help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0732
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Füzesi, T.
Right arrow Articles by Fekete, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Füzesi, T.
Right arrow Articles by Fekete, C.
Endocrinology Vol. 148, No. 11 5442-5450
Copyright © 2007 by The Endocrine Society

Contribution of Noradrenergic and Adrenergic Cell Groups of the Brainstem and Agouti-Related Protein-Synthesizing Neurons of the Arcuate Nucleus to Neuropeptide-Y Innervation of Corticotropin-Releasing Hormone Neurons in Hypothalamic Paraventricular Nucleus of the Rat

Tamás Füzesi, Gábor Wittmann, Zsolt Liposits, Ronald M. Lechan and Csaba Fekete

Department of Endocrine Neurobiology (T.F., G.W., Z.L., C.F.), Institute of Experimental Medicine, Hungarian Academy of Sciences, and Department of Neuroscience (Z.L.), Faculty of Information Technology, Pázmány Péter Catholic University, Budapest 1083, 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, and Department of Neuroscience (R.M.L.), Tufts University School of Medicine, Boston, Massachusetts 02111

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, Budapest, Hungary 1083. E-mail: feketecs{at}koki.hu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN) integrate neuronal and hormonal inputs and serve as a final common pathway to regulate the hypothalamic-pituitary-adrenal axis. One of the neuronal regulators of CRH neurons is neuropeptide Y (NPY) contained in axons that densely innervate CRH neurons. The three main sources of NPY innervation of the PVN are the hypothalamic arcuate nucleus and the noradrenergic and adrenergic neurons of the brainstem. To elucidate the origin of the NPY-immunoreactive (NPY-IR) innervation to hypophysiotropic CRH neurons, quadruple-labeling immunocytochemistry for CRH, NPY, dopamine-β-hydroxylase, and phenylethanolamine-N-methyltransferase was performed. Approximately 63% of NPY-IR varicosities on the surface of CRH neurons were catecholaminergic (22% noradrenergic and 41% adrenergic), and 37% of NPY-IR boutons were noncatecholaminergic. By triple-labeling immunofluorescence detection of NPY, CRH, and agouti-related protein, a marker of NPY axons projecting from the arcuate nucleus, the noncatecholaminergic, NPY-ergic axon population was shown to arise primarily from the arcuate nucleus. When NPY was administered chronically into the cerebral ventricle of fed animals, a dramatic reduction of CRH mRNA was observed in the PVN (NPY vs. control integrated density units, 23.9 ± 2.7 vs. 77.09 ± 15.9). We conclude that approximately two thirds of NPY-IR innervation to hypophysiotropic CRH neurons originates from catecholaminergic neurons of the brainstem, whereas the remaining one third arises from the arcuate nucleus. The catecholaminergic NPY innervation seems to modulate the activation of CRH neurons in association with glucoprivation and infection, whereas the NPY input from the arcuate nucleus may contribute to inhibition of CRH neurons during fasting.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPOPHYSIOTROPIC CRH-SYNTHESIZING neurons located in the paraventricular nucleus of the hypothalamus (PVN) control the hypothalamic-pituitary-adrenal (HPA) axis (1). They integrate humoral and neuronal signals and mediate the effects of these inputs on the pituitary and adrenal glands. Several neurotransmitters and neuropeptides have been proposed to regulate the HPA axis at the level of CRH neurons (2). Neuropeptide Y (NPY) has been detected in axon terminals densely innervating hypophysiotropic CRH neurons (3) and believed to activate the HPA axis through direct effects on CRH gene expression in the hypothalamus (4, 5). The origin of the NPY-immunoreactive (NPY-IR) axons innervating the hypophysiotropic CRH neurons, however, is unknown.

The NPY innervation of the PVN originates from multiple anatomical loci: neurons of the arcuate nucleus (6, 7) that also express agouti-related protein (AGRP) (8) and catecholamine-producing cells of the brainstem (9) that synthesize either adrenaline (C1–C3 cell groups) or noradrenaline (A1, A2, and A6 cell groups). To elucidate the putative involvement of the different NPY-synthesizing cell groups in the innervation of hypophysiotropic CRH neurons, multiple-labeling immunofluorescence was performed on hypothalamic sections of intact and arcuate nucleus-ablated rats using antisera against CRH and NPY in combination either with AGRP or the specific catecholamine-synthesizing enzymes, dopamine-β-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT).

During fasting, NPY neurons are activated in the arcuate nucleus (10), whereas CRH gene expression is inhibited in the PVN (11). To clarify the role of NPY neurons of the arcuate nucleus in the regulation of the hypophysiotropic CRH cells, we studied the effects of chronic, central NPY administration on CRH gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The experiments were carried out on adult male rats (Wistar and Sprague Dawley), weighing 280–320 g, housed under standard environmental conditions (light between 0600 and 1800 h, temperature 22 ± 1 C, rat chow and water ad libitum). All experimental protocols were reviewed and approved by the Animal Welfare Committee at the Institute of Experimental Medicine of the Hungarian Academy of Sciences and Tufts-New England Medical Center.

Monosodium glutamate treatment
To eliminate NPY axons arising from the arcuate nucleus, a chemical lesion of this region was performed by monosodium glutamate (MSG) treatment of six neonatal rats. Neonatal animals were injected sc with a MSG solution (dissolved in water), using a treatment paradigm adapted from Légrádi and Lechan (7). On postnatal d 2 and 4, MSG was administered at a dose of 2 mg/g body weight, and on postnatal d 6, 8, and 10, at a dose of 4 mg/g body weight. Intact animals were treated with the same volume of saline. The animals were studied as adults when they reached a body weight of 280–320 g.

Tissue preparation for immunocytochemistry
Because colchicine treatment is necessary to visualize the perikarya and dendrites of hypophysiotropic CRH neurons and our preliminary studies indicated that a low dose of colchicine (40 µg/animal) does not alter the staining pattern of DBH, PNMT, AGRP, or NPY axons in the PVN, we used colchicine-treated rats for our studies. Three intact animals for the PNMT/DBH/NPY/CRH staining and three MSG-treated rats for NPY/DBH/CRH staining were deeply anesthetized with sodium pentobarbital (35 mg/kg body weight, ip) and injected intracerebroventricularly (icv) with 40 µg colchicine in 2 µl 0.9% saline under stereotaxic control. After 20 h, the animals were perfused transcardially with 20 ml 0.01 M PBS (pH 7.4), followed sequentially by 100 ml 2% paraformaldehyde/4% acrolein in 0.1 M phosphate buffer (pH 7.4) and 50 ml 2% paraformaldehyde in the same buffer. For triple-labeling immunofluorescence using AGRP, NPY, and CRH, three animals were perfused transcardially with 20 ml 0.01 M PBS followed by 150 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were rapidly removed, and blocks containing the hypothalamus were cryoprotected in 30% sucrose in 0.01 M PBS (pH 7.4) overnight at 4 C and snap frozen on dry ice. Serial 30-µm-thick coronal sections through the PVN were cut on a freezing microtome (Leica Microsystems, Wetzlar, Germany), collected in freezing solution (30% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) and stored at –20 C until used.

Quadruple-labeling immunofluorescence for DBH, PNMT, NPY, and CRH
To elucidate the putative contribution of noradrenergic and adrenergic cell groups of the brainstem in the NPY-ergic innervation of hypophysiotropic CRH neurons, quadruple-labeling immunocytochemistry was performed on every third hypothalamic section of animals possessing an intact arcuate nucleus. The sections from each brain were treated with 1% sodium borohydride in distilled water for 30 min and 0.5% Triton X-100/0.5% H2O2 in PBS for 20 min. An additional 10-min incubation in 0.5% Triton X-100 was applied to further improve antibody penetration. To reduce nonspecific antibody binding, the sections were treated with 2% normal horse serum in PBS for 20 min. The sections were then incubated in the following mixture of primary antibodies for 3 d at 4 C: murine monoclonal antibody to DBH (Chemicon, Temecula, CA) at 1:150, guinea pig anti-CRH serum (Peninsula Laboratories Inc., San Carlos, CA) at 1:3500, sheep anti-NPY serum (gift from István Merchenthaler) at 1:8000, and rabbit anti-PNMT serum (a gift from Martha C. Bohn, Northwestern University Medical School, Chicago, IL) at 1:500. After rinses in PBS, the sections were incubated in a mixture of secondary antibodies for 1 d at 4 C. The secondary antibodies were as follows: CY3-conjugated donkey antimouse IgG (Jackson ImmunoResearch, West Grove, PA) at 1:200 dilution, biotinylated donkey anti-guinea pig IgG (Jackson) at 1:250 dilution, CY5-conjugated donkey antisheep IgG (Jackson) at 1:100 dilution, and fluorescein isothiocyanate (FITC)-conjugated donkey antirabbit IgG (Jackson) at 1:50 dilution. The sections were then rinsed in PBS and incubated in 7-amino-4-methyl-coumarin-3-acetic acid (AMCA)-conjugated avidin D (Vector Laboratories, Burlingame, CA), diluted at 1:250 for 1 d at 4 C. Primary and secondary antibodies and AMCA-avidin D were diluted in PBS that contained 2% normal horse serum and 0.2% sodium azide. The sections were mounted onto glass slides and coverslipped with Vectashield (Vector) mounting medium.

Validation of the arcuate nucleus ablation
The effectiveness of the chemical ablation of the arcuate nucleus was assessed by examination of hypothalamic sections from intact and MSG-treated rats (Fig. 1Go). After the same tissue pretreatment, the sections were incubated in sheep anti-NPY serum (gift from István Merchenthaler) at 1:100,000 for 1 d at 4 C. After rinsing in PBS, the sections were incubated first in biotinylated donkey antirabbit IgG (Jackson) diluted to 1:500 and, after additional washes in PBS, in avidin-biotin complex (ABC, 1:1000; Vector) for 1 h. The peroxidase reaction was developed in 0.05 M Tris buffer (pH 7.6) containing 0.025% diaminobenzidine and 0.0036% H2O2. Animals with more than three to five NPY cells per section were excluded from additional studies. The effectiveness of the ablation was also confirmed by Nissl staining.


Figure 1
View larger version (116K):
[in this window]
[in a new window]

  		FIG. 1. Effect of chemical ablation of the arcuate nucleus on NPY-IR neurons of the arcuate nucleus. A, Distribution of NPY-IR neurons in the arcuate nucleus of control rats; B, neonatal MSG treatment results in a dramatic reduction of NPY perikarya and almost complete disappearance of NPY fibers in the arcuate nucleus. Arc, Arcuate nucleus; III, third ventricle. Scale bar, 100 µm.

 
Triple-labeling immunofluorescence for DBH, NPY, and CRH
To elucidate the relative proportion of NPY-containing axons that innervate hypophysiotropic CRH neurons and derive either from the arcuate nucleus or the brainstem, triple-labeling immunofluorescence was performed on every third hypothalamic section of MSG-treated rats. Antisera against DBH, NPY, and CRH were used following the immunocytochemical protocol described above for quadruple labeling except that the PNMT immunolabeling was excluded.

Triple-labeling immunofluorescence for AGRP, NPY, and CRH
To determine the number of NPY-IR axons arising from the arcuate nucleus, we performed a triple-labeling immunofluorescence on every third hypothalamic section of animals with an intact arcuate nucleus. The sections were pretreated according to the method described above. Sections were incubated in a cocktail of primary antisera that contained antibodies against NPY and CRH as described above, and rabbit anti-AGRP serum (H-003-57; Phoenix Pharmaceuticals, Inc., Burlingame, CA) was used at 1:2500 for 3 d at 4 C. For NPY and CRH labeling, the same secondary antibodies and AMCA-avidin D was used as written above, whereas CY3-conjugated donkey antirabbit IgG (Jackson) was used at 1:200 dilution for 1 d at 4 C for AGRP.

Image analysis of immunofluorescence
Both quadruple-labeled and triple-labeled sections were examined using a Radiance 2100 confocal microscope (Bio-Rad Laboratories, Hemel Hempstead, UK). From each brain, at least four sections were analyzed from different rostrocaudal levels of the medial parvocellular subdivision of the PVN in which hypophysiotropic CRH neurons reside. The atlas by Paxinos and Watson (12) was used to identify the subdivisions of the PVN (Fig. 2Go).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 2. Schematic illustration of the subdivisions in the PVN at rostral (A), mid (B), and caudal (C) levels of the nucleus. AP, Anterior parvocellular subdivision; DP, dorsal parvocellular subdivision; LP, lateral parvocellular subdivision; MN, magnocellular division; MP, medial parvocellular subdivision; PV, periventricular parvocellular subdivision; VP, ventral parvocellular subdivision.

 
With a x60 oil lens, serial optical sections of 180 x 180 µm areas covering the entire medial parvocellular subdivision of the PVN were recorded in each section. The sections subjected to quadruple-labeling immunofluorescence were scanned in two steps such that two, consecutive scans were recorded from each area. The first scan was for FITC, CY3, and CY5 (laser excitation lines 488 nm for FITC, 543 nm for CY3, and 637 nm for CY5; dichroic/emission filters, 560/500–530 nm for FITC, 650/565–625 nm for CY3, and a 660-nm-long pass filter for CY5). The second scan was for AMCA (laser excitation line 405 nm and emission filter 420–480 nm). The sections subjected to triple-labeling immunofluorescence were scanned in one step for CY3, CY5, and AMCA. Pinhole sizes were set to obtain optical slices less than 0.8 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 LaserVox (Bio-Rad) and Image Pro Plus (Media Cybernetics Inc., Bethesda, MD) software and an IBM-compatible personal computer. Perikarya and proximal dendrites of the CRH neurons were traced through the optical slices, and the different types of boutons juxtaposed to the CRH neurons were counted. The regional heterogeneity of AGRP-IR boutons were analyzed with one-way ANOVA followed by Newman-Keuls test. P < 0.05 was considered significant. All data are presented as mean ± SEM.

To illustrate quadruple labeling, the three basic colors (red, green, and blue) were used to show pairs of triple-colored images in the same field and at the same magnification in adjacent figures. Thus, CRH-, DBH-, and PNMT-IR are displayed in one image, whereas CRH-, DBH-, and NPY-IR are shown in the second image. Accordingly, DBH- and CRH-IR are displayed in red and blue, respectively, in both images, whereas the green represents either PNMT- or NPY-IR. Therefore, DBH/PNMT- and DBH/NPY-IR double-labeled axons appear yellow due to red and green color mixing. All images represent single optical slices. Images captured through a x20 objective are less than 2.1 µm thick, whereas images captured through the x60 oil lens are less than 0.8 µm thick.

Boutons that contained only DBH-IR without PNMT-IR were considered noradrenergic, whereas fibers containing both DBH- and PNMT-IR were considered adrenergic axons. To examine the regional differences in the AGRP-IR innervation of the CRH neurons, the CRH neurons were analyzed at rostral, mid, and caudal levels of the PVN. The mid level was further divided into dorsal and ventral compartments (Fig. 3Go).


Figure 3
View larger version (32K):
[in this window]
[in a new window]

  		FIG. 3. Distribution of CRH neurons in the PVN. Localization of the CRH cells in rostrocaudal planes of the PVN. A, Rostral population, –1.6 mm from bregma; B, middorsal and midventral populations, –1.8 mm; C and D, caudal population, –2.0 and –2.2 mm. Scale bar, 100 µm.

 
Animal preparation for NPY infusion
Adult male Sprague Dawley rats were implanted with a 22-gauge stainless steel guide cannula (Plastics One Inc., Roanoke, VA) into the lateral cerebral ventricle under stereotaxic control (coordinates from Bregma anterior to posterior, –0.8; lateral, 1.2; dorsal to ventral, 3.;) through a burr hole in the skull under anesthesia. The cannula was secured to the skull with three stainless steel screws and dental cement and temporarily occluded with a dummy cannula. Bacitracin ointment was applied daily to the interface of the cement and the skin. One week after icv cannulation, under general anesthesia, an osmotic minipump (Alzet model 1003D; Alza Pharmaceuticals, Palo Alto, CA) was implanted sc between scapulae and connected with polyethylene tubing to a 28-gauge needle that was permanently inserted into and extended 1 mm below the external guide cannula. The animals had free access to food and were divided in two groups. The osmotic minipumps delivered artificial cerebrospinal fluid (140 mM NaCl, 3.35 mM KCl, 1.15 mM MgCl2, 1.26 mM Ca Cl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, 0.1% BSA, pH 7.4) (group 1, n = 8), or 10 µg/24 h NPY (Peninsula) (group 2, n = 7) in artificial cerebral spinal fluid for 3 d at a rate of 1 µl/h. These doses have been previously shown to induce pronounced orexigenic activity when administered centrally (13). The weight of the animals and food intake were monitored daily.

Tissue preparation for in situ hybridization histochemistry
At completion of the NPY administration experiment, the animals were anesthetized with sodium pentobarbital and perfused transcardially with 20 ml 0.01 M PBS (pH 7.4) containing 15,000 U/liter heparin sulfate, followed by 150 ml 4% paraformaldehyde in PBS. The brains were removed and postfixed by immersion in the same fixative for 2 h at room temperature. Tissue blocks containing the hypothalamus were cryoprotected in 20% sucrose in PBS at 4 C overnight and then frozen on dry ice. Serial, 18-µm-thick coronal sections through the rostrocaudal extent of the PVN were cut on a cryostat (Reichert-Jung 2800 Frigocut-E) and adhered to Superfrost/Plus glass slides (Fisher Scientific Co., Pittsburgh, PA) to obtain four sets of slides, each set containing every fourth section through the PVN. Cannula placement was confirmed by light microscopic examination, and animals with cannulas outside the lateral ventricle were excluded from further study. The tissue sections were desiccated overnight at 42 C and stored at –80 C until prepared for in situ hybridization histochemistry.

In situ hybridization histochemistry
Every fourth section of the PVN was hybridized with a 976-bp single-stranded [35S]UTP-labeled cRNA probe for CRH as previously described (14). Specificity of the probe for CRH mRNA has been demonstrated in previous studies from our laboratories (15). The hybridization was performed under plastic coverslips in a buffer containing 50% formamide, a 2-fold concentration of standard sodium citrate, 10% dextran sulfate, 0.5% sodium dodecyl sulfate, 250 µg/ml denatured salmon sperm DNA, and 6 x 105 cpm radiolabeled probe for 16 h at 56 C. Slides were dipped into Kodak NTB2 autoradiography emulsion (Eastman Kodak, Rochester, NY), and the autoradiograms were developed after 2 wk of exposure at 4 C.

Image analysis
Autoradiograms were visualized under dark-field illumination using a COHU 4910 video camera (COHU, Inc., San Diego, CA). The images were captured with a PCI frame grabber board (Scion Corp., Frederick, MD) and analyzed with a Macintosh G3 computer using Scion Image. Background density points were removed by thresholding the image, and integrated density values (OD x area of distribution of silver grains on each side of the PVN) were measured in four consecutive sections for each animal, which represent the majority of neurons containing CRH mRNA in the PVN. The integrated density values were summed to yield total integrated density values for each animal, and the means and SE of the total integrated density values were calculated for each experimental group. Nonlinearity of radioactivity in the emulsion was evaluated by comparing density values with a calibration curve created from autoradiograms of known dilutions of the radiolabeled probes immobilized on glass slides in 2% gelatin fixed with 4% formaldehyde and exposed and developed simultaneously with the in situ hybridization autoradiograms.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Involvement of the brainstem noradrenergic and adrenergic cell groups in the NPY-IR innervation of CRH neurons
NPY-, DBH-, and PNMT-IR axons densely innervated the parvocellular subdivisions of the PVN (Fig. 4AGo). However, the distribution of the three fiber networks showed regional differences (Fig. 4AGo). NPY-IR axons and slightly less intensely DBH-IR axons inundated the ventral parvocellular subdivision, whereas the PNMT-IR axons were rare in this location (Fig. 4AGo). Furthermore, NPY-IR axons more densely innervated the periventricular parvocellular subdivision and the medial part of the medial parvocellular subdivision than PNMT-IR or DBH-IR fibers (Fig. 4AGo).


Figure 4
View larger version (62K):
[in this window]
[in a new window]

  		FIG. 4. Distribution of, DBH, PNMT, and NPY axons and CRH perikarya in the PVN of intact rats. A1 and A2, Low-magnification confocal images of the same field demonstrate the distribution of NPY (green), DBH (red), and CRH (blue) (A1) and PNMT (green), DBH (red), and CRH (blue) (A2) containing elements in the PVN. A1, NPY-IR is present in DBH-IR axons in the area of CRH neurons. Note that single-labeled NPY-IR axons, which contain only NPY without DBH-IR, are much less dense in the region of CRH neurons, whereas a dense, single-labeled NPY-IR network is present in the ventral parvocellular subdivision and in the ventral part of the medial parvocellular subdivision. A2, CRH neurons in the medial parvocellular subdivision are embedded in a dense network of adrenergic fibers. Noradrenergic (DBH-IR but not PNMT-IR) fibers appear red, adrenergic fibers (both DBH- and PNMT-IR) appear yellow. B and C, High-magnification images of the same field demonstrate the uneven distribution of NPY-IR boutons juxtaposed to CRH neurons in the dorsal (B1 and B2) and the ventral (C1 and C2) parts of the medial parvocellular subdivision of the PVN. Note that whereas single-labeled NPY-IR boutons are relatively rare in the dorsal part of the medial parvocellular subdivision (B1 and B2), these noncatecholaminergic NPY fibers are more frequently seen in the ventral part of the subdivision (C1 and C2). White arrow, NPY/DBH/PNMT-IR; white arrowhead, DBH/NPY-IR; open arrow, single-labeled NPY boutons; open arrowhead, single-labeled DBH-IR boutons juxtaposed to CRH neurons. III, Third ventricle. Scale bars, 100 µm (shown in A2) for A1 and A2 and 10 µm (shown in C2) for B1, B2, C1, and C2.

 
The area of the medial parvocellular subdivision, where the majority of the hypophysiotropic CRH neurons are located, was heavily innervated by all three afferent systems (Fig. 4AGo). As expected, the vast majority of PNMT-IR axons were also labeled for DBH (Fig. 4Go, A2, B2, and C2). The majority of adrenergic (PNMT-IR) and a subpopulation of noradrenergic (DBH-IR but not PNMT-IR) axons also showed NPY-IR (Fig. 4Go, B and C). In the area of hypophysiotropic CRH neurons, the density of catecholaminergic NPY-IR fibers was much higher than the density of single-labeled NPY-IR axons (Fig. 4BGo). However, in the ventral and most caudal part of the medial parvocellular subdivision, single-labeled NPY-IR fibers exceeded the density of catecholaminergic NPY-IR fibers (Fig. 4CGo).

NPY/PNMT-IR axon varicosities were found in juxtaposition to the vast majority of CRH neurons (94.2 ± 1.1%) (Fig. 4Go, B and C). An average of 5.5 ± 0.4 NPY/PNMT boutons per CRH cell was observed. Noradrenergic NPY/DBH-IR boutons were also found juxtaposed to the 82.8 ± 6.2% of CRH neurons with an average of 3.5 ± 0.8 NPY/DBH boutons per cell (Fig. 4Go, B and C). Table 1Go shows the results of the quantitative analysis.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Quantitative analysis of quadruple-labeling immunofluorescence (PNMT/DBH/NPY/CRH) in intact rats

 
Of all NPY-containing axon varicosities located on the surface of CRH neurons, 41.2 ± 5.6% contained both PNMT- and DBH-IR, whereas 22.2 ± 3.0% were only DBH-IR. An additional 36.6 ± 3.1% of NPY-IR axon varicosities were only single labeled, indicating that these fibers do not originate from catecholaminergic sources. However, the single-labeled NPY-IR fibers were found in juxtaposition to 89.0 ± 5.3% of CRH neurons. These single-labeled NPY-IR axon varicosities were unevenly distributed and more frequently contacted the most posterior CRH neurons, and the CRH neurons located laterally and in the ventral part of the medial parvocellular subdivision (Fig. 4Go, B and C). Conversely, 86.5 ± 5.6% of PNMT-IR boutons and 47.8 ± 12.0% of DBH-IR, PNMT-immunonegative boutons on the surface of CRH neurons contained NPY.

With respect to the catecholaminergic boutons on the surface of CRH neurons, 48.5 ± 6.2% contained DBH but not PNMT, suggesting that these varicosities produce noradrenaline, whereas 51.6 ± 6.2% contained both DBH and PNMT, indicating their adrenergic phenotype.

Effect of arcuate nucleus ablation on the NPY-IR innervation of CRH neurons
Immunostaining against NPY in MSG-treated animals demonstrated that the vast majority of NPY-IR cells in the arcuate nucleus were ablated (Fig. 1Go). Only a small number of NPY-IR neurons remained in the most caudal part of the arcuate nucleus.

Catecholaminergic axons more densely inundated the medial parvocellular subdivision, and fewer catecholaminergic/NPY-IR fibers were found in the ventral and caudal parts of this subdivision (Fig. 5AGo1). In the ventral part of the medial parvocellular subdivision where the density of the single-labeled NPY axons was the heaviest in intact rats, only sparse remaining fibers were found in MSG-treated animals (Fig. 5Go, A2 and A3). In triple-labeled preparations, the density of single-labeled NPY axons dramatically decreased throughout the PVN. Only 2.3 ± 0.3 single-labeled NPY boutons were found juxtaposed to CRH neurons, and these boutons comprised only 8.2 ± 2.3% of the total number of NPY varicosities. The vast majority (91.8 ± 2.3%) of the NPY boutons on the surface of the CRH neurons also contained DBH-IR (Fig. 5Go, A2 and A3). Table 2Go shows the results of the quantitative analysis.


Figure 5
View larger version (61K):
[in this window]
[in a new window]

  		FIG. 5. Involvement of the arcuate nucleus neurons in the NPY innervation of the CRH neurons in the PVN. A1–3, Low-power magnification image illustrates the distribution of NPY-IR (green), DBH-IR (red), and CRH-IR (blue) elements in the PVN of MSG-treated rats. A1, DBH-IR is present in the vast majority of NPY-IR fibers (yellow), suggesting that these fibers originate from the catecholaminergic neurons of the brainstem. Note the dramatic reduction in the ventral part of the parvocellular division in the density of single-labeled NPY fibers after the chemical ablation of the arcuate nucleus (compare Fig 4A1Go). A2 and A3, High-level magnification images demonstrate NPY-IR varicosities juxtaposed to the CRH neurons in different parts of the parvocellular division in MSG-treated rats. Note that the vast majority of the NPY-IR boutons contain DBH-IR. In the dorsal part of the medial parvocellular subdivision (A2), more intensive catecholaminergic innervation is seen compared with the ventral part (A3). B1, Low-power magnification image illustrates the distribution of AGRP-IR (red), NPY-IR (green), and CRH-IR (blue) elements in the PVN of intact rats. Note that a dense AGRP-IR network is present in the ventral part of the medial parvocellular subdivision, whereas single-labeled NPY-IR fibers mainly innervate the dorsal part (B1). B2 and B3, High-power magnification images demonstrate NPY- and AGRP-IR varicosities juxtaposed to the CRH neurons. The majority of the boutons contain only NPY-IR in the dorsal part of the medial parvocellular subdivision, suggesting that these fibers arise from the brainstem (B2). In the images taken from the caudal part of the PVN, a notable portion of the NPY-IR varicosities contain AGRP, identifying the arcuate nucleus as source of origin (B3). White arrow, DBH/NPY-IR; open arrow, AGRP/NPY-IR; white arrowhead, single-labeled NPY boutons; open arrowhead, single-labeled DBH boutons juxtaposed to CRH neurons. III, Third ventricle. Scale bars, 100 µm (shown in B1) for A1 and B1 and 10 µm (shown in B3) for A2, A3, B2, and B3.

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. Quantitative analysis of triple-labeling immunofluorescence (DBH/NPY/CRH) in MSG-treated rats

 
Relative involvement of the arcuate nucleus in the NPY-IR innervation of CRH neurons
AGRP/NPY-IR fibers arising from the arcuate nucleus densely innervated the parvocellular subdivisions of the PVN (Fig. 5BGo1). The distribution of the AGRP/NPY fibers was similar to the distribution of single-labeled NPY-IR fibers in the quadruple-labeling studies: the dorsal part of the medial parvocellular subdivision of the PVN was less intensely innervated, whereas the ventral part of the medial parvocellular subdivision was covered by a dense network of AGRP-IR axons (Fig. 5Go, B1 and B2). Quantitative analysis of the regional heterogeneity of the AGRP innervation showed that in the caudal region, significantly more AGRP boutons were juxtaposed to CRH neurons than in the other regions of the parvocellular subdivision (Fig. 5BGo3). The relative contribution of AGRP/NPY-IR varicosities to the innervation of the CRH neurons was significantly higher in the midventral region than in the middorsal region but significantly lower than in the caudal region. Table 3Go shows the regional heterogeneity in the density of the AGRP-IR boutons juxtaposed to the hypophysiotropic CRH neurons. The topography of analyzed regions is shown in Fig. 3Go.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Regional heterogeneity of NPY-IR and AGRP/NPY-IR innervation in the PVN of intact rats

 
Double-labeled NPY/AGRP-IR axon varicosities were found in juxtaposition to the vast majority of CRH neurons (94%) (Fig. 5Go, B2 and B3). An average of 7.1 NPY/AGRP boutons per CRH cell was observed. Of all NPY-containing axon varicosities on the surface of CRH neurons, 33.7% contained AGRP-IR. Table 4Go shows the results of the quantitative analysis.


View this table:
[in this window]
[in a new window]

  		TABLE 4. Quantitative analysis of triple-labeling immunofluorescence (AGRP/NPY/CRH) in intact rats

 
Effect of central NPY administration on food intake, body weight, and CRH mRNA level in the PVN
NPY-treated animals consumed significantly more food (control vs. NPY, 64.2 ± 1.8 vs. 119.07 ± 5.6 g) and gained considerably more weight than controls (control vs. NPY, 14.8 ± 1.2 vs. 31.00 ± 5.3%) during the 3 d of infusion.

In control animals, neurons containing CRH mRNA were readily visualized by in situ hybridization histochemistry, symmetrically distributed in the medial parvocellular subdivision of the PVN on both sides of the third ventricle (Fig. 6AGo). A 3-d central infusion of NPY resulted in a uniform decrease in the hybridization signal over the CRH neurons throughout the anterior-posterior extent of the PVN (Fig. 6BGo). By image analysis, the mean of integrated density values of CRH mRNA in the PVN of NPY-treated animals was approximately 30% of that of the control animals (control vs. NPY, 77.1 ± 15.9 vs. 23.9 ± 2.7 integrated density units).


Figure 6
View larger version (95K):
[in this window]
[in a new window]

  		FIG. 6. Effect of central NPY administration on CRH mRNA in the PVN. Dark-field illumination micrographs depicting CRH mRNA expression in the parvocellular subdivision of the PVN in control rats (A) and in rats receiving an icv infusion of 10 µg/d NPY for 3 d (B). Note the marked reduction in the accumulation of silver grains over the PVN in NPY-treated animals compared with controls. III, Third ventricle. Scale bar, 200 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data demonstrate that the three main functional sources of the NPY innervation of hypophysiotropic CRH neurons are noradrenergic and adrenergic neuron populations of the brainstem and the arcuate nucleus. Other regions may have only a minor role in the NPY innervation of these cells. Approximately two thirds of the NPY innervation of CRH neurons of the PVN seems to originate from the brainstem, 41% from adrenergic, and 22% from noradrenergic neurons. The adrenergic NPY innervation originates from the C1–3 regions of the medulla, where the vast majority of the adrenergic neurons are known to cosynthesize NPY and project to the medial parvocellular subdivision of the PVN (9, 16). The noradrenergic/NPY innervation of the PVN originates from the A1, A2, and A6 noradrenergic cell populations of the brainstem (9). The noradrenergic A1 region contains the largest population of the NPY-synthesizing, DBH-IR neurons that innervate the PVN, but it projects primarily to the magnocellular part of the PVN and sends only scattered NPY fibers to parvocellular subnuclei (17). Only 15% of the noradrenergic neurons of the A2 region contain NPY-IR (9), but these neurons innervate the parvocellular subnuclei of the PVN including the medial parvocellular subdivision (17). The A6 region (locus ceruleus) also contains an NPY-synthesizing noradrenergic subpopulation that projects to the PVN, but the locus ceruleus projects primarily to the periventricular parvocellular subdivision and sends only scattered fibers to the medial parvocellular subdivision (17). Accordingly, because the CRH neurons are largely localized in the medial parvocellular subdivision of the PVN, we assume that the noradrenergic/NPY innervation of the CRH neurons mainly originates from the A2 region, and the A1 and A6 regions contribute only in a minor way to this innervation.

One of the known functions of the brainstem catecholaminergic neurons is the mediation of the neuronal response to glucoprivation, and several lines of evidence indicate that the catecholaminergic neurons of the brainstem innervating the PVN are activated by glucoprivation. Glucoprivation increases c-fos expression in the C1–3 regions (18), significantly increases NPY mRNA level in the C1–3 and A1 regions (19), and stimulates DBH gene expression in the C1, A1, and A2 regions (20). Because glucoprivation activates CRH neurons in the PVN and increases circulating corticosterone levels that can be prevented by the ablation of the catecholaminergic projection neurons to the PVN (21), it is presumed that brainstem catecholamine neurons are important for this response. It has also been shown that brainstem catecholamine neurons are necessary for increased food intake in response to hypoglycemia (22, 23, 24, 25).

Brainstem catecholaminergic neurons also contribute to activation of the HPA axis in response to infection and inflammation. After the systemic administration of IL-1, c-fos expression was observed in neurons of C1, C2, A1, and A2 regions projecting to the PVN, and lesioning these regions decreased the number of the IL-1-activated CRH neurons in the PVN (26, 27). Moreover, the CRH mRNA increase induced by IL-1 or bacterial lipopolysaccharide was markedly attenuated by transection of ascending brainstem pathways (26, 28).

On the basis of these data, therefore, it is conceivable that catecholaminergic NPY neurons may contribute to the activating effects of hypoglycemia and immune stress on hypophysiotropic CRH neurons. In support of this possibility, the acute icv administration of NPY markedly stimulates CRH neurons (4, 5, 29). Although CRH neurons are directly innervated by NPY axons, and contain Y1 receptors (30), the stimulating effects of NPY on CRH gene activity could be mediated indirectly because all NPY receptors are coupled to inhibitory G proteins (31). In support of this possibility, Pronchuk et al. (32) demonstrated that acutely administered NPY markedly suppresses the GABAA-mediated inhibitory postsynaptic currents in neurons in the PVN, suggesting that NPY may stimulate CRH neurons by disinhibition of the GABA-ergic tone. In addition, CRH neurons are embedded in a network of Y1-IR fibers, suggesting a presynaptic location of Y1 receptor in the afferents of CRH neurons (33). Furthermore, NPY increases the number of presynaptic {alpha}2 catecholaminergic receptors, potentiating the presynaptic effects of noradrenaline (34).

To determine the origin of the noncatecholaminergic NPY axons that innervate CRH neurons in the PVN, we performed triple-labeling immunocytochemistry on sections of arcuate nucleus-ablated rats. MSG treatment eradicated the vast majority of arcuate nucleus including the NPY-IR neurons, leading to a marked decrease in the number of NPY-IR axons lacking DBH-IR that were in contact with hypophysiotropic CRH neurons. In addition, triple-labeling immunocytochemistry using AGRP as a marker for NPY axons of arcuate nucleus origin showed that 34% of the NPY boutons in contact with CRH neurons contained AGRP and that 94% of hypothalamic CRH neurons were contacted by these AGRP/NPY fibers. However, most of the NPY axons originating from the arcuate nucleus tended to innervate ventromedially and caudally located CRH neurons in the PVN. In the caudal parts of the PVN, more than half of the NPY boutons juxtaposed to CRH neurons contained AGRP-IR. These data not only assign an important role for the arcuate nucleus in the NPY-IR innervation of the CRH neurons in the PVN, but because of the regional heterogeneity of the arcuate innervation pattern, they also raise the possibility of functional segregation of paraventricular CRH neurons.

The arcuate nucleus plays a crucial role in the integration of peripheral metabolic signals, such as leptin, ghrelin, and insulin (35, 36, 37). During fasting, the circulating level of leptin is decreased, resulting in a marked increase in NPY synthesis in the arcuate nucleus neurons (38). Despite the marked activation of NPY neurons in the arcuate nucleus, however, CRH gene expression in the PVN decreases during fasting (38, 39). Given the observations that NPY can increase circulating levels of ACTH, corticosterone (40), and CRH mRNA levels in the PVN (4, 5) as well as phosphorylate cAMP response element-binding protein (CREB) in the nucleus of CRH neurons in the PVN (29), an inhibitory effect of NPY on CRH neurons would seem paradoxical. However, in contrast to the studies in the literature where the effect of an acute increase of NPY concentration was studied, NPY is chronically elevated during fasting (38).

To determine whether chronically elevated levels of NPY may regulate CRH synthesis during fasting, we examined the effect of a 3-d, continuous, central infusion of NPY on CRH gene expression in the PVN. In contrast to the acute effect of NPY, the chronic administration of NPY over 3 d by continuous infusion resulted in a marked reduction in CRH mRNA in the PVN. Because all NPY receptors are coupled to inhibitory G proteins (31), we presume that the effect of NPY is exerted directly on these neurons to decrease cAMP synthesis through the activation of one or more NPY receptors.

We conclude that three major cell populations give rise to the origin of the NPY-IR innervation of hypophysiotropic CRH neurons, adrenergic and noradrenergic NPY-IR neurons of the brainstem, and NPY/AGRP neurons of the arcuate nucleus. We propose that NPY pathways ascending from the brainstem have an acute activating effect on CRH neurons in response to glucoprivation, infection, and inflammation, whereas neurons located in the arcuate nucleus exert a chronic inhibitory effect on CRH gene expression in association with fasting. The concept that NPY could exert both inhibitory and stimulatory effects on CRH neurons raises the possibility that NPY may exert diverse effects on the HPA axis depending upon the origin of the NPY input and nature of the specific physiological stimuli.


   Acknowledgments
 
We are grateful to Drs. Martha C. Bohn and Dr. István Merchenthaler for the generous donation of PNMT and NPY antibodies.


   Footnotes
 
This work was supported by grants from the National Science Foundation of Hungary (OTKA T046492 and T046574), NKFP 1A/002/2004, the Sixth EU Research Framework Programme (contract LSHM-CT-2003-503041), and National Institutes of Health Grant DK37021.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 9, 2007

Abbreviations: AGRP, Agouti-related protein; AMCA, 7-amino-4-methyl-coumarin-3-acetic acid; DBH, dopamine-β-hydroxylase; FITC, fluorescein isothiocyanate; HPA, hypothalamic-pituitary-adrenal; IR, immunoreactive; MSG, monosodium glutamate; NPY, neuropeptide Y; PNMT, phenylethanolamine-N-methyltransferase; PVN, hypothalamic paraventricular nucleus.

Received June 1, 2007.

Accepted for publication August 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sawchenko PE, Imaki T, Potter E, Kovacs K, Imaki J, Vale W 1993 The functional neuroanatomy of corticotropin-releasing factor. Ciba Found Symp 172:5–21; discussion 21–29[Medline]
  2. Liposits Z 1990 Ultrastructural immunocytochemistry of the hypothalamic corticotropin releasing hormone synthesizing system. Anatomical basis of neuronal and humoral regulatory mechanisms. Prog Histochem Cytochem 21:1–98[Medline]
  3. Liposits Z, Sievers L, Paull WK 1988 Neuropeptide-Y and ACTH-immunoreactive innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamus of the rat. An immunocytochemical analysis at the light and electron microscopic levels. Histochemistry 88:227–234[CrossRef][Medline]
  4. Suda T, Tozawa F, Iwai I, Sato Y, Sumitomo T, Nakano Y, Yamada M, Demura H 1993 Neuropeptide Y increases the corticotropin-releasing factor messenger ribonucleic acid level in the rat hypothalamus. Brain Res 18:311–315[CrossRef]
  5. Brunton PJ, Bales J, Russell JA 2006 Neuroendocrine stress but not feeding responses to centrally administered neuropeptide Y are suppressed in pregnant rats. Endocrinology 147:3737–3745[Abstract/Free Full Text]
  6. Bai FL, Yamano M, Shiotani Y, Emson PC, Smith AD, Powell JF, Tohyama M 1985 An arcuato-paraventricular and -dorsomedial hypothalamic neuropeptide Y-containing system which lacks noradrenaline in the rat. Brain Res 331:172–175[CrossRef][Medline]
  7. Legradi G, Lechan RM 1998 The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. Endocrinology 139:3262–3270[Abstract/Free Full Text]
  8. Hahn TM, Breininger JF, Baskin DG, Schwartz MW 1998 Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1:271–272[CrossRef][Medline]
  9. Sawchenko PE, Swanson LW, Grzanna R, Howe PR, Bloom SR, Polak JM 1985 Colocalization of neuropeptide Y immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricular nucleus of the hypothalamus. J Comp Neurol 241:138–153[CrossRef][Medline]
  10. Beck B, Jhanwar-Uniyal M, Burlet A, Chapleur-Chateau M, Leibowitz SF, Burlet C 1990 Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status. Brain Res 528:245–249[CrossRef][Medline]
  11. Schwartz MW, Woods SC, Porte Jr D, Seeley RJ, Baskin DG 2000 Central nervous system control of food intake. Nature 404:661–671[Medline]
  12. Paxinos G, Watson C 1998 The rat brain in stereotaxic coordinates. 4th ed. San Diego: Academic Press
  13. Fekete C, Kelly J, Mihaly E, Sarkar S, Rand WM, Legradi G, Emerson CH, Lechan RM 2001 Neuropeptide Y has a central inhibitory action on the hypothalamic-pituitary-thyroid axis. Endocrinology 142:2606–2613[Abstract/Free Full Text]
  14. Thompson RC, Seasholtz AF, Herbert E 1987 Rat corticotropin-releasing hormone gene: sequence and tissue-specific expression. Mol Endocrinol 1:363–370[Abstract/Free Full Text]
  15. Kakucska I, Qi Y, Clark BD, Lechan RM 1993 Endotoxin-induced corticotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus is mediated centrally by interleukin-1. Endocrinology 133:815–821[Abstract/Free Full Text]
  16. Cunningham Jr ET, Bohn MC, Sawchenko PE 1990 Organization of adrenergic inputs to the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J Comp Neurol 292:651–667[CrossRef][Medline]
  17. Cunningham Jr ET, Sawchenko PE 1988 Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol 274:60–76[CrossRef][Medline]
  18. Ritter S, Llewellyn-Smith I, Dinh TT 1998 Subgroups of hindbrain catecholamine neurons are selectively activated by 2-deoxy-D-glucose induced metabolic challenge. Brain Res 805:41–54[CrossRef][Medline]
  19. Li AJ, Ritter S 2004 Glucoprivation increases expression of neuropeptide Y mRNA in hindbrain neurons that innervate the hypothalamus. Eur J Neurosci 19:2147–2154[CrossRef][Medline]
  20. Li AJ, Wang Q, Ritter S 2006 Differential responsiveness of dopamine-β-hydroxylase gene expression to glucoprivation in different catecholamine cell groups. Endocrinology 147:3428–3434[Abstract/Free Full Text]
  21. Ritter S, Watts AG, Dinh TT, Sanchez-Watts G, Pedrow C 2003 Immunotoxin lesion of hypothalamically projecting norepinephrine and epinephrine neurons differentially affects circadian and stressor-stimulated corticosterone secretion. Endocrinology 144:1357–1367[Abstract/Free Full Text]
  22. Ritter S, Bugarith K, Dinh TT 2001 Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation. J Comp Neurol 432:197–216[CrossRef][Medline]
  23. Ste Marie L, Palmiter RD 2003 Norepinephrine and epinephrine-deficient mice are hyperinsulinemic and have lower blood glucose. Endocrinology 144:4427–4432[Abstract/Free Full Text]
  24. Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S 2005 Basomedial hypothalamic injections of neuropeptide Y conjugated to saporin selectively disrupt hypothalamic controls of food intake. Endocrinology 146:1179–1191[Abstract/Free Full Text]
  25. Sindelar DK, Ste Marie L, Miura GI, Palmiter RD, McMinn JE, Morton GJ, Schwartz MW 2004 Neuropeptide Y is required for hyperphagic feeding in response to neuroglucopenia. Endocrinology 145:3363–3368[Abstract/Free Full Text]
  26. Ericsson A, Kovacs KJ, Sawchenko PE 1994 A functional anatomical analysis of central pathways subserving the effects of interleukin-1 on stress-related neuroendocrine neurons. J Neurosci 14:897–913[Abstract]
  27. Buller K, Xu Y, Dayas C, Day T 2001 Dorsal and ventral medullary catecholamine cell groups contribute differentially to systemic interleukin-1β-induced hypothalamic pituitary adrenal axis responses. Neuroendocrinology 73:129–138[CrossRef][Medline]
  28. Fekete C, Singru PS, Sarkar S, Rand WM, Lechan RM 2005 Ascending brainstem pathways are not involved in lipopolysaccharide-induced suppression of thyrotropin-releasing hormone gene expression in the hypothalamic paraventricular nucleus. Endocrinology 146:1357–1363[Abstract/Free Full Text]
  29. Sarkar S, Lechan RM 2003 Central administration of neuropeptide Y reduces {alpha}-melanocyte-stimulating hormone-induced cyclic adenosine 5'-monophosphate response element binding protein (CREB) phosphorylation in pro-thyrotropin-releasing hormone neurons and increases CREB phosphorylation in corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. Endocrinology 144:281–291[Abstract/Free Full Text]
  30. Dimitrov EL, Dejoseph MR, Brownfield MS, Urban JH 2007 Involvement of neuropeptide Y Y1 receptors in the regulation of neuroendocrine corticotropin releasing hormone neuronal activity. Endocrinology 148:3666–3673[Abstract/Free Full Text]
  31. Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, Westfall T 1998 XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 50:143–150[Abstract/Free Full Text]
  32. Pronchuk N, Beck-Sickinger AG, Colmers WF 2002 Multiple NPY receptors Inhibit GABAA synaptic responses of rat medial parvocellular effector neurons in the hypothalamic paraventricular nucleus. Endocrinology 143:535–543[Abstract/Free Full Text]
  33. Li C, Chen P, Smith MS 2000 Corticotropin releasing hormone neurons in the paraventricular nucleus are direct targets for neuropeptide Y neurons in the arcuate nucleus: an anterograde tracing study. Brain Res 854:122–129[CrossRef][Medline]
  34. Agnati LF, Fuxe K, Benfenati F, Battistini N, Harfstrand A, Tatemoto K, Hokfelt T, Mutt V 1983 Neuropeptide Y in vitro selectivity increases the number of {alpha}2-adrenergic binding sites in membranes of the medulla oblongata of the rat. Acta Physiol Scand 118:293–295[Medline]
  35. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG 1996 Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101–1106[Medline]
  36. Schwartz MW, Sipols AJ, Marks JL, Sanacora G, White JD, Scheurink A, Kahn SE, Baskin DG, Woods SC, Figlewicz DP 1992 Inhibition of hypothalamic neuropeptide Y gene expression by insulin. Endocrinology 130:3608–3616[Abstract/Free Full Text]
  37. Chen HY, Trumbauer ME, Chen AS, Weingarth DT, Adams JR, Frazier EG, Shen Z, Marsh DJ, Feighner SD, Guan XM, Ye Z, Nargund RP, Smith RG, Van der Ploeg LH, Howard AD, MacNeil DJ, Qian S 2004 Orexigenic action of peripheral ghrelin is mediated by neuropeptide Y and agouti-related protein. Endocrinology 145:2607–2612[Abstract/Free Full Text]
  38. Brady LS, Smith MA, Gold PW, Herkenham M 1990 Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinology 52:441–447[Medline]
  39. Fekete C, Legradi G, Mihaly E, Tatro JB, Rand WM, Lechan RM 2000 {alpha}-Melanocyte stimulating hormone prevents fasting-induced suppression of corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus. Neurosci Lett 289:152–156[CrossRef][Medline]
  40. Wahlestedt C, Skagerberg G, Ekman R, Heilig M, Sundler F, Hakanson R 1987 Neuropeptide Y (NPY) in the area of the hypothalamic paraventricular nucleus activates the pituitary-adrenocortical axis in the rat. Brain Res 417:33–38[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
P. S. Singru, E. Sanchez, R. Acharya, C. Fekete, and R. M. Lechan
Mitogen-Activated Protein Kinase Contributes to Lipopolysaccharide-Induced Activation of Corticotropin-Releasing Hormone Synthesizing Neurons in the Hypothalamic Paraventricular Nucleus
Endocrinology, May 1, 2008; 149(5): 2283 - 2292.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Füzesi, T.
Right arrow Articles by Fekete, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Füzesi, T.
Right arrow Articles by Fekete, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals