2P stimulation MAGslow 2P F can also be used in vivo to photomanipulate neuronal of MAG460 (ΔF/F < 0.1)34, we obtained herein calcium-induced 8 NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 ARTICLE a Dorsal ALMR PMV PLMR ALML AVM PLML Ventral b Top Section c GCaMP6s e MAGs2lPo_wF mCherry 1 1P 2P 0.8 0.6 ΔF/F 0.5 0.4 ΔF/F 0.2 1P 2P d Vehicle 15 s 0 –0.2 –0.4 MAGs2lPo_wF Vehicle Fig. 7 In vivo calcium induced photoresponses by two-photon (2P) stimulation of MAGslow 2P F in C. elegans. a Schematics of C. elegans in which touch receptor neurons (TRNs) are depicted. Squared region is magnified in b. b Microphotograph of an animal expressing LiGluR-mCherry (red) and GCaMP6s (green) (scale bar = 5 μm). Top and lateral section view of TRN from the tail. c, d Average traces of one-photon (1P)- and 2P-induced photoactivation of TRNs in 2P F (n = 5 and 6 cells from four different animals experiments for 1P and 2P traces, respectively) or d with vehicle (n = 5 and 6 animals treated with c MAGslow cells from four different animals experiments for 1P and 2P traces, respectively). Continuous line trace indicates GCaMP6s fluorescence signal and dashed trace mCherry fluorescence. In c and d 2P stimulation was performed at 780 nm (red bar, 2.8 mW mm−2) and 1P stimulation at 405 nm (purple bar, 15 µW mm−2) and 514 nm (green bar, 1.21 µW mm−2). e Quantification of photoresponses (fluorescence enhancement) in animals injected with MAGslow 2P F (red bars, n = 5 cells from four different animals experiments) and vehicle (black bars, n = 5 cells from four different animals experiments). Error bars are SEM. Source data for c–e are provided as a source Data file fluorescence enhancement values higher than 1 for the same MAGslow 2P F cause receptor photoactivation, channel opening, and indicator in HEK cells upon excitation of MAGslow 2P F -tethered closing by desensitization, which limits the cytotoxic effects of LiGluR with NIR light. calcium influx. The stability of cis isomers also allows shortening Other advantages derive from the use of MAGslow 2P and illumination pulses and reducing phototoxicity. The repetitive MAGslow 2P F over MAG2P and MAG460 for the 2P stimulation of photoresponses that we obtained in slices and in vivo are in LiGluR. Technically, cis isomer stability allows maintaining the agreement with the low toxicity of our compounds. These robust glutamate moiety bound to the receptor in the dark, which best calcium responses mediated by desensitizing receptors could be mimics the presence of a high neurotransmitter concentration in related to triggering of intracellular processes50 and/or changes in extracellular medium during presynaptic release. This approach is synaptic receptor mobility51. These processes are involved in in contrast to the use of Concanavalin A to block receptor neuronal plasticity and are currently under investigation. desensitization thus keeping the channel open (MAG2P)33, and to Photocontrol of endogenous neuronal receptors52 and intra- mutation K456A used in GluK2 to quicken receptor recovery cellular presynaptic proteins53 has been recently shown in C. from desensitization34, which results in higher and longer-lasting elegans using 1P excitation. Our 2P excitation experiments in vivo currents during illumination of a cis-unstable photoswitch with MAGslow 2P F conjugated to GluK2-L439C take advantage of (MAG460). Such sustained calcium influx facilitates photo- genetic manipulation for demonstration purposes (including the response detection in imaging experiments but could be toxic for expression of fluorescent calcium reporters), but the robust the neurons, and is not physiological. In our case, we used photoresponses obtained (Fig. 7, Supplementary Movies 6-7) Concanavalin A in order to quantify steady-state currents in suggest that introducing our ortho-fluorosubstituted azobenzene recordings with cell lines (Figs. 3–5), and to obtain clear cores in freely diffusible photoswitches would also allow photo- responses in the first in vivo studies (Fig. 7), but physiological controlling endogenous neuronal receptors and signaling proteins desensitization was always preserved in all the experiments with with high efficacy and spatial confinement. 2P excitation is cur- rat brain slices (Fig. 6). Likely, the cis isomers of MAGslow2P and rently the only method that allows photostimulation with axial NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 plane selectivity at micrometer resolution48 and thus has an GAUSSIAN09 program64. For energy barrier calculations of thermal cis-trans advantage over 1P excitation to activate individual neurons in isomerization, we investigated both the rotation and inversion mechanisms. In the case of asymmetric azobenzenes, the two feasible paths leading to inversion were clustered 3D structures like ganglia and brain tissue. Thus, pho- analyzed. toswitches that combine high 2P excitation efficacy and phar- macological selectivity will be an invaluable tool to investigate Synthesis. A detailed description of the synthesis of MAGslow slow 2P and MAG2P F is intact neuronal circuits and subcellular signaling pathways, and a given in the Supplementary Methods. powerful complement to optogenetic manipulation techniques54. Thus, we have shown that 2P-optimized azobenzene photo- Photochemical characterization. Trans-cis isomerization of MAGslow slow 2P , MAG2P F , switches are an important complement to 2P-enabled caged and their separated azobenzene photochromes in solution was investigated by: (i) ligands like MNI-glutamate49 or optogenetic tools like C1V148 1H NMR for the elucidation of the photostationary state mixtures in organic and ChR2-H134R55, but they have advantages of their own. solvents; and (ii) steady-state UV-vis absorption spectroscopy for trans-cis pho- toisomerization in aqueous media and slow cis-trans thermal back-isomerization Tethered MAG derivatives allow reversible activation of gluta- processes. mate receptors without the need of perfusing high concentrations of caged glutamate compounds and the disadvantages of gluta- Cell culture. Tsa201 cells were purchased from the European Collection of mate and byproduct spillover upon uncaging. Moreover, gluta- Authenticated Cell Culture. HEK293 tsA201 cells were plated on glass coverslips mate receptors have larger cation conductances than and transfected with GluK2-L439C-eGFP or co-transfected with GluK2-L439C channelrhodopsins56, do not require illuminating large cellular and GCaMP6s or R-GECO1. Prior to each experiment, coverslips were incubated with the PTL of choice to allow chemical conjugation to the receptor channel. regions57, and can be targeted by MAG-like photoswitches at A second incubation with Concavalin A was done to inhibit desensitization of their physiological location58. Finally, the 2P-optimized azo- the glutamate receptor. Cells were mounted on the recording chamber filled with benzene core reported here entails minimal structural modifica- a bath solution composed of (in mM): 140 NaCl, 1 MgCl2, 2.5 KCl, 10 4-(2- tions that can be grafted into other azobenzene-based hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2.5 CaCl2, and 10–20 photoswitches, provided that their pharmacological properties are glucose to fix osmolarity to 310 mOsm kg−1. NaOH was added to adjust the pH to 7.42. To activate GluK2-L439C, 300 μM glutamate in bath solution was not altered, and thus these findings have direct and general perfused. application to light-regulated ligands2–4. In conclusion, we rationally designed azobenzene chromo- Electrophysiology. Voltage-clamp recordings under whole-cell configuration were phores based on theoretical calculations to present both high 2P acquired at 1 kHz. Borosilicate glass pipettes were pulled with a typical resistance of absorptivity of NIR light and long cis state lifetime, which were 4–6 MOhm and filled with a solution containing (in mM): 120 cesium methane- then exploited in the synthesis of PTLs MAGslow slow sulfonate, 10 tetraethylamonium chloride, 5 MgCl2, 3 Na2ATP, 1 Na3GTP, 20 2P and MAG2P F . HEPES, 0.5 EGTA; osmolarity was 290 mOsm kg−1 and pH 7.2 was adjusted with Optimized 2P stimulation of light-gated ionotropic glutamate CsOH. Cell membrane voltage was held at −70 mV. Photostimulation during receptors was accomplished with these compounds, which far electrophysiological recordings was induced by illuminating the entire focused field surpassed the performance of other azobenzene-based photo- with monochromatic light in an inverted microscope (power density: 22.0 μW mm−2 at 380 nm, 45.9 μW mm−2 at 460 nm, and 47.4 μW mm−2 at 500 nm). switches previously assayed for the manipulation of neuronal tissues under multiphoton excitation conditions. Taking advan- Calcium imaging and 1P stimulation. Cells were imaged on an inverted fully tage of the high 2P excitability of MAGslow slow 2P and MAG2P F with motorized digital microscope at room temperature with a frame rate of 2 s and NIR radiation, reliable and sustained photocontrol over the exciting GCaMP6s at 490 nm during 10 ms. Photoisomerization was achieved by activity of neurons could be attained in light-scattering tissue illuminating the focused sample with flashes of violet (380 nm, 0.5 s duration) and both in vitro and in vivo, such as in brain slices under nearly green (500 nm, 0.5 s duration) light for activation and deactivation, respectively. Calcium imaging activation spectra under 1P stimulation ranged from 280 to physiological conditions and C. elegans TRNs. The results pre- 480 nm at 20 nm steps. Light flashes were nested in between GCaMP6s fluores- sented here constitute a proof of concept that paves the way cence measurements. Photostimulation intervals lasted a total of 3.2 min for acti- toward all-optical experiments of neuronal activity imaging and vation and 2.4 min for deactivation. At the end of this protocol, 300 μM free manipulation in vivo using azobenzene photoswitches, which glutamate solution was added and the calcium imaging response was measured. ultimately require the use of multiphoton excitation with NIR light59. Calcium imaging and 2P stimulation in HEK cells. 2P experiments were per- formed in the Advanced Digital Microscopy Core Facility of IRB Barcelona with a confocal multiphoton microscope equipped with a 80 MHz Ti:Sapphire for 2P Methods stimulation with NIR light (710–990 nm), and cw diode (405 nm) and Ar (514 nm) Theoretical calculations. All calculations were carried out at the DFT (for ground lasers for 1P stimulation and calcium imaging with visible light. In these experi- electronic states) and TDDFT (for excited electronic states) levels using the long- ments, R-GECO1 was used as a Ca2+ fluorescent indicator instead of GCaMP6s range corrected hybrid CAM-B3LYP functional, which is known to correctly because it does not absorb at 405 nm, the excitation wavelength used to test the 1P describe excited states of charge-transfer type60. 6–31G(d), a split-valence basis set activity of LiGluR. Imaging of R-GECO1 was done at 514 nm with a frame rate of with polarization functions in heavy atoms, was used in all the cases. Twenty 4 s. Photostimulation was achieved by raster-scanning the tightly focused laser of excited electronic states of the same multiplicity as the ground electronic state choice over a selected area of the field of view: 405 nm (0.37 mW μm−2) for 1P (singlet) were converged in the TDDFT calculations. Solvent (water) effects were activation, 514 nm (0.35 mW μm−2) for 1P deactivation, and 720–840 nm introduced through the self-consistent polarizable continuum model continuum (2.8 mW μm−2) for 2P activation of LiGluR. Photostimulation scans were fit to method61. To compute 2P absorption properties, the density functional response keep imaging interval, and illumination periods at a given wavelength lasted in theory was employed to calculate the 2P transition matrix elements. 2P absorption total 1 min. Interstimulus imaging periods also lasted 1 min. cross sections were then estimated through the following expression62: Calcium imaging and 2P stimulation in hippocampal slices. All procedures were 8π 2 αa50 ω2 σ 2 ðωÞ ¼ δðωÞ ð1Þ conducted in accordance with the European Guidelines for Animal Care and Use cΓ in Research and were approved by the Animal Experimentation Ethics Committee at the University of Barcelona (Spain). Hippocampal organotypic slices of 400 μm where α is the fine structure constant, a0 the Bohr radius, c is the speed of light in in thickness were obtained from postnatal day 6–8 rats and cultured for 5–7 days vacuum, Γ is the full width at half maximum of the Lorentzian line-shape until biolistically transfected with RCaMP2a and GluK2-L439C-eGFP, as described broadening, and δ(ω) is the 2P absorption transition probability calculated through in ref. 46. Although transfection with R-GECO1 was also assayed, RCaMP2a was the response theory assuming linearly polarized excitation light. We set Γ = 0.2 eV, finally used as a fluorescent indicator in hippocampal slice cultures because of the which reasonably agrees with the value experimentally determined for trans- higher level of expression achieved. Before each experiment, slices were incubated MAG460 (~0.15 eV34). Here we also considered the most common case of a with the PTL of choice for 10 min in artificial cerebrospinal fluid (ACSF) con- degenerate 2P absorption process where ω is half of the transition frequency of the taining 119 mM NaCl, 2.5 mM KCl, 3 mM CaCl2, 0.2 mM MgCl2, 26.2 mM excited state. All TDDFT calculations and evaluation of 2P absorption properties NaHCO3, 1 mM NaH2PO4, and 11 mM glucose, equilibrated with 5% CO2/95% O2. were calculated using the Dalton suite of programs63. Optimizations and evaluation After washes with fresh ACSF, slices were placed on the recording chamber and of energy barriers in the ground electronic state were carried out with the were continuously perfused with ACSF. RCaMP2a was excited at 561 nm with an 10 NATURE COMMUNICATIONS | (2019)10:907 | https://doi.org/10.1038/s41467-019-08796-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-08796-9 ARTICLE imaging interval of 4 s. eGFP was excited at 488 nm. Photostimulation was 8. Hansen, M. J., Lerch, M. M., Szymanski, W. & Feringa, B. L. Direct and achieved by raster-scanning the tightly focused laser of choice over the whole field versatile synthesis of red-shifted azobenzenes. Angew. Chem. Int. Ed. 55, of view at 400 Hz: 405 nm (0.81 mW μm−2) for 1P activation, 514 nm (0.35 mW 13514–13518 (2016). μm−2) for 1P deactivation, and 780 nm (2.8 mW μm−2) for 2P activation of 9. Bléger, D., Schwarz, J., Brouwer, A. M. & Hecht, S. o-Fluoroazobenzenes as LiGluR. Photostimulation scans were fit to keep imaging interval, and illumination readily synthesized photoswitches offering nearly quantitative two-way periods at a given wavelength lasted in total 1 min. Interstimulus imaging periods isomerization with visible light. J. Am. Chem. Soc. 134, 20597–20600 (2012). lasted 1.5 min. 10. Hammerich, M. et al. Heterodiazocines: synthesis and photochromic properties, trans to cis switching within the bio-optical window. J. Am. Chem. Calcium imaging and 2P stimulation in vivo in C. elegans. Animal experiments Soc. 138, 13111–13114 (2016). carried out have been approved by the ethics committee of the ERC and the animal 11. Dong, M. et al. Near-Infrared photoswitching of azobenzenes under welfare has been respected. C. elegans are nematodes and therefore not covered by physiological conditions. J. Am. Chem. Soc. 139, 13483–13486 (2017). EU Directive 2010/63/EU, which only covers vertebrates and cephalopods. Like- 12. Wegener, M., Hansen, M. J., Driessen, A. J. M., Szymanski, W. & Feringa, B. L. wise, worms are not covered by the Spanish “Código de protección y bienestar Photocontrol of antibacterial activity: shifting from UV to red light activation. animal”. For these reasons, there is no need for authorizations, personal licenses, J. Am. Chem. Soc. 139, 17979–17986 (2017). standards for procedures or detailed description of number of animals to be used, 13. Siewertsen, R. et al. Highly efficient reversible Z-E photoisomerization of a nature of experiments or anticipated impact, and minimization thereof. We gen- bridged azobenzene with visible light through resolved S(1)(n pi*) absorption erated strain MSB104 [mirEx22(mec-17p::iGluR6::mCherry;myo-2p::mCherry); bands. J. Am. Chem. Soc. 131, 15594–15595 (2009). ljSi123(mec-7p:GCaMP6s::SL2::tagRFP);lite-1(ce314)X]. Transgenesis was per- 14. Pawlicki, M., Collins, H. A., Denning, R. G. & Anderson, H. L. Two-photon formed according to standard methods for microinjection65 by microinjecting a absorption and the design of two-photon dyes. Angew. Chem. Int. Ed. 48, DNA mix containing 50 ng μl−1 pNMSB18 (mec-17p::iGluR6::mCherry, Supple- 3244–3266 (2009). mentary Table 7), 1.5 ng μl−1 myo-2p:mCherry as a coinjection marker, and 15. Bort, G., Gallavardin, T., Ogden, D. & Dalko, P. I. From one-photon to two- 50 ng μl−1 1Kb Plus DNA ladder (Invitrogen) as a carrier into the gonad of GN692 photon probes: “caged” compounds, actuators, and photoswitches. Angew. young adult worms66. MAGslow −1 ConA) was 2P F (10 mM in M9 buffer and 0.3 mg ml Chem. Int. Ed. 52, 4526–4537 (2013). administered to the animals by microinjection into the body cavity. Control group 16. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning was performed microinjecting with vehicle (10% DMSO and 0.3 mg/ml ConA in fluorescence microscopy. Science 248, 73–76 (1990). M9 buffer), and allowed to recover. Only roaming worms surviving the treatment 17. Papagiakoumou, E. et al. Functional patterned multiphoton excitation deep were considered for the following imaging experiments. After 4 h of compound inside scattering tissue. Nat. Photon. 7, 274–278 (2013). administration, TRN neurons co-expressing GluK2-L439C-mCherry and 18. Oron, D., Papagiakoumou, E., Anselmi, F. & Emiliani, V. 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Inter-synaptic lateral diffusion of GABAA receptors shapes inhibitory synaptic currents. Neuron 95, 63–69.e5 (2017). Additional information 52. Damijonaitis, A. et al. AzoCholine enables optical control of alpha 7 nicotinic Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- acetylcholine receptors in neural networks. ACS Chem. Neurosci. 6, 701–707 019-08796-9. (2015). 53. Frank, J. A. et al. Photoswitchable diacylglycerols enable optical control of Competing interest: The authors declare no competing interest. protein kinase C. Nat. Chem. Biol. 72, 510–516 (2016). 54. Rost, B. R., Schneider-Warme, F., Schmitz, D. & Hegemann, P. Optogenetic Reprints and permission information is available online at http://npg.nature.com/ tools for subcellular applications in neuroscience. Neuron 96, 572–603 (2017). reprintsandpermissions/ 55. dal Maschio, M., Donovan, J. C., Helmbrecht, T. O. & Baier, H. 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