The rats were sacrificed 3 h after the 1st access session on their 15th day of drinking. Brains were rapidly extracted and flash-frozen in isopentane in dry ice and stored at −80 C until sectioning. Brains were sectioned (300 μm) and the DRN micro-punched using procedures previously described (McBride et al., 2014). The DRN was punched from coronal sections between −7.30 mm to −8.30 mm post bregma according to the rat brain atlas by Paxinos and Watson (1998). All equipment used to obtain tissue was treated with RNase Zap (Life Technologies, Carlsbad, CA). Other brain regions of these animals have been studied (McBride et al., 2014). The animals used in these experiments were maintained in facilities fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC). All research protocols were approved by the institutional animal care and use committee and are in accordance with the guidelines of the Institutional Care and Use Committee of the National Institute on Drug Abuse, National Institutes of Health, and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council 1996).
