Basal forebrain, olfactory bulb, anterior commissure, and hippocampus were manually segmented by blinded investigators to refine segmentation volumes. Manual segmentation reduced variation and mean differences between groups. For example, control hippocampal automated segmentation volumes ranged from 22 to 27.5 mm3 (coefficient of variation = 0.66) compared to manual hippocampal volumes that ranged from 24.5 to 28 mm3 (coefficient of variation = 0.45), likely due to the difficulty in resolving the most anterior and ventral regions of the hippocampus. Both automated and blinded manual segmentation indicated AE treated animals have slightly smaller, but not significantly different hippocampal volumes (Figure S4A). Though automatic segmentation reported significantly smaller anterior commissure in mice that underwent adolescent binge, no significant difference was found when segmented manually (p=0.31), indicating that AE treated animals may have slightly smaller, but not significantly different anterior commissure volumes (FigureS4B). However, manual segmentation of the olfactory bulb (Figure 7) and the basal forebrain/septum (Figure 8) confirmed significant reductions in volume in adult mice that received adolescent binge ethanol treatment. The mouse olfactory bulb is a large frontal brain region representing over
