ventromedial arcuate nucleus communicates peripheral ......ventromedial arcuate nucleus communicates...

Ventromedial Arcuate Nucleus CommunicatesPeripheral Metabolic Information to theSuprachiasmatic Nucleus

Chun-Xia Yi, Jan van der Vliet, Jiapei Dai, Guangfu Yin, Liqiang Ru, and Ruud M. Buijs

Netherlands Institute for Brain Research (C.-X.Y., J.v.d.V., J.D., R.M.B.) 1105AZ, Amsterdam, The Netherlands; and TongjiMedical College of Huazhong University of Science and Technology (J.D., G.Y., L.R.), 430030 Wuhan, Peoples Republic ofChina

The arcuate nucleus (ARC) is crucial for the maintenance ofenergy homeostasis as an integrator of long- and short-termhunger and satiety signals. The expression of receptors formetabolic hormones, such as insulin, leptin, and ghrelin, al-lows ARC to sense information from the periphery and signalit to the central nervous system. The ventromedial ARC(vmARC) mainly comprises orexigenic neuropeptide agouti-related peptide and neuropeptide Y neurons, which are sen-sitive to circulating signals. To investigate neural connectionsof vmARC within the central nervous system, we injected theneuronal tracer cholera toxin B into vmARC. Due to variationof injection sites, tracer was also injected into the subependy-mal layer of the median eminence (seME), which showed sim-ilar projection patterns as the vmARC. We propose that thevmARC forms a complex with the seME, their reciprocal con-nections with viscerosensory areas in brain stem, and other

circumventricular organs, suggesting the exchange of meta-bolic and circulating information. For the first time, thevmARC-seME was shown to have reciprocal interaction withthe suprachiasmatic nucleus (SCN). Activation of vmARC neu-rons by systemic administration of the ghrelin mimetic GH-releasing peptide-6 combined with SCN tracing showedvmARC neurons to transmit feeding related signals to theSCN. The functionality of this pathway was demonstrated bysystemic injection of GH-releasing peptide-6, which inducedFos in the vmARC and resulted in a reduction of about 40% ofearly daytime Fos immunoreactivity in the SCN. This obser-vation suggests an anatomical and functional pathway forperipheral hormonal feedback to the hypothalamus, whichmay serve to modulate the activity of the SCN. (Endocrinology147: 283–294, 2006)

ALTHOUGH MOST RESEARCH on nonphotic input tothe suprachiasmatic nucleus (SCN) has focused on the

intergeniculate leaflet and raphe nuclei, signals from otherparts of the brain are also possible. The secretion of manyhormones is synchronized by the SCN, and feedback fromperipheral hormones to the hypothalamus could thus alsoinfluence this central clock and adjust its output. Althoughmany metabolic active peripheral hormones/substanceshave transport systems to pass the blood-brain barrier, thereare selected areas in the central nervous system (CNS) withspecial sensory functions for circulating substances, such asthe arcuate nucleus (ARC). Several studies analyzing thepossible ability of the ARC to sense blood-borne substancesindicate that the ventromedial ARC (vmARC) may sense

signals directly from the general circulation via the medianeminence (ME) (1–3) and thus functions as an importantsensory “window” for blood-born substances to the brain(4–6). This is firmly supported by the presence of receptorswith high expression levels for nearly all known circulatingmetabolically active hormones, such as leptin, insulin, glu-cocorticoid, and ghrelin in the vmARC (7–10). These hor-mones and also glucose are able to modulate the electricalactivity of vmARC neurons (11–13) and thus affect the outputof the vmARC in energy homeostasis. This special role of thevmARC in integrating hormonal signaling was confirmed bythe fact that leptin signaling to the CNS crucially depends onnormal ARC functioning (14, 15).

The anatomical mapping of ARC in the CNS is still up-dated with a variety of methods, such as transsynaptic (16)or monosynaptic tracing studies (14, 17). Because a specificvmARC tracing has not yet been performed, a systematicretrograde and anterograde tracing study is necessary tounderstand the anatomical relationship of the vmARCwithin the CNS.

We therefore studied the connections of vmARC with theCNS and used injection of fluorophore-conjugated choleratoxin B (CTB; CTB-Alexa Fluor 555) as retrograde and an-terograde tracers into this area. The specificity of the pro-jections of the vmARC to the SCN and nucleus of the solitarytract (NTS) were confirmed by control tracing from SCN andNTS, respectively.

The functional interaction between vmARC and SCN was

First Published Online September 29, 2005Abbreviations: AGRP, Agouti-related peptide; AMC, arcuate-median

eminence complex; AP, area postrema; ARC, arcuate nucleus; CNS,central nervous system; CTB, cholera toxin B; CVO, circumventricularorgan; DMH, dorsomedial hypothalamic nucleus; DMV, dorsal motornucleus of the vagus; GHRP-6, GH-releasing peptide-6; ME, medianeminence; NPY, neuropeptide Y; NTS, nucleus of the solitary tract;OVLT, organum vasculosum of the lamina terminals; PB, parabrachialnucleus; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus;seME, subependymal layer of the median eminence; SFO, subfornicalorgan; SON, supraoptic nucleus; TBS, Tris-buffered saline; vmARC,ventromedial arcuate nucleus; VMH, ventromedial hypothalamic nu-cleus; ZT, Zeitgeber time.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/06/$15.00/0 Endocrinology 147(1):283–294Printed in U.S.A. Copyright © 2006 by The Endocrine Society

doi: 10.1210/en.2005-1051

283

demonstrated by the use of the natural leptin antagonist, thegut-brain peptide ghrelin (18), which is the only metabolicmolecule found to date that is able to activate vmARC neu-rons by specifically targeting agouti-related peptide(AGRP)/neuropeptide Y (NPY) neurons and that stimulatesfood ingestion (19). With systemic administration of the gh-relin mimetic, GH-releasing peptide-6 (GHRP-6), combinedwith SCN tracing and AGRP/Fos staining, the present datashowed that GHRP-6 activated vmARC neurons projectingto the SCN. Finally, the influence of systemic GHRP-6 stim-ulation on diurnal Fos immunoreactivity in the SCN wasinvestigated.

Materials and MethodsCTB tracing

All the following experiments were conducted under the approval ofthe animal care committee of the Royal Netherlands Academy of Sci-ences. Male Wistar rats, weighing 300 � 10 g (Harlan Nederland, Horst,The Netherlands), were housed at room temperature with a 12-h light,12-h dark schedule (lights on at 0700 h). For tract tracing, rats wereanesthetized [0.8 ml/kg Hypnorm (Janssen, High Wycombe, Bucking-hamshire, UK), im, and 0.4 ml/kg Dormicum (Roche, Almere, TheNetherlands), sc], mounted with their heads in a standard stereotaxicapparatus, tooth bar set at �3.4 mm, and received tracer injection.Because, in contrast to CTB, fluorophore-labeled CTB cannot be appliedby iontophoresis, we used pressure injection. Thus, fluorophore-conju-gated CTB (with Alexa Fluor 555, Molecular Probes, Eugene, OR; CTBwill be used in the text as the abbreviation of CTB-Alexa Fluor 555 forconvenience) was used for purpose of combination of neurotransmitterand tracer detection in target area. Finally, in 30 cases CTB injectionswere aimed at the vmARC. To confirm the reciprocity of connection,injections with CTB were also aimed at the SCN (n � 30) or NTS (n �10); all coordinates for these regions were adapted from the atlas ofPaxinos (20).

CTB was injected with pressure (10 mbar, 5 sec) using a glass pipettewith a tip of maximally 50 �m in diameter to minimize damage ofpassing fibers; the glass pipette was filled with an injection volume of50–100 nl. The coordinates for the vmARC injection were 3.3 mm caudalto bregma, 0.4 mm lateral to the midline, and 10.4 mm below the dura.The coordinates for SCN injection followed those reported by Buijs et al.(21); for NTS injection, the coordinate was 0.4 lateral to the midline, 13.8mm caudal to bregma, and 5 mm ventral from dura. After the tracer wasinjected by pressure injection, the pipettes were fixed with dental cementto the skull and left in the brain to minimize leakage from the tract untilthe animals were killed. This procedure did not lead to visible extradiscomfort of the animals, whereas the reduction in leakage along thepipette track is substantial. Fifteen rats of the SCN tracing were alsocombined with the placement of a jugular venous catheter for the ivinjection of GHRP-6. See below for details on this operation protocol.

GHRP-6 iv infusion

For the iv infusion of GHRP-6, a silicone catheter was implanted inthe right jugular vein according to the method of Steffens (22). Theoperation of the first group of rats (n � 15) was combined with SCNtracing, such that both operations were carried out at same time. Thesecond group of rats (n � 8) received only catheter implantation for ivinfusion of GHRP-6 (n � 4) or vehicle (Ringer solution; n � 4) to examineFos immunoreactivity in the vmARC and SCN. After surgery, all ratswere given 10 d to recover. After the operation, the animals were han-dled every day and connected to the jugular catheter to familiarize therats with the experimental procedures. On d 9, the rats were connectedto a drug administration catheter for 24 h, which was kept out of reachof the rats by means of counterbalanced beam. This allowed all manip-ulations to be carried out outside the cages without handling the rat. Allexperiments were performed in the rat home cage.

The drug administration catheter was connected to a syringe con-taining vehicle with or without GHRP-6. Infusion into the catheter wasinitiated at Zeitgeber time 1 (ZT1), with ZT0 being defined as the onset

of the light period. GHRP-6 was used at a dose of 5 �g/rat dissolved in0.4 ml Ringer solution, and 0.4 ml Ringer solution was used as the vehiclecontrol infusion. The infusion speed was kept at 0.1 ml/min. All ratswere killed by perfusion 100 min after the infusion.

Food deprivation

We found that neurons in the subependymal layer of ME (seME) havea similar projection pattern as those in the vmARC. Previous studiessupposed them to be an extension of vmARC (23); therefore, we exam-ined whether seME neurons show a comparable increase in the expres-sion of NPY/AGRP after food deprivation as observed in vmARC neu-rons (24). Food, but not water, was removed at ZT1 from four intact ratsfor 48 h; subsequently, animals were killed by perfusion.

Immunocytochemical staining

All rats were deeply anesthetized with a lethal dose of sodium pen-tobarbital and perfused with saline, followed by a solution of 4% para-formaldehyde in 0.1 m PBS (pH 7.4) at 4 C. The brains were removed andkept in fixative at 4 C for overnight postfixation, equilibrated 48 h with30% sucrose in 0.1 m Tris-buffered saline (TBS; pH 7.2). Brains werecoronally cut in a cryostat into 30-�m sections; sections used for im-munolabeling were collected and rinsed in 0.1 m TBS.

With ARC, ME, SCN, and NTS tracing, whole brain sections wereincubated with rabbit anti-CTB primary antibody (Sigma-Aldrich Corp.,St. Louis, MO) at a 1:100,000 dilution. Sections of fasted rats were in-cubated with rabbit anti-AGRP (1:1500; Phoenix Pharmaceuticals, Bel-mont, CA) or NPY (1:2000; Niepke 091188, Netherlands Institute forBrain Research) primary antibody. Sections of second group of GHRP-6infusion rats were incubated with goat anti-Fos (1:1500; Santa CruzBiotechnology, Inc., Santa Cruz, CA). After all these primary antibodyincubations overnight at 4 C, sections were rinsed and incubated inbiotinylated secondary antibody (goat antirabbit or horse antigoat IgG)for 1 h, then rinsed and incubated in avidin-biotin complex (VectorLaboratories, Inc., Burlingame, CA) for 1 h, and the reaction product wasvisualized by incubation in 1% diaminobenzidine (0.05% nickel ammo-nium sulfate was added to the diaminobenzidine solution to darken thereaction product) with 0.01% hydrogen peroxide for 5–7 min. Sectionswere mounted on gelatin-coated glass slides, dried, run through ethanoland xylene, and covered for observation by light microscope.

Confocal microscopy

To characterize the functionality of neurons in the vmARC thatproject to the SCN, we used triple-labeling immunofluorescence to showthe colocalization of GHRP-6-induced Fos with AGRP in retrogradelyCTB-labeled neurons in vmARC from SCN tracing. Sections were in-cubated overnight at 4 C with goat anti-Fos and rabbit anti-AGRPprimary antibody, rinsed in 0.1 m TBS, incubated 1 h in biotinylatedhorse antigoat IgG, rinsed, incubated with streptoavidin-Cy5 and don-key antirabbit-Cy2 for 1 h, rinsed, mounted on gelatin-coated glassslides, dried, covered with glycerol in 0.1 m PBS (pH 9.0), and observedby confocal laser scanning microscopy.

Analysis of Fos immunoreactivity

The effect of GHRP-6 treatment on Fos immunoreactivity in the SCNwas determined in four GHRP-6-infused or four Ringer solution-infusedrats. From SCN sections, tiled images were captured by a computerizedimage analysis system consisting of an Axioskop 9811� Sony XC77 blackand white video camera (Sony Corp., Tokyo, Japan). In these images, theright side of middle portion of the SCN was manually outlined. TheFos-positive nuclear profiles were automatically (no user interaction)segmented by a dedicated macro written within the ImagePro program-ming environment. For each rat, three sections were measured 90 �mapart (from bregma �1.20 to �1.40 mm); the mean number of Fos-positive nuclear profiles from these three sections was calculated. Allvalues are expressed as the mean � sem, and data were analyzed usingone-way ANOVA. Statistical significance was set at P � 0.01.

284 Endocrinology, January 2006, 147(1):283–294 Yi et al. • vmARC Communicates Hormonal Information to SCN

ResultsCTB injections into the vmARC and seME

Of all the animals injected, two rats had a successful in-jection within the middle part of the vmARC along its ros-trocaudal extension, and two rats had a successful injectionwithin the seME. Due to the difficulties of accurate vmARCinjection, we only focused on the middle part (in the anteriorposterior direction) of the vmARC, which guaranteed max-imal vmARC space for tracing. Misplaced injections weremainly located in a different coronal plane, but usually at thesame distance from the bregma. The misplaced injections inthe parenchymal area, including the external zone of the MEand the dorsal or ventrolateral ARC, served as controls forthe specificity of the projections of the vmARC and seME.Misplacement and injection in the third ventricle served ascontrol for leakage of tracer into cerebrospinal fluid, whichis almost inevitable. The injections in which the small tip ofglass pipette was blocked by brain tissue and the tracer couldnot be pushed out by pressure were used as specificity con-trols of the used antibodies. In none of these latter cases wasany staining observed, which shows that all the stainingobserved was due to CTB.

Tracer injections into the vmARC resulted in the spread ofCTB not only limited to the ventromedial part of the nucleus,but expanding also into the ipsilateral and contralateral sub-ependymal layers of the ME (Fig. 1A). After seME injection,

the CTB distribution covered the dorsal aspect of the ME,between the ependymal layer and the fibrous layer (Fig. 1D).AGRP- and NPY-containing cell bodies can more easily bedetected in the seME as well as in the vmARC after 48 h offasting (Fig. 1, C and F), whereas with a normal feedingschedule, we only observed AGRP or NPY fibers and ter-minals in these two areas. This observation together with thedistribution pattern of the tracer could support the previoussuggestion based on development that seME neurons are anextension of the ARC (23). Consequently, Table 1 showsretrograde and anterograde labeling in different areas of theCNS with CTB injections into the vmARC and seME.

Control injections

Injections in the ventrolateral ARC (Fig. 1B), dorsal ARC,external zone of the ME (Fig. 1E), and third ventricle wereused as controls for specificity of the projections of thevmARC and seME and leakage of the tracer. Three rats hadtracing into the external zone of the ME, three rats hadventricle injection only, and two rats received ventrolateralARC and dorsal ARC tracing, respectively.

The labeling pattern of external zone tracing was con-sistent with those from Lechan et al. (25) and Wiegand andPrice (26). In contrast with the seME injection, it revealedmainly retrogradely labeled neurons in the parvocellularparaventricular nucleus (PVN; Fig. 3F) and several mag-

FIG. 1. Coronal sections of the hypothalamus show the injection sites of the ARC and ME and immunocytochemical staining for NPY and AGRPin the vmARC and seME. A, CTB was injected into the vmARC (arrow) and illustrates the spread of tracer into the ipsilateral part of the seME(double arrows). B, Control injection into ventrolateral ARC resulted in strong terminal labeling in the vmARC area, which confirms theinteraction between the lateral proopiomelanocortin-cocaine- and amphetamine-regulated transcript neuron group and the ventromedialAGRP-NPY neuron group (68). Yet, unlike vmARC injection, they did not result in labeling of fibers in SCN or SON (see Fig 2C). D, CTB wasinjected into the middle part of the seME (arrow), and the distribution area covers the entire dorsal aspect of the ME. E, Injection site of CTBin the external zone of ME. C and F, Forty-eight hours of fasting resulted in incremental visualization of AGRP (C) or NPY (F) in the ARC neurons,the AGRP- and NPY-positive neurons are also clearly visible in the seME area (arrows). III, Third ventricle. Scale bar: A and B, 100 �m; Cand F, 50 �m; D and E, 200 �m.

Yi et al. • vmARC Communicates Hormonal Information to SCN Endocrinology, January 2006, 147(1):283–294 285

nocellular neurons in PVN and supraoptic nucleus (SON;Fig. 3I) as well as the periventricular nucleus and mag-nocellular dorsal ARC. The absence of detectable antero-grade tracing confirms the external zone of ME as a pureneurosecretory pathway of the hypothalamus. Three con-trol injections into the third ventricle resulted in labelingof the ependymal plexus and tanycytes or cerebrospinalfluid contacting neurons along the third and lateral ven-tricles, some neurons in the dorsal raphe nucleus were alsolabeled. This pattern was consistent with ventricle tracingexperiments aiming at detecting cerebrospinal fluid con-tacting neurons in the CNS by CTB-horseradish peroxi-dase injection (27). Injections into the ventrolateral or dor-sal part of the ARC had projection patterns distinct fromthat of injection into the vmARC, e.g. with ventrolateralARC injection even when very close to the vmARC, weonly observed some neurons present in the dorsal andmedial parts of the SCN (Fig. 3G) and no projections to theSON or NTS.

Retrograde and anterograde labeling in the CNS with CTBinjection into the vmARC and seME

The injection cases R122 and R173 had their injection tipslocalized within the vmARC, whereas R121 and R144 hadtheir injection tips in the seME. Cases R122–R173 and R121–R144 showed some similar pattern of retrograde and antero-grade labeling in the CNS, although with several differencesexemplified by the presence of retrogradely filled neuronsand anterogradely filled fibers in a number of additionalareas after injection in the vmARC as compared with injec-tion in the seME (see Table 1). The projections from the seMEarea as compared to the projections from the vmARC are lessdense than vmARC.

Areas with reciprocal interaction with vmARC and seME

CTB tracer injection into the vmARC and seME revealedseveral sites that have extensive reciprocal interaction withthis area. In the anterior part of the CNS, reciprocal connec-

TABLE 1. Anterograde and retrograde labeling with CTB injection into vmARC and seME

R122 R173 R121 R144

vmARC seME

N F N F N F N F

DPi dorsal endopiriform nucleus 00 � 00 � 0 � * �Acb nucleus accumbens 00 � 00 � 0 � * �LSV lateral septal nucleus ventral part 000 � 000 � 00 � 0 �CLA Claustrum 00 � 00 � 0 � � �OVLT organum vasculosum lamina terminalis 0 �� 0 � 0 � 0 ��VLPO ventrolateral preoptic nucleus 0 * 0 � � * * �AVPe anteroventral periventricular nucleus 00 �� 000 �� 0 � * ��MPOL medial preoptic nucleus, lateral part 00 � 00 � 00 � * *VMPO ventromedial preoptic nucleus 0 �� 00 �� 0 �� * ��BST bed nucleus of the stria terminalis 0 � 0 � 0 � 0 �SFO subfornical organ 000 �� 000 � 00 � 00 �Pe periventricular hypothalamic nucleus 0 �� 0 �� 0 � 0 *PVP paraventricular thalamic nucleus, posterior part 0 � � � 0 � � �LH lateral hypothalamus 0 � 00 � � � � �SCN suprachiasmatic nucleus 00 � 00 �� 0 �� 0 ��SON supraoptic nucleus � �� RCh retrochiasmatic area � �� TC tuber cinerum area � �� *PVN paraventricular nucleus 0 �� 00 �� 0 � 0 �Sub PVN sub paraventricular nucleus 000 � 00 �� ARCdm arcuate nucleus, dorsomedial part 00 �� 00 �� 00 � 00 �ARCvl arcuate nucleus, ventrolateral part 0 �� 0 �� 0 � 0 �A amygdaloid complex 00 � 00 � � � � �DMH dorsomedial hypothalamic nucleus 0 �� 00 �� 00 � 0 *VMH ventromedial hypothalamic nucleus 0 � 0 � 0 � 0 �PMV premamillary nucleus, ventral part 000 � 000 � 00 � 0 �VTM ventral tuberomamillary nucleus 0 � 00 � 0 � � �DTM dorsal tuberomamillary nucleus 0 � 0 �� 0 �IGL intergeniculate nucleus � � � � � � � �PAG periaqueductal grey 0 � 0 � 0 � � �LC locus coeruleus 00 �� 0 � 0 � 0 �Bar Barrington’s nucleus 0 �� 0 � 0 � � �Pr/C1 prepositus nucleus/C1 adrenaline cells 00 � 0 � 0 � 0PBr parabrachial nucleus, rostral part 00 �� 000 �� 00 �� 0 *PBc parabrachial nucleus, caudal part 00 �� 00 �� 0 *NTS nucleus of the solitary tract 00 �� 0 � 00 �� 00 �DMV dorsal motor nucleus of vagus 0 � 0 � 0 � 0 �AP area postrema 00 �� 00 � * * 000 �

Column N, Retrograde labeling neurons; column F, anterograde labeling fibers and terminals. N: �, no neurons; *, faint; 0, a few; 00, modest;000, strong. F: �, no fiber; *, faint; �, a few: ��, modest; ��, strong.


tions with this area arise from the anteroventral PVN, ven-tromedial preoptic nucleus, and medial preoptic nucleus(lateral part), with labeled cells and fibers scattered through-out these areas, and the labeled neurons were mainly smallin size. Both lateral septum and accumbens nucleus connectwith vmARC reciprocally, but labeled fibers were not visibleafter seME tracing.

We observed that all parts of the SCN show labeled neu-rons, with the highest density in the medial and dorsal parts.Moreover, abundant labeling of fibers and terminals werefound in the dorsal and ventral parts. A few labeled neuronsand fibers were also observed in the area around the SCN(Fig. 2). From rostral to caudal, the whole extension of theperiventricular hypothalamic nucleus is labeled by scatteredneurons and fibers.

Consistent with previously published data about the pro-jection from the ARC to the PVN (28, 29), with vmARC andseME tracing, the triangular shape of the PVN was full oflabeled fibers and terminals (Fig. 3, A and B); in contrast withthe dense labeling of fibers and terminals, only few PVNneurons were labeled. Although when the injection was inthe external zone of the ME, PVN labeling was characterizedby the presence of a large number of neurons that by locationand appearance can easily be recognized as neuroendocrine(Fig. 3F). Also in the SON the contrast is obvious; vmARCand seME injections result in dense innervations in SON (Fig.3H), whereas injection in the external zone only shows ret-rogradely filled neurons (Fig. 3I). After vmARC injection, aconsiderable number of fibers and small neurons in the sub-PVN (30) were also labeled (Fig. 3A). More laterally, thereciprocal connection also only exists between lateral hypo-thalamus and vmARC, but not with the seME.

Within the whole ARC, many anterogradely labeled fibersas well as a few retrogradely labeled neurons were presentin the dorsal and ventrolateral parts of the ARC near theborder that separates the ARC and ventromedial hypotha-lamic nucleus (VMH; Fig. 1). This labeling pattern of ARCwas consistent from rostral to caudal.

Another important target of the vmARC and seME is thedorsomedial hypothalamic nucleus (DMH). A large numberof labeled neurons, fibers, and terminals visualized through-out the DMH demonstrate the reciprocal projection betweenthe vmARC and DMH (Fig. 3D). Interestingly, the vmARC

and seME tracing revealed a very weak anatomical relation-ship with the VMH, with just a few neurons and fibers beinglabeled. In the posterior hypothalamus, numerous labeledneurons were present in the ventral premamillary nucleus,whereas fibers were relatively sparse. Of the tuberomamil-lary nucleus, the dorsal part received a denser innervationfrom the vmARC and seME than the ventral area.

In addition, neurons and some axonal terminals were ob-served in the other three circumventricular organs (CVOs)after vmARC and seME injection. In the subfornical organ(SFO), neurons and fibers were spread over the vascular area(Fig. 4A). In the organum vasculosum of the lamina terminals(OVLT), abundant labeling was present in neurons and fibersbilaterally located in the vascular zone and perinucleus area.A strong labeling of fibers, as in the ependymal plexus, wasseen along the wall of the ventricle (Fig. 4B). Similarly, in thearea postrema (AP), several fibers and neurons were ob-served in the vascular area (Fig. 4C). At the border areabetween the AP and NTS, we visualized strongly labeledfibers, probably derived from the ependymal plexus that isoften labeled after injection in the seME (Fig. 4C), this label-ing pattern in AP as well as in OVLT, however, was also seenwith the third ventricle control injection, whereas with con-trol incubation of anti-CTB antibody with sections from non-tracing, we did not see any such staining, and we also havenot found such a labeling pattern with lateral ARC and SCNCTB tracing animals.

In the brain stem, almost all subdivisions of the parabra-chial nucleus (PB) contain labeled neurons and terminalsfrom the most rostral to the caudal part of the nucleus (Fig.4, E and F). In addition, scattered neurons and terminals wereseen in the periaqueductal gray, locus coeruleus, Bar-rington’s nucleus, and prepositus nucleus.

Similar to the PB, another visceral integration nucleus, theNTS, contained several labeled neurons and fibers from thecaudal to the middle part. Consistent with a previous study,after vmARC injection, we observed a dense labeling of fibersin a limited area at ipsilateral part in the caudal NTS (Fig. 4,H and I); in view of the paucity of retrogradely labeledneurons, we consider these fibers be largely due to antero-grade transport and not derived from retrogradely labeledneurons as has been suggested to occur with CTB tracing(31). Interestingly, some small labeled neurons were alsofound in the dorsal motor nucleus of the vagus (DMV) (Fig.4H). These finding corroborate our previous observation thatthe area of the DMV incorporates neurons without an au-tonomic motor function (32).

Areas with only input to or output from the vmARCand seME

A few regions seem to only provide input to the vmARCand seME, as demonstrated by the presence of merely ret-rogradely labeled cell bodies and no clear staining of fibers.Because other brain regions at the same level do show labeledfibers, this does not seem to be a technical limitation of theanterograde tracing. Only retrogradely filled neurons weredetected in the dorsal endopiriform nucleus, claustrum, bednucleus of the stria terminalis, and posterior part of the PVN.

FIG. 2. Coronal sections of the SCN show reciprocal connection be-tween the vmARC and SCN after CTB injection in the vmARC. A,Several CTB-labeled neurons are present in different parts of theSCN, fibers and terminals cover the whole nucleus, whereas aroundthe SCN the labeling is less dense. Details of labeled fibers in theventral part are shown with high magnification in B; double arrowsindicate the same group of CTB labeled fibers. III, Third ventricle.Scale bar: A, 100 �m; B, 50 �m.


The whole extension of most of the medial amygdaloid com-plex was also strongly retrogradely labeled (Fig. 4D).

With vmARC and seME injections, a dense innervation ofthe SON, retrochiasmatic area, and tuber cinereum area, butwithout retrogradely labeled cell bodies, indicates that theseareas only get input from the vmARC and seME. We alsofound that the vmARC and seME tracing resulted in pro-jections to the intergeniculate leaflet (Fig. 4G).

Retrograde tracing from SCN and NTS

To provide additional evidence for the projections of thevmARC and seME to the SCN and NTS and to ensure thatthe projections are not due to uptake from passing fibers orto anterograde labeling by retrogradely labeled neurons (33,34), CTB was injected into the SCN and NTS.

Injections of CTB into both the SCN and NTS revealed not

FIG. 3. Coronal sections of the hypothalamus illustrating anterograde and retrograde labeling in the hypothalamus after vmARC andseME injections or ventrolateral ARC and external zone of ME injections. A, vmARC injection results at the midlevel of the PVN in manylabeled terminals and few small, oval or fusiform neurons in the medial and dorsal parvocellular (p) subdivision, whereas the magnocellularaspect (m) mostly contained fibers and a single large neuron. Note just under the triangular shape of the PVN, very abundant small neuronsare distributed over the whole subPVN (arrows). High magnification in B shows more details of the PVN. Nissl counterstaining of thesame section (C) revealed that the magnocellular (m) and parvocellular (p) parts of the PVN are innervated by CTB-labeled fibers (compareB and C). Asterisks in A–C indicate the same blood vessel. The external zone of ME control tracing resulted in an abundant labeling ofthe parvocellular PVN and several neurons in magnocellular PVN and SON (F and I). D, VmARC tracing results in densely labeled fibers,terminals, and cell bodies in the DMH. The central part of the DMH (c) has fewer labeled fibers and terminals than the dorsal and ventralparts, and the labeled neurons are denser in the ventral part. Using Nissl counterstaining of the same section (E), the area that filledwith CTB-labeled fibers overlapped with the Nissl-stained DMH. Note the area between the DMH and VMH where Nissl-stained cellsare less evident; the asterisks in D and E indicate the same CTB-labeled neuron in this cell-sparse area. G, With ventrolateral ARC controlinjection, only a few neurons could be detected in the SCN (arrows). Labeling of fibers was absent. H, VmARC injection results in stronglylabeled terminals and fibers in the SON; labeled neurons are only observed in the perinucleus area (arrow). III, Third ventricle. Scalebar: A and D, 200 �m; B, C, E, F, G, H, and I, 100 �m.


only neurons in the vmARC (Fig. 5, A, B, and D), but alsoretrogradely labeled neurons in the subependymal layer ofthe ME with a location just under the ependymal lining of thethird ventricle (Fig. 5C). We also considered the abundantpresence of fibers as evidence for the reciprocity of the con-nection of the vmARC and seME with the SCN and NTS. Thelabeled neurons in the seME area were different from glialcell. Firstly, the location of these subependymal neurons isjust under and very close to the ependymal layer lining thebottom of the third ventricle. In Fig. 5C we can see the

difference with ependymal cells that were not labeled byCTB. Secondly, the orientation of the oval or fusiform-shapedlabeled neurons was parallel with the border of the thirdventricle, whereas glia cells have many more processes andmay extend to every orientation, including the external zone.Thirdly, the size of subependymal neurons was far largerthan glial cell, either tanycytes or astrocytes.

After CTB injection into the SCN, fibers and terminalswere abundant in the lateral and dorsal parts of the ARCwhere few retrogradely labeled neurons were present (Fig.

FIG. 4. Coronal sections of the brain illustrate anterograde and retrograde tracing in the CVOs, amygdaloid complex, intergeniculate nucleus,and brain stem after vmARC and seME injection. A, Many labeled neurons with their processes are found at the border of the SFO and extendto the vascular core of this organ; the core area was rarely labeled. Similarly, CTB-labeled terminals are present more heavily in the borderarea. Abundant processes within the OVLT (B) and AP (C) are oriented on both sides (double arrows) with almost the same orientation on theborder. In the AP, the labeled fibers in the vascular area were also visible (arrowhead). A moderate number of neurons were scattered in thevascular area (arrows) of both OVLT and AP. D, Many retrogradely labeled neurons distributed over the whole extension of the medialamygdaloid complex, with sparse labeling in the central part (Ac). E and F, Along the whole extension of the PB, almost all subdivisions of thenucleus are labeled with CTB. E, The pattern in lateral PB subnucleus (LPB), the central part of the LPB (with high magnification in F; asteriskshows the same blood vessel) containing the most strongly labeled neurons (arrow), fibers and terminals (double arrows). G, Labeled fibers inintergeniculate leaflet from injection case R173 is indicated by double arrows. H, Labeled neurons are found on both sides of the NTS. Somelabeled neurons were also found in the contralateral DMV (arrows). Densely labeled fibers and terminals are present in the ipsilateral part(right side) of the medial NTS. I, High magnification of H; the arrow shows labeled neuron, double arrows show fibers and terminals in NTS,and the asterisk shows the same blood vessel in H and I. ON, Optic nerve. Scale bar: A, 50 �m; B, C, F, and I, 100 �m; D, E, G, and H, 200�m.


5, A and B). This projection pattern confirmed the vmARCand seME tracing, which resulted in labeling of neurons,fibers, and terminals in the SCN, and the ventrolateral anddorsal ARC tracing, which resulted in only labeled neuronsin the SCN without fibers or terminals (Fig. 3G).

Functionality of the connections between vmARC and SCN

We addressed the possible role of vmARC neurons in theregulation of metabolism in relation to their projection to theSCN by examining the induction of Fos in AGRP neuronsafter systemic administration of GHRP-6 combined withSCN tracing. In addition, we observed that GHRP-6 stimu-lation allowed an easier detection of AGRP in vmARC neu-rons, which is normally difficult. Most AGRP-positive neu-rons also expressed Fos, and some of these Fos-positiveAGRP neurons were shown to project to the SCN, as revealedby colocalization with CTB from SCN tracing (Fig. 6). Inter-estingly, in both ad libitum-fed and feeding-restricted rats, itwas difficult to detect AGRP fibers in the SCN. We observedonly very thin AGRP-positive fibers in the ventral SCN after2 h of refeeding following 48-h fasting (data not shown),which suggests that although AGRP neurons are the targetof peripheral GHRP-6 and project to the SCN, the detectionof this peptide is limited, probably because of the low quan-tity of peptide present in these nerve terminals.

After SCN tracing, there was no obvious Fos immunore-activity in ARC neurons or in intact rat ARC that werehoused and perfused under the same conditions as SCN

tracing rats, which means that SCN tract tracing has no effecton Fos immunoreactivity in the ARC. Fos immunoreactivityin vmARC induced by GHRP-6 is apparent after a 5-�g ivinfusion, whereas the Ringer solution infusion had no de-tectable Fos in the vmARC (Fig. 7, A and B). In addition, Fos

FIG. 7. Coronal sections of the middle portion of the ARC and SCNillustrate the pattern of Fos immunoreactivity in vmARC and SCNafter Ringer (A and C) or 5 �g GHRP-6 systemic administration (Band D). A, Low Fos immunoreactivity in ARC after iv Ringer solutioninfusion. B, With 5 �g GHRP-6 iv infusion, Fos is strongly expressedby vmARC neurons. C, Diurnal Fos immunoreactivity pattern in SCNwith Ringer control infusion at ZT1. D, Five-microgram GHRP-6infusion resulted in an obvious decrease in Fos immunoreactivity inthe SCN. III, Third ventricle. Scale bar: A and B, 150 �m; C and D,100 �m.

FIG. 5. Coronal sections of the hypothalamus illustrate the labelingpattern in vmARC and seME after tracer injection into the SCN (A–C)or NTS (D). A, Labeled neurons in vmARC after SCN injection aremore abundant than in dorsal and ventrolateral ARC, whereas fibersand terminals show the opposite labeling pattern. Double arrowsshow the border of the vmARC and lateral ARC. B, High magnifica-tion gives details of the labeled neurons (arrow shows the same neu-ron in B), fibers and terminals in vmARC (double arrows). C, Labeledneurons (arrow) are present in the seME area after CTB injection intoSCN. The outline of the ependymal layer is visible just over the seMEarea; labeled fibers not only exist in the seME area (double arrows),but also scatter in the fibrous layer of the ME (arrowhead). D, Labeledneurons (arrows) and fibers (double arrows) in the vmARC and seMEarea after NTS control injection. III, Third ventricle. Scale bar: A, 100�m; B–D, 50 �m.

FIG. 6. Confocal laser scanning micrograph of the vmARC revealsthat CTB (red) retrogradely labeled neurons after SCN injection areactivated by systemic GHRP-6 administration. With GHRP-6 stim-ulation, Fos (Cy5; blue) was only strongly expressed in vmARC cells,many of them colocalized with AGRP neurons (Cy2; green) and/or CTBneurons. Arrows show AGRP colocalized with Fos; arrowhead showsCTB colocalized with Fos. Double arrows show AGRP colocalized withCTB and Fos; the yellow represents the colocalization of CTB andAGRP. III, Third ventricle. Scale bar, 100 �m.


immunoreactivity was checked in the geniculate area andraphe nucleus, which are responsible for nonphotic input tothe SCN, and there was no significant difference betweenRinger control and 5 �g GHRP-6 administration in bothnuclei.

The number of detectable Fos-positive nuclear profiles inthe middle portion SCN was counted in controls as well asGHRP-6-infused animals. Negatively correlated with thesame dose of 5 �g GHRP-6 action on the vmARC, the numberof Fos-positive nuclei in the middle portion SCN was de-creased by about 40% compared with that after Ringer so-lution infusion (195.33 � 14.45 vs. 116.89 � 4.7 in Ringersolution and 5 �g GHRP-6, respectively; P � 0.01; Fig. 7, Cand D).

Discussion

In the present study the properties of CTB as anterogradeand retrograde tracer were confirmed by the presence oftracer-labeled terminals and cell bodies, for example, in theNTS after injection into the vmARC, which is consistent withprevious tracing studies on the ARC and NTS (17) and con-sequently allowed us to study the input as well as the outputof one area simultaneously.

To avoid uptake of tracer by damage of fibers of passageas much as possible, glass pipettes with a tip approximately50 �m in diameter were used for pressure injection insteadof a Hamilton syringe (400 �m), which minimized the pos-sibility of tracer uptake by passing fibers.

This is demonstrated by the fact that only injection in theexternal zone of the median eminence resulted in retrogradelabeling of neuroendocrine neurons in the PVN and SON,which confirms previous studies (25, 26). Injections into otherareas where neuroendocrine fibers pass, such as the sub-ependymal layer-internal zone and the lateral ARC, did notresult in cell body labeling. In contrast, injection into thesubependymal layer-internal zone resulted only in labelingof fiber terminals in the SON and PVN. These findings sug-gest not only a high specificity of projections between the

different layers of the median eminence, but also a minimaluptake of fibers of passage by the method used.

The present results confirm and extend previous anatom-ical studies of ARC and ME and provide a more compre-hensive view of the organization of the ARC, including itsinteraction with the biological clock (Fig. 8).

Moreover, we demonstrate for the first time that both thevmARC and seME areas have a similar extensive interactionwith the SCN, the sensory CVOs, and other parts of the brain.In both the vmARC and seME, the blood-brain barrier op-erates in a modified manner comparable with that of otherCVOs that also have permeable capillary networks for pen-etration of circulating substances (35). Moreover, both areasnot only share the same projection patterns, but also containNPY and AGRP neurons, which respond similarly to, forinstance, fasting. Considering these aspects, we propose thatthe vmARC-seME should be viewed as an arcuate-medianeminence complex (AMC).

It is proposed that three typical sensory CVOs, SFO, OVLT,and AP, have active transport systems and receptors forblood-borne molecules and play a key role in sensing andrelaying humoral information to other CNS areas (36). Ourpresent data demonstrate that the AMC has similar specificneuronal connections to hypothalamic neuroendocrine andautonomic centers, such as the SFO, OVLT, and AP. Earlystudies have already suggested that the blood-brain barrierborder could reside at the area between the ventromedial (orproximal) part and dorsolateral (or distal) part of the ARC,and that the perivascular spaces of vmARC could thus com-municate with the neurohemal area of the ME, where fenes-trated capillaries allow the passage of larger molecules (1–3).Therefore, the present finding that the SCN receives directinput from the AMC suggests a route of communicationbetween the general circulation and the SCN. Intravenousinjection of the ghrelin mimetic GHRP-6 results in Fos-pos-itive neurons in a narrow band in the AMC. Of all tracerinjections in the ARC (n � 30), many were very close to thevmARC; only four injections that were inside and covered

FIG. 8. Sagittal scheme illustrates the main connections of vmARC within the CNS. Reciprocal connections are shown in blue; yellow areasrepresent input to vmARC; red areas represent output from vmARC. For abbreviations, see Table 1.


the same area that is known to express Fos after GHRP-6stimulation had reciprocal interaction with the SCN. Thissuggests that a relatively small area is involved in this ex-change of information. Furthermore, injections into the SCNresulted in the combination of retrogradely labeled neuronsand fibers only in this ventromedial area of the ARC, whichconfirms the reciprocity of this connection. In view of the(daily) time-dependent organization of metabolism, whichdepends so crucially on the presence of the SCN (37), theinteraction of the AMC with the SCN could serve this timingpurpose and provide the SCN with information about glu-cose concentration and the associated level of hormones.

That there are innervations from vmARC to SCN is a novelfinding. However, previous electrophysiological studies onARC (38) indicated this connection. Other proof for the func-tional interaction between vmARC and SCN comes from anumber of studies indicating that physiological and behav-ioral circadian rhythms are also affected by ARC malfunc-tion. The fact that ARC lesions by neonatal monosodiumglutamate treatment, which mainly destroys NPY cells (39),is able to compromise the circadian activity while the SCNremains intact (40, 41), could be interpreted, together withthe present data, to mean that the innervations from vmARCto the SCN may relay peripheral hormonal information to theSCN and may thus affect the circadian activity of the SCN.At the same time, input from the SCN to the ARC (38, 42, 43)may time the activity of the ARC, as shown by rhythmic Fosexpression in the ARC (44) and the rhythmic activity ofdopaminergic ARC neurons negatively driven by vasoactiveintestinal peptide from the SCN (45). Actually, such an SCN-CVO reciprocal connection has already been demonstratedfor the SCN-SFO and SCN-OVLT interaction (46, 47). Addi-tional evidence for a more general interaction between SCNand CVOs, including the ARC, can be found in studies de-scribing the action of prokineticin 2, which is synthesized inthe SCN and for which receptors have been shown in the SFOand the vmARC (48, 49). Together, these studies suggest thecapacity of the SCN to modulate the properties of the CVOs,probably to control or regulate the sensitivity of CVOs toperipheral signals, and the present study indicates that thesesignals may be relayed to the SCN as well not only via theSFO and OVLT, but also via the vmARC and seME. In viewof the high sensitivity to peripheral metabolic hormones inthe vmARC, the possible feedback pathway for these hor-mones to the SCN is proposed to be via this area.

Systemic administration of the ghrelin mimetic, GHRP-6,combined with SCN tracing showed that AGRP neuronsactivated by GHRP-6 project to the SCN; at the same time, thediurnal Fos immunoreactivity in the SCN was reduced by40%, suggesting that the ghrelin mimetic, GHRP-6, couldinterfere with the activity of the SCN via the vmARC. Theobvious decrease in Fos in the ventrolateral SCN suggests apossible regulation of the light-receiving part of the SCN. Thefact that the staining of AGRP fibers in the SCN comparedwith the PVN is much less intense may indicate that a certainselection of neurons in vmARC projects to the SCN, or thosecollaterals of vmARC neurons to SCN and PVN may containtransmitters in different proportions.

It is well known that most nonphotic inputs to the SCNhave the ability to reset the clock when they are provided

during the day (50, 51). Nonphotic inputs to the SCN wereshown to arise from the intergeniculate leaflet (52) and raphenucleus (53). In addition, nonphotic inputs are known toinhibit photic-responsive Fos expression in the SCN at night(54). In the present study with peripheral GHRP-6 admin-istration, there is no obvious variation in Fos immunoreac-tivity in the geniculate area and raphe nucleus, which indi-cates that these areas are not activated. Consequently, theGHRP-6 information relayed by the vmARC to the SCN, asindicated by Fos diminishment in the SCN, is another func-tional support for the anatomical connection between theAMC and SCN, and suggests that pharmacological effectsof ghrelin may inhibit the activity of the SCN during thelight period. This observation agrees with the fact thatwith a normal feeding schedule, ghrelin peaks after darkonset (39), suggesting another central function of ghrelin,such as interaction between locomotion and metabolism(55, 56).

Because the SCN is essential for the organization of dailymetabolic activity (the daily rhythm of plasma glucose andmetabolic hormones is driven by the SCN) (37), the presentresults provide an anatomical basis for our previous findingsthat metabolic cues could indeed influence circadian activityof, for example, vasopressin neurons in the SCN (57). Ad-ditional research is needed to elucidate the effect of the me-tabolism-related nonphotic input on SCN functionality andthe possible neurotransmitters that are involved in thesignaling.

Naturally the neuronal connections of AMC with otherareas in the CNS may also implicate those areas as potentialparticipants in metabolism. In this study we stress the role ofinteraction between AMC and CVOs; not only are the classicCVOs engaged in monitoring changes in metabolic, osmotic,ionic, and hormonal compositions, they also show neuronalactivation or inhibition by cytokines (58–60). In this study weshow that the SFO, OVLT, and AP have reciprocal connec-tions with the vmARC and seME, centers for the control offood intake, which suggests a functional interaction of CVOs,for example, in cytokine activity and sickness behavior (61).This could be the anatomical basis for anorexia and weightloss during inflammations. In addition, the present findingthat the amygdala complex and nucleus accumbens provideinput to the AMC supports the significance of limbic struc-tures in energy homeostasis. This possibility is supported bythe fact that chemical manipulation of the nucleus accum-bens induced high Fos expression in ARC NPY neurons (62).

Moreover, the AMC area has reciprocal connections withvisceral sensory integration sites such as the PB and NTS, aswas shown in part previously (17, 63). This indicates that thevmARC and seME may integrate hormonal information withsignals from first order visceral sensory centers (64, 65). Log-ically, the outcome of this interaction should be signaled tomany sites, including the SCN, and is necessary for the mul-tilevel central control of visceral activities.

Our present data provide the anatomical and functionalbasis for the hypothesis that both humoral and neuronalmetabolic information may be relayed back to the SCN viathe AMC. Herein the AMC area is responsible for the firstorder integration of circulating signals. When the normalmetabolic balance is seriously disturbed, e.g. by night-eating


syndrome (66) or stress, this may influence the integrativeactivity of the AMC as well as the organizational activity ofthe SCN. Thus, a slight disturbance at the level of theseessential integration sites raises a serious risk of an unbal-anced energy state and possibly obesity, diabetes, or othermetabolic diseases (67).

Acknowledgments

We thank Drs. Carolina Escobar and Marco van der Top for theirvaluable comments on the manuscript, Wilma Verweij for correction ofthe English, and Henk Stoffels for the art work.

Received August 17, 2005. Accepted September 22, 2005.Address all correspondence and requests for reprints to: Dr. Chun-

Xia Yi, Netherlands Institute for Brain Research, Meibergdreef 33, Am-sterdam, The Netherlands. E-mail: [email protected].

This work was supported by a grant from the Royal NetherlandsAcademy of Arts and Sciences for a joint project between The Nether-lands and China (03CDP014).

References

1. Broadwell RD, Balin BJ, Salcman M, Kaplan RS 1983 Brain-blood barrier? Yesand no. Proc Natl Acad Sci USA 80:7352–7356

2. Krisch B, Leonhardt H 1978 The functional and structural border of theneurohemal region of the median eminence. Cell Tissue Res 192:327–339

3. Shaver SW, Pang JJ, Wainman DS, Wall KM, Gross PM 1992 Morphology andfunction of capillary networks in subregions of the rat tuber cinereum. CellTissue Res 267:437–448

4. Berthoud HR 2002 Multiple neural systems controlling food intake and bodyweight. Neurosci Biobehav Rev 26:393–428

5. Horvath TL, Diano S 2004 The floating blueprint of hypothalamic feedingcircuits. Nat Rev Neurosci 5:662–667

6. Elmquist JK, Elias CF, Saper CB 1999 From lesions to leptin: hypothalamiccontrol of food intake and body weight. Neuron 22:221–232

7. Aronsson M, Fuxe K, Dong Y, Agnati LF, Okret S, Gustafsson JA 1988Localization of glucocorticoid receptor mRNA in the male rat brain by in situhybridization. Proc Natl Acad Sci USA 85:9331–9335

8. Unger J, McNeill TH, Moxley III RT, White M, Moss A, Livingston JN 1989Distribution of insulin receptor-like immunoreactivity in the rat forebrain.Neuroscience 31:143–157

9. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG 1996 Identi-fication of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101–1106

10. Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ,Smith RG, Van der Ploeg LH, Howard AD 1997 Distribution of mRNAencoding the growth hormone secretagogue receptor in brain and peripheraltissues. Brain Res Mol Brain Res 48:23–29

11. Pinto S, Roseberry AG, Liu H, Diano S, Shanabrough M, Cai X, FriedmanJM, Horvath TL 2004 Rapid rewiring of arcuate nucleus feeding circuits byleptin. Science 304:110–115

12. van den Top M, Lee K, Whyment AD, Blanks AM, Spanswick D 2004Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic ar-cuate nucleus. Nat Neurosci 7:493–494

13. Spanswick D, Smith MA, Mirshamsi S, Routh VH, Ashford ML 2000 Insulinactivates ATP-sensitive K� channels in hypothalamic neurons of lean, but notobese rats. Nat Neurosci 3:757–758

14. Bouret SG, Draper SJ, Simerly RB 2004 Formation of projection pathwaysfrom the arcuate nucleus of the hypothalamus to hypothalamic regions im-plicated in the neural control of feeding behavior in mice. J Neurosci 24:2797–2805

15. Dawson R, Pelleymounter MA, Millard WJ, Liu S, Eppler B 1997 Attenuationof leptin-mediated effects by monosodium glutamate-induced arcuate nucleusdamage. Am J Physiol 273:E202–E206

16. DeFalco J, Tomishima M, Liu H, Zhao C, Cai X, Marth JD, Enquist L,Friedman JM 2001 Virus-assisted mapping of neural inputs to a feeding centerin the hypothalamus. Science 291:2608–2613

17. Sim LJ, Joseph SA 1991 Arcuate nucleus projections to brainstem regionswhich modulate nociception. J Chem Neuroanat 4:97–109

18. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature402:656–660

19. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K,Matsukura S 2001 A role for ghrelin in the central regulation of feeding. Nature409:194–198

20. Paxinos G, Watson C 1998 The rat brain in stereotaxic coordinates. San Diego:Academic Press

21. Buijs RM, Markman M, Nunes-Cardoso B, Hou YX, Shinn S 1993 Projectionsof the suprachiasmatic nucleus to stress-related areas in the rat hypothalamus:a light and electron microscopic study. J Comp Neurol 335:42–54

22. Steffens AB 1969 A method for frequent sampling blood and continuousinfusion of fluids in the rat without disturbing the animals. Physiol Behav4:833–836

23. Rethelyi M 1975 Neurons in the subependymal layer of the rat median em-inence. Neuroendocrinology 17:330–339

24. Hahn TM, Breininger JF, Baskin DG, Schwartz MW 1998 Coexpression ofAgrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci1:271–272

25. Lechan RM, Nestler JL, Jacobson S 1982 The tuberoinfundibular system of therat as demonstrated by immunohistochemical localization of retrogradelytransported wheat germ agglutinin (WGA) from the median eminence. BrainRes 245:1–15

26. Wiegand SJ, Price JL 1980 Cells of origin of the afferent fibers to the medianeminence in the rat. J Comp Neurol 192:1–19

27. Zhang LC, Zeng YM, Ting J, Cao JP, Wang MS 2003 The distributions andsignaling directions of the cerebrospinal fluid contacting neurons in the pa-renchyma of a rat brain. Brain Res 989:1–8

28. Baker RA, Herkenham M 1995 Arcuate nucleus neurons that project to thehypothalamic paraventricular nucleus: neuropeptidergic identity and conse-quences of adrenalectomy on mRNA levels in the rat. J Comp Neurol 358:518–530

29. Bell ME, Bhatnagar S, Akana SF, Choi S, Dallman MF 2000 Disruption ofarcuate/paraventricular nucleus connections changes body energy balanceand response to acute stress. J Neurosci 20:6707–6713

30. Watts AG, Swanson LW 1987 Efferent projections of the suprachiasmaticnucleus. II. Studies using retrograde transport of fluorescent dyes and simul-taneous peptide immunohistochemistry in the rat. J Comp Neurol 258:230–252

31. Chen S, Aston-Jones G 1998 Axonal collateral-collateral transport of tracttracers in brain neurons: false anterograde labelling and useful tool. Neuro-science 82:1151–1163

32. Buijs RM, Chun SJ, Niijima A, Romijn HJ, Nagai K 2001 Parasympatheticand sympathetic control of the pancreas: a role for the suprachiasmatic nucleusand other hypothalamic centers that are involved in the regulation of foodintake. J Comp Neurol 431:405–423

33. Luppi PH, Fort P, Jouvet M 1990 Iontophoretic application of unconjugatedcholera toxin B subunit (CTb) combined with immunohistochemistry of neu-rochemical substances: a method for transmitter identification of retrogradelylabeled neurons. Brain Res 534:209–224

34. Aston-Jones G, Chen S, Zhu Y, Oshinsky ML 2001 A neural circuit forcircadian regulation of arousal. Nat Neurosci 4:732–738

35. Gross PM 1992 Circumventricular organ capillaries. Prog Brain Res 91:219–23336. Cottrell GT, Ferguson AV 2004 Sensory circumventricular organs: central

roles in integrated autonomic regulation. Regul Pept 117:11–2337. la Fleur SE, Kalsbeek A, Wortel J, Fekkes ML, Buijs RM 2001 A daily rhythm

in glucose tolerance: a role for the suprachiasmatic nucleus. Diabetes 50:1237–1243

38. Saeb-Parsy K, Lombardelli S, Khan FZ, McDowall K, Au-Yong IT, DyballRE 2000 Neural connections of hypothalamic neuroendocrine nuclei in the rat.J Neuroendocrinol 12:635–648

39. Meister B, Ceccatelli S, Hokfelt T, Anden NE, Anden M, Theodorsson E 1989Neurotransmitters, neuropeptides and binding sites in the rat mediobasalhypothalamus: effects of monosodium glutamate (MSG) lesions. Exp Brain Res76:343–368

40. Mistlberger RE, Antle MC 1999 Neonatal monosodium glutamate alters cir-cadian organization of feeding, food anticipatory activity and photic maskingin the rat. Brain Res 842:73–83

41. Schoelch C, Hubschle T, Schmidt I, Nuesslein-Hildesheim B 2002 MSGlesions decrease body mass of suckling-age rats by attenuating circadian de-creases of energy expenditure. Am J Physiol 283:E604–E611

42. Horvath TL 1997 Suprachiasmatic efferents avoid phenestrated capillaries butinnervate neuroendocrine cells, including those producing dopamine. Endo-crinology 138:1312–1320

43. Saeb-Parsy K, Dyball RE 2003 Responses of cells in the rat suprachiasmaticnucleus in vivo to stimulation of afferent pathways are different at differenttimes of the light/dark cycle. J Neuroendocrinol 15:895–903

44. Jamali KA, Tramu G 1999 Control of rat hypothalamic pro-opiomelanocortinneurons by a circadian clock that is entrained by the daily light-off signal.Neuroscience 93:1051–1061

45. Gerhold LM, Sellix MT, Freeman ME 2002 Antagonism of vasoactive intes-tinal peptide mRNA in the suprachiasmatic nucleus disrupts the rhythm ofFRAs expression in neuroendocrine dopaminergic neurons. J Comp Neurol450:135–143

46. Lind RW, Van Hoesen GW, Johnson AK 1982 An HRP study of the connec-tions of the subfornical organ of the rat. J Comp Neurol 210:265–277

47. Trudel E, Bourque CW 2003 A rat brain slice preserving synaptic connectionsbetween neurons of the suprachiasmatic nucleus, organum vasculosum laminaterminalis and supraoptic nucleus. J Neurosci Methods 128:67–77

48. Cheng MY, Bullock CM, Li C, Lee AG, Bermak JC, Belluzzi J, Weaver DR,


Leslie FM, Zhou QY 2002 Prokineticin 2 transmits the behavioural circadianrhythm of the suprachiasmatic nucleus. Nature 417:405–410

49. Cottrell GT, Zhou QY, Ferguson AV 2004 Prokineticin 2 modulates the ex-citability of subfornical organ neurons. J Neurosci 24:2375–2379

50. Horikawa K, Yokota S, Fuji K, Akiyama M, Moriya T, Okamura H, ShibataS 2000 Nonphotic entrainment by 5-HT1A/7 receptor agonists accompaniedby reduced Per1 and Per2 mRNA levels in the suprachiasmatic nuclei. J Neu-rosci 20:5867–5873

51. Maywood ES, Mrosovsky N, Field MD, Hastings MH 1999 Rapid down-regulation of mammalian period genes during behavioral resetting of thecircadian clock. Proc Natl Acad Sci USA 96:15211–15216

52. Card JP, Moore RY 1989 Organization of lateral geniculate-hypothalamicconnections in the rat. J Comp Neurol 284:135–147

53. Meyer-Bernstein EL, Morin LP 1996 Differential serotonergic innervation ofthe suprachiasmatic nucleus and the intergeniculate leaflet and its role incircadian rhythm modulation. J Neurosci 16:2097–2111

54. Bradbury MJ, Dement WC, Edgar DM 1997 Serotonin-containing fibers in thesuprachiasmatic hypothalamus attenuate light-induced phase delays in mice.Brain Res 768:125–134

55. Carlini VP, Monzon ME, Varas MM, Cragnolini AB, Schioth HB, ScimonelliTN, de Barioglio SR 2002 Ghrelin increases anxiety-like behavior and memoryretention in rats. Biochem Biophys Res Commun 299:739–743

56. Wellman PJ, Davis KW, Nation JR 2005 Augmentation of cocaine hyperac-tivity in rats by systemic ghrelin. Regul Pept 125:151–154

57. Kalsbeek A, van Heerikhuize JJ, Wortel J, Buijs RM 1998 Restricted daytimefeeding modifies suprachiasmatic nucleus vasopressin release in rats. J BiolRhythms 13:18–29

58. Elmquist JK, Scammell TE, Jacobson CD, Saper CB 1996 Distribution ofFos-like immunoreactivity in the rat brain following intravenous lipopolysac-charide administration. J Comp Neurol 371:85–103

59. Ericsson A, Kovacs KJ, Sawchenko PE 1994 A functional anatomical analysis

of central pathways subserving the effects of interleukin-1 on stress-relatedneuroendocrine neurons. J Neurosci 14:897–913

60. Kelly JF, Elias CF, Lee CE, Ahima RS, Seeley RJ, Bjorbaek C, Oka T, SaperCB, Flier JS, Elmquist JK 2004 Ciliary neurotrophic factor and leptin inducedistinct patterns of immediate early gene expression in the brain. Diabetes53:911–920

61. Hart BL 1988 Biological basis of the behavior of sick animals. Neurosci Biobe-hav Rev 12:123–137

62. Zheng H, Corkern M, Stoyanova I, Patterson LM, Tian R, Berthoud HR 2003Peptides that regulate food intake: appetite-inducing accumbens manipulationactivates hypothalamic orexin neurons and inhibits POMC neurons. Am JPhysiol 284:R1436–R1444

63. Ricardo JA, Koh ET 1978 Anatomical evidence of direct projections from thenucleus of the solitary tract to the hypothalamus, amygdala, and other fore-brain structures in the rat. Brain Res 153:1–26

64. Berthoud HR, Patterson LM, Neumann F, Neuhuber WL 1997 Distributionand structure of vagal afferent intraganglionic laminar endings (IGLEs) in therat gastrointestinal tract. Anat Embryol 195:183–191

65. Schemann M, Grundy D 1992 Electrophysiological identification of vagallyinnervated enteric neurons in guinea pig stomach. Am J Physiol 263:G709–G718

66. Qin LQ, Li J, Wang Y, Wang J, Xu JY, Kaneko T 2003 The effects of nocturnallife on endocrine circadian patterns in healthy adults. Life Sci 73:2467–2475

67. Kreier F, Yilmaz A, Kalsbeek A, Romijn JA, Sauerwein HP, Fliers E, BuijsRM 2003 Hypothesis: shifting the equilibrium from activity to food leads toautonomic unbalance and the metabolic syndrome. Diabetes 52:2652–2656

68. Dhillo WS, Small CJ, Stanley SA, Jethwa PH, Seal LJ, Murphy KG, GhateiMA, Bloom SR 2002 Hypothalamic interactions between neuropeptide Y,agouti-related protein, cocaine- and amphetamine-regulated transcript and�-melanocyte-stimulating hormone in vitro in male rats. J Neuroendocrinol14:725–730

Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.


ventromedial arcuate nucleus communicates peripheral ......ventromedial arcuate nucleus communicates...

Documents