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. Author manuscript; available in PMC: 2011 Jan 27.
Published in final edited form as: Neuropathol Appl Neurobiol. 2009 Dec 8;36(4):285–299. doi: 10.1111/j.1365-2990.2009.01057.x

Figure 5.

Figure 5

No evidence of loss of orexin (OreA) or melanin-concentrating hormone (MCH) neurones, but early-onset reactive gliosis in Ubb−/− mice. (A) Colocalization of OreA and MCH immunoreactive neurones in the lateral hypothalamic area to Ubb-expressing neurones. Coronal brain sections were stained for OreA and MCH, DNA was visualized using TO-PRO-3 iodide, and green fluorescent protein (GFP) fluorescence was visualized directly from GFP-Puror protein. Representative confocal images from 4-month-old male Ubb+/− mice (n = 3) are shown. Scale bar, 200 μm; inset, 100 μm. (B) Loss of α-melanocyte-stimulating hormone (α-MSH), but not OreA and MCH, immunoreactive neurones in the hypothalamic sections of Ubb−/− mice. Sleep abnormalities in Ubb−/− mice are not attributable to the loss of OreA or MCH neurones. For quantification of neurones, cell counts were obtained from three coronal sections (200 μm interval) generated from each brain and compared with wild-type (+/+) controls. Images are representative of 5-month-old male mice for each group (+/+, n = 3; −/−, n = 3). Representative sections are from bregma −1.75 mm for α-MSH (second set), −1.6 mm for OreA (first set) and −1.85 mm for MCH (second set). Scale bar, 200 μm. Data are expressed as mean ± SEM from the indicated number of mice. *P < 0.05 vs. wild-type (+/+) mice. (C) Coronal brain sections were stained for glial fibrillary acidic protein (GFAP) and DNA was visualized using TO-PRO-3 iodide. Representative confocal images from 1-month-old wild-type and Ubb−/− mice (n = 3 per genotype) are shown. GFAP immunoreactivity was increased throughout the brain sections from the Ubb−/− mice, except for the subcortical regions (top). In the hypothalamus, GFAP immunoreactivity was most prominent in the arcuate nucleus (bottom). Scale bar, 200 μm. (D) Coronal brain sections were stained for NeuN to visualize neurones. Representative images from 4-month-old wild-type and Ubb−/− mice (n = 3 per genotype) are shown. NeuN-positive neurone number is similar between two genotypes in various brain regions such as HPC, CTX, and BLA. Scale bar, 500 μm. 3V, third ventricle; ARC, arcuate nucleus; BLA, basal amygdala; CTX, cerebral cortex; DMH, dorsomedial hypothalamus; HPC, hippocampus; LHA, lateral hypothalamic area; VMH, ventromedial hypothalamus.