Prilocaine

30 mice were weighed and anesthetized using a ketamine (66.7 mg/kg; Henry Schein, Dublin, OH) and xylazine (6.67 mg/kg) cocktail administered by i.p. injection. 0.1 mL 1% Lidocaine HCl (Hospira, Inc., Lake Forest, IL) was subcutaneously applied above the skull. Green and red Retrobeads (0.2 – 0.3 μL/site; Lumafluor, Durham, NC), directed at ipsilateral BNST (AP +0.15, ML −1.00, DV −4.8; no angle) and VTA (AP −3.1, ML −0.50, DV −4.6; no angle) structures, were infused via Hamilton syringes. RetroBeads were intended to be used in combination with c-Fos IR to study neurocircuitry activated in response to binge-like drinking. However, since RetroBeads failed to exhibit measurable retrograde labeling these data were not quantified and are not described further. Following surgery conclusion, animals received topical analgesic cream containing Lidocaine and Prilocaine (25mg/g of each; Akorn Pharmaceuticals, Gurnee, IL). Subjects recovered for two weeks before undergoing testing (i.e., age at testing onset = 9 – 11 weeks). N=15 mice consumed only tap water during DID while n=15 consumed 20% ethanol. We used a larger sample size in this cohort relative to the surgery-naïve cohort described below as we anticipated loss of subjects due to missed RetroBead placement, but since we abandoned Retrobead assessment there was no need to remove animals.