Volume 7, Issue 2, Pages 179-185 (February 2008) Hypothalamic Neural Projections Are Permanently Disrupted in Diet-Induced Obese Rats  Sebastien G. Bouret, Judith N. Gorski, Christa M. Patterson, Stephen Chen, Barry E. Levin, Richard B. Simerly  Cell Metabolism  Volume 7, Issue 2, Pages 179-185 (February 2008) DOI: 10.1016/j.cmet.2007.12.001 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Disruption of Projection Pathways from ARH to the PVH in Offspring of Diet-Induced Obese Dams Confocal images (A and C) and quantitative comparisons (B and D) of arcuate DiI-labeled fibers in the paraventricular nucleus of the hypothalamus (PVH) of P12 (A and B) and P16 (C and D) offspring of diet-induced obese (DIO) and diet-resistant (DR) dams fed either a chow or high-energy (HE) diet throughout pregnancy and lactation. Pups born to DIO dams showed a significant reduction in the density of DiI-labeled fibers innervating the PVH relative to DR pups, regardless of maternal diet. Scale bar = 200 μm. Data are presented as mean ± SEM. p < 0.05 between a and b. Cell Metabolism 2008 7, 179-185DOI: (10.1016/j.cmet.2007.12.001) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Arcuate AgRP Projections to the PVH Are Altered in Adult DIO Rats Confocal images (A) and quantitative comparisons (B) of AgRP-immunoreactive (AgRP-IR) fibers innervating the PVH of adult DIO and DR offspring of dams fed chow or HE diet during pregnancy and lactation. DIO animals showed a significant reduction in the density of AgRP-IR fibers innervating the PVH relative to DR rats, regardless of maternal diet. V3, third ventricle. Scale bar = 160 μm. Data are presented as mean ± SEM. p < 0.05 between a and b. Cell Metabolism 2008 7, 179-185DOI: (10.1016/j.cmet.2007.12.001) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 αMSH Projections to the PVH in Adult DIO and DR Rats Confocal images (A) and quantitative comparisons (B) of αMSH-IR fibers innervating the PVH of adult DIO and DR offspring of dams fed chow or HE diet during pregnancy and lactation. No differences in αMSH-IR fiber density in the PVH were found between DIO and DR rats overall. However, offspring of chow-fed DIO animals showed a significant reduction in the density of αMSH-IR fibers innervating the PVH relative to DIO HE-diet offspring. V3, third ventricle. Scale bar = 160 μm. Data are presented as mean ± SEM. p < 0.05 between a and b. Cell Metabolism 2008 7, 179-185DOI: (10.1016/j.cmet.2007.12.001) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Response of ARH Neurons to Leptin Is Reduced in DIO Neonates (A) Leptin-induced phospho-STAT3 immunoreactivity (pSTAT3-IR) in the arcuate nucleus of the hypothalamus (ARH) of DR and DIO pups on P10. DIO neonates show a 24% reduction in the number of pSTAT3-IR cells in the ARH following leptin administration as compared to DR pups. (B and C) Isolated organotypic cultures of ARH explants from P6 DR (A) and DIO (B) rats were incubated for 36 hr with leptin (5 μg/ml) or vehicle and then immunostained with β III-tubulin, a marker of neurites. Leptin induces an approximately 2- to 3-fold increase in fiber density from ARH explants from DR rats. This effect was abolished in ARH explants derived from DIO rats. Scale bar = 100 μm. Data are presented as mean ± SEM. ∗p < 0.001 between control and leptin-treated explants. Cell Metabolism 2008 7, 179-185DOI: (10.1016/j.cmet.2007.12.001) Copyright © 2008 Elsevier Inc. Terms and Conditions