Animals

The experiment was carried out on 406 naïve adult male Swiss albino mice (25–30 g) purchased from the licensed breeder (Kołacz, Warsaw, Poland). The animals were housed in the environmentally controlled rooms with a 12-h light/dark cycle, in groups of 10 in standard cages under strictly controlled laboratory conditions: temperature maintained at 22–23 °C with relative humidity in a range of 45–55%. Throughout the study, the animals were given ad libitum access to water and food. The experiment began after at least a 1-week acclimation period to the laboratory conditions and was conducted between 8 a.m. and 3 p.m. to minimize circadian influences. Separate groups of animals were used for the forced swim test (FST), tail suspension test (TST), locomotor activity test, and pharmacokinetic studies. Because the locomotor activity test is noninvasive, following its assessment, the tissues were collected for quantitative real-time PCR (qRT-PCR) analysis. All procedures were approved by the Local Ethics Committee at the Medical University of Lublin (license no. 29/2015) and were performed in accordance with binding European standards related to the experimental studies on animal models. Each mouse was used only once.

Drug administration

Caffeine (5 mg/kg; Sigma-Aldrich, Poznań, Poland) and mianserin hydrochloride (10 mg/kg; Sigma-Aldrich) were dissolved in saline (0.9% sodium chloride (NaCl)). Agomelatine (20 mg/kg; Sigma-Aldrich) was suspended in a 1% aqueous solution of Tween 80 (POCH, Gliwice, Poland). The doses and treatment schedules were selected based on our previous experiments (Poleszak et al. 2016). The mice were randomly assigned to groups that received the solution of caffeine or saline intraperitoneally (i.p.), twice a day (at 8 a.m. and 8 p.m.), for 14 days. Both groups were further divided into subgroups that received on the 15th day caffeine or saline 40 min before behavioral testing and the solutions/suspensions of antidepressants or saline 60 min before behavioral testing. All solutions/suspensions were prepared fresh before administration and were administered at a volume of 0.1 ml per 10 g of body weight. The number of animals used per group was 10 in the FST, TST, and pharmacokinetic studies and 8 in the locomotor activity test.

Forced swim test

The FST was performed 40 min following caffeine administration and 60 min following drug administration. The procedure was carried out according to the method of Porsolt et al. (1977). Each mouse was placed individually into the glass cylinders (height 25 cm, diameter 10 cm) containing 10 cm of water at 23–25 °C. The animals were left in the cylinder for 6 min. The total duration of immobility was recorded during the last 4 min of the 6-min long testing period. The mouse was judged immobile when it ceased struggling and remained floating motionless in the water, making only the movements necessary to keep its head above the water level. The immobility time was scored in real time by two blind observers. The results are shown as the arithmetic mean of immobility time of animals given in seconds ± standard error of the mean (SEM) for each experimental group.

Tail suspension test

The TST was performed 40 min following caffeine administration and 60 min following drug administration. The procedure was carried out according to the method of Steru et al. (1985). Each mouse was individually suspended by its tail to a vertical bar in wooden box (30 × 30 cm). The animals were fastened by means of an adhesive tape fixed 2 cm from the end of the tails for 6 min. The total duration of immobility was recorded during the last 4 min of the 6-min long testing period. The mouse was judged immobile when it ceased moving its limbs and body, making only the movements necessary to breathe. The results are shown as the arithmetic mean of immobility time of animals given in seconds ± SEM for each experimental group.

Spontaneous locomotor activity

The spontaneous locomotor activity was assessed 40 min following caffeine administration and 60 min following drug administration. The spontaneous locomotor activity was measured in an animal activity meter Opto-Varimex-4 Auto-Track (Columbus Instruments, USA). This device consists of four transparent cages with a lid (43 × 43 × 32 cm), a set of four infrared emitters (each emitter has 16 laser beams), and four detectors monitoring animal movements. Mice were placed individually into the cages for 10 min. Spontaneous locomotor activity was evaluated between the 2nd and the 6th min, which corresponds to the time interval analyzed in the FST and the TST. The results are presented as the arithmetic average distance traveled by a mouse in centimeters ± SEM for each experimental group.

Tissue processing for pharmacokinetic studies

Forty minutes following caffeine administration and 60 min following drug administration, mice were decapitated to collect biological material for pharmacokinetic studies. The blood was collected into Eppendorf tubes and allowed to clot at room temperature. Subsequently, the blood was centrifuged at 5000×g for 10 min and serum was collected into polyethylene tubes and frozen at − 25 °C. Immediately after the decapitation, the brains were dissected from the skull, washed with saline, and also frozen at − 25 °C. Serum and brain concentrations of caffeine and the tested antidepressants were assayed by the high-performance liquid chromatography (HPLC) method.

Determination of antidepressants in serum and brain tissue

The brains were homogenized in distilled water (1:4, w/v) with a TH220 tissue homogenizer (Omni International, Inc., Warrenton, VA, USA). For agomelatine, 1 ml of brain homogenate or 200 μl of serum was spiked with carbamazepine (100 ng/ml) as an internal standard (IS). Before the extraction, 1 ml of the concentrated NaCl solution (10 g/50 ml) was added to brain homogenate and the samples were vortexed for 15 s. The extraction of agomelatine from brain homogenate was performed using 5 ml of a mixture of dichloromethane/hexane/isoamyl alcohol (39.5:59.5:1 v/v/v), whereas that from serum with 1 ml of dichloromethane. The samples were shaken for 20 min and centrifuged for 15 min at 1000×g. After the centrifugation, the organic layers were transferred into conical glass tubes and evaporated to dryness at 37 °C under a gentle stream of nitrogen in a water bath. The residues were dissolved with 100 μl of methanol, and aliquots of 50 μl were injected into the HPLC system. For mianserin, the extraction from serum and brain homogenates was performed using a mixture of ethyl acetate/hexane (30:70, v/v). Amitriptyline (1 μg/ml) used as IS was added to serum (200 μl) and brain homogenate (0.5 ml) containing mianserin, and the samples were alkalized with 100 and 250 μl of 4 M NaOH, respectively. Next, the samples were extracted with 5 ml of the extraction reagent by shaking for 20 min (IKA Vibrax VXR, Germany). After centrifugation at 1000×g for 20 min (Universal 32, Hettich, Germany), the organic layers were transferred to new tubes containing a 150 μl solution of 0.1 M H 2 SO 4 and methanol (90:10, v/v), shaken for 0.5 h, and then centrifuged for 15 min (1000×g). Then, the organic layers were discarded and 50-μl aliquots of acidic solutions were injected into the HPLC system.

The HPLC system consisted of an isocratic pump (model L-7100) and an autosampler (model L-7200), both from Merck Hitachi (Darmstadt, Germany), and a UV variable-wavelength K-2600 detector (Knauer, Berlin, Germany). Data acquisition and processing were carried out using the D-7000 HSM software (Merck Hitachi). Analysis of agomelatine was performed on a 250 × 4 mm LiChrospher1100 RP-18 column with a particle size of 5 mm (Merck, Darmstadt, Germany) protected with a guard column (4 × 4 mm) with the same packing material, whereas mianserin was determined using a 250 × 4.6 mm Supelcosil LC-CN column with a particle size of 5 μm (Sigma-Aldrich, Steinheim, Germany) protected with a guard column (20 × 4 mm) with the same packing material. The mobile phase consisting of acetonitrile and 50 mM potassium dihydrogen phosphate was mixed at a ratio of 37:63 (v/v) for agomelatine and 25:75 (v/v) for mianserin and run at 1 ml/min. Chromatographic analysis was carried out at 21 °C and an analytical wavelength of 230 nm for agomelatine and 214 nm for mianserin.

The calibration curves constructed by plotting the ratio of the peak heights of the studied drug to IS vs. the concentration of the drug were linear in the tested concentration ranges. No interfering peaks were observed in the chromatograms. The assays were reproducible with low intra- and inter-day variation (coefficients of variation less than 10%). The extraction efficiencies of the analyzed compounds and IS ranged from 70 to 92%. Antidepressant concentrations were expressed in nanograms/milliliter of serum or nanograms/gram of wet brain tissue.

Determination of caffeine in serum and brain tissue

The brains were homogenized in phosphate buffer (pH 7.2; 1:2, w/v) with a tissue homogenizer (Ultra Turrax T8, IKA, Germany). Caffeine extraction from serum (200 μl) and brain homogenate (300 μl) was performed by 6% perchloric acid protein precipitation. The mixture was vortexed for 30 s and centrifuged at 2500×g for 15 min. The supernatants were filtered through a cellulose filter (nominal pore diameter 0.20 μm), and a 20 μl volume of each sample solution was injected into the HPLC system. The undertaken sample preparation method was the modification of the procedure described by Novitskayaa et al. (2013).

The HPLC system (PerkinElmer Series 200, Shelton, CT, USA) consisted of an isocratic pump, a variable-wavelength UV-Vis detector, and an autosampler. All analyses were performed on a 250 × 4.6 mm Hypersil® column with a particle size of 5 μm (Thermo Electron Corporation, Waltham, MA, USA) protected with a guard column (4 × 4 mm) with the same packing material. The mobile phase consisting of water (brought to pH 4.0 with 1% formic acid)/acetonitrile/methanol (80:8:14, v/v/v) was run at 1 ml/min. Chromatographic analysis was carried out at 21 °C and an analytical wavelength of 273 nm.

The calibration curves constructed based on the analysis of samples containing caffeine at concentrations covering the range of 1 to 24 μg/ml prepared for murine serum and brain homogenates were linear in the tested concentration ranges. No interference from the matrix at the retention time of caffeine was observed in the chromatograms. Caffeine concentrations were expressed in micrograms/milliliter of serum or nanograms/gram of wet brain tissue.

The quantitative real-time PCR analysis

Because locomotor activity is a noninvasive test, following its assessment, mice were decapitated; their brains were rapidly dissected and immersed in cooled (2–8 °C) saline. The Cx was dissected on a cold plate, immediately frozen on dry ice, and stored at − 80 °C until analysis.

The qRT-PCR method was used to evaluate the expression of the selected genes in the Cx. RNA was isolated from 30 mg of tissue using Syngen Tissue RNA Mini Kit (Syngen Biotech, Poland), and reverse transcription was performed by means of NG dART RT-PCR kit (EURx, Poland) according to the manufacturer’s instructions. The relative expression of the following genes: Slc6a15, Comt, and Adora1 (Mn00558415_m1, Mn00514377_m1, Mn01308023_m1, respectively (TaqMan Gene Expression Assays, Life Technologies, USA)) was determined by qRT-PCR and the ΔΔCt method using hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (Mn00446968_m1) as an endogenous control. The reaction was carried out in quadruplicate using the SmartChip Real-Time PCR System (WaferGen Biosystems) and TaqMan Fast Universal PCR Master Mix (2×) (Applied Biosystems, USA) according to the manufacturer’s instructions. The data quality screening based on amplification curves and Ct values was performed to remove any outlier data before ΔΔCt calculations and to determine fold change in messenger RNA (mRNA) levels. Statistical analysis was performed on RQ values (RQ = 2 − ΔΔCt).

Statistical analysis

The results obtained in the FST, TST, and locomotor activity tests and relative gene expression levels of Slc6a15, Comt, and Adora1 were analyzed using a two-way ANOVA with chronic treatment (saline chronic vs. caffeine chronic) and 15th-day treatment as factors, followed by a Tukey’s post hoc test (for the comparison of effects within chronic treatment) or a Sidak’s post hoc test (for the comparison of effects within 15th-day treatments). The concentrations of caffeine and the tested antidepressants in serum and brains of mice were analyzed using a Student’s t test. The statistical analysis was carried out using GraphPad Prism for Windows, version 7.04 (GraphPad Software, San Diego, CA, USA). All results are presented as the mean ± SEM. A p value < 0.05 was considered as statistically significant with 95% confidence.