On the day of preparation of micro-punch samples, brains were transferred to a cryostat set at −6 to −10°C at least 2 hours prior to sectioning. Sections (300 μm) were obtained and transferred to glass slides that had been pre-cooled in the cryostat. Micro-punch sampling was done on a frozen stage (−25 to −35°C) with an anatomic microscope equipped with a cool microscope lamp. The stereotaxic atlas of Paxinos and Watson (1998) was used to identify the VTA. Micro-dissection needles (Fisher Scientific) with an inner diameter of 0.77 mm were used to obtain the VTA. This inner diameter fits within the entire region and minimizes contamination from adjacent tissue. Punches were taken bilaterally from 2–3 sections. A different fresh sterile micro-punch needle was used for each animal. After withdrawing the micro-punch sample, a distinct demarcated hole remained; this hole was used to validate the micro-dissection method. All equipment used to obtain tissue was treated with RNAse Zap (Ambion, Inc. Austin, TX) to prevent RNA degradation. A second trained individual independently verified the quality of the micro-punch dissections.
