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Endocrinology Vol. 144, No. 1 281-291
Copyright © 2003 by The Endocrine Society

ARTICLE

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

Sumit Sarkar and Ronald M. Lechan

Tupper Research Institute and Department of Medicine (S.S., R.M.L.), Division of Endocrinology, Diabetes, Metabolism and Molecular Medicine, Tufts-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: Ronald M. Lechan M.D., Ph.D., Professor of Medicine, Division of Endocrinology, Box No. 268, Tufts-New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111. E-mail: rlechan{at}lifespan.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptide Y (NPY) has a potent inhibitory effect on TRH gene expression in the paraventricular nucleus (PVN) and contributes to the fall in circulating thyroid hormone levels during fasting mediated by a reduction in serum leptin levels. Because {alpha}-MSH activates the TRH gene by increasing the phosphorylation of CREB in the nucleus of these neurons, we raised the possibility that at least one of the mechanisms by which NPY reduces TRH mRNA in hypophysiotropic neurons is by antagonizing the ability of {alpha}-MSH to phosphorylate CREB. As NPY increases CRH mRNA in the hypothalamus, we further determined whether intracerebroventricular (icv) administration of NPY regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. NPY [10 µg in artificial CSF (aCSF)] was administered into the lateral ventricle icv 30 min before the icv administration of aCSF or {alpha}-MSH (10 µg in aCSF), the latter in a dose previously demonstrated to increase proTRH mRNA and phosphorylate CREB in TRH neurons. By double-labeling immunocytochemistry, only few TRH neurons in the PVN contained phosphoCREB (PCREB) in animals treated only with aCSF (4 ± 0.2%) or with NPY followed by aCSF (9.7 ± 2.5), whereas {alpha}-MSH-infused animals dramatically increased the percentage of TRH neurons containing PCREB (75.3 ± 6.9%). Pretreatment with NPY before {alpha}-MSH infusion, however, significantly reduced the percentage of TRH neurons containing PCREB (40.8 ± 3.5%) compared with {alpha}-MSH infused animals (P = 0.01). Only 12.2 ± 0.9% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle-treated animals, whereas 30 min following NPY infusion, the number of CRH neurons containing PCREB increased dramatically to 88 ± 2.9%. Whereas {alpha}-MSH infusion increased the percentage of CRH neurons that contained PCREB to 56 ± 2.2% compared with control, animals pretreated with NPY further increased the number of CRH neurons colocalizing with PCREB to 87 ± 2.5%. These data demonstrate a functional interaction between NPY and {alpha}-MSH in the regulation of proTRH neurons in the PVN, suggesting that NPY can antagonize {alpha}-MSH induced activation of the TRH gene by interfering with melanocortin signaling at the postreceptor level, preventing the phosphorylation of CREB. In contrast, NPY infusion increases the phosphorylation of CREB in CRH neurons, indicating that NPY has independent effects on discrete populations of neurons in the PVN, presumably mediated through different signaling mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FASTING IS ASSOCIATED with reduction in circulating thyroid hormone and TSH levels, largely orchestrated by indirect actions of leptin on TRH-producing neurons in the hypothalamic paraventricular nucleus (PVN) (1, 2). The hypothalamic arcuate nucleus is a primary target for the action of leptin on the brain (3), and it mediates the regulatory effect of leptin on the hypothalamic-pituitary-thyroid axis (4) through its extensive monosynaptic and parallel multisynaptic projections to the PVN (5, 6, 7). We have recently demonstrated that the arcuate nucleus derived peptide, {alpha}-MSH, contributes to the effects of leptin on gene regulation of hypophysiotropic TRH (8). Thus, the intracerebroventricular (icv) administration of {alpha}-MSH to fasting animals restores proTRH mRNA to normal levels selectively in hypophysiotropic neurons of the PVN despite continuation of the fast (8).

The phosphorylation of the cAMP response element binding protein (CREB) is likely a mandatory component for the activation of hypophysiotropic TRH and CRH by {alpha}-MSH. Melanocortin receptors couple in a stimulatory fashion to cAMP, which, by activating protein kinase A, can phosphorylate CREB at serine-133 and induce the transcription of genes containing a cAMP response element (CRE) (9). The TRH contains a nearly perfect consensus cAMP response element (CRE) (10, 11). Indeed, recent in vitro evidence indicates that phosphorylated CREB binds to the CRE in the TRH promoter to activate the gene, and when deleted, lowers basal activity of the gene and reduces activation by {alpha}-MSH in vitro (12). In addition, recent in vivo studies from our laboratory in rats have demonstrated that {alpha}-MSH induces phosphorylation of CREB in the majority of TRH neurons (13). The CRH gene also contains a palindromic sequence in its promoter that allows the binding of phosphoCREB (PCREB) (14, 15). This sequence is important for the activation of the CRH gene as in vitro deletion completely abolishes the stimulatory affect of cAMP in several different cell lines (16). Furthermore, more than 50% of the CRH neurons show CREB phosphorylation in the PVN following {alpha}-MSH administration into the cerebrospinal fluid (13).

During fasting, gene expression for proopiomelanocortin (POMC), the precursor protein from which {alpha}-MSH is derived (17), is inhibited in arcuate nucleus neurons, simultaneously with an up-regulation of neuropeptide Y (NPY) mRNA (18), the latter expressed in a distinct arcuate nucleus neuronal population (19). NPY-producing neurons also send monosynaptic projections to TRH neurons in the PVN (20, 21) but inhibit TRH gene expression when centrally administered to ad libitum-feeding animals (22). Because NPY receptors can couple to inhibitory G(i/o) proteins, effectively reducing intracellular concentrations of cAMP (23), we raised the possibility that the increase in NPY during fasting may contribute to inhibition of the hypothalamic-pituitary-thyroid axis by interfering with melanocortin signaling at a postreceptor level, reducing the phosphorylation of CREB. Thus, we determined whether the phosphorylation of CREB following {alpha}-MSH administration could be altered by pretreatment with NPY, specifically in TRH neurons in the PVN.

NPY is also considered to be a major regulator of CRH because NPY fibers have been observed in synaptic association with CRH producing neurons in the PVN (24). Furthermore, icv administration of NPY has been shown to stimulate the gene expression and immunoreactivity of CRH in the PVN (25, 26) and to increase ACTH secretion (27). Therefore, we also determined the effect of NPY on phosphorylation of CREB in the CRH neurons in the medial parvocellular divisions of the PVN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Experiments were performed on adult male Sprague Dawley rats (Taconic Farms, Inc., Germantown, NY) weighing 210–230 g. The animals were housed individually in cages under standard environmental conditions (light between 0600 and 1800 h; temperature, 22 ± 1 C; rat chow and water available ad libitum). All experimental protocols were reviewed and approved by the Animal Research Committee at Tufts-New England Medical Center and Tufts University School of Medicine.

Animal preparation and {alpha}-MSH infusion
Ten days before experimentation, a 22-gauge stainless steel guide cannula (Plastic One, Roanke, VA) was placed into the lateral cerebral ventricle under stereotaxic control (coordinates from bregma, antero-posterior, -0.8 mm; lateral, -1.2 mm; and ventral, 3.2 mm) through a burr hole in the skull. The cannula was secured to the skull with three stainless steel screws and dental cement and temporarily occluded with a dummy cannula. The rats were made accustomed to handling by daily mock injections consisting of removal of the dummy cannula and connecting to an empty cannula connector for at least 1 wk before experimentation to reduce stress. In addition, 5 d before the experiments, all rats were jugular vein-cannulated to ensure rapid and stress-free anesthesia at the termination of the experiment. All icv injections were made in freely moving animals through a 28-gauge needle that extended 1 mm below the guide cannula. The needle was connected by polyethylene tubing to a 1-cc GlasPak syringe, and injections were made over 2 min by a microprocessor controlled infusion pump (Bee Electronic Minipump; Bioanalytical Systems Inc., West Lafayette, IN). All animals were fasted for 65 h and subdivided into 4 groups (n = 4 per group). The first group was administered 6 µl artificial CSF (aCSF) (140 mM NaCl; 3.35 mM KCl; 1.15 mM MgCl2; 1.26 mM CaCl2; 1.2 mM Na2HPO4; and 0.3 mM NaH2PO4, pH 7.4) containing 0.05% BSA icv and the brain fixed by intracardiac perfusion10 min later. The second group received 10 µg of porcine NPY (Peninsula Laboratories, Inc., Belmont, CA) in 6 µl of aCSF and the brain fixed 30 min later. The third group received aCSF and after 30 min, a second injection of 10 µg of {alpha}-MSH (Peninsula Laboratories, Inc.) in 6 µl of aCSF through the same cannula, and the brain fixed by intracardiac perfusion 10 min later. The fourth group was prepared similar to the third group except that the animals received an initial injection of 10 µg of NPY icv instead of aCSF. At conclusion of the experiments, the animals were deeply anesthetized with sodium pentobarbital (50 mg/kg) through the jugular catheters and within 2 min of anesthesia, perfused transcardially with 10 ml of heparinized saline for 10–20 sec followed by a mixture of 1% acrolein and 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10–15 min. Following perfusion, the brains were dissected and postfixed by immersion in 3% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for several hours at 4 C. A hypothalamic block was removed from the rest of the brain using a coronal Rat Brain Matrix (Ted Pella, Inc., Redding, CA) and transferred into 20% sucrose in 0.01 M PBS at 4 C for 2 d to promote cryoprotection.

Immunocytochemical detection of PCREB
To determine the extent of CREB phosphorylation in the nucleus of cells within the parvocellular division of the PVN after infusion of aCSF, {alpha}-MSH, NPY, or NPY + {alpha}-MSH, a series of 25-µm coronal sections were cut on cryostat through the PVN and collected in PBS (pH 7.4). The sections were washed in PBS, treated with 1% sodium borohydride in deionized water for 30 min, and then rinsed several times in deionized water and PBS until the sections became free of bubbles. The sections were then placed in 0.5% H2O2 in PBS for 15 min to remove endogenous peroxidase activity from the tissues, washed in PBS, and treated in 0.5% Triton X-100 in PBS for 1 h to improve antibody penetration. Following preincubation in 10% normal horse serum for 30–60 min, the sections were incubated in the primary antiserum against phosphorylated CREB128–141, diluted 1:12,000 (gift of Dr. Marc R. Montminy, The Salk Institute, La Jolla, CA) for 3 d at 4 C with continuous agitation on a rotary shaker. All primary antisera dilutions were made in 1% normal horse serum in PBS containing 0.08% sodium azide and 0.2% Kodak (Rochester, NY) Photo-Flo. After thorough rinsing in PBS, sections were incubated in biotinylated goat antirabbit IgG (1:200, Vector Laboratories, Burlingame, CA) for 2 h. The sections were then washed three times in PBS and incubated in avidin-peroxidase complex (1:100, ABC Elite Kit, Vector Laboratories) for 1 h. After three washes in PBS and a rinse in 0.05 M Tris buffer (pH 7.8), the color reaction was developed in 0.025% diaminobenzidine containing 0.06% nickel ammonium sulfate and 0.0027% H2O2 for 7 min to yield a dark purple labeling in the cell nucleus. A rinse in Tris buffer (pH 7.6) was used to stop the reaction. Sets of PCREB labeled sections were then mounted onto slides, dehydrated in graded series of ethanol, followed by three changes in Histosol and coverslipped from DPX mountant for light microscopy.

Light microscopic double-labeling immunocytochemistry of PCREB-immunoreactive (IR) nuclei and proTRH and CRH neurons in the PVN
Sets of PCREB-labeled PVN sections were rinsed in Tris-buffered saline and then incubated in either rabbit antiserum recognizing rat preproTRH 53–74 (gift of Dr. Ivor Jackson, Rhode Island Hospital, Providence, RI) at a dilution of 1:20,000 or in rabbit antiserum raised against rat CRH (gift of Dr. Paul E. Sawchenko, Salk Institute, La Jolla, CA) at a dilution of 1: 20,000 for 2 d at 4 C. After washing in Tris-buffer saline, the tissue sections were incubated in donkey antirabbit IgG (1:400, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and the ABC Elite Complex (1:100). The immunolabeling was visualized by 0.025% diaminobenzidine and 0.03% H2O2 in Tris buffer (pH 7.6) alone, to yield a contrasting brown cytoplasmic labeling. After development, the sections were rinsed in Tris buffer (pH 7.6), mounted into slides, air-dried, and dehydrated in ethanol followed by Histosol and coverslipped.

Quantitative analysis of PCREB-labeled nuclei and proTRH or CRH immunoreactive perikarya in the PVN
Double-labeled sections containing PCREB and proTRH or CRH immunoreactivity were used for quantitative analysis. ProTRH immunostaining, which is confined exclusively to perikarya and proximal dendrites, was developed with a light brown chromogen, allowing the determination whether the nucleus contained the previously developed dark purple label for PCREB. Only cells with an intense or medium density of nuclear labeling for PCREB and distinct cytoplasmic reaction product for proTRH were counted as double labeled. The percentage of double-labeled neurons was determined from three distinct rostro-caudal levels of the PVN at 300-µm intervals (rostral, mid-level, and caudal PVN) in all animal groups. The subdivisions of the PVN were identified based on the rat brain atlas of Paxinos and Watson (28) and projected from a Zeiss microscope equipped with a COHU video camera (San Diego, CA) onto the monitor of a Macintosh computer using Image 1.54 software (NIH). The percentage of proTRH immunoreactive neurons containing PCREB nuclei were determined and statistically analyzed. The percentage of CRH neurons containing PCREB-labeled nuclei were determined from the mid-level of the PVN.

Antisera characterization
PCREB antiserum.
Antiserum to PCREB was raised in rabbits against synthetic CREB 128–141, containing a phosphorylated Ser133 residue, and purified by removing antibodies to the unphosphorylated peptide using affinity chromatography with phosphopeptide resin (29). Immunoblot analysis of nuclear extracts from the hypothalamus showed a single band corresponding to PCREB and no cross-reactivity with unphosphorylated CREB (29). Immunostaining in the PVN is completely abolished when the antiserum is preabsorbed with excess of the corresponding phosphopetide (10 µm) (30).

ProTRH antiserum.
Antiserum to rat preproTRH (53–74) was raised in New Zealand White rabbits and its specificity characterized both by RIA and immunocytochemistry (31, 32). Preabsorption of this antiserum with excess (10-6 M) preproTRH (53–74) (Peninsula Laboratories, Inc.) results in the complete loss of immunostaining in the PVN (32).

CRH antiserum.
Antiserum to rat CRH (code C70) was generated in rabbits against synthetic CRH and has been characterized in detail previously (33). Preabsorption of the CRH antiserum by 10 µM of synthetic rat CRH (American Peptide Co., Sunnyvale, CA) results in the complete loss of immunolabeling in the PVN (30).

Statistical analysis
The results are presented as mean ± SEM. Statistical significance was determined by ANOVA followed by post hoc Newman-Keuls test. Differences were considered to be significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ICV aCSF, NPY,{alpha}-MSH, and NPY+{alpha}-MSH on PCREB immunoreactivity in the PVN
The effects of aCSF, NPY, {alpha}-MSH, and NPY + {alpha}-MSH on CREB phosphorylation in PVN neurons are shown in Table 1Go. In the aCSF-treated animals, PCREB immunolabeling was confined to the nucleus of magnocellular neurosecretory neurons, and only very few immunopositive nuclei were present in the parvocellular subdivision of the PVN (Fig. 1Go, A–C). NPY treatment increased PCREB immunoreactivity primarily in the nuclei of medial parvocellular subdivision PVN neurons, particularly in lateral portions at mid-level regions (Fig. 1EGo), but little increase was observed in the nuclei of anterior and periventricular parvocellular subdivision neurons (Fig. 1Go, D–F). The magnitude of CREB phosphorylation in NPY-treated animals, however, did not equal that observed following {alpha}-MSH infusion, in which a dramatic increase in PCREB immunoreactivity was observed in parvocellular neurons of the PVN, particularly in anterior and mid and caudal portions of the medial parvocellular subdivision (Fig. 1Go, G–I). Animals pretreated with NPY before {alpha}-MSH administration showed reduced PCREB immunoreactivity in all parvocellular subdivisions compared with animals treated only with {alpha}-MSH, resembling animals treated with NPY alone (compare Fig. 1Go, G–I, with J–L). Thus, neurons in the most lateral portions of the medial parvocellular subdivision retained immunoreactivity for PCREB when NPY was administered before {alpha}-MSH without apparent attenuation (Fig. 1KGo).


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  		Table 1. Number of PCREB-IR neurons in the hypothalamic PVN following icv administration of aCSF, NPY, {alpha}-MSH, and NPY+{alpha}-MSH

 


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  		Figure 1. Distribution of PCREB-labeled nuclei in the anterior, mid, and caudal levels of the PVN in fasted animals. Animals were treated with aCSF (A–C), NPY (D–F), {alpha}-MSH (G–I), or pretreated with NPY before {alpha}-MSH infusion (J–L). Note the moderate increase of PCREB immunolabeling in NPY infused animals primarily in the lateral part of the medial parvocellular subdivision and very few PCREB containing nuclei in the ventral and periventricular subdivisions of the PVN (E). A dramatic increase of PCREB immunolabeled nuclei was observed in {alpha}-MSH-treated animals in the mid and caudal part of the medial parvocellular subdivisions of the PVN (H, I). Pretreatment with NPY before {alpha}-MSH infusion resulted decrease of PCREB nuclei in the ventral and periventricular parvocellular subdivisions of the PVN (compare H with K). PVNa, anterior parvocellular subdivision of the PVN, PVNmp, medial parvocellular subdivision of the PVN, PVNpv, periventricular parvocellular subdivision of the PVN, PVNvp, ventral parvocellular subdivision of the PVN, PVNm, magnocellular division of the PVN. Scale bar, 100 µm.

 
Double labeling PCREB-proTRH immunostaining in the PVN
ProTRH-immunoreactive neurons were distributed primarily in anterior, medial, and periventricular parvocellular subdivisions of the PVN. The concentration of TRH neurons in the anterior PVN was relatively low and the neurons more widely dispersed than in the medial and periventricular parvocellular subdivisions, particularly in the caudal part of the medial parvocellular subdivision where proTRH neurons were highly concentrated and densely packed. Mid and caudal levels of the medial parvocellular PVN, which contain the highest numbers of hypophysiotropic proTRH neurons (34), were used for detailed analysis. Only few proTRH neurons contained PCREB following aCSF administration (Figs. 2Go, A and B, and 3Go). Following NPY infusion, 10.9 ± 1.1% of the proTRH neurons in the medial and periventricular parvocellular subdivisions of the PVN at mid levels and 10.5 ± 1.7% in caudal portions contained PCREB (Figs. 2Go, C and D, and 3Go), but not significantly different than the aCSF-treated control group (P = 0.08 for mid level, P = 0.07 for caudal level). In contrast, animals pretreated with aCSF and then infused with {alpha}-MSH, showed a dramatic increase in the number of proTRH neurons that contained PCREB in the parvocellular neurons in both the mid (69.7 ± 3.6%, P < 0.001) and caudal (78.9 ± 2.3%, P < 0.001) levels of the PVN (Figs. 2Go, E and F, and 3Go). The percentage of proTRH neurons containing PCREB in aCSF, NPY, {alpha}-MSH, and NPY + {alpha}-MSH infused groups are shown in the graph in Fig. 3Go. Pretreatment with NPY before {alpha}-MSH infusion led to a significantly reduced number of doubly labeled neurons in both parvocellular regions of the PVN (P < 0.001 for both mid and caudal levels) compared with animals treated with {alpha}-MSH, alone (Figs. 2Go, G and H, and 3Go).



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  		Figure 2. Medium (A, C, E, G) and high-power magnification (B, D, F, H) light microscopic photomicrographs showing double immunolabeling for proTRH neurons (brown cytoplasm) and PCREB positive cells (black nuclei) in the medial parvocellular subdivision of the PVN in control- (A, B), NPY- (C, D), {alpha}-MSH- (E, F) and NPY + {alpha}-MSH- (G, H) treated animals. Double-labeled neurons are rarely found in the aCSF- (A, B) and NPY- (C, D) treated animals. Maximum double immunolabeling was observed in the {alpha}-MSH-treated animals (E, F), whereas the percentage of proTRH neurons containing PCREB nuclei was significantly reduced in animals treated with NPY before {alpha}-MSH infusion (G, H). Scale bars: A, C, E, G, 200 µm; B, D, F, H, 50 µm.

 


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  		Figure 3. Percentage of TRH neurons containing PCREB immunoreactivity in the nuclei of aCSF-, NPY-, {alpha}-MSH-, and NPY + {alpha}-MSH-treated animals.

 
Double labeling PCREB-CRH immunostaining in the PVN
Midlevel PVN sections that contain the highest numbers of CRH neurons in the lateral portion of the medial parvocellualr subdivision were used for detailed analysis. In PCREB-labeled sections of the PVN sequentially labeled with CRH antiserum, approximately 35–40 neurons were observed in each animal. Only 12.2 ± 0.9% of CRH neurons of the medial parvocellular neurons contained PCREB following aCSF administration (Figs. 4Go, A and B, and 5Go). Following NPY infusion, the number of CRH neurons that contained PCREB was dramatically increased (88.2 ± 2.9%, P < 0.001) (Figs. 4Go, C and D, and 5Go), and even higher than that following {alpha}-MSH infusion in which 56.5 ± 2.2% of the CRH neurons contained PCREB (Fig. 4Go, E and F). Animals pretreated with NPY before {alpha}-MSH infusion had a significantly increased percentage of CRH neurons containing PCREB (87.2 ± 2.5%) (Fig. 4Go, G and H) compared with {alpha}-MSH-infused animals (P < 0.001), but not significantly different from animals treated with NPY, alone (P = 0.08). The percentage of CRH neurons containing PCREB in aCSF, NPY, {alpha}-MSH, and NPY + {alpha}-MSH infused groups are shown in the graph in Fig. 5Go. With respect to the total number of PCREB-positive cells induced by NPY in the parvocellular PVN, CRH-producing neurons comprised approximately 60% of these neurons.



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  		Figure 4. Medium (A, C, E, G) and high-power magnification (B, D, F, H) light microscopic photomicrographs showing double immunolabeling for CRH neurons (brown cytoplasm) and PCREB positive cells (black nuclei) in the medial parvocellular subdivision of the PVN in control- (A, B), NPY- (C, D), {alpha}-MSH- and NPY + {alpha}-MSH- (G, H) treated animals. Note numerous CRH neurons containing PCREB nuclei after NPY infusion (C, D). {alpha}-MSH infusion (E, F) increased the percentage of CRH neurons containing PCREB compared with control animals (A, B), but the percentage of CRH neurons containing PCREB in NPY + {alpha}-MSH animals (G, H) was essentially same as NPY-treated animals (compare C, D with G, H). Scale bars: A, C, E, G, 200 µm; B, D, F, H, 50 µm.

 


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  		Figure 5. Percentage of CRH neurons containing PCREB immunoreactivity in nuclei of aCSF-, NPY-, {alpha}-MSH-, and NPY+ {alpha}-MSH-treated animals.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The neuroendocrine adaptation to fasting is dependent upon endocrine, behavioral, and autonomic responses mediated by the circulating adipose-derived protein, leptin (35). Included among these responses is a reduction in thyroid hormone levels secondary to inhibition of proTRH mRNA in neurons of the hypothalamic PVN (1, 2) as a way to promote energy conservation by reducing thyroid thermogenesis (2, 36). Because pharmacological ablation of the arcuate nucleus results in a reduction in TSH and thyroid hormone levels (4), we have proposed that the arcuate nucleus exerts a net stimulatory effect on TRH neurons in the PVN. One of the peptides of arcuate nucleus origin that stimulates proTRH gene expression is {alpha}-MSH, a 13 amino acid peptide derived from the posttranslational processing of POMC (17). When administered exogenously into the CSF, {alpha}-MSH increases proTRH mRNA selectively in hypophysiotropic neurons in the PVN (8). Thus, during fasting, when circulating levels of leptin decline (37), POMC gene expression is suppressed (38), thereby contributing to the fall in thyroid hormone levels.

Simultaneous with suppression of POMC mRNA in the arcuate nucleus during fasting (38), there is a marked up-regulation of agouti-related peptide (AGRP) mRNA (39) and NPY mRNA (18) in a separate population of arcuate nucleus neurons. Because AGRP is a melanocortin receptor antagonist (40), the increase in AGRP gene expression during fasting presumably cooperates with the suppression of {alpha}-MSH to inhibit hypophysiotropic TRH by antagonizing the action of {alpha}-MSH at melanocortin receptors (40) and/or by exerting independent inhibitory effects as an inverse agonist at constitutively active melanocortin receptors (41, 42). Indeed, icv administration of AGRP (83–132) is capable of suppressing proTRH mRNA in ad libitum-fed animals and significantly reduces circulating thyroid hormone levels (43, 44). Similarly, the icv administration of NPY to fed animals causes profound reduction in proTRH gene expression and circulating thyroid hormone levels even though endogenous AGRP gene expression in arcuate nucleus neurons is suppressed (22). The mechanism(s) by which NPY inhibits hypophysiotropic TRH neurons, however, is not known.

At least two NPY receptors, Y1 and Y5, contribute to the inhibitory effects of NPY on the thyroid axis. When administered icv, the selective Y1 agonist, [Phe7, Pro34]pNPY, and Y5 agonist, [cPP1–7, NPY19–23 Ala31, Aib32, Q34]hPP, individually cause profound suppression of proTRH mRNA and circulating thyroid hormone levels in otherwise normal, ad libitum feeding animals (45). Y1 and Y5 receptors are coupled to pertussis toxin-sensitive G proteins that result in the inhibition of cAMP in several tissues (46). Because production of intracellular cAMP can result in activation of the protein kinase A cascade and hence, the phosphorylation of CREB protein (29), we reasoned that NPY might antagonize the activating effect of {alpha}-MSH on proTRH gene expression in hypophysiotropic neurons by reducing the phosphorylation of CREB. Previous studies in our laboratory have demonstrated that the administration of {alpha}-MSH induces the phosphorylation of CREB in the majority of hypophysiotropic TRH neurons in the PVN (13), suggesting that CREB phosphorylation may mediate the activating effects of {alpha}-MSH on the TRH gene. This concept is supported by the observation that in a cell culture system, activation of the TRH gene by {alpha}-MSH can be abolished by mutating the CRE in the promoter of the gene or by expressing a dominant inhibitor of CREB that cannot be phosphorylated (12). In this study, therefore, the effect of NPY treatment on the ability of exogenous {alpha}-MSH to induce the phosphorylation of CREB in hypophysiotropic TRH neurons was determined.

As previously observed (13), fasting animals showed little CREB phosphorylation in parvocellular neurons, with PCREB present almost exclusively in magnocellular neurons. NPY resulted in a modest increase in the number of parvocellular neurons in the PVN containing PCREB compared with aCSF-treated controls, similar to observations previously made by Sheriff et al. (47). The majority of the PCREB-labeled cells induced by NPY were located in mid-portions of the PVN in the lateral most aspects of the medial parvocellular subdivision, reminiscent of the location of hypophys-iotropic CRH (48), but with fewer cells in caudal portions of the medial parvocellular subdivision where hypophysiotropic TRH neurons are most densely concentrated (49). Whereas an increase in the number of PVN neurons that contain PCREB following NPY administration might seem paradoxical given the ability of NPY to inhibit the production of cAMP (23), recent studies by Sheriff et al. (47) in vitro suggest that NPY can phosphorylate CREB in cells that use the calcium/calmodulin-dependent protein kinase (Cam-kinase) pathway. This is supported by the observations that intrahypothalamic NPY administration increases total hypothalamic Cam-kinase II activity by approximately 63% (47) and NPY-receptor interaction can lead to the elevation of intracellular calcium (50, 51). Among the eight known Cam kinases, Cam-kinase II is the major enzyme present in synaptic junctions in many brain regions and is present in high concentration in the hypothalamus (52). Although Cam-kinase II {alpha} immunoreactive neurons are prevalent in the PVN, very few of these neurons have been observed in the medial parvocellular subdivision (53) where most of the NPY-induced CREB phosphorylated cells were located. Therefore, either other Cam-kinases are involved in the phosphorylation of CREB in this region of the PVN, or the phosphorylation of CREB in these neurons by NPY is mediated by another mechanism.

By double-labeling immunocytochemistry, the vast majority of TRH neurons in the PVN contained nuclei immunoreactive for PCREB following {alpha}-MSH administration, whereas in aCSF-treated control animals, only approximately 5% of the TRH neurons contained PCREB. Despite the increase in NPY-induced CREB phosphorylation in the mid portion of the medial parvocellular PVN, no significant increase in PCREB was observed in TRH neurons in the PVN in these animals compared with aCSF-treated controls. These data suggest that if Cam kinase mediates CREB phosphorylation by NPY, the enzyme may not be expressed in hypophysiotropic TRH neurons under these conditions, or that its effects are overridden by other mechanisms. Animals pretreated with NPY before {alpha}-MSH infusion, however, had a significant reduction in the percentage of TRH neurons containing PCREB when compared with {alpha}-MSH-treated animals, falling more than 40% of that induced by {alpha}-MSH, alone. This suppressive effect of NPY on {alpha}-MSH-induced CREB phosphorylation in TRH neurons may be the direct effect of inhibiting adenyl cyclase and the decline in production of cAMP as described above. However, other mechanisms should also be considered including dephosphorylation of CREB at Ser 133 by activation of a specific phosphodiesterase or by modulating the activity of the protein phosphatase 2B, an enzyme implicated in the dephosphorylation of CREB (54) and inhibition of CRE activity by the binding of inhibitory transcription factors such as the cAMP-responsive modulator and/or cAMP early repressor (55). The ability of NPY to affect any of these mechanisms in TRH neurons, however, is unknown. In addition, NPY might inhibit a separate population of neurons such as {alpha}-MSH-producing neurons in the hypothalamic arcuate nucleus, that through mono- or multisynaptic projections, activate TRH neurons in the PVN (8). The latter mechanism, however, is unlikely to explain the results observed in this study because the exogenous administration of {alpha}-MSH would be expected to act directly on melanocortin receptors expressed on TRH neurons (12).

The medial parvocellular subdivision of the PVN is also the major source of tuberoinfundibular CRH neurons (56) that regulate the secretion and synthesis of ACTH in the anterior pituitary gland (57). Whereas CRH is regulated by a negative feedback effect of circulating glucocorticoids (58), it can also be affected by a number of other centrally mediated responses (59, 60). Included among the substances known to activate CRH is NPY. NPY-IR axons heavily innervate the CRH-synthesizing neurons in the PVN (24) and the central administration of NPY into the PVN or lateral ventricles activates the hypothalamic-pituitary-adrenal axis (25) and stimulates the gene expression and immunoreactivity of CRH in the PVN (25, 26).

By double labeling immunocytochemistry, the vast majority (88.2 ± 2.9%) of CRH neurons in the PVN contained nuclei immunoreactive for PCREB following NPY administration, whereas in aCSF-treated control animals, only approximately 13% of the CRH neurons contained PCREB. In addition, approximately 60% of the total number of NPY-induced CREB-activated neurons in the PVN contained CRH, indicating that CRH neurons comprise the major cell type activated by NPY infusion, but not the only cell type. Because colchicine was not used in this study, however, we may have underestimated the total number of PCREB-positive cells that also contain CRH. Whereas {alpha}-MSH also increased CREB phosphorylation in CRH neurons, consistent with our previous results (13), the magnitude of the response was significantly greater following NPY administration and the combination of NPY and {alpha}-MSH was essentially the same as NPY infusion alone.

As CRH mRNA in PVN neurons is reduced during fasting when the gene expression of NPY in the hypothalamic arcuate nucleus is increased (18), it might seem intuitively inconsistent that the exogenous administration of NPY should induce the phosphorylation of CREB in CRH neurons because PCREB would be expected to activate these neurons (14, 15). In addition, CREB phosphorylation in PVN neurons was observed in only the minority of CRH neurons in the fasting animals receiving aCSF in this study when endogenous NPY secretion in the PVN should be increased (61). Exogenous administration of NPY into the CSF would be expected to influence all neurons in the PVN that express NPY receptors, however, whereas activation of endogenous NPY systems may be more selective. Indeed, the NPY systems involved in the innervation of the PVN originate from diverse locations within the brain including the hypothalamic arcuate nucleus (62, 63, 64, 65), bed nucleus of the stria terminalis (66), and lateral geniculate (67), and each region may contact discrete neuronal populations in the PVN. In particular, NPY-containing axon terminals that contact CRH neurons derive primarily from neurons outside of the arcuate nucleus (65). This conclusion is based on the demonstration that whereas the majority of CRH perikarya and proximal dendrites in the PVN are enveloped by NPY-containing axon terminals, only a small proportion of these neurons are contacted by axon terminals containing AGRP (65). Because the majority of NPY-producing neurons in the arcuate nucleus of the rat and human hypothalamus also contain AGRP (65, 68) and axons containing AGRP densely innervate the PVN and arise exclusively from arcuate nucleus neurons (69), it is inferred that the arcuate nucleus contributes little to the NPY innervation of CRH neurons. Rather, a prominent source of NPY containing axons in the PVN arises from the catecholaminergic A1 and C1–3 neuronal groups in the medulla (70). Because the majority of NPY contacts on CRH neurons in the rat PVN also contain the catecholamine synthesizing enzyme, phenylethanolamine N-methyltransferase (71), the main source of the NPY input to CRH neurons in the PVN may be shared with the ascending catecholaminergic pathways. In contrast, the arcuate nucleus is the major source for the NPY innervtion of TRH neurons in the PVN (21). Thus, increased NPY signaling in the PVN induced by fasting may have a selective effect on TRH neurons as well as other specific neuronal populations that receive monosynaptic projections from the arcuate-paraventricular pathway, whereas CRH neurons may be influenced by NPY primarily when brain stem catecholaminergic neurons are activated, perhaps as part of the acute stress response.

We conclude that NPY has selective and diverse actions in the PVN to regulate the phosphorylation of CREB. In TRH neurons, NPY derived primarily from hypothalamic arcuate nucleus neurons, can antagonize the central effects of {alpha}-MSH to induce the phosphorylation of CREB. Thus, during fasting, NPY may participate in inhibiting TRH hypophysiotropic gene expression by antagonizing melanocortin signaling through effects at the postreceptor level. In contrast, NPY, derived primarily from neurons outside of the arcuate nucleus, is capable of inducing the phosphorylation of CREB in CRH neurons, and under the appropriate physiologic conditions, may contribute to activation of the hypothalamic-pituitary-adrenal axis.


   Footnotes
 
This work was supported by NIH Grant DK-37021 and a grant from the Tufts-New England Medical Center Research Council.

Abbreviations: aCSF, Artificial CSF; AGRP, agouti-related peptide; Cam-kinase, calcium/calmodulin-dependent protein kinase; CRE, cAMP response element; CREB, cAMP response element binding protein; icv, intracerebroventricular; IR, immunoreactive; NPY, neuropeptide Y; PCREB, phosphoCREB; POMC, proopiomelanocortin; PVN, paraventricular nucleus.

Received July 3, 2002.

Accepted for publication September 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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