To harvest brain tissue, mice were given a lethal injection of ketamine/xylazine/ acepromazine. After loss of the pedal reflex, mice were transcardially perfused with 15 ml of saline and 15 ml of 10% buffered formalin. The brain was then extracted and stored in 10% buffered formalin for at least 1 day, and then, the brain was switched to 30% sucrose solution and stored for at least 1 more day. Brains were frozen with dry ice on a microtome and sectioned into 30 μM slices. The slices were mounted onto microscope slides and coverslipped with Fluoromount (Diagnostic BioSystems Inc., Sigma-Aldrich). A confocal microscope was used to image the slices (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany). For immunohistochemistry, brains were washed and placed in a blocking solution for 1 hour (10% donkey serum, 0.3% Triton X-100 dissolved in phosphate-buffered saline), and then incubated overnight at 4°C with the appropriate primary antibody. Then, after washing, they were incubated overnight at 4°C with the appropriate secondary antibody. Purple and green, rather than red and green, were used for ease of visual discrimination. The following antibodies were used:

Primary. c-Fos rabbit monoclonal antibody (mAb) (9F6) (#2250, Cell Signaling Technology)

Orexin A rabbit mAb (#H-003-30, Phoenix Pharmaceuticals)

Melanin-concentrating hormone rabbit mAb (#H-070-47, Phoenix Pharmaceuticals)

Secondary. Alexa Fluor 647–conjugated AffiniPure Donkey (H+L) anti-rabbit immunoglobulin G (Jackson ImmunoResearch)

Distance from bregma in millimeters is written next to the scale bar in each image; these are approximated with reference to Franklin and Paxinos (44).

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