2.5. Measurement of brain monoaminergic neurotransmitters and their metabolites using in vivo microdialysis

Mice were anesthetized using 50 mg/kg pentobarbital (Nembutal^®) and fixed in a stereotaxic apparatus equipped with a mouse adapter (David Kopf, CA, USA). A dialysis probe (D-I-6-02, cut-off 50,000 Da, Eicom, Kyoto) was stereotaxically implanted into the brain region determined to be involved in the ambulatory response to BUP by the c-Fos study according to the mouse brain atlas [42] and fixed with dental cement. After surgery, the animals were individually housed in cages and allowed to recover for 2–3 days.

Two to three days after surgery, online measurement of the extracellular monoaminergic neurotransmitters, DA, NE, and 5-HT, and their metabolites, (3,4-dihydroxy-phenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxy-4-hydroxy-phenyl-ethylene glycol (MHPG), and 5-hydroxyindolacetic acid (5-HIAA)) was performed using in vivo microdialysis coupled with high-performance liquid chromatography (HPLC). The probe-implanted mouse was placed in a cage for the microdialysis experiment and allowed to move freely in the cage. Food and water were available ad libitum throughout the microdialysis measurement. Ringer’s solution (147 mM Na⁺, 4 mM K⁺, and 2.3 mM Ca⁺, 155.6 mM Cl⁻) was perfused at a rate of 2.0 μl/min through the probe using a syringe pump (ESP-64, Eicom). Each dialysate sample was collected for 25 min using an auto injector (EAS-2, Eicom) that automatically injected the dialysate sample into HPLC immediately after the end of each 25-min collection. After at least four dialysate samples were collected to establish baseline levels, saline or 10 mg/kg BUP was administered to the mouse, followed by continuous collection of six dialysate samples. The lag time from the probe tip to the auto injector was considered to determine the time point for the administration. After the end of the microdialysis measurement, eosin solution was perfused through the probe, the mouse was deeply anesthetized using pentobarbital (Nembutal^®), and the brain was removed and fixed using 10% formaldehyde neutral buffer solution (Nacalai Tesque). The placement of the probe was verified histologically (Appendix E).

Monoaminergic neurotransmitters and their metabolites in dialysate samples were analyzed using HPLC (HTEC-500, Eicom). Each sample was analyzed for 25 min using HPLC during which all analytes of interest appeared in a chromatogram. Before starting the microdialysis experiment for each mouse, HPLC was calibrated using authentic standard substances for monoaminergic neurotransmitters and their metabolites. The column used was SC-50DS (Eicom), and the mobile phase (pH 3.5) consisted of 83% 0.1 M acetic acid-citric acid buffer, 17% methanol (Nacalai Tesque), 190 mg/L octanesulfonic acid (Nacalai Tesque), and 5 mg/L Na₂EDTA (Wako Pure Chemical Ind. Ltd.). The flow rate of the mobile phase was 0.23 ml/min. Monoaminergic neurotransmitters and their metabolites were detected using an electrochemical detector (ECD-300, Eicom) that included a graphite electrode (WE-3G, Eicom). The applied voltage was +700 mV against an Ag/AgCl reference electrode. Data were collected from HPLC via an interface (EPC-300, Eicom) to a personal computer and analyzed using software (PowerChrom, AD instruments Japan, Nagoya).