Specific inactivation of orexinergic neurons in wild-type mice by exposure to anesthetizing doses of isoflurane and sevoflurane. Coronal sections through the perifornical hypothalamus depict c-Fos staining (red nuclei) in orexinergic neurons (green cytoplasm, A–C) or in MCH neurons (green cytoplasm, D and E) prepared from adjacent sections of perifornical hypothalamus. (A) Nonanesthetized oxygen control mouse. (B) Isoflurane-anesthetized mouse. (C) Sevoflurane-anesthetized mouse. (D) Nonanesthetized oxygen control mouse. (E) Isoflurane-anesthetized mouse. (F) Bar graphs summarizing c-Fos expression in both neuronal populations. Arrows depict examples of double-positive neurons, and arrowheads mark MCH or orexinergic neurons that lack c-Fos expression. (Scale bar: A–E, 100 μm.) Insets D-1 and E-1 show higher magnification of MCH-positive, c-Fos-negative neurons, and Insets D-2, D-3, E-2, and E-3 show higher-power views of strong or weak c-Fos signals above background, which were all scored as c-Fos positive. (Scale bar: Insets, 20 μm.) All bar graphs reveal mean ± SEM counts. Cell counts were analyzed by ANOVA with post hoc Bonferroni correction for multiple comparisons. *, P < 0.05; ***, P < 0.001; both relative to nonanesthetized oxygen control group.