In situ hybridization was performed (on 20 µm-thick cryosections as previously described to measure PENK mRNA expression levels) [29], [30]. The PENK riboprobe was an EcoRI/Pvu 792 bp fragment complementary to the full coding region of the PENK human gene [31]. Briefly, brain sections were hybridized with 20×103 CPM/ µl [35S]-αUTP PENK riboprobe solution overnight at 55°C and following post-hybridization washes, exposed to Kodak Biomax MR film for 5 days. Optical density values measurements were taken over subregions of the striatum and the central amygdala and the values converted to DPM (disintegrations per minute)/mg by reference to co-exposed C14 standards (American Radiolabeled Chemicals, Inc., St. Louis, MO). DPM/mg values from duplicate slides were averaged.
