Photoactivatable drugs and peptides can drive quantitative studies into receptor signaling with high spatiotemporal precision, yet few are compatible with behavioral studies in mammals. We developed CNV-Y-DAMGO—a caged derivative of the mu opioid receptor-selective peptide agonist DAMGO. Photoactivation in the mouse ventral tegmental area produced an opioid-dependent increase in locomotion within seconds of illumination. These results demonstrate the power of in vivo photopharmacology for dynamic studies into animal behavior.
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The data supporting the findings of this study are available within the paper and its Extended Data files. Source data are provided with this paper.
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We thank E. Berg for technical support, J. Isaacson for helpful discussions and J. Chang-Weinberg for manuscript editing. This work was supported by the National Institute of Neurological Disorders and Stroke and the National Institute of Mental Health (U01NS113295 to M.R.B., including Diversity Supplements to D.A.J. and A.E.L.), the National Institute of General Medical Sciences (R35GM133802 to M.R.B., T32GM007240 to X.J.H.), the Brain and Behavior Research Foundation (M.R.B.), the Esther A. and Joseph Klingenstein Fund and Simons Foundation (M.R.B.), the Rita Allen Foundation (M.R.B.) and by Plan Nacional Sobre Drogas (Spanish Ministerio de Sandidad, 2021I070 to J.B.).
The authors declare no competing interests.
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Extended Data Fig. 1 Synthesis of CNV-Y-DAMGO (4).
The N-terminal amine in DAMGO (3) was Boc-protected prior to phenol alkylation with 6. Global deprotection with TFA produced CNV-Y-DAMGO (4) in good overall yield.
Extended Data Fig. 2 In vitro characterization of CNV-Y-DAMGO activity at opioid receptors.
(A) Dose-response curves at the delta opioid receptor (DOR) using a GloSensor assay of cAMP signaling in HEK293T cells (n = 5 wells per data point, 1 representative independent experiment shown). Data were normalized to the maximal response to leucine-enkephalin (LE, 1 µM) and are expressed as the mean ± SEM. (B) Same as A, but using the kappa opioid receptor (KOR) and dynorphin A(1-8) (Dyn8, 1 µM) for normalization. (C) Same as A, but using the nociceptin/orphanin FQ receptor (NOP) and nociception/orphanin FQ (N/OFQ, 1 µM) for normalization. (D) Dose-response curves at the mu opioid receptor (MOR) using a NanoBiT-based luminescence complementation assay of β-arrestin signaling in HEK293T cells (n = 4 wells per data point, 3 independent experiments averaged). Data were normalized to the maximal response to DAMGO (10 µM) and are expressed as the mean ± SEM.
Extended Data Fig. 3 In vitro characterization of CNV-Y-DAMGO (4) photolysis and dark stability.
(A) Waterfall plot of HPLC chromatograms of CNV-Y-DAMGO (0.4 mM) during illumination with 375 nm light (10 mW) in PBS, pH 7.2. (B) Enlarged HPLC chromatogram after 2 min of illumination (top) and mass spectrograms corresponding to the indicated peaks (bottom). (C) HPLC chromatograms of a sample of CNV-Y-DAMGO (1 mM) in PBS left for 24 hours in the dark. (D) Summary of MNI-Glutamate and CNV-Y-DAMGO photouncaging reactions over time, as measured by HPLC (n = 3 samples per condition). Samples were optical density-matched at 375 nm in PBS and illuminated with 375 nm laser irradiation. Data are expressed as the mean ± SEM. (E) UV-VIS spectrum of samples of CNV-Y-DAMGO (0.4 mM) in PBS before and after irradiation with 375 nm light (10 mW) for the indicated time periods.
Extended Data Fig. 4 DAMGO infusion into the VTA and location of optofluidic cannula placements.
(A) Schematic of the experimental configuration for DAMGO infusion through an optofluidic cannula. (B) Experimental timeline. (C) Movement velocity vs. time in the open field for a cohort of mice after DAMGO (200 µM, 0.5 uL) infusion. (D) Average data from C (n = 6 mice). Data are expressed as the mean ± SEM. (E) Example maps of open field locomotor activity from a single mouse after infusion of either saline or DAMGO. (F) Change in distance traveled (after-before uncaging) mapped to the implant location for the mice shown in Fig. 2g,h.
Representative formalin-treated mouse infused with CNV-Y-DAMGO in the NAc and exposed to light: minutes 29–31 postinjection.
Representative formalin-treated control mouse exposed to light: minutes 29–31 postinjection.
Representative mouse infused with CNV-Y-DAMGO in the VTA and exposed to light.
Representative mouse infused with CNV-Y-DAMGO in the VTA and exposed to light after treatment with naloxone.
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Ma, X., Johnson, D.A., He, X.J. et al. In vivo photopharmacology with a caged mu opioid receptor agonist drives rapid changes in behavior. Nat Methods 20, 682–685 (2023). https://doi.org/10.1038/s41592-023-01819-w