On the day of preparation of micro-punch samples, brains were transferred to a cryostat set at -6 to -10° C at least 2 hr 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. Micropunch samples (0.77 mm dia.) were obtained bi-laterally; usually samples of the ACB-shell could be obtained from 2-3 sections from each rat. The stereotaxic atlas of Paxinos and Watson (1998) was used to identify the ACB-shell. After withdrawing the micro-punch sample, a distinct demarcated hole remained; this hole was used to validate the micro-dissection method. Two trained individuals independently verified the dissections. The micro-punched samples from the same animal were pooled and stored at -70° C until samples from all rats had been collected. Samples from one animal were not pooled with samples from another animal.
