Involvement of Neuropeptide Y Y1 Receptors in the Regulation of ...

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Endocrinology 148(8):3666 –3673 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1730

Involvement of Neuropeptide Y Y1 Receptors in the Regulation of Neuroendocrine Corticotropin-Releasing Hormone Neuronal Activity Eugene L. Dimitrov, M. Regina DeJoseph, Mark S. Brownfield, and Janice H. Urban Department of Physiology and Biophysics (E.L.D., M.R.D., J.H.U.), Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064; and Department of Comparative Biosciences (M.S.B.), School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706 The neuroendocrine parvocellular CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus are the main integrators of neural inputs that initiate hypothalamic-pituitary-adrenal (HPA) axis activation. Neuropeptide Y (NPY) expression is prominent within the PVN, and previous reports indicated that NPY stimulates CRH mRNA levels. The purpose of these studies was to examine the participation of NPY receptors in HPA axis activation and determine whether neuroendocrine CRH neurons express NPY receptor immunoreactivity. Infusion of 0.5 nmol NPY into the third ventricle increased plasma corticosterone levels in conscious rats, with the peak of hormone levels occurring 30 min after injection. This increase was prevented by pretreatment with the Y1 receptor antagonist BIBP3226. Immunohistochemistry showed that CRH-immunoreactive neurons co-

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HE PARAVENTRICULAR NUCLEUS of the hypothalamus (PVN) is the final common pathway linking excitatory stress response signals to activation of the pituitaryadrenocortical axis (1). The stress response is a reflex that incorporates both neuronal and endocrine components. In the CNS, the role of integrating stress signals and producing a neuroendocrine output to the hypothalamic-pituitary-adrenal (HPA) axis belongs to CRH neurons (2, 3). This 41amino-acid peptide is the most powerful ACTH secretogogue that also causes a wide spectrum of neuroendocrine, autonomic, and behavioral changes typical of a stress reaction. This diverse functionality is achieved by CRH actions not only as a hormone but also as a neurotransmitter and a neuromodulator in a number of brain structures (3, 4). The efferent fibers of neuroendocrine CRH neurons arise from cell bodies solely in the PVN, which project to the external layer of the median eminence, from which CRH circulates to the anterior pituitary and stimulates ACTH release from the corticotropes. ACTH in the blood ultimately stimulates gluFirst Published Online April 26, 2007 Abbreviations: CRF, Corticotropin-releasing factor; FG, FluoroGold; HPA, hypothalamic-pituitary-adrenal; ICC, immunocytochemistry; icv, intracerebroventricular; ir, immunoreactivity; NPY, neuropeptide Y; pCREB, phosphorylated cAMP response element-binding protein; pCREB, phosphorylated cAMP response element-binding protein; PVN, paraventricular nucleus; SON, supraoptic nucleus; VP, vasopressin; Y1r, Y1 receptor. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

expressed Y1 receptor immunoreactivity (Y1r-ir) in the PVN, and a majority of these neurons (88.8%) were neuroendocrine as determined by ip injections of FluoroGold. Bilateral infusion of the Y1/Y5 agonist, [leu31pro34]NPY (110 pmol), into the PVN increased c-Fos and phosphorylated cAMP response element-binding protein expression and elevated plasma corticosterone levels. Increased expression of c-Fos and phosphorylated cAMP response element-binding protein was observed in populations of CRH/Y1r-ir cells. The current findings present a comprehensive study of NPY Y1 receptor distribution and activation with respect to CRH neurons in the PVN. The expression of NPY Y1r-ir by neuroendocrine CRH cells suggests that alterations in NPY release and subsequent activation of NPY Y1 receptors plays an important role in the regulation of the HPA. (Endocrinology 148: 3666 –3673, 2007)

cocorticoid release from the adrenal gland. The glucocorticoids (mainly cortisol in humans and corticosterone in rats) produce a large array of metabolic and nonmetabolic effects including, but not restricted to, gluconeogenesis, glycogenolysis, lipolysis, proteinolysis, and immunosuppression and the actions of epinephrine, norepinephrine, and glucagons. All of these processes are aimed at helping the organism cope with adverse conditions (stress reaction) (5). Stress, in general, activates a number of neural inputs to the PVN (6). One neuropeptide with a prominent input to the PVN is neuropeptide Y (NPY). NPYergic inputs to the PVN arise from the arcuate nucleus of the hypothalamus and the brain stem in which NPY is colocalized within catecholaminergic cells (7–9). Numerous anatomical studies demonstrate that NPY projections from both the brain stem and arcuate nucleus are in close apposition to CRH cell bodies and fibers (1, 7–9). The activity of NPY neurons projecting to the PVN is increased by food deprivation (10, 11), cold (12), immobilization (13), hemorrhage (14), and foot shock (15), indicating that NPY neurons could transmit different signals (food intake, stress, cardiovascular tone) to the PVN. Previous studies showed that administration of NPY into the PVN is able to modulate the activity of cells within the different divisions of the PVN and increase corticosterone release (16). Central administration of NPY into the PVN increases c-Fos expression in the PVN, but the cell types expressing c-Fos have not been identified (17). Complementary to these findings are data demonstrating that hypothalamic CRH mRNA levels are increased in response to NPY

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Dimitrov et al. • NPY Regulation of CRH Neuronal Activity

(18), as are plasma ACTH and corticosterone levels in anesthetized animals (18, 19). Some of the most important functions of NPY in the PVN are its participation in feeding and sympathomimetic transmission (20, 21). Therefore, identifying the role that NPY plays in the modulation of PVN function is important to further understand the pleiotropic role of NPY as a modulator of neural and endocrine stress responses. The present studies were designed to identify sites of action of NPY within the PVN as it relates to the regulation of HPA axis activity. Multiple-label immunohistochemistry was used to identify the distribution of Y1 receptors (Y1rs) in populations of neuroendocrine cells in general, CRH neurons specifically, and determine the expression of activational markers [c-Fos and phosphorylated cAMP response element-binding protein (pCREB)] after intra-PVN injections of the Y1/Y5 receptor agonist [leu31pro34]NPY. The functional neuroendocrine implication of these cytochemical results was determined by measurement of plasma corticosterone concentration in conscious animals after intracerebral PVN injection of NPY and [leu31pro34]NPY. Materials and Methods Animals Male Sprague-Dawley rats (Charles River, Wilmington, MA; 250 –300 g) were housed three per cage in an Association Assessment and Accreditation of Laboratory Animal Care accredited facility with standard lighting and temperature conditions (14 h light, 10 h dark cycle, 22 C room temperature) and allowed free access to food and water. The animals were given a 5-d acclimatization period before beginning any experiments. All studies were conducted according to National Institutes of Health guidelines and with the approval of the Rosalind Franklin University Animal Care and Use Committee.

Identification of neuroendocrine neurons To identify populations of hypothalamic neuroendocrine neurons that expressed CRH and Y1r immunoreactivity (ir), we used ip injection of 5 mg FluoroGold (FG) dissolved in 0.9% saline (Fluorochrome, LLC, Denver, CO) as described elsewhere (22, 23). On the fifth day after FG injection, the animals were perfused with 4% paraformaldehyde and the brains removed and processed for CRH/Y1r-ir using immunocytochemistry (ICC).

Implantation of third ventricle and PVN cannula Animals were anesthetized with ketamine (90 mg /kg, ip) and xylazine (10 mg/kg, ip) and placed in a Stoelting stereotaxic apparatus. Longitudinal skin incision and removal of pericranial connective tissue exposed bregma and lambda sutures of the skull. A 26-gauge stainless steel guide cannula (Plastics One, Roanoke, VA) was implanted into the third ventricle (anteroposterior: ⫺1.0 mm, lateral: ⫺0.05 mm, dorsoventral: ⫺8.2 mm from bregma) or a double 26-gauge stainless steel guide cannula (Plastics One) was implanted into the PVN (anteroposterior: ⫺1.8 mm, lateral: ⫾ 0.5 mm, dorsoventral: ⫺7.5 mm from bregma). The cannulae were secured to the skull with three screws and acrylic dental cement. Stylets were placed in each cannula to maintain patency. Animals were handled daily, and the stylets were manipulated to acclimate the animals to the microinjection procedure.

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atrium. The catheter was secured in the vein with silk ligatures. The distal end of the catheter was tunneled under the skin and exteriorized through the nape of the neck. The catheter was stabilized with a plastic cuff and trimmed to ensure that the animal could not gain access to it. To ensure patency, the dead space of the catheter was filled with heparinized saline (100 U/ml) and closed with a plug.

Drug infusion On the day of the experiment, the animals were brought to the experiment room in which they acclimated to the surroundings. Two hours before the start of the experiment, 33-gauge internal injectors were inserted into the intracerebral cannula, which extended 1 mm beyond the tip of the guide cannula. A cannula connector was used to attach the injectors to a syringe pump for delivery of equal volumes of 0.5 ␮l vehicle (0.1% BSA in 0.9% saline) or 110 pmol (250 ng) of [leu31pro34]NPY (AnaSpec, San Jose, CA) into the PVN over a period of 5 min. For third ventricle injections, to ascertain the role of Y1r in regulating HPA activity, animals were injected with either 0.9% saline vehicle (5 ␮l) or BIBP3226 (1 ␮g; Peninsula Laboratories, Belmont, CA) 20 min before injection with either 1% BSA or NPY (0.5 nmol). The injectors remained in place for the duration of both experiments. Blood samples of 0.2 ml were collected at predetermined times before and after drug infusion and placed in cold microcentrifuge tubes containing 5 ␮l of 100 IU heparin in saline. Blood was centrifuged for 20 min at 1800 rpm; the plasma was collected and frozen (⫺20 C) until determination of plasma corticosterone levels. The volume of withdrawn blood was replaced with an equal volume of heparinized saline (50 IU/ml). Hematocrit was measured at the beginning and end of the experiment. The animals for pCREB immunostaining were killed 15 min, and for c-Fos 90 min, after the start of the infusion. Probe placement was verified histologically and only those animals that had cannula inserted above the PVN or into the third ventricle were included in the study (Fig. 1).

ICC Animals were anesthetized with pentobarbital (50 mg/kg, ip) and transcardially perfused with PBS containing 0.1 g procainamide and 5000 IU heparin per 100 ml of solution, followed by cold 4% paraformaldehyde in PBS (pH 7.4). The brains were removed and placed in fixative at 4 C overnight. Forty-micrometer-thick coronal sections throughout the PVN were obtained using a vibratome.

Double-label ICC After sectioning, free-floating sections were washed with PBS, incubated in 1% H2O2 for 15 min, and blocked with 5% normal donkey serum (Sigma, St. Louis, MO) in ICC buffer [0.2% gelatin, 0.01% thimerosal, 0.002% neomycin in PBS (pH 7.5)] for 30 min, and then incubated for 48 h at 4 C in primary antibody (CRH, 1:15,000; Biogenesis, Kingston, NH) diluted in ICC buffer. Next, the sections were rinsed with ICC buffer and incubated in donkey antimouse biotinylated antiserum (Jackson ImmunoResearch Labs, West Grove, PA; 1:2,500) for 1 h at room temperature.

Jugular catheters After 5–7 d of recuperation from the stereotaxic surgery, a cohort of animals received atrial catheters for blood sampling for determination of plasma corticosterone concentration. Animals were anesthetized with 4% halothane, the jugular vein was exposed, and a silastic catheter (0.025 in. inner diameter; 0.047 in. outer diameter) was inserted into the right

FIG. 1. Photomicrograph of a coronal brain section (bregma ⫺1.8 mm) showing bilateral injection cannula tracts above CRH immunoreactive cells in the PVN. Probe tracts are designated with an asterisk. 3v, Third ventricle.

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After washes with ICC buffer, the sections were incubated in Vectastain Elite ABC; (Vector Laboratories, Burlingame, CA; 1:500) for 30 min. After rinses with PBS, they were incubated with biotinylated tyramide (3 ␮g/ml) for 10 min. The tissue was washed again with PBS and incubated in CY3-conjugated streptavidin (Jackson ImmunoResearch Labs; 1:250) for 3 h. After additional PBS washes, the sections were incubated in Y1r antibody (ImmunoStar, Inc., Hudson, WI; 1:800) (31) for 48 h at 4 C. After PBS rinses, the sections were incubated in CY5 donkey antirabbit serum (Jackson ImmunoResearch Laboratories; 1:250) for 3 h. The last washes included Tris-buffered saline [100 mm Tris base, 150 mm NaCl (pH 7.5)]. The sections were mounted on gelatin-subbed slides, air dried, and coverslips applied with 2.5% polyvinyl alcohol-1,4-diazabicyclo[2.2.2]octane (DABCO) antifade mounting medium.

Triple-label ICC After the staining for CRH as indicated above, the sections were incubated at 4 C for 48 h with c-Fos (Oncogene, San Diego, CA; 1:12,000) or pCREB (Cell Signaling, Beverly, MA; 1:1,000) rabbit antibodies. After washes with PBS, the sections were incubated for 5 h in CY5-conjugated donkey antirabbit secondary antibody. The last staining in the triplelabel ICC was for Y1r-ir as described above using fluorescein isothiocyanate donkey antirabbit secondary antibody. The last washes included TBS [100 mm Tris base, 150 mm NaCl (pH 7.5)]. The sections were mounted on gelatin-subbed slides, air dried, and coverslips applied with 2.5% PVA-1,4-diazabicyclo[2.2.2]octane mounting medium.

Assessment of ICC The specificity of all immunoreactive signals was determined by either omitting the primary antibody or by preadsorbing the primary antibody with saturating amounts (10 –15 ␮g/ml) of the immunogenic peptide. Sections through the PVN were atlas matched according to Paxinos and Watson (24). For assessment of FG, CRH, and Y1r-ir immunolabeling, the staining was visualized in representative sections at various levels relative to bregma using a Eclipse E600 microscope (Nikon, Melville, NY). Photomicrographs of the PVN sections were obtained with a Spot II camera and the number of FG/Y1r-ir, CRH/Y1r-ir, and FG/CRH/Y1r-ir cells was counted at different rostral-caudal levels of the PVN using MetaMorph software program (Universal Imaging, Downingtown, PA). The presence of FG in hypothalamic neurons was directly visualized using an UV filter (excitation: 360 nm). For the analysis of pCREB or c-Fos and Y1r-ir/CRH immunoreactivity, confocal microscopy (Fluoview; Olympus, Center Valley, PA) was used to evaluate colocalization. The wavelength of the three channels is 488 nm for fluorescein isothiocyanate, 543 nm for CY3, and 633 nm for CY5. The expression of CRH/Y1/c-Fos and CRH/Y1/pCREB immunoreactivity was assessed on three sections per level relative to bregma.

FIG. 2. Plasma corticosterone levels after infusion of 0.5 nmol NPY into the third ventricle with or without pretreatment with BIBP3226. *, Significantly different from 0 time point and 30-min vehicle (P ⬍ 0.05, repeated ANOVA, Student-Newman-Keuls test).

To assess the presence of Y1r-ir on hypothalamic neuroendocrine CRH neurons, animals were injected with FG ip, followed by ICC for CRH-ir and Y1r-ir. The ip injection of FG generated intense labeling of neurons in the hypothalamic nuclei that correlated with the known distribution of neuroendocrine neurons in the PVN and supraoptic nucleus (SON; Fig. 3). FG particles were visible as fine granules in the cytoplasm and fibers. FG labeling was also noted in the median eminence, subfornical organ, and endothelial cells of blood vessels. In the PVN, both parvo- and magnocellular neurons showed robust FG labeling throughout the rostrocaudal extent of the nucleus. Numerous parvo- and magnocellular neurons of the PVN also demonstrated a strong Y1r-ir signal. In most of the cells, Y1r-ir was confined mainly to the cytoplasm and cytoplasmic membrane. Intensely stained Y1r-ir fibers were also observed in the PVN. The majority of Y1r-ir expressing cells in the magnocellular division of the PVN were FG positive. There also was a con-

RIA Plasma corticosterone levels were measured using the ImmuChem double-antibody 125I RIA kit manufactured by MP Biomedicals (Orangeburg, NY). All samples from each experiment were processed together and the sensitivity of the assay was 7.7 ng/ml.

Statistics Data are reported as mean ⫾ sem. Data were analyzed using a Student’s t test or a two-way ANOVA followed by Student-Newman-Keuls test. The results were considered significantly different when P ⬍ 0.05.

Results

To examine the effect of NPY receptor activation on the activity of the HPA axis, NPY was injected into the third ventricle either after vehicle or pretreatment with the Y1 receptor antagonist, BIBP3226 (25). Infusion of 0.5 nmol NPY significantly increased plasma corticosterone levels after 30 min (Fig. 2). Prior treatment with BIBP3226 effectively prevented the NPY-induced corticosterone secretion but did not alter corticosterone levels on its own.

FIG. 3. Photomicrograph of FG labeling of neuroendocrine cells of PVN and SON 5 d after ip injection of FG. 3V, Third ventricle; dp, paraventricular hypothalamic nucleus, dorsomedial cap; pm, lateral magnocellular; mp, medial parvocellular.


siderable degree of colocalization of Y1r-ir and FG in cells within the parvocellular PVN. Immunohistochemistry was applied to identify whether neuroendocrine CRH cells express Y1r-ir within the PVN. Overall, the distribution of hypothalamic CRH-ir neurons was restricted to the parvocellular division of the nucleus as previously discovered (3). The most rostral cluster of CRH neurons appeared as a small group in the center of the anterior parvocellular subdivision. The largest congregation of CRH neurons was distributed evenly between the mediodorsal, medioventral, and the dorsal parvocellular sections of PVN. The most posterior group consisted of horizontally oriented, fusiform neurons in the lateral parvocellular subdivision that exhibit long varicose fibers. Neurons containing CRH/FG, CRH/Y1, FG/ Y1, and CRH/FG/Y1-ir were found throughout the PVN (Fig. 4). FG was visible in an average of 82.8 ⫾ 4.5% (113 ⫾ 7 cells) of the CRH neurons (137 ⫾ 10 total CRH-ir cells), indicating that these were neuroendocrine neurons (Fig. 5A). There was a higher degree of FG/CRH colocalization within the anterior aspect of the nucleus (92.9 ⫾ 3.7% at ⫺1.4 mm to bregma), and this percentage gradually declined in the posterior direction (76.8 ⫾ 4.2% at ⫺2.12 mm to bregma; Fig. 5B). Of the total number of CRH neurons, 80.2 ⫾ 2.3% (110 ⫾ 8 cells) also coexpressed Y1r-ir within their perikarya (Fig. 5A). The pattern of CRH/Y1r-ir distribution within the PVN was similar to that of CRH/FG-labeled cells (Fig. 5B). Triple-labeled cells (CRH/ FG/Y1; 101 ⫾ 6 cells) represented 73.5 ⫾ 5.8% of the total CRH population and 88.8 ⫾ 6.5% of the neuroendocrine CRH/FG neurons (Fig. 5A). The highest degree of coexpression of all three markers was detected in the anterior parts of PVN and waned posteriorly (Fig. 5B). Activation of Y1 receptors in the PVN by local infusion of 110 pmol of [leu31pro34]NPY led to a significant increase of c-Fos expression throughout PVN detected 90 min after the injection (Fig. 6, B and D). The patterns of c-Fos expression were confined within the borders of the PVN with a few scattered c-Fosexpressing cells outside the PVN area. Both parvo- and magnocellular neurons in the PVN were activated, as assessed by c-Fos immunoreactivity. Approximately 89.5% of the total number of c-Fos immunoreactive-positive cells also expressed Y1r-ir. Small numbers of magno- as well as parvocellular Y1r-ir cells without c-Fos were observed in each section. Negligible c-Fos staining was found after BSA injection, mainly in an area

FIG. 4. Photomicrograph of CRH neurons (red) (A), FG (green) (B), and Y1r-ir (blue) (C) in the dorsal parvocellular division of the PVN (bregma ⫺1.8 mm). D, Merged images. Arrows indicate triplelabeled cells; arrowheads indicate double-labeled cells. Horizontal arrowhead points to CRH neuron without Y1r-ir; vertical arrowhead indicates FG/Y1r-ir neurons. Yellow denotes FG/CRH colocalization; light blue, FG/Y1r colocalization; pink/purple, triple-labeled cell. dp, Dorsal parvocellular division; mp, medial parvocellular division. Scale bar, 100 ␮m.


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FIG. 5. A, Percentage of CRH-immunoreactive neurons expressing FG, Y1r-ir or FG/Y1r-ir. B, Rostral-caudal distribution of CRH and FG or Y1r-ir cell populations in the PVN (n ⫽ 4/group). *, Significantly different from the corresponding value at ⫺1.4 mm from bregma (ANOVA followed by Student-Neuman-Keuls test).

around the tip of the injector. CRH neurons coexpressing c-Fos were estimated to be 28.4 ⫾ 3.8% of the total number of CRH-ir cells in the [leu31pro34]NPY group vs. 1.3 ⫾ 0.3% in the control group (t ⫽ 5.278, df ⫽ 6, P ⫽ 0.0019; Figs. 6, A–D, and 7); about 83.8% of the CRH/c-Fos neurons were Y1r-ir positive as well (Fig. 8, A–C). The same dose of [leu31pro34]NPY also increased pCREB expression in the PVN 15 min after the infusion (Fig. 6, F and H). Activated neurons were evident in parvo- as well as magnocellular subdivisions. CRH neurons showing pCREB coexpression were 11.7 ⫾ 0.6 vs. 0.5 ⫾ 0.3% in the control

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FIG. 7. Percent of CRH neurons expressing c-Fos 1.5 h after infusion of 110 pmol [leu31pro34]NPY. **, Significantly different from BSAtreated control (P ⫽ 0.0019, Student’s t test, n ⫽ 5/group).

groups. Infusion of BSA into the PVN did not increase corticosterone levels. Discussion

FIG. 6. Patterns of c-Fos and pCREB expression in the PVN after local infusion of 110 pmol [leu31pro34]NPY. CRH (A) and c-Fos ir (B) in a BSA-injected control animal (90 min after injection) are shown. CRH (C) and c-Fos ir (D) in the PVN 90 min after infusion of [leu31pro34]NPY are shown. CRH (E) and pCREB ir (F) in the PVN are shown 15 min after BSA injection. CRH (G) and pCREB ir (H) in the PVN are shown 15 min after infusion of [leu31pro 34]NPY. Arrows indicate double-labeled cells. dp, Dorsal parvocellular division; mp, medial parvocellular division; pm, posterior magnocellular division. Scale bar, 200 ␮m.

group (t ⫽ 16.25, df ⫽ 6, P ⬍ 0.0001; Figs. 6, E–H, and 9). Triple-labeled neurons (CRH/Y1/pCREB) were 85.1% of the total number of CRH/pCREB cells (Fig. 8, D–F). Infusion of [leu31pro34]NPY but not 0.1% BSA into the PVN significantly elevated plasma corticosterone 15 min after the injection (Fig. 10). This was the peak of corticosterone secretion, and the values returned to baseline and were not significantly different from BSA controls 90 min after injection (BSA:168.4 ⫾ 33.7 vs.[leu31pro34]NPY: 261.8 ⫾ 25.2 ng/ ml). Baseline plasma corticosterone levels were not different between the vehicle (BSA) control and [leu31pro34]NPY

The results of the present studies demonstrate that activation of NPY receptors in the hypothalamus stimulates CRH neurons in the PVN and increases plasma corticosterone levels. The elevation of plasma corticosterone levels after intracerebroventricular (icv) injection of NPY was blocked by administration of the Y1 receptor antagonist, BIBP3226, suggesting a strong influence of Y1 receptor activation over the regulation of the HPA axis. The expression of both c-Fos and pCREB was increased by the Y1/Y5 receptor agonist [leu31pro34]NPY, suggesting that the downstream effects of NPY can be mediated by different or complementary signaling mechanisms. The high level of NPY Y1 receptor expression within neuroendocrine cells, and specifically CRH immunoreactive cells, demonstrates the ability of NPY to directly influence neuroendocrine function. The involvement of NPY in the control of hypothalamic/ endocrine function has been intensely studied, and the focus of NPY and CRH interactions is important from the aspect of the control of stress responses (26). Numerous studies demonstrate the close apposition of NPY fibers to CRH neurons (1) and that the brain stem and arcuate nucleus of the hypothalamus provide the source of these fibers (7, 9). Light and electron microscopy have shown that CRH neurons have multiple contacts with NPY-positive fibers and receive NPY axon terminals from multiple sources (1, 8, 27, 28). These histological details indicate a possibility that CRH neurons in the PVN are equipped with NPY receptors. Early studies show that NPY delivery into the PVN increases the firing rate of neurons within the PVN and through the PVN produces increases in plasma corticosterone levels (16). NPY-induced increases in plasma corticosterone levels are well accepted, yet the contribution of the NPY receptor subtype to this effect has not been elucidated. Using anatomical methodologies, either the protein or mRNA for the different NPY receptor subtypes (Y1, Y2, Y4, and Y5) has been detected in the PVN (29 –31). Activation of ACTH and/or corticosterone release has been observed after icv administration of Y2 and Y5 (32, 33) but not Y4 agonists (33). Studies by Hastings et al. (34) showed that NPY-induced increases in CRH release from hypothalamic explants can be blocked by prior treatment with the Y1 antagonist GR231118. Tebbe et al. (35) supported the role for PVN NPY Y1 receptors in CRH-mediated



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FIG. 8. Photomicrograph of CRH- (red)/ c-Fos-ir (blue) (A); Y1r-ir (green) (B) and CRH/c-Fos/Y1r-ir (C) in the PVN 90 min after injection of 110 pmol [leu31pro 34 ]NPY. Photomicrographs of CRH- (red)/ pCREB-ir (blue) (D); Y1r-ir (green) (E) and CRH/pCREB/Y1r-ir cells (F) in the PVN 15 min after intra-PVN injection of 110 pmol [leu31pro34]NPY. Arrows point to triple-labeled cells. Horizontal arrowheads indicate CRH neurons without cFos- or pCREB-ir. Vertical arrowheads point to Y1r-ir cells expressing c-Fos or pCREB. Yellow denotes CRH/Y1r colocalization; light blue, pCREB or cfos/Y1r colocalization; pink/purple, triple-labeled cell. Mp, Medial parvocellular division; pm, posterior magnocellular division. Scale bar, 50 ␮m.

inhibition of gastric acid secretion. We have shown that administration of the Y1 antagonist, BIBP3226, attenuates the increase in plasma corticosterone induced by NPY injection into the third ventricle just anterior to the PVN. Administration of the Y1/Y5 agonist, [leu31pro34]NPY, directly into the PVN also produces elevations in plasma corticosterone levels. Together these data support the predominant involvement of the Y1 receptor in the regulation of HPA axis activity. However, we cannot rule out other NPY receptors in contributing to this response because the ligand used these studies can activate Y5 receptors, and Y5r-ir has been detected on CRH neurons within the PVN (36). The peak of corticosterone release in response to activation of NPY receptors occurs 15 min earlier when the peptide is injected into the PVN than the icv infusion. This recapitulates the time course previously reported (19) and would be expected because the peptide would need more time to diffuse to sites of action when injected icv. Previous studies by Small et al. (33) suggested that icv NPY stimulates ACTH release via a novel NPY receptor. In that study, the administration of BIBP3226 failed to block NPY-stimulated ACTH release; however, the peptides were administered into the lateral ventricles. This more generalized infusion would result in the wider spread of the drug than the third ventricle injection used here, which could stimulate an additional number of pathways influencing hormone secretion. The present immunohistochemical experiments indicate a

direct link between NPY circuitry and HPA axis activity in the rat brain. Different parts of the hypothalamus including the PVN contain Y1 and Y5 receptors in cell perikarya as well as on neuronal fibers (29, 31). Whereas the above studies support the involvement of the Y1r in the regulation of HPA axis activation, we used double-label ICC to demonstrate the presence of Y1-ir on populations of CRH-expressing cells. One technical issue that caused concern was the use of colchicine to visualize CRH-immunoreactive cells. In previous studies we found that colchicine, while increasing CRH immunoreactivity, significantly decreased Y1r-ir (our unpublished results). Colchicine treatment also decreases galanin receptor expression as reported by others (37). To visualize the populations of hypothalamic CRH-ir cells in the present studies, we used biotinylated tyramide amplification for ICC (38). This method increased the immunohistochemical signal so that we could easily visualize CRH cells without disrupting Y1r-ir expression. To identify populations of neuroendocrine CRF neurons, FG was administered peripherally as previously demonstrated (22, 23) and labeled neuroendocrine populations within the hypothalamus (including PVN, SON, periventricular and arcuate nuclei). Colabeling with the Y1 antibody demonstrated a strong coexpression of FG and the Y1r throughout the PVN, particularly within the magnocellular division of the PVN and SON coincident with Y1r-ir expression in vasopressin (VP) and oxytocin immunoreactive neurons (39). Additionally, Y1r-ir and FG were

FIG. 9. Percent of CRH neurons expressing pCREB 15 min after infusion of 110 pmol [leu31pro34]NPY. ***, Significantly different from BSA-treated control (P ⬍ 0.0001, Student’s t test, n ⫽ 4/group).

FIG. 10. Plasma corticosterone levels in animals receiving 0.1% BSA or 110 pmol [leu31pro34]NPY into PVN. *, Significantly different from preinjected control and BSA-treated animals (P ⫽ 0.018, two-way repeated-measures ANOVA and Student-Neuman-Keuls test, n ⫽ 5/group).

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present throughout the parvocellular PVN in which we further identified colocalization within CRH neurons and a number of other unidentified neurons that could be TSHreleasing factor neurons (40). We also observed a small number of CRH neurons without FG (17.25%). These neurons likely belong to a class of CRH projection neurons, which comprise the anatomic arm of the stress response projecting to the sympathetic as well as parasympathetic divisions of the autonomic nervous system (41, 42). The observation that hypothalamic neuroendocrine CRH neurons express the postsynaptic Y1r supports the possibility of direct signal transmission from NPY fibers onto CRH cells. Studies from Pronchuk et al. (43) showed that activation of NPY receptors in the PVN inhibits presynaptic ␥-aminobutyric acid release in the PVN. Inhibition of ␥-aminobutyric acid results in an activation of CRH neurons (44). Whereas it is difficult to separate out the presynaptic from postsynaptic mechanisms in vivo, it is very likely that the NPY signaling to CRH neurons is an admixture of these two mechanisms. Stimulation of CRH neurons by NPY has been illustrated through the use of pCREB and c-Fos activational markers. The rationale for using c-Fos and pCREB as markers is that they have a broad application in assessing neuronal activation after exposure to different stress paradigms, neurochemicals, and receptor agonists. Y1rs are known to couple to a pertussis toxin-sensitive Gi/o protein, and the downstream intracellular signaling pathway includes inhibition of cAMP production, activation of phospholipase C, increase of cytoplasmic Ca 2⫹ levels with subsequent activation of calcium/calmodulin-dependent kinase II, and phosphorylation of CREB (45). pCREB has previously been shown to bind to the promoter of the CRH gene, thus initiating gene transcription (46). Whereas the increased expression of these cellular markers by NPY has been shown previously (45), the effect of local injection of NPY analogs into the PVN and the phenotype of these cells has not been addressed. Injection of NPY into the lateral ventricle increases the phosphorylation of CREB in CRH-immunopositive cells in the PVN (47). In the current studies, injection of [leu31pro34]NPY into the PVN dramatically increased c-Fos and pCREB in the PVN, and the majority of these cells also expressed immunoreactivity for the Y1r (85%). The observation that activated CRH neurons highly coexpress Y1r-ir further supports the model of a possible direct link between NPY and CRH neurons. c-Fos and pCREB expression was observed after the appropriate times for intracellular signal transmission with both markers: 90 min for c-Fos and 15 min for pCREB, respectively. The difference in the percent of activated CRH cells with c-Fos vs. cells with pCREB could be attributed to the fact that in the first occasion [leu31pro 34]NPY had more time to diffuse into the tissue, thus stimulating a larger area of PVN. Whereas these numbers are modest, it no less suggests that locally released NPY in the PVN could stimulate CRF cells via two different/complementary signaling pathways. With respect to activation of the HPA axis, Kovacs and Sawchenko (48) have postulated that the increase in CREB phosphorylation follows a time course that parallels the stimulation of CRH heteronuclear RNA expression, whereas immediate early gene expression is closely related to changes


observed in parvocellular VP gene expression. Although no reports have suggested a role for NPY in increasing parvocellular VP gene expression, NPY does increase CRH mRNA levels (18), and this may occur, in part, through pCREB. Despite the strong c-Fos expression, the infusion of [leu31pro 34 ]NPY into PVN failed to increase c-Fos coexpression in the VP parvocellular subdivision of the nucleus (our unpublished observations). Whether NPY increases c-Fos and pCREB within the same cells is not evident by these studies, and it is therefore interesting to postulate that NPY may activate different signaling pathways in subsets of PVN cells. The results of our studies favor the hypothesis that NPY functions as an integrator between different stress signals and a neuroendocrine response to stress. CRH/Y1r-ir neurons represent the anatomical substrate of such integration. Stressors such as water and food deprivation (49), immobilization, cold, hypoglycemia, hemorrhage, and pain raise plasma corticosterone levels in rats (50). The same stressors evoke substantial increase of NPY or Y1r signal in the hypothalamus (10 –15, 39). The feeding circuitry could serve as a particular illustration of the possible bond between NPY and the HPA axis. The circuitry consists of several interconnected brain structures in which NPY is a major neurotransmitter. Disruption of energy homeostasis is a strong command for glucocorticoid release (51, 52). NPY is in position to transmit a signal of this nature and evoke HPA axis activation directly via neuroendocrine CRH neurons expressing the postsynaptic Y1r, thus connecting the feeding to stress response. Further investigation is required to determine what implications neuroendocrine CRH/Y1r-ir neurons have in different stressful paradigms as well as the projection sites of nonendocrine neurons of the same phenotype. Acknowledgments Confocal images were obtained using the Rosalind Franklin University of Medicine and Science Multiuser Imaging Facility under the direction of Dr. Daniel A. Peterson. Received December 22, 2006. Accepted April 13, 2007. Address all correspondence and requests for reprints to: Janice H. Urban, Ph.D., Department of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, Illinois 60064. E-mail: [email protected]. This work was supported by National Institutes of Health Grant MH62621 (to J.H.U. and M.S.B.). Disclosure Statement: E.L.D., M.R.D., and J.H.U. have nothing to disclose. M.S.B. consults for Immunostar, Inc.

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