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Cocaine- and Amphetamine-Regulated Transcript (CART) Is Colocalized with the Orexigenic Neuropeptide Y and Agouti-Related Protein and Absent from the Anorexigenic -Melanocyte-Stimulating Hormone Neurons in the Infundibular Nucleus of the Human Hypothalamus

Department of Endocrine Neurobiology (J.M., G.W., Z.L., C.F.), Institute of Experimental Medicine, Hungarian Academy of Sciences, 1083 Budapest, Hungary; Tupper Research Institute and Department of Medicine (R.M.L., C.F.), Division of Endocrinology, Diabetes, Metabolism, and Molecular Medicine, New England Medical Center, Boston, Massachusetts 02111; Department of Neuroscience (R.M.L.), Tufts University School of Medicine, Boston, Massachusetts 02111; Department of Forensic Medicine (É.K.), Semmelweis University, 1085 Budapest, Hungary; and Department of Neuroscience (Z.L.), Faculty of Information Technology, Pázmány Péter Catholic University, 1083 Budapest, Hungary

Address all correspondence and requests for reprints to: Csaba Fekete M.D., Ph.D., Department of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, 43 Szigony Street, 1083 Budapest, Hungary. E-mail: feketecs{at}koki.hu.

	Abstract

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Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered anorexigenic peptide. In rodents, CART inhibits food intake and is expressed in the anorexigenic -MSH- but not in the orexigenic neuropeptide Y (NPY)- and agouti-related protein (AGRP)-synthesizing neurons of the arcuate nucleus. To understand whether CART is similarly expressed in feeding-related neuronal groups of the human hypothalamus as observed in rodents, colocalization of CART with -MSH, NPY, AGRP, and melanin-concentrating hormone was studied using double-labeling immunofluorescence and confocal microscopy on human hypothalamic tissues obtained at autopsy. Unlike in rodents, we observed that CART is absent from the perikarya and axons of -MSH-synthesizing neurons, but expressed in approximately one third of NPY/AGRP neurons in the human infundibular nucleus. In the lateral hypothalamus of the humans, colocalization of CART and melanin-concentrating hormone was observed, similar to that described in rodents. The anatomy of CART-containing neurons in the human infundibular nucleus differs markedly from that observed in the rodent brain, raising the question whether the colocalization of CART with orexigenic NPY and AGRP neurons is associated with an orexigenic role of CART in the human brain.

	Introduction

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COCAINE- AND AMPHETAMINE-regulated transcript (CART) was first identified as a transcript up-regulated in the striatum by both cocaine and amphetamine administration (1). Later, CART was described as the third most abundant transcript in the rodent hypothalamus (2) and implicated in the regulation of several hypothalamic functions including the control of energy homeostasis (3, 4, 5). Central administration of CART decreases food intake and prevents the orexigenic effects of neuropeptide Y (3, 4). In addition, central blockade of CART by administration of CART antiserum results in hyperphagia (3), and CART knockout animals have increased weight (6, 7). Therefore, CART is considered as an anorexigenic peptide. Indeed, in rodents, CART is expressed in the anorexigenic, -MSH-synthesizing cell population of the arcuate nucleus, and it is critical for the maintenance of energy homeostasis (8, 9). In contrast, CART is absent from the orexigenic neurons of the arcuate nucleus that express neuropeptide Y (NPY) and agouti-related protein (AGRP) (9, 10). Interestingly, however, CART is also present in a population of melanin-concentrating hormone (MCH)-synthesizing neurons of the lateral hypothalamus (10, 11). Because MCH has a potent stimulatory effect on food intake (12), it is currently not understood why orexigenic and anorexigenic peptides are colocalized in this neuronal group.

CART is also abundantly expressed in the human hypothalamus (11), but little is known about the relationship of CART to feeding-related neuronal groups in the human hypothalamus and whether CART also plays an anorexigenic role in humans. Therefore, using double-labeling immunofluorescence, we studied whether CART is expressed in the anorexigenic -MSH- or orexigenic NPY/AGRP-synthesizing neurons of the human infundibular nucleus, the analog structure of rodent arcuate nucleus. In addition, we also determined whether CART is colocalized with MCH in neurons of the lateral hypothalamus and whether CART-containing fibers innervate any of these three feeding-related neuronal groups in the human hypothalamus.

	Materials and Methods

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Preparation of human tissue samples
Diencephalic samples of four adult human individuals with no history of neurological or endocrinological disorders were obtained at autopsy (Table 1). Tissue samples were taken within 6–24 h after death with the permission and in accordance with regulations of the Regional Committee of Science and Research Ethics, Budapest, Hungary (permission number TUKEB 49/1999). The diencephalic blocks were fixed in a mixture of 4% acrolein and 2% paraformaldehyde for 48 h at 4 C and then cryoprotected in 30% sucrose and frozen on dry ice. Serial 30-µm-thick coronal sections were cut parallel to the lamina terminalis with a freezing microtome (Leica Microsystem Nussloch GmbH, Nussloch, Germany) and stored in a freezing solution (30% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) at –20 C until used.

Double-labeling immunofluorescence studies in the human hypothalamus
Double-labeling fluorescent immunocytochemistry was performed to study the colocalization of CART with -MSH-, NPY-, AGRP-, and MCH-immunoreactive (IR) elements in the infundibular nucleus and the CART innervation of the three feeding-related neuronal groups. Sections were pretreated with 1% sodium borohydride in distilled water for 30 min. After rinsing in PBS (pH 7.4), the sections were treated in 0.5% hydrogen peroxide and 0.5% Triton X-100 in PBS for 15 min. Then they were immersed in 1% sodium acetate for 1 min, treated in graded dilutions of acetone (50, 70, 90, 100, and 90%) for 5 min each, washed in 70% ethanol, and treated with 0.3% Sudan black in 70% ethanol for 30 min to reduce autofluorescence (13). After differentiation in 70% ethanol, sections were washed in PBS, pretreated with 2% normal horse serum in PBS, and then incubated in the following mixtures: 1) murine monoclonal antibody against CART 1:9000 (gift from Dr. Jes Thorn Clausen, Novo Nordisk A/S, Bagsvaerd, Denmark) and sheep anti--MSH serum 1:6000 (gift from Jeffrey B. Tatro, Tufts-New England Medical Center, Boston, MA), 2) murine monoclonal antibody against CART 1:9000 and sheep anti-NPY serum 1:16,000 (gift from István Merchenthaler, University of Maryland, Baltimore, MD), 3) murine monoclonal antibody against CART 1:9000 and rabbit anti-AGRP serum 1:1500 (Phoenix Pharmaceuticals Inc., Burlingame, CA), and 4) murine monoclonal antibody against CART 1:9000 and rabbit anti-MCH 1:6000 (gift from Eleftheria Maratos-Flier, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA) for 48 h at 4 C. After additional washes in PBS, tissues were immersed overnight in one of the following cocktails: 1) Cy3-conjugated donkey-antimouse IgG for the detection of CART-IR elements (1:200; Jackson Immunoresearch, West Grove, PA) and fluorescein isothiocyanate (FITC)-conjugated donkey-antisheep IgG for detection of -MSH and NPY 2), Cy3-conjugated donkey-antimouse IgG to detect CART and FITC-conjugated donkey-antirabbit IgG for detection of AGRP and MCH (1:100; Jackson Immunoresearch). The sections were mounted and then coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA).

Specificity of primary antibodies has been described elsewhere (13, 14, 15). Omitting any of the primary antibodies from the double-label immunostainings resulted in loss of the immunostaining specific for the omitted primary antibody, demonstrating the lack of the cross-reactivity in the double-labeling immunofluorescence technique.

Image analysis
Double-labeled preparations were analyzed using a confocal laser microscope (Bio-Rad Laboratories, Hemel Hempstead, UK). At least five sections were analyzed from each brain, from different rostrocaudal levels of the infundibular nucleus, hypothalamic paraventricular nucleus (PVN), and lateral hypothalamus. For quantification, 296 x 296-µm areas of the entire infundibular nucleus, PVN, or lateral hypothalamus were recorded with a x40 oil immersion objective. For illustrations, the images were taken with x40 or x60 oil immersion objectives. The fluorochromes were detected with the following laser lines and filters: 488 nm for FITC and 543 nm for Cy3 and dichroic/emission filters 560 nm/500–530 nm for FITC and 560–625 nm for Cy3. Pinhole sizes were set to obtain optical slices less than 0.7 µm thick, and the series of optical slices were recorded with a 0.6-µm Z step. The series of optical sections were merged and displayed with Laser Vox software and an IBM-compatible personal computer. Colocalization of CART and the other peptides were examined in perikarya and fibers. All single- and double-labeled perikarya were counted on five sections from different rostrocaudal levels from each brain to determine the degree of colocalization between CART and the other examined neuropeptides (-MSH, NPY, AGRP, and MCH).

	Results

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In the infundibular nucleus of the human hypothalamus, numerous -MSH-IR neurons and relatively fewer CART-IR neurons were found in a dense network of axons containing -MSH or CART immunoreactivity (Fig. 1, A and B). No colocalization of these peptides was observed in neurons of the infundibular nucleus (Fig. 1, A and B). In addition, only single-labeled -MSH and CART axons were observed in both the infundibular nucleus and the PVN (Fig. 1, A–C). In contrast, in the rat brain processed similar to the human hypothalamic samples, the majority of -MSH-IR neurons in the arcuate nucleus and -MSH-IR axons in the arcuate and PVN also contained CART immunoreactivity (Fig. 1, D and E).

View larger version (90K): [in this window] [in a new window]   FIG. 1. Relationship of CART-IR (red) and -MSH-IR (green) neuronal elements in the human hypothalamus. Low (A) and medium (B) power micrographs illustrate the distribution of CART- and -MSH-IR elements in the human infundibular nucleus. CART is present both in the perikarya (arrows, A) and axons in the infundibular nucleus. There is no apparent colocalization of the two peptides. The PVN is densely innervated by both CART- and -MSH-IR fibers, but the two peptides are observed in separate populations of axons (C). Although -MSH immunoreactivity is present only in fibers in the PVN, CART immunoreactivity can be observed in both perikarya and axons (C). In contrast, in the rat arcuate nucleus, CART is present in nearly all -MSH-IR perikarya and axons (D; yellow profiles). In addition, all -MSH-IR axons contain CART in the PVN of rats (E). High-magnification micrographs illustrate the juxtaposition of CART-IR terminals to -MSH-IR neurons in the human infundibular nucleus (arrows) (F–I). Unlike rodents, no CART/-MSH colocalization was observed in the infundibular nucleus in humans, but numerous CART-IR boutons were seen in juxtaposition to -MSH perikarya (arrows). F1–F3 represent the same neuron in different confocal plans. Scale bars, 40 µm (A) and 20 µm (B–F).

View larger version (89K): [in this window] [in a new window]   FIG. 2. Relationship of CART-IR (red) and NPY-IR (green; A–C, G, I, and J) and CART-IR (red) and AGRP-IR (green; D–F, H, and K–M) elements in the human hypothalamus. A, Colocalization of CART and NPY in neurons of the infundibular nucleus. Cells containing both CART and NPY appear in yellow (arrows). Single-labeled CART (arrowhead) and single-labeled NPY neurons (open arrowhead) can also be seen in the infundibular nucleus. CART-IR boutons are present in juxtaposition to both NPY/CART (B) and single-labeled NPY neurons (C; arrows). CART and NPY are also colocalized in axons in the infundibular nucleus (G; arrows) and in the PVN (I and J; arrows). D, Colocalization of CART and AGRP in the perikarya of the infundibular nucleus. Double-labeled AGRP/CART cells (arrows), single-labeled CART (arrowhead), and single-labeled AGRP neurons (open arrowhead) are seen in the human infundibular nucleus. Both AGRP (E) and AGRP/CART neurons (F) are surrounded by CART-IR varicosities in the infundibular nucleus (arrows). Colocalization of CART and AGRP is also detected in axons within the infundibular nucleus (H; arrows) and the PVN (K–M; arrows). Scale bars, 20 µm; scale bar on E corresponds to E and F; scale bar on G corresponds to G and H; scale bar on I corresponds to I–M.

View larger version (45K): [in this window] [in a new window]   FIG. 3. Relationship of CART-IR (red) and MCH-IR (green) elements in the human hypothalamus. A, Low-magnification micrograph illustrates that CART immunoreactivity is present in a population of MCH-IR neurons (arrows) in the perifornical area; B, medium-power micrograph shows colocalization of CART and MCH in a neuron from the perifornical region; C, a portion of CART-IR axons in the infundibular nucleus contains MCH immunoreactivity (arrows) indicating the lateral hypothalamic origin of these fibers; D–F, high-magnification micrographs illustrate the juxtaposition of CART-IR axon varicosities (arrows) to the perikarya of CART/MCH neurons (D and F) and a single-labeled MCH cell (E) in the perifornical area. Scale bars, 40 µm; scale bar on D corresponds to D–F.

All studied parameters were highly similar in the four studied cases independently from the cause of death.

	Discussion

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The hypothalamic arcuate/infundibular nucleus plays a pivotal role in the regulation of energy homeostasis (16). Neurons of this nucleus are regulated by peripheral feeding-related hormones such as leptin, insulin, and ghrelin and mediate the effects of these peripheral signals to other brain centers regulating food intake and energy metabolism (16). In the rodent arcuate nucleus, CART is expressed in the anorexigenic cell population that also expresses -MSH (8, 9, 11). According to the anorexigenic role of CART, fasting inhibits whereas leptin administration stimulates CART synthesis in arcuate nucleus neurons (17, 18).

In the present study, we demonstrate that contrary to the rat, in the human infundibular nucleus, CART and -MSH do not colocalize. Because rapid axonal transport of CART into the axon terminals of -MSH neurons could explain the absence of CART in these neurons, we also determined whether CART and -MSH were present in axons of two brain regions known to be heavily innervated by -MSH fibers of arcuate nucleus origin, the infundibular nucleus itself and the hypothalamic PVN (13). Although CART and -MSH fibers densely innervated both regions, the two peptides were present in separate populations of axons without any apparent colocalization. To exclude the possibility that the discrepancies between the rodent and human data are due to different processing of the brain tissues, we performed double-labeling immunocytochemistry on rat hypothalamic tissues processed similarly to the human diencephalic blocks. In rat hypothalamic samples that were immersion fixed 6 h after death, the vast majority of the -MSH-IR elements also contained CART immunoreactivity. Together, these data suggest that in contrast to the rodent brain, the expression of CART in -MSH neurons of the infundibular nucleus is not necessary to mediate the effects of peripheral satiety signals in the human hypothalamus. Similar observations were made by Grayson and co-workers (19) in the hypothalamus of nonhuman primates.

Although CART expression is not observed in NPY/AGRP neurons in the rodent hypothalamus (20), approximately one third of the NPY/AGRP neurons detected by the immunofluorescent labeling were found to contain CART in the human samples, and colocalization of CART with NPY and AGRP was also observed in axons in the known projection fields of infundibular NPY neurons. Although CART/NPY fibers may originate from both the arcuate nucleus and adrenergic neurons of the brainstem that synthesize both CART and NPY and project to the PVN (10, 14, 21, 22), the exclusive source of CART/AGRP fibers are NPY/AGRP neurons in the infundibular nucleus, because AGRP is synthesized exclusively in the arcuate/infundibular nucleus (23, 24).

The preferential colocalization of CART with orexigenic peptides in the human hypothalamus raises the possibility that contrary to the rodent, CART may play an orexigenic role in the human infundibular nucleus or subserves other functions. The potential orexigenic role of CART is supported by data showing that CART injection to the PVN increases food intake even in rodents (25). Alternatively, infundibular CART neurons may have a role in prolactin regulation. We have previously demonstrated that CART is capable of inhibiting TRH-induced prolactin secretion (26), and Baranowska et al. (27) have shown direct prolactin-inhibiting effects of CART in cell culture. Although we presume that the origin of CART neurons involved in prolactin regulation in the rat derives primarily from the hypothalamic PVN (28), this may not be the case in the human brain.

Nevertheless, there is some physiological evidence that CART may have an anorexigenic role in the human brain despite the observation that CART does not colocalize with -MSH. A point mutation of the CART gene that leads to altered processing and decreased activity of CART peptides is associated with severe obesity in humans (29), suggesting that CART exerts a constant anorexigenic tone in the human brain. Indeed, starvation decreases CART mRNA levels in the arcuate nucleus of monkeys (30), where the -MSH neurons also do not synthesize CART (19), suggesting that infundibular CART is regulated like an anorexigenic peptide in primates.

The coexpression of CART with NPY and AGRP in the human brain is distinctly different from the rodent where these peptides do not colocalize. The explanation for this difference is not clear but by itself, does not exclude the possibility that CART exerts anorexic actions. There are many examples where neurons synthesize substances that exert opposite actions on target neuronal populations. For example, the two, main, excitatory and inhibitory neurotransmitters, glutamate and GABA, respectively, have been shown to colocalize in nearly all neurons of the anteroventral periventricular area (31) and in hippocampal mossy fibers of young animals (32). In addition, both CART and NPY are synthesized by adrenergic neurons of the C1–3 area in the rat brain (10, 14, 21, 22) and contained in adrenergic axons innervating the hypophysiotropic TRH neurons (14, 22). Although CART stimulates TRH gene expression, NPY exerts a potent inhibitory effect on TRH synthesis in the same neurons (33). Presumably, differential regulation of the opposing transmitters may enable the neurons to regulate their postsynaptic targets in a more precise manner.

As noted previously, it is also possible that CART synthesized in NPY neurons of the infundibular nucleus is not involved in the regulation of food intake but rather regulates other neuronal functions. Indeed, fasting has no effect on the NPY expression in the arcuate nucleus of the monkey hypothalamus (30). Because both CART and NPY are known to stimulate the hypothalamic-pituitary-adrenal axis through hypophysiotropic CRH neurons and these paraventricular neurons are innervated by AGRP/NPY fibers (34), it is feasible that the AGRP/NPY/CART neurons of the human infundibular nucleus may be involved in the regulation of the hypothalamic-pituitary-adrenal axis under certain stress conditions.

In contrast to the infundibular nucleus, the colocalization of CART with MCH in the lateral hypothalamus is similar to that observed in the rodent brain (11). Unfortunately, little is known about the role of CART synthesized in this neuronal population. Although it is well known that MCH gene expression is markedly up-regulated by fasting (35) and MCH has a potent orexigenic effect (12), CART gene expression seems to be unaltered by changes in energy homeostasis in the MCH neurons of rodents (36). These data suggest that MCH and CART may be involved in the regulation of different physiological functions executed by the same neurons. Indeed, it has been suggested that CART synthesis in lateral hypothalamic neurons is involved in the regulation of dopaminergic reward pathways (37). Lateral hypothalamic CART neurons are known to project to the ventral tegmental area where CART exerts psychostimulant-like effects through the stimulation of dopaminergic neurons (38, 39, 40).

Dense CART innervation of feeding-related neurons in both the infundibular nucleus and in the lateral hypothalamus suggest that CART, originating from multiple brain sources, may be involved in the control of food intake through the regulation of these neuronal groups.

In summary, in contrast to the rodent brain, CART is synthesized in a population of NPY/AGRP neurons and it is absent from the -MSH-synthesizing neurons in the infundibular nucleus of the human hypothalamus. These data indicate that there are important differences between the hypothalamic feeding regulatory systems of humans and rodents and raise the possibility that CART may play a different role in the regulation of feeding in humans than described in rodents.

	Footnotes

 
This work was supported by the National Science Foundation of Hungary (OTKA T046492, T046574), NKFP 1A/002/2004, the Sixth European Union Research Framework Programme (Contract LSHM-CT-2003-503041).

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 24, 2007

Abbreviations: AGRP, Agouti-related protein; CART, cocaine- and amphetamine-regulated transcript; FITC, fluorescein isothiocyanate; IR, immunoreactive; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; PVN, paraventricular nucleus.

Received March 26, 2007.

Accepted for publication May 16, 2007.

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