(A) Brain schematic of optogenetic surgery whereby Agrp-IRES-Cre animals were unilaterally injected with Cre-dependent ChR2-tdTomato virus and unilaterally implanted with an optical fiber over the ARC (left). Intruder animals were tested under five discrete appetite conditions (right). (B) Intruders for the assay were group-housed until the experiments. Each sexually-mature female was used only once to ensure virgin status and avoid variables associated with pregnancy, ensuing maternal experience, and social stress related to cagemates’ exposure to the female after the mating experience. Similarly, each male intruder was used only once to avoid further injury and prevent development of social defeat stress or learned helplessness, depressive-like occurrences resulting from repeated bouts of aggression from more dominant mice. (C) All virgin female intruders used tested positive for estrus. Arrows represent different cells in standard vaginal cytology samples throughout the estrous cycle (black arrows, nucleated epithelial cells; red arrows, cornified epithelial cells; blue arrows, leukocytes). (D) Each intruder animal was tested for 1 hr over 3 different 20 min phases; a Pre-Phase (grey color) in the absence of food and an intruder (left panel), a Trial Phase (blue color) which could occur in the intruder homecage in the presence of food but the absence of an intruder (middle panel, top), in the occupied, territorialized resident cage in the presence of both food and an intruder (middle panel, center) or in the empty, territorialized resident cage in the presence of food but the absence of an intruder (middle panel, bottom) and a Post-Phase (pink color) in the empty, territorialized residents cage in the presence of food but the absence of an intruder (right panel). Food intake was assessed first in the intruder homecage, then the empty resident cage and finally in the occupied resident cage over 2 weeks. Red and green circles on the feeding device indicate food inaccessibility and accessibility, respectively.