Rapid sensing of circulating ghrelin by hypothalamicappetite-modifying neuronsMarie 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; bInstitutNational 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, UnitéMixte de Recherche 5221, Laboratoire Charles Coulomb, F-34095 Montpellier, France; gCisbio Bioassays, 30200 Codolet, France; hCentre National de laRecherche 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 arcuatenucleus must dynamically sense and integrate a multitude ofperipheral signals. Blood-borne molecules must therefore be ableto circumvent the tightly sealed vasculature of the blood–brainbarrier to rapidly access their target neurons. However, how in-formation encoded by circulating appetite-modifying hormones isconveyed to central hypothalamic neurons remains largely unex-plored. Using in vivo multiphoton microscopy together with fluo-rescently 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 feedingstatus. Thus, by virtue of its vascular connections, the hypothala-mus is able to directly sense peripheral signals, modifying energystatus accordingly.

hormone diffusion | in vivo imaging | median eminence | metabolism

Continuous integration of peripheral signals by neurons be-longing to the arcuate nucleus of the hypothalamus (ARH) is

critical for central regulation of energy balance and neuroendo-crine function (1). To dynamically report alterations to homeostasisand ensure an appropriate neuronal response, blood-borne factorssuch as hormones must rapidly access the central nervous system(CNS). This is particularly evident in the case of food intake, whichis regulated by a plethora of circulating satiety signals (2) whoselevels fluctuate in an ultradian manner. Despite this, it remainsunclear how key energy status-signaling hormones such as ghrelincan be rapidly sensed by target neurons to alter feeding re-sponses (3). Elucidation of the mechanisms underlying mole-cule entry into the brain is important for understanding not onlynormal maintenance of homeostasis but also how this is perturbedduring common pathologies such as obesity and diabetes (4, 5).Although molecule transport mechanisms within the ARH

are poorly characterized, they likely assume one of two forms.First, chronic feedback may be accomplished by uptake of cir-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 morphologicallylocated in close apposition to the median eminence (ME),a circumventricular organ composed of fenestrated capillaries.Because these vessels project toward the ventromedial ARH(vmARH), they could represent a direct vascular input for pas-sive diffusion of peripheral molecules into the hypothalamus (10–13). So far, study of the functional importance of fenestratedcapillaries in molecule entry into the metabolic brain has beenimpeded by lack of appropriate tools.To evaluate the role of fenestrated ME/ARH capillaries in

rapid detection of peripheral signals by the hypothalamus, weused a recently developed in vivo imaging approach to visualize

in real time the extravasation of fluorescent molecules (14). Ghrelinwas chosen as a candidate hormone because its acute effects uponfeeding behavior (15), together with a short circulating half-life,demand the presence of rapid and precise sensing mechanisms.Although ghrelin’s orexigenic effects on hypothalamic feedingcenters are well documented (16, 17), it remains unclear how pe-ripherally secreted hormone accesses this BBB-protected site, asa specialized transport system from the circulation to the brain isyet to be identified (18). Here, we show that circulating fluorescentlylabeled ghrelin diffuses through fenestrated capillaries of the ME,which project to the vmARH before rapidly binding nearby neu-ropeptide Y (NPY)- and proopiomelanocortin (POMC)-expressingneurons, the two functionally opposing neuron populations impli-cated in regulation of food intake in the ARH. Thus, our datasupport a role for ARH-residing neurons in eliciting ghrelin’seffects on feeding behavior through direct and rapid sensing ofcirculating ghrelin. Furthermore, we demonstrate that this processis inherently plastic as it can be manipulated in a nutrient-dependentmanner by simply using a controlled fasting–refeeding paradigm.As such, hypothalamic neurons are able to monitor peripheralenergy balance directly through their vascular inputs, allowingrapid organismal adaptation to prevailing metabolic state.

ResultsIn Vivo Permeability of Fenestrated Vessels in the Median Eminence.To access deep structures on the ventral surface of the brain anddirectly visualize vessels of the ME in vivo, surgical approachesdeveloped for functional imaging of the pituitary (14) werecombined with a multiphoton microscope adapted with long-working distance (2 cm) objectives (Fig. 1A). A coronal view ofthe ME and the ARH is schematized in Fig. 1A (Left), and arepresentative image of the median eminence vasculature (ven-tral view) is shown in Fig. 1A (Right). Fluorescence intensityvariations in the ME parenchyma were recorded in vivo follow-ing i.v. injection of fluorescent dextrans (Fig. 1B and Movie S1),and a transient increase in fluorescence intensity could be detected

Author contributions: M.S., B.D., P.-F.M., V.P., and P.M. designed research; M.S., F.L., andC.L. performed research; T.R., L.L., P.V., E.B., J.-L.B., J. Martinez, J. Marie, E.T., and J.-A.F.contributed new reagents/analytic tools; M.S., F.L., C.L., F.M., and D.J.H. analyzed data;and M.S., D.J.H., and P.M. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1M.S. and F.L. contributed equally to this work.2Present address: Section of Cell Biology, Division of Diabetes Endocrinology and Metab-olism, Department of Medicine, Imperial College London, London SW7 2AZ,United Kingdom.

3To whom correspondence should be addressed. E-mail: patrice.mollard@igf.cnrs.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1212137110/-/DCSupplemental.

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in the parenchyma when molecules were able to diffuse throughthe capillaries (Fig. 1B). Fluorescence intensity variations overtime postinjection in selected regions of interest were measured(Fig. 1B, color bars), allowing calculation of extravasation ratesacross fenestrated capillaries for each fluorescent molecule (Fig.1C). Molecule size cutoff was below 70 kDa, with a step-likedecrease in permeability rate between 20 and 40 kDa (Fig. 1C)(n = 4–16 movies from three to eight animals per molecule).To investigate the transfer rate of ghrelin (3.3 kDa), a recently

developed bioactive fluorescently labeled ghrelin derivative (3.8kDa), capable of specifically binding and activating growth hor-mone secretagogue receptor-1a (GHS-R-1a) (19, 20), was used.The extravasation rate of fluorescent ghrelin across the MEcapillary barrier was comparable to that of 4-kDa FITC-conju-gated dextran (∼0.8 μm/s) (Fig. 1C and Movie S2) (n = 8 moviesfrom three animals), suggesting that ghrelin extravasates pas-sively but rapidly (second range) through fenestrated capillarybranches of the ME, which are known to project to the vmARH(Fig. 2A, asterisk) (12, 13).

Ghrelin Rapidly and Specifically Binds to Neurons in the Vicinity ofFenestrated Capillaries in the ARH. We assessed rapid peptidebinding in the vicinity of fenestrated capillaries projecting deeperwithin the ARH (Fig. 2A, asterisk), by injecting a bolus of bio-active fluorescent ghrelin i.v. before euthanizing animals 5–10 minlater. Fluorescent ghrelin binding was localized to cell bodies in

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 fasthormone entry into the ARH was the observation that ghrelin-labeled neurons were located significantly closer to capillarybranches, 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 = 4animals) (Fig. 2 C and D). No ghrelin-labeled cells could bedetected under the same conditions in other BBB-protectedareas of the brain, such as the CA3 and CA1 regions (n = 3animals), 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 taggedhormone (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). Specificityof fluorescent ghrelin binding was assessed by competition ex-periments using pretreatment with excess native hormone orGHS-R-1a agonist; complete displacement of fluorescent ghrelinfrom neurons could be achieved (Fig. 3C; n = 3 animals percondition). In addition, i.v. injection of 25 nmol of either fixable3-kDa rhodamine-labeled dextran or inactive FITC-labeledghrelin before killing failed to label any cells in the ME/ARHregion (n = 3 animals per condition).

Ghrelin Labels Primarily Appetite-Modifying Neurons in a MetabolicState-Dependent Manner. To determine specificity of fluorescentghrelin binding in known ghrelin-responsive neuron populations,we used NPY-eGFP transgenic mice (21) and performed immu-nostaining for β-endorphin, a marker of POMC-neurons (25)(Fig. S1A). Of the 340 ± 19 (mean ± SEM) ghrelin-labeledneurons detected in the ARH (1,200 μm in length; divided into 24slices, 50 μm thick) (n = 11 animals, fed on standard chow), 35 ±4% and 41 ± 3% corresponded to NPY- and β-endorphin–expressing neurons, respectively (n = 4 animals per group) (Fig.S1B). Of note, only a small proportion of the total NPY- andβ-endorphin–expressing neuron population was labeled (up to3%, n= 4 animals/group), suggesting that only a subset of neuronswas targeted by ghrelin.Finally, we investigated whether feeding status of the animals

could alter fluorescent ghrelin binding, by subjecting animals toa controlled fasting–refeeding schedule. A significant increase innumbers of both total and NPY fluorescent ghrelin-positiveneurons was detected in 24-h–starved animals, and this could bereversed by refeeding the mice over a 24-h period (Fig. 4) (n = 11and 4 animals, respectively). By contrast, the number of ghrelin-labeled POMC neurons did not significantly vary (Fig. 4) (n =4 animals).

DiscussionThe regulation of a variety of homeostatic functions dependsupon central integration of peripheral feedback signals. Brain–periphery cross talk therefore requires tight control of moleculeentry and/or dynamic sensing to adapt responses to physiologicalstate. With obesity and diabetes reaching epidemic proportions,research efforts have focused on understanding how hormonessuch as ghrelin, insulin, or leptin functionally target appetite-regulating neurons of the hypothalamus. However, the funda-mental mechanisms by which peripherally secreted hormonesaccess hypothalamic neurons remain poorly characterized. Byusing tractable bioactive ghrelin together with in vivo multiphoton

Fig. 1. In vivo extravasation of molecules through fenestrated vessels in themedian 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 twotime points after i.v. injection of fluorescent ghrelin (Top), 4-kDa dextran(Middle), and 70-kDa dextran (Bottom). Fluorescence variations were mea-sured 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).

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imaging techniques, we have uncovered a functional role forfenestrated vessels of the ME/ARH in rapid blood-borne mole-cule sensing by the metabolic brain. It is anticipated that thesefindings will not only be valuable for understanding the basicmechanisms underlying appetite regulation but can also be ex-tended to a range of other homeostatic functions that rely onintegration of peripheral signals by the CNS.Fenestrated capillaries of the ME possess pores of 50–80 nm

in diameter, encompassed by a permeable diaphragm composedof radial fibrils (26, 27). Passive molecule diffusion throughfenestrae can be both size and charge limited (28), and it isreasonable to assume that the ME displays similar selectivity. Tostudy passive diffusion of molecules and exclude other potentialrate-limited transport mechanisms, it is necessary to follow thetime course of events at the blood–brain interface in real time.Using our approaches, we were able to show that relatively smallcirculating molecules (≤40 kDa in size) could freely and rapidlydiffuse through fenestrated capillaries of the ME, a finding withimplications for the study of non–receptor-dependent mecha-nisms of hormone and drug uptake into the CNS. Furthermore,as saturable transporters are modulated by physiological statusand pathological conditions (29, 30), modifications in vesselpermeability could constitute yet another level of regulation formolecule diffusion into the metabolic brain.

Because diffusion dynamics for inert sugars may differ fromthose for bioactive hormones, we investigated the diffusion offunctional fluorescently labeled ghrelin, a pleiotropic hormonefor which mechanism of entry into the ARH remains elusive(31). Our results demonstrate that ghrelin also crosses fenes-trated capillaries in the ME through passive diffusion. Althoughwe cannot exclude a role for additional receptor-mediated trans-port processes in the ARH, no specific ghrelin uptake mecha-nisms could be identified in the murine BBB (18), and the rateof transport of ghrelin across the BBB was much lower whenstudied by brain perfusion vs. i.v. injection (32). Although sys-temic administration of ghrelin has previously been shown toinduce hypothalamic c-fos expression (17), the current studiesdemonstrate sensing of hormone by ARH neurons over time-scales (5 vs. 30 min) that are more compatible with the acuteorexigenic effects of ghrelin on food intake (15). In addition, theclose proximity of ghrelin-labeled neurons to fenestrated capil-laries projecting from the ME into the ARH supports an im-portant functional role for this vascular route in rapid moleculeentry into the metabolic brain. In accordance with previousobservations (22), the absence of labeling in BBB-protectedareas distant from circumventricular organs suggests that directghrelin effects in other brain regions involved in nonhomeostaticfeeding behaviors (33–35) may be mediated through either indirect

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 minafter 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/ARHexpress 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 markerGlut1 (green). (D) Distances between ghrelin-labeled neurons and nearest Glut1-negative or Glut1-positive vessel (>600 neurons, n = 4 animals). Wilcoxonrank-sum test of medians (Left) and Kolmogorov–Smirnov analysis of cumulative frequency distributions (bin size, 2.5 μm) (Right) indicate ghrelin-labeledneurons are in closer vicinity to Glut1-negative vessels (***P < 0.001).

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actions (e.g., via neuronal relays) or slower transport mechanisms.Given that ME/ARH fenestrated capillaries are permissive formolecules as large as 20–40 kDa, it is not unfeasible that otherkey hormones involved in energy homeostasis such as leptin(16 kDa), insulin (5.8 kDa), or GLP-1 (4.1 kDa) might also usethis vascular route to access metabolic-sensing neurons.Reflecting its role in food intake regulation, fluorescent ghrelin

primarily bound appetite-modifying NPY and POMC neurons,and cytoplasmic labeling was consistent with the rapid internali-zation of a receptor–hormone complex (21). Although nearly allNPY neurons express GHS-R-1a receptors (36), and one-thirdup-regulate c-fos mRNA expression following GHS-R-1a agonisttreatment (37), we found that numbers of ghrelin-labeled NPYneurons were relatively small (∼3% of the population). The dif-ferent treatment timescales, marked species divergence in GHS-R-1a expression (rat vs. mouse) (38), and detection methods used(in situ hybridization vs. fluorescence) could explain the lowerproportion of detected NPY neurons. These numbers were none-theless within the range of that recently shown to be sufficient tostimulate food intake (∼300 neurons and above) (39). By contrast,labeling of POMC neurons was more surprising given that ghrelinis generally acknowledged to indirectly target this population viaGHS-R-1a–expressing presynaptic NPY neurons (25, 40). To-gether with ex vivo studies demonstrating direct effects of ghrelinon POMC neuron electrical activity (25), our results clearlysuggest that POMC neurons may be a direct target for ghrelinin vivo.Feeding status dynamically regulated the number of ghrelin-

labeled neurons. Although the number of POMC ghrelin-labeledneurons remained unchanged, as would be expected from a neu-ronal population that displays minimal activity in the fasted state(41), we observed a reversible increase in the number of NPY

ghrelin-labeled neurons following 24-h fasting. These resultsappear to be consistent with the physiological necessity to securerobust feeding responses following a fasting period, the directcorrelation between quantity of ingested food and number ofstimulated NPY neurons (39), the increase in GHS-R mRNA ex-pression in the ARH following short-term fasting (42, 43), and theregulation by nutritional state of the effects of ghrelin on Fosprotein expression in the ARH (17, 44). The unique vmARHmilieu irrigated by fenestrated capillaries may therefore not onlyprovide a niche for neurogenic tanycytes (45) but may also supporta population of “scout neurons” that are able to rapidly senseperipheral signals and coordinate more global responses.In summary, we have demonstrated that (i) circulating hor-

mones such as ghrelin can freely and rapidly diffuse throughfenestrated capillaries of the ME and into their site of actionwithin the ventromedial ARH; (ii) hypothalamic neurons mayplay an important role in eliciting ghrelin’s effects on feedingbehavior through direct and rapid sensing of circulating ghrelinlevels; (iii) the number of labeled NPY neurons is compatiblewith that recently shown to underlie acute ghrelin effects on foodintake (39); and (iv) feeding status modifies the capacity of NPYneurons to bind ghrelin. The use of in vivo imaging in combi-nation with the development of fluorescently labeled ligands thatretain biological activity could therefore help unveil access routesand in vivo neuronal targets not only for endogenous moleculesbut also for novel appetite-modifying drugs intended for weightmanagement.

Materials and MethodsA brief outline is provided; for full details, see SI Materials and Methods.

Mice and in Vivo Surgery. C57BL/6 mice were purchased from Janvier-SAS.NPY-eGFP mice (46) on a C57BL/6 background were sourced from The

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 frommice 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 slicefollowing 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 percondition). White, active fluorescent ghrelin.

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Jackson Laboratory. In all experiments, 7- to 15-wk-old male mice were used.All animal studies complied with the animal welfare guidelines of the Eu-ropean Community and were approved by the Direction of VeterinaryDepartments of Hérault and Nord, France (59-350134), and the LanguedocRoussillon Institutional Animal Care and Use Committees (CE-LR-0818). TheME was exposed using a surgery approach previously described for in vivostudies of the pituitary gland (14).

Long-Working Distance Multiphoton Imaging of Fluorescent Molecule Extravasationin Vivo. Imaging offluorescentmolecule extravasation in theME’s parenchymain vivo was performed using a multiphoton microscope (Zeiss 7MP) adaptedwith a long-working distance objective M Plan Apo NIR 20×, 0.4 NA, 2.0-cmWD (Mitutoyo). Fluorescently labeled molecules were injected through anindwelling jugular catheter. Recordings in areas rich in tortuous vessels atthe level of the ME and at a depth between 15 and 40 μm below the me-ninges, were started at the time of injection. Details of extravasation ratescalculation are provided in SI Materials and Methods.

Fluorescent Hormones Synthesis and Binding in Vivo. Both bioactive and in-active fluorescent ghrelin derivatives were developed by Cisbio Bioassays incollaboration with the Institut des Biomolécules Max Mousseron (Mont-pellier, France) (19). Active fluorescent ghrelin (25 nmol per animal) wasinjected i.v. into the tail vein or into the jugular vein under ketamine/xyla-zine anesthesia, and animals were killed 5–10 min postinjection, to assess in

vivo binding. To test the influence of metabolic state on ghrelin binding,active fluorescent ghrelin (25 nmol) was injected in either wild-type or NPY-eGFP mice (46) fed on standard chow (RM3; Special Diet Services), after 24 hof fasting, or after 24 h of fasting followed by a 24-h refeeding period, andanimals were killed 5–10 min postinjection.

Confocal Imaging. Terminally anesthetized mice were perfused via the heartwith 10 mL of PBS followed by 30 mL of 4% paraformaldehyde solution. Insome experiments, vessels were labeled using rhodamine-labeled lectin (400μg per mouse; Vector Laboratories) diluted in the perfusate (PBS). Brainswere collected and prepared for confocal imaging. Details are provided in SIMaterials and Methods.

ACKNOWLEDGMENTS. We thank Dr. B. Engelhardt for antibodies to MECA-32; Dr. P. Le Tissier and Prof. D. G. Grattan for useful comments onthe manuscript; and P. Fontanaud, M. Asari, G. Osterstock, M. Granier,P. Samper, and A. Guillou for technical assistance. We were supported byAgence Nationale de la Recherche (Gliodiabesity, PCV08_323168), InstitutNational de la Santé et de la Recherche Médicale, Centre National de laRecherche Scientifique, Universities of Montpellier 1 and 2, University of Lille2, National Biophotonics and Imaging Platform (Ireland), Infrastructures enBiologie, Santé et Agronomie, the Fondation pour la Recherche Medicale,Diabetes UK (R. D. Lawrence Fellowship), Institut Fédératif de Recherches3 and 114, and Région Languedoc Roussillon (Imagerie du Petit Animal deMontpellier).

Fig. 4. Fluorescent ghrelin labels NPY but not β-endorphin–expressing neurons in a metabolic state-dependent manner. (A) Confocal images of ME/ARHregion following i.v. injection of fluorescent ghrelin; NPY-eGFP transgenic mouse (Top) (green, GFP) and β-endorphin immunostaining (Bottom) (red). Theorange 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 undercontrol 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 werenormalized 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 NPYghrelin-labeled neurons, which was reversed by refeeding (one-way ANOVA, ***P < 0.001), whereas fasting had no significant effect on the total number ofghrelin-labeled β-endorphin–expressing neurons (one-way ANOVA, P > 0.05).

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