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Altered Expression of Agouti-Related Protein and Its Colocalization with Neuropeptide Y in the Arcuate Nucleus of the Hypothalamus during Lactation¹

Division of Neuroscience (P.C., C.L., M.S.S.), Oregon Regional Primate Research Center, Beaverton, Oregon 97006; Department of Physiology & Pharmacology (P.C., C.L., M.S.S.), Vollum Institute (C.H.L., R.O.C.), Oregon Health Sciences University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. M. Susan Smith, Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Avenue, Beaverton, Oregon 97006. E-mail: smithsu{at}ohsu.edu

	Abstract

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During lactation, the levels of neuropeptide Y (NPY), which plays an important role in mediating food intake, are significantly elevated in a number of hypothalamic areas, including the arcuate nucleus (ARH). To identify additional hypothalamic systems that might be important in mediating the increase in food intake and alterations in energy homeostasis during lactation, the present studies examined the expression of agouti-related protein (AGRP), a recently described homologue of the skin agouti protein. AGRP is found in the hypothalamus and has been suggested to play an important role in the regulation of food intake. In the first experiment, animals were studied during diestrus of the estrous cycle, a stage of the cycle when estrogen levels are basal and similar to lactation, or during days 12–13 postpartum. Lactating animals had their litters adjusted to eight pups on day 2 postpartum. Brain tissue sections were used to measure AGRP messenger RNA (mRNA) levels by in situ hybridization. AGRP mRNA signal was found mostly in the ventromedial portion of the ARH, which has been shown to contain a high density of NPY neurons. A significant increase in AGRP mRNA content was observed in the mid- to caudal portion of the ARH of lactating animals compared with diestrous females. No difference was found in the rostral portion of the ARH. In the second experiment, double-label in situ hybridization for AGRP and NPY was performed in lactating animals to determine the extent of colocalization of the two peptides in the ARH, using ³⁵S-labeled and digoxigenin-labeled antisense complementary RNA probes. It was found that almost all of the NPY-positive neurons throughout the ARH also expressed AGRP mRNA signal. Furthermore, AGRP expression was confined almost exclusively to NPY-positive neurons. Thus, the present study showed that during lactation, AGRP gene expression was significantly elevated in a subset of the AGRP neurons in the ARH. The high degree of colocalization of AGRP and NPY, coupled with previous reports from our laboratory demonstrating increased NPY expression in the ARH in response to suckling, suggests that AGRP and NPY are coordinately regulated and may be involved in the increase in food intake during lactation.

	Introduction

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DURING LACTATION, there is increased energy demand due to milk production, exceeding the whole-body nutrient requirements of nonlactating animals. In addition, there are hypertrophy and increased synthetic activity of the gastrointestinal tract and liver (1). The energy demands of lactation are met primarily by increased food intake, which rises several folds over the amount observed in nonlactating animals (2, 3, 4). The mechanism that drives hyperphagia during lactation is still not completely understood. It has been suggested that the mechanism may reside in the hypothalamus because of its important role in the regulation of food intake and energy homeostasis. Furthermore, the levels of neuropeptide Y (NPY), which plays an important role in mediating food intake, are significantly elevated in a number of hypothalamic areas in lactating animals (4, 5, 6); this is further confirmed by an increase in NPY messenger RNA (mRNA) levels in the hypothalamus (7, 8).

Recently, agouti-related protein (AGRP), a 132-amino acid protein, was cloned from mouse as well as human (9, 10). Its carboxyl terminus shares a high degree of homology to the skin agouti protein (9, 10). Pharmacological studies have shown that it is a potent antagonist of melanocortin receptor-3 and -4 (MC3-R and MC4-R) (10, 11). Transgenic animal studies (10, 12) showed that mice overexpressing the protein exhibit an obese phenotype that mimics that of mutant A^y mice (13) and MC4-R knockout mice (14). In addition, the expression of AGRP mRNA is significantly elevated in ob/ob obese mutant mice compared with nonmutant lean mice (9). These results suggest that AGRP may play an important role in regulating food intake and energy homeostasis. The distribution of the mRNA encoding for AGRP has been examined in mouse brain (9) and was found to be restricted to the ventromedial portion of the ARH, an area known to contain abundant NPY neurons (15). Furthermore, the mRNA signals for AGRP and NPY were increased in response to fasting and found to be expressed in the same neurons (16). The potential importance of AGRP in food intake and energy balance raises the possibility that AGRP may be another important system in the hypothalamus involved in mediating the hyperphagia and energy adaptation during lactation.

Thus, in the present study, the expression of AGRP in the ARH was examined by in situ hybridization to determine whether lactation induces changes in AGRP gene expression. In addition, double-label in situ hybridization was performed to determine whether NPY and AGRP are colocalized in the same neurons in the ARH.

	Materials and Methods

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Animals
Cycling (n = 9) and day 18–19 pregnant (n = 9) Sprague Dawley rats (B & K Universal, Inc., Kent, WA) were housed individually and maintained under 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. The pregnant rats were checked for the birth of the pups every morning; the day of delivery was considered as day 0 postpartum. Litters were adjusted to 8 pups on day 2 postpartum. Stage of the estrous cycle for the cycling rats was determined by vaginal smears. The animals were killed by decapitation on day 12–13 postpartum for lactating animals and on diestrus for the cycling animals. Diestrus was used as a basis for comparison because it is characterized by low basal levels of estrogen, similar to those observed during lactation. The brains were quickly removed, frozen on dry ice, and sectioned with a cryostat microtome. One in three series of 20 µm coronal brain sections were collected through the forebrains. The fresh frozen sections were stored at -80 C until used for in situ hybridization. All of the animal procedures were approved by the Oregon Regional Primate Research Center Institutional Animal Care and Use Committee.

In situ hybridization of AGRP
Probe synthesis, the specificity of the complementary RNA (cRNA) probes, and procedures for in situ hybridization have been described previously (17). Briefly, the sense and antisense human AGRP cRNA probes (see reference 9 for description of the probe) were transcribed from a 400-bp cDNA in which 30% of the UTP was ³⁵S-labeled (NEN Life Science, Boston, MA). The saturating concentration of the probe used in the assay was 0.45 µg/ml·kb and the specific activity was 6–7 x 10⁸ dpm/µg. Two sets of rostral-caudal series of tissue sections from a lactating animal were used for the comparison of sense and antisense probes. The fresh frozen brain sections 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 2x SSC, dehydrated through a graded series of alcohols, delipidated in chloroform, rehydrated through a second series of alcohols, and then air dried. Sense or antisense probe (100 µl) was applied to each slide. Slides were coverslipped and incubated in moist chambers at 55 C for 15 h. After incubation, the slides were washed in SSC that increased in stringency, in RNase A, and then in 0.1x SSC at 60 C and rehydrated through graded series of 6% ammonium acetate-alcohols. Slides were exposed to autoradiography films (Hyperfilm-ßmax, Amersham, Arlington Heights, IL) for 6 days at 4 C and developed. The sections were counterstained with cresyl violet and coverslipped with histomount.

Double-label in situ hybridization for NPY and AGRP
Tissue sections from two lactating and two diestrous rats were used in this study. The AGRP cRNA probe was prepared as described above. NPY cRNA probe was transcribed from a 511-bp rat cDNA (see Ref. 18 for a description of the probe) in which digoxigenin-UTP (Boehringer Mannheim, Indianapolis, IN) was used. The concentration of digoxigenin-labeled NPY cRNA in the hybridization mixture was 0.1 µg/ml. The slides were exposed to the mixture of ³⁵S-hAGRP and digoxigenin-NPY cRNA probes in moist chambers for 15 h at 55 C. After incubation and posthybridization washes, slides were incubated in alkaline phosphatase (AP) conjugated goat antidigoxigenin antibody (1:1000, Boehringer Mannheim) at 4 C overnight. The AP-complexes were visualized with a mixture of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-inodyl phosphate toluidinum. Slides were then dipped in 3% parlodion followed by NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 14 days at 4 C and developed.

Data analysis
AGRP mRNA in the ARH. The ARH was divided from rostral to caudal into four subdivisions as described in a previous study (7), using the rat brain atlas of Paxinos and Watson (19). Briefly, ARH-A corresponded to the retrochiasmatic area rostrally, to the elongation of the third ventricle caudally [coronal plate 19, Paxinos and Watson rat brain atlas (19)]; ARH-B continued caudally to the beginning of DMH (coronal plate 20); ARH-C contained the compact zone of the DMH (coronal plate 21); ARH-D began with the disappearance of the DMH, to the end of the ARH (coronal plate 22). The coronal brain sections were anatomically matched across animals from all groups. The AGRP mRNA signal in the ARH was analyzed on the ß-max film using the VIDEK HARMONY image analysis system by VIDEK (Rochester, NY). An individual brain section image was captured by a CCD camera (Cohu) and displayed on a computer monitor. A marked area (1.0 mm x 1.6 mm) was drawn to include the entire ARH. The marked area was constant for all the sections analyzed. The optical density of AGRP mRNA signal in the marked area was then determined after background subtraction.

AGRP and NPY double-labeled neurons. NPY-positive neurons were visualized under bright field as dark blue deposits in the cytoplasm, whereas AGRP-positive neurons in the same area were identified under dark field as clusters of silver grains. An NPY neuron was considered to be double-labeled with AGRP if the number of silver grains on top of the cell body was greater than three times the background level (20).

Statistical analysis
The optical density value of AGRP mRNA signal in the ARH was expressed as arbitrary units per section. The mean value from each subdivision of the ARH was determined for each animal. Data are presented as mean ± SEM. Differences between groups within each subdivision of the ARH were evaluated using Student’s t test. Differences were considered significant if P < 0.05.

	Results

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Validation of antisense cRNA probe
The AGRP mRNA signal was mostly restricted to the ARH in the brain sections that were hybridized with the antisense cRNA probe (Fig. 1). No specific signal was observed in the corresponding sections that were hybridized with the sense probe. Nonspecific signal in the hippocampal area was observed in both sense and antisense cRNA probe hybridized sections (Fig. 1).

View larger version (79K): [in this window] [in a new window]   Figure 1. Pseudocolor film autoradiographic images of representative brain sections incubated with 35S-labeled antisense (a) and sense (b) AGRP cRNA probes. Note that only the ARH (arrow) exhibits specific hybridization signals. Scale bar, 1 mm.

View larger version (132K): [in this window] [in a new window]   Figure 2. High power magnification of representative film autoradiographs showing AGRP gene expression in the caudal ARH of diestrous (a) and lactating (b) rats. The AGRP mRNA signal in the lactating animal is more intense and covers a greater area. Scale bar, 100 µm.

View larger version (18K): [in this window] [in a new window]   Figure 3. Comparison of AGRP mRNA levels between diestrous and lactating rats in different regions of the ARH. AGRP mRNA levels were significantly increased in ARH-B, -C, and -D areas in the lactating animals compared with the same areas in the diestrous rats. There was no difference in the ARH-A area between the two groups. *, P < 0.01 vs. corresponding area in the diestrous animals.

View larger version (141K): [in this window] [in a new window]   Figure 4. NPY and AGRP double labeling in the ARH. Representative photomicrographs showing the AGRP mRNA signal (a, silver grain clusters, dark field) and the NPY mRNA signal (b, dark blue staining, bright field) in the same area in the caudal ARH. Under low magnification (a, b), the pattern of distribution of the two signals was very similar. Under higher magnification (c, bright and dark field double exposure), most NPY positive neurons (dark brown staining) also showed AGRP mRNA signals (silver grain clusters). Arrows indicate representative NPY/AGRP double-labeled cells. Scale bars, 200 µm (a) and 50 µm (c). 3V, Third ventricle; ME, median eminence.

View larger version (21K): [in this window] [in a new window]   Figure 5. Representative schematic drawings showing the distribution of neuronal profiles expressing NPY (), AGRP (•) or both () in the rostral and caudal portion of the ARH. The majority of the NPY neurons examined throughout the ARH were double-labeled for AGRP mRNA. Very few NPY or AGRP single-labeled cells were observed.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Based on the results from double-label in situ hybridization, almost all NPY and AGRP expression appears to be colocalized to the same neurons in the ARH, results that are in agreement with those of Hahn et al. (16). However, because of the small number of animals used in the present studies, it cannot be ruled out that AGRP also may be expressed in other cell types in the ARH during lactation. The colocalization of NPY and AGRP was only observed in the ARH, as the NPY neurons in the rest of the brain areas did not express AGRP mRNA, including the suckling-activated NPY neurons in the DMH.

The pattern of differential expression of AGRP mRNA in the ARH observed in the lactating animals in the present study, that is, increased expression only in the middle and caudal portions, is similar to that of NPY in the ARH during lactation (7, 17). We and others have reported that NPY mRNA is increased only in the caudal portion of the ARH during lactation (7, 8). Compared with the pattern of increased NPY expression, AGRP expression is increased in a greater portion of the ARH (ARH-B, -C, and -D) than NPY (ARH-C). It has been suggested that the increase in expression of NPY is mediated by the neural signals activated by the suckling stimulus (17) and is not dependent on the suckling-induced hyperprolactinemia (21). The colocalization of NPY and AGRP and the somewhat similar pattern of activation between the two peptides suggest that the increased expression of AGRP observed in the present study also may be mediated by the suckling stimulus. More studies are needed to resolve this issue. In addition, it also remains to be determined whether the increased expression of AGRP and NPY in the same neurons is mediated by the same cellular mechanisms.

The importance of AGRP in the regulation of food intake during physiological and pathological conditions is still under intensive investigation. Ubiquitous expression of the AGRP gene in transgenic mice results in an obesity syndrome (10, 12). Intracerebroventricular injection of the C-terminal fragment of AGRP causes an increase in food intake and antagonizes the inhibitory effect of -MSH on feeding (22). The detailed mechanism by which AGRP may regulate feeding also remains to be elucidated. It has been shown that AGRP exerts its function by binding to two of the receptors (MC3-R and MC4-R) for the POMC peptides (POMC) and preventing their activation by POMC peptides (10, 11, 22). Thus, AGRP may represent an endogenous system in the hypothalamus that mediates food intake and energy homeostasis by antagonizing the binding of POMC peptides to the MC-Rs.

Recently, the distribution of AGRP-positive fibers in the brain was studied in the rat and monkey (23). AGRP fibers were observed mainly in the hypothalamus, including the medial preoptic area, the paraventricular nucleus, the DMH and the ARH. The distribution of fibers was very similar to that of ARH NPY neurons (unpublished observation), which further indicates that the two ARH peptide systems are colocalized and have the same target areas. Thus, the elevated expression of AGRP during lactation may result in increased release into these target areas, which could antagonize the MC-Rs and positively modulate the increase in food intake during lactation. In support of this notion, anatomical mapping has shown that MC4-R mRNA, the receptor that is important in mediating the ability of POMC peptides to inhibit food intake, was expressed in some of the AGRP- projecting areas, such as the medial preoptic area and the paraventricular nucleus (24). Interestingly, POMC gene expression in the ARH, the major resource of POMC peptides that interact with the MC-Rs to modulate food intake and energy homeostasis, is also reduced during lactation (7, 25). The suppression of POMC expression, together with the increase in AGRP expression, would ensure that activation of MC-Rs is prevented during lactation, which would allow the lactating female rat to escape from the inhibitory effect of MC-Rs on feeding. Thus, the POMC and NPY/AGRP neurons may function coordinately in the control of energy homeostasis. Furthermore, ARH NPY neurons may modulate ARH POMC neuronal activity (26, 27, 28, 29), an effect that may be mediated by the NPY Y1 receptor (30). The colocalization of NPY and AGRP and the elevation of AGRP expression in the present study suggest that AGRP may also modulate ARH POMC activity during lactation. The expression of MC3-R in the ARH indirectly supports this notion (31). However, more studies are needed to determine whether POMC neurons in the ARH express the MC3-R.

The colocalization of AGRP and NPY in the ARH raises an interesting issue. The importance of the NPY system in the hypothalamus has been challenged by the NPY knockout mouse study (32). Apparently, the mouse without NPY is still fertile, can maintain normal body weight and food intake, and metabolic parameters appear to be normal (32, 33). The only defect observed was that the mouse was more susceptible to neuronal damage by seizure (32, 34), which is related to the lack of NPY in the hippocampus. The coexpression of AGRP and NPY in the same neurons in the ARH suggests that the neurons would still be able to respond to incoming signals to release AGRP into the target areas, even when NPY is absent. The released AGRP could then act on common downstream pathways for both NPY and AGRP to modulate downstream neuronal activity, thus explaining why removal of NPY has a less serious effect on regulation of food intake than originally hypothesized.

In conclusion, the present study demonstrated that during lactation, the expression of AGRP mRNA in the ARH is differentially altered, with only mid- to caudal portions of the AGRP-positive neurons showing increased mRNA expression. Almost complete colocalization of NPY and AGRP was observed throughout the ARH, suggesting that the two systems are regulated in parallel. A high degree of coordination between the NPY and the POMC systems in the hypothalamus may be involved in the increase in food intake during lactation.

	Acknowledgments

 
Thanks to Dr. Steve Sabol at NIH for providing the rat NPY cDNA plasmid (pBLNPY1).

	Footnotes

 
¹ This work was supported by NIH Grants DK-51730 (to R.D.C.), DK-09231 (to C.H.L.), HD-14643, HD-18185, and the Oregon Regional Primate Research Center Grant RR-00163 (to M.S.S.).

Received October 15, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Widdowson EM 1976 Changes in the body and its organs during lactation: nutritional implications. Ciba Found Symp 45:103–118
2. Vernon RG, Flint DJ 1984 Adipose tissue: metabolic adaptation during lactation. Symp Zool Soc Lond 51:119–145
3. Flint DJ, Vernon RG 1998 Effects of food restriction on the responses of the mammary gland and adipose tissue to prolactin and growth hormone in the lactating rat. J Endocrinol 156:299–305[Abstract]
4. Malabu UH, Kilpatrick A, Ware M, Vernon RG, Williams G 1994 Increased neuropeptide Y concentrations in specific hypothalamic regions of lactating rats: possible relationship to hyperphagia and adaptive changes in energy balance. Peptides 15:83–87[CrossRef][Medline]
5. Ciofi P, Fallon JH, Croix D, Polak JM, Tramu G 1991 Expression of neuropeptide Y precursor-immunoreactivity in the hypothalamic dopaminergic tubero-infundibular system during lactation in rodents. Endocrinology 128:823–834[Abstract/Free Full Text]
6. Pickavance L, Dryden S, Hopkins D, Bing C, Frankish H, Wang Q, Vernon RG, Williams G 1996 Relationships between hypothalamic neuropeptide Y and food intake in the lactating rat. Peptides 17:577–582[CrossRef][Medline]
7. Smith MS 1993 Lactation alters neuropeptide-Y and proopiomelanocortin gene expression in the arcuate nucleus of the rat. Endocrinology 133:1258–1265[Abstract/Free Full Text]
8. Pape J-R, Tramu G 1996 Suckling-induced changes in neuropeptide Y and proopiomelanocortin gene expression in the arcuate nucleus of the rat: evaluation of a putative intervention of prolactin. Neuroendocrinology 63:540–549[Medline]
9. Shutter JR, Graham M, Kinsey AC, Scully S, Luthy R, Stark KL 1997 Hypothalamic expression of ART, a novel gene related to agouti, is upregulated in obese and diabetic mutant mice. Genes Dev 11:593–602[Abstract/Free Full Text]
10. Ollmann MM, Wilson BD, Yang Y-K, Kerns JA, Chen Y, Gantz I, Barsh GS 1997 Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science 278:135–137[Abstract/Free Full Text]
11. Fong TM, Mao C, MacNeil C, Kalyani R, Smith T, Weinberg D, Tota MR, Van der Ploeg LH 1997 ART (protein product of agouti-related transcript) as an antagonist of MC-3 and MC-4 receptors. Biochem Biophys Res Commun 237:629–631[CrossRef][Medline]
12. Graham M, Shutter JR, Sarmiento U, Sarosi I, Stark KL 1997 Overexpression of ART leads to obesity in transgenic mice. Nat Genet 17:273–274[CrossRef][Medline]
13. Wolff GL 1990 Obesity, cancer and the viable yellow Avy/a mouse. Discrimination among pathological effect of the expression of the Avy/a genotype in obese and lean mice. In: Oomura Y, Tarui S, Shimazu T (eds) Progress in obesity research. John Libbey & Company Ltd., vol 70:445–448
14. Huszar D, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F 1997 Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88:131–141[CrossRef][Medline]
15. De Quidt ME, Kiyama H, Emson PC 1990 Pancreatic polypeptide, neuropeptide Y and peptide YY in central neurons. In: Björklund A, Hökfelt T, Kuhar MJ (eds) Handbook of chemical neuroanatomy. Vol 9: Neuropeptide in the CNS, part II. Elsevier, Amsterdam, pp 287–358
16. Hahn TM, Breninger JF, Baskin DG, Schwartz MW 1998 Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nature Neurosci 1:271–272[CrossRef][Medline]
17. Li C, Chen P, Smith MS 1998 The acute suckling stimulus induces expression of neuropeptide Y (NPY) in cells in the dorsomedial hypothalamus and increases NPY expression in the arcuate nucleus. Endocrinology 139:1645–1652[Abstract/Free Full Text]
18. Higuchi H, Yang H-Y T, Sabol SL 1988 Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester. J Biol Chem 263:6288–6295[Abstract/Free Full Text]
19. Paxinos G, Watson C 1982 The Rat Brain in Stereotaxic Coordinates. Academic Press, New York
20. Standish LJ, Adams LA, Vician L, Clifton DK, Steiner RA 1987 Neuroanatomical localization of cells containing gonadotropin-releasing hormone messenger ribonucleic acid in the primate brain by in situ histochemistry. Mol Endocrinol 1:371–376[Abstract/Free Full Text]
21. Li C, Chen P, Smith MS 1999 Neuropeptide Y and tuberoinfundibular dopamine activities are altered during lactation: role of prolactin. Endocrinology 140:118–123[Abstract/Free Full Text]
22. Rossi M, Kim MS, Morgan DGA, Small CJ, Edwards CMB, Sunter D, Abusnana S, Goldstone AP, Russell SH, Stanley SA, Smith DM, Yagaloff K, Ghatei MA, Bloom SR 1998 A C-terminal fragment of agouti-related protein increases feeding and antagonizes the effects of -melanocyte stimulating hormone in vivo. Endocrinology 139:4428–4431[Abstract/Free Full Text]
23. Haskell-Luevano C, Chen P, Li C, Chang K, Smith MS, Cameron JL, Cone RD 1999 Characterization of the neuroanatomical distribution of agouti-related protein (AGRP) immunoreactivity in the rhesus monkey and the rat. Endocrinology 140:1408–1415[Abstract/Free Full Text]
24. Mountjoy KG, Mortrud MT, Low MJ, Simerly RB, Cone RD 1994 Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 8:1298–1308[Abstract/Free Full Text]
25. Kim EM, Kotz CM, Welch CC, Grace MK, Billington CJ, Levine AS 1997 Lactation decreases mRNA levels of opioid peptides in the arcuate nucleus of the rat. Brain Res 769:303–308[CrossRef][Medline]
26. De Yebenes EG, Songyun Li, Fournier A, St-Pierre S, Pelletier G 1995 Regulation of proopiomelanocortin gene expression by neuropeptide Y in the rat arcuate nucleus. Brain Res 674:112–116[CrossRef][Medline]
27. Horvath TL, Naftolin F, Kalra SP, Leranth C 1992 Neuropeptide-Y innervation of ß-endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis. Endocrinology 131:2461–2467[Abstract/Free Full Text]
28. Csiffary A, Gorcs TJ, Palkovits M 1990 Neuropeptide Y innervation of ACTH-immunoreactive neurons in the arcuate nucleus of rats: a correlated light and electron microscopic double immunostaining study. Brain Res 506:215–222[CrossRef][Medline]
29. Kalra SP, Kalra PS 1996 Nutritional infertility: the role of the interconnected hypothalamic neuropeptide Y-galanin-opioid network. Front Neuroendocrinol 17:371–401[CrossRef][Medline]
30. Broberger C, Landry M, Wong H, Walsh JN, Hàkfelt T 1997 Subtype Y1 and Y2 of the neuropeptide Y receptor are respectively expressed in proopiomelanocortin- and neuropeptide-Y-containing neurons of the rat hypothalamic arcuate nucleus. Neuroendocrinol 66:393–408[Medline]
31. Roselli-Rehfuss L, Mountjoy KG, Robbins LS, Mortrud MT, Low MJ, Tatro JB, Entwistle ML, Simerly RB, Cone RD 1993 Identification of a receptor for melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system. Proc Natl Acad Sci USA 90:8856–8860[Abstract/Free Full Text]
32. Erickson JC, Clegg KE, Palmiter RD 1996 Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature 381:415–421[CrossRef][Medline]
33. Erickson JC, Ahima RS, Hollopeter G, Flier JS, Palmiter RD 1997 Endocrine function of neuropeptide Y knockout mice. Regul Pept 70:199–202[CrossRef][Medline]
34. Baraban SC, Hollopeter G, Erickson JC, Schwartzkroin PA, Palmiter RD 1997 Knock-out mice reveal a critical antiepileptic role of neuropeptide Y. J Neurosci 17:8927–8936[Abstract/Free Full Text]

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				X. Q. Xiao, K. L. Grove, S. Y. Lau, S. McWeeney, and M. S. Smith Deoxyribonucleic Acid Microarray Analysis of Gene Expression Pattern in the Arcuate Nucleus/Ventromedial Nucleus of Hypothalamus during Lactation Endocrinology, October 1, 2005; 146(10): 4391 - 4398. [Abstract] [Full Text] [PDF]

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				R G P Denis, C Bing, S Brocklehurst, J A Harrold, R G Vernon, and G Williams Diurnal changes in hypothalamic neuropeptide and SOCS-3 expression: effects of lactation and relationship with serum leptin and food intake J. Endocrinol., October 1, 2004; 183(1): 173 - 181. [Abstract] [Full Text] [PDF]

				P. Chen, S. M. Williams, K. L. Grove, and M. S. Smith Melanocortin 4 Receptor-Mediated Hyperphagia and Activation of Neuropeptide Y Expression in the Dorsomedial Hypothalamus during Lactation J. Neurosci., June 2, 2004; 24(22): 5091 - 5100. [Abstract] [Full Text] [PDF]

				L. Huo, H. Munzberg, E. A. Nillni, and C. Bjorbaek Role of Signal Transducer and Activator of Transcription 3 in Regulation of Hypothalamic trh Gene Expression by Leptin Endocrinology, May 1, 2004; 145(5): 2516 - 2523. [Abstract] [Full Text] [PDF]

				C. Bjorbaek and B. B. Kahn Leptin Signaling in the Central Nervous System and the Periphery Recent Prog. Horm. Res., January 1, 2004; 59(1): 305 - 331. [Abstract] [Full Text]

				J. W. Hill and J. E. Levine Abnormal Response of the Neuropeptide Y-Deficient Mouse Reproductive Axis to Food Deprivation But Not Lactation Endocrinology, May 1, 2003; 144(5): 1780 - 1786. [Abstract] [Full Text] [PDF]

				H. Munzberg, L. Huo, E. A. Nillni, A. N. Hollenberg, and C. Bjorbaek Role of Signal Transducer and Activator of Transcription 3 in Regulation of Hypothalamic Proopiomelanocortin Gene Expression by Leptin Endocrinology, May 1, 2003; 144(5): 2121 - 2131. [Abstract] [Full Text] [PDF]

				S. Brugman, D. J. Clegg, S. C. Woods, and R. J. Seeley Combined Blockade of Both {micro}- and {kappa}-Opioid Receptors Prevents the Acute Orexigenic Action of Agouti-Related Protein Endocrinology, November 1, 2002; 143(11): 4265 - 4270. [Abstract] [Full Text] [PDF]

				A. M. Mistry and D. R. Romsos Intracerebroventricular Leptin Administration Reduces Food Intake in Pregnant and Lactating Mice Experimental Biology and Medicine, September 1, 2002; 227(8): 616 - 619. [Abstract] [Full Text] [PDF]

				K. L. Grove, R. S. Brogan, and M. S. Smith Novel Expression of Neuropeptide Y (NPY) mRNA in Hypothalamic Regions during Development: Region-Specific Effects of Maternal Deprivation on NPY and Agouti-Related Protein mRNA Endocrinology, November 1, 2001; 142(11): 4771 - 4776. [Abstract] [Full Text] [PDF]

				J. G. Mercer, K. M. Moar, T. J. Logie, P. A. Findlay, C. L. Adam, and P. J. Morgan Seasonally Inappropriate Body Weight Induced by Food Restriction: Effect on Hypothalamic Gene Expression in Male Siberian Hamsters Endocrinology, October 1, 2001; 142(10): 4173 - 4181. [Abstract] [Full Text] [PDF]

				G.S.H. Yeo, I.S. Farooqi, B.G. Challis, R.S. Jackson, and S. O'Rahilly The role of melanocortin signalling in the control of body weight: evidence from human and murine genetic models QJM, January 1, 2000; 93(1): 7 - 14. [Abstract] [Full Text] [PDF]

				A. Sorensen, C. L. Adam, P. A. Findlay, M. Marie, L. Thomas, M. T. Travers, and R. G. Vernon Leptin secretion and hypothalamic neuropeptide and receptor gene expression in sheep Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2002; 282(4): R1227 - R1235. [Abstract] [Full Text] [PDF]

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