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. 2013 Jan 22;110(4):1512-7.
doi: 10.1073/pnas.1212137110. Epub 2013 Jan 7.

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons

Marie Schaeffer  1 , Fanny Langlet, Chrystel Lafont, François Molino, David J Hodson, Thomas Roux, Laurent Lamarque, Pascal Verdié, Emmanuel Bourrier, Bénédicte Dehouck, Jean-Louis Banères, Jean Martinez, Pierre-François Méry, Jacky Marie, Eric Trinquet, Jean-Alain Fehrentz, Vincent Prévot, Patrice Mollard
Affiliations

Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons

Marie Schaeffer et al. Proc Natl Acad Sci U S A. .

Abstract

To maintain homeostasis, hypothalamic neurons in the arcuate nucleus must dynamically sense and integrate a multitude of peripheral signals. Blood-borne molecules must therefore be able to circumvent the tightly sealed vasculature of the blood-brain barrier to rapidly access their target neurons. However, how information encoded by circulating appetite-modifying hormones is conveyed to central hypothalamic neurons remains largely unexplored. Using in vivo multiphoton microscopy together with fluorescently labeled ligands, we demonstrate that circulating ghrelin, a versatile regulator of energy expenditure and feeding behavior, rapidly binds neurons in the vicinity of fenestrated capillaries, and that the number of labeled cell bodies varies with feeding status. Thus, by virtue of its vascular connections, the hypothalamus is able to directly sense peripheral signals, modifying energy status accordingly.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In vivo extravasation of molecules through fenestrated vessels in the median eminence (ME). (A) Schematic representation of the imaging setup (Left), and representative image of the ME vasculature acquired in vivo (Right). Z-projection of a 100-µm stack. Green, The 150-kDa dextran-FITC. (Scale bar: 130 µm.) (B) Fluorescence variation in the ME parenchyma at two time points after i.v. injection of fluorescent ghrelin (Top), 4-kDa dextran (Middle), and 70-kDa dextran (Bottom). Fluorescence variations were measured in regions of interest (color bars). (Scale bar: 20 µm.) Green, FITC. (C) Molecule extravasation rate in vivo as a function of molecular weight (mean ± SEM, n = 4–16 movies from 3 to 8 animals per molecule).
Fig. 2.
Fig. 2.
Fluorescent ghrelin binds to neurons in the ME/ARH region in proximity to BBB-free vessels. (A–C) Confocal images of brain frozen sections 5–10 min after i.v. injection of fluorescent ghrelin (B and C). (Scale bar: 100 µm.) (A–C) Blue, Nuclei (Hoechst). (A) Capillaries (rhodamine-lectin, red) in the ME/ARH express the fenestration marker MECA-32 (green) and project to the ARH (asterisk). (B) Fluorescent ghrelin (white) labels hypothalamic neurons (HuC/D, red). (C) Ghrelin-labeled neurons (white) are primarily located in the vicinity of vessels (CD31, red) (orange arrows) not expressing the blood–brain barrier marker Glut1 (green). (D) Distances between ghrelin-labeled neurons and nearest Glut1-negative or Glut1-positive vessel (>600 neurons, n = 4 animals). Wilcoxon rank-sum test of medians (Left) and Kolmogorov–Smirnov analysis of cumulative frequency distributions (bin size, 2.5 µm) (Right) indicate ghrelin-labeled neurons are in closer vicinity to Glut1-negative vessels (***P < 0.001).
Fig. 3.
Fig. 3.
Fluorescent ghrelin activity and binding specificity. (A and C) Representative confocal images of coronal sections of the ME/ARH region. Twenty-micrometer z-projections are shown. (Scale bar: 150 µm.) (A) Fluorescent bioactive ghrelin induces c-Fos expression in the ARH. Images of brain slices from mice killed 2 h after i.v. injection of 0.9% NaCl (Left), 25 nmol of inactive fluorescent ghrelin (Center), or 25 nmol of active fluorescent ghrelin (Right). Blue, Hoechst, red, c-Fos. The white arrows indicate c-Fos–positive nuclei. (B) Quantification of the number of c-Fos–positive nuclei in the ARH per 20-µm-thick slice following treatment with different GHS-R-1a agonists. All active agonists induced c-Fos in a significant manner compared with saline or inactive ghrelin (one-way ANOVA, ***P < 0.001, n = 12–30 slices from three animals per condition). (C) Competition experiment. Injection (i.v.) of commercial ghrelin (rat, mouse) 15 min before i.v. injection of active fluorescent ghrelin prevented labeling of cell bodies in the ARH (right panel vs. control left panel) (n = 3 animals per condition). White, active fluorescent ghrelin.
Fig. 4.
Fig. 4.
Fluorescent ghrelin labels NPY but not β-endorphin–expressing neurons in a metabolic state-dependent manner. (A) Confocal images of ME/ARH region following i.v. injection of fluorescent ghrelin; NPY-eGFP transgenic mouse (Top) (green, GFP) and β-endorphin immunostaining (Bottom) (red). The orange arrowheads indicate ghrelin-labeled (white) NPY (top) or β-endorphin (bottom) neurons under control conditions (Left), following 24-h fasting (Center), and 24-h fasting plus 24-h refeeding (Right). (Scale bar: 100 µm.) (B) Quantification of ghrelin-labeled neurons in the whole ME/ARH region under control conditions, following 24-h fasting, and 24-h fasting plus 24-h refeeding (15–24 slices per animal, n = 4–11 animals per condition). Numbers were normalized to correspond to total ME length (1,200 µm; 24 slices, 50 µm thick). Fasting induced a significant increase in both total number and number of NPY ghrelin-labeled neurons, which was reversed by refeeding (one-way ANOVA, ***P < 0.001), whereas fasting had no significant effect on the total number of ghrelin-labeled β-endorphin–expressing neurons (one-way ANOVA, P > 0.05).
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