Escitalopram oxalate

Tramadol hydrochloride was gifted by Nippon Shinyaku Co., Ltd (Kyoto, Japan); morphine hydrochloride was purchased from Takeda Pharmaceutical Company, Ltd. (Osaka, Japan); maprotiline hydrochloride was purchased from Wako Pure Chemical Ind., Ltd (Osaka, Japan); and escitalopram oxalate was purchased from SIGMA Chemical Co. (St. Louis, MO).

Ketamine hydrochloride (Vet One, MWI Animal Health, Boise, ID, USA), fluoxetine hydrochloride (Sigma Aldrich, St. Louis, MO, USA) and reboxetine mesylate hydrate (Sigma Aldrich, St. Louis, MO, USA), were individually dissolved in sterile saline (0.9% NaCl solution, Hospira, Mountainside Medical Equipment, Marcy, NY, USA) and administered via intraperitoneal injection (i.p.) at 10 mg kg⁻¹ and a volume of 5 mL kg⁻¹ body weight. Escitalopram oxalate (Sigma Aldrich, St. Louis, MO, USA) was prepared and administered following the same method at the following doses: 1 mg kg⁻¹, 3 mg kg⁻¹, 10 mg kg⁻¹ and 30 mg kg⁻¹. Urethane (Sigma Aldrich, St. Louis, MO, USA) was dissolved in sterile saline at 25% w/v and administered at 7 μLg⁻¹ mouse body weight for surgical anesthesia. Post-calibration solutions for FSCAV were prepared by dissolving serotonin hydrochloride (Sigma–Aldrich Co., St. Louis, MO, United States) in Tris buffer to make solution concentrations of 10, 25, 50, and 100 nM. Tris buffer consisted of: 15 mM H₂NC(CH₂OH)₂•HCl, 140 mM NaCl, 3.25 mM KCl, 1.2 mM CaCl₂, 1.25 mM NaH₂PO₄•H₂O, 1.2 mM MgCl₂, and 2.0 mM Na₂SO₄ (Sigma–Aldrich Co., St. Louis, MO, United States) in deionized water; the pH adjusted to 7.4 (± 0.03).

Donepezil hydrochloride (Figure 1a) was purchased from Tokyo Chemical Industry (Tokyo, Japan), and escitalopram oxalate (Figure 1b) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). DMN patches were prepared in the Laboratory of Pharmaceutics, College of Pharmacy, Gyeongsang National University (Jinju, South Korea). Fisher Scientific (Hampton, NH, USA) provided HPLC-grade methanol, acetonitrile, and water (Seoul, South Korea). Methyl tert-butyl ether was purchased from Avantor Performance Materials (Center-Valley, PA, USA). Tegaderm™ film and Micropore™ surgical tape were purchased from 3M Healthcare (Saint Paul, MN, USA).

The active metabolite of the SSRI escitalopram, (S)‐citalopram, has a therapeutic window of between 50 and 130 ng/ml (Kumar, Kung, & Shinozaki, 2014), which corresponds to doses of between 120 and 313 nM in vitro. Subsequently, cells were treated with a range of doses, incorporating this therapeutically‐relevant window. Drug doses were achieved by dissolving escitalopram oxalate (Sigma) in molecular grade ethanol (Sigma) to form a 10 mM stock solution. Drug doses (145 nM, 290 nM, and 1160 nM) were then formed by dilution of the stock with media; with the relative proportion of ethanol kept constant across all dose groups including a vehicle control (0 nM).

Escitalopram oxalate, o-phthalaldehyde, reduced glutathione and Bradford reagent were from Sigma-Aldrich Chemicals GmbH (Munich, Germany), while EDTA-Na₂ and 2-thiobarbituric acid were purchased from Merck KGaA (Darmstadt, Germany). Absolute ethanol, hydrogen peroxide and n-butanol were obtained from Chimopar (Bucharest, Romania). Antibodies against BDNF, MeCP2, β actin and secondary peroxidase-linked antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while phosphorylated histone. ELISA tests for caspase-3 were purchased from Elabscience (Houston, TX, USA) and Bradford total protein concentration assay was from BioRad (Hercules, CA, USA).