2.3. Drinking in the dark (DID)

Binge-like drinking was measured using the two-bottle DID procedure as previously described using 20% ethanol (Dutton et al., 2017). Mice were individually housed for 2 weeks in the reverse-dark cycle room prior to testing ethanol consumption. Three days before testing ethanol consumption, mice drank water from two tubes made from 10 ml polycarbonate pipettes connected to stainless steel sipper tubes containing ball-bearings to prevent leakage (Rhodes et al., 2005) for one day, in order to acclimate them to the tubes. Three days later mice were given a choice between 20% ethanol and water in the sipper tubes. Fluid consumption was measured every day for 4 days by measuring the volume of fluid in the tubes. The position of the bottles (left or right) was changed every day to control for side preference. On the first 3 days of testing, mice were given access to the ethanol and water tubes 3 h into the dark cycle for a period of 2 h. On the fourth day, mice were given access to ethanol and water tubes for 4 h and the volume consumed was measured at 4 h. All mice were given vehicle (0.1 ml) by oral gavage on days 1 & 2. On the third and fourth days, mice were administered 60 mg/kg MY10, MY33-3 or vehicle (0.1 ml) by oral gavage 1 hour before the drinking session in the DID test (n = 12/group). Preference score was calculated as the ratio of the volume of ethanol consumed over the volume of total fluid consumed (Chen et al., 2017; Dutton et al., 2017). For the sucrose consumption test, a separate group of mice were tested exactly as in the ethanol consumption test, except that mice were provided with two tubes containing 2% sucrose in water and water instead of 20% ethanol and water.

Blood samples (20 μl) were collected immediately after the 4-hour drinking session on day 4 to measure blood ethanol concentrations (BECs). Blood was collected in heparinized capillary tubes via tail vein puncture. BECs were determined using a nicotinamide adenine dinucleotide-alcohol dehydrogenase enzymatic assay (Zapata et al., 2006).