In vivo microdialysis for striatal DA release

Nomifensine (DAT and NET inhibitor) is dissolved in artificial cerebrospinal fluid (aCSF in mM: NaCl, 125; KCl, 2.5; CaCl2, 1.26 and MgCl2 1.18)

Concentrated solutions (1 mM; pH adjusted to 6.5–7 with NaHCO3 when necessary) are stored at -80ºC and working solutions are prepared daily by dilution in aCSF.

One concentric dialysis probe (Cuprophan membrane; 6000 Da molecular weight cut-off; 1.5 mm-long) is implanted in the striatum (AP, 0.5; ML, -1.7; DV, -4.5 in mm) of isoflurane-anesthetized mice

Microdialysis experiments are performed in freely-moving mice 24h after surgery.

Probes are perfused with aCSF at 1.5 μL/min

Following an initial 100-min stabilization period, 5 or 7 baseline samples are collected (20 min each) before local drug application

Nomifensine is administered by reverse dialysis at 10 and 50µM (uncorrected for membrane recovery) and then successive dialysate samples are collected

The concentration of DA in dialysate samples is determined by HPLC coupled to electrochemical detection (+0.7 V, Waters 2465), with 3-fmol detection limit.

The mobile phase containing 0.15 M NaH2PO4.H2O, 0.9 mM PICB8, 0.5 mM EDTA (pH 2.8 adjusted with orthophosphoric acid), and 10 % methanol is pumped at 1 ml/min (Waters 515 HPLC pump)

DA is separated on a 2.6 mm particle size C18 column (7.5 x 0.46 cm, Kinetex, Phenomenex) at 28°C.

Microdialysis data is expressed as femtomoles per fraction (uncorrected for recovery) and are shown as percentages of basal values (individual means of 5-7 pre-drug fractions).