Experiment 8: In vivo Brain Microdialysis in Rats

Microdialysis experiments were performed in six additional groups of rats to evaluate the effects of vehicle (5% Kolliphor solution) or BCP (25, 50 mg/kg) alone on basal levels of extracellular DA or BCP pretreatment on METH-enhanced NAc dopamine (DA). Microdialysis protocols and probe construction were as reported previously (Xi et al., 2006). Guide cannulae (20 gauge; Plastics One, Roanoke,VA) were surgically implanted into the NAc (anteroposterior, +1.6 mm; mediolateral, ±1.8 mm; dorsoventral,—4.3 mm, angled 6° from vertical) using standard surgical and stereotaxic techniques. Microdialysis probes were inserted into the NAc 12 h before the experiment to minimize damage-induced neurotransmitter release. During the experiment, microdialysis buffer was perfused through the probe (2.0 ml/min) for at least 2 h before sampling started. Samples were collected every 20 min into 10 μl of 0.5 M perchloric acid to prevent neurotransmitter degradation. After 1 h baseline collection, one of the two doses of BCP (25, 50 mg/kg, i.p.) or vehicle (5% Kolliphor solution) were administered 40 min prior to METH administration. All samples were frozen at 80°C until analyzed. After microdialysis experiments were completed, rats were anesthetized with a high dose of pentobarbital (>100 mg/kg i.p.) and perfused transcardially with 0.9% saline followed by 10% formalin. Brains were removed and placed in 10% formalin for histological verification of microdialysis probe locations in rat brain.

Microdialysate DA was measured by high performance liquid chromatography (HPLC) with an ESA (ESA Biosciences, Chelmsford, MA) electrochemical (EC) detection system as described previously (Xi et al., 2006), upgraded by a Coulochem III EC detector. Areas under the curve (AUC) for DA were measured and quantified with external standard curves. The minimum detection limit for DA was 1–10 fmol.