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Endocrinology Vol. 140, No. 3 1408-1415
Copyright © 1999 by The Endocrine Society

ARTICLES

Characterization of the Neuroanatomical Distribution of Agouti-Related Protein Immunoreactivity in the Rhesus Monkey and the Rat1

Carrie Haskell-Luevano2, Peilin Chen2, Chien Li1, Kang Chang, M. Susan Smith, Judy L. Cameron and Roger D. Cone

Vollum Institute (C.H.-L., R.D.C.), Oregon Regional Primate Research Center (P.C., C.L., M.S.S., J.C.) Oregon Health Sciences University, Portland, Oregon 97201; and Phoenix Pharmaceuticals, Inc. (K.C.), Mountain View, California 94043

Address all correspondence and requests for reprints to: Dr. Roger D. Cone, Vollum Institute L-474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201. E-mail: cone{at}ohsu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
Agouti-related protein (AGRP) is a recently described homolog of the skin agouti protein. AGRP is transcribed primarily in the adrenal and hypothalamus and is a high affinity antagonist of the neural melanocortin-3 and melanocortin-4 receptors. The perikarya expressing AGRP messenger RNA are found in the arcuate nucleus of the rat and rhesus monkey. Using a polyclonal antibody against the pharmacologically active domain of AGRP (amino acids 83–132), we have also characterized the distribution of AGRP-immunoreactive neurons in both species. The major fiber tracts are conserved in both species, with dense projections originating in the arcuate nucleus and proceeding along the third ventricle. Dense fiber bundles are also visible in the paraventricular, dorsomedial, and posterior nuclei in the hypothalamus, in the bed nucleus of the stria terminalis, and in the lateral septal nucleus of the septal region. AGRP-containing neurons are not visualized in a number of areas, including portions of the amygdala, thalamus, and brain stem, that express MC3-R and MC4-R messenger RNA and receive innervation from POMC neurons that serve as the source of melanocortin agonists. Thus, AGRP is most likely to be involved in modulating a conserved subset of the physiological functions of central melanocortin peptides. Based on the particular distribution of AGRP neurons, those functions are likely to include the central control of energy homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
POMC, THE precursor of the melanocortin peptides ({alpha}-MSH, ß-MSH, {gamma}-MSH, and ACTH), is expressed in the brain exclusively in neuronal cell bodies throughout the rostrocaudal extent of the arcuate nucleus (ARC) and periarcuate area of the hypothalamus and within the caudal half of the commissural nucleus of the solitary tract (1, 2, 3, 4, 5). Proceeding from rostral to caudal, arcuate POMC cells send a dense bundle of fibers ventral to the anterior commissure, to a number of nuclei in the septal region, including the bed nucleus of the stria terminalis and lateral septal nucleus (LS), as well as to the nucleus accumbens in the caudate putamen. More caudally, fibers are seen projecting to the periventricular region of the thalamus and to the medial amygdala. Within the hypothalamus, the densest fibers project to the periventricular nucleus, the paraventricular nucleus (PVH), and the perifornical region, with some fibers seen in almost all hypothalamic regions. Finally, arcuate POMC fibers also project to several regions of the brain stem, including the periaquaductal gray, reticular formation, and parabrachial nucleus.

In situ binding studies using [125I]Nle4,D-Phe7-{alpha}-MSH have demonstrated that {alpha}-MSH binding sites correlate well with POMC terminal fields in the forebrain (6) and brain stem (7). Furthermore, the distribution of expression of the MC3-R and MC4-R in the rat central nervous system (CNS), determined by in situ hybridization (8, 9, 10, 11), can easily account for the majority, if not all, of the melanocortin binding demonstrated in the CNS.

The central melanocortin system, as defined above by POMC neurons and the central MC3-R and MC4-R receptors, was recently given an added level of complexity by the discovery of agouti-related protein (AGRP) (12, 13). This 132-amino acid peptide is a homolog of the skin agouti peptide. The skin agouti peptide has previously been demonstrated to be an antagonist of the melanocortin-1 receptor (14) on melanocytes, where it is involved in the inhibition of eumelanin, or brown-black pigment, synthesis. The existence of a brain-specific agouti was suggested by the observation that the skin agouti peptide was also a high affinity antagonist of the neural MC4-R receptor (14) and caused an obesity syndrome when expressed ectopically in mouse strains containing certain dominant alleles of the gene (15, 16, 17). This obesity syndrome appears to result specifically from blockade of MC4-R signaling, as shown both pharmacologically and by gene knockout of the MC4-R (18, 19), suggesting that one function of POMC neurons involves the control of energy homeostasis.

In addition to being expressed in the arcuate nucleus, AGRP is also a potent antagonist of both the MC3-R and MC4-R (13, 20). As might be predicted, ubiquitous expression of a ß-actin promoter/AGRP gene in transgenic mice (13, 21) results in an obesity syndrome comparable to that seen in mice with dominant agouti alleles such as AVY (22) and in MC4-RKO mice (19). POMC neurons and the MC4-R have been demonstrated to be involved in a number of processes other than energy homeostasis, however, including grooming behavior (23), control of fever (24), and cardiovascular homeostasis (25). Thus, determination of the potential physiological roles for AGRP requires characterization of the distribution of this protein in the CNS to identify MC3-R- and MC4-R-containing sites that may be coregulated by melanocortin agonists and the AGRP antagonist. Using a specific antibody to the C-terminal portion of this protein, we show here the distribution of AGRP immunoreactivity in the rat and rhesus monkey.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Animals
Rats. Female Sprague-Dawley rats (n = 8; B & K Universal, Inc., Kent, WA), weighing 240–260 µg, were housed in a room with a 12-h light, 12-h dark cycle (lights on at 0700 h) and constant temperature (23 ± 2 C). Food and water were provided ad libitum. Vaginal smears were examined daily to check the stage of the estrous cycle for each animal. Four animals were killed by decapitation during the diestrous stage of the cycle. Brains were removed, frozen on dry ice, and stored at -80 C until used for in situ hybridization. The remaining four animals were killed by perfusion as described below. All animal procedures involving rats were approved by the Oregon Regional Primate Research Center Institutional Animal Care and Use Committee.

Perfusion and tissue sectioning: Rats were anesthetized with a lethal dose of pentobarbital (125 mg/kg BW, ip) and perfused transcardially with 150 ml 2% sodium nitrite in saline followed by 150 ml 2.5% acrolein (EM grade, Polysciences, Warrington, PA) in phosphate-buffered 4% paraformaldehyde (pH 7.4). The brain was removed, blocked, and immersed in 25% sucrose at 4 C overnight. Coronal sections (25 µm) were cut through the whole brain on a sliding microtome and collected in a one in six series. The tissue sections were stored at -20 C in multiwell tissue culture plates containing cryoprotectant until use.

Rhesus monkeys. Hypothalamic and brain stem tissues from adult male rhesus monkeys (Macaca mulatta), weighing 8.9–10.2 kg, were used for this study. Monkeys were maintained in single animal cages in a room with a controlled lighting schedule (lights on, 0700–1900 h) and were fed one meal a day at approximately 1100 h of about 600 Cal high protein monkey chow (Ralston Purina Co., St. Louis, MO), with water available at all times. At least 1 month before death, monkeys had indwelling iv and intragastric catheters implanted using sterile surgical techniques, as previously described (26, 27). Catheters exited the body at the midscapular region of the back, and monkeys were maintained in jacket/tether/swivel systems (27), with the catheters running from the top of the swivel through a hole in the wall behind the monkey’s cage to enter an adjacent room. Catheter patency was maintained by a constant saline infusion through the catheters of 100 ml/day, with the venous drip containing 400 U sodium heparin. Before death, all monkeys had been fasted at least one time and had received at least one meal via the gastric cannula, and blood samples had been collected and assayed for insulin and T3 to ensure that the metabolic changes expected to occur with fasting and feeding were detectable. This study was approved by the institutional animal care and use committee of the University of Pittsburgh.

Perfusion and tissue sectioning: Monkeys were fasted the day before death, and on the morning of death a fed monkey received an infusion of 200 ml nutrients (containing 600 Cal, with the same percentage of protein, carbohydrate, and fat as the standard monkey chow) (26) via the gastric cannula from 0630–0730 h, whereas the fasted animal did not. At 1025 h monkeys were sedated with ketamine HCl (10 mg/kg, im), and deeply anesthetized with sodium pentobarbital (~15 mg/kg, iv). The chest was then opened by a midline incision, and each monkey was perfused transcardially with 0.9% NaCl containing 2% sodium nitrite (800–1000 ml) to flush blood from the vascular system, followed by 4% paraformaldehyde in 0.1 M potassium phosphate buffer solution (KPBS; pH 6.8–7.2; 1000–1200 ml). The brain and upper portion of the spinal cord were removed, hypothalamic and brain stem tissue blocks were cut, and blocks were immersed in a postperfusion fixative of 2.5% acrolein and 4% paraformaldehyde-KPBS solution for 2 h at 25 C, followed by placement in a 25% sucrose solution in distilled water at 4 C for 4–6 days until they sunk to the bottom of the sucrose solution. Fresh sucrose solution was replaced daily. The hypothalamus was sectioned (30-µm sections) on a coronal plane using a freezing microtome, and sections were stored in cryoprotectant at -20 C until immunocytochemical and in situ hybridization procedures were performed. Due to the limited number of animals available for this study, the data presented here result from analysis of one fasted and one fed rhesus monkey.

Immunohistochemistry
Both monkey and rat sections were processed at the same time to ensure uniformity of immunostaining. Tissue sections were removed from cryoprotectant and rinsed in 0.05 M KPBS followed by treatment with 1% NaBH4-KPBS solution (Sigma Chemical Co., St. Louis, MO). Sections were incubated in rabbit anti-AGRP antibody (G-003–53; 1:35,000; Phoenix Pharmaceuticals, Inc., Mountain View, CA) in KPBS with 0.4% Triton X-100 at 4 C for 48 h. Control sections were preabsorbed with 20 µg AGRP-(83–132) peptide to 1 µg antibody to test the specificity of the antibody. After rinsing in KPBS, the sections were incubated for 1 h at room temperature in biotinylated goat antirabbit IgG (1:600, Vector Laboratories, Inc., Burlingame, CA), followed by 10-min incubation in tyramide signal amplification solution according to the manufacturer’s instruction (TSA-Indirect kit, New England Nuclear Life Science, Boston, MA). The antibody-peroxidase complex was stained with a mixture of 3,3'-diaminobenzidine and H2O2 in 0.05 M Tris buffer-saline solution. After the staining, tissue sections were mounted on gelatin-coated glass slides and air-dried. The 3,3'-diaminobenzidine staining on the tissue sections was further enhanced by osmium tetroxide (OsO4), as described previously (28), before being coverslipped with Histomount (Fisher Scientific, Pittsburgh, PA).

In situ hybridization
Monkey brain. A 399-bp human AGRP complementary DNA was generated by PCR and subcloned into the pBSII SK(±) vector (Stratagene, La Jolla, CA). The human (h) AGRP complementary RNA (cRNA) probe was transcribed from the vector with 40% of the UTP provided in a 33P-labeled form (New England Nuclear Life Science, Boston, MA) (29). The specific activity of the probe ranged from 1–2 x 109 dpm/µg. Brain sections were rinsed in PBS and treated with 1% NaBH4-PBS solution. After washing with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) and in 2 x SSC, sections were incubated in prehybridization solution containing 50% formamide, 6.25% dextran sulfate, 0.7% Ficoll, 0.7% polyvinyl pyrolidone, and 2 mg/ml yeast transfer RNA for 2 h at 55 C. Subsequently, sections were incubated in the same solution, with the addition of 1.5 x 105 cpm/µl antisense (or sense) cRNA probe for 15 h at 55 C. After hybridization, sections were washed in 4 x SSC (standard saline citrate), digested with ribonuclease A for 30 min at 37 C, and washed through decreasing concentrations of SSC to a final stringency of 0.1 x SSC at 60 C for 30 min. Sections were mounted on gelatin-coated glass slides and allowed to dry. The slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 7–8 days at 4 C, and developed. After development, the slides were dehydrated, counterstained with cresyl violet, and coverslipped in Histomount.

Rat brain. Human AGRP cRNA probe was synthesized and used for in situ hybridization as described above. Briefly, the hAGRP cRNA probe was transcribed from a 399-bp complementary DNA in which 40% of the UTP was 35S labeled (New England Nuclear Life Science, Boston, MA). The saturating concentration of the probe used in the assay was 1.0 µg/ml. The specific activity of the probe ranged from 8–9 x 108 dpm/µg. In situ hybridization was performed as described previously (29). Frozen brain sections (20 µM) were fixed in 4% paraformaldehyde and treated with a fresh solution containing 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), followed by a rinse in 2 x SSC, dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air-dried. The slides were exposed to the hAGRP cRNA probe overnight in moist chambers at 55 C. After incubation, the slides were washed in ribonuclease-containing SSC that increased in stringency up to a wash in 0.1 x SSC at 60 C, then dehydrated through a graded series of alcohols and air-dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 7–8 days at 4 C, and developed. After development, the slides were coverslipped with Histomount.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Expression of AGRP messenger RNA (mRNA) in the rat and rhesus brain
Using in situ hybridization, AGRP-positive signals in the rat were observed predominantly in the ARC (Fig. 1aGo). AGRP-positive signals in the rhesus were detected in the comparable structure in the primate, known as the infundibular nucleus, in the fasted animal (Fig. 1bGo). No signal was detected in tissue from the fed animal (not shown).



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  		Figure 1. In situ hybridization demonstrating that AGRP mRNA is expressed in the ARC of the rat (a) and the infundibular nucleus (In) of the rhesus monkey (b). A 400-bp human AGRP probe was transcribed in the presence of [33P]UTP and hybridized to sections from the diestrous rat and fasted male rhesus monkey. Bar, 20 µm in a and 50 µm in b.

 
AGRP immunoreactivity in the Rat CNS
The studies described below were performed with a polyclonal antibody against a biologically active C-terminal fragment (amino acids 83–132) of the human AGRP prepared using synthetic methods (30). AGRP gene expression has been demonstrated to be elevated in the ob/ob mouse (12), and high levels of AGRP mRNA were also observed in the diestrous rat (M. S. Smith, unpublished); the latter model was used for the rodent studies reported here. Immunohistochemical staining in the rat revealed a dense network of AGRP-positive fibers throughout the hypothalamus (Fig. 2Go, a and b). The specificity of the staining was confirmed by preabsorbing the hAGRP antibody with the peptide corresponding to the C-terminal residues 83–132 before the antibody was incubated with tissue sections (Fig. 2cGo). Dense AGRP fibers were found projecting from the arcuate to only two divisions of the neuraxis, the hypothalamus and the septal region. In the hypothalamus, the densest fiber staining was observed originating in the arcuate and proceeding along the third ventricle as well as in the anterioperiventricular preoptic area, the periventricular nucleus, the parvocellular portion of the PVH, the dorsomedial nucleus (DMH), and the rostral end of the posterior nucleus (PH). Dense fiber staining was seen throughout the lateral and medial ARC. Scattered fiber staining was observed in the anterior and lateral hypothalamus (data not shown). Hypothalamic areas devoid of AGRP-positive fibers include the magnocellular portion of the PVH, the supraoptic nucleus, the suprachiasmatic nucleus (SCN), the ventromedial nucleus, and the compact zone of the DMH. The main extrahypothalamic projections led to the bed nucleus of the stria terminalis and the ventral part of the LS. Scattered fiber staining was also observed in the paraventricular nucleus of thalamus. No fibers were detected in the corpus striatum or the rostral portion of the brain stem. The caudal portion of the rat brain stem was not examined. Table 1Go summarizes the regions in the CNS of the diestrous rat containing AGRP immunoreactivity.



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  		Figure 2. Immunohistochemistry demonstrates dense hypothalamic neuronal fibers expressing AGRP in the diestrous rat. AGRP immunoreactivity is found in hypothalamic fibers projecting from the ARC as well as in the PVH (a) and DMH and PH nuclei (b). Preabsorption with the immunizing peptide AGRP-(83–132) blocks the staining reaction (c). Bars, 100 µm.

 

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  		Table 1. Distribution of AGRP immunoreactivity in the rat central nervous system

 
AGRP immunoreactivity in the primate hypothalamus
Hypothalamic sections from fed and fasted male rhesus monkeys provided materials for the analysis of AGRP immunoreactivity. AGRP immunoreactivity was undetectable in the hypothalamus of the fed rhesus monkey (data not shown), but was readily visible in the fasted animal. As in the rat, dense hypothalamic fiber bundles were detected in the infundibular nucleus and projecting from there along the third ventricle. Dense fibers were observed in the PVH (Fig. 3aGo) and more caudally in the DMH (Fig. 3bGo). Also, as in the rat, a dense caudal fiber bundle was found in the septal region, predominantly in the stria terminalis (Fig. 4aGo) and LS region (Fig. 4bGo), passing through the medial preoptic area (Fig. 4cGo). In the rhesus, very dense fiber staining could be seen in the infundibulum (equivalent to the rodent median eminence, Fig. 4dGo), and cell bodies could be detected in the infundibular nucleus (Fig. 4eGo). Table 2Go summarizes the AGRP-immunoreactive fibers distributed throughout the hypothalamus of the fasted male rhesus monkey. AGRP antiserum preabsorbed with control peptide, corresponding to the amino acids 83–132 mapping to the C-terminus of hAGRP, abolished all immunoreactivity (data not shown).



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  		Figure 3. Immunohistochemistry demonstrates dense hypothalamic neuronal fibers expressing AGRP in a fasted male rhesus monkey. AGRP immunoreactivity is found in hypothalamic fibers projecting from the infundibular nucleus (In) as well as in the PVH (a) and DMH nuclei (b). Bars, 100 µm.

 


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  		Figure 4. Immunohistochemistry reveals the distribution of AGRP-immunoreactive fibers and cell bodies in the fasted male rhesus monkey. Dense fiber staining is demonstrated in the nucleus of the stria terminalus (ST), and moderate fiber densities are found in the ventral portion of the LS and the medial preoptic area (MPA). Very dense fiber staining is seen in the infundibulum (inf; d), and immunoreactive cell bodies are detectable in the infundibular nucleus (In; e). Bars, 50 µm (a–d) and 25 µm (e).

 

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  		Table 2. Distribution of AGRP immunoreactivity in the rhesus central nervous system

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
AGRP-immunoreactive fibers in rat and rhesus monkey appear primarily in a subset of the same hypothalamic and septal brain regions containing dense POMC innervation (2, 31, 32, 33), with the densest fibers found innervating the PVH, DMH, posterior hypothalamus, and septal regions around the anterior commissure (Fig. 5Go). Within the rat hypothalamus, POMC fibers have a much wider distribution than AGRP, with moderate fiber density seen in just about every nucleus, with the possible exceptions of the VMH and supraoptic nucleus. A notable lack of AGRP fibers was observed in the VMH. These initial studies will, of course, need to be tempered by studies using additional antibodies, as it is possible that smaller amounts of immunoreactivity may be below the sensitivity of detection here, that the carboxyl-terminal epitope is specifically blocked in some brain regions, or that the immunoreactive epitope is unrecognizable in some brain regions as a consequence of proteolytic processing. Additionally, although the distributions reported here in the diestrous rat and fasted male rhesus are highly conserved, it will be important to determine whether the same distribution of immunoreactivity in the diestrous rat is seen in the fasted rat.



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  		Figure 5. Schematic diagram of a sagittal view of the rat brain, illustrating the comparative distributions of POMC and AGRP neurons. AAA, Anterior amygdaloid area; AC, anterior commissure; ACB, nucleus accumbens; Aci, anterior commissure; intrabulbar, ARH; arcuate nucleus of the hypothalamus; BST, bed nucleus of the stria terminalus; CA1–3, field CA1–CA3 of the hippocampus; CeA, central nucleus of the amygdala; CP, caudate putamen; DMX, dorsal motor nucleus of the vagus; LHA, lateral hypothalamic area; LSd, lateral septal area, dorsal aspect; MeA, medial amygdala; MH, medial habenula; MPO, medial preoptic area; OT, olfactory tubercle; PAG, periaquaductal gray; PH, posterior hypothalamus; Pir, piriform cortex; PV, periventricular zone; PVT, paraventricular nucleus of the thalamus; RN, red nucleus; SC, superior colliculus; SN, substantia nigra; SON, supraoptic nucleus; Subv, subiculum, ventral; VTA, ventral tegmental area; ZI, zona incerta. The locations of AGRP-immunoreactive fibers and cell bodies are based on data from the rat; fiber termini remain hypothetical. AGRP fiber distribution in the caudal brainstem was not examined in this study.

 
AGRP-immunoreactive fibers were also notably absent in the rat from additional divisions of the neuraxis receiving POMC fibers such as the rostral portion of the brain stem, hippocampus, amygdala, corpus striatum, and olfactory cortex tract (1, 2, 3, 4, 5). Remarkable conservation was seen in the distribution of AGRP fibers in rat and monkey; however, one striking difference was noted. Although AGRP fiber staining in the rat median eminence was limited and restricted to the lateral regions, very heavy fiber staining was seen in the rhesus infundibulum, suggesting the potential secretion of AGRP into the hypophyseal-portal circulation. Alternatively, this peptide could be acting at MSH{alpha}-binding sites in the infundibulum proper, as high levels of [125I]NDP-{alpha}-MSH binding are observed in the rat median eminence (6) and thus may exist in the primate as well.

The overall distribution of AGRP-immunoreactive fibers represents a subset of regions that contain POMC projections and express MC3-R and MC4-R receptor mRNA hybridization (8, 9). These data thus further support the general hypothesis that AGRP serves as a locally delivered functional antagonist of the MC3-R and MC4-R (8, 9, 34). Although MC4-R is very widely distributed in the brain and has been demonstrated to be involved in feeding behavior and metabolism (18, 19), control of somatic growth (19), grooming behavior (23), control of the hypothalamic-pituitary-adrenal axis (23), febrile responses (24), and cardiovascular homeostasis (25), the data shown here suggest that AGRP may serve as a counterregulatory force in a more limited subset of these functions. In particular, the dense expression of AGRP in the PVH and DMH strongly imply a role for the peptide in energy homeostasis, as further supported by the observation that AGRP mRNA levels are regulated by leptin. In fact, preliminary data suggest that the melanocortin antagonist AGRP is much more robustly regulated by metabolic state than is the source of melanocortin agonist, POMC. For example, AGRP mRNA is up-regulated 5- to 10-fold in the ob/ob mouse (12, 13) compared with the 40–60% down-regulation reported for POMC mRNA in this model (35, 36). It is thus tempting to speculate that the main site for hormonal input to the melanocortin system in regard to regulation of energy homeostasis is at the AGRP neuron rather than the POMC neuron. A comparable example is seen in the pigmentary system, in which variable levels of agouti gene expression in the hair follicle act to modulate the effects of constitutive {alpha}-MSH on eumelanin synthesis in the melanocyte.


   Note Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
While this manuscript was in proof, a detailed analysis of AGRP distribution in the mouse was reported (39).


   Acknowledgments
 
The authors thank Linda Cordilia for her work on the illustrations.


   Footnotes
 
1 This work was supported by NIH Grants DK-51730 (to R.D.C.), RR-00163 and HD-14643 (to M.S.S.), and HD-26888 (to J.L.C.). Back

2 These authors contributed equally to this manuscript. Back

Received August 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

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