Brain Research 965 (2003) 45–50www.elsevier.com/ locate/brainres

Research report

H ypothalamic cocaine-amphetamine regulated transcript (CART) isregulated by glucocorticoids

a , a a a*Niels Vrang , Philip J. Larsen , Mads Tang-Christensen , Leif Kongskov Larsen ,bPeter Kristensen

aRheoscience, Glerupvej 2, 2610Rodovre, DenmarkbHealth Care Discovery, Novo Nordisk A /S, Bagsværd, Denmark

Accepted 19 November 2002

Abstract

Cocaine-amphetamine-regulated transcript (CART) is one of the most abundantly expressed mRNAs in the rat hypothalamus. CARTmRNA expression in the arcuate nucleus has been shown to be regulated by leptin, and CART peptides have been implicated in feedingbehavior and in the regulation of the HPA-axis. To more fully understand the physiological regulation of CART gene expression, we haveexamined the effects of adrenalectomy and different types of glucocorticoid substitution (corticosterone and dexamethasone) onhypothalamic CART and POMC mRNA levels. In situ hybridization revealed a reduction in CART mRNA levels in both thehypothalamic paraventricular and arcuate nuclei in adrenalectomized rats, which was fully restored upon dexamethasone treatment but notby a subcutaneous 25% corticosterone pellet. Unlike CART mRNA levels hypothalamic POMC expression was unaltered byadrenenalectomy. The present results show that the CART gene is influenced by glucocorticoids, presumably via a GR dependentmechanism. 2002 Published by Elsevier Science B.V.

Keywords: In situ hybridization; HPA-axis; GR receptor; Arcuate nucleus; Parventricular nucleus; POMC

1 . Introduction ment of CART has been previously identified in porcinehypothalami [24]. Albeit isolated from striatal extracts, the

Cocaine-amphetamine-regulated transcript (CART) was CART gene is most abundantly expressed in the hypo-discovered in 1995 by polymerase chain reaction (PCR) thalamus [12]. CART mRNA and CART peptide immuno-differential display in a search for mRNAs in the striatum reactivity are located in a number of hypothalamic struc-acutely up-regulated by psychostimulants [9]. In the rat the tures involved in the control of feeding behavior, such asprimary CART transcript is differentially spliced and the the lateral hypothalamic area and the paraventricular andtwo different mRNAs encode peptides of either 116 or 129 arcuate nuclei [7,14–16,30]. Consistent with these ana-amino acids [9]. The leader sequence consists of 27 amino tomical observations we and others have shown thatacids and mature CART peptides therefore contain either intracerebroventricular administration of CART peptides89 or 102 residues [9]. Since only the short form of CART inhibit food intake in rats and mice [1,16,17,27,32].(89 amino acids long) is present in human tissue [8], we Additionally, intracerebroventricularly administeredhave chosen to number CART peptides after this form CART(42–89) induces c-Fos expression in areas of the rat[CART(1–89)]. CART peptide processing is tissue depen- brain that are involved in feeding behavior [32].dent and two forms predominate in the rat brain: The hypothalamic arcuate nucleus (Arc) contains aCART(42–89) and CART(49–89) [26]. The former frag- population of CART containing neurons that also co-

express pro-opiomelanocortin (POMC) [10,30]. Interest-ingly, both CART and POMC expression in the Arc is

*Corresponding author. Laboratory of Obesity Research, CCBR,regulated by leptin and fasting, pointing to an importantBallerup Byvej 222, 2750 Ballerup, Denmark. Tel.:145-4470-4404; fax:role of these neurons in appetite regulation [16,19,23].145-4468-4220.

E-mail address: nv@ccbr.dk(N. Vrang). Besides leptin and fasting, a number of experiments have

0006-8993/02/$ – see front matter 2002 Published by Elsevier Science B.V.PI I : S0006-8993( 02 )04064-7

46 N. Vrang et al. / Brain Research 965 (2003) 45–50

showed effects of adrenal steroids on arcuate POMC morning of the 11th day (between 08:00 a.m. and 10:00expression [4,5,20,22,28,33]. There is indirect evidence to a.m.) animals were sacrificed by decapitation. Trunk bloodsupport a glucocorticoid effect on POMC/CART neurons. was collected in heparinized tubes and brains removed andPro-opiomelanocortin neurons contain glucocorticoid re- snap frozen on dry ice. Blood samples were subsequentlyceptor immunoreactivity [6,11], and fibers containing centrifuged at 3000 rpm for 10 min and plasma frozenPOMC and CART immunoreactivity are found in close until corticosterone analysis. Corticosterone was measuredapposition to the hypothalmic–pituitary–adrenal (HPA)- using a commercially available RIA kit (Rat Corticos-axis motorneurons [13,21,31] suggesting that CART and terone, Diagnostic Products, San Diego, CA, USA).POMC derived peptides influence the HPA-axis and there-fore possibly could be part of a feed-back loop involvingadrenal steroids. Finally, CART peptides injected in- 2 .2. In situ hybridizationtracerebroventricularly [31] or directly into the PVN [25]activate the HPA-axis, presumably via a direct action on Twelve-micrometer thick frontal sections of the freshlyCRH motorneurons. frozen brains were cut in a freezing microtome and

The aim of the present study was to gain further insight mounted directly on Superfrost Plus slides. Slides wereinto the regulation of the CART gene in the hypothalamus allowed to dry and sections were kept at280 8C until inby examining the effects of adrenal steroids on CART/ situ hybridization experiments were performed. In situPOMC expression. hybridization was performed using a protocol described in

detail previously [30]. Pro-opiomelanocortin and CARTmRNA was detected using RNA probes directed against

2 . Materials and methods the rat CART cDNA (bp 226–411; GenBank accessionnumber U10071) and the rat POMC cDNA (bp 162–626;

2 .1. Animals and surgery GenBank accession number J00759). Sense RNA probeswere used as negative controls. Probes were labeled

35A total of 33 male Wistar rats weighing 200–250 g at radioactively by the use of S labeled UTP (Amershamthe time of surgery were used for the experiments. The Pharmacia). Notably, in order to avoid overexposure of theanimals were kept on a 12:12 light–dark cycle (lights on at autoradiograms (both CART and POMC mRNAs are06.00 h) in a temperature controlled environment (22– expressed at high levels) both probes were transcribed with24 8C) with free access to food and water. All experiments a 3:1 mix of radioactive and non-radioactive UTP. The usewere conducted in accordance with internationally ac- of a probe with a lower specific activity should facilitatecepted principles for the care and use of laboratory animals detection of changes in highly abundant mRNAs and avoidand were approved by the Danish Committee for Animal overexposure of the film (used previously by us toResearch. Animals were kept three per cage upon arrival to demonstrate changes in CART and POMC mRNAthe laboratory and until the end of the experiments. [16,29]). Posthybridization washes were performed at 62

The rats were anesthetized using Hypnorm Dormicum and 678C in 50% formamide. Following hybridization,(1.0 ml /kg; containing 0.08 mg fentanyl, 2.5 mg sections were exposed onb-max film (Amersham, Buckin-fluanisone and 5.0 mg medazolam per ml) and sham ghamshire, UK) for 1 week (both probes). Finally, sectionsadrenalectomized (sham-ADX;n56) or adrenalectomized were dipped in fluid emulsion (K2, AgFA) exposed for 2(ADX; n527) using the dorsal approach. Following weeks and developed in Kodak D-19 (Kodak). Autoradio-surgery all ADX animals were substituted with 0.9% saline grams were analyzed on a computerized image analysisin their drinking water. ADX rats were further subdivided system (Image 1.60b, NIH, USA), and the signal quanti-into three groups: one group received no glucocorticoid tated as the area of expression with a mean density abovereplacement (ADX group;n59), one group was substi- threshold (area3mean5arbitrary units). Two sections fromtuted with dexamethasone in the drinking water (0.6 mg/ l; each animal were measured and the average calculated.ADX1DEX group; n59, to examine the possible role of Images of emulsion-dipped sections were acquired using aGR receptors) and one group received a corticosterone/ Nikon Coolpix 990 attached to a Nikon E1000 microscope.cholesterol pellet (placed subcutaneously in the neck Images were adjusted for brightness and contrast in Adoberegion before suturing the wound) consisting of 25% Photoshop 5.0 and mounted into plates in Adobe Illustratorcorticosterone (Sigma–Aldrich, St. Louis, MO, USA) and 7.0.75% cholesterol (Sigma–Aldrich) and weighing approxi-mately 200 mg. This size of pellet is based on previousexperiments [2,18] giving rise to plasma levels of corticos- 2 .3. Statisticsterone around 50 ng/ml (24 h mean levels) in order toprimarily engage the mineralocorticoid receptor (MR). Experiments were evaluated using two-way analysis ofFollowing surgery, food intake (measured per cage) and variance (ANOVA) followed by Fisher’s post-hoc analysis.bodyweight was recorded daily for 11 days. On the Values (hormone concentrations, arbitrary units, body-

N. Vrang et al. / Brain Research 965 (2003) 45–50 47

weight) are expressed as the mean6S.E.M. P,0.05 was dipped sections of both the Arc (Fig. 2, top row) and PVNconsidered significant. (Fig. 2, bottom row).

3 . Results 4 . Discussion

Some key variables related to the metabolic state of the In this study, we have shown using in situ hybridizationrats as well as hormonal measurements performed in techniques that CART expression is influenced byexperiment 2 are shown in Table 1. To assure occupation glucocorticoids. Interestingly, only subtle non-significantof type II glucocorticoid receptors, a high dose of dexa- effects of glucocorticoids on POMC expression in the Arcmethasone that clearly caused catabolism was used. No- was observed, pointing to a possible differential regulationtably, ADX1DEX rats lost bodyweight and showed a of the two co-localised neurotransmitters.reduction in retroperitoneal fat mass over the course of the Adrenalectomy reduced CART levels in both the PVNtreatment period (Table 1). On day 2 following surgery all and Arc to approximately 50% of levels in sham-operatedADX-rats consumed significantly less food than sham- controls. Dexamethasone treatment abolished ADX in-operated rats (Table 1). However on the 10th day of the duced decreases of PVN and Arcuate CART mRNAstudy the average daily food intake per 100 g bodyweight expression suggesting that the adrenal signal to hypo-was identical across experimental groups (Table 1). Cor- thalamic CART gene expression is of glucocorticoid originticosterone measurements showed that sham-operated con- and mediated by glucocorticoid receptors (GRs). The hightrol had plasma levels of 59619 ng/ml. Adrenalectomized doses of dexamethasone used in the present study (clearlyrats and dexamethasone treated rats both had undetectable in the catabolic range) failed to increase CART mRNA tolevels of circulating corticosterone, whereas the group levels above control levels suggesting that the effect onreceiving the corticosterone pellet had circulating levels CART gene transcription is mainly permissive. In furtherthat were comparable to the values of the sham group support of a GR mediated mechanism is the fact that the(Table 1). corticosterone treated ADX rats—with low levels of

The effects of adrenalectomy and different types of circulating corticosterone (3963 ng/ml)—were unable toglucocorticoid replacement therapy on the expression of restore CART mRNA levels in the hypothalamus. At thisCART and POMC expression were examined by in situ level of corticosterone—comparable to nadir values of thehybridization experiments. Quantification by image analy- sham group—circulating corticosterone occupy predomi-sis showed that CART expression in both the Arc and the nantly high affinity MR receptors [31]. Our data are in linePVN was downregulated in ADX rats to approximately with a recent paper examining CART-immunoreactivity60% of the level found in sham operated controls (Arc, (ir) in ADX rats [3]. CART-ir was examined in the PVNFig. 1A: ADX vs. sham-ADX; 2826762296 vs. and in the supraoptic nucleus (SON) and found to be4837666864 arbitrary units; PVN, Fig. 1B: ADX vs. decreased in the former and unchanged in the lattersham-ADX: 125792616433 vs. 185823616034). Dexa- following ADX. Corticosterone replacement (30mg/ml inmethasone treatment prevented this fall in mRNA expres- the drinking water) only partially counteracted this effectsion, whereas substitution with corticosterone to baseline [3].cort levels only caused a non-significant increase towards The presence of glucocorticoid receptor immunoreactivi-baseline values (Fig. 1A and B). POMC mRNA levels in ty within POMC/CART neurons in the Arc suggest athe arcuate nucleus were unaffected by adrenalectomy as direct action of corticosterone on these neurons [24,25]. Towell as by dexamtheasone/corticosterone treatment (Fig. look for a possible direct effect of corticosterone on CART1C). The differences in CART hybridization signals across gene expression we performed a search for glucocorticoidgroups were also visible when examining the emulsion response elements (GRE) in the promotor region of the

Table 1Summary of metabolic parameters from the four treatment groups

sham-ADX ADX ADX1DEX ADX1Cort.

Bodyweight day 1 (g) 29864 29063 29565 29666Bodyweight day 11 (g) 36169 33767 27167* 34969Food/100 g bodyweight day 2 (g) 8.360.2 7.660.3* 6.260.2*§¶ 7.560.4*Food/100 g bodyweight day 10 (g) 7.560.2 7.760.2 7.860.2 7.960.2Epididymal fat (g) 3.060.2 2.960.1 3.060.1 3.160.2Retroperitoneal fat (g) 3.760.3 2.160.2* 1.760.2*¶ 2.660.3*Corticosterone (ng/ml) 57619 x x 3963

Data are mean6S.E.M. * P,0.05 versus sham-ADX; §P,0.05 versus ADX; ¶P,0.05 versus ADX1Cort. ADX and ADX1DEX groups hadcorticosterone values (x) below detection limit of the assay (10 ng/ml).

48 N. Vrang et al. / Brain Research 965 (2003) 45–50

human CART gene using the TRANSFAC database [32].A putative GRE element (gtcacctttTGTCctt) is locatedapproximately 6.2 kb upstream of exon 1 in the genomicCART sequence. Whether this rather distant GRE is of anyfunctional significance in the corticosteroidal regulation ofCART gene, or corticosterone influences the CART genevia other pathways remains to be determined.

Much more is known about the molecular mechanismsunderlying POMC expression and results from numerousexperiments show that corticosterone exert a powerfulfeedback inhibition on POMC gene expression in theanterior pituitary [33–35]. It has been shown that theglucocorticoid repression of POMC mRNA levels in theanterior pituitary is mediated by a negative GRE elementlocated within the first 100 base pairs of the POMCpromotor region [36]. Interestingly, appropriate expressionof the POMC gene in anterior pituitary corticotrophs andmelanotrophs depends on only 769 59-flanking base pairsof the gene [37]. In the hypothalamus, however, POMCexpression is dependent on a much larger portion of thepromotor region, and the negatively active GRE element isnon-functional [36,37]. In fact, the role of glucocorticoidsin the regulation of hypothalamic POMC expression is notat all clear. As mentioned previously, the POMC neuronsin the Arc contain glucocorticoid receptor indirectly point-ing to sensitivity to steroids, but possible effects ofglucocorticoids on hypothalamic POMC expression remainelusive. Adrenalectomy has been reported to increase [18],decrease [20,21] or produce no change [22,23] in POMCmRNA levels in the Arc. A variety of different RNAquantification methods and different periods of survivalhave been used making direct comparison between studiesdifficult. Piekut used ribo probe in situ hybridizationhistochemistry on 14 day ADX rats and found a reductionin POMC mRNA expression at all levels of the arcuatenucleus [21] and a dexamethasone induced increase in thecentral and caudal portions of the Arc. Wardlaw et al. [33]used solution hybridization S1 nuclease protection assayand radioimmunoassay to examine POMC mRNA andb-endorphin/a-MSH levels, respectively. In line withPiekut, they observed a reduction in POMC mRNA andb-endorphin/a-MSH protein levels following ADX and areversal (of mRNA but not protein levels) by dexametha-sone substitution. In our study we examined the centralpart of the the Arc—the level where Piekut observed the

Fig. 1. Relative abundance of CART (A, B) and POMC (C) mRNAlevels in adrenalectomized and glucocorticoid substituted rats. (A) and(B) CART mRNA levels in the mid-arcuate nucleus and paraventricularnucleus of the hypothalamus, respectively. (C) POMC mRNA levels inthe mid-arcuate nucleus. Data are mean6S.E.M. Asterisks indicatesignificant differences between groups (P,0.05, ANOVA factorial fol-lowed by Fisher’s posthoc analysis).

N. Vrang et al. / Brain Research 965 (2003) 45–50 49

Fig. 2. Microphotographs of emulsion dipped sections showing CART expression in the arcuate (top row) and paraventricular (bottom row) nuclei. Alower in situ hybridization signal (density of silver grains) is seen in the Arc and PVN in ADX and ADX1B groups.

greatest differences in POMC mRNA levels between A cknowledgementsgroups—but failed to find any significant effects. This

This study was supported by grants from the Danishapparent discrepancy is not readily explained, but could beMedical Research Council (9701798), The Danish Dia-due to one or a combination of small experimentalbetes Association, The Novo Nordisk Foundation anddifferences: slightly different survival times (11 vs. 14Fonden til Lægevidenskabens Fremme.days), different administration route or dose of dexametha-

sone (not given in study by Piekut), different strains ofrats. Due to the different techniques used it is difficult to

R eferencesexplain the discrepancy between our data and thosereported by Wardlaw et al. [33]. Which method is more

[1] S. Aja, S. Sahandy, E.E. Ladenheim, G.J. Schwartz, T.H. Moran,sensitive is difficult to say. Both techniques have beenIntracerebroventricular CART peptide reduces food intake and altersused to detect relatively small changes (0.5–23 relativemotor behavior at a hindbrain site, Am. J. Physiol.

de/ increases) in mRNA levels, and in this study we were [2] S.F. Akana, C.S. Cascio, J. Shinsako, M.F. Dallman, Corticosterone:able to detect changes in CART mRNA levels. In line with narrow range required for normal body and thymus weight andour results, a recent study of adrenalectomized lean Ob/? ACTH, Am. J. Physiol. 249 (1985) R527–R532.

[3] B. Balkan, E.O. Koylu, M.J. Kuhar, S. Pogun, The effect ofmice showed no effect of ADX on POMC expression inadrenalectomy on cocaine and amphetamine-regulated transcriptthe Arc [38]. One other study has suggested that CART(CART) expression in the hypothalamic nuclei of the rat 1, Brain

and POMC genes could be differentially regulated in the Res. 917 (2001) 15–20.Arc [39]. This group reported an elevation in CART [4] S. Beaulieu, B. Gagne, N. Barden, Glucocorticoid regulation ofmRNA expression in the Arc coinciding with the onset of proopiomelanocortin messenger ribonucleic acid content of rat

hypothalamus, Mol. Endocrinol. 2 (1988) 727–731.short day anorexia in the hibernating Siberian hamster[5] N.C. Birnberg, J.C. Lissitzky, M. Hinman, E. Herbert, Glucocor-[39]. If indeed the CART and POMC genes are differen-

ticoids regulate proopiomelanocortin gene expression in vivo at thetially regulated within the same neurons this suggests that levels of transcription and secretion, Proc. Natl. Acad. Sci. USA 80the transmitters derived from the gene products could (1983) 6982–6986.subserve different roles in hypothalamic integration. [6] A. Cintra, F. Bortolotti, Presence of strong glucocorticoid receptor

immunoreactivity within hypothalamic and hypophyseal cells con-In conclusion, we have shown that the effect oftaining pro-opiomelanocortic peptides, Brain Res. 577 (1992) 127–glucocorticoids on CART mRNA levels appears to be133.

permissive and mediated by GR receptors since the [7] P.R. Couceyro, E.O. Koylu, M.J. Kuhar, Further studies on thereduction in CART mRNA in ADX rats could be pre- anatomical distribution of CART by in situ hybridisation, J. Chem.vented—but not further increased—by catabolic doses of Anat. 12 (1997) 229–241.

[8] J. Douglass, S. Daoud, Characterization of the human cDNA andthe GR selective agonist dexamethasone. Whether cortico-genomic DNA encoding CART: a cocaine- and amphetamine-steroidal influence on CART gene expression is involvedregulated transcript, Gene 169 (1996) 241–245.

in HPA-axis regulation or modulates the appetitive effects [9] J. Douglass, A.A. McKinzie, P. Couceyro, PCR differential displayof CART peptides will have to be examined in future identifies a rat brain mRNA that is transcriptionally regulated byexperiments. cocaine and amphetamine, J. Neurosci. 15 (1995) 2471–2481.

50 N. Vrang et al. / Brain Research 965 (2003) 45–50

[10] C.F. Elias, C. Lee, J. Kelly, C. Aschkenasi, R.S. Ahima, P.R. Seal, D.G. Morgan, D. Sunter, C.L. Dakin, M.S. Kim, R. Hunter, M.Couceyro, M.J. Kuhar, C.B. Saper, J.K. Elmquist, Leptin activates Kuhar, M.A. Ghatei, S.R. Bloom, Actions of cocaine- and amphet-hypothalamic CART neurons projecting to the spinal cord, Neuron amine-regulated transcript (CART) peptide on regulation of appetite21 (1998) 1375–1385. and hypothalamo–pituitary axes in vitro and in vivo in male rats,

¨[11] K. Fuxe, A.C. Wikstrom, S. Okret, L.F. Agnati, A. Harfstrand, Z.Y. Brain Res. 893 (2001) 186–194.Yu, S. Granholm, M. Zoli, W. Vale, J. Gustafsson, Mapping of [26] L. Thim, P. Kristensen, P.F. Nielsen, B.S. Wulff, J.T. Clausen, Tissueglucocorticoid receptor immunoreactive neurons in the rat tel- and specific processing of cocaine and amphetamine regulated transcriptdiencephalon using a monoclonal antibody against rat liver peptides in the rat, Proc. Natl. Acad. Sci. USA 96 (1999) 2722–glucocorticoid receptor, Endocrinology 117 (1985) 1803–1812. 2727.

[12] K.M. Gautvik, L. De Lecca, V.T. Gautvik, P.E. Danielson, P. [27] L. Thim, P.F. Nielsen, M.E. Judge, A.S. Andersen, I. Diers, M.Tranque, A. Dopazo, F.E. Bloom, J.G. Sutcliffe, Overview of the Egel-Mitani, S. Hastrup, Purification and characterisation of a newmost prevalent hypothalamus-specific mRNAs, as identified by hypothalamic satiety peptide, cocaine and amphetamine regulateddirectional tag PCR subtraction, Proc. Natl. Acad. Sci. 93 (1996) transcript (CART), produced in yeast, FEBS Lett. 428 (1998)8733–8738. 263–268.

[13] J. Guy, H. Vaudry, G. Pelletier, Differential projections of two [28] Y. Tong, H.F. Zhao, F. Labrie, G. Pelletier, Regulation ofimmunoreactive alpha-melanocyte stimulating hormone (alpha- proopiomelanocortin messenger ribonucleic acid content by sexMSH) neuronal systems in the rat brain, Brain Res. 220 (1981) steroids in the arcuate nucleus of the female rat brain, Neurosci.199–202. Lett. 112 (1990) 104–108.

[14] E.O. Koylu, P.R. Couceyro, P.D. Lambert, M.J. Kuhar, Cocaine- and [29] N.Vrang, P. Kristensen, M. Tang-Christensen, P.J. Larsen, Effects ofamphetamine-regulated transcript peptide immunohistochemical leptin on arcuate POMC and CART expression are independent oflocalization in the rat brain, J. Comp. Neurol. 391 (1998) 115–132. circulating levels of corticosterone, J. Neuroendocrinol., in press.

[15] E.O. Koylu, P.R. Couceyro, P.D. Lambert, N.C. Ling, E.B. DeSouza, [30] N. Vrang, P.J. Larsen, J.T. Clausen, P. Kristensen, NeurochemicalM.J. Kuhar, Immunohistochemical localization of novel CART characterization of hypothalamic cocaine-amphetamine-regulatedpeptides in rat hypothalamus, pituitary and adrenal gland, J. transcript neurons, J. Neurosci. 19 (RC5) (1999) 1–8.Neuroendocrinol. 9 (1997) 823–833. [31] N. Vrang, P.J. Larsen, P. Kristensen, M. Tang-Christensen, Central

[16] P. Kristensen, M.E. Judge, L. Thim, U. Ribel, K.N. Christjansen, administration of cocaine-amphetamine-regulated transcript activatesB.S. Wulff, J.T. Clausen, P.B. Jensen, O.D. Madsen, N. Vrang, P.J. hypothalamic neuroendocrine neurons in the rat, Endocrinology 141Larsen, S. Hastrup, Hypothalamic CART is a new anorectic peptide (2000) 794–801.regulated by leptin, Nature 393 (1998) 72–76. [32] N.Vrang, M. Tang-Christensen, P.J. Larsen, P. Kristensen, Recombi-

[17] P.D. Lambert, P.R. Couceyro, K.M. McGirr, S.E. Dall Vechia, Y. nant CART peptide induces c-Fos expression in central areasSmith, M.J. Kuhar, CART peptides in the central control of feeding involved in control of feeding behaviour, Brain Res. 818 (1999)and interactions with neuropeptide Y, Synapse 29 (1998) 1–6. 499–509.

[18] J.S. Meyer, D.J. Micco, B.S. Stephenson, L.C. Krey, B.S. McEwen, [33] S.L. Wardlaw, K.C. McCarthy, I.M. Conwell, Glucocorticoid regula-Subcutaneous implantation method for chronic glucocorticoid re- tion of hypothalamic proopiomelanocortin, Neuroendocrinology 67placement therapy, Physiol. Behav. 22 (1979). (1998) 51–57.

[19] T.M. Mizuno, S.P. Kleopoulos, H.T. Bergen, J.L. Roberts, C.A. [34] J.R. Lundblad, J.L. Roberts, Regulation of proopiomelanocortinPriest, C.V. Mobbs, Hypothalamic pro-opiomelanocortin mRNA is gene expression in pituitary, Endocr. Rev. 9 (1988) 135–158.reduced by fasting in ob/ob and db/db mice, but is stimulated by [35] J.L. Roberts, C.L. Chen, J.H. Eberwine, M.J. Evinger, C. Gee, E.leptin 3, Diabetes 47 (1998) 294–297. Hergert, B.S. Schachter, Glucocorticoid regulation of

[20] G. Pelletier, Regulation of proopiomelanocortin gene expression in proopiomelanocortin gene expression in rodent pituitary, Recentrat brain and pituitary as studied by in situ hybridization, Ann. N. Y. Prog. Horm. Res. 38 (1982) 227–256.Acad. Sci. 680 (1993) 246–259. [36] J. Drouin, Y.L. Sun, M. Chamberland, Y. Gauthier, A. De Lean, M.

[21] D.T. Piekut, Relationship of ACTH1–39-immunostained fibers and Nemer, T.J. Schmidt, Novel glucocorticoid receptor complex withmagnocellular neurons in the paraventricular nucleus of rat hypo- DNA element of the hormone-repressed POMC gene, Embo J. 12thalamus, Peptides 6 (1985) 883–890. (1993) 145–156.

[22] M.N. Scanlon, E. Lazar-Wesley, T. Csikos, G. Kunos, Rat hypo- [37] Y. Tremblay, I. Tretjakoff, A. Peterson, T. Antakly, C.X. Zhang, J.thalamic proopiomelanocortin messenger RNA is unaffected by Drouin, Pituitary-specific expression and glucocorticoid regulationadrenalectomy, Biochem. Biophys. Res. Commun. 186 (1992) 418– of a proopiomelanocortin fusion gene in transgenic mice, Proc. Natl.425. Acad. Sci. USA 85 (1988) 8890–8894.

[23] M.W. Schwartz, R.J. Seeley, S.C. Woods, D.S. Weigle, L.A. Cam- [38] H. Makimura, T.M. Mizuno, J. Roberts, J. Silverstein, J. Beasley,pfield, P. Burn, D.G. Baskin, Leptin increases hypothalamic pro- C.V. Mobbs, Adrenalectomy reverses obese phenotype and restoresopiomelanocortin mRNA expression in the rostral arcuate nucleus, hypothalamic melanocortin tone in leptin-deficient ob/ob mice,Diabetes 46 (1997) 2119–2123. Diabetes 49 (2000) 1917–1923.

[24] J. Spiess, J. Villarreal, W. Vale, Isolation and sequence analysis of a [39] C.L. Adam, K.M. Moar, T.J. Logie, A.W. Ross, P. Barrett, P.J.somatostatin-like polypeptide from ovine hypothalamus 20 (1981) Morgan, J.G. Mercer, Photoperiod regulates growth, puberty and1982–1988. hypothalamic neuropeptide and receptor gene expression in female

[25] S.A. Stanley, C.J. Small, K.G. Murphy, E. Rayes, C.R. Abbott, L.J. Siberian hamsters, Endocrinology 141 (2000) 4349–4356.