Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation

Abstract

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 photoisomerization 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 theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds 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.

Fig. 1

Strategy toward optimized azobenzene photoswitches for the two-photon (2P) excitation of light-gated glutamate receptors (LiGluR). a Operating mode of MAG-type photoswitchable tethered ligands (PTLs) on LiGluR, which are composed of three covalently tethered units: a glutamate ligand, an azobenzene core, and a maleimide group that binds to a cysteine residue genetically engineered in the receptor. Ultraviolet-visible (one-photon; 1P) or near-infrared (2P) light excitation induces glutamate recognition and channel opening via trans → cis isomerization, which results in ion flow across the membrane. This process is reverted by illumination with visible light excitation (1P) or thermal back-isomerization of the cis state of the switch. b Structures of the azobenzene cores of MAG, MAG₀, MAG_2P, and MAG₄₆₀ PTLs proposed for the photoswitching of LiGluR under 1P and 2P excitation conditions. c Structures of PTLs ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$ and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$

Fig. 2

Synthesis of ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$ and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$. 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 ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$, quantitative yield for ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$)

Fig. 3

Photochemical and physiological characterization of ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$ and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ under one-photon (1P) stimulation. a Normalized absorption spectra of trans-MAG, trans-${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, trans-${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$, and trans-MAG_2P in 99% phosphate-buffered solution (PBS):1% dimethylsulfoxide (DMSO). b Thermal lifetimes of cis-MAG, cis-${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, cis-${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$, and cis-MAG_2P at room temperature in 99% PBS:1% DMSO. Errors from the monoexponentials fits to obtain τ_cis 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, ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$, and MAG_2P (n = 5, 7, 3, and 8 biologically independent cells, respectively). Errors are 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, ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ (n = 33, 20, and 25 biologically independent cells, respectively). Errors are SEM. In c and 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

Fig. 4

One-photon (1P) and two-photon (2P) stimulation of MAG, ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ in cultured cells. a Individual (thin lines) and average (thick lines) calcium imaging fluorescence traces recorded for human embryonic kidney 293 (HEK293) cells co-expressing GluK2-L439C and R-GECO1 after conjugation to MAG (n = 16 biologically independent cells), ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$ (n = 14 biologically independent cells), and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ (n = 34 biologically independent cells). The bands around average traces plot the corresponding SEM. Both 1P (violet, 405 nm, power density = 0.37 mW μm⁻²) and 2P excitation scans (red, 780 nm, power density = 2.8 mW μm⁻²) were applied to open LiGluR channels and trigger calcium-induced R-GECO1 fluorescence enhancement, while 1P excitation scans (green, 514 nm, power density = 0.35 mW μm⁻²) were applied to revert back the process. b Repetitive 2P-induced calcium imaging fluorescence responses recorded in five different HEK293 cells co-expressing GluK2-L439C and R-GECO1 after conjugation to ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$. Source data for a are provided as a source Data file

Fig. 5

Average two-photon (2P) activity of MAG, ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ in cultured cells. a 2P action spectra of MAG, ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ after conjugation to GluK2-L439C-expressing human embryonic kidney 293 (HEK293) cells. Fluorescence calcium responses were measured using R-GECO1. Before averaging over different cells, the 2P responses of each cell 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 independent cells, respectively; ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$: 740, 760, 780, 800, and 820 nm; n = 20, 9, 12, 16, and 17 biologically independent cells, respectively; and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$: 720, 740, 760, 780, 800, 820, and 840 nm; n = 14, 17, 18, 86, 15, 23, and 17 biologically independent cells, respectively). Errors are SEM. b Ratio between the 2P and 1P responses of MAG, ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ for the same cells excited at 780 and 405 nm, respectively. Errors are SEM. c Reliability of the 2P calcium imaging response elicited in GluK2-L439C-expressing HEK293 cells after conjugation to MAG, ${\mathbf{MAG}}_{\mathbf{2P}}^{\mathbf{slow}}$, and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ (n = 72, 25, and 86 biologically independent cells, respectively). Reliability is expressed as the percentage of transfected cells showing measurable 2P stimulation signals. Source data for a and b are provided as a source Data file

Fig. 6

Two-photon (2P) Ca²⁺ photoresponses in rat hippocampal 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 ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$. d, e Average one-photon (1P) and 2P responses of neurons incubated with d MAG (n = 3 biologically independent cells) or e ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ (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 biologically independent cells) and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ (red bars, n = 6 biologically independent cells): f fluorescence enhancement; g ratio between the 2P and 1P responses of MAG and ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ for the same cells. Error bars are SEM. Source data for d–g are provided as a source Data file

Fig. 7

In vivo calcium induced photoresponses by two-photon (2P) stimulation of ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ 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 animals treated with c ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ (n = 5 and 6 cells from four different animals experiments for 1P and 2P traces, respectively) or d with vehicle (n = 5 and 6 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 ${\mathbf{MAG}}_{{\mathbf{2P}}_{-}\mathbf{F}}^{\mathbf{slow}}$ (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