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. 2022 Sep 4;23(17):10114.
doi: 10.3390/ijms231710114.

Reversible Photocontrol of Dopaminergic Transmission in Wild-Type Animals

Carlo Matera  1   2   3 , Pablo Calvé  4 , Verònica Casadó-Anguera  5 , Rosalba Sortino  1   2 , Alexandre M J Gomila  1   2 , Estefanía Moreno  5 , Thomas Gener  4 , Cristina Delgado-Sallent  4 , Pau Nebot  4 , Davide Costazza  1 , Sara Conde-Berriozabal  6 , Mercè Masana  6 , Jordi Hernando  7 , Vicent Casadó  5 , M Victoria Puig  4 , Pau Gorostiza  1   2   8
Affiliations

Reversible Photocontrol of Dopaminergic Transmission in Wild-Type Animals

Carlo Matera et al. Int J Mol Sci. .

Abstract

Understanding the dopaminergic system is a priority in neurobiology and neuropharmacology. Dopamine receptors are involved in the modulation of fundamental physiological functions, and dysregulation of dopaminergic transmission is associated with major neurological disorders. However, the available tools to dissect the endogenous dopaminergic circuits have limited specificity, reversibility, resolution, or require genetic manipulation. Here, we introduce azodopa, a novel photoswitchable ligand that enables reversible spatiotemporal control of dopaminergic transmission. We demonstrate that azodopa activates D1-like receptors in vitro in a light-dependent manner. Moreover, it enables reversibly photocontrolling zebrafish motility on a timescale of seconds and allows separating the retinal component of dopaminergic neurotransmission. Azodopa increases the overall neural activity in the cortex of anesthetized mice and displays illumination-dependent activity in individual cells. Azodopa is the first photoswitchable dopamine agonist with demonstrated efficacy in wild-type animals and opens the way to remotely controlling dopaminergic neurotransmission for fundamental and therapeutic purposes.

Keywords: GPCR; azobenzene; behavior; brainwave; dopamine; in vivo electrophysiology; optogenetics; optopharmacology; photochromism; photopharmacology; photoswitch; zebrafish.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Design, structure, and photochromism of azodopa. (a) Chemical structure of dopamine and representative (semi)rigidified derivatives: rotigotine (nonselective agonist), A68930 (D1 agonist), and dinapsoline (D1 agonist). (b) 2D and 3D chemical structure of the photochromic dopamine ligand azodopa (trans and cis isomers). Essential pharmacophoric features for D1 receptor binding are highlighted: blue spheres represent cationic site points and H-bond donors, i.e., the protonated amino function that can form a salt bridge and a hydrogen bond with Asp103 and Ser107, respectively; red spheres represent H-bond acceptors, i.e., the hydroxyl groups of the catechol ring that can interact with Ser198, Ser199, and Ser202; yellow spheres represent hydrophobic elements, i.e., the aromatic ring that can form π–π interactions with Phe288 and Phe289. The mutual position and orientation of such pharmacophoric features in the receptor-bound conformation should affect binding affinity and efficacy of the ligands. (c) Photochromic behavior of azodopa (50 µM) studied with steady-state spectroscopy in aqueous (PBS, pH 7.4) and organic (DMSO) solutions. As lifetimes of cis-hydroxyazobenzenes are very short in polar protic solvents, no changes in the absorption spectrum of aqueous solutions of azodopa could be observed after illumination with 365 nm light (3 min). (d) Photochromic behavior of azodopa (30 µM) investigated by transient absorption spectroscopy in water (only representative traces are shown for the sake of clarity; see Figure S7 for the full experiment). Transient absorption time traces were measured at different wavelengths upon excitation of trans-azodopa with a 5 ns pulsed laser at λ = 355 nm (3 mJ/pulse energy) and 25 °C. Thermal relaxation half-life of the cis isomer (200 μs) was estimated by applying an exponential one-phase decay model (GraphPad Prism 6). Inset: Transient absorption spectrum of trans-azodopa upon pulsed irradiation at λ = 355 nm recorded at t = 0 μs. X-values represent wavelength (nm), Y-values represent ∆A (arbitrary units, AU).
Figure 2
Figure 2
In vitro pharmacological characterization of azodopa. (a) Effect on D1-mediated adenylyl cyclase activation. cAMP accumulation experiments in HEK-293T cells transiently transfected with D1 and treated with different concentrations of azodopa, in the dark (black bars) or under illumination (purple bars), in the presence (gray area) or not (white area) of a D1-like receptor antagonist (SKF83566). Values are represented in percentage vs. basal levels of cAMP. Data are mean ± S.E.M. (6 experiments performed in quadruplicate). Statistical differences were analyzed by two-way ANOVA followed by Tukey’s post hoc test (*** p < 0.001 vs. basal; * p < 0.05 vs. basal; ^^^ p < 0.001 vs. dark; ^^ p < 0.01 vs. dark; $$$ p < 0.001 vs. controls non-pretreated with the antagonist). (b) Effect on D1-mediated ERK1/2 activation. ERK1/2 phosphorylation was determined in HEK-293T cells transiently transfected with D1 and treated with different concentrations of azodopa, in the dark (black bars) or under illumination (purple bars), in the presence (gray area) or not (white area) of a D1-like antagonist (SKF83566). Values are represented in percentage vs. basal levels of ERK1/2 phosphorylation. Data are mean ± S.E.M. (3 or 4 experiments performed in triplicate or quadruplicate). Statistical differences were analyzed by two-way ANOVA followed by Tukey’s post hoc test (*** p < 0.001 vs. basal; ^^ p < 0.01 vs. dark; $ p < 0.05 vs. controls non-pretreated with the antagonist). (c–e) Effect on D1-mediated intracellular calcium release compared to dopamine. (c) Real-time calcium imaging response (averaged traces, black line, n = 24 cells) in HEK-293T cells co-expressing D1 receptors and R-GECO1 as calcium indicator. Traces were recorded upon direct application of azodopa (50 µM, orange bars) in the dark (white area) and under illumination (purple area). Shadow represents “± S.E.M.”. Gray and green bars indicate the application of vehicle (control) and dopamine (reference agonist), respectively. Light blue bars indicate wash-out periods. See example frames and raw data traces of individual cells in supplementary Figure S10, and supplementary Video S1 for the entire movie. Two values of the calcium responses generated by azodopa were calculated (Origin 8 software) and compared: the peak amplitude ΔF/F0 (d), calculated as the difference between the maximal and the minimal intensity of each response (**** p < 0.0001 for vehicle vs. dopamine; **** p < 0.0001 for vehicle vs. azodopa/dark; **** p < 0.0001 for azodopa/365 nm vs. azodopa/dark; ** p = 0.035 for dopamine vs. azodopa/365 nm), and the area under the curve (AUC) (e), calculated as the integral over the entire application time of vehicle or drugs (**** p < 0.0001 vehicle vs. dopamine; **** p < 0.0001 for vehicle vs. azodopa/dark; **** p < 0.0001 for dopamine vs. azodopa dark; **** p < 0.0001 for azodopa/365 nm vs. azodopa/dark; *** p = 0.001 for vehicle vs. dopamine; ** p = 0.0025 for dopamine vs. azodopa/365 nm). Data are mean ± S.E.M. (n = 40 cells from 3 independent experiments). Data were normalized over the maximum response obtained with the saturating concentration of dopamine (50 μM) and were analyzed by one-way ANOVA followed by Tukey’s post hoc test for statistical significance. All statistical analyses (panels (a,b,d,e)) were performed with GraphPad Prism 6.
Figure 3
Figure 3
Behavioral effects of azodopa in zebrafish. (a–e) Experiments with normal zebrafish. (a) Swimming activity (distance/time) in larvae exposed to vehicle (control, gray line) or 100 μM azodopa (treatment, orange line) in the dark (white areas) or under illumination with 365 nm light (purple bars). Data are mean ± S.E.M. (n = 11–12 individuals/group). (b) Representative time frame (40–41.5 min) of the swimming activity integrated every 5 s, showing how the effect of azodopa can be completely shut down upon illumination. The spike of activity observed for the control group upon illumination represents the startle response to the light stimulus. (c) Exemplary trajectories of individual larvae in one well containing the vehicle and two wells containing 100 μM azodopa in the dark (40–40.5 min) and under illumination (40.5–41 min). Green lines and red lines indicate slow and fast swimming periods, respectively. The remarkable and light-dependent difference in behavior between untreated and azodopa-treated fish can be appreciated by observing these trajectories and the supplementary Video S2. (d) Quantification of the total distances swum by the control group (vehicle) and the treatment group (100 μM azodopa) during 4 consecutive cycles of illumination (30 s) and dark (30 s before and after illumination). Data are mean ± S.E.M. (n = 11–12 individuals/group) and were analyzed by two-way ANOVA followed by Tukey’s post hoc test (**** p < 0.0001; ** p = 0.0037). (e) Dose–response study of azodopa (white area) and effect of a co-administered D1-like antagonist (gray box). Different groups of larvae were exposed to increasing concentrations of azodopa. For quantification, the average distance swum by each group during the last 4 consecutive dark–light cycles (30 s integration) was considered. The graph shows that trans-azodopa (black bars) increases the fish locomotor activity in a dose-dependent manner, but its effects are abolished by the co-administration of a potent and selective D1-like antagonist (SKF83566, 50 μM). Data are mean ± S.E.M. (n = 12 individuals/group) and were analyzed by two-way ANOVA followed by Sidak’s post hoc test (**** p < 0.0001; ** p = 0.0063). (f,g) Experiments with blinded zebrafish. (f) Swimming activity (distance/time) in larvae exposed to vehicle (control, gray line) or 100 μM azodopa (treatment, orange line) in the dark (white areas) or under illumination with 365 nm light (purple bars). Data are mean ± S.E.M. (n = 12 individuals/group). (g) Quantification of the total distances swum by the control group (vehicle) and the treatment group (100 μM azodopa) during 4 consecutive cycles of illumination (30 s) and dark (30 s before and after illumination). Data are mean ± S.E.M. (n = 12 individuals/group) and were analyzed by two-way ANOVA followed by Tukey’s post hoc test (**** p < 0.0001; * p = 0.0232). All statistical analyses (panels (d,e,g)) were performed with GraphPad Prism 6.
Figure 4
Figure 4
Effect of cortical administration of trans-azodopa on electrophysiological recordings in anesthetized mice. (a) Animals were anesthetized with isoflurane and placed in a stereotaxic apparatus. A craniotomy was drilled above the secondary motor cortex (M2) and an octrode was inserted in the superficial layers. Analogic signals were bandpass filtered and digitized by a preamplifier, amplified by an Open Ephys data acquisition system (green arrow), and finally visualized and recorded in a PC (blue arrow). Neural activity was recorded during baseline conditions and after administration of trans-azodopa on the cortical surface (3 μM concentration in 10 μL volume). See the Supplementary Materials for further details of the setup and supplementary Figures S17–S19 for the effect of illumination on azodopa. (b) Neural activity during baseline conditions in Mouse 1. Raster plot of spiking activity in the cortex of one mouse for 10 min. Each row depicts the spiking activity of a single neuron (unit), each tick representing an action potential. We used arrays of 8 electrodes (octrodes) in each animal, from which several units could be recorded. Neurons are labeled by their electrode number (E1 to E8). Firing rates were stable and followed the UP and DOWN slow fluctuations typical of anesthesia. The quantification of firing rates and average time–frequency spectrogram of the power of neural oscillations (n = 8 electrodes) are shown below. (c) Neural activity after the administration of 3 μM trans-azodopa in Mouse 1 (zero indicates the time of administration). Azodopa boosted the firing rate of neurons and increased the power of neural oscillations. (d) Azodopa increased spiking activity of cortical neurons. Mean firing rate of neurons before and after the administration of trans-azodopa. Data are mean ± S.E.M. (n = 44 neurons during baseline vs. 43 neurons after azodopa in two mice) and were analyzed with an unpaired T-test (*** p = 0.0002). (e) Azodopa increased the power (1–10 Hz) of neural oscillations in the two animals. Due to the large differences in the baseline power of the two mice, we normalized the power to its baseline for visualization purposes only. Data are mean ± S.E.M. (n = 16 channels per condition from two mice) and were analyzed with a paired T-test (** p = 0.0011).
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