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Recombinant CART peptide induces c-Fos expression in central areas involved in control of feeding behaviour
Brain Research 818 Ž1999. 499–509
Research report
Recombinant CART peptide induces c-Fos expression in central areas involved in control of feeding behaviour Niels Vrang a
a, )
, Mads Tang-Christensen
a,c
, Philip J. Larsen b , Peter Kristensen
c
Institute of Medical Anatomy, Department B, The Panum Institute, UniÕersity of Copenhagen, BlegdamsÕej 3 DK-2200, Copenhagen, Denmark b Neuroendocrine Pharmacology, NoÕo Nordisk, Copenhagen, Denmark c Histology, NoÕo Nordisk, Copenhagen, Denmark Accepted 8 December 1998
Abstract We have recently shown that the hypothalamic neuropeptide CART Žcocaine-amphetamine-regulated-transcript. is a leptin dependent endogenous satiety factor in the rat. In the present study we confirm and extend our previous observations by showing that intracerebroventricular Ži.c.v.. administered CARTŽ42–89. dose-dependently inhibits 3-h food intake in food restricted rats with a lowest effective dose of 0.5 mg. CART also potently inhibits NPY-induced food intake in satiated rats as well as nighttime food intake in free feeding animals. To identify brain areas potentially involved in mediating the anorectic effects of CART, the temporal expression pattern of the immediate early gene c-fos was examined in the central nervous system by immunohistochemistry in rats receiving recombinant CART. Compared to vehicle, CART induced c-Fos expression in several hypothalamic and brainstem structures implicated in the central control of food intake. In the hypothalamus, high numbers of c-Fos immunoreactive Ž-ir. cells were observed in the medial parvocellular part of the paraventricular nucleus and in the posterior part of the dorsomedial nucleus. Lower numbers of c-Fos positive nuclei were found in the supraoptic and arcuate nuclei. A relatively high number of c-Fos-ir cells was found in the central nucleus of the amygdala. In the brainstem, c-Fos-positive nuclei were found in the parabrachial nucleus, and in the nucleus of the solitary tract. Notably both the area postrema and the dorsal motor nucleus of the vagus were virtually devoid of c-Fos-ir cells. The present experiments suggest that CART peptide exerts its inhibitory effects on appetite by activating hypothalamic and brainstem neurones implicated in the central control of feeding behaviour and metabolism. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Regulation of food intake; c-Fos induction; i.c.v. Injection; Paraventricular nucleus of the hypothalamus
1. Introduction Cocaine-amphetamine-regulated-transcript ŽCART . mRNA was isolated from rat brain striatum by PCR differential display as an mRNA transcriptionally regulated by acute administration of cocaine or amphetamine w6x. In the rat brain, two transcripts of different length can be found and these are generated by differential splicing. The short mRNA gives rise to a peptide of 116 amino acid residues with a common hydrophobic leader sequence of 27 amino acids resulting in a putative mature CART peptide of 89 amino acids w6x. In humans, only this short form is present and except for one amino acid ŽVal-42-Ile. it is identical to the rat protein w5x. In the rat, the additional
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longer transcript is generated by insertion of 39 nucleotides encoding an insertion of 13 aminoacids in the N-terminal part of the peptide. CART mRNA is expressed in several areas throughout the rat and human brain as well as in the rat anterior pituitary and adrenal medulla w4,6x. In a systematic analysis of mRNAs whose expression is restricted or enriched in the hypothalamus, CART was among those most heavily expressed w7x. Interestingly, CART mRNA is expressed within hypothalamic structures implicated in the central control of feeding behaviour and metabolism, i.e., the paraventricular, arcuate and dorsomedial hypothalamic nuclei and the lateral hypothalamus w4,6,16x. The distribution of CART peptide immunoreactivity in the hypothalamus has been mapped using antibodies generated against synthetic fragments of CART w14,15x or a CART fusion protein w16x and shows an almost perfect overlap of CART immunoreactive cell bodies and CART mRNA.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 4 9 - 3
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We and others have recently provided evidence that the CART peptide Ž42–89. is involved in the control of food intake, since intracerebroventricular Ži.c.v.. administration of CARTŽ42–89. peptide w16x or CART peptide fragments w18x potently inhibits food intake. Furthermore, fasting reduces the expression of CART mRNA in the arcuate nucleus supporting a physiological role of endogenous CART in the control of feeding behaviour w16x. In farfa rats and obrob mice the CART mRNA is virtually absent from the arcuate nucleus and in obrob mice the expression is increased upon leptin treatment suggesting that leptin-induced anorexia is at least partially mediated via CART neurones w16x Žfor a review on leptin, see Ref. w41x.. At present, the central sites of action of CART are unknown. Using c-Fos immunohistochemistry, we have studied the distribution of sites potentially activated by CARTŽ42–89.. As c-Fos is also induced by cellular stress Žother than specific protein–receptor interaction. we sought to use the lowest dose of CARTŽ42–89. effective of inhibiting food intake. In initial experiments, the anorectic effects of increasing doses of CART were tested on both food-restricted rats and in rats subjected to NPY-induced feeding. Guided by the results of these two experiments, the lowest effective anorectic dose of CART was injected i.c.v., and c-Fos expression was examined at 60, 120 and 240 min post-injection.
with food ŽStandard Altromin Mix for rats a1314; C. Petersen, Ringsted Denmark. and water ad libitum. All experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Danish Committee for Animal Research. Under tribromethanol anaesthesia ŽAvertin, Merck, 50 mgrkg. rats were fitted with a 22 gauge guide cannula aimed at the lateral ventricle Žcoordinates: 1 mm posterior and 1.3 mm lateral to bregma and 4 mm below the surface of the skull, Ref. w37x. The cannula was fixed in place by acrylic dental cement and two anchoring screws. During the following 6 day recovery period rats were handled daily to get accustomed to the injection procedure. 2.3. Feeding behaÕiour experiments (Experiments 1, 2 and 3) Six days after surgery, rats were transferred to individual metabolic cages to allow precise measurement of food intake. No experiments were conducted until the 24-h food intake had stabilised Ž5–6 days.. During this period the rats were handled daily to accustom them to the injection paradigm. All experiments were conducted in the morning Ž0900–1100 h.. 2.4. Experiment 1 (restricted feeding)
2. Materials and methods 2.1. Peptides and nomenclature Due to the presence of the splice forms and putative hydrophobic leader sequence different nomenclature has been used with regard to the CART peptides. Here, we base our numbering on the aminoacids in the human peptide. The C-terminal part of CART probably comprising residue 42–89,was isolated from ovine hypothalamus during the isolation of somatostatin, suggesting, together with the existence of several potential di-basic cleavage sites in the CART peptide, that CART is post-translationally modified in vivo w35x. Recently, several CART peptides have been synthesised using a yeast expression system w40x. One of these, the C-terminal CARTŽ42–89. probably corresponds to a naturally occurring form of CART w35x. CARTŽ42–89. was used in all experiments and prepared as previously described w40x. Porcine NPY was obtained from Peninsula. 2.2. Animals and surgery Seventy-two adult male Wistar rats weighing 200–250 g at the time of surgery were used for the experiments. The rats were maintained on a 12:12 light–dark cycle Žlights on 0600 h. in a temperature controlled environment Ž22–248C.
After cannulation, animals were acclimatised in metabolic cages for 5 days before a restricted feeding schedule, with access to food for 3 h in the morning Ž0900–1200 h. was initiated w38x. Experiments were started when 3-h food intake had stabilised Ž5–6 days.. After stabilisation of daily 3-h food intake Ž5–6 days., the experiments were started. On each day of experimentation, rats were randomised into groups receiving i.c.v. injections of either 5 ml vehicle ŽKPBS: 50 mM phosphate buffered saline containing 0.02% potassium. or different doses of recombinant rat CARTŽ42–89. dissolved in 5 ml vehicle. Animals were injected immediately prior to the feeding session and food and water intake was measured every 30 min for 3 h. Animals were used in three consecutive experiments each separated by three days. 2.5. Experiment 2 (NPY-induced feeding) A separate group of cannulated rats were accustomed to the metabolic cage with food and water ad libitum as described above. Food was removed from the cages at 0800 h and animals injected at 0900 h. All animals received two 5 ml i.c.v. injections separated by 15 min. One group received vehicle followed by vehicle, another vehicle followed by 5 mg NPY and the last CARTŽ42–89. Ž0.5 or 1.0 mg. followed by NPY Ž5 mg.. Food intake was measured every 30 min for 3 h. Only two sets of experi-
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ments were conducted on the same rats each separated by three days. 2.6. Experiment 3 (oÕernight feeding) Another group of cannulated rats were accustomed to the metabolic cage and feeding and drinking ad lib as described above. Animals were injected with either vehicle or 0.5 mg CART immediately prior to lights out Žlights out at 0600 h.. Food intake was measured after 1 and 2 h Žthe use of a nightscope avoided light disturbing the animals. and in the morning immediately after lights on Ž12 h after the injection.. 2.7. Experiment 4 (c-Fos induction) Intracerebroventricularly cannulated rats were kept in individual rodent cages with free access to food and water. Rats were handled daily in the morning. On the day of the study, rats were injected i.c.v. with either 5 ml vehicle Ž n s 5. or 0.5 mg CARTŽ42–89. in 5 ml vehicle Ž n s 19. and the food removed from the cage. The animals injected with CARTŽ42–89. were sacrificed either 60 Ž n s 7., 120 Ž n s 7. or 240 Ž n s 5. min after the injection. The vehicle group was sacrificed 120 min after the injection. At the designated time-point following the injection, the animals were anaesthetised with tribromethanol Ž50 mgrkg. and perfused transcardially, first with heparinised Ž15.000 IUrl. KPBS, followed by Stephanini fixative Ž2% paraformaldehyde in KPBS containing picric acid, pH: 7.4. for 10 min. The brains were removed and post-fixed overnight in the same fixative then transferred for two days to a 30% sucrose–KPBS solution for cryoprotection. One-in-six series of 40 mm thick frontal sections were cut on a freezing microtome and collected in KPBS.
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KBPS and 10 min in a 0.05 M Tris–HCl buffer ŽpH 7.6.. Finally, the sections were developed in a solution containing 0.125% of the chromogen diaminobenzidine ŽDAB. and 0.01% H 2 O 2 in 0.05 M Tris- -HCl buffer. Sections were rinsed 2 = 10 min in distilled water before being mounted and coverslipped in Depex. Counting of c-Fos positive cell nuclei was performed unilaterally on single non-counterstained sections. All areas were counted at the same level in each individual animal making direct comparison between individual animals possible without counting all the c-Fos-ir cells in a given area. Counting was performed using a computerised image analysis system ŽImage 1.60 b, courtesy of NIH, Bethesda, MD., and the c-Fos cells were counted using a macro designed for this specific purpose. All images were acquired in the same session, and the validity of the computerised counts was tested by comparisons to manual counts of the same sectionsrareas. Photographs were taken at a Zeiss Axiophot microscope and negatives scanned into a computer using a Polaroid w slidescanner. Image editing software ŽAdobe Photoshop w . was used to combine photographs into plates and figures were printed on a Sony w dye sublimation printer. 2.9. Statistics Statistical evaluation of the experiments used two-way analysis of variance ŽANOVA. followed by Fisher’s posthoc analysis. Values Žfood intake in grams, number of c-Fos positive cells. are expressed as mean " S.E.M. P values - 0.05 were considered significant.
3. Results
2.8. Immunohistochemistry
3.1. Experiment 1 (restricted feeding)
All reactions were carried out on free-floating sections. Initially sections were washed 3 = 10 min in KPBS followed by incubation for 10 min in 1% H 2 O 2 in KPBS to block endogenous peroxidase activity. After 20 min in 5% swine serum diluted in BST ŽKPBS containing 1% bovine serum albumin ŽBSA. and 0.3% Triton-X-100 ŽTX.. the sections were incubated overnight at 58 with a c-Fos antibody diluted 1:1000 in BST. The c-Fos antibody has been described previously w45x. The sections were next washed for 3 = 10 min in KPBS containing 0.25% BSA and 0.1% TX ŽKPBS-BT. followed by 60 min of incubation at room temperature in a biotinylated swine anti-rabbit antibody ŽaE353, Dakopatts, Copenhagen. diluted 1:600 in BST. The sections were then rinsed 3 = 10 min in KPBS-BT, incubated for 60 min in an avidin–biotin–peroxidase complex ŽVector Elite Kite. diluted 1:250 in KPBS-BT and rinsed 10 min in KPBS-BT, 10 min in
Initially, we aimed at determining the lowest effective anorectic dose of CARTŽ42–89.. Doses ranging from 0.1 mg to 2.0 mg were injected into the lateral ventricle of rats kept on a restricted feeding scheme and food and water intake was subsequently monitored. In keeping with earlier studies, rats subjected to this feeding regimen showed an initial drop in bodyweight followed by a slower weight gain than free-feeding controls Žnot shown. w38,39x. Central administration of CARTŽ42–89. dose dependently inhibited food intake. The highest dose of CARTŽ42–89 significantly inhibited food intake throughout the observation period when compared to control rats, whereas the lowest dose of CART had no effect on food intake ŽFig. 1.. The lowest effective dose of CART with a clear effect on food intake was 0.5 mg Ž P - 0.05 at all time points except 180 min. ŽFig. 1.. Fig. 2 shows the cumulative food intake during the first 60 min as a function of CART dose
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Fig. 1. Effect of i.c.v. injections of either 5 ml vehicle or different doses of recombinant CARTŽ42–89. in 5 ml vehicle on cumulative 3-h food intake in food restricted rats. Vehicle Ž n s 12.; CART 0.1 mg Ž n s 8.; CART 0.5 mg Ž n s 15.; CART 2.0 mg Ž n s 13.. Data are means" S.E.M. Asterisks indicate significant differences between CART and vehicle group and † indicate significant differences between CART 0.5 mg and CART 2.0 mg Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
and demonstrates a dose-dependent inhibition of food intake by CART.
In our previous studies we observed altered locomotor behaviour in response to i.c.v. injections of CARTŽ42–89.
Fig. 2. CARTŽ42–89. dose dependently decrease food intake in rats. Cumulative food intake during the first 60 min after i.c.v. CART injection in food restricted rats. Vehicle Ž n s 12.; CART 0.1 mg Ž n s 8.; CART 0.25 mg Ž n s 12.; CART 0.5 mg Ž n s 15.; CART 1.0 mg Ž n s 7.; CART 2.0 mg Ž n s 13.. Data are means" S.E.M. Asterisks indicate significant differences between CART and vehicle groups, † indicate significant differences between CART Ž0.25 mg. and CART Ž0.5, 1.0 and 2.0 mg. and †† indicate significant differences between CART Ž0.5 mg. and CART Ž2.0 mg. groups Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
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Fig. 3. Central administration of CART inhibits NPY-induced food intake in satiated rats. Both doses of CART significantly reduced food intake induced by prior administration of 5 mg NPY throughout the 3-h observation period. The difference between 0.5 mg and 1.0 mg was non-significant at all time points. Vehicleq vehicle Ž n s 5.; vehicleq NPY Ž n s 8.; CART0.5 mg q NPY Ž n s 8.; CART 1.0 mg q NPY Ž n s 5.. Data are means" S.E.M. Asterisks indicate significant differences between CART and vehicle groups, † indicate significant differences between CARTq NPY and vehicleq NPY groups. Vehicleq vehicle was significantly different from all other groups at all time points Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
Fig. 4. Effect of 0.5 m CART i.c.v. on nighttime feeding. Freely fed rats were injected immediately prior to lights out ŽTime 0. and food intake was measured at 1, 2 and 12 h after the injection of either 5 ml vehicle or 0.5 mg CARTŽ42–89. in 5 ml vehicle. Vehicle Ž n s 9.; CART 0.5 mg Ž n s 11.. Data are means " S.E.M. Asterisks indicate significant differences between CART and vehicle group Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
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Fig. 5. Photomicrographs of c-Fos like immunoreactivity in selected areas of one animal that received 0.5 mg CARTŽ42–89. i.c.v. Žleft columns; A, C, E, G, I, K, M. and one control animal injected with vehicle Žright columns; B, D, F, H, J, L, N. i.c.v. Both rats were sacrificed at 120 min following the injection. A and B: the supraoptic nucleus; C and D: the paraventricular nucleus of the hypothalamus; E and F: the arcuate nucleus and the ventromedial nucleus; G and H: the posterior part of the dorsomedial nucleus; I and J: the central nucleus of the amygdala; K and L: the parabrachial nucleus; M and N: the area postrema and the nucleus of the solitary tract.
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Fig. 5 Žcontinued..
w16x. These side-effects were also observed in the present study, but only at the highest dose of CARTŽ42–89. Ž2 mg.. 3.2. Experiment 2 (NPY-induced feeding) Fig. 3 shows the results from this experiment in which the ability of CART to inhibit NPY-induced food intake was tested. Measurements were started at the time of the second injection Ž t 0 .. In rats injected with NPY, food intake increased over the first 90 min and this increase was significantly suppressed by both 0.5 mg and 1.0 mg of CART. Apparently, 1.0 mg of CART suppressed food intake more than 0.5 mg, but this difference failed to reach statistical significance. The suppression of NPY induced feeding was evident throughout the observation period for
both doses of CART tested Ž t 180 : 7.66 " 0.70 Žvehicleq NPY.; 5.35 " 0.69 Ž0.5 mg CARTq NPY.; 3.90 " 0.62 Ž1.0 mg CARTq NPY... 3.3. Experiment 3 (oÕernight feeding) In this experiment, the ability of CART to inhibit normal nighttime feeding in freely fed animals was tested. Measurements were started at the time of injection Žimmediately before the dark period in the animal care facility. and food intake measured at 1 Ž t 1 ., 2 Ž t 2 . and 12 Ž t 12 . h. Fig. 4 shows the results from this experiment, and demonstrates that CART significantly inhibited food intake at t 12 Ž t 12 : 18.29 " 0.55 Žvehicle.; 13.18 " 1.08 Ž0.5 mg CART., but not at t 1 or t 2 Žsee Fig. 4..
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Table 1 Number of c-Fos positive cell nuclei in selected brain areas Žmean " S.E.M..
SON PVN Arc CeAl DMHp VMH LPB AP NTS
Saline, 120 min Ž n s 4.
0.5 mg CART, 60 min Ž n s 6.
0.5 mg CART, 120 min Ž n s 7.
0.5 mg CART, 240 min Ž n s 5.
0.5 " 0.3 8.3 " 1.9 3.5 " 0.9 21.3 " 7.6 0.75 " 0.25 19.3 " 2.5 3.2 " 0.25 3.0 " 2.7 1.25 " 0.3
16.0 " 5.2 186.8 " 19.5) 36.0 " 5.9) 50.1 " 15.1 19.3 " 6.0) 26.0 " 5.6 45.7 " 5.1) 3.3 " 2.3 36.3 " 8.9)
26.9 " 5.0) 195.7 " 21.8) 33.3 " 6.2) 89.9 " 10.1)† 45.6 " 7.4) 28.6 " 7.6 46.6 " 4.5) 4.5 " 1.8 70.3 " 5.8)†
29.4 " 11.2) 194.6 " 19.5) 34.2 " 6.3) 39.2 " 5.3 32.8 " 5.6) 24.8 " 3.6 31.8 " 3.8) 4.6 " 2.0 48.8 " 6.8)
Asterisks indicate significant differences between CART and vehicle groups and † indicate significant differences between CART, 120 min and CART 60, 240 min. Significance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis. Abbreviations: SON s supraoptic nucleus; PVN s paraventricular nucleus of the hypothalamus; Arc s arcuate nucleus; CeAl s the central nucleus of the amygdala, lateral part; DMHps the posterior part of the dorsomedial nucleus of the hypothalamus; VMHs ventromedial nucleus of the hypothalamus; LPB s lateral parabrachial nucleus; AP s area postrema; NTS s nucleus of the solitary tract.
3.4. Experiment 4 (c-Fos induction) In comparison to vehicle, i.c.v. administration of 0.5 mg CARTŽ42–89. resulted in induction of c-Fos like immunoreactivity in several distinct regions of the fore- and hindbrain Žsee Fig. 5 and Table 1.. The time course of the emerging pattern and number of c-Fos-ir cells was studied by examining sections from animals sacrificed 60, 120 and 240 min following CART administration. Time dependent c-Fos expression was observed only in the central nucleus of the amygdala and in the NTS Žsee Table 1.. In Fig. 5, the distribution of CART-induced c-Fos immunoreactive cells is shown from areas of interest. In the hypothalamus, a high number of c-Fos immunoreactive Ž-ir. cells was observed in the paraventricular nucleus ŽPVN. and in the posterior part of the dorsomedial nucleus ŽDMHp.. In the central part of the PVN, the majority of c-Fos-ir cells were located in the dorsal aspect of the medial parvocellular subdivision of the nucleus. Few c-Fos-ir cells were seen in the perimeter of the posterior magnocellular subnucleus, while the central core of the posterior magnocellular subnucleus was virtually devoid of c-Fos-ir nuclei Žsee Fig. 5.. The majority of c-Fos positive elements in the PVN were observed in the area where most of the CRH containing neurones are located ŽFig. 5.. A lower number of c-Fos positive nuclei was found in the arcuate nucleus ŽArc. and supraoptic nucleus ŽSON.. In the SON the c-Fos positive nuclei were distributed in the dorsolateral portion of the nucleus, an area corresponding to the region where the oxytocinergic neurones are located. CART peptide also induced c-Fos expression in the lateral part of the bed nucleus of the stria terminalis ŽBSTL. and in the central nucleus of the amygdala Žlateral subnucleus, CeAl.. In the brainstem c-Fos-positive nuclei were found in the lateral parabrachial nucleus ŽLPB. Ždorsal and central subnuclei., and throughout the rostrocaudal extent of the nucleus of the solitary tract ŽNTS.. Quantification of c-Fos-ir cells in
the NTS was carried out in sections taken at the level of the area postrema. At this level, c-Fos-ir cells were concentrated in the medial subnuclei of the NTS and to a lesser degree in the subpostreme area. In contrast, both the area postrema and the dorsal motor nucleus of the vagus were virtually devoid of c-Fos-ir cells ŽFig. 5..
4. Discussion We have confirmed and extended our previous observations that central administration of recombinant CARTŽ42–89. dose-dependently inhibits food intake in food restricted rats as well as in rats subjected to NPY-induced food intake w16x. Also, we have demonstrated, that i.c.v. CARTŽ42–89. at a minimal effective dose of 0.5 mg is capable of inhibiting overnight food intake in free feeding animals. This dose of CART induces c-Fos expression in several brain regions believed to be involved in the central control of food intake and metabolism. At present, it is uncertain whether CART mediates its actions via one or several receptors and possible interactions with various second messenger systems remain speculative. Therefore, c-Fos immunohistochemistry remains a versatile method to unravel the circuits influenced by this novel anorectic peptide. Central administration of 0.5 mg CART also effectively suppressed night time feeding suggesting that CART belongs to the class of intermediaterlong term regulators of food intake. However, longer observation periods Žto examine potential rebound effects. and chronic administration of CART peptide is necessary in order to determine the time course and potential long-term body regulatory effects of CART peptide. This is in keeping with our previous observation that central administration of antiCART serum elevates the amount of food eaten during a night time feeding session w16x. The lowest effective dose
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of CART Ž0.5 mg. inhibiting food intake was used in the c-Fos experiments. We have previously observed that although 1 and 2 mg doses of CART i.c.v. causes some movement-associated tremor, these doses have no effect on spontaneous locomotor activity w16x. Thus, with the low dose of CART used in the present study, it is unlikely that c-Fos-ir occurred in central areas as a consequence of altered locomotor behaviour, although it is impossible to assess whether some of the c-Fos-ir emerged in areas controlling motor activities upstream to cortical projection neurones. Expression of c-Fos and other immediate early genes are primarily induced by cellular excitation w32x. As CART serves as an inhibitor of food intake, and therefore possibly as an inhibitor of neuronal activity, it is possible that neurones sensitive to CART Ži.e., containing the as yet unidentified CART receptor. remain electrophysiologically silent and, consequently, do not express c-Fos. However, if the main action of CART peptide is inhibitory, c-Fos induction probably still reflects specific but indirect action Ži.e., removal of an inhibitory input. upon neuronal circuits responsive for mediating the anorectic effects of CART. Expression of c-Fos in hypothalamic nuclei is largely distributed as CART immunoreactive fibers w15x although the density of CART innervation may not necessarily reflect the number of Fos-positive nuclei. This overlap suggest that CART exerts its effects locally, either directly or indirectly via adjacent interneurons, but it should be noted that CART induced c-Fos expression was absent in many areas that have been shown to contain CART-immunopositive fibers w14x. Notably, no c-Fos induction was observed in the induseum griseum, hippocampus, septal nuclei, or nucleus accumbens, areas that have all been found to contain moderate to high densities of CART immunopositive fibers. Also no c-Fos induction was observed in the striatum. Within the hypothalamus, the most prominent induction of c-Fos was observed in the PVN ŽFig. 5.. This nucleus is a key component in the neural circuitry involved in the regulation of feeding behaviour. Lesions of the PVN have profound effects on food intake and body weight regulation w8x and several neurotransmitters with orexigenic as well as anorectic properties have been shown to exert their influence on feeding behaviour most potently when injected directly into this nucleus. For example, neuropeptide Y ŽNPY., galanin and noradrenaline all stimulate feeding when injected into the PVN w17,21,36x, whereas GLP-1 and CRF inhibits food intake when injected here w25,31x. CART induced c-Fos expression in the PVN was most prominent in the medial parvocellular parts of the nucleus. This pattern of activation is very similar to that observed in studies of c-Fos expression following central administration of other anorectic peptides Že.g., GLP-1 w20x, leptin w43x and CCK w30x.. In the magnocellular subnuclei of the PVN as well as the SON, CART induced fos-expression overlapped with the localisation of oxytocinergic ŽOT. neurons, suggesting
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that CART activates these neurones. A number of centrally acting anorectic agents including CCK and GLP-1 trigger c-Fos expression in OT neurones w20,30x. However, it remains to be established whether central application of CART has any effect upon circulating levels of OT. The arcuate nucleus houses neurons containing a variety of peptides, several of which are known to affect feeding behaviour w2x. The effect of NPY on feeding, for example, is believed to be mediated in large by an arcuato-PVN NPY-ergic connection w1,28x. The orexigenic property of NPY is emphasised by the ability of repeated injections of NPY to cause obesity w12,28x. It is generally accepted that NPY acts on neurons in the PVN area to initiate feeding behaviour, but the nature of these NPY-sensitive feedinginducing neurons needs to be determined. The PVN receives dense projections from the different parts of the DMH including the posterior region where CART-induced c-Fos expression predominates w24x. Similarities between arcuate and DMH neurones exist. Both nuclei contain NPYergic neurones projecting to the PVN w1x and central injection of NPY elicits c-Fos in both the arcuate and the DMH w22x. However, selective destruction of either the arcuate or DMH has opposite effects on energy balance. Destruction of the arcuate nucleus by neonatal monosodium glutamate produce obesity and hyperglycaemia w26,29x while selective DMH lesions lower the body weight set point w3x. Also NPY mRNA synthesis in the arcuate and DMH is differentially regulated, with high levels of expression in the arcuate in response to adrenal steroids w19x, while NPY mRNA levels in the DMH is selectively increased under lactation and in Agouti mice w13,23,33x. The BSTL and the CeA constitute part of a network of autonomic nuclei that are of importance in regulating various autonomic functions w34x. The presence of CART induced c-Fos expression in both of these nuclei suggests that several autonomic functions may be regulated by CART. Both the CeA and BSTL are reciprocally connected with the PVN, the midbrain central grey, the parabrachial nucleus, the NTS, the dorsal vagal nucleus and the ventrolateral medulla w10,11x. Interestingly, all of these areas contain c-Fos-ir nuclei triggered by central CART application supporting a rather ubiquitous action on this functional continuum. CRH containing neurons of the CeA are main contributors to the descending pathways that innervate the aforementioned target areas w27x. The CeA is centrally involved in regulating responses associated in learned aversion w9,44x and is also involved in the control of food and energy balance. Thus, it is possible that CART influences food intake via activation of descending CRH neurones of the CeA and BSTL which interferes with PB and NTS neurones. An alternative explanation would be that ascending pathways from the brain stem regions including the NTS and PB activate CeA neurones. In the brainstem, a high number of c-Fos immunoreactive cells were seen in the NTS. The NTS is a major
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sensory relay nucleus receiving afferent input from the 7th, 9th and 10th cranial nerve and is reciprocally connected with numerous brainstem and forebrain structures. Among these the PVN, lateral parabrachial nucleus and central nucleus of the amygdala, nuclei also express c-Fos following CART i.c.v. Interestingly i.c.v. CARTŽ42–89. had no effect on c-Fos expression in the area postrema suggesting that the chemosensitive neurones here are not excited by CART and indicating that the induction of c-Fos within the NTS is not a consequence of non-specific chemo-activation of AP-neurones connected to the NTS. One likely target for CART in the NTS could be the GLP-1 containing cells. These neurons project to the hypothalamus Žmost notably the PVN. w20x and have been proposed to constitute the endogenous system mediating GLP-1 induced satiety w38,39,42x.
5. Conclusion We have shown that centrally administered recombinant CARTŽ42–89. potently inhibits food intake in both fasted rats and satiated rats induced to eat with NPY. Furthermore CART peptide induces c-Fos expression in hypothalamic and brainstem areas that are believed to be involved in the regulation of feeding behaviour, metabolism and other autonomic functions, suggesting that CART inhibits food intake by activating neurones in one or more of these sites. Thus, the present study support—together with our previous observations—a role for CART as yet another player within the complex neuronal circuitry linking hypothalamus and brainstem regulating food intake and metabolism.
Acknowledgements This study was supported by grants from the Danish Medical Research Council Ž9701798., The Danish Diabetes Foundation, The Novo Nordisk Foundation, The Danish Research Foundation to the Biotechnology Centre for Cellular Communication. N.V. is supported by a research grant from the P. Carl Petersen Foundation. The authors wish to thank G. Hahn for excellent photographic assistance.
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Research report
Recombinant CART peptide induces c-Fos expression in central areas involved in control of feeding behaviour Niels Vrang a
a, )
, Mads Tang-Christensen
a,c
, Philip J. Larsen b , Peter Kristensen
c
Institute of Medical Anatomy, Department B, The Panum Institute, UniÕersity of Copenhagen, BlegdamsÕej 3 DK-2200, Copenhagen, Denmark b Neuroendocrine Pharmacology, NoÕo Nordisk, Copenhagen, Denmark c Histology, NoÕo Nordisk, Copenhagen, Denmark Accepted 8 December 1998
Abstract We have recently shown that the hypothalamic neuropeptide CART Žcocaine-amphetamine-regulated-transcript. is a leptin dependent endogenous satiety factor in the rat. In the present study we confirm and extend our previous observations by showing that intracerebroventricular Ži.c.v.. administered CARTŽ42–89. dose-dependently inhibits 3-h food intake in food restricted rats with a lowest effective dose of 0.5 mg. CART also potently inhibits NPY-induced food intake in satiated rats as well as nighttime food intake in free feeding animals. To identify brain areas potentially involved in mediating the anorectic effects of CART, the temporal expression pattern of the immediate early gene c-fos was examined in the central nervous system by immunohistochemistry in rats receiving recombinant CART. Compared to vehicle, CART induced c-Fos expression in several hypothalamic and brainstem structures implicated in the central control of food intake. In the hypothalamus, high numbers of c-Fos immunoreactive Ž-ir. cells were observed in the medial parvocellular part of the paraventricular nucleus and in the posterior part of the dorsomedial nucleus. Lower numbers of c-Fos positive nuclei were found in the supraoptic and arcuate nuclei. A relatively high number of c-Fos-ir cells was found in the central nucleus of the amygdala. In the brainstem, c-Fos-positive nuclei were found in the parabrachial nucleus, and in the nucleus of the solitary tract. Notably both the area postrema and the dorsal motor nucleus of the vagus were virtually devoid of c-Fos-ir cells. The present experiments suggest that CART peptide exerts its inhibitory effects on appetite by activating hypothalamic and brainstem neurones implicated in the central control of feeding behaviour and metabolism. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Regulation of food intake; c-Fos induction; i.c.v. Injection; Paraventricular nucleus of the hypothalamus
1. Introduction Cocaine-amphetamine-regulated-transcript ŽCART . mRNA was isolated from rat brain striatum by PCR differential display as an mRNA transcriptionally regulated by acute administration of cocaine or amphetamine w6x. In the rat brain, two transcripts of different length can be found and these are generated by differential splicing. The short mRNA gives rise to a peptide of 116 amino acid residues with a common hydrophobic leader sequence of 27 amino acids resulting in a putative mature CART peptide of 89 amino acids w6x. In humans, only this short form is present and except for one amino acid ŽVal-42-Ile. it is identical to the rat protein w5x. In the rat, the additional
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longer transcript is generated by insertion of 39 nucleotides encoding an insertion of 13 aminoacids in the N-terminal part of the peptide. CART mRNA is expressed in several areas throughout the rat and human brain as well as in the rat anterior pituitary and adrenal medulla w4,6x. In a systematic analysis of mRNAs whose expression is restricted or enriched in the hypothalamus, CART was among those most heavily expressed w7x. Interestingly, CART mRNA is expressed within hypothalamic structures implicated in the central control of feeding behaviour and metabolism, i.e., the paraventricular, arcuate and dorsomedial hypothalamic nuclei and the lateral hypothalamus w4,6,16x. The distribution of CART peptide immunoreactivity in the hypothalamus has been mapped using antibodies generated against synthetic fragments of CART w14,15x or a CART fusion protein w16x and shows an almost perfect overlap of CART immunoreactive cell bodies and CART mRNA.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 3 4 9 - 3
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We and others have recently provided evidence that the CART peptide Ž42–89. is involved in the control of food intake, since intracerebroventricular Ži.c.v.. administration of CARTŽ42–89. peptide w16x or CART peptide fragments w18x potently inhibits food intake. Furthermore, fasting reduces the expression of CART mRNA in the arcuate nucleus supporting a physiological role of endogenous CART in the control of feeding behaviour w16x. In farfa rats and obrob mice the CART mRNA is virtually absent from the arcuate nucleus and in obrob mice the expression is increased upon leptin treatment suggesting that leptin-induced anorexia is at least partially mediated via CART neurones w16x Žfor a review on leptin, see Ref. w41x.. At present, the central sites of action of CART are unknown. Using c-Fos immunohistochemistry, we have studied the distribution of sites potentially activated by CARTŽ42–89.. As c-Fos is also induced by cellular stress Žother than specific protein–receptor interaction. we sought to use the lowest dose of CARTŽ42–89. effective of inhibiting food intake. In initial experiments, the anorectic effects of increasing doses of CART were tested on both food-restricted rats and in rats subjected to NPY-induced feeding. Guided by the results of these two experiments, the lowest effective anorectic dose of CART was injected i.c.v., and c-Fos expression was examined at 60, 120 and 240 min post-injection.
with food ŽStandard Altromin Mix for rats a1314; C. Petersen, Ringsted Denmark. and water ad libitum. All experiments were conducted in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Danish Committee for Animal Research. Under tribromethanol anaesthesia ŽAvertin, Merck, 50 mgrkg. rats were fitted with a 22 gauge guide cannula aimed at the lateral ventricle Žcoordinates: 1 mm posterior and 1.3 mm lateral to bregma and 4 mm below the surface of the skull, Ref. w37x. The cannula was fixed in place by acrylic dental cement and two anchoring screws. During the following 6 day recovery period rats were handled daily to get accustomed to the injection procedure. 2.3. Feeding behaÕiour experiments (Experiments 1, 2 and 3) Six days after surgery, rats were transferred to individual metabolic cages to allow precise measurement of food intake. No experiments were conducted until the 24-h food intake had stabilised Ž5–6 days.. During this period the rats were handled daily to accustom them to the injection paradigm. All experiments were conducted in the morning Ž0900–1100 h.. 2.4. Experiment 1 (restricted feeding)
2. Materials and methods 2.1. Peptides and nomenclature Due to the presence of the splice forms and putative hydrophobic leader sequence different nomenclature has been used with regard to the CART peptides. Here, we base our numbering on the aminoacids in the human peptide. The C-terminal part of CART probably comprising residue 42–89,was isolated from ovine hypothalamus during the isolation of somatostatin, suggesting, together with the existence of several potential di-basic cleavage sites in the CART peptide, that CART is post-translationally modified in vivo w35x. Recently, several CART peptides have been synthesised using a yeast expression system w40x. One of these, the C-terminal CARTŽ42–89. probably corresponds to a naturally occurring form of CART w35x. CARTŽ42–89. was used in all experiments and prepared as previously described w40x. Porcine NPY was obtained from Peninsula. 2.2. Animals and surgery Seventy-two adult male Wistar rats weighing 200–250 g at the time of surgery were used for the experiments. The rats were maintained on a 12:12 light–dark cycle Žlights on 0600 h. in a temperature controlled environment Ž22–248C.
After cannulation, animals were acclimatised in metabolic cages for 5 days before a restricted feeding schedule, with access to food for 3 h in the morning Ž0900–1200 h. was initiated w38x. Experiments were started when 3-h food intake had stabilised Ž5–6 days.. After stabilisation of daily 3-h food intake Ž5–6 days., the experiments were started. On each day of experimentation, rats were randomised into groups receiving i.c.v. injections of either 5 ml vehicle ŽKPBS: 50 mM phosphate buffered saline containing 0.02% potassium. or different doses of recombinant rat CARTŽ42–89. dissolved in 5 ml vehicle. Animals were injected immediately prior to the feeding session and food and water intake was measured every 30 min for 3 h. Animals were used in three consecutive experiments each separated by three days. 2.5. Experiment 2 (NPY-induced feeding) A separate group of cannulated rats were accustomed to the metabolic cage with food and water ad libitum as described above. Food was removed from the cages at 0800 h and animals injected at 0900 h. All animals received two 5 ml i.c.v. injections separated by 15 min. One group received vehicle followed by vehicle, another vehicle followed by 5 mg NPY and the last CARTŽ42–89. Ž0.5 or 1.0 mg. followed by NPY Ž5 mg.. Food intake was measured every 30 min for 3 h. Only two sets of experi-
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ments were conducted on the same rats each separated by three days. 2.6. Experiment 3 (oÕernight feeding) Another group of cannulated rats were accustomed to the metabolic cage and feeding and drinking ad lib as described above. Animals were injected with either vehicle or 0.5 mg CART immediately prior to lights out Žlights out at 0600 h.. Food intake was measured after 1 and 2 h Žthe use of a nightscope avoided light disturbing the animals. and in the morning immediately after lights on Ž12 h after the injection.. 2.7. Experiment 4 (c-Fos induction) Intracerebroventricularly cannulated rats were kept in individual rodent cages with free access to food and water. Rats were handled daily in the morning. On the day of the study, rats were injected i.c.v. with either 5 ml vehicle Ž n s 5. or 0.5 mg CARTŽ42–89. in 5 ml vehicle Ž n s 19. and the food removed from the cage. The animals injected with CARTŽ42–89. were sacrificed either 60 Ž n s 7., 120 Ž n s 7. or 240 Ž n s 5. min after the injection. The vehicle group was sacrificed 120 min after the injection. At the designated time-point following the injection, the animals were anaesthetised with tribromethanol Ž50 mgrkg. and perfused transcardially, first with heparinised Ž15.000 IUrl. KPBS, followed by Stephanini fixative Ž2% paraformaldehyde in KPBS containing picric acid, pH: 7.4. for 10 min. The brains were removed and post-fixed overnight in the same fixative then transferred for two days to a 30% sucrose–KPBS solution for cryoprotection. One-in-six series of 40 mm thick frontal sections were cut on a freezing microtome and collected in KPBS.
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KBPS and 10 min in a 0.05 M Tris–HCl buffer ŽpH 7.6.. Finally, the sections were developed in a solution containing 0.125% of the chromogen diaminobenzidine ŽDAB. and 0.01% H 2 O 2 in 0.05 M Tris- -HCl buffer. Sections were rinsed 2 = 10 min in distilled water before being mounted and coverslipped in Depex. Counting of c-Fos positive cell nuclei was performed unilaterally on single non-counterstained sections. All areas were counted at the same level in each individual animal making direct comparison between individual animals possible without counting all the c-Fos-ir cells in a given area. Counting was performed using a computerised image analysis system ŽImage 1.60 b, courtesy of NIH, Bethesda, MD., and the c-Fos cells were counted using a macro designed for this specific purpose. All images were acquired in the same session, and the validity of the computerised counts was tested by comparisons to manual counts of the same sectionsrareas. Photographs were taken at a Zeiss Axiophot microscope and negatives scanned into a computer using a Polaroid w slidescanner. Image editing software ŽAdobe Photoshop w . was used to combine photographs into plates and figures were printed on a Sony w dye sublimation printer. 2.9. Statistics Statistical evaluation of the experiments used two-way analysis of variance ŽANOVA. followed by Fisher’s posthoc analysis. Values Žfood intake in grams, number of c-Fos positive cells. are expressed as mean " S.E.M. P values - 0.05 were considered significant.
3. Results
2.8. Immunohistochemistry
3.1. Experiment 1 (restricted feeding)
All reactions were carried out on free-floating sections. Initially sections were washed 3 = 10 min in KPBS followed by incubation for 10 min in 1% H 2 O 2 in KPBS to block endogenous peroxidase activity. After 20 min in 5% swine serum diluted in BST ŽKPBS containing 1% bovine serum albumin ŽBSA. and 0.3% Triton-X-100 ŽTX.. the sections were incubated overnight at 58 with a c-Fos antibody diluted 1:1000 in BST. The c-Fos antibody has been described previously w45x. The sections were next washed for 3 = 10 min in KPBS containing 0.25% BSA and 0.1% TX ŽKPBS-BT. followed by 60 min of incubation at room temperature in a biotinylated swine anti-rabbit antibody ŽaE353, Dakopatts, Copenhagen. diluted 1:600 in BST. The sections were then rinsed 3 = 10 min in KPBS-BT, incubated for 60 min in an avidin–biotin–peroxidase complex ŽVector Elite Kite. diluted 1:250 in KPBS-BT and rinsed 10 min in KPBS-BT, 10 min in
Initially, we aimed at determining the lowest effective anorectic dose of CARTŽ42–89.. Doses ranging from 0.1 mg to 2.0 mg were injected into the lateral ventricle of rats kept on a restricted feeding scheme and food and water intake was subsequently monitored. In keeping with earlier studies, rats subjected to this feeding regimen showed an initial drop in bodyweight followed by a slower weight gain than free-feeding controls Žnot shown. w38,39x. Central administration of CARTŽ42–89. dose dependently inhibited food intake. The highest dose of CARTŽ42–89 significantly inhibited food intake throughout the observation period when compared to control rats, whereas the lowest dose of CART had no effect on food intake ŽFig. 1.. The lowest effective dose of CART with a clear effect on food intake was 0.5 mg Ž P - 0.05 at all time points except 180 min. ŽFig. 1.. Fig. 2 shows the cumulative food intake during the first 60 min as a function of CART dose
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Fig. 1. Effect of i.c.v. injections of either 5 ml vehicle or different doses of recombinant CARTŽ42–89. in 5 ml vehicle on cumulative 3-h food intake in food restricted rats. Vehicle Ž n s 12.; CART 0.1 mg Ž n s 8.; CART 0.5 mg Ž n s 15.; CART 2.0 mg Ž n s 13.. Data are means" S.E.M. Asterisks indicate significant differences between CART and vehicle group and † indicate significant differences between CART 0.5 mg and CART 2.0 mg Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
and demonstrates a dose-dependent inhibition of food intake by CART.
In our previous studies we observed altered locomotor behaviour in response to i.c.v. injections of CARTŽ42–89.
Fig. 2. CARTŽ42–89. dose dependently decrease food intake in rats. Cumulative food intake during the first 60 min after i.c.v. CART injection in food restricted rats. Vehicle Ž n s 12.; CART 0.1 mg Ž n s 8.; CART 0.25 mg Ž n s 12.; CART 0.5 mg Ž n s 15.; CART 1.0 mg Ž n s 7.; CART 2.0 mg Ž n s 13.. Data are means" S.E.M. Asterisks indicate significant differences between CART and vehicle groups, † indicate significant differences between CART Ž0.25 mg. and CART Ž0.5, 1.0 and 2.0 mg. and †† indicate significant differences between CART Ž0.5 mg. and CART Ž2.0 mg. groups Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
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Fig. 3. Central administration of CART inhibits NPY-induced food intake in satiated rats. Both doses of CART significantly reduced food intake induced by prior administration of 5 mg NPY throughout the 3-h observation period. The difference between 0.5 mg and 1.0 mg was non-significant at all time points. Vehicleq vehicle Ž n s 5.; vehicleq NPY Ž n s 8.; CART0.5 mg q NPY Ž n s 8.; CART 1.0 mg q NPY Ž n s 5.. Data are means" S.E.M. Asterisks indicate significant differences between CART and vehicle groups, † indicate significant differences between CARTq NPY and vehicleq NPY groups. Vehicleq vehicle was significantly different from all other groups at all time points Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
Fig. 4. Effect of 0.5 m CART i.c.v. on nighttime feeding. Freely fed rats were injected immediately prior to lights out ŽTime 0. and food intake was measured at 1, 2 and 12 h after the injection of either 5 ml vehicle or 0.5 mg CARTŽ42–89. in 5 ml vehicle. Vehicle Ž n s 9.; CART 0.5 mg Ž n s 11.. Data are means " S.E.M. Asterisks indicate significant differences between CART and vehicle group Žsignificance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis..
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Fig. 5. Photomicrographs of c-Fos like immunoreactivity in selected areas of one animal that received 0.5 mg CARTŽ42–89. i.c.v. Žleft columns; A, C, E, G, I, K, M. and one control animal injected with vehicle Žright columns; B, D, F, H, J, L, N. i.c.v. Both rats were sacrificed at 120 min following the injection. A and B: the supraoptic nucleus; C and D: the paraventricular nucleus of the hypothalamus; E and F: the arcuate nucleus and the ventromedial nucleus; G and H: the posterior part of the dorsomedial nucleus; I and J: the central nucleus of the amygdala; K and L: the parabrachial nucleus; M and N: the area postrema and the nucleus of the solitary tract.
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Fig. 5 Žcontinued..
w16x. These side-effects were also observed in the present study, but only at the highest dose of CARTŽ42–89. Ž2 mg.. 3.2. Experiment 2 (NPY-induced feeding) Fig. 3 shows the results from this experiment in which the ability of CART to inhibit NPY-induced food intake was tested. Measurements were started at the time of the second injection Ž t 0 .. In rats injected with NPY, food intake increased over the first 90 min and this increase was significantly suppressed by both 0.5 mg and 1.0 mg of CART. Apparently, 1.0 mg of CART suppressed food intake more than 0.5 mg, but this difference failed to reach statistical significance. The suppression of NPY induced feeding was evident throughout the observation period for
both doses of CART tested Ž t 180 : 7.66 " 0.70 Žvehicleq NPY.; 5.35 " 0.69 Ž0.5 mg CARTq NPY.; 3.90 " 0.62 Ž1.0 mg CARTq NPY... 3.3. Experiment 3 (oÕernight feeding) In this experiment, the ability of CART to inhibit normal nighttime feeding in freely fed animals was tested. Measurements were started at the time of injection Žimmediately before the dark period in the animal care facility. and food intake measured at 1 Ž t 1 ., 2 Ž t 2 . and 12 Ž t 12 . h. Fig. 4 shows the results from this experiment, and demonstrates that CART significantly inhibited food intake at t 12 Ž t 12 : 18.29 " 0.55 Žvehicle.; 13.18 " 1.08 Ž0.5 mg CART., but not at t 1 or t 2 Žsee Fig. 4..
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Table 1 Number of c-Fos positive cell nuclei in selected brain areas Žmean " S.E.M..
SON PVN Arc CeAl DMHp VMH LPB AP NTS
Saline, 120 min Ž n s 4.
0.5 mg CART, 60 min Ž n s 6.
0.5 mg CART, 120 min Ž n s 7.
0.5 mg CART, 240 min Ž n s 5.
0.5 " 0.3 8.3 " 1.9 3.5 " 0.9 21.3 " 7.6 0.75 " 0.25 19.3 " 2.5 3.2 " 0.25 3.0 " 2.7 1.25 " 0.3
16.0 " 5.2 186.8 " 19.5) 36.0 " 5.9) 50.1 " 15.1 19.3 " 6.0) 26.0 " 5.6 45.7 " 5.1) 3.3 " 2.3 36.3 " 8.9)
26.9 " 5.0) 195.7 " 21.8) 33.3 " 6.2) 89.9 " 10.1)† 45.6 " 7.4) 28.6 " 7.6 46.6 " 4.5) 4.5 " 1.8 70.3 " 5.8)†
29.4 " 11.2) 194.6 " 19.5) 34.2 " 6.3) 39.2 " 5.3 32.8 " 5.6) 24.8 " 3.6 31.8 " 3.8) 4.6 " 2.0 48.8 " 6.8)
Asterisks indicate significant differences between CART and vehicle groups and † indicate significant differences between CART, 120 min and CART 60, 240 min. Significance level P - 0.05 determined by ANOVA followed by Fisher post-hoc analysis. Abbreviations: SON s supraoptic nucleus; PVN s paraventricular nucleus of the hypothalamus; Arc s arcuate nucleus; CeAl s the central nucleus of the amygdala, lateral part; DMHps the posterior part of the dorsomedial nucleus of the hypothalamus; VMHs ventromedial nucleus of the hypothalamus; LPB s lateral parabrachial nucleus; AP s area postrema; NTS s nucleus of the solitary tract.
3.4. Experiment 4 (c-Fos induction) In comparison to vehicle, i.c.v. administration of 0.5 mg CARTŽ42–89. resulted in induction of c-Fos like immunoreactivity in several distinct regions of the fore- and hindbrain Žsee Fig. 5 and Table 1.. The time course of the emerging pattern and number of c-Fos-ir cells was studied by examining sections from animals sacrificed 60, 120 and 240 min following CART administration. Time dependent c-Fos expression was observed only in the central nucleus of the amygdala and in the NTS Žsee Table 1.. In Fig. 5, the distribution of CART-induced c-Fos immunoreactive cells is shown from areas of interest. In the hypothalamus, a high number of c-Fos immunoreactive Ž-ir. cells was observed in the paraventricular nucleus ŽPVN. and in the posterior part of the dorsomedial nucleus ŽDMHp.. In the central part of the PVN, the majority of c-Fos-ir cells were located in the dorsal aspect of the medial parvocellular subdivision of the nucleus. Few c-Fos-ir cells were seen in the perimeter of the posterior magnocellular subnucleus, while the central core of the posterior magnocellular subnucleus was virtually devoid of c-Fos-ir nuclei Žsee Fig. 5.. The majority of c-Fos positive elements in the PVN were observed in the area where most of the CRH containing neurones are located ŽFig. 5.. A lower number of c-Fos positive nuclei was found in the arcuate nucleus ŽArc. and supraoptic nucleus ŽSON.. In the SON the c-Fos positive nuclei were distributed in the dorsolateral portion of the nucleus, an area corresponding to the region where the oxytocinergic neurones are located. CART peptide also induced c-Fos expression in the lateral part of the bed nucleus of the stria terminalis ŽBSTL. and in the central nucleus of the amygdala Žlateral subnucleus, CeAl.. In the brainstem c-Fos-positive nuclei were found in the lateral parabrachial nucleus ŽLPB. Ždorsal and central subnuclei., and throughout the rostrocaudal extent of the nucleus of the solitary tract ŽNTS.. Quantification of c-Fos-ir cells in
the NTS was carried out in sections taken at the level of the area postrema. At this level, c-Fos-ir cells were concentrated in the medial subnuclei of the NTS and to a lesser degree in the subpostreme area. In contrast, both the area postrema and the dorsal motor nucleus of the vagus were virtually devoid of c-Fos-ir cells ŽFig. 5..
4. Discussion We have confirmed and extended our previous observations that central administration of recombinant CARTŽ42–89. dose-dependently inhibits food intake in food restricted rats as well as in rats subjected to NPY-induced food intake w16x. Also, we have demonstrated, that i.c.v. CARTŽ42–89. at a minimal effective dose of 0.5 mg is capable of inhibiting overnight food intake in free feeding animals. This dose of CART induces c-Fos expression in several brain regions believed to be involved in the central control of food intake and metabolism. At present, it is uncertain whether CART mediates its actions via one or several receptors and possible interactions with various second messenger systems remain speculative. Therefore, c-Fos immunohistochemistry remains a versatile method to unravel the circuits influenced by this novel anorectic peptide. Central administration of 0.5 mg CART also effectively suppressed night time feeding suggesting that CART belongs to the class of intermediaterlong term regulators of food intake. However, longer observation periods Žto examine potential rebound effects. and chronic administration of CART peptide is necessary in order to determine the time course and potential long-term body regulatory effects of CART peptide. This is in keeping with our previous observation that central administration of antiCART serum elevates the amount of food eaten during a night time feeding session w16x. The lowest effective dose
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of CART Ž0.5 mg. inhibiting food intake was used in the c-Fos experiments. We have previously observed that although 1 and 2 mg doses of CART i.c.v. causes some movement-associated tremor, these doses have no effect on spontaneous locomotor activity w16x. Thus, with the low dose of CART used in the present study, it is unlikely that c-Fos-ir occurred in central areas as a consequence of altered locomotor behaviour, although it is impossible to assess whether some of the c-Fos-ir emerged in areas controlling motor activities upstream to cortical projection neurones. Expression of c-Fos and other immediate early genes are primarily induced by cellular excitation w32x. As CART serves as an inhibitor of food intake, and therefore possibly as an inhibitor of neuronal activity, it is possible that neurones sensitive to CART Ži.e., containing the as yet unidentified CART receptor. remain electrophysiologically silent and, consequently, do not express c-Fos. However, if the main action of CART peptide is inhibitory, c-Fos induction probably still reflects specific but indirect action Ži.e., removal of an inhibitory input. upon neuronal circuits responsive for mediating the anorectic effects of CART. Expression of c-Fos in hypothalamic nuclei is largely distributed as CART immunoreactive fibers w15x although the density of CART innervation may not necessarily reflect the number of Fos-positive nuclei. This overlap suggest that CART exerts its effects locally, either directly or indirectly via adjacent interneurons, but it should be noted that CART induced c-Fos expression was absent in many areas that have been shown to contain CART-immunopositive fibers w14x. Notably, no c-Fos induction was observed in the induseum griseum, hippocampus, septal nuclei, or nucleus accumbens, areas that have all been found to contain moderate to high densities of CART immunopositive fibers. Also no c-Fos induction was observed in the striatum. Within the hypothalamus, the most prominent induction of c-Fos was observed in the PVN ŽFig. 5.. This nucleus is a key component in the neural circuitry involved in the regulation of feeding behaviour. Lesions of the PVN have profound effects on food intake and body weight regulation w8x and several neurotransmitters with orexigenic as well as anorectic properties have been shown to exert their influence on feeding behaviour most potently when injected directly into this nucleus. For example, neuropeptide Y ŽNPY., galanin and noradrenaline all stimulate feeding when injected into the PVN w17,21,36x, whereas GLP-1 and CRF inhibits food intake when injected here w25,31x. CART induced c-Fos expression in the PVN was most prominent in the medial parvocellular parts of the nucleus. This pattern of activation is very similar to that observed in studies of c-Fos expression following central administration of other anorectic peptides Že.g., GLP-1 w20x, leptin w43x and CCK w30x.. In the magnocellular subnuclei of the PVN as well as the SON, CART induced fos-expression overlapped with the localisation of oxytocinergic ŽOT. neurons, suggesting
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that CART activates these neurones. A number of centrally acting anorectic agents including CCK and GLP-1 trigger c-Fos expression in OT neurones w20,30x. However, it remains to be established whether central application of CART has any effect upon circulating levels of OT. The arcuate nucleus houses neurons containing a variety of peptides, several of which are known to affect feeding behaviour w2x. The effect of NPY on feeding, for example, is believed to be mediated in large by an arcuato-PVN NPY-ergic connection w1,28x. The orexigenic property of NPY is emphasised by the ability of repeated injections of NPY to cause obesity w12,28x. It is generally accepted that NPY acts on neurons in the PVN area to initiate feeding behaviour, but the nature of these NPY-sensitive feedinginducing neurons needs to be determined. The PVN receives dense projections from the different parts of the DMH including the posterior region where CART-induced c-Fos expression predominates w24x. Similarities between arcuate and DMH neurones exist. Both nuclei contain NPYergic neurones projecting to the PVN w1x and central injection of NPY elicits c-Fos in both the arcuate and the DMH w22x. However, selective destruction of either the arcuate or DMH has opposite effects on energy balance. Destruction of the arcuate nucleus by neonatal monosodium glutamate produce obesity and hyperglycaemia w26,29x while selective DMH lesions lower the body weight set point w3x. Also NPY mRNA synthesis in the arcuate and DMH is differentially regulated, with high levels of expression in the arcuate in response to adrenal steroids w19x, while NPY mRNA levels in the DMH is selectively increased under lactation and in Agouti mice w13,23,33x. The BSTL and the CeA constitute part of a network of autonomic nuclei that are of importance in regulating various autonomic functions w34x. The presence of CART induced c-Fos expression in both of these nuclei suggests that several autonomic functions may be regulated by CART. Both the CeA and BSTL are reciprocally connected with the PVN, the midbrain central grey, the parabrachial nucleus, the NTS, the dorsal vagal nucleus and the ventrolateral medulla w10,11x. Interestingly, all of these areas contain c-Fos-ir nuclei triggered by central CART application supporting a rather ubiquitous action on this functional continuum. CRH containing neurons of the CeA are main contributors to the descending pathways that innervate the aforementioned target areas w27x. The CeA is centrally involved in regulating responses associated in learned aversion w9,44x and is also involved in the control of food and energy balance. Thus, it is possible that CART influences food intake via activation of descending CRH neurones of the CeA and BSTL which interferes with PB and NTS neurones. An alternative explanation would be that ascending pathways from the brain stem regions including the NTS and PB activate CeA neurones. In the brainstem, a high number of c-Fos immunoreactive cells were seen in the NTS. The NTS is a major
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sensory relay nucleus receiving afferent input from the 7th, 9th and 10th cranial nerve and is reciprocally connected with numerous brainstem and forebrain structures. Among these the PVN, lateral parabrachial nucleus and central nucleus of the amygdala, nuclei also express c-Fos following CART i.c.v. Interestingly i.c.v. CARTŽ42–89. had no effect on c-Fos expression in the area postrema suggesting that the chemosensitive neurones here are not excited by CART and indicating that the induction of c-Fos within the NTS is not a consequence of non-specific chemo-activation of AP-neurones connected to the NTS. One likely target for CART in the NTS could be the GLP-1 containing cells. These neurons project to the hypothalamus Žmost notably the PVN. w20x and have been proposed to constitute the endogenous system mediating GLP-1 induced satiety w38,39,42x.
5. Conclusion We have shown that centrally administered recombinant CARTŽ42–89. potently inhibits food intake in both fasted rats and satiated rats induced to eat with NPY. Furthermore CART peptide induces c-Fos expression in hypothalamic and brainstem areas that are believed to be involved in the regulation of feeding behaviour, metabolism and other autonomic functions, suggesting that CART inhibits food intake by activating neurones in one or more of these sites. Thus, the present study support—together with our previous observations—a role for CART as yet another player within the complex neuronal circuitry linking hypothalamus and brainstem regulating food intake and metabolism.
Acknowledgements This study was supported by grants from the Danish Medical Research Council Ž9701798., The Danish Diabetes Foundation, The Novo Nordisk Foundation, The Danish Research Foundation to the Biotechnology Centre for Cellular Communication. N.V. is supported by a research grant from the P. Carl Petersen Foundation. The authors wish to thank G. Hahn for excellent photographic assistance.
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