the 66th generation of the second replicate were used. Replicate lines are used to ensure that the alcohol preference selection phenotype could be produced in multiple lines. Our choice to only test replicate two here was based on the similar physiological measures between replicates two and three that we found previously (Fritz et al. 2019) and that the first LAP replicate is currently extinct. To minimize pain and distress of animals, animals were anesthetized under isoflurane by placement into a desiccator containing a gauze pad with approximately 200 uL of isoflurane. After approximately 30 seconds in the desiccator, animals were removed and rapid decapitation for tissue collection was performed after animals were assessed for the absence of a pedal withdrawal reflex. Isoflurane was chosen over other anesthetics due to the rapid onset of action and minimally invasive method (i.e. no injections) for deeply anesthetizing animals prior to decapitation (Gargiulo et al. 2012). Following longer periods of isoflurane exposure usually intended to model a surgical procedure, others have reported a significant impact of isoflurane on the brain phosphoproteome (Kohtala et al. 2016), protein kinases (Tao et al. 2016), and phosphatases (Cao et al. 2014). Given our very brief period of isoflurane
