Microdialysis study

Rats were anaesthetised with ketamine (75 mg/kg) and xylazine (10 mg/kg) and secured in a stereotaxic frame (Stoelting, USA). Vertical microdialysis guide cannulas (Intracerebral Guide Cannula with stylet; BAS Bioanalytical, USA) were implanted in the striatum (STR) according to the following stereotaxic coordinates: A/P + 1.0, L/M + 2.5 and V/D − 3.5 mm from bregma and the dura (G. Paxinos and C.H. Watson). Seven days after surgery, microdialysis probes (length 4 mm) were inserted into the cannulas, and the STR was perfused with artificial cerebrospinal fluid (aCSF), which consisted of 140 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl₂, 1 mM MgCl₂, 0.3 mM NaH₂PO₄ and 1.7 mM Na₂HPO₄ (pH 7.4), at a flow rate of 1.5 μl/min maintained with a microinfusion pump (Stoelting, IL USA). Samples were collected from freely moving rats in 20-min intervals after a 3-h wash-out period. All dialysates were immediately frozen on dry ice (− 70 °C) until they were used in a biochemical assay.

Dopamine (DA), serotonin (5-HT), noradrenaline (NA) levels and the levels of their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), homovanillic acid (HVA) and normetanephrine (NM) in dialysates (20 μl) were analysed by HPLC with electrochemical detection. Chromatography was performed using an LC-10 AD pump (Shimadzu Europa GmbH, Warszawa, Poland), an LC-4B amperometric detector with a cross-flow detector cell (BAS, IN, USA), and a BDS-Hypersil C18 analytical column (3 × 100 mm², a 3 μm, Thermo Electron Corp., UK). The mobile phase was composed of 0.1 M monochloracetic acid adjusted to pH = 3.7 with 3 M sodium hydroxide, 0.5 mM EDTA, 25 mg/l 1-octanesulfonic acid sodium salt, 5.7% methanol and 2.5% acetonitrile. The flow rate was 0.5 ml/min, and the applied potential of a 3 mm glassy carbon electrode was + 600 mV with a sensitivity of 2 nA/V. The chromatographic data were processed by Chromax 2001 (Pol-Lab, Warszawa, Poland) software run on a personal computer. The values were not corrected for an in vitro probe recovery, which was approximately 15%.