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Endocrinology, doi:10.1210/en.2006-0377
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Endocrinology Vol. 147, No. 9 4122-4131
Copyright © 2006 by The Endocrine Society

Influence of Dehydration on the Expression of Neuropeptide Y Y1 Receptors in Hypothalamic Magnocellular Neurons

Janice H. Urban, Randy J. Leitermann, M. Regina DeJoseph, Suwit J. Somponpun, Michael L. Wolak and Celia D. Sladek

Department of Physiology and Biophysics (J.H.U., R.J.L., M.R.D., M.L.W.), The Chicago Medical School/Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064; and Department of Physiology and Biophysics, University of Colorado at Denver and Health Sciences Center (S.J.S., C.D.S.), Aurora, Colorado 80045

Address all correspondence and requests for reprints to: Janice H. Urban, Ph.D., Department of Physiology and Biophysics, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, Illinois 60064. E-mail: janice.urban{at}rosalindfranklin.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of vasopressin (VP) and oxytocin (OT) secretion involves integration of neural signals from hypothalamic osmoreceptors, ascending catecholaminergic and peptidergic cell groups in the brain stem, and local and autoregulatory afferents. Neuropeptide Y (NPY) is one factor that stimulates the release of VP and OT from the supraoptic (SON) and paraventricular nuclei of the hypothalamus via activation of Y1 receptors (Y1R). The current studies were designed to assess the regulation and distribution of NPY Y1R expression in the SON of male rats that were either given 2% NaCl drinking water (24–72 h) or water deprived (48 h). Subjecting male rats to these conditions resulted in significant increases in both the number of cells expressing Y1R immunoreactivity (ir) and the amount of Y1R protein per cell within the SON. Y1R immunoreactivity was increased in the magnocellular but not medial parvocellular paraventricular nuclei, and Y1R mRNA levels were increased in the SON of salt-loaded rats. Subpopulations of both VP and OT cells in the hypothalamus express Y1R immunoreactivity and a greater percentage of VP-ir cells express Y1R after salt loading. To control for potential effects of dehydration-induced anorexia, a group of euhydrate animals was pair fed with animals consuming 2% NaCl. No detectable change in Y1R expression was observed in the SON of pair-fed animals, even though body weights were significantly lower than controls. These data demonstrate that NPY Y1R gene and protein expression are increased in the SON of salt-loaded and water-deprived animals and provide a mechanism whereby NPY can support VP/OT release during prolonged challenges to fluid homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASOPRESSIN (VP) AND oxytocin (OT) secretion from the neural lobe of the pituitary is essential for the proper maintenance of plasma volume and osmolarity. These nonapeptides are contained in cell bodies present within the supraoptic (SON) and paraventricular (PVN) nuclei and accessory magnocellular cell groups (1). VP maintains blood pressure through increasing water reabsorption from the renal collecting ducts and through its actions as a vasoconstrictor. OT plays an integral role in the processes of parturition and lactation yet also has natriuretic properties (2). The regulation of VP and OT gene expression in the PVN and SON and secretion of VP and OT from the neural lobe is influenced by changes in plasma osmolarity and blood volume and pressure. Alterations in blood volume and pressure are transduced to the hypothalamus via multiple pathways. Information about decreases in blood pressure is transmitted via the ascending A1 catecholaminergic neurons from the ventrolateral medulla (3). Although there has been some controversy about the role of the A1 pathway in carrying information about hypovolemia (4, 5), more recent studies have clarified that this pathway is activated by both hypovolemia and hypotension (6). The A1 fibers, although providing a dense catecholamine innervation to the SON and PVN, also release other neurotransmitters, notably ATP and neuropeptide Y (NPY) (7, 8). The importance of these other neurotransmitters is evidenced by the fact that whereas stimulation of the A1 pathway increases the release of VP and OT, blockade of adrenoreceptors in the SON fail to inhibit cell responses to A1 stimulation (9).

The regulation of the activity of the neurohypophyseal system by NPY has been well documented. Injection of NPY into the PVN and SON increases the firing rate of magnocellular neurons (10) and the release of VP and OT from axon terminals of these cells in posterior pituitary (11, 12, 13). NPY directly activates VP/OT neurons and/or modulates the response of the magnocellular neurons to {alpha}1-adrenergic stimulation (12, 13, 14, 15). The stimulatory effects of NPY on VP/OT release are mediated via activation of Y1 (Y1R) and/or Y5 receptors, whereas activation of Y2 receptors may cause suppression of magnocellular neural activity (10, 12, 14). Direct activation of VP and OT neurons by NPY is supported by the demonstration of both Y1 and Y5 receptor expression in the SON and magnocellular division of the PVN (16, 17).

Under normotensive conditions, the expression of NPY immunoreactivity within the magnocellular divisions of the hypothalamus is primarily within fibers in the PVN and SON. There are few NPY immunoreactive cells within the magnocellular system. Subjecting animals to osmotic stressors, either water deprivation or replacing the drinking water with hypertonic saline, significantly increases NPY mRNA and peptide expression within the PVN/SON (18, 19). Additionally, hemorrhage increases NPY gene expression within the medulla [C1 and A1 cell groups (20)]. These data suggest that the activity of NPY neurons projecting to, and within, the hypothalamus is activated under conditions of hyperosmolarity or decreased blood pressure. If activation of NPY neural systems is important to the regulation of magnocellular VP/OT then increases in NPY receptor expression may also occur within these cells. The present studies were therefore designed to test the hypothesis that NPY Y1R expression within magnocellular neurons is increased with 2% NaCl ingestion or water deprivation. NPY receptor mRNA and protein expression within VP and OT neurons was assessed, using in situ hybridization and immunocytochemistry, in male rats after ingestion of 2% sodium chloride for 24–72 h or water deprivation for 48 h.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats (250–300 g; Charles River Laboratories, Portage, MI) were housed in an Association for the Assessment of Laboratory Animal Care-accredited facility and allowed free access to food and water. All animal procedures were approved by the institutional animal care and use committee. After 7 d equilibration in the animal facility, rats were assigned to different groups including a control (euhydrate), 2% NaCl (2% NaCl drinking water for 24, 48, or 72 h), and water deprivation (48 h) group; all animals had access to rat chow. Control animals were allowed free access to water. Rats were weighed daily. Animals in the pair-fed group were given equal amounts of rat chow based on the amount of food consumed by the 2% NaCl group. To assess the amount of rat chow given to the pair-fed animals, the amount of food eaten over a 24-h period by the 2% NaCl animals (72 h) was measured, and the pair-fed animals received only that amount of chow per 24 h and water ad libitum. After treatment, animals were anesthetized with pentobarbital (50 mg/kg, ip), and an aliquot of blood was taken via cardiac puncture for determination of plasma osmolality (vapor pressure osmometer; Wescor, Logan, UT). The animals were transcardially perfused with warm (37 C) PBS containing 0.1% procaine and heparin, followed by cold 4% paraformaldehyde in PBS. After perfusion, the brains were removed and placed in fixative at 4 C overnight. Tissues were sectioned on a vibratome (40 µm) and placed in PBS.

Immunohistochemistry protocol
Single-label immunohistochemistry was performed on free-floating sections as described previously (16) and Y1R immunoreactivity was detected using a modification of the biotinylated tyramide signal enhancement method previously described (21). In brief, sections were rinsed in PBS (pH 7.4), treated with 1% H2O2 for 15 min, and further rinsed in 3 x 5 min PBS washes. The sections were blocked for 30 min in 4% normal donkey serum (NDS) in PBS-gelatin and then incubated for 48 h in primary antibody (Y1: 1:1,500, 3,000, 6,000, or 12,000 for serial dilution study; 1:2,000 for all other studies) in 4% NDS. After incubation, the sections were rinsed in PBS-gelatin and incubated in biotinylated donkey antirabbit antibody (1:2000) for 1 h. After washes in PBS, the tissues were incubated in avidin-biotin complex (2 µl/ml; ABC reagent; Vector Laboratories, Burlingame, CA) for 30 min and rinsed in PBS-gelatin. For amplification, the tissues were incubated in biotinylated tyramide in 0.01% H2O2/PBS for 10 min, rinsed, and incubated with Cy2-streptavidin (1:250; Jackson ImmunoResearch Laboratories, West Grove, PA) for 3 h at room temperature. Sections were mounted on gelatin-subbed slides and air dried. Coverslips were applied using polyvinyl alcohol-1,4 diazabicyclo(2.2.2.)octane. The NPY Y1R antibody (ImmunoStar, Inc., Hudson, WI) is directed against the last 20 amino acids of the C-terminal tail. Specificity of the signal was assessed in tissue from both euhydrate and salt loaded animals by incubating the Y1R antiserum with an excess of peptide (100-fold excess) used to generate the antiserum and by omitting primary antibody.

Double-label immunohistochemistry was conducted using the Y1R antibody combined with monoclonal antibodies for VP-neurophysin (VP-NP; PS41) and OT-NP (PS38) generously provided by Dr. Hal Gainer (22). Sections were processed for the Y1R as indicated above and then incubated with either VP-NP (1:3000) or OT-NP (1:1000) in 4% NDS for 24 h. After rinses (5 x 5 min) in PBS-gelatin, the sections were incubated with donkey antimouse Cy3 (1:250; Jackson ImmunoResearch Laboratories) for 3 h at room temperature and rinsed in Tris-buffered saline (pH 7.4). Sections were mounted on gelatin-subbed slides, air-dried and coverslips were applied with polyvinyl alcohol-1,4 diazabicyclo(2.2.2.)octane. Specificity of immunoreactivity for double label experiments was determined by omitting each or both antibodies during the processing and assessing signal intensity for each fluorophore. No signal was observed when the primary antibodies were omitted from the incubations.

Analysis of signal.
To maintain consistency in the analysis of cell counts among the different groups, tissue sections from each condition were processed in the same assay. Sections through the SON and PVN of salt-loaded, water-deprived, and euhydrate animals were atlas matched [1.30–2.12 mm caudal to bregma; (23)] and immunoreactive staining patterns were visualized using epifluorescence microscopy (Eclipse C600; Nikon, Melville, NY), captured using a Spot II camera and analyzed using MetaMorph software (Universal Imaging, Downingtown, PA). The total number of Y1R-immunopositive cells represents unilateral cell counts through four sections of the SON and three sections through the PVN. Magnocellular and parvocellular neurons of the PVN were distinguished based on their overall distribution within the nucleus (23). Magnocellular neurons were located more laterally, and the Y1R staining pattern is specifically on the cell body and not on processes. The medial parvocellular neurons (PaMP) were located immediately adjacent to the third ventricle and exhibit Y1R immunoreactivity on both the cell body and processes. Because the PVN contains multiple divisions of parvocellular groups, a standardized outline was derived in MetaMorph (Universal Imaging) for the PaMP at each atlas level. This ensured that we were counting cells within a defined region of the PVN. For double-label analysis, Y1R and VP- and OT-NP immunopositive and double-labeled cells were counted at the same atlas levels, and double-labeled cells are reported as percent of VP-NP or OT-NP cells expressing Y1R-immunoreactivity.

In situ hybridization protocol
Frozen sections (10 µm) through the SON in brains from control (euhydrate) and salt-loaded (2% NaCl) animals were cut on a microtome and mounted on gelatin-subbed RNase-free slides. The tissues were postfixed in 4% paraformaldehyde in PBS (pH 7.4) at 4 C followed by 2 x 5 min washes in PBS. Tissues were treated with acetic anhydride, rinsed in PBS, and dehydrated through a graded series of ethanol (70, 80, 95%). The sections were prehybridized in a humidified chamber at 50 C for 2 h with prehybridization buffer [50% formamide, 0.6 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1x Denhardt’s solution, 0.5 mg/ml salmon sperm DNA, and 0.1 mg/ml yeast tRNA]. After the prehybridization solution was decanted from the slides, sections were hybridized with a 35S-uridine 5-triphosphate-labeled riboprobe complementary to the coding region of the Y1R mRNA (cDNA generously provided by Dr. Claes Wahlestedt; Karolinska Institutet, Sweden). Probe (1 x 107 cpm/ml) was applied to the tissues in a hybridization mix [10% dextran sulfate, 50% formamide, 0.6 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1x Denhardt’s solution, 0.5 mg/ml salmon sperm DNA, and 0.1 mg/ml yeast tRNA], and the tissues were hybridized in a humidified chamber overnight at 50 C. The following day, hybridization buffer was rinsed off the sections with 2x standard saline citrate (SSC), and the sections were subjected to a series of posthybridization washes: RNase A for 60 min at 37 C; 2x SSC for 30 min, and two washes in 0.1x SSC for 90 min at 60 C. The sections were dehydrated in a graded series of ethanols (50, 70, 90%) containing 0.3 M ammonium acetate and air dried. Slides were coated with emulsion (NTB-2; Kodak, Rochester, NY) and exposed for 6 months. Slides were developed (D-19 and Kodak developer) and counterstained with 1% pyronin-Y, and coverslips were applied with DPX mountant.

Analysis of hybridization signal.
Slides were coded so that the experimenter was unaware of the different conditions. Silver grains over the SON were visualized using bright-field microscopy (AX70; Olympus, Center Valley, PA) and quantified using National Institutes of Health Image software. Signal was assessed over cells (six per section) in four representative atlas-matched sections through the SON. Background measurements of signal over adjacent tissue was made and subtracted from total signal. The specificity of the hybridization signal for Y1R mRNA was assessed using hybridization with sense-strand RNA probes.

Statistics
Data are reported as mean ± SEM. Statistical analysis was completed using either a Student’s t test to compare group differences or a one-way ANOVA followed by a Student-Neuman-Keuls test for post hoc comparisons. Differences between groups were considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of plasma osmolality and body weight in euhydrate and 2% NaCl-loaded and water-deprived male rats
Animals that were subjected to either 72 h of 2% NaCl ingestion or 48 h water deprivation had increases in plasma osmolality (F(2, 12) = 8.964; P = 0.0042) and decreases in body weight (F(2, 19) = 12.88; P < 0.0001) that were significantly different from their corresponding controls (Table 1Go; baseline values for body weight was 277 ± 13 g). There were no differences in body weight or plasma osmolality in the euhydrate controls for the 2% NaCl ingestion and water-deprivation groups. Therefore, controls were combined in the experiments in which all three groups were compared; otherwise the description of the control group is provided in the text. An increase in hematocrit is a hallmark of water deprivation indicative of a decrease in extracellular fluid. Similarly, hematocrit was significantly increased by 72 h of 2% NaCl ingestion from 41.2 ± 0.5% in euhydrate controls to 45.1 ± 1.1% in rats drinking 2% NaCl for 72 h (P < 0.05). Animals deprived of water for 48 h had increased hematocrit values from 48.9 ± 1.1 to 57.5 ± 1.0% (P < 0.002). Therefore, both manipulations resulted in dehydration as characterized by a decrease in plasma volume and an increase in osmolality of the extracellular fluid.


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  		TABLE 1. Effect of 48 h water deprivation or 3 d of 2% sodium chloride ingestion on plasma osmolality and body weight in adult male rats

 
Changes in NPY Y1R expression in the SON with water deprivation and 2% saline ingestion
NPY Y1R immunoreactivity was present on cells in the SON as demonstrated previously (16). The intensity of overall staining for the Y1R in control (euhydrate) animals was light and predominantly localized to the cell body (Fig. 1AGo). There was no remarkable receptor immunoreactivity present on fibers. However, after either 48 h water deprivation or 72 h 2% saline ingestion, there was a dramatic increase in the intensity and number of cells expressing Y1R immunoreactivity (Fig. 1Go, B and C). The rostral-caudal distribution of Y1R-positive cells indicated that there were slightly more Y1R-immunopositive cells at atlas levels 1.40 and 1.80 mm caudal to bregma, although this difference was not significant from other regions in the same animal (Fig. 2AGo). At the individual atlas levels, there were significantly more Y1R immunoreactive cells in the sections (1.3–1.8 mm caudal to bregma) from water-deprived and salt-loaded animals when compared with the euhydrate controls. Total cell counts through representative sections of the SON (Fig. 2BGo) show a significant increase in the total number of cells expressing Y1R immunoreactive in animals that were either salt loaded (72 h) or water deprived (48 h) when compared with euhydrate controls [F(2, 20) = 20.81, P < 0.0001]. Visual inspection of these sections also suggested that the intensity of immunoreactive staining in each cell was increased, which would reflect more protein expressed per cell. To assess this more empirically, dilution curves of the Y1R antiserum (1:1,500; 1:3,000; 1:6,000, and 1:12,000) were run on serial sections through the SON of euhydrate, water-deprived, and salt-loaded animals (Fig. 1Go). At higher concentrations of the Y1R antibody (1:1500), distinct cells can be seen in all three groups (Fig. 1Go, A–C). However, as the antibody becomes more dilute, the signal becomes lighter (1:3000, Fig. 1Go, D–F). At the 1:6000 dilution, cells are no longer detectable in the control (euhydrate) group (Fig. 1GGo), but immunoreactive signal can still be detected in tissue sections from osmotically challenged animals (Fig. 1Go, H and I). At 1:12,000 dilution of the antiserum, no Y1R immunoreactivity was present within the SON from any of the groups (data not shown) but was still detected in other brain regions that express high levels of the Y1R (i.e. arcuate nucleus of the hypothalamus).


Figure 1
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  		FIG. 1. Photomicrographs of NPY Y1R immunoreactivity in the SON from animals that received ad libitum water (euhydrate; A, D, and G) or 2% NaCl drinking water (72 h; B, E, and H) or were water deprived (48 h; C, F, and I). Serial sections through animals from each condition were processed using different dilutions of the Y1R antisera (1:1500, 1:3000, 1:6000) to demonstrate increased amounts of immunoreactive protein in the SON of osmotically challenged animals. Sections are representative at 1.30–1.40 mm caudal to bregma. Scale bar, 100 µm.

 

Figure 2
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  		FIG. 2. A, Rostral-caudal representation of Y1R immunoreactive cell counts in the SON of male rats exposed to control (euhydrate), 2% NaCl (72 h) ingestion, and water deprivation (48 h). B, Total cell counts of Y1R immunoreactive (ir) cells within the SON of euhydrate control (euh; n = 8), 2% NaCl (72 h; n = 6) ingestion, and water deprivation (48 h; n = 5) groups. *, P < 0.05, **, P < 0.01, significantly different from corresponding euhydrate control (ANOVA, Student-Neuman-Keuls test).

 
The ability to detect Y1R immunoreactivity in the SON of osmotically challenged animals at the higher dilutions of antibody suggests that there is more protein available per cell to be detected in these groups. In situ hybridization for the NPY Y1R in the SON from control and animals given 2% NaCl drinking water indicates that there are more grains per cell in the salt-loaded animals, which is reflective of a greater expression of Y1R mRNA per cell (Fig. 3AGo). There are light-clustered grain densities over the SON, which are reflective of Y1R gene expression in the euhydrate animal (Fig. 3BGo). Augmented Y1R mRNA expression is observed as an increase in grain density within the SON of salt loaded animals (Fig. 3CGo).


Figure 3
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  		FIG. 3. A, Y1R mRNA levels in the SON from control animals and those receiving 2% NaCl in their drinking water for 72 h. **, P < 0.01, significantly different from euhydrate (euh) control, Student t test; n = 4 and 6, respectively. Dark-field photomicrographs of emulsion-coated sections showing Y1 mRNA expression in the SON of a euhydrate (B) and salt-loaded (C) (72 h 2% NaCl) male rat. Note the increased area of grain density over the SON in C. Ox, Optic chiasm. Scale bar, 100 µm.

 
To assess the rapidity of the effect of 2% NaCl ingestion on the expression of Y1R immunoreactivity, a time course at 24, 48, and 72 h was performed. At 24 h, hematocrit was significantly increased, compared with the euhydrate controls (Table 2Go). Plasma osmolality was significantly elevated after 48 and 72 h 2% NaCl ingestion. Whereas there was a small increase in Y1R immunoreactivity at 24 and 48 h, this was not significantly different from euhydrate controls. There was a significant increase in the number of cells expressing Y1R immunoreactivity at the 72-h time point [Fig. 4Go; F(3, 20) = 21.51, P < 0.001].


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  		TABLE 2. Time-dependent changes in plasma osmolality and hematocrit in male rats subjected to 2% NaCl drinking water for 24–72 h

 

Figure 4
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  		FIG. 4. NPY Y1R immunoreactivity in the SON of animals subjected to 2% NaCl ingestion for 24, 48, and 72 h. ***, P < 0.001, significantly different from all other groups (n = 5/group; ANOVA, Student-Neuman-Keuls test).

 
NPY Y1R immunoreactivity in VP and OT neurons
The generalized increase in the expression of Y1R that occurs throughout the SON corresponds and overlaps with the distribution of both VP and OT cells. Double-label immunohistochemistry was used to examine the relative change in expression of the Y1R in VP-NP vs. OT-NP immunoreactive cells. In control (euhydrate) animals, 34% of VP-NP cells and 33% of OT-NP cells coexpress the Y1R (Fig. 5Go). After 72 h of saline ingestion, 63% of VP-NP cells and 52% of OT-NP cells express Y1R immunoreactivity. Examples of double label immunohistochemistry for Y1R and VP-NP and OT-NP are represented in Figs. 6Go and 7Go. Whereas there was a significantly greater expression of the Y1R in VP-NP immunoreactive cells after salt loading (P < 0.01), the relatively equal distribution of Y1R immunoreactivity within both VP and OT cell populations corresponds well with the generalized increased expression of Y1R immunoreactivity throughout the SON (Figs. 1Go, B and C, 6Go, and 7Go). However, the increase in colocalization within the OT-NP cell population did not reach statistical significance.


Figure 5
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  		FIG. 5. Percent of VP-NP and OT-NP cells in the SON expressing Y1R immunoreactivity in euhydrate and salt-loaded animals (2% NaCl for 72 h). **, P < 0.01, significantly different from euhydrate group, Student’s t test; n = 5 per group.

 

Figure 6
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  		FIG. 6. Photomicrograph of Y1R immunoreactive (A) and VP-NP immunoreactive (B) cells in the SON (bregma –1.30 mm) from an animal given 2% NaCl drinking water (72 h). C, Merged image of A and B demonstrating colocalization of VP-NP and Y1R immunoreactivities as indicated by arrow. Arrowhead indicates single-labeled cells. D, Higher magnification of Y1R and VP-NP double-labeled cells in the SON from C. Note the characteristic distribution of VP-NP within cells in the ventral portion of the SON. Scale bar, 100 µm. ox, Optic chiasm.

 

Figure 7
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  		FIG. 7. Photomicrograph of Y1R immunoreactive (A) and OT-NP immunoreactive (B) cells in the SON (bregma –1.30 mm) from an animal given 2% NaCl drinking water (72 h). C, Merged image of A and B demonstrating colocalization of OT-NP and Y1R immunoreactivities as indicated by arrow. Arrowhead indicates single-labeled cells. Note the characteristic distribution of OT-NP within cells in the dorsal portion of the nucleus. Scale bar, 100 µm. ox, Optic chiasm.

 
Changes in expression of NPY Y1R immunoreactivity in magnocellular neurons
Other areas that demonstrated obvious changes in the number of cells that expressed Y1R immunoreactivity were the magnocellular division of the PVN (Fig. 8Go, A and B), the nucleus circularis (Fig. 8Go, C and D), and the retrochiasmatic nucleus (data not shown). In each of these regions, there was an apparent increase in the number of cells per area, and the intensity of the immunoreactive staining patterns after 2% NaCl ingestion was increased when compared with the euhydrate group. There was a significant increase in the number of cells expressing Y1R immunoreactivity in the magnocellular PVN (P = 0.012), whereas no significant change in cell number was observed within the medial parvocellular division of the PVN (PaMP; Figs. 8Go, A and B, and 9Go). In summary, changes in Y1R immunoreactivity were observed in cell groups of the hypothalamic magnocellular system: nuclei involved in the regulation of fluid homeostasis.


Figure 8
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  		FIG. 8. Photomicrographs of NPY Y1R immunoreactivity in the PVN of animals that received ad libitum water (euhydrate; A) or 2% NaCl drinking water (72 h; B). Y1R immunoreactivity in the n. circularis from a euhydrate (C) and salt-loaded (D) animal. E, Inset demonstrates VP-NP-immunoreactive cells in a euhydrate animal to verify the presence of the cell group in C. Scale bar, 100 µm. 3v, Third ventricle; PaLM, magnocellular PVN.

 

Figure 9
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  		FIG. 9. Effect of 72 h 2% NaCl ingestion on the number of Y1R immunoreactive cells in the magnocellular and parvocellular divisions of the PVN. **, P < 0.01, ANOVA, Student-Neuman-Keuls test, n = 4–8/group.

 
Y1R expression in the SON after food restriction
Animals that are water deprived or given 2% saline in their drinking water lose weight, in part due to the decrease in water intake and the resultant anorexia (24). Animals that are food deprived have increased hypothalamic NPY levels (25) and alterations in NPY receptor levels (26). To ensure that the elevations in Y1R immunoreactivity in the SON are due to changes in plasma osmolality and not alterations in food intake, a group of pair-fed animals were allowed free access to drinking water but given the same amounts of rat chow to match the amount that the 2% NaCl ingestion group consumed within a similar timeframe. This allowed the approximation of caloric intake and the degree of weight loss without disturbances to plasma osmolality (Table 3Go). Animals that ingested 2% NaCl for 72 h had plasma osmolality that was significantly elevated when compared with the euhydrate or pair-fed controls [F(2, 12) = 9.247; P = 0.0147]. The control rats gained weight (7.6%) over the 72 h, whereas the salt-loaded and pair-fed animals had net weight losses of 7.7and 0.9%, respectively. The differences in weight loss between the pair-fed and salt-loaded animals were not statistically different. The degree of further weight loss in the salt-loaded animals could be accounted for by the fact that they were not consuming as much fluid as the pair-fed animals. Animals given 2% NaCl had significantly more Y1R-immunoreactive cells in the SON than did either the euhydrate or pair-fed control animals (Fig. 10Go). The expression of Y1R immunoreactivity in the SON of euhydrate and pair-fed animals is indistinguishable between the two groups.


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  		TABLE 3. Effect of 72 h of 2% NaCl ingestion and pair feeding on plasma osmolality and body weight in adult male rats

 

Figure 10
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  		FIG. 10. Number of Y1R immunopositive cells in the SON of euhydrate, salt-loaded (2% NaCl for 72 h), or pair-fed animals (72 h). *, P < 0.05, significantly different from all other groups (ANOVA, Student-Neuman-Keuls test; n = 4/group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies demonstrate that NPY Y1R mRNA and protein expression in the SON is increased in animals subjected to dehydration via 48 h of water deprivation or 2% NaCl drinking water (72 h). The results from in situ hybridization and immunohistochemistry studies indicate that there is an increase in both the number of cells expressing the Y1R and the amount of Y1R per cell. The increased expression of Y1R is reflective of dehydration, and not decreases in food intake, because Y1R expression was not different between control and pair-fed animals. The greater percentage of VP and OT cells coexpressing Y1R immunoreactivity observed after 72 h of 2% NaCl ingestion provides evidence that these cells may become more responsive to NPY. This combined with the prior evidence that hypovolemia induced by dehydration or hemorrhage increases NPY peptide in SON and PVN magnocellular neurons and in the A1 cells projecting to these neurons suggests that NPY may be important in mounting a sustained elevation in VP and OT secretion during extended periods of dehydration. Future studies will focus on the role of up-regulated Y1 receptors and dynamic changes in NPY peptide expression in regulating dehydration-induced vasopressin and oxytocin secretion.

NPY has long been implicated in the regulation of VP and OT secretion (7, 11, 27), and more recently it has been determined that the stimulatory effects of NPY are mediated primarily through activation of the Y1R and/or Y5 receptor subtypes in the SON and PVN (12, 13). In these studies, NPY directly stimulates VP and OT release from the neurohypophyseal system as well as enhances the stimulatory actions of an {alpha}1-agonist on hormone release. This establishes a stimulatory role for NPY in the control of neurohypophyseal function and supports the presence of functional NPY receptors in these brain regions. NPY Y1R expression has been noted in the SON using both in situ hybridization (28) and immunohistochemical (16, 17) methods. The present studies demonstrate Y1R immunoreactivity on both VP- and OT-immunoreactive cells, providing additional, anatomical evidence that activation of NPY receptors can directly influence the activity of VP and OT neurons. That there is no detectable Y1R immunoreactivity on fibers within the SON suggests that the stimulatory effects of NPY are directly on the VP or OT cell body and not through a presynaptic mechanism of action like that for the NPY Y2 receptor subtype (10). Because cell bodies within the SON also contain {alpha}-adrenergic receptors, it is likely that both the stimulatory and modulatory effects of NPY (12) occurs at the level of the cell body.

The function of neuropeptides, such as NPY, is often to amplify and reinforce the actions of other neurotransmitters regulating important physiological processes (29, 30). This role can be further enhanced by dramatic increases in peptide and/or receptor expression under conditions of sustained stimulation (25) such as the stimulation of VP and OT secretion induced by chronic dehydration or salt loading. Whereas the expression of NPY is relatively low within the magnocellular hypothalamus under euhydrate conditions, dehydration, or hypovolemia induced by hemorrhage significantly increases the expression of NPY mRNA and peptide within cell bodies and fibers in the SON and PVN (18, 31). This increased NPY expression in magnocellular neurons is consistent with an autoregulatory role for NPY as has been shown for VP and OT as well as other peptides expressed in these neurons (32, 33). Additionally, hemorrhage induces NPY gene expression in cell groups projecting to the SON (20, 34). Thus, there may be more NPY released from adjacent nerve terminals within the SON. The current studies demonstrate that NPY Y1Rs are also elevated in animals subjected to either 48 h of water deprivation or 72 h of 2% NaCl ingestion. Both stressors induced similar increases in receptor expression. This is not surprising because the increase in plasma osmolality and the decrease in body weight was similar in both groups. The increase in Y1R expression did not occur until 72 h of 2% NaCl ingestion, which also coincided with the time course of increased NPY in the magnocellular neurons (18). The longer time course change in receptor expression suggests that NPY may not be involved in the moment to moment regulation of VP/OT release but may be necessary to support release over longer-term physiological challenges. The concurrent increase in NPY and Y1R expression during conditions of dehydration and lowered blood volume sets up a circuitry for a more sensitized response of VP/OT neurons to NPY and suggests that heightened NPY responsiveness may be important to achieve the sustained elevation in VP and OT release that occurs during times of extended dehydration stress.

The observed increase in Y1R immunoreactive staining in the SON could reflect either an increase in receptor synthesis or a decrease in receptor protein turnover. That mRNA levels are increased as detected by in situ hybridization for the Y1R suggests that the increase in immunoreactivity reflects increased synthesis of the receptor protein. Using a combination of similar techniques, elevated plasma osmolality and/or dehydration also increases N-methyl-D-aspartate receptor subunit R1 (35), apelin receptor (36), galanin R1 (37), and substance P receptor (19) expression. Whereas the expression of these receptors is elevated after either water deprivation or salt loading, each receptor has a time course that is different from the others. N-methyl-D-aspartate R1 receptor expression was elevated after 7–10 d of 2% saline ingestion (35), whereas apelin gene expression was significantly elevated after exposure to 48 h of either stimulus (36). Recent studies in our laboratory have shown that the number of neurokinin 3 receptor immunoreactive cells in the SON are not altered after 48 h of water deprivation [euhydrate: 87 ± 2 cells vs. water deprivation: 98 ± 13 cells (P = 0.44, t test)]. These data indicate that not all receptors are regulated to the same extent in response to perturbations in fluid intake. However, in general, these increases in receptor expression demonstrate that receptor levels are dynamically regulated and that multiple systems likely participate in the physiological responses of the magnocellular-neurohypophyseal system to water deprivation or hypertonic saline ingestion.

Examination of the pattern of immunostaining within the SON shows that there is a generalized increase in Y1R cell number throughout the rostral-caudal and dorsal-ventral aspects of the nucleus. This suggested equal expression within OT- and VP-containing cells within the nucleus. Double-label immunohistochemistry with VP-NP and OT-NP antibodies verified this observation in that approximately 30–36% of OT and VP immunoreactive cells coexpressed the Y1R in euhydrate controls and these values were increased to 52–63% for OT and VP, respectively, in salt-loaded animals.

Whereas these current studies provide a detailed analysis of Y1R expression in the SON, similar increases in Y1R immunoreactivity were also observed in other magnocellular nuclei: PVN, retrochiasmatic SON, and the nucleus circularis after either water deprivation or NaCl ingestion. However, other regions of the hypothalamus, such as the arcuate nucleus or the parvocellular PVN, in which NPY is important in the regulation of food intake, did not have detectable increases in the expression of Y1R immunoreactivity. Because these increases in Y1R expression occurred in neurons intimately involved with the maintenance of fluid balance, it lends further credence to the hypothesis that these changes in Y1R expression are specifically related to changes in fluid balance. However, one concern was that the increase in Y1R expression could be due to not only changes in plasma osmolality but also decreases in body weight as a result of the anorexia experienced by these animals (24). NPY peptide and receptor are increased in response to food deprivation (26, 38, 39). These findings, combined with other reports suggesting that the magnocellular neurons may be involved in feeding responses (40), led us to examine Y1R expression in a group of pair-fed animals to control for dehydration-induced anorexia (41). This was an important control because the pair-fed animals had significantly decreased body weights, compared with the control animals, yet their plasma osmolality was within the normal range, unlike the animals provided 2% NaCl in their drinking water. Interestingly, no significant increases in Y1R immunoreactivity were observed within any magnocellular cell group in the pair-fed group. Therefore, the observed increases in Y1R expression in response to salt loading or water deprivation are specific to dehydration. This difference in the regulation of Y1R expression in subdivisions of the PVN with different physiological challenges underscores the different roles of NPY within the PVN.

The mechanisms underlying the regulation of Y1R expression, specifically in the hypothalamus, are not fully elucidated. The observed increases in Y1R gene expression in the SON, although likely resulting from increased de novo mRNA synthesis, could also reflect increases in mRNA stability. The proximal signals increasing Y1R expression in the SON and PVN could be contributed by one of the many neurotransmitter signals to these regions that are elevated during salt loading or dehydration (42). NPY expression, among others, is one neurotransmitter that is elevated within the SON and PVN during salt loading. Because both NPY peptide and Y1R show a similar time course of induction within the SON after saline ingestion, it is possible that NPY might induce its own receptor expression. Additionally, elevated Y1R expression after saline ingestion or water deprivation could result from an increase in the expression of transcription factors such as cfos (43), which drives the expression of a large complement of genes. Equally likely is the possibility that Y1R expression is elevated as a result of the down-regulation of factors that are inhibitory to the VP/OT neurosecretory system. Activation of estrogen receptor (ER)-ß in the SON inhibits the secretion of vasopressin (44), and ERß expression within the magnocellular hypothalamus decreases after 2% NaCl ingestion, thereby removing a level of inhibition from the cell (45). Whereas the transcription of the Y1R gene is increased on activation of ER{alpha} in vitro, stimulation of ERß inhibits this process (46). The contributory role of ERß on Y1R expression in the SON in vivo is unknown.

In conclusion, changes in fluid balance and not food restriction induce the expression of the Y1R, stressing the importance of the Y1R in the regulation of water balance and vasopressin and oxytocin secretion. Using these paradigms of water deprivation and salt loading, there is a general increase in the expression of Y1R within both VP and OT neural populations. Further studies will need to more fully examine the role of NPY and its receptors in regulating osmotic- and hemodynamic-induced changes in VP and OT release as well as the interaction of Y1R with other neurotransmitter systems. Additionally, it will be important to identify the mechanisms underlying the increase in Y1R expression in the magnocellular system.


   Footnotes
 
This work was supported by National Institutes of Health Grants NS044835 (to C.D.S., J.H.U.) and MH62121 (to J.H.U.).

Current address for S.J.S.: Department of Clinical Investigation, MCKH-CI1 Jarrett White Road, Tripler Army Medical Center, Honolulu, Hawaii 96859.

Disclosure statement: The authors have nothing to disclose.

First Published Online May 25, 2006

Abbreviations: ER, Estrogen receptor; NDS, normal donkey serum; NP, neurophysin; NPY, neuropeptide Y; OT, oxytocin; PaMP, medial parvocellular neuron; PVN, paraventricular nucleus; SON, supraoptic nucleus; SSC, standard saline citrate; VP, vasopressin; Y1R, Y1 receptor.

Received March 23, 2006.

Accepted for publication May 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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