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Am J Physiol Regul Integr Comp Physiol 290: R1251-R1261, 2006. First published November 23, 2005; doi:10.1152/ajpregu.00727.2005
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Neurohypophyseal Hormones: From Genomics and Physiology to Disease

Differential effects of water and saline intake on water deprivation-induced c-Fos staining in the rat

Helmut B. Gottlieb, Lisa L. Ji, Heath Jones, Maurice L. Penny, Tiffany Fleming, and J. Thomas Cunningham

Department of Pharmacology and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas

Submitted 13 October 2005 ; accepted in final form 22 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We studied c-Fos staining in adult male rats after 48 h of water deprivation and after 46 h of water deprivation with 2 h of access to water or physiological saline. Controls were allowed ad libitum access to water and physiological saline. For immunocytochemistry, anesthetized rats were perfused with a commercially available antibody for c-Fos. Dehydration significantly increased plasma vasopressin (AVP), osmolality, plasma renin activity (PRA), hematocrit, and sodium concentration and decreased urinary volume. Fos staining was significantly increased in the median preoptic nucleus, organum vasculosum of the lamina terminalis, supraoptic nucleus (SON), and magnocellular and parvocellular paraventricular nucleus (PVN), as well as the area postrema, nucleus of the solitary tract (NTS), and rostral ventrolateral medulla (RVL). Rehydration with water significantly decreased AVP levels and Fos staining in the SON, PVN, and RVL and significantly increased Fos expression in the perinuclear zone of the SON, NTS, and parabrachial nucleus. Rehydration with water was associated with decreased urinary sodium concentration and hypotonicity, and hematocrit and PRA were comparable to levels seen after dehydration. After rehydration with saline, plasma osmolality, hematocrit, and PRA were not different from control, but plasma AVP and urinary sodium concentration were increased. In the SON, Fos staining was significantly increased, with a great percentage of the Fos cells also stained for oxytocin compared with water deprivation. Changes in Fos staining were also observed in the NTS, RVL, parabrachial nucleus, and PVN. Rehydration with water or saline produces differential effects on plasma AVP, Fos staining, and sodium concentration.

vasopressin; thirst; anteroventral third ventricle; dehydration

IN THE RAT, WATER DEPRIVATION is a progressive physiological condition associated with increased plasma osmolality, hypovolemia, and activation of the renin-angiotensin system. In addition, dehydration results in release of neurohypophyseal hormones, activation of the sympathetic nervous system, and thirst and sodium appetite. The central mechanisms responsible for these effects are not completely understood.

Immunocytochemistry for the protein Fos, the product of the gene c-fos, has been widely used to map the activation of specific regions of the central nervous system associated with acute homeostatic challenges (9, 10, 12, 13, 20). In addition, several studies have demonstrated that water deprivation results in apparently persistent Fos staining in a variety of brain areas related to body fluid regulation, including neurohypophyseal neurons located in the supraoptic (SON) and the magnocellular subdivisions of the paraventricular (PVN) nuclei of the hypothalamus, parvocellular neurons in the PVN, sympathetic premotor neurons in the rostral ventrolateral medulla (RVL), and areas related to the lamina terminalis that are involved in water and sodium intake (15, 25, 31, 34, 37, 47, 48).

Water intake rapidly decreases plasma vasopressin (AVP) in a variety of species (5, 16, 23, 52). Electrophysiological studies have shown that the decrease in circulating AVP associated with water intake after dehydration is likely mediated by a rapid inhibition of magnocellular neurons in the SON and PVN (4, 38, 46, 57). Several studies also have observed that Fos staining in the SON produced by a variety of challenges, including dehydration, is rapidly decreased after water intake (15, 25, 34, 36, 58). In the rat, the decrease in plasma AVP does not occur when the animals are given a saline solution to drink (23).

In the present study, we used c-Fos immunocytochemistry to study the areas of the central nervous system that are activated during dehydration and to test the hypothesis that rehydration with water, but not physiological saline, will decrease circulating AVP and Fos staining in neurohypophyseal neurons in the SON and PVN. In addition, we also tested the effects of rehydration with water or saline on Fos staining in other regions of the brain that might mediate the effects of dehydration on sympathetic activation and water and salt intake. In some experiments, we also measured the effects of these treatments on urinary output and sodium concentration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Adult male Sprague-Dawley rats (250–350 g body wt; Charles River) were individually housed and maintained in a temperature-controlled (23°C) environment on a 14:10-h light-dark cycle. The rats had ad libitum access to food, water, and 0.9% NaCl before the experiments. All experimental protocols were approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the US Public Health Service, American Physiological Society, and Society for Neuroscience.

Experimental Protocol

All rats had ad libitum access to food throughout the experiments, except during the 2 h before perfusion. The control group was allowed ad libitum access to water and 0.9% NaCl throughout the experiment. One group of rats was water deprived for 48 h. Two additional groups were water deprived for 46 h and given water or 0.9% NaCl for 2 h before the experimental procedures. After the treatment protocols, plasma renin activity (PRA), AVP, osmolality, and hematocrit were measured in four separate groups of rats. The rats were lightly anesthetized with thiobutabarbital (Inactin; 100 mg/kg ip) and immediately decapitated, and trunk blood was rapidly collected into chilled centrifuge tubes containing EDTA. A separate sample was collected into a 1.5-ml microcentrifuge tube that did not contain EDTA. Two hematocrit tubes (Fisher) were filled from the microcentrifuge tube for measurement of hematocrit (Micro-Hematocrit capillary tube reader; Lancer, St. Louis, MO). The remainder of the blood in the microcentrifuge tube was centrifuged for 5 min (at 10,000 rpm). After the blood was centrifuged, a 200-µl sample of plasma was removed for plasma osmolality measurement using a vapor pressure osmometer (Wescor, Logan, UT).

The blood collected in tubes containing EDTA (1 mg/ml) was centrifuged at 10,000 rpm for 10 min at 4°C. Plasma (2 ml) was removed from the sample, placed in a conical centrifuge tube, and kept in an ultracold (–80°C) freezer until it was shipped on dry ice to the RIA Core at the University of Iowa for measurement of PRA and AVP. PRA was determined using a commercially available kit that measures the conversion of angiotensin I (Gammacoat PRA kit, DiaSorin). Plasma AVP concentration was determined using a specific RIA after an acetone-petroleum ether extraction, as previously described (29, 50), with use of an anti-AVP antibody donated by Dr. Willis Samson (St. Louis University) (43).

Fos Immunocytochemistry

Four separate groups of rats were used for histology. Some of the rats were tested in commercially available metabolism cages (Lab Products, Maywood, NJ) for collection of urine samples to measure urinary volume and sodium concentration. To habituate the rats to the protocol, we placed them in the metabolism cages for 2–3 h for 5 days before the experiment. During this habituation period and the testing period, water and saline intake were recorded and urine samples were collected for determination of baseline urinary rate and sodium concentration. No attempt was made to void the bladders before or after the treatment period, so the volume of urine collected is only an approximation of the actual output. On the day of the experiment, fluid intake was measured for 2 h before perfusion for all rats that had access to water or saline, whether the rats were in their home cage or a metabolism cage. Food was withheld during the habituation trials and during testing. All experiments and habituation trials were conducted between 0900 and 1200.

All rats were deeply anesthetized with Inactin (100 mg/kg ip) and perfused with 0.1 M PBS followed by 300–500 ml of 4% paraformaldehyde in PBS. After the perfusion, the brains were removed from the skull and placed in PBS with 30% sucrose for 3–4 days. The forebrain and hindbrain were separately sectioned at 40 µm in a cryostat. Three serial sets of coronal sections from each region were placed in cryoprotectant and stored at –20°C until they were processed for immunocytochemistry as previously described (8, 21).

A commercially available antibody directed at amino acid residues 4–17 in human c-Fos (rabbit anti-c-Fos Ab5, Calbiochem, San Diego, CA) was used to stain one set of free-floating sections from each rat for Fos. After 72 h of incubation in the primary antibody (1:30,000 dilution) at 4°C, the sections were incubated in biotinylated horse anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:200 in PBS for 2 h at room temperature. After an additional 60-min rinse in PBS, the sections were reacted with an avidin-peroxidase conjugate (Vectastain ABC kit; Vector Laboratories) and PBS containing 0.04% 3,3'-diaminobenzidine hydrochloride and 0.04% nickel ammonium sulfate. Forebrain sections were rinsed for 30 min in PBS and incubated in antioxytocin antibody (courtesy of A. J. Silverman) diluted 1:1,000 in PBS for 5 days at 4°C. After a 60-min rinse in PBS, the sections were incubated in a Cy3-labeled anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) for 3 h at room temperature (8). A commercially available antibody (mouse antidopamine beta-hydroxylase, MAb 308, Chemicon, Temecula, CA) and a Cy3-labeled anti-mouse secondary antibody (Jackson ImmunoResearch) were used to stain hindbrain sections for dopamine beta-hydroxylase (DBH). After the staining was complete, the sections were mounted on gelatin-coated slides and air-dried for 1–2 days, and coverslips were applied with Permount.

Sections were examined using light microscopy to identify c-Fos-positive cells in the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus (MnPO), perinuclear zone of the SON (PNZ), PVN, and SON from the forebrain and the nucleus of the solitary tract (NTS), caudal ventrolateral medulla (CVL), rostral ventrolateral medulla (RVL), and parabrachial nucleus (PBN) from the hindbrain. Tissue sections containing regions of interest were examined with a microscope (model IX 50, Olympus) equipped for epifluorescence. Digital images were acquired using a Spot camera (SPOT RT Slider, Diagnostic Instruments, Sterling Heights, MI) connected to a Pentium computer running Spot imaging software (version 3.24). Regions of the forebrain were identified on the basis of the rat brain stereotaxic atlas of Paxinos and Watson (41) as previously described (21, 25). For analysis, three images were obtained from the SON of each set of stained sections. The oxytocin immunofluorescence was used to ensure that sections were obtained from the same rostral-caudal plane for each set of sections from each rat, and the number of oxytocin- and Fos-positive cells was recorded for each section. The magnocellular and parvocellular portions of the PVN were analyzed separately. For parvocellular PVN, counts from the different parvocellular subnuclei were pooled for analysis. The number of oxytocin- and Fos-stained cells in the magnocellular PVN was included in the analysis. For the OVLT, analysis included dividing the nucleus into two regions, the dorsal cap and the lateral margins, on the basis of the work of McKinley et al. (32) and Oldfield and colleagues (40). The portion of the NTS used for analysis extends from 300 µm caudal to obex and 300–400 µm past the rostral edge of the area postrema (11, 42). The CVL was defined posteriorly by the pyramidal decussation and anteriorly by the appearance of the principal nucleus of the inferior olive (11, 42). The anterior border of the RVL was defined by the caudal pole of the facial nucleus, and the rostral hypoglossal nucleus was used for the posterior border (11, 42). Analysis of the PBN included the ventral lateral, central medial, external lateral, and external medial regions, which included the pontine taste area (19, 39, 54). Analysis of the NTS and RVL included the number of cells that were labeled for Fos and DBH. Manual cell counts were performed by individuals who were blinded to the experimental conditions associated with the samples. Counters were instructed to include cells that appeared to be positively labeled, regardless of the intensity of the staining. Counts from each area were averaged for each animal.

Statistics

Data were analyzed by one-way analysis of variance with Student-Newman-Keuls t-test for post hoc analysis of significant main effects (SigmaStat, version 2.03, Systat Software, Point Richmond, CA). Alternatively, Kruskal-Wallis analysis of variance on ranks and Dunn's multiple comparison procedure were used when data were not normally distributed (28). Significance was set at P < 0.05. Values are means ± SE.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
During the 2-h test period, the rats given access to water drank an average of 26.1 ± 2 ml and the rats given access to saline drank 48.8 ± 3 ml. This difference was statistically significant (P < 0.001).

Plasma Measurements

The results of the plasma measurements are listed in Table 1. Water deprivation for 48 h significantly increased plasma AVP, osmolality, hematocrit, and PRA. In rats given access to water for 2 h, plasma AVP was significantly decreased compared with water-deprived rats and not significantly different from control. Plasma osmolality was significantly decreased compared with control, whereas PRA and hematocrit were significantly higher than control. In rats given saline for 2 h, plasma AVP levels were still significantly increased compared with control but significantly lower than in 48-h water-deprived rats. Plasma osmolality, hematocrit, and PRA of the rats given access to saline were not different from control.


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  		Table 1. Plasma AVP, osmolality, hematocrit, and PRA in control and water-deprived rats

 
Urine Measurements

During the 2-h test period, urinary volume of the 48-h water-deprived group was significantly decreased compared with the other groups (Fig. 1). Urinary sodium concentration was significantly decreased in the 48-h water-deprived rats and water-deprived rats given access to water compared with control rats (Fig. 1). In contrast, urinary sodium concentration was significantly increased in the rats that were dehydrated and given access to saline (Fig. 1).


Figure 1
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  		Fig. 1. Changes in urinary flow (V) and sodium concentration (UNa) measured for 2 h during control (Con, n = 6), 46 h of water deprivation with 2 h of access to 0.9% NaCl (46 h + S, n = 9), 46 h of water deprivation with 2 h of access to water (46 h + W, n = 7), and 48 h of water deprivation (48 h, n = 7). *Significantly different from all other groups (P < 0.05 by Student-Newman-Keuls test). +Significantly different from Con and 46 h + W. #Significantly different from Con.

 
c-Fos Immunocytochemistry

Forebrain. Dehydration and rehydration with water or 0.9% saline produced significant changes in Fos staining in all forebrain regions included in the analysis (Fig. 2). Water deprivation for 48 h significantly increased Fos staining in the SON, MnPO, dorsal cap and lateral margins of the OVLT, and magnocellular and parvocellular PVN. Water deprivation did not significantly affect Fos staining in the PNZ.


Figure 2
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  		Fig. 2. Changes in c-Fos staining in forebrain associated with control (n = 7), 46 h of water deprivation and 2 h of access to water (n = 8), 46 h of water deprivation with 2 h of access to 0.9% NaCl (n = 10), and 48 h of water deprivation (n = 7). SON, supraoptic nucleus; PVPm and PVNp, magnocellular and parvocellular paraventricular nuclei; PNZ, perinuclear zone of SON; MnPO, median preoptic nucleus; OVLTdc and OVLTlm, dorsal cap and lateral margin of organum vasculosum of lamina terminalis. *Significantly different from Con (P < 0.05). #Significantly different from Con and 46 h + W (P < 0.05). +Significantly different from all other groups.

 

REHYDRATION WITH WATER. Rehydration with water significantly increased Fos staining in the PNZ. In the SON, Fos staining after rehydration with water was significantly different from 48 h of water deprivation and not different from control. After rehydration with water, staining for Fos in the parvocellular and magnocellular PVN was significantly decreased compared with 48 h of water deprivation and still significantly increased compared with control. Fos staining in the MnPO and OVLT after rehydration with water was significantly increased compared with control and not different from 48 h of water deprivation.


REHYDRATION WITH SALINE. After rehydration with saline, Fos staining in the SON, MnPO, and OVLT was significantly increased compared with control and not statistically different from 48 h of water deprivation (Fig. 2). In the parvocellular PVN, rehydration with saline was associated with a decrease in Fos staining significantly lower than that in rats subjected to 48 h of water deprivation but still significantly higher than control. In the PNZ, Fos staining after rehydration with saline was significantly decreased compared with rehydration with water and not significantly different from control.

Although we did not observe significant differences in the average number of Fos-positive cells in the OVLT between dehydration and rehydration with saline, the pattern of labeling suggested that rehydration with saline was associated with more Fos staining in the dorsal cap of the OVLT (Fig. 3). When the percentage of the Fos-positive cells located in the dorsal cap of the OVLT after rehydration with saline was tested against 48 h of water deprivation and rehydration with water, rehydration with saline was associated with a significantly higher percentage than the other two conditions: 44.2 ± 1.7% for 48 h, 42.3 ± 4% for 46 h + water, and 51.1 ± 3.6% for 46 h + saline (P < 0.04 by Student-Newman-Keuls test).


Figure 3
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  		Fig. 3. Digital images of representative examples of Fos staining in OVLT from control, 46 h of water deprivation and 2 h of access to water, 46 h of water deprivation with 2 h of access to saline, and 48 h of water deprivation. DC, dorsal cap; LM, lateral margin.

 
Oxytocin double labeling. Further analysis of the SON and magnocellular PVN revealed significant changes in the number of Fos-positive oxytocin cells produced by the different treatment conditions. An example of Fos staining in the medial SON is shown in Fig. 4. Both 48 h of water deprivation and rehydration with saline were associated with significant Fos staining throughout the dorsal and ventral aspect of the nucleus, suggesting that AVP and oxytocin cell types are Fos positive. However, when the sections are analyzed for oxytocin immunofluorescence, sections from rats that were allowed to rehydrate with saline showed more oxytocin and Fos double labeling than sections from dehydrated rats (Fig. 5, top). The average number of oxytocin- and Fos-labeled cells was significantly increased in the SON of rats rehydrated with saline compared with 48-h water-deprived rats (28.5 ± 2.6 vs. 17.0 ± 2.1, P < 0.001), whereas the average number of magnocellular PVN neurons that were stained for oxytocin and Fos was not significantly different between these treatments (14.2 ± 2.7 for 46 h + saline and 17.7 ± 3.8 for 48 h, P < 0.4). However, the percentage of c-Fos-positive cells in the SON and magnocellular PVN that are also labeled for oxytocin was significantly increased in rats rehydrated with saline compared with 48-h water-deprived rats (Fig. 5, bottom).


Figure 4
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  		Fig. 4. Digital images of representative examples of Fos staining in SON and PNZ from control, 46 h of water deprivation and 2 h of access to water, 46 h of water deprivation with 2 h of access to saline, and 48 h of water deprivation. ot, Optic tract.

 

Figure 5
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  		Fig. 5. Top: digital images of representative examples of Fos immunostaining (A and C) and oxytocin immunofluorescence (B and D) in SON from rats subjected to 46 h of water deprivation and 2 h of access to saline (A and B) and 48 h of water deprivation (C and D). Scale bars, 50 µm. Bottom: percentage of c-Fos-labeled cells that were also labeled with oxytocin in SON and magnocellular portion of PVN (PVNm) from rats subjected to 46 h of water deprivation with access to saline and 48 h of water deprivation. Arrows indicate cells labeled for both c-Fos and oxytocin. *Significantly different (P < 0.05 by Student-Newman-Keuls test).

 
Hindbrain. Dehydration and rehydration with water or saline had differential effects on Fos staining in the hindbrain regions (Fig. 6). Dehydration significantly increased Fos staining in the AP, NTS, and RVL. In contrast, Fos staining in the CVL and PBN was not significantly influenced after dehydration.


Figure 6
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  		Fig. 6. Changes in Fos staining in hindbrain associated with control (n = 7), 46 h of water deprivation and 2 h of access to water (n = 8), 46 h of water deprivation with 2 h of access to 0.9% NaCl (n = 10), and 48 h of water deprivation (n = 7). AP, area postrema; NTS, nucleus of solitary tract; CVL and RVL, caudal and ventrolateral medulla; PBN, parabrachial nucleus. *Significantly different from Con (P < 0.05). #Significantly different from Con and 46 h + W (P < 0.05). +Significantly different from all other groups.

 

REHYDRATION WITH WATER. In the AP, rehydration with water was associated with Fos staining that was not different from staining associated with 48 h of water deprivation and significantly greater than control. Rehydration with water significantly increased Fos staining in the PBN and NTS compared with control and 48 h of water deprivation. In the RVL, Fos staining after rehydration with water was significantly decreased compared with 48 h of water deprivation but still significantly higher than control, whereas Fos staining in the CVL was not significantly affected.


REHYDRATION WITH SALINE. In the AP, rehydration with physiological saline was associated with Fos staining that was increased compared with control but not different from 48 h of water deprivation or rehydration with water. In the NTS, RVL, and PBN, Fos staining after rehydration with saline was significantly increased compared with control and significantly decreased compared with 48 h of water deprivation and rehydration with water. Staining for Fos in the CVL was not significantly affected by rehydration with saline.

Fos staining in the NTS after dehydration was located primarily in the caudal and subpostremal parts of the nucleus (Fig. 7). Rehydration with water or saline was associated with increased staining for Fos in these parts of the nucleus and portions of the NTS rostral to the AP (Fig. 8). In the PBN, rehydration with water was associated with increased Fos staining in the medial subnucleus ventral to the superior cerebellar penduncle (Fig. 9). In addition, Fos-positive cells were also located in the ventral, central, and external portions of the lateral PBN. After rehydration with saline, Fos staining in the PBN was located primarily in the ventral and central portions of the lateral subnuclei, with some staining in the medial PBN (Fig. 9).


Figure 7
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  		Fig. 7. Digital images of representative examples of c-Fos staining in caudal NTS from control rats and rats subjected to 46 h of water deprivation and 2 h of access to water, 46 h of water deprivation with access to saline, and 48 h of water deprivation. DMV, dorsal motor nucleus of vagus.

 

Figure 8
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  		Fig. 8. Digital images of representative examples of c-Fos staining in medial NTS from control rats and rats subjected to 46 h of water deprivation and 2 h of access to water, 46 h of water deprivation with access to saline, and 48 h of water deprivation.

 

Figure 9
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  		Fig. 9. Digital images of representative examples of c-Fos staining in parabrachial nucleus from control rats and rats subjected to 46 h of water deprivation and 2 h of access to water, 46 h of water deprivation with access to saline, and 48 h of water deprivation. LPBN and MPBN, lateral and medial parabrachial nuclei; c, el, and v, central, external, and ventral LPBN; scp, superior cerebellar peduncle.

 

DBH DOUBLE LABELING. In the caudal NTS, the number of DBH- and Fos-positive cells was significantly increased after dehydration and rehydration with water or saline: 2.5 ± 0.6 for control, 9.0 ± 1.1 for 48 h, 20.7 ± 1.9 for 46 h + water, and 25.3 ± 2.4 for 46 h + saline (all P < 0.01 by Student-Newman-Keuls test; Fig. 10). Furthermore, the increases in DBH- and Fos-stained cells were significantly higher after rehydration with water or saline than after 48 h of water deprivation (P < 0.001 by Student-Newman-Keuls test). The percentage of Fos-positive cells that were also labeled with DBH was higher after rehydration with water than in the other treatment groups, but the apparent increase was not statistically significant (Fig. 10). In the RVL, the average number of Fos- and DBH-stained cells was significantly increased after water deprivation compared with the other treatments: 4.5 ± 0.9 for control, 28.4 ± 2.1 for 48 h, 14.4 ± 1.4 for 46 h + water, and 10.2 ± 1.1 for 46 h + saline (all P < 0.001 by Student-Newman-Keuls test). Rehydration with water or saline also was associated with significant increases in the numbers of DBH- and Fos-stained cells (all P < 0.05 by Student-Newman-Keuls test). The percentage of Fos-stained cells that were also DBH positive was not significantly different among the groups (Fig. 10).


Figure 10
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  		Fig. 10. Top: digital images of representative examples of c-Fos immunostaining (A and C) and dopamine beta-hydroxylase immunofluorescence (B and D) in caudal NTS from rats subjected to 46 h of water deprivation and 2 h of access to water (A and B) and 48 h of water deprivation (C and D). Scale bars, 25 µm. Bottom: percentage of c-Fos-labeled cells that were also labeled with dopamine beta-hydroxylase in caudal NTS and RVL from rats subjected to 46 h of water deprivation with access to water, 46 h of water deprivation with access to saline, and 48 h of water deprivation. Arrows indicate cells that contained both c-Fos and dopamine beta-hydroxylase staining.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Rehydration with water or saline produced differential effects on plasma AVP and PRA, urinary output, and Fos staining. Dehydration significantly increased plasma AVP, osmolality, hematocrit, and PRA, whereas urinary rate decreased and urinary sodium concentration increased compared with controls. Significant increases in Fos staining were observed in a variety of areas, including the MnPO, OVLT, SON, and PVN. In the SON and PVN, dehydration increased Fos staining in AVP and oxytocin neurons. In the hindbrain, Fos staining was increased in the NTS and RVL. In the RVL, Fos staining after 48 h of water deprivation was significantly increased in catecholamine and noncatecholamine neurons. These findings are consistent with previous studies (15, 25, 30, 31, 34, 37, 48, 56).

Access to water alone after dehydration produced hypoosmolality and significantly decreased AVP and urinary sodium concentration without affecting PRA and hematocrit. As expected, rehydration with water was associated with significant decreases in Fos staining in the SON and magnocellular PVN that were not different from control. This decrease in Fos staining in magnocellular neurons of the neurohypophyseal systems is consistent with the decrease in circulating AVP and the increase in urinary volume and decrease in urinary sodium concentration. In the parvocellular PVN and the RVL, Fos staining was significantly attenuated by water intake but still significantly higher than control. These results suggest that the effects of water intake were greater on neurohypophyseal neurons than in regions associated with autonomic function and regulation of anterior pituitary function. These results are consistent with previous studies in the rat that suggest neurohypophyseal function is subject to rapid inhibition by visceral afferents (1, 3, 23, 25, 38, 46, 49), as opposed to autonomic pathways, which may be regulated more by changes in plasma osmolality (6, 53). This hypothesis would suggest that the inhibitory signal for neurohypophyseal neurons would be generated during ingestion, whereas postabsorption cues that might regulate the parvocellular PVN and the RVL might take longer to develop and be reflected by changes in Fos staining. Also, the decreases in Fos observed in these regions occurred despite the fact that PRA and hematocrit were still significantly increased. This could suggest that the increases in Fos staining in each of these areas after dehydration are related to plasma osmolality or that the inhibitory effects of water intake can override any excitatory drive provided by hypovolemia or activation of the renin-angiotensin system.

The NTS, PBN, and PNZ demonstrated significant increases in Fos staining after rehydration with water. The increase in Fos staining in the NTS after rehydration with water was significantly higher than the increase after dehydration, and this increase occurred in the caudal and medial portions of the NTS. In addition, the number of Fos-positive cells that were also labeled for DBH also was increased after rehydration with water. In the PBN, Fos staining after rehydration with water occurred in the medial and lateral parts of the nucleus, including regions that have been described as receiving projections from the caudal and medial NTS (19), as well as from the hypothalamus and the limbic system (35). Staining for Fos in the MnPO, OVLT, and AP after water intake was not different from the increases observed after dehydration. In general, the results of the Fos staining were similar to those we reported previously (25), despite the fact that the rats in the present study did not have access to food during the rehydration period.

When rats were allowed access to saline for 2 h after dehydration, plasma osmolality, hematocrit, and PRA were significantly reduced to levels comparable to controls. Plasma AVP was significantly decreased compared with levels produced by dehydration but was still significantly higher than in control rats or rats rehydrated with water. Urinary sodium concentration was also significantly increased in rats given saline to drink after dehydration, whereas urinary volume was not different from control. This increase in sodium concentration, along with the change in urinary output, compared with dehydrated rats may have contributed to the observed change in plasma osmolality.

In the SON and magnocellular PVN, Fos staining after rehydration with saline was significantly higher than control and, in the SON, comparable to levels seen after dehydration. However, in both regions, the numbers and percentage of Fos-positive cells that were also labeled with oxytocin were significantly increased compared with the water-deprived group, suggesting that more of the Fos-stained cells were oxytocinergic. Previous experiments suggest that oxytocin has natriuretic effects in the rat (18, 22), and the Fos staining observed in oxytocin neurons in the SON and magnocellular PVN could be related to increased levels of circulating oxytocin that contribute to the natriuresis associated with dehydration and rehydration with saline. These results also demonstrate that rehydration with saline was not associated with the same decrease in Fos staining in magnocellular neurons or circulating AVP observed after rehydration with water. Overall, the results are consistent with previous studies which demonstrated that the reduction in plasma AVP associated with water intake in the rat does not occur after saline ingestion (23), although the attenuation in plasma AVP may have been related to the time course used in the present study.

The levels of Fos staining in the RVL and parvocellular PVN after rehydration with saline were significantly decreased compared with dehydration but still significantly higher than after rehydration with water and control. Thus the magnitude of the decrease in Fos staining in these two regions was differentially influenced by whether the rats had access to water or saline. These intermediate results could be due to the time course associated with Fos staining and return of plasma osmolality to levels that were not significantly different from control. If the increase in urinary sodium concentration was required for plasma osmolality to approach normal values, this change should have occurred later in the 2-h test period than the point at which the rats had water to drink. Also, it is possible that the decreases in Fos staining in these regions after rehydration reflect the contribution of the renin-angiotensin system and/or volume receptors to the regulation of these areas.

The average number of Fos-positive neurons in the AP, MnPO, and OVLT was not decreased after rehydration with water or saline and, in each case, was not statistically different from the Fos staining observed after dehydration. However, in the OVLT, the anatomic distribution of Fos staining was different when the rats were rehydrated with saline. A greater proportion of the Fos-positive cells in the OVLT were located in the dorsal cap region after rehydration with saline than after dehydration or rehydration with water. The dorsal cap of the OVLT has been described as containing osmosensitive neurons that project to the SON (31, 33). The other major subdivisions of the OVLT, the lateral zones, are characterized as having cells that are sensitive to circulating angiotensin (33). The change in the distribution of Fos-positive cells in the OVLT could be related to the significant decrease in PRA after rehydration with saline. The decrease in PRA would be associated with decreased circulating angiotensin, which could reduce excitatory drive to neurons in the lateral zone of the OVLT. Alternatively, the sodium in the saline could be responsible for greater activation of neurons in the dorsal cap, even though plasma osmolality was not different from control. In contrast to the OVLT, Fos staining in the MnPO and AP does not appear to be differentially affected by rehydration with water or saline. Previously, it had been suggested that Fos staining in the MnPO and OVLT after rehydration with water alone was due to sustained activation of the renin-angiotensin system (15). On the basis of the present results, this could be true for the OVLT, whereas the MnPO and AP may be less dependent on the renin-angiotensin system or do not respond with the same time course.

The NTS and PBN showed significant increases in Fos staining after rehydration with saline that were less than the increases observed after rehydration with water. These results occurred despite the fact that the rats drank more saline than water during the test period. In the NTS, there were fewer Fos-stained cells in the caudal and medial regions of the nucleus and fewer Fos-positive catecholamine cells in the A2 region of the caudal NTS after rehydration with saline than after rehydration with water. However, the percentage of Fos-positive cells that were DBH positive was not different among the conditions. In the PBN, Fos staining after rehydration with saline tended to be more localized in the lateral parts of the nucleus. The NTS and PBN are involved in gustatory processing (54), so it could be expected that both regions would show increases in Fos staining associated with water or saline intake. The rapid decrease in circulating neurohypophyseal hormones is hypothesized to be mediated by visceral afferents that are activated during water intake, but not during saline ingestion (23). The NTS receives afferent projections from a variety of organ systems, which likely include these visceral afferents (27). The NTS also receives afferents from other brain regions, including the PBN, hypothalamus, and limbic system (44, 45), which may also play a role in the differences in Fos staining that were observed after dehydration and rehydration with water or saline.

In contrast to rehydration with water, Fos staining in the PNZ was not affected by rehydration with saline. The PNZ is located dorsal to the SON and reported to contain a population of interneurons that participate in the inhibition of AVP neurons by increases in blood pressure and blood volume (2, 7). Increased Fos staining in the PNZ was only observed after rehydration with water, which also was associated with significant decreases in circulating AVP and Fos staining in magnocellular neurons. This occurred despite the fact that the rats were volume depleted before rehydration and that, after rehydration with water, plasma volume was not different from control. It could be that the increase in Fos staining in the PNZ associated with rehydration with water is related to the activation of visceral afferents associated with water intake.

Perspectives

The results of the present study support the hypothesis that water intake decreases plasma AVP levels and Fos staining in the SON and magnocellular PVN and that these effects are not associated with saline intake. On the basis of the results of the Fos staining, it could be speculated that the population of putative inhibitory neurons in the PNZ could participate in the rapid inhibition of neurohypophyseal hormone release associated with water intake. In addition, the NTS and PBN also showed increased Fos staining after water intake. The NTS has a well-described projection to the PBN (19, 54), and the NTS and PBN are reported to project to the PNZ region (55). Thus the NTS and PBN could contribute to the increased Fos staining observed in the PNZ and/or the decrease in neurohypophyseal hormones after rehydration with water. Nevertheless, the role of the PBN in regulating AVP release has been difficult to define (14, 24, 51). The significance of the differential effects of water and sodium intake on Fos staining in the NTS and PBN remains to be determined.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Institutes of Health Grants R01 HL-67529 and R01 DK-57822 (to J. T. Cunningham).


   ACKNOWLEDGMENTS
 
We acknowledge the technical assistance of M. A. Martinez, A. Ferguson, J. Little, and D. Farley (University of Iowa RIA Core). We thank R. L. Cunningham for assistance with the preparation of the manuscript.

Preliminary results have been presented in abstract form (17).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. T. Cunningham, Dept. of Pharmacology, UTHSCSA, 7703 Floyd Curl Dr., San Antonio, TX 78229 (e-mail: cunninghamt{at}uthscsa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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