The Journal of Neuroscience, 1999, Vol. 19 RC26 1of7 Anatomy of an Endogenous Antagonist: Relationship between Agouti-Related Protein and Proopiomelanocortin in Brain Didier Bagnol,1,4 Xin-Yun Lu,1 Christopher B. Kaelin,3 Heidi E. W. Day,1 Michael Ollmann,3 Ira Gantz,2 Huda Akil,1 Gregory S. Barsh,3 and Stanley J. Watson1 1Mental Health Research Institute and 2Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109-0720, 3Departments of Pediatrics and Genetics, Howard Hughes Medical Institute, Stanford, California 94305, and 4Centre National de la Recherche Scientifique, Neurobiologie des Fonctions Ve´ge´ tatives, Universite´ d’Aix-Marseille, Marseille 3, Cedex 20, France Agouti-related protein (AGRP) is a recently discovered orexi- situ hybridization. Although deriving from distinct cell groups, genic neuropeptide that inhibits the binding and action of AGRP and melanocortin terminals project to identical brain a-melanocyte-stimulating hormone derived from proopiomela- areas. Both AGRP and melanocortin neurons selectively ex- nocortin (POMC) at the melanocortin 3 receptor (MC3R) and press the MC3R, which provides a neuroanatomical basis for a melanocortin 4 receptor (MC4R) and has been proposed to dual-input circuit with biological amplification and feedback function primarily as an endogenous melanocortin antagonist. inhibition. These studies highlight a broader complexity in To better understand the interplay between the AGRP and POMC-mediated behavior in the brain. melanocortin signaling systems, we compared their nerve fiber distributions with each other by immunohistochemistry and Key words: Agouti-related protein; proopiomelanocortin; in- their perikarya distribution with MC3R and MC4R by double in gestive behavior; MC3R; MC4R; arcuate nucleus Naturally occurring antagonists can act either by binding to and intake and obesity (Graham et al., 1997; Ollmann et al., 1997; sequestering a ligand or by binding to a receptor to prevent its Grill et al., 1998; Rossi et al., 1998). response to another molecule. Unique advantages for biological Because the action of AGRP has only been tested on melano- regulation are provided by the latter mechanism, of which Agouti cortin receptors, the question remains, does AGRP work primar- protein and Agouti-related protein (AGRP) are prime examples ily as a melanocortin antagonist, or might it have other functions? (Ollmann et al., 1997, 1998; Shutter et al., 1997). These proteins One approach to this question is to examine its anatomy vis-a-vis inhibit the activity of melanocortins, small peptides such as the anatomy of the melanocortins and to determine whether a-melanocyte-stimulating hormone (a-MSH) or adrenocortico- AGRP only exists where melanocortins are found, or whether it is trophic hormone derived from a large precursor, proopiomelano- also expressed at other sites independent of either the ligands or cortin (POMC), that also gives rise to b-endorphin. AGRP binds the receptor(s) that it is purported to antagonize. To investigate directly to melanocortin receptors but has little intrinsic signaling the potential for presynaptic and/or direct crosstalk between activity, and instead functions primarily by inhibiting a-MSH AGRP and POMC systems, we examined the colocalization of binding (Ollmann et al., 1997; Shutter et al., 1997). Indeed, the AGRP or POMC with the MC3R and MC4R using double in situ melanocortin receptors were originally identified by their ability hybridization. to activate adenylate cyclase in response to a-MSH. However, recent studies have suggested that physiological modulation of MATERIALS AND METHODS receptor signaling may be accomplished mainly by alteration in Animals. Male Sprague Dawley rats (Charles River Laboratories, Wil- a mington, MA) weighing 300–350 gm were used in this study. Rats were the levels of AGRP rather than -MSH. Starvation and leptin housed in groups of two or three per cage with food and water available deficiency cause a predominant rise in levels of AGRP mRNA ad libitum in a 12 hr light/dark cycle (lights on at 7 A.M.) under rather than a decrease in POMC mRNA levels in the hypothal- conditions of constant temperature and humidity. Animals were allowed amus (Thornton et al., 1997; Mizuno et al., 1998; Mizuno Mobbs, to habituate for 1 week before experiments. Protocols for animal exper- 1999; Wilson et al., 1999). Artificial increases in AGRP achieved pharmacologically or in transgenic animals cause elevated food This article is published in The Journal of Neuroscience, Rapid Received May 21, 1999; revised July 15, 1999; accepted July 21, 1999. Communications Section, which publishes brief, peer- This work was supported by National Institutes of Health Grants P01 MH-42251 reviewed papers online, not in print. Rapid Communications (from National Institute of Mental Health; to S.J.W.), DK28506 (to G.S.B.), and R01 are posted online approximately one month earlier than they DK-54032–01 (to I.G.). We thank Sharon Burke and Robert Pavlic for technical assistance and Drs. Serge Campeau and Manuel Lopez-Figueroa for advice and would appear if printed. They are listed in the Table of comments. Contents of the next open issue of JNeurosci. Cite this article D.B. and X.-Y.L. contributed equally to this work. as: JNeurosci, 1999, 19:RC26 (1–7). The publication date is Correspondence should be addressed to Stanley J. Watson, Mental Health Re- search Institute, The University of Michigan, 205 Zina Pitcher Place, Ann Arbor, the date of posting online at www.jneurosci.org. MI 48109-0720. Copyright © 1999 Society for Neuroscience 0270-6474/99/190001-07$05.00/0 http://www.jneurosci.org/cgi/content/full/3484 2of7 J. Neurosci., 1999, Vol. 19 Bagnol et al. • Anatomy of an Endogenous Melanocortin Antagonist, AGRP Table 1. Distribution and relative abundance of AGRP- and POMC- Table 1. Continued immunoreactive fibers and terminals in the rat CNS Anatomical sites Agrp g-MSH Anatomical sites Agrp g-MSH Compact 22 Telencephalon Ventral part 111 1111 1 21 Anterior olfactory nucleus, posterior part / Dorsal hypothalamic area 11 111 1 21 Olfactory tubercle / Lateroanterior hypothalamic nucleus 1 111 212 Cingulate cortex / Lateral hypothalamic area 111 111 1 212 Infralimbic cortex / / Ventrolateral hypothalamic nucleus 11 11 212 Ventral orbital cortex / Perifornical nucleus 1111 111 1 212 Endopiriform nucleus / / Posterior hypothalamic area 111 Accumbens nucleus Arcuate nucleus 11111 1111 111 Shell Median eminence, internal part 111 111 1 212 Core / / Median eminence, external part 11 11 111 Substantia innominata Medial tuberal nucleus 11 111 11 Anterior amygdaloid area Supramammillary nucleus 111 1 212 Anterior cortical amygdaloid nucleus / / Mesencephalon Central nucleus Substantia nigra compact 211 11 11 Medial division Ventral tegmental area 1/211 11 Lateral division Intrafascicular nucleus 1/21 11 Medial amygdaloid nucleus Interpeduncular nucleus 1/21 Basomedial amygdaloid nucleus Rostral linear nucleus raphe 1/21 11 Anterior part Periacqueductal gray 1(1) 11(1) 212 Ventral part / Edinger–Westphal nucleus 1/21 Bed nucleus of stria terminalis Dorsal raphe nucleus 1(1) 111 1111 111 Ventral division Precommissural nucleus 1 111 111 1111 Medial division Commissure of the superior colliculus 11 1 1 111 Lateral division ( ) Medial pretectal nucleus 21 112 Supracapsular division / Deep mesencephalic nucleus 211 Lateral septum: Anterior pretectal nucleus, ventral part 21 11 111 Ventral part Peripedoncular nucleus 21 1 212 Dorsal division / / Metencephalon 111 Medial septum ventral Cerebellum 22 11 Nucleus of the diagonal band Parabrachial nucleus, lateral part 11 111 11 Subfornical organ Parabrachial nucleus, medial part 1/21(1) Diencephalon Locus coeruleus 11 111 21 Medial habenular nucleus Subcoeruleus nucleus 1/21 11 1 111 Paraventricular thalamic nucleus ( ) Motor trigeminal nucleus 11 111 1 211 Paratenial thalamic nucleus / Mesencephalic trigeminal nucleus 11 111 212 Laterodorsal thalamic nucleus, ventrolateral part / Pontine reticular nucleus 1/211 112 Stria terminalis / Pontine reticular nucleus caudal 21 1 21 Nucleus of stria medullaris / Principal sensory trigminal nucleus, ventrolateral 211 111 Zona incerta ( ) Myelencephalon 11 111 Strial part of the preoptic area Raphe magnus nucleus 21(1) 11 1 11 1 Striohypothalamic nucleus ( ) ( ) A5 Noradrenaline cells 21 1111 111 Organum vasculosum of the lamina terminalis Gignatocellular reticular nucleus 21 1111 111 Medial preoptic area Nucleus of solitary tract, median part 111 111 1 111 Median preoptic nucleus ( ) Nucleus of solitary tract, lateral part (1) 1(1) 111 11 1 Anteroventral preoptic nucleus ( ) Ambigus nucleus 11 11 11 11 Lateral preoptic area Spinal cord (cervical) 111 Supraoptic nucleus Spinal cord layers 1–7, 10 21/2 11 1 Suprachiasmatic nucleus Lateral funiculus of the spinal cord 211 11 111 Anterior hypothalamic nucleus Pituitary gland Paraventricular hypothalamic nucleus Posterior pituitary 12 Magnocellular division 11111 111 111 11 The density of immunoreactive fibers is estimated and indicated as 2, undetectable Parvocellular division 1 2 1 11 1111 1111 immunoreactivity; / , occasional single fibers; , light density; , moderate; Periventricular hypothalamic nucleus 111, dense; 1111, heavy; and 11111, compact, with parentheses represent- Retrochiasmatic area 1111 1111 ing intermediate levels. Ventromedial hypothalamic nucleus 11 Dorsomedial hypothalamic nucleus Dorsal part 11 111 Bagnol et al. • Anatomy of an Endogenous Melanocortin Antagonist, AGRP J. Neurosci., 1999, Vol. 19 3of7