The ventromedial nucleus of the hypothalamus (VMN) and the arcuate nucleus (ARC) are two centres regulating energy balance and food intake, but inter-connectivity of these nuclei is not well defined in non-rodent species. In this study, we performed retrograde tracing and immunohistochemistry in the ovine brain with ewes receiving FluoroGold® (FG) injections into either ARC or VMN for the mapping of retrogradely labelled cells. Strong reciprocal connections were found between the two regions. The distribution of the FG labelled neurons in other regions of the hypothalamus and brain stem was also mapped. Some of the cells projecting from ARC to VMN were immunopositive for neuropeptide Y, galanin, adrenocorticotropin (marker of pro-opiomelanocortin cells) or tyrosine hydroxylase (marker of dopaminergic cells). Melanin-concentrating hormone and orexin neurons in the lateral hypothalamic area were also found to provide input to the VMN and ARC. This observed interconnectivity between regions important for metabolic regulation and other neuroendocrine functions presumably allows coordinated functions. Input to both the ARC and VMN from other brain regions, such as brain stem cell groups, provides a further level of regulation. These data provide a substrate upon which further understanding of appetite regulation and neuroendocrine function can be derived in this species.

Keywords:
Body weight, Food intake, Appetite, Neuropeptides
1.
Hillebrand JJ, de Wied D, Adan RA: Neuropeptides, food intake and body weight regulation: a hypothalamic focus. Peptides 2002;23:2283–2306.
2.
Kimura H, Kuriyama K: Distribtuion of gamma-aminobutyric acid (GABA) in the rat hypothalamus: functional correlates of GABA with activities of appetite controlling mechanisms. J Neurochem 1975;24:903–907.
3.
Harlan RE, Shivers BD, Romano GJ, Howells RD, Pfaff DW: Localization of preproenkephalin mRNA in the rat brain and spinal cord by in situ hybridization. J Comp Neurol 1987;258:159–184.
4.
Routh VH: Glucosensing neurons in the ventromedial hypothalamic nucleus (VMN) and hypoglycemia-associated autonomic failure (HAAF). Diabetes Metab Res Rev 2003;19:348–356.
5.
Cone RD, Cowley MA, Butler AA, Fan W, Marks DL, Low MJ: The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis. Int J Obes Relat Metab Disord 2001;25(suppl 5):S63–S67.
6.
Elmquist JK: Hypothalamic pathways underlying the endocrine, autonomic, and behavioral effects of leptin. Physiol Behav 2001;74:703–708.
7.
Luiten PG, Room P: Interrelations between lateral, dorsomedial and ventromedial hypothalamic nuclei in the rat: an HRP study. Brain Res 1980;190:321–332.
8.
Bernardis LL, Bellinger LL: The lateral hypothalamic area revisited: neuroanatomy, body weight regulation, neuroendocrinology and metabolism. Neurosci Biobehav Rev 1993;17:141–193.
9.
Elmquist JK, Elias CF, Saper CB: From lesions to leptin: hypothalamic control of food intake and body weight. Neuron 1999;22:221–232.
10.
Elias CF, Saper CB, Maratos-Flier E, Tritos NA, Lee C, Kelly J, Tatro JB, Hoffman GE, Ollmann MM, Barsh GS, Sakurai T, Yanagisawa M, Elmquist JK: Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. J Comp Neurol 1998;402:442–459.
11.
Elias CF, Aschkenasi C, Lee C, Kelly J, Ahima RS, Bjorbaek C, Flier JS, Saper CB, Elmquist JK: Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 1999;23:775–786.
12.
Peruzzo B, Pastor FE, Blazquez JL, Schobitz K, Pelaez B, Amat P, Rodriguez EM: A second look at the barriers of the medial basal hypothalamus. Exp Brain Res 2000;132:10–26.
13.
Breen TL, Conwell IM, Wardlaw SL: Effects of fasting, leptin, and insulin on AGRP and POMC peptide release in the hypothalamus. Brain Res 2005;1032:141–148.
14.
Cummings DE, Foster-Schubert KE, Overduin J: Ghrelin and energy balance: focus on current controversies. Curr Drug Targets 2005;6:153–169.
15.
Kita H, Oomura Y: An HRP study of the afferent connections to rat medial hypothalamic region. Brain Res Bull 1982;8:53–62.
16.
Li C, Chen P, Smith MS: Identification of neuronal input to the arcuate nucleus (ARH) activated during lactation: implications in the activation of neuropeptide Y neurons. Brain Res 1999;824:267–276.
17.
Beltt BM, Keesey RE: Hypothalamic map of stimulation current thresholds for inhibition of feeding in rats. Am J Physiol 1975;229:1124–1133.
18.
Yoshimatsu H, Niijima A, Oomura Y, Yamabe K, Katafuchi T: Effects of hypothalamic lesion on pancreatic autonomic nerve activity in the rat. Brain Res 1984;303:147–152.
19.
Shimazu T: Innervation of the liver and glucoregulation: roles of the hypothalamus and autonomic nerves. Nutrition 1996;12:65–66.
20.
Henry BA, Rao A, Tilbrook AJ, Clarke IJ: Chronic food-restriction alters the expression of somatostatin and growth hormone-releasing hormone in the ovariectomised ewe. J Endocrinol 2001;170:R1–R5.
21.
Perkins MN, Rothwell NJ, Stock MJ, Stone TW: Activation of brown adipose tissue thermogenesis by the ventromedial hypothalamus. Nature 1981;289:401–402.
22.
Fahrbach SE, Morrell JI, Pfaff DW: Studies of ventromedial hypothalamic afferents in the rat using three methods of HRP application. Exp Brain Res 1989;77:221–233.
23.
Clarke IJ, Henry B, Iqbal J, Goding JW: Leptin and the regulation of food intake and the neuroendocrine axis in sheep. Clin Exp Pharmacol Physiol 2001;28:106–107.
24.
Henry BA, Goding JW, Tilbrook AJ, Dunshea FR, Blache D, Clarke IJ: Leptin-mediated effects of undernutrition or fasting on luteinizing hormone and growth hormone secretion in ovariectomized ewes depend on the duration of metabolic perturbation. J Neuroendocrinol 2004;16:244–255.
25.
Tillet Y, Thibault J: Catecholamine-containing neurons in the sheep brainstem and diencephalon: immunohistochemical study with tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) antibodies. J Comp Neurol 1989;290:69–104.
26.
Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M: Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proc Natl Acad Sci USA 1999;96:748–753.
27.
Iqbal J, Pompolo S, Murakami T, Grouzmann E, Sakurai T, Meister B, Clarke IJ: Immunohistochemical characterization of localization of long-form leptin receptor (OB-Rb) in neurochemically defined cells in the ovine hypothalamus. Brain Res 2001;920:55–64.
28.
Bittencourt JC, Presse F, Arias C, Peto C, Vaughan J, Nahon JL, Vale W, Sawchenko PE: The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization. J Comp Neurol 1992;319:218–245.
29.
Carey DG, Iismaa TP, Ho KY, Rajkovic IA, Kelly J, Kraegen EW, Ferguson J, Inglis AS, Shine J, Chisholm DJ: Potent effects of human galanin in man: growth hormone secretion and vagal blockade. J Clin Endocrinol Metab 1993;77:90–93.
30.
Christie MJ, Beart PM, Jarrott B, Maccarrone C: Distribution of neuropeptide/immunoreactivity in the rat basal ganglia: effects of excitotoxin lesions to caudate-putamen. Neurosci Lett 1986;63:305–309.
31.
Iqbal J, Manley TR, Yue Q, Namavar MR, Clarke IJ: Noradrenergic regulation of hypothalamic cells that produce growth hormone-releasing hormone and somatostatin and the effect of altered adiposity in sheep. J Neuroendocrinol 2005;17:341–352.
32.
Reddy S, Oliver JR, Elliott RB: Immunohistochemical demonstration of somatostatin in the pancreas of fetal and adult sheep. Aust J Biol Sci 1984;37:25–29.
33.
Willoughby JO, Oliver JR, Fletcher TP, Clarke IJ: Distribution of somatostatin immunoreactivity in sheep hypothalamus: a comparison with that of the rat. Arch Histol Cytol 1995;58:31–36.
34.
Berod A, Hartman BK, Keller A, Joh TH, Pujol JF: A new double labeling technique using tyrosine hydroxylase and dopamine-beta-hydroxylase immunohistochemistry: evidence for dopaminergic cells lying in the pons of the beef brain. Brain Res 1982;240:235–243.
35.
Kitahama K, Denoroy L, Goldstein M, Jouvet M, Pearson J: Immunohistochemistry of tyrosine hydroxylase and phenylethanolamine N-methyltransferase in the human brain stem: description of adrenergic perikarya and characterization of longitudinal catecholaminergic pathways. Neuroscience 1988;25:97–111.
36.
Herz A, Gramsch C, Hollt V, Meo T, Riethmuller G: Characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides. Life Sci 1982;31:1721–1724.
37.
Iqbal J, Pompolo S, Dumont LM, Wu CS, Mountjoy KG, Henry BA, Clarke IJ: Long-term alterations in body weight do not affect the expression of melanocortin receptor-3 and -4 mRNA in the ovine hypothalamus. Neuroscience 2001;105:931–940.
38.
Richard P: Atlas stéréotaxique du cerveau de brebis Pré-Alpes-du-Sud. Paris, Institut national de la recherche agronomique, 1967.
39.
Iqbal J, Manley TR, Yue Q, Namavar MR, Clarke IJ: Noradrenergic regulation of hypothalamic cells that produce growth hormone-releasing hormone and somatostatin and the effect of altered adiposity in sheep. J Neuroendocrinol 2005;17:341–352.
40.
Kita H, Oomura Y: An HRP study of the afferent connections to rat lateral hypothalamic region. Brain Res Bull 1982;8:63–71.
41.
Luiten PG, ter Horst GJ, Steffens AB: The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism. Prog Neurobiol 1987;28:1–54.
42.
Bernardis LL, Bellinger LL: The lateral hypothalamic area revisited: ingestive behavior. Neurosci Biobehav Rev 1996;20:189–287.
43.
Pompolo S, Rawson JA, Clarke IJ: Projections from the arcuate/ventromedial region of the hypothalamus to the preoptic area and bed nucleus of stria terminalis in the brain of the ewe; lack of direct input to gonadotropin-releasing hormone neurons. Brain Res 2001;904:1–12.
44.
Scott CJ, Clarke IJ, Tilbrook AJ: Neuronal inputs from the hypothalamus and brain stem to the medial preoptic area of the ram: neurochemical correlates and comparison to the ewe. Biol Reprod 2003;68:1119–1133.
45.
Rivalland ET, Tilbrook AJ, Turner AI, Iqbal J, Pompolo S, Clarke IJ: Projections to the preoptic area from the paraventricular nucleus, arcuate nucleus and the bed nucleus of the stria terminalis are unlikely to be involved in stress-induced suppression of GnRH secretion in sheep. Neuroendocrinology 2006;84:1–13.
46.
Scott CJ, Rawson JA, Pereira AM, Clarke IJ: Oestrogen receptors in the brainstem of the female sheep: relationship to noradrenergic cells and cells projecting to the medial preoptic area. J Neuroendocrinol 1999;11:745–755.
47.
Pompolo S, Pereira A, Scott CJ, Fujiyma F, Clarke IJ: Evidence for estrogenic regulation of gonadotropin-releasing hormone neurons by glutamatergic neurons in the ewe brain: an immunohistochemical study using an antibody against vesicular glutamate transporter-2. J Comp Neurol 2003;465:136–144.
48.
Quan N, Xin L, Ungar AL, Blatteis CM: Preoptic norepinephrine-induced hypothermia is mediated by alpha 2-adrenoceptors. Am J Physiol 1992;262:R407–R411.
49.
Barker-Gibb ML, Clarke IJ: Effect of season on neuropeptide Y and galanin within the hypothalamus of the ewe in relation to plasma luteinizing hormone concentrations and the breeding season: an immunohistochemical analysis. J Neuroendocrinol 2000;12:618–626.
50.
Hashimoto H, Fukui K, Noto T, Nakajima T, Kato N: Distribution of vasopressin and oxytocin in rat brain. Endocrinol Jpn 1985;32:89–97.
51.
Hou-Yu A, Lamme AT, Zimmerman EA, Silverman AJ: Comparative distribution of vasopressin and oxytocin neurons in the rat brain using a double-label procedure. Neuroendocrinology 1986;44:235–246.
52.
Anand BK, Brobeck JR: Localization of a ‘feeding center’ in the hypothalamus of the rat. Proc Soc Exp Biol Med 1951;77:323–324.
53.
Ter Horst GJ, Luiten PG: Phaseolus vulgaris leuco-agglutinin tracing of intrahypothalamic connections of the lateral, ventromedial, dorsomedial and paraventricular hypothalamic nuclei in the rat. Brain Res Bull 1987;18:191–203.
54.
Backberg M, Hervieu G, Wilson S, Meister B: Orexin receptor-1 (OX-R1) immunoreactivity in chemically identified neurons of the hypothalamus: focus on orexin targets involved in control of food and water intake. Eur J Neurosci 2002;15:315–328.
55.
Abbott CR, Kennedy AR, Wren AM, Rossi M, Murphy KG, Seal LJ, Todd JF, Ghatei MA, Small CJ, Bloom SR: Identification of hypothalamic nuclei involved in the orexigenic effect of melanin-concentrating hormone. Endocrinology 2003;144:3943–3949.
56.
Canteras NS, Simerly RB, Swanson LW: Organization of projections from the ventromedial nucleus of the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat. J Comp Neurol 1994;348:41–79.
57.
McShane TM, May T, Miner JL, Keisler DH: Central actions of neuropeptide-Y may provide a neuromodulatory link between nutrition and reproduction. Biol Reprod 1992;46:1151–1157.
58.
Cunningham MJ: Galanin-like peptide as a link between metabolism and reproduction. J Neuroendocrinol 2004;16:717–723.
59.
Gottsch ML, Clifton DK, Steiner RA: Galanin-like peptide as a link in the integration of metabolism and reproduction. Trends Endocrinol Metab 2004;15:215–221.
60.
Schioth HB, Watanobe H: Melanocortins and reproduction. Brain Res Brain Res Rev 2002;38:340–350.
61.
Herbison AE: Neurochemical identity of neurones expressing oestrogen and androgen receptors in sheep hypothalamus. J Reprod Fertil Suppl 1995;49:271–283.
62.
Scott CJ, Tilbrook AJ, Simmons DM, Rawson JA, Chu S, Fuller PJ, Ing NH, Clarke IJ: The distribution of cells containing estrogen receptor-alpha (ERalpha) and ERbeta messenger ribonucleic acid in the preoptic area and hypothalamus of the sheep: comparison of males and females. Endocrinology 2000;141:2951–2962.
63.
Herbison AE, Pape JR: New evidence for estrogen receptors in gonadotropin-releasing hormone neurons. Front Neuroendocrinol 2001;22:292–308.
64.
Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA: Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 2005;146:3686–3692.
65.
Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, Clifton DK, Steiner RA: Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 2005;146:2976–2984.
66.
Franceschini I, Lomet D, Cateau M, Delsol G, Tillet Y, Caraty A: Kisspeptin immunoreactive cells of the ovine preoptic area and arcuate nucleus co-express estrogen receptor alpha. Neurosci Lett 2006;401:225–230.
67.
Smith JT, Acohido BV, Clifton DK, Steiner RA: KiSS-1 neurones are direct targets for leptin in the ob/ob mouse. J Neuroendocrinol 2006;18:298–303.
68.
Schwartz MW, Woods SC, Porte D Jr, Seeley RJ, Baskin DG: Central nervous system control of food intake. Nature 2000;404:661–671.
69.
Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P: Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett 1996;387:113–116.
70.
Kurose Y, Iqbal J, Rao A, Murata Y, Hasegawa Y, Terashima Y, Kojima M, Kangawa K, Clarke IJ: Changes in expression of the genes for the leptin receptor and the growth hormone-releasing peptide/ghrelin receptor in the hypothalamic arcuate nucleus with long-term manipulation of adiposity by dietary means. J Neuroendocrinol 2005;17:331–340.
71.
Dhillon H, Zigman JM, Ye C, Lee CE, McGovern RA, Tang V, Kenny CD, Christiansen LM, White RD, Edelstein EA, Coppari R, Balthasar N, Cowley MA, Chua S Jr, Elmquist JK, Lowell BB: Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis. Neuron 2006;49:191–203.
72.
Yamano M, Inagaki S, Kito S, Tohyama M: An enkephalinergic projection from the hypothalamic paraventricular nucleus to the hypothalamic ventromedial nucleus of the rat: an experimental immunohistochemical study. Brain Res 1985;331:25–33.
73.
Matthews SG, Parrott RF, Sirinathsinghji DJ: Isolation- and dehydration-induced changes in neuropeptide gene expression in the sheep hypothalamus. J Mol Endocrinol 1993;11:181–189.
74.
Commons KG, Kow LM, Milner TA, Pfaff DW: In the ventromedial nucleus of the rat hypothalamus, GABA-immunolabeled neurons are abundant and are innervated by both enkephalin- and GABA-immunolabeled axon terminals. Brain Res 1999;816:58–67.
75.
Henry BA, Tilbrook AJ, Dunshea FR, Rao A, Blache D, Martin GB, Clarke IJ: Long-term alterations in adiposity affect the expression of melanin-concentrating hormone and enkephalin but not proopiomelanocortin in the hypothalamus of ovariectomized ewes. Endocrinology 2000;141:1506–1514.
76.
Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG: Identification of targets of leptin action in rat hypothalamus. J Clin Invest 1996;98:1101–1106.
77.
Lawrence CB, Snape AC, Baudoin FM, Luckman SM: Acute central ghrelin and GH secretagogues induce feeding and activate brain appetite centers. Endocrinology 2002;143:155–162.
78.
Chaillou E, Baumont R, Tramu G, Tillet Y: Effect of feeding on Fos protein expression in sheep hypothalamus with special reference to the supraoptic and paraventricular nuclei: an immunohistochemical study. Eur J Neurosci 2000;12:4515–4524.
79.
Chaillou E, Baumont R, Tramu G, Tillet Y: Long-term undernutrition followed by short-term refeeding effects on the corticotropin-releasing hormone containing neurones in the paraventricular nucleus: an immunohistochemical study in sheep. J Neuroendocrinol 2002;14:269–275.
80.
Segar JL, Ellsbury DL, Smith OM: Inhibition of sympathetic responses at birth in sheep by lesion of the paraventricular nucleus. Am J Physiol 2002;283:R1395–R1403.
81.
Kenney MJ, Weiss ML, Mendes T, Wang Y, Fels RJ: Role of paraventricular nucleus in regulation of sympathetic nerve frequency components. Am J Physiol 2003;284:H1710–H1720.
82.
Rinaman L: Postnatal development of catecholamine inputs to the paraventricular nucleus of the hypothalamus in rats. J Comp Neurol 2001;438:411–422.
83.
Cunningham ET Jr, Sawchenko PE: Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol 1988;274:60–76.
84.
Sawchenko PE, Arias C, Bittencourt JC: Inhibin beta, somatostatin, and enkephalin immunoreactivities coexist in caudal medullary neurons that project to the paraventricular nucleus of the hypothalamus. J Comp Neurol 1990;291:269–280.
85.
Ohman LE, Johnson AK: Lesions in lateral parabrachial nucleus enhance drinking to angiotensin II and isoproterenol. Am J Physiol 1986;251:R504–R509.
86.
Ohman LE, Johnson AK: Role of lateral parabrachial nucleus in the inhibition of water intake produced by right atrial stretch. Brain Res 1995;695:275–278.
87.
Hosoi T, Kawagishi T, Okuma Y, Tanaka J, Nomura Y: Brain stem is a direct target for leptin’s action in the central nervous system. Endocrinology 2002;143:3498–3504.
88.
Panksepp J, Rossi J 3rd: D-Glucose infusions into the basal ventromedial hypothalamus and feeding. Behav Brain Res 1981;3:381–392.
89.
Huo L, Maeng L, Bjorbaek C, Grill HJ: Leptin and the control of food intake: Neurons in the nucleus of the solitary tract (NTS) are activated by both gastric distension and leptin. Endocrinology 2007;148:2189–2197.
© 2007 S. Karger AG, Basel
2007
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.