Mice
All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Chinese Institute for Brain Research, Beijing (CIBR), and complied with the national guidelines for the housing and care of laboratory animals set by the Ministry of Health, China. Mice were housed in a specific pathogen-free facility on a 12-h light–dark cycle with ad libitum access to food and water. All experiments were conducted on male and female mice aged 8–16 weeks.
The study used WT C57BL/6J mice, Adora1−/− mice (NM-KO-225140, Shanghai Model Organisms Center), Adora2a−/− mice (NM-KO-200018, Shanghai Model Organisms Center), Nt5e−/− mice (provided by J. Chen, Wenzhou Medical University) and Rosa26-Cas9-GFP mice (Gt(ROSA)26Sortm1.1(CAG-cas9*, -EGFP) Fezh/J; The Jackson Laboratory, 024858). All mouse strains were subjected to CRS to induce depression, and WT, Nt5e–/–, Adora1−/− and Adora2a−/− mice were used for fibre photometry experiments.
Chemical reagents
Ketamine analogues were synthesized and provided by the Changchun Institute of Applied Chemistry, CAS, China (see below). Additional key chemicals were purchased from commercial sources, including norketamine hydrochloride (Tocris, 1970), (2R,6R)-HNK (Tocris, 6094), ticlopidine (Selleck, S0721), ketoconazole (Selleck, S1353), ritonavir (Selleck, S1185), dipyridamole (Selleck, S1895), LPS from Escherichia coli O127:B8 (LPS; Sigma, L3129), PSB36 (MCE, HY-103175), ZM241385 (Selleck, S8105), CHA (MCE, HY-18939), CGS21680 hydrochloride (MCE, HY-13201A), adenosine (MCE, HY-B0228), sodium [13C 3 ]pyruvate (MCE, HY-W015913S), ADP (MCE, HY-W010918) and wheat germ agglutinin (Alexa Fluor 555; Thermo Scientific, W32464).
Compound synthesis and characterization
Full experimental procedures, compound characterization data (1H NMR and 13C NMR) and analytical spectra are provided in the Supplementary Information. A summary of the synthesis for the two key compounds (DCK and 2C-DCK) is presented below.
For the general procedure for the synthesis of 2-aryl-2-bromo-cycloketones, N-bromosuccinimide (1.5 equiv.) and dimethyl sulfoxide (2.0 equiv.) were added to a solution of 2-arylcyclohexan-1-one (1.0 equiv.) in CHCl 3 . The reaction mixture was stirred at room temperature and monitored by thin-layer chromatography. After completion, the reaction was quenched with saturated aqueous Na 2 S 2 O 3 and water. The aqueous phase was extracted with CH 2 Cl 2 (3 times). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography (petroleum ether/ethyl acetate) to afford the desired 2-aryl-2-bromo-cycloketone.
For the general procedure for the synthesis of ketamine derivatives (DCK and 2C-DCK), a solution of the appropriate 2-aryl-2-bromo-cycloketone (1.0 mmol) in anhydrous THF was cooled to the specified temperature (−25 °C) under a nitrogen atmosphere. The corresponding amine (methylamine for DCK; ethylamine for 2C-DCK; 4.0 equiv.) was added, and the reaction was stirred until thin-layer chromatography indicated complete consumption of the starting material. The reaction was quenched by the addition of saturated aqueous Na 2 CO 3 and water. The mixture was extracted with CH 2 Cl 2 (3 times), and the combined organic layers were dried over anhydrous Na 2 SO 4 . The solvent was removed in vacuo, and the residue was treated with diethyl ether and aqueous HCl. The aqueous layer was washed with diethyl ether, neutralized with saturated aqueous Na 2 CO 3 and extracted with CH 2 Cl 2 (3 times). The final organic layers were combined, dried over anhydrous Na 2 SO 4 and concentrated to dryness under vacuum to produce the final product.
Below are the characterizations of DCK and 2C-DCK using NMR spectroscopy.
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