Microdialysis probe

A microdialysis probe (4-mm guide cannula length, 0.22-mm membrane outer diameter, 1-mm membrane length, MW cutoff of 50 kD; Eicom Corp) was stereotaxically inserted into the mPFC (15° angle, 1.75 mm anterior and 0.75 mm lateral from bregma, and 1.5 mm ventral to the dura) through the cannula guide. Artificial cerebrospinal fluid (ACSF) (124 mM NaCl; 4.4 mM KCl; 2 mM CaCl₂; 2 mM MgSO₄; 25 mM NaHCO₃; 1 mM KH₂PO₄; and 10 mM glucose; pH 7.4) was perfused at a flow rate of 1 μl/min using a microinjection pump. After 1 h of equilibrium, the mouse brain interstitial fluid was continuously collected into microvials for 4 h, and these interstitial fluid samples were subsequently lyophilized and redissolved in 20 μl of ACSF.

A guide cannula was implanted in the left hippocampus (2.7 mm posterior, 3.3 mm left, and 1.8 mm below from the bregma) of ddY mice (male, 5 weeks old). After 5–7 days of recovery, a microdialysis probe (Eicom) was inserted into the left hippocampus through the guide cannula. The tip of the microdialysis probe reached 4.8 mm below the bregma. The next day, the probe was connected to a perfusion tube and perfused with Ringer solution (containing 147.0 mM NaCl, 4.7 mM KCl, 0.6 mM MgSO₄, 2.5 mM CaCl₂, 5.0 mM HEPES, pH 7.4) at a flow rate of 1.5 μL/min. After ≥160 minutes of perfusion, collection of the perfusate (30 μL/20 min/fraction) was initiated, and 12 fractions per animal were collected for 240 minutes. E2730 (10–100 mg/kg), tiagabine (3–30 mg/kg), or vehicle was orally administered 72 minutes after initiating sample collection. Tiagabine was used as a reference ASM of a non‐competitive GAT1 inhibitor. Perfusate was switched from Ringer solution to high‐potassium Ringer solution (51.7 mM NaCl, 100.0 mM KCl, 0.6 mM MgSO₄, 2.5 mM CaCl₂, 5.0 mM HEPES, pH 7.4) after 127 minutes following the collection. GABA concentration in each sample was measured by a high‐performance liquid chromatography with tandem mass spectrometry method.

Extracellular histamine was collected using in vivo microdialysis system as previously described^{4 (link)}. Briefly, a guide cannula (EICOM, Kyoto, Japan) was implanted stereotactically into the hypothalamus (AP: −1.5 mm, ML: +0.5 mm, DV: −3.5 mm from bregma). After 1 week from surgery, a 2-mm membrane length of the microdialysis probe (EICOM) was inserted into the guide cannula and used to perfuse artificial CSF solution. Dialysate was collected every 30 min and applied to the HPLC system.

Mice were anesthetized using pentobarbital (Somnopentyl; Kyoritsu, Tokyo, Japan). Lidocaine (Xylocaine Jelly; AstraZeneca, London, UK) was used for calvarial local anesthesia. Each mouse was placed in a stereotaxic frame (Narishige, Tokyo, Japan). A guide cannula (Eicom) was inserted into the hippocampal region (bregma −3.5 mm anterior, −3.0 mm lateral, and 1.8 mm vertical) after drilling a port through the calvaria. A dummy cannula was inserted into the guide cannula, and allowed to recover for at least 5 days. A microdialysis probe (Eicom) was inserted into hippocampal region via the guide cannula. The probe was perfused with a Ringer’s solution, containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl₂, and 1.2 mM MgCl₂, at a flow rate of 1.0 μL/min. MHBA were administered orally at a dose of 10 mg/kg. Microdialysis samples were collected every 20 min before and after administration. NE was quantified using HPLC-ECD (Eicom).

Microdialysis was performed as previously described^{29 (link)}. Briefly, under pentobarbital anesthesia (60 mg kg⁻¹, intraperitoneal), we implanted bilaterally guide cannulas (0.40 mm inner diameter, 0.50 mm outer diameter; Eicom) above the NAc 1.4 mm anterior and 1.2 mm lateral to the bregma and 2.9 mm below the dura⁵⁷ . At the time of the behavioral experiment, the mouse was quickly anesthetized using isoflurane and the dummy cannula was removed followed by insertion of the microdialysis probe (1 mm membrane length; Eicom) into the guide cannula. The probe was infused continuously with Ringer’s solution using an infusion pump at a flow rate of 0.5 μl min⁻¹. Two hours after inserting the probe, dialysates were continuously collected from the probe for 3 h. The dialysates were kept at −20 °C until the UPLC analysis was performed.
A Shimadzu UPLC system equipped with a UV detection system and a TSKgel ODS-100V UPLC column (Tosoh Bioscience) and maintained with an aqueous acetonitrile mobile phase containing 100 mM monosodium phosphate (aqueous to organic solvent ratio 96:4) at a flow rate of 1 ml min^-1 was used for the UPLC separation. 80 μL of each dialysate or adenosine standard was injected into the UPLC system and calibration curves were constructed from the peak area ratio of adenosine to determine the adenosine concentration in each sample.