Surgery and Experimental Procedures

Rats were anesthetized by intraperitoneal (i.p.) injection of chloral hydrate (0.4 g/kg), and a concentric dialysis probe was inserted at the level of the mPFC (A +3.2, ML +0.8, V −5.3 relative to the bregma), according to the Paxinos and Watson (1982) Atlas. The active length of the dialysis membrane (Hospal Dasco, Bologna, Italy) was restricted to 4 mm. As previously described (Dazzi et al., 2014), the length of the dialitycal membrane allowed to sample from both infralimbic and prelimbic cortices. Experiments were performed in freely moving rats, 24 hafter probe implantation to allow recovery from surgery procedures. Ringer’s solution [3 mM KCl, 125 mM NaCl, 1.3 mM CaCl₂, 1 mM MgCl₂, 23 mM NaHCO₃, 1.5 mM potassium phosphate (pH 7.3)] was pumped through the dialysis probe at a constant rate of 2 μl/min. Samples of dialysate were collected every 20 min from 8:30 to 15:00 h and immediately analyzed for dopamine by high performance liquid chromatography (HPLC) with electrochemical detection as previously described (Dazzi et al., 2002b); the detection limit for dopamine was 2 fmol per injection. The average neurotransmitter concentration in the first two samples was taken as 100%, and all subsequent values were expressed as mean ± SEM relative to the basal value. The mean in vitro recovery of the probes was 15 ± 3%. All probes were tested before implantation, and those with a recovery value outside of this range were not used. The absolute concentration of dopamine was not corrected for this value. At the end of each experiment, the placement of the probe was verified histologically. All rats in which the probe was located outside of the target region were excluded from the analysis. A group of rats were subjected to an acute treatment with ethanol (0.5, 2 g/kg, i.p., 20% solution v/v); the drug was injected after three stable samples (variation in dopamine concentrations less than 20%). For the acute ethanol administration experiments the average neurotransmitter concentration in the first three samples was taken as 100%, and all subsequent values were expressed as mean ± SEM relative to the basal value.