Rapid Sensing of Circulating Ghrelin by Hypothalamic Appetite-Modifying Neurons
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Rapid sensing of circulating ghrelin by hypothalamic appetite-modifying neurons Marie Schaeffera,b,c,1, Fanny Langletd,e,1, Chrystel Lafonta,b,c, François Molinoa,b,c,f, David J. Hodsona,b,c,2, Thomas Rouxg, Laurent Lamarqueg, Pascal Verdiéh, Emmanuel Bourrierg, Bénédicte Dehouckd,e,i, Jean-Louis Banèresh, Jean Martinezh, Pierre-François Mérya,b,c, Jacky Marieh, Eric Trinquetg, Jean-Alain Fehrentzh, Vincent Prévotd,e, and Patrice Mollarda,b,c,3 aCentre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France; bInstitut National de la Santé et de la Recherche Médicale, Unité 661, F-34000 Montpellier, France; cUniversities of Montpellier 1 and 2, Unité Mixte de Recherche 5203, F-34000 Montpellier, France; dInstitut National de la Santé et de la Recherche Médicale, Jean-Pierre Aubert Research Center, Unité 837, F-59000 Lille, France; eFaculté de Médecine, Université Droit et Santé de Lille, F-59000 Lille, France; fUniversity Montpellier 2, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5221, Laboratoire Charles Coulomb, F-34095 Montpellier, France; gCisbio Bioassays, 30200 Codolet, France; hCentre National de la Recherche Scientifique, Unité Mixte de Recherche 5247, Institut des Biomolécules Max Mousseron, Faculty of Pharmacy, Universities of Montpellier 1 and 2, F-34093 Montpellier Cedex 5, France; and iUniversité d’Artois, F-62800 Liévin, France Edited* by Tomas G. M. Hökfelt, Karolinska Institutet, Stockholm, Sweden, and approved December 7, 2012 (received for review July 24, 2012) To maintain homeostasis, hypothalamic neurons in the arcuate in real time the extravasation of fluorescent molecules (14). Ghrelin nucleus must dynamically sense and integrate a multitude of was chosen as a candidate hormone because its acute effects upon peripheral signals. Blood-borne molecules must therefore be able feeding behavior (15), together with a short circulating half-life, to circumvent the tightly sealed vasculature of the blood–brain demand the presence of rapid and precise sensing mechanisms. barrier to rapidly access their target neurons. However, how in- Although ghrelin’s orexigenic effects on hypothalamic feeding formation encoded by circulating appetite-modifying hormones is centers are well documented (16, 17), it remains unclear how pe- conveyed to central hypothalamic neurons remains largely unex- ripherally secreted hormone accesses this BBB-protected site, as plored. Using in vivo multiphoton microscopy together with fluo- a specialized transport system from the circulation to the brain is rescently labeled ligands, we demonstrate that circulating ghrelin, yet to be identified (18). Here, we show that circulating fluorescently a versatile regulator of energy expenditure and feeding behavior, labeled ghrelin diffuses through fenestrated capillaries of the ME, rapidly binds neurons in the vicinity of fenestrated capillaries, which project to the vmARH before rapidly binding nearby neu- and that the number of labeled cell bodies varies with feeding ropeptide Y (NPY)- and proopiomelanocortin (POMC)-expressing status. Thus, by virtue of its vascular connections, the hypothala- neurons, the two functionally opposing neuron populations impli- mus is able to directly sense peripheral signals, modifying energy cated in regulation of food intake in the ARH. Thus, our data status accordingly. support a role for ARH-residing neurons in eliciting ghrelin’s effects on feeding behavior through direct and rapid sensing of hormone diffusion | in vivo imaging | median eminence | metabolism circulating ghrelin. Furthermore, we demonstrate that this process is inherently plastic as it can be manipulated in a nutrient-dependent ontinuous integration of peripheral signals by neurons be- manner by simply using a controlled fasting–refeeding paradigm. Clonging to the arcuate nucleus of the hypothalamus (ARH) is As such, hypothalamic neurons are able to monitor peripheral critical for central regulation of energy balance and neuroendo- energy balance directly through their vascular inputs, allowing crine function (1). To dynamically report alterations to homeostasis rapid organismal adaptation to prevailing metabolic state. and ensure an appropriate neuronal response, blood-borne factors such as hormones must rapidly access the central nervous system Results (CNS). This is particularly evident in the case of food intake, which In Vivo Permeability of Fenestrated Vessels in the Median Eminence. is regulated by a plethora of circulating satiety signals (2) whose To access deep structures on the ventral surface of the brain and levels fluctuate in an ultradian manner. Despite this, it remains directly visualize vessels of the ME in vivo, surgical approaches unclear how key energy status-signaling hormones such as ghrelin developed for functional imaging of the pituitary (14) were can be rapidly sensed by target neurons to alter feeding re- combined with a multiphoton microscope adapted with long- sponses (3). Elucidation of the mechanisms underlying mole- working distance (2 cm) objectives (Fig. 1A). A coronal view of cule entry into the brain is important for understanding not only the ME and the ARH is schematized in Fig. 1A (Left), and a normal maintenance of homeostasis but also how this is perturbed representative image of the median eminence vasculature (ven- during common pathologies such as obesity and diabetes (4, 5). tral view) is shown in Fig. 1A (Right). Fluorescence intensity Although molecule transport mechanisms within the ARH variations in the ME parenchyma were recorded in vivo follow- fl are poorly characterized, they likely assume one of two forms. ing i.v. injection of uorescent dextrans (Fig. 1B and Movie S1), fl First, chronic feedback may be accomplished by uptake of cir- and a transient increase in uorescence intensity could be detected culating molecules into the ARH via saturable receptor-medi- ated transport at the level of the choroid plexus and/or blood– brain barrier (BBB) (6–9). Second, the ARH is morphologically Author contributions: M.S., B.D., P.-F.M., V.P., and P.M. designed research; M.S., F.L., and C.L. performed research; T.R., L.L., P.V., E.B., J.-L.B., J. Martinez, J. Marie, E.T., and J.-A.F. located in close apposition to the median eminence (ME), contributed new reagents/analytic tools; M.S., F.L., C.L., F.M., and D.J.H. analyzed data; a circumventricular organ composed of fenestrated capillaries. and M.S., D.J.H., and P.M. wrote the paper. Because these vessels project toward the ventromedial ARH The authors declare no conflict of interest. (vmARH), they could represent a direct vascular input for pas- – *This Direct Submission article had a prearranged editor. sive diffusion of peripheral molecules into the hypothalamus (10 1M.S. and F.L. contributed equally to this work. 13). So far, study of the functional importance of fenestrated 2Present address: Section of Cell Biology, Division of Diabetes Endocrinology and Metab- capillaries in molecule entry into the metabolic brain has been olism, Department of Medicine, Imperial College London, London SW7 2AZ, impeded by lack of appropriate tools. United Kingdom. To evaluate the role of fenestrated ME/ARH capillaries in 3To whom correspondence should be addressed. E-mail: [email protected]. rapid detection of peripheral signals by the hypothalamus, we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. used a recently developed in vivo imaging approach to visualize 1073/pnas.1212137110/-/DCSupplemental. 1512–1517 | PNAS | January 22, 2013 | vol. 110 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1212137110 Downloaded by guest on September 26, 2021 both the ME and vmARH, corresponding to hypothalamic HuC/ D-positive neurons (100%; n = 18 slices from three animals) (Fig. 2B). Intracellular labeling was consistent with rapid in- ternalization of GHS-R-1a bound to fluorescent ghrelin (21). Supporting a functional role of fenestrated capillaries in fast hormone entry into the ARH was the observation that ghrelin- labeled neurons were located significantly closer to capillary branches, which did not express the BBB marker Glut1 com- pared with BBB-protected Glut1-positive vessels (26.6 ± 1.6 vs. 100.8 ± 2.9 μm, mean ± SEM, 123–250 neurons per animal, n = 4 animals) (Fig. 2 C and D). No ghrelin-labeled cells could be detected under the same conditions in other BBB-protected areas of the brain, such as the CA3 and CA1 regions (n = 3 animals), which densely express GHS-R-1a (22). Because expression levels of the immediate-early genes, c-fos, are up-regulated in the ARH following systemic ghrelin admin- istration (17), the induction of Fos protein was measured in hy- pothalamic neurons following in vivo treatment with tagged hormone (Fig. 3A). Two hours after fluorescent ghrelin injection, an increase in c-Fos expression was observed in the ARH, com- parable with that obtained following i.v. injection of native rat/ mouse ghrelin and GHS-R-1a agonists JMV4336 and MK-0677 (23, 24) (Fig. 3 A and B; n = 3 animals per condition). Specificity of fluorescent ghrelin binding was assessed by competition ex- periments using pretreatment with excess native hormone or GHS-R-1a agonist; complete displacement of fluorescent ghrelin from neurons could be achieved (Fig. 3C; n = 3 animals per condition). In addition, i.v. injection of 25 nmol of either fixable NEUROSCIENCE 3-kDa rhodamine-labeled dextran or inactive FITC-labeled ghrelin before killing failed to label any cells in the ME/ARH = Fig. 1. In vivo extravasation of molecules through fenestrated vessels in the region (n 3 animals per condition). median eminence (ME). (A) Schematic representation of the imaging setup (Left), and representative image of the ME vasculature acquired in vivo Ghrelin Labels Primarily Appetite-Modifying Neurons in a Metabolic (Right). Z-projection of a 100-μm stack. Green, The 150-kDa dextran-FITC. State-Dependent Manner. To determine specificity of fluorescent (Scale bar: 130 μm.) (B) Fluorescence variation in the ME parenchyma at two ghrelin binding in known ghrelin-responsive neuron populations, time points after i.v.