Figure 5. Immunohistochemical evidence for activation of orexin neurons by handling stress.
A, schematic drawing of a coronal section of the mouse brain showing structure of the hypothalamus. A rectangle denotes examined area (711 × 944 μm). Both sides were examined in the actual experiment although only a right side window was depicted for simplicity. DMH, dorsomedial hypothalamus; f, fornix; LHA, lateral hypothalamic area; mt, mammillothalamic tract; PeF, perifornical area. B, representative photograph of double immunostaining for orexin and c-Fos in the hypothalamus of a wild-type (WT) mouse sampled after 2 h of repetitive handling stress. Orexin was stained in red and c-Fos was stained in green. Yellow designates cells stained for both orexin and c-Fos. Filled triangles indicate double-stained cells and open triangles indicate orexin-containing but not c-Fos-expressing cells. C, representative photograph of double immunostaining for LacZ (β-galactosidase) and c-Fos in the hypothalamus of an orexin knockout (ORX-KO) mouse sampled after 2 h of repetitive handling stress. LacZ gene was introduced into the ORX-KO mice instead of the normal orexin gene. LacZ was stained in red and c-Fos was stained in green. Yellow designates cells stained for both LacZ and c-Fos. Filled triangles indicate double-stained cells and open triangles indicate LacZ-containing but not c-Fos expressing cells. Bar, 100 μm in B and C. D, typical distribution of double-stained cells (filled circle), orexin-positive but not c-Fos positive cells (open circle), and c-Fos-positive but not orexin-positive cells (×) in the examination window of a WT mouse. E, typical distribution of double-stained cells (filled circle), LacZ-positive but not c-Fos-positive cells (open circle), and c-Fos positive but not LacZ-positive cells (×) in the examination window of a ORX-KO mouse. F, typical distribution of c-Fos-positive cells (×) in the examination window of a orexin neuron-ablated (ORX-AB) mouse. Note that there is no apparent difference in the distribution of c-Fos-positive cells among D, E and F. Bar, 200 μm in D–F. G, numbers of c-Fos-, orexin (ORX)- and LacZ-immunopositive cells and double-stained cells (c-Fos and ORX in WT or in ORX-AB, and c-Fos and LacZ in ORX-KO) in the hypothalamus. Every 4th section in an animal (6 sections per mouse) was examined. Data are presented as mean ±s.e.m. of 4 mice in a group except for handling-stressed ORX-KO mice (n = 3). *P < 0.05 vs. naïve. No orexin-immunopositive cell was detected (n.d.) in ORX-AB mice.