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Endocrinology, doi:10.1210/en.2006-0048
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Endocrinology Vol. 147, No. 8 3737-3745
Copyright © 2006 by The Endocrine Society

Neuroendocrine Stress But Not Feeding Responses to Centrally Administered Neuropeptide Y Are Suppressed in Pregnant Rats

Paula J. Brunton, Juliana Bales and John A. Russell

Laboratory of Neuroendocrinology, Centre for Integrative Physiology, University of Edinburgh, Edinburgh EH8 9XD, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Paula J. Brunton, Laboratory of Neuroendocrinology, Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, Scotland, United Kingdom. E-mail: p.j.brunton{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Metabolic peptides such as orexin and neuropeptide Y (NPY) exert profound effects on feeding but also act centrally to stimulate the hypothalamo-pituitary-adrenal (HPA) axis. In late pregnancy the HPA axis is hyporesponsive to centrally administered orexin-A, which signals to the HPA axis, in part, via arcuate NPY neurones. We investigated whether reduced HPA axis responses to orexin may be a consequence of down-regulated NPY signaling to the paraventricular nucleus (PVN) in pregnancy. Pregnant (d 21) and virgin rats were blood sampled for ACTH, corticosterone, and oxytocin (also a stress hormone in rats) before and after intracerebroventricular NPY or vehicle. Behavior was monitored. Rats were killed 4 h after NPY and brains removed for in situ hybridization. In another experiment rats were given vehicle or NPY, perfuse fixed 90 min later, and brain sections processed for Fos and oxytocin immunocytochemistry. NPY significantly increased ACTH, corticosterone and oxytocin secretion in the virgins but had no such effect on ACTH or oxytocin in the pregnant rats; the corticosterone response to NPY was markedly attenuated in pregnant rats. NPY increased CRH and vasopressin mRNA expression in the parvocellular PVN and stimulated Fos expression in magnocellular supraoptic and PVN oxytocin neurones of virgin but not pregnant rats. NPY increased food intake and drinking similarly in virgin and pregnant rats. Thus, neuroendocrine stress responses to central NPY are absent in late pregnancy, whereas ingestive behavioral responses are intact. These changes may explain the similarly attenuated HPA response to centrally administered orexin-A and will favor anabolic adaptations in pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMO-PITUITARY-adrenal (HPA) axis aids restoration of homeostasis after perturbation by stressful stimuli and is essential in maintaining glucose homeostasis (1). The HPA axis is excited by acute glucopenia and peptides known to regulate appetite, resulting in glucocorticoid secretion as part of the compensatory response to reduced glucose availability. Orexins (A and B) are hypothalamic neuropeptides that have stimulatory actions on feeding and arousal and also activate the HPA axis by stimulating the CRH neurones in the parvocellular division of the paraventricular nucleus (pPVN) (2).

Another orexigenic peptide, neuropeptide Y (NPY) is involved in mediating the effects of orexin-A on the HPA axis (3, 4, 5). Both orexin-A (6, 7) and NPY (8, 9) stimulate feeding and activate the HPA axis in a dose-dependent manner. Orexigenic NPY neurones are located primarily in the arcuate nucleus of the hypothalamus and to a lesser extent in the nucleus of the tractus solitarius (NTS) in the brain stem (10) and project to the parvocellular division of the PVN in which they synapse on CRH cell bodies and dendrites (11). NPY stimulates CRH release in the median eminence (12, 13), increases CRH mRNA expression in the pPVN (9), and increases circulating levels of ACTH (13) and corticosterone (14). NPY neurone cell bodies receive substantial synaptic contacts from orexin-containing boutons and orexin-A stimulates NPY release from hypothalamic explants (5). Moreover, pretreatment with a NPY antagonist (4, 5) or NPY antiserum (4) abolishes the corticosterone response to centrally administered orexin-A in vivo.

In late pregnant rats, the excitatory effect of orexin-A on HPA activity is suppressed as a result of reduced activation of the CRH neurones in the pPVN (2). Here we investigated whether the responsiveness of the HPA axis to NPY is reduced in late pregnancy, which may explain the reduced activation of the HPA axis seen after intracerebroventricular (i.c.v.) orexin-A at this time.

In addition to its roles in parturition and lactation, oxytocin is a stress hormone in rats (15), and its secretion is increased in response to both emotional and physical stressors (15). However, in late pregnancy, oxytocin secretion stimulated by stress is markedly attenuated (16). Because NPY stimulates oxytocin neurones in nonpregnant rats (17), we also monitored the activity of the oxytocin system after centrally administered NPY in pregnant and virgin rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and housing
Female Sprague Dawley rats (body weights at the start of the experiments were 250–280 g; Bantin and Kingman, Hull, UK) were used. Rats, initially housed in groups of five to six, were maintained under standard conditions of temperature (20–22 C), humidity, and lighting (12-h light, 12-h dark cycle; lights on at 0700 h) with free access to food and water. Pregnant rats were obtained by mating virgin females overnight with a sexually experienced male; pregnancy was confirmed by the presence of a vaginal plug of semen in the breeding cage the following morning (designated d 1 of pregnancy; d 22 is the expected day of parturition). All procedures were performed in accordance with current U.K. Home Office regulations under the Animals (Scientific Procedures) Act 1986.

Surgery
Five days before the experiment, virgin and pregnant rats (d 16 of pregnancy) were fitted with a silastic jugular vein cannula (filled with sterile heparinized 0.9% saline, 50 U/ml) and an i.c.v. guide cannula (22-gauge; Bilaney Consultants Ltd., Kent, UK; stereotaxic coordinates: 1.6 mm lateral and 0.6 mm posterior to bregma, inserted to a depth of 4 mm from the skull surface and secured with stainless steel screws and dental acrylic) under halothane anesthesia (2–3% in 1200 ml/min of O2) for blood sampling and drug administration, respectively. After surgery, rats were caged individually and handled daily to familiarize them with the i.c.v. infusion procedure.

Experiment 1: blood sampling and behavioral measures
On the day of experiment (d 21 of pregnancy) the iv cannula was connected to PVC extension tubing led out of the cage and connected to a 1-ml syringe filled with sterile heparinized saline (as above) between 0730 and 0830 h. At the same time, food was removed from the food hoppers, weighed, and replaced. Rats were left undisturbed for 2 h, and then two basal blood samples (0.6ml) were taken 30 min apart. Immediately after the second basal blood sample was collected, rats were gently held while an i.c.v. infusion cannula (connected to a 10 µl Hamilton syringe via polythene tubing and prefilled with injectate) was inserted into the guide cannula. Rats were administered either 5 µg of porcine NPY (1–36; in 2 µl; Tocris Bioscience, Avonmouth, UK) or vehicle [2 µl artificial cerebrospinal fluid (aCSF) (pH 7.2), composition in millimoles: NaCl, 138; KCl, 3.36; NaHCO3, 9.52; Na2HPO4, 0.49; urea, 2.16; NaH2PO4, 0.49; CaCl2, 1.26; MgCl2, 1.18] i.c.v. over 30 sec. Further blood samples were taken 15, 30, 60, 90, and 120 min after the i.c.v. infusion. Blood samples were withdrawn into syringes containing 50 µl chilled 5% EDTA and replaced with 0.9% sterile saline. Plasma was separated by centrifugation and stored at –20 C until RIA.

The following behavioral events were noted continually during the 2-h period after the i.c.v. injection: inactivity, grooming, feeding, and drinking (an event was scored if an animal spent ≥ 10 consecutive seconds exhibiting a behavior). Rats were killed by conscious decapitation 4 h after the i.c.v. infusion [this time point has previously been shown to be optimal for demonstrating increased CRH/arginine vasopressin (AVP) mRNA expression in the pPVN after a variety of stressors (18, 19)]. Trunk blood was collected (in tubes containing 0.5 ml chilled 5% EDTA) and plasma separated as before for RIA. Brains were rapidly removed, frozen on dry ice, and stored at –70 C. The food in the hoppers was reweighed to establish food intake.

Experiment 2
Additional groups of virgin and pregnant rats were fitted with an i.c.v. cannula as above (5 d before the experiment). On the morning (between 0730 and 1030 h) of the experiment (d 21 of pregnancy) rats were administered either 5 µg NPY (in 2 µl) or vehicle (aCSF) as before. Ninety minutes after the i.c.v. injection, rats were deeply anesthetized with sodium pentobarbitone (60 mg/ml, 0.7 ml/kg ip; Ceva Animal Health Ltd., Chesham, Buckinghamshire, UK) and perfused transcardially with heparinized saline (50 ml, 20 U/ml 0.9% saline) followed by 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer (350–400 ml). Brains were removed, postfixed overnight at 4 C in 4% (wt/vol) paraformaldehyde in 0.1 m phosphate buffer (PB) containing 15% (wt/vol) sucrose and cryoprotected in 30% sucrose in 0.1 M PB for 24–48 h. Brains were frozen on dry ice and stored at –70 C until coronal sectioning and immunocytochemistry for Fos (the protein product of the immediate early gene, c-fos, used here as an indicator of neuronal activation) and oxytocin.

RIAs
Plasma ACTH and corticosterone concentrations were determined in single RIAs, using commercially available kits (IDS Ltd., Boldon, Tyne and Wear, UK). Plasma ACTH concentrations were measured in unextracted plasma samples using a two-site immunoradiometric assay, which uses two polyclonal antibodies recognizing different binding sites on the intact ACTH molecule (20). Corticosterone concentrations were measured directly in unextracted plasma (diluted 1:10 in assay buffer) using a double-antibody RIA with 125I-corticosterone as the tracer (21). Sensitivities were 5 pg/ml and 0.5 ng/ml and the intraassay variation less than 8 and less than 6%, for the ACTH and corticosterone assays, respectively.

Plasma oxytocin concentration was determined using an in-house assay. The method used was adapted from that described by Higuchi et al. (22). Briefly, rabbit antioxytocin antibody THF-3 (diluted 1:200,000 in assay buffer; kindly donated by Dr. T. Higuchi, Fukui Medical School, Fukui, Japan) was added to tubes containing standard oxytocin (National Institute for Biological Standards and Controls, range 2.4–1500 pg/ml; in triplicate) or plasma (in duplicate). After 24 h incubation, radioiodinated oxytocin (125I, 3.7 kBq/ml, 50 µl; NEN Life Science Products, Boston, MA) was added to all of the tubes. After a further 48 h incubation, a second antibody, donkey antirabbit gammaglobulin (diluted 1:25 in assay buffer; IDS Ltd., Boldon, Tyne and Wear, UK) was added, and the resulting aggregate was precipitated with pansorbin cells (diluted 1:25 in assay buffer; Calbiochem, Nottingham, UK). Radioactivity of the precipitate was measured with a {gamma}-counter and unknown oxytocin concentrations read directly from the standard curve automatically produced by the Ultraterm 2 software package (Wallac Oy, Turku, Finland). The sensitivity of the assay was 2.4 pg/ml and the intraassay variation less than 9%.

In situ hybridization
Coronal cryostat brain sections (15 µm) cut through the hypothalamus were thaw-mounted onto Polysine slides, fixed in 4% (wt/vol) paraformaldehyde, washed in PBS (0.1 M), acetylated in triethanolamine/acetic anhydride solution, dehydrated in an ascending ethanol series (70 to 100%), delipidated in chloroform, and partially rehydrated in 95% (vol/vol) ethanol. All sections from a particular experiment were processed in the same hybridization reaction, and in each case, synthetic oligonucleotide probes were used (MWG Biotech, Ebersberg, Germany). To detect CRH mRNA expression, a 42-mer probe (5'-CCT GTT GCT GTG AGC TTG CTG AGC TAA CTG CTC TGC CCT GCC-3'), complementary to bases 496–537, which encode amino acids 22–35 of the rat CRH peptide (23) was used. The AVP mRNA probe was a 36-mer oligonucleotide (5'-GAC CCG GGG CTT GGC AGA ATC CAC GGA CTC TTG TGT-3') complementary to bases 486–521 of rat AVP mRNA (24).

Probes were 3'-labeled with 35S-dATP using terminal deoxynucleotidyl transferase and purified using spin columns (QIAquick nucleotide removal kit; QIAGEN, Crawley West Sussex, UK). Sections were hybridized with radiolabeled probe (105 cpm labeled probe/ section) in hybridization buffer [BSA, 1% (wt/vol); dextran sulfate, 5% (wt/vol); diothiothreitol, 15 mM; EDTA, 2 mM; ficoll, 1% (wt/vol); formamide, 50% (vol/vol); PolyA, 0.1 mg/ml; polyvinylpyrrolidone, 1% (wt/vol); salmon testes DNA, 0.2 mg/ml; sodium chloride, 1.2 M; sodium pyrophosphate, 2.5% (wt/vol); Tris (pH 7.6), 20 mM; yeast tRNA, 0.1 mg/ml; yeast total RNA, 0.1 mg/ml] overnight at 37 C in humidified chambers. Sections were rinsed three times in 1x saline sodium citrate at room temperature, washed 4 x 15 min in 1x saline sodium citrate at 58 C (for CRH mRNA) or 56 C (for AVP mRNA), and 2 x 30 min at room temperature. Sections were air dried and dipped in liquid autoradiographic emulsion (Ilford K-5; Knutsford, Cheshire, UK), and exposed at 4 C for either 10 wk for CRH mRNA or 7 d for AVP mRNA. Slides were developed (Kodak D-19 developer; Sigma, Poole, UK), fixed (Ilford rapid fixer), counterstained with hematoxylin and eosin, and coverslipped with dinbutyl phthalate in xylene (DPX).

All in situ hybridization analyses were performed on coded slides. AVP mRNA hybridization was quantified from emulsion-dipped sections using a computerized image analysis system (objective magnification, x4; NIH Image, version 1.62). Grain area was measured over a minimum of six dorsomedial parvocellular PVN profiles in three consecutive sections (scattered magnocellular neurones expressing AVP mRNA among the parvocellular neurones in the dorsomedial PVN were not excluded from the measurements). The area of each profile was measured to calculate grain area/pPVN (square millimeters/square millimeters). Background measurements were made over areas adjacent to the region of interest, converted to grain area/ square millimeters and subtracted. CRH mRNA hybridization was quantified using two methods. The number of cells expressing CRH mRNA in the pPVN was manually counted in emulsion-dipped sections (at x40 magnification; in six to 12 pPVN profiles per rat). A positive cell was defined as one with more overlying silver grains than the mean over 10 cells lateral to the PVN (background) + 3 SD. Data are presented as number of positive cells per pPVN profile. In addition, silver grain area was measured in the pPVN over 120–240 individual cells/rat that were positively hybridized with the CRH mRNA probe. Background measurements were made over the equivalent of 20 cells in neuropil adjacent to the PVN, divided by 20, and subtracted. Data are presented as silver grain area per cell. For all in situ hybridization measurements, average values for each rat were used to calculate group means ± SEM. Total CRH mRNA expression per pPVN section was calculated by multiplying the number of positive cells per pPVN by the silver grain area/neurone.

Immunocytochemistry
Brains were coronally sectioned at 52 µm on a freezing microtome, and the free-floating sections were processed for immunocytochemistry as described previously (25). Briefly, sections were washed for 10 min in 0.1 M PB containing 0.2% Triton X-100 (PB-T), followed by another 10 min wash in 0.1 M PB. Endogenous brain peroxidases were quenched by incubating sections in 0.1 M PB-T containing 20% methanol and 1.5% H2O2 for 15 min. Nonspecific labeling was blocked by preincubating sections for 60 min in 0.1 M PB-T containing 2% normal sheep serum (NSS) before incubation with a polyclonal antibody, rabbit anti-Fos (diluted 1:1000 in PB-T containing 2% NSS; raised against amino acid residues 4–17 of the protein product of the human c-fos gene; Calbiochem) at 4 C for 24 h.

The following day, sections were washed in 0.1 M PB-T for 3 x 5 min and 2 x 10 min before incubation with the second antibody, biotinylated antirabbit Ig (Vectastain Elite kit; Vector Laboratories, Bretton, Peterborough, UK), diluted 1:100 in 0.1 M PB-T containing 3% normal goat serum for 60 min at room temperature. Next, sections were washed in 0.1 M PB-T for 3 x 5 min and 2 x 10 min before incubation in the antibody-antigen complex (avidin DH, 20 µl/ml; biotinylated horseradish peroxidase, 20 µl/ml in 0.1 M PB-T; Vectastain Elite kit, Vector Laboratories) for 60 min at room temperature. Sections were then washed in 0.1 M PB-T for 2 x 10 min and rinsed for 5 min in 0.1 M sodium acetate buffer. Staining was visualized using the nickel-intensified-glucose oxidase-3,3'-diaminobenzidine method (25) and the reaction stopped by rinsing sections in 0.1 M sodium acetate for 5 min. Sections for double labeling were washed 6 x 5 min in 0.1 M PB, followed by 2 x 5 min in PB-T. Sections were then incubated in 0.3% hydrogen peroxide for 15 min and washed 2 x 5 min in PB-T before incubation in a rabbit antioxytocin antibody (diluted 1:1000 in PB-T containing 2% NSS; Calbiochem) at 4 C for 24 h. The next day sections were washed 3 x 10 min in PB-T before 60 min incubation in biotinylated antirabbit Ig (as above) at room temperature. Next, sections were washed in 0.1 M PB-T for 3 x 10 min before incubation in the antibody-antigen complex (avidin-biotinylated horseradish peroxidase complex, as above, for 60 min at room temperature. Sections were washed 2 x 10 min in PB-T, and oxytocin immunoreactivity was visualized by incubating sections in 0.1 M PB containing 0.025% 3,3'-diaminobenzidine and 0.03% hydrogen peroxide. The reaction was stopped by washing sections 6 x 5 min in 0.1 M PB.

Sections were mounted onto gelatinized microscope slides, air dried, dehydrated through an ascending ethanol series, cleared in xylene, and coverslipped with DPX. Fos immunoreactive cells were manually counted (at x40 magnification) bilaterally in the supraoptic nucleus (SON) and magnocellular division of the PVN (mgPVN) in three to six sections per rat. The mean number of Fos immunoreactive nuclei per SON/mgPVN profile for each rat was used to calculate the group means. In sections processed for both Fos and oxytocin immunoreactivity, the number of double-labeled cells and the total number of oxytocin-labeled cells in both the SON and mgPVN were counted (bilaterally in two to five sections/rat). The data are presented as the percentage of the total number of oxytocin neurones in the SON/mgPVN containing Fos immunoreactivity.

Statistical analysis
Two-way repeated-measures ANOVA (RM ANOVA) followed by Student-Newman-Keuls multiple comparison tests were used for statistical analysis (Sigmastat; Systat Software UK Ltd., Hounslow, London, UK) of the ACTH, corticosterone, and oxytocin data. The CRH mRNA, AVP mRNA, Fos/oxytocin immunoreactivity, and behavioral data were analyzed using a two-way ANOVA. Data are presented as group means ± SEM. P < 0.05 was considered statistically significant in each case.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stress hormone secretion
Basal plasma concentrations of ACTH were not different among any of the groups (mean basal values were 24.3 ± 2.3 and 23.2 ± 3.4 pg/ml in virgin and pregnant groups, respectively). Central administration of vehicle (aCSF) had no effect on ACTH secretion in either the virgin or pregnant groups (Fig. 1AGo). Intracerebroventricular infusion of NPY caused a rapid (within 15 min) increase in ACTH secretion in the virgin rats (P < 0.001; two-way RM ANOVA), which peaked at 30 min (4.3-fold increase vs. basal) and was still significantly elevated 2 h after the infusion (Fig. 1AGo). However, in the pregnant rats, i.c.v. NPY had no effect on ACTH secretion; thus, plasma ACTH levels were significantly greater in the virgin group than the pregnant group at 15, 30, 60, 90, and 120 min after i.c.v. NPY (P < 0.001 at 15, 30, and 60 min and P < 0.005 at 90 and 120 min; two-way RM ANOVA).


Figure 1
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  		FIG. 1. Effect of i.c.v. NPY on stress hormone secretion. After collection of basal (B) blood sample(s), rats were administered either vehicle (aCSF) or NPY (5 µg) i.c.v. Further blood samples were withdrawn at various time points after the i.c.v. infusion. Rats were killed 4 h after the i.c.v. infusion and trunk blood was collected. Plasma was assayed for ACTH (A), corticosterone (B), and oxytocin (C). Values are group means ± SEM. Group numbers are: virgin/aCSF, n = 5; virgin/NPY, n = 7; pregnant/aCSF, n = 5; pregnant/NPY, n = 8. Statistical analyses: two-way RM ANOVA followed by Student-Newman-Keuls multiple comparison tests. *, P < 0.001 vs. basal values in the same group; #, P < 0.03 vs. all other groups at the same time point (A); *, P < 0.04 vs. basal values in the same group; #, P < 0.04 vs. all other groups at the same time point (B); *, P < 0.001 vs. basal values in the same group; #, P < 0.01 vs. all other groups at the same time point (C).

 
There was no significant difference in basal plasma corticosterone concentrations between the pregnant (mean basal, 121.9 ± 13.1 ng/ml) and virgin groups (mean basal, 109.7 ± 17.1 ng/ml; P = 0.28, Student’s t test). Central administration of vehicle caused a transient, nonsignificant increase in corticosterone secretion in both virgin and pregnant rats (Fig. 1BGo). NPY administration caused a significant increase in corticosterone secretion in both the virgin (mean increase from basal, 112.8 ± 26.6 ng/ml; P < 0.001, two-way RM ANOVA) and pregnant (mean increase from basal, 44.6 ± 14.8 ng/ml; P < 0.04, two-way RM ANOVA) groups within 30 min; however, the response in the pregnant rats was significantly less than in the virgin rats (Fig. 1BGo; P < 0.001, two-way RM ANOVA) and was not different vs. either the vehicle-treated virgin or pregnant rats.

Basal plasma oxytocin concentrations were not significantly different among any of the groups (Fig. 1CGo). Vehicle i.c.v. had no significant effect on oxytocin secretion in either the virgin or pregnant groups. Centrally administered NPY significantly increased oxytocin secretion within 15 min in the virgin rats, with levels peaking at 30 min (6.1-fold increase vs. basal levels) and remaining elevated 60 min after the infusion (Fig. 1CGo; P < 0.001, two-way RM ANOVA). In contrast, in the late pregnant rats NPY had no significant effect on oxytocin secretion. Consequently plasma oxytocin concentrations were significantly lower in NPY-treated pregnant compared with NPY-treated virgin rats at 15, 30, and 45 min (Fig. 1CGo; P < 0.001, two-way RM ANOVA).

CRH and AVP mRNA in the pPVN
The number of cells in the pPVN hybridized with the CRH mRNA probe was not significantly different between the virgin and pregnant control groups (Fig. 2Go, A and C) nor was silver grain density per neurone (Fig. 2Go, B and C). Four hours after i.c.v. NPY, both the number of cells expressing CRH mRNA (Fig. 2AGo; P < 0.05, two-way ANOVA) and the level of CRH mRNA expression per cell (Fig. 2BGo; P < 0.05, two-way ANOVA) were significantly increased in the pPVN of virgin but not late pregnant rats. Thus, the number of cells expressing CRH mRNA and the level of expression per cell in the pPVN was greater in NPY-treated virgin rats than NPY-treated late pregnant rats (Fig. 2Go, A–C; P < 0.01, two-way ANOVA). Total CRH mRNA expression per pPVN section after NPY treatment relative to respective control was 166 ± 13% in the virgins (P < 0.002, Student’s t test) and 117 ± 16% in the pregnant rats (n.s.; P = 0.424, Student’s t test; data not shown).


Figure 2
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  		FIG. 2. Effect of i.c.v. NPY on CRH and AVP mRNA expression in the pPVN. Rats were killed 4 h after i.c.v. vehicle (aCSF) or NPY (5 µg). Values are group means ± SEM. Quantification of CRH mRNA in situ hybridization: A, number of CRF mRNA-expressing cells per pPVN profile; B, CRF mRNA silver grain area/cell. Group numbers are: virgin/aCSF, n = 9; virgin/NPY, n = 9; pregnant/aCSF, n = 7; pregnant/NPY, n = 9. C, Autoradiographs of coronal sections cut through the paraventricular nucleus hybridized with the CRH mRNA probe. Dark field: (i) virgin/aCSF; (ii) virgin/NPY; (iii) pregnant/aCSF; (iv) pregnant/NPY. Scale bar, 100 µm. Asterisks indicate third ventricle. Bright field: (v) autoradiograph demonstrating positively labeled CRH mRNA-expressing neurones in the pPVN. Filled arrows indicate examples of positively labeled cells and open arrows indicate examples of unlabeled cells. Scale bar, 25 µm. Quantification of AVP mRNA in situ hybridization: D, AVP mRNA grain area/pPVN profile. Group numbers are: virgin/aCSF, n = 8; virgin/NPY, n = 9; pregnant/aCSF, n = 9; pregnant/NPY, n = 9. E, Bright-field autoradiographs of coronal sections cut through the PVN and hybridized with the AVP mRNA probe. (i) virgin/aCSF; (ii) virgin/NPY; (iii) pregnant/aCSF; (iv) pregnant/NPY. Scale bar, 100 µm. 3V, Third ventricle. Statistical analyses: two-way ANOVA followed by Student-Newman-Keuls multiple comparison tests: *, P < 0.05 vs. virgin/aCSF group; #, P < 0.05 vs. all other groups.

 
AVP mRNA expression in the dorsomedial pPVN was not different between the virgin and pregnant control groups. After centrally administered NPY, AVP mRNA expression was increased in the virgin group (Fig. 2Go, D and E; P < 0.05, two-way ANOVA) but not in the late pregnant group (Fig. 2Go, D and E).

Fos expression in supraoptic oxytocin neurones
Fos expression in the SON was not different between the vehicle-treated virgin and late pregnant rats. Centrally administered NPY evoked a 5-fold increase in Fos expression in the SON in virgin rats (Fig. 3Go, A and B; P < 0.01, two-way ANOVA) but in contrast had no effect in the pregnant rats. After i.c.v. NPY, the number of supraoptic oxytocin neurones expressing Fos significantly increased from 9.5 to 59.6% in the virgin rats (Fig. 3CGo; P < 0.001, two-way ANOVA; mean number of SON oxytocin neurones measured was 128/rat), but no such effect was observed in the pregnant rats.


Figure 3
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  		FIG. 3. Effect of i.c.v. NPY on Fos expression in magnocellular SON and PVN oxytocin neurones. Rats were perfuse fixed 90 min after i.c.v. vehicle (aCSF) or NPY (5 µg), and brains were processed for Fos or Fos and oxytocin immunocytochemistry. A, Quantification of Fos immunoreactivity in the SON. Values are group means ± SEM. Group numbers are: virgin/aCSF, n = 4; virgin/NPY, n = 5; pregnant/aCSF, n = 6; pregnant/NPY, n = 5. Statistical analyses: two-way ANOVA followed by Student-Newman-Keuls multiple comparison tests: *, P < 0.01 vs. virgin/aCSF group; #, P < 0.02 vs. all other groups. B, Photomicrographs showing Fos immunoreactive cells in the SON of virgin/aCSF (i); virgin/NPY (ii); pregnant/aCSF (iii); and pregnant/NPY-treated rats (iv). OC, Optic chiasm. Scale bar, 100 µm. C, Quantification of cells immunoreactive for Fos and oxytocin in the SON. Data are presented as percent of the total number of oxytocin neurons in the SON expressing Fos. Values are group means ± SEM. Group numbers are: virgin/aCSF, n = 6; virgin/NPY, n = 4; pregnant/aCSF, n = 5; pregnant/NPY, n = 3. Statistical analyses: two-way ANOVA followed by Student-Newman-Keuls multiple comparison tests: *, P < 0.001 vs. virgin/aCSF group; #, P < 0.02 vs. pregnant groups. D, Quantification of Fos immunoreactivity in the mgPVN. Values are group means ± SEM. Group numbers are: virgin/aCSF, n = 6; virgin/NPY, n = 4; pregnant/aCSF, n = 5; pregnant/NPY, n = 4. Statistical analyses: two-way ANOVA followed by Student-Newman-Keuls multiple comparison tests: *, P < 0.001 vs. virgin/aCSF group; #, P < 0.001 vs. pregnant groups. E, Photomicrograph showing Fos immunoreactive cells in the mgPVN of virgin/aCSF (i); virgin/NPY (ii); pregnant/aCSF (iii); pregnant/NPY-treated rats (iv). 3V, Third ventricle. Scale bar, 100 µm. (v) High-magnification photomicrograph showing examples of mgPVN oxytocin neurones (brown, cytoplasm and processes). Scale bar, 25 µm. (vi) High-magnification photomicrograph showing an example of a mgPVN oxytocin neurone (brown, cytoplasm and processes) expressing Fos (black, nuclear) from a virgin rat treated with NPY. Scale bar, 25 µm. F, Quantification of cells immunoreactive for Fos and oxytocin in the magnocellular subdivision of the PVN. Data are presented as percent of the total number of oxytocin neurons in the mgPVN expressing Fos. Values are group means ± SEM. Group numbers are: virgin/aCSF, n = 6; virgin/NPY, n = 4; pregnant/aCSF, n = 5; pregnant/NPY, n = 3. Statistical analyses: two-way ANOVA followed by Student-Newman-Keuls multiple comparison tests: *, P < 0.001 vs. aCSF groups; #, P < 0.001 vs. pregnant/NPY group.

 
Fos expression in oxytocin neurones in the magnocellular division of the PVN
Fos expression in the mgPVN was not different between the vehicle-treated virgin and late pregnant rats. Centrally administered NPY evoked a 6.7-fold increase in Fos expression in the mgPVN in virgin rats (Fig. 3Go, D and E; P < 0.01, two-way ANOVA) but, however, had no effect in the pregnant rats. There was a significant increase (15.9-fold) in the number of oxytocin neurones in the mgPVN-expressing Fos after i.c.v. NPY in the virgin rats (Fig. 3FGo; P < 0.001, two-way ANOVA; mean number of mgPVN oxytocin neurones measured was 255/rat) but not in the pregnant rats. The proportion of oxytocin neurones expressing Fos after i.c.v. NPY in virgin rats was much less in the mgPVN than the SON.

Behavior
Food intake was not different between the vehicle-treated virgin and pregnant rats (Fig. 4AGo; P = 0.23, two-way ANOVA). Centrally administered NPY induced a significant increase in food intake in both the virgin (3.52 ± 0.38 g; P < 0.001, two-way ANOVA) and pregnant (3.33 ± 0.66 g; P < 0.002, two-way ANOVA) rats, compared with vehicle-treated controls. There was no difference in food consumption between the virgin and pregnant NPY-treated rats (Fig. 4AGo; P = 0.80, two-way ANOVA). Drinking behavior was not different between vehicle-treated virgin and pregnant rats. NPY significantly induced drinking behavior in both groups (P < 0.005, two-way ANOVA), compared with vehicle-treated controls, and to a similar extent in both the virgin and pregnant rats (Fig. 4BGo). There was no significant effect of i.c.v. vehicle or NPY on grooming behavior in either the virgin or pregnant rats (Fig. 4CGo).


Figure 4
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  		FIG. 4. Behavioral effects of i.c.v. NPY. A, Food intake was measured by weighing the contents of the food hoppers at the beginning and end of the experiment. The number of drinking (B) and grooming (C) events was continually noted over the 120-min period immediately after the i.c.v. infusion of aCSF or NPY (5 µg). Values are group means ± SEM. Group numbers are: virgin/aCSF, n = 9; virgin/NPY, n = 9; pregnant/aCSF, n = 9; pregnant/NPY, n = 9. Statistical analyses: two-way ANOVA followed by Student-Newman-Keuls multiple comparison tests: *, P < 0.002 vs. respective aCSF control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we have shown that neuroendocrine stress responses to centrally administered NPY are essentially absent in late pregnant rats, although ingestive behavioral responses are intact. Specifically we have shown that the ACTH secretory response to i.c.v. NPY is abolished in late pregnancy and the corticosterone response markedly attenuated. This is likely to be a consequence of reduced activation of the hypothalamic neurones that regulate the HPA axis because CRH mRNA and AVP mRNA (which is coexpressed in CRH neurones) expression in the pPVN was not increased by NPY in the late pregnant rats, in contrast with virgin rats. Similarly the lack of a significant oxytocin secretory response after NPY in the late pregnant rats appears to be a result of a failure to activate the oxytocin neurones in the hypothalamus because Fos (a marker of neuronal activation) expression in the SON and mgPVN oxytocin neurones was up-regulated after NPY in the virgin but not in the pregnant rats. The findings are consistent with other studies demonstrating attenuated responsiveness of the HPA axis and the neurohypophysial oxytocin system in response to emotional and physical stressors at the end of pregnancy (2, 16, 26, 27).

The reduced responsiveness of the hypothalamic CRH- or AVP-expressing pPVN and magnocellular oxytocin neurones to centrally administered NPY in late pregnancy could be a result of reduced excitatory drive by inputs on which NPY has a net stimulatory action or enhanced opposing inhibitory inputs to these neurones in the PVN and SON. With respect to the HPA axis, there is no reduced capacity to secrete CRH or ACTH (28), and there is a large increase in neurohypophysial oxytocin stores near the end of pregnancy (29). We have previously shown that enhanced rapid negative feedback inhibition by glucocorticoids does not underlie the reduced responsiveness of the HPA axis to stress at the end of pregnancy (30). Because NPY can mediate the actions of orexin-A on the HPA axis, reduced HPA axis responses to centrally administered orexin-A in late pregnancy (2) could be a result of the reduced capacity of the CRH neurones to respond to NPY at this time.

NPY neurones located in the arcuate nucleus and the NTS of the brain stem innervate the PVN and SON as well as the medial preoptic area, ventromedial hypothalamus, dorsomedial hypothalamus, and perifornical region (31). NPY exerts its effects on the HPA axis by acting centrally at the level of the CRH neurones to evoke CRH release (13, 32). Similarly NPY has been shown to act in the SON to trigger oxytocin release into the general circulation (17). Although the mechanisms are unclear, Y1 and Y5 receptors are widely distributed throughout the brain (33), and both these receptor subtypes have been identified in the PVN and SON (33). However, to date there have been no reports about NPY receptor expression in the SON or PVN in pregnancy, changes in which might explain the absent or greatly attenuated responses of the CRH/AVP and oxytocin neurones and hence of the HPA axis and oxytocin system to NPY in late pregnancy.

Endogenous opioid mechanisms are activated to inhibit supraoptic oxytocin neurones in late pregnancy (34). NTS neurones may be the source of this opioid because proenkephalin-A mRNA expression is up-regulated in the NTS in late pregnancy. Furthermore, opioid(s) inhibit CRH neurone responses after cytokine challenge in late pregnancy, by acting presynaptically on noradrenergic inputs to the CRH neurones (27). NPY-containing catecholaminergic neurones in the medulla project to the PVN (35); however, it is not known whether in late pregnancy opioids also interfere with NPY signaling to pPVN CRH and/or AVP or SON oxytocin neurones.

In late pregnancy the {gamma}-aminobutyric acid (GABA) input to magnocellular oxytocin neurones is more effective (36). Increased levels of allopregnanolone (a progesterone metabolite with neuroactive properties) in the brain in pregnancy enhance the actions of GABA through actions at the GABAA receptor (36), and there are increased GABAergic synaptic contacts with oxytocin neurones (37). Moreover, allopregnanolone restrains IL-1ß-stimulated ACTH (38) and oxytocin (39) secretion in late pregnancy, possibly through its actions on GABAA receptors. This mechanism might also stand for suppressed NPY-stimulated oxytocin secretion and HPA axis activation in late pregnancy.

We found no differences between virgin and pregnant rats in the stimulatory effect of NPY on feeding and drinking behavior, suggesting that reduced stimulation of the HPA axis and oxytocin system observed in late pregnancy is not the result of a generalized loss of central responses to NPY at this time [e.g. in brain regions involved in appetite regulation (40, 41, 42)] and instead is selective for neuroendocrine stress responses. These findings are comparable with the retention of orexin-stimulated eating in pregnancy despite loss of HPA axis responses (2).

Stimulation of the HPA axis by i.c.v. NPY in nonpregnant rats results in increased circulating glucocorticoid concentration, which is expected to act to mobilize glucose stores (43). In late pregnancy, reduced corticosterone secretion after i.c.v. NPY administration may reflect a switch to anabolic metabolism, which would help meet the energy demands of the fetuses without causing a negative energy balance in the pregnant dam. The functional significance of oxytocin release in response to NPY is not clear other than in lactation (17), although oxytocin released from magnocellular neurones can enter the hypothalamohypophysial portal blood to potentiate CRH-induced ACTH secretion (15, 44, 45). In late pregnancy it is likely that the suppression of NPY-stimulated oxytocin release will minimize the risk of preterm labor and preserve the accumulated neurohypophysial stores of oxytocin for when they are required at parturition.

In summary, we have shown that neuroendocrine stress responses to centrally administered NPY are suppressed in late pregnant rats, although ingestive behavioral responses are intact. Reduced HPA axis responses to NPY may promote anabolic adaptations in pregnancy. The absence of an oxytocin secretory response to NPY in late pregnancy should reduce the risk of preterm labor and preserve the accumulated neurohypophysial stores for parturition. The pregnancy model could prove useful for dissecting the pathways that are involved in the feeding response to NPY without the complication of activated neuroendocrine stress pathways.


   Acknowledgments
 
The authors thank Helen Cameron, Laura Wylie (funded by a Biotechnology and Biological Sciences Research Council vacation studentship), and Caroline McGown (who received a Wellcome Trust vacation studentship) for technical assistance.


   Footnotes
 
This work was supported by Biotechnology and Biological Sciences Research Council. J.B. is in receipt of a Medical Research Council Ph.D. studentship.

Disclosure: P.J.B., J.B., and J.A.R. have nothing to declare.

First Published Online May 4, 2006

Abbreviations: aCSF, Artificial cerebrospinal fluid; AVP, arginine vasopressin; GABA, {gamma}-aminobutyric acid; HPA, hypothalamo-pituitary-adrenal; i.c.v., intracerebroventricular; mgPVN, magnocellular division of the PVN; NPY, neuropeptide Y; NSS, normal sheep serum; NTS, nucleus of the tractus solitarius; PB, phosphate buffer; PB-T, PB containing Triton X-100; pPVN, parvocellular division of the paraventricular nucleus; RM ANOVA, repeated-measures ANOVA; SON, supraoptic nucleus.

Received January 13, 2006.

Accepted for publication April 24, 2006.


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 Introduction
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 Results
 Discussion
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