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Am J Physiol Regul Integr Comp Physiol 288: R311-R321, 2005. First published September 30, 2004; doi:10.1152/ajpregu.00399.2004
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WATER AND ELECTROLYTE HOMEOSTASIS

Effects of water deprivation and rehydration on c-Fos and FosB staining in the rat supraoptic nucleus and lamina terminalis region

Lisa L. Ji,1 Tiffany Fleming,1 Maurice L. Penny,1 Glenn M. Toney,2 and J. Thomas Cunningham1

Departments of 1Pharmacology and 2Physiology, and the Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas

Submitted 15 June 2004 ; accepted in final form 23 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We studied cFos and FosB staining in the supraoptic nucleus (SON) the organum vasculosum of the lamina terminalis (OVLT) and the median preoptic nucleus (MnPO) in adult male rats after water deprivation (24 h, n = 11; 48 h, n = 12) and water deprivation with rehydration (22 h + water, n = 11; 46 h + water, n = 10). Control rats (n = 15) had water available ad libitum. Separate sets of serial sections from each brain were processed for immunocytochemistry using primary antibodies against either c-Fos or FosB protein. Plasma osmolality, vasopressin, hematocrit, and plasma proteins were measured in separate groups (n = 6–7). The number of c-Fos-positive cells in the SON was significantly increased after 24 and 48 h of water deprivation. In contrast, rehydrated groups were not different from control. Water deprivation significantly increased c-Fos staining in both the OVLT and the MnPO, but c-Fos staining was not altered by rehydration. FosB staining in the SON was significantly increased only by 48-h water deprivation, and this effect was significantly decreased by rehydration. In the MnPO and OVLT, FosB staining was significantly increased by water deprivation, and, like c-Fos staining, these increases were not affected by rehydration. Water deprivation significantly increased osmolality and hematocrit, as well as plasma protein and vasopressin concentrations. Plasma measurements from rehydrated rats were not different from control. We conclude that water deprivation and rehydration differentially affect c-Fos and FosB staining in a region-dependent manner.

vasopressin; thirst; anteroventral region of third ventricle; dehydration

THE INDUCIBLE transcription factor c-fos has been widely used as an indicator of neural activation after a variety of homeostatic challenges (7, 9–11, 16). Fos protein is a member of the AP-1 family of transcription regulatory proteins, which also includes Jun and Fos-related proteins. Recent studies (15, 16, 33) have demonstrated that different AP-1 proteins have distinct levels of constitutive expression, as well as different time courses of activation. For example, c-Fos has a low level of constitutive expression in the central nervous system peak expression occurring 90–120 min after central stimulation (15, 16, 33). Fos-related proteins, such as FosB (and its splice variant {Delta}FosB), also have low levels of constitutive expression but peak much later. Indeed, Cunningham and colleagues (19) have recently observed FosB staining lasting 24 h after acute volume expansion. According to some reports (15, 33), FosB staining may remain elevated for weeks after acute stimulation of the CNS.

The neurohypophyseal system originates from cell bodies located in the supraoptic nucleus (SON) and paraventricular nucleus of the hypothalamus (2). In this system, c-Fos staining is induced under a variety of acute conditions (7, 10, 28). It has also been demonstrated that chronic stimulation by salt loading stimulates c-Fos and FosB staining in the SON (30). The increase in c-Fos staining-induced by salt loading has been reported to return to baseline 2 h after the rats are given access to water. In contrast, FosB staining remains elevated for up to 8 h (30). The salt loading protocol used in this study appears to be primarily an osmotic stimulus because plasma osmolality is significantly increased without a change in blood volume. Another cumulative stimulus that can be used to activate the neurohyphoseal system is water deprivation. Unlike salt loading, hypovolemia accompanies the increase in plasma osmolality during water deprivation. Thus water deprivation could activate neurohypophyseal neurons by stimulating central osmoreceptors, unloading cardiopulmonary receptors and central actions of the renin-angiotensin system. In this study, we examined the effects of water deprivation and rehydration on c-Fos and FosB staining in the neurohypophyseal neurons of the SON. In addition, c-Fos and FosB staining in the median preoptic nucleus (MnPO) and the organum vasculosum of the lamina terminalis (OVLT) was determined. These were included because both have been reported to contain ANG II and osmosensitive neurons and project to the SON and the paraventricular nucleus (3, 5, 27, 28, 35). Moreover, lesions of the anteroventral region of the third ventricle (AV3V), which include the OVLT and ventral MnPO, inhibit vasopressin release and drinking behavior associated with osmotic and nonosmotic stimuli (5, 27, 28). Finally, electrophysiological studies have shown that both the OVLT and the MnPO are capable of influencing the discharge of neurohypophyseal neurons in the SON (34, 47). Recent studies (28, 35) using c-Fos staining by McKinley and colleagues suggest that the OVLT contains distinct functional regions. Neurons in the dorsal cap of the OVLT are reported to respond to changes in plasma osmolality and project to the SON, whereas circulating ANG II acts primarily on cells in the lateral margins (28, 35). Because water deprivation is associated with increases in both plasma osmolality and ANG II, we separately analyzed these two regions of the OVLT.

Our hypothesis was that water deprivation would increase c-Fos and FosB staining in the SON, the MnPO, and throughout the OVLT. Rehydration produced by providing access to water for 2 h before perfusion was predicted to restore body fluid balance and plasma vasopressin (AVP) and to significantly decrease c-Fos staining without significantly affecting FosB staining.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Experiments were conducted on adult male Sprague-Dawley rats that weighed 250–350 g (Charles River). The rats were individually housed and maintained in a temperature-controlled environment on a 12:12-h light-dark cycle. Rats had ad libitum access to food and water before the experiments. All experimental protocols were approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the Public Health Service, American Physiological Society, and Society for Neuroscience.

Experimental protocol. All rats had ad libitum access to food throughout the experiments. The control group was allowed ad libitum access to water throughout the experiment. Two groups of rats were water deprived for either 24 h or 48 h and not allowed access to water. Two additional groups were water deprived for 22 and 46 h and given water for 2 h before performing the experimental procedures. After the treatment protocols, AVP, osmolality, hematocrit, and plasma proteins were measured in five separate groups of rats. The rats were lightly anesthetized (100 mg/kg ip Inactin), immediately decapitated, and trunk blood was rapidly collected into chilled centrifuge tubes containing ETDA. 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 measuring hematocrit (Micro-Hematocrit capillary tube reader; Lancer, St. Louis, MO) and plasma protein by refactometry (National Protometer; National Instruments, Baltimore, MD). 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 measuring plasma osmolality using a vapor pressure osmometer (Wescor, Logan, UT).

The blood collected in EDTA (1 mg/ml) was centrifuged at 10,000 g for 10 min at 4°C. Two milliliters of plasma were removed from the sample, placed in a conical centrifuge tube, and kept in an ultracold freezer (–80°C) until it was shipped on dry ice to the RIA core at the University of Iowa for measurement of AVP concentration. AVP concentration was determined using a specific radioimmunoassay after an acetone-petroleum ether extraction, as previously described (23, 43). The antibody to AVP (courtesy Dr. Willis Samson, St. Louis University; Ref. 39) showed <0.1% cross-reactivity with oxytocin, ANG I, ANG II, corticotropin-releasing factor, and atrial natriuretic peptide. Sensitivity of the AVP RIA was 0.087 pg, which corresponded to 0.170 pg/ml when factoring in the volume of plasma extracted, extraction efficiency, and the volume of extract assayed. The intra- and interassay coefficients of variation averaged 11% and 17%, respectively.

c-Fos and FosB immunocytochemistry. Five separate groups of rats were used for histology. All rats were deeply anesthetized with pentobarbital sodium (50 mg/kg ip) and perfused with 0.1 M phosphate-buffered saline (PBS), followed by 300–500 ml of 4% paraformaldehyde in PBS. The brains were removed and placed in PBS with 30% sucrose for 3–4 days. Each brain was sectioned in a cryostat or with a sliding microtome with a freezing stage. Three serial sets of coronal sections from each brain were placed in cryoprotectant and stored at –20°C until they were processed for immunocytochemistry as previously described (6, 19).

One set of free-floating sections from each rat was stained for c-Fos with the use of a commercially available antibody directed at the amino acid residues 4–17 in human c-Fos (rabbit anti-c-Fos Ab5, Calbiochem, San Diego, CA). The sections were incubated in the primary antibody (1:30,000) for 72 h at 4°C. Next, the sections were incubated in biotyinlated 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, 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. Sections then were rinsed for 30 min in PBS and incubated in anti-oxytocin antibody (courtesy of A. J. Silverman) diluted 1:1,000 in PBS diluent for 5 days at 4°C. After a 60-min PBS rinse, sections were incubated in a Cy3-labeled anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) for 3 h at room temperature (6).

A second set of serial sections from each rat was stained with a commercially available antibody raised against FosB [Rabbit anti-FosB (102), Santa Cruz Biotechnology, Santa Cruz, CA], as previously described (19). The sections were incubated with the primary antibody (1:1,000) for 72 h at 4°C. Tissue incubated with the FosB primary antibody was subsequently processed as described above for c-Fos immunocytochemistry. After the 3,3'-diaminobenzidine hydrochloride and 0.04% nickel ammonium sulfate reaction, the sections were rinsed and processed for oxytocin immunofluorescence, as described above. After the staining was completed, sections were mounted on gelatin-coated slides and air dried for 1–2 days; then the slides were coverslipped with Permount.

Sections were examined using light microscopy to identify FosB- and c-Fos-positive cells in the OVLT, MnPO, and SON. Oxytocin immunofluorescence was used to anatomically define the SON. Tissue sections containing regions of interest were recorded with the use of microscope (model IX 50, Olympus), equipped for epifluorescence. The images were acquired with the use of a digital camera (SPOT RT Slider, Diagnostic Instruments, Sterling Heights, MI) connected to a Pentium computer running Spot imaging software (version 3.24). Regions of the brain were identified on the basis of the rat brain stereotaxic atlas of Paxinos and Watson (37). 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. For the MnPO, 3 to 4 images were obtained that included both the dorsal and ventral regions of the nucleus. Counts of c-Fos and FosB-positive nuclei taken from the dorsal and ventral MnPO were pooled for statistical analysis. One or two images of the OVLT from each set of sections were used for analysis. Analysis of the OVLT included dividing the nucleus into two regions, the dorsal cap and the lateral margins, based on the work of McKinley and colleagues (28, 35). The subfornical organ (SFO) was not included because too few images could be obtained from each set of sections for valid statistical comparison.

Statistics. Data were analyzed by one-way ANOVA with Student-Newman-Keuls t-test for post hoc analysis of significant main effects (SigmaStat version 2.03, Systat Software, Point Richmond, CA). Significance was set at P < 0.05. All values are presented as means ± SE.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Plasma measurements. The results of the plasma measurements are listed in Table 1. AVP was significantly increased after both 24 and 48 h of water deprivation (Table 1). Although the average AVP level of the 48-h water-deprivation group was slightly higher than that of the 24-h water deprivation group, this difference was not statistically significant. The AVP levels in the two groups that had access to water for 2 h before decapitation was significantly lower than the 24- and 48-h water deprivation groups and was not different from control (Table 1).


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  		Table 1. Measurements of AVP osmolality, hematocrit and plasma proteins from control, 22 H + W, 24 H, 46 H + W, and 48 H rats

 
The results of the plasma osmolality measurements were consistent with the results of vasopressin measurements. Plasma osmolality was significantly increased by 24 and 48 h of water deprivation (Table 1), and whereas there was a trend for the 48 h water deprivation group to have a greater increase in plasma osmolality, osmolality values were not statistically different from those of the 24-h water deprivation group. Plasma osmolalities of the two groups that were given access to water for 2 h after water deprivation were not different from control and were significantly lower than the two water deprivation groups that did not have access to water (Table 1). Hematocrit and plasma proteins were significantly increased in the 24- and 48-h water deprivation groups only, and the increases observed after 48-h water deprivation were significantly greater than the 24 h water deprivation group (Table 1).

During the 2-h period of access to water, the 22 -h water deprivation group drank 21.5 ± 1.0 ml of water and the 46-h water-deprivation group drank 22.8 ± 2.8 ml.

c-Fos immunocytochemistry. In the SON, 24- and 48-h water deprivation significantly increased the number of c-Fos-positive cells compared with control (Fig. 1; Table 2). The increase in c-Fos staining in the 48-h water deprivation group was significantly greater than in the 24-h water deprivation group (Table 2). When rats were given access to water 2 h before perfusion, the c-Fos staining in the SON was significantly less than that observed in the SON of the 24- and 48-h water deprivation groups (Table 2). The number of c-Fos-positive cells in the SON of rats given 2-h access to water after either 22 or 46 h of water deprivation was not statistically different from control.



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  		Fig. 1. Digital images showing representative examples of c-Fos staining in the supraoptic nucleus (SON) from control (Con), 22-h water deprivation, and 2-h access to water (22 h + water), 24-h water deprivation (24 h), 46-h water deprivation and 2-h access to water (46 h + water) and 48-h water deprivation (48 h). ot, optic tract.

 

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  		Table 2. c-Fos staining in SON, MnPO, and OVLT with separate counts for DC and LM of OVLT from control, 22 H + W, 24 H, 46 H + W, and 48 H rats

 
Results for the OVLT were in contrast to those observed for the SON. In the 24- and 48-h water deprivation groups, there was a significant increase in c-Fos staining (Fig. 2; Table 2). However, unlike in the SON, c-Fos staining in the OVLT was also significantly increased in water-deprived rats that were given access to water for 2 h (Fig. 2 and Table 2). The average number of c-Fos-positive cells in the 46-h water deprivation group given access to water was not only significantly greater than control but also significantly greater than either the 24-h water deprivation group or the group deprived of water for 22-h and given access to water for 2 h (Table 2). The c-Fos staining in the later two groups were not different from each other but were significantly greater than control (Table 2). When separate regions of the OVLT were examined, c-Fos staining in the dorsal cap was similar to the overall results, i.e., c-Fos staining increased in all four treatment groups compared with the control group, and staining in the 46 h ± water group was greater that the 22 h ± water group (Table 2). However, in the lateral margins of the OVLT, c-Fos staining in the 46 h ± water and 48-h water deprivation groups was significantly greater than control, 22 h ± water and 24-h water deprivation (Table 2). Significant increases from control were also observed in the 22 h ± water and 24-h water-deprivation groups (Table 2).



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  		Fig. 2. Digital images showing representative examples of c-Fos staining in the organum vasculosum of the lamina terminalis (OVLT) from control, 22 h of water deprivation and 2-h access to water (22 h + water), 24-h water deprivation (24 h), 46-h water deprivation, and 2-h access to water (46 h + water) and 48-h water deprivation (48 h). DC, dorsal cap of the OVLT; LM, lateral margins of the OVLT.

 
Results from the MnPO were similar to the overall results of the OVLT. Staining for c-Fos was significantly increased in the MnPO of all four treatment groups compared with the control group (Fig. 3 and Table 2). The c-Fos staining in the 48-h water deprivation group and the 46-h water deprivation plus water group was also significantly higher than that observed in either the 24-h water deprivation group or the 22-h water deprivation plus water group (Table 2).



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  		Fig. 3. Digital images showing representative examples of c-Fos staining in the median preoptic nucleus (MnPO) from control, 22-h water deprivation, and 2-h access to water (22 h + water), 24-h water deprivation (24 h), 46-h water deprivation and 2-h access to water (46 h + water) and 48-h water deprivation (48 h). III, third ventricle; ac, anterior commissure.

 
FosB immunocytochemistry. In the SON, a significant increase in FosB staining was observed after 48 h of water deprivation (Fig. 4; Table 3). A significant increase in FosB staining was also observed in the SON of rats subjected to 46 h of water deprivation, followed by 2 h of access to water (Fig. 4; Table 3). The latter increase, however, was significantly less than that observed after 48-h water deprivation alone (Table 3). FosB staining in the SON after 24-h water deprivation and 22-h water deprivation plus access to water was not different from control (Table 3).



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  		Fig. 4. Digital images showing representative examples of FosB staining in SON from control, 22-h water deprivation and 2-h access to water (22 h + water), 24-h water deprivation (24 h), 46-h water deprivation and 2-h access to water (46 h + water) and 48-h water deprivation (48 h).

 

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  		Table 3. FosB staining in SON, MnPO, and OVLT with separate counts for DC and LM of OVLT from control, 22 H + W, 24 H, 46 H + W, and 48 H rats

 
Compared with controls, FosB staining in the OVLT (Fig. 5) and MnPO (Fig. 6) was significantly increased in all treatment groups (Table 3). However, there was no statistically significant difference between treatment groups; including those which were allowed access to water for 2 h. Staining for FosB in the dorsal cap and the lateral margins of the OVLT was similar to the results from the total analysis of the OVLT (Table 3).



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  		Fig. 5. Digital images showing representative examples of FosB staining in the OVLT from control, 22-h water deprivation and 2-h access to water (22 h + water), 24-h water deprivation (24 h), 46-h water deprivation and 2-h access to water (46 h + water) and 48-h water deprivation (48 h).

 


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  		Fig. 6. Digital images showing representative examples of FosB staining in MnPO from control, 22-h water deprivation and 2-h access to water (22 h + water), 24-h water deprivation (24 h), 46-h water deprivation and 2-h access to water (46 h + water) and 48-h water deprivation (48 h).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Whereas several studies have examined c-Fos staining in the SON, MnPO, and OVLT after water deprivation (13, 26, 32, 42, 45), this study examined both c-Fos and FosB staining after two different periods of water deprivation. In addition, we determined the effects of rehydration on the expression of these two inducible transcription factors. In all three brain regions examined, 24 and 48 h of water deprivation induced a significant increase in c-Fos staining as previously described (13, 26, 32, 42, 45). Twenty-four- and forty-eight-hour water deprivation were also accompanied by a significant increase in plasma osmolality and hematocrit levels, as well as plasma protein and AVP concentrations, indicating that water-deprived rats were volume depleted in addition to being hyperosmotic.

When rats were allowed access to water for 2 h before perfusion, plasma osmolality, specific gravity, hematocrit, and AVP were significantly reduced to levels comparable to those of the ad libitum controls. Rehydration had differential effects on c-Fos staining that were region specific. In the SON, c-Fos staining was reduced to control levels, consistent with the reduction in AVP. This suggests that the increase in plasma AVP and the increase in c-Fos staining in the SON were the result of increases in plasma osmolality and/or hypovolemia associated with dehydration.

The increase in c-Fos staining in the SON is consistent with water deprivation leading to an increase in synaptic activity to magnocellular neurons in the SON. This effect may be countered by rehydration because water intake appeared to reduce the number of c-Fos immunoreactive neurons. Watanabe et al. (44a) provide evidence to support this hypothesis. In their study, extracellular recordings from neurons in the paraventricular nucleus of conscious rats showed that the activity of phasically active neurons (a characteristic of putative vasopressin magnocellular neurons; Ref. 2) was increased by water deprivation and decreased by allowing the rats to drink water (44). Water intake has been shown to rapidly inhibit vasopressin release (1, 4, 20). This inhibition appears to occur before the water is absorbed by the gastrointestinal system and changes in plasma osmolality or volume can take place (1, 4, 20). Although the mechanism responsible for this rapid inhibition of vasopressin release by drinking has not been determined, it appears to be related to the activation oropharyngeal afferents (1, 4, 20). Thus changes in c-Fos staining in the SON that were produced by water deprivation and rehydration are consistent with the hypothesis that c-Fos expression is related to increased synaptic activation associated with dehydration and the active inhibition of vasopressin release by drinking.

In contrast to the results observed in the SON, c-Fos staining in the OVLT and MnPO was still increased after rats were given 2 h of access to water. Functionally, both the OVLT and MnPO are involved in the production of drinking behavior (8, 27–29) and are reported to contain osmosenstive neurons (5, 17, 38). Furthermore, it has been hypothesized that neurons in the OVLT and MnPO may provide excitatory input to SON neurons that mediate AVP release under a variety of physiological circumstances, including water deprivation (28). A previous study (26) that combined retrograde labeling from the SON with c-Fos immunocytochemistry found that ~60% of the retrogradely labeled cells in the OVLT were c-Fos-positive after water deprivation. Lesions of the anteroventral region of the third ventricle, which includes the OVLT and the ventral portion of the MnPO, significantly attenuate c-Fos staining in the SON after 24-h water deprivation (45). Taken together, these functional data indicate that the OVLT and MnPO each provide a major source of afferent drive to SON neurons during water deprivation.

The increase in c-Fos staining in the OVLT and MnPO following rehydration appears to be at odds with the proposed role of these regions in regulating AVP release. However, while the SON is dedicated to neurosecretion, the OVLT and MnPO are involved in several functions, including drinking behavior and sodium appetite (8, 21, 27, 29). It could be that differences in c-Fos staining between these two regions and the SON after access to water is related to differences in the visceral mechanisms that terminate drinking behavior as opposed to vasopressin release. In addition to drinking and salt appetite, studies in the rat have demonstrated that dehydration is associated with increased sodium excretion, (24, 41), and this natriuresis may be mediated by neurons in the lamina terminalis region (25). This increase in sodium excretion, associated with water deprivation, is likely to buffer osmotic disturbance in the face of progressive dehydration. Therefore, it seems plausible that c-Fos staining in the OVLT and MnPO is related to dehydration-induced natriuresis.

Several studies using c-Fos staining to investigate the neural substrates of drinking behavior and sodium appetite have reported results that are consistent with those of the present study. For example, Herbert and colleagues (14, 46) showed that c-Fos staining in the OVLT, MnPO, and SON induced by central injections of angiotensin was differentially affected by water availability. Rats that did not have access to water showed a significant increase in c-Fos staining in all three regions, whereas those that had access to water had a significantly blunted c-Fos response only in the SON, not the OVLT or MnPO (14). A follow-up study (46) demonstrated that intragastric water loading also blocks angiotesin-induced c-Fos staining in the SON but, not in the OVLT. Similar results were obtained in a study that examined c-Fos staining induced by central ANG II in rats instrumented with gastric fistulas (31). The results of this study showed that drinking with the fistulas closed, sham drinking, and intragastric infusion of water significantly decreased c-Fos staining in the SON. However, c-Fos staining in the OVLT and MnPO were not reduced after any of the treatment conditions. This led the authors to conclude that c-Fos staining in the OVLT and MnPO, which was produced by centrally injected ANG II was not influenced by oral or gastric cues associated with drinking (31).

De Luca et al. (13) used water deprivation as a model for sodium appetite while examining c-Fos staining in the forebrain. They also found that 30 h of water deprivation, followed by 2 h access to water, but not salt, blocked c-Fos staining in the SON. In contrast, the same treatment only attenuated c-Fos staining in the OVLT and MnPO. In a separate group of rats, the authors showed that water deprivation increased plasma renin activity and osmolality, while plasma volume decreased. Rats with access to water for 2 h without a source of sodium ingested enough water to return osmolality to control levels but not plasma volume or plasma renin activity. They suggested that the residual c-Fos staining in the MnPO and OVLT could be related to elevated levels of circulating angiotensin, which mediates sodium appetite after water deprivation. In the present study, we also observed a decrease in plasma volume after water deprivation, but rehydration for 2 h returned plasma osmolality, hematocrit, and specific gravity to control levels, suggesting that both the hyperosmolality and hypovolemia had been corrected. This might be because, in contrast to the study by De Luca et al., the rats in the current study had access to food as a source of salt during the rehydration period. Although we did not measure food intake during the 2-h rehydration period, we did observe that rats in the two rehydration groups ingested food during this time period. Studies by Schoorlemmer and Evered (40, 41) demonstrate that in the rat, water deprivation is associated with decreased food intake and that once water is returned to the rats via intragastric infusion, normal food intake resumes (40). Thus food was a source of sodium for rats in the present study, and this might have allowed them to more effectively compensate for their hypovolemia. These results suggest that c-Fos staining in the OVLT and MnPO persisted in the absence of hyperosmolality and hypovolemia. This is consistent with other studies using different models of salt appetite that showed c-Fos staining in the OVLT and SFO is maintained for 2 h (36) and may last for 3 to 12 h after the rats are allowed to correct their fluid imbalance by drinking water and saline solutions (18, 44). This suggests that the expression of c-Fos in the MnPO, OVLT, and SFO persists after a variety of experimental manipulations that induce drinking and sodium appetite even after the animals are given access to water- and sodium-containing solutions.

In light of our evidence that both osmolality and blood volume were restored by rehydration, it seems unlikely that plasma ANG II would have remained elevated to account for the maintenance of c-Fos staining in the OVLT and MnPO. Whether signaling mechanisms downstream from the Gq coupled AT1 receptor remain active and support elevated c-Fos expression in OVLT and MnPO neurons after rehydration is an intriguing possibility.

FosB staining was also effected by water deprivation and rehydration in a region-specific manner. In the MnPO and OVLT, 24- and 48-h water deprivation increased FosB staining. Providing the rats with access to water for 2 h did not significantly influence FosB staining in the MnPO or OVLT compared with the results of water deprivation. In contrast to the results obtained for c-Fos staining, we did not observe any differences in FosB staining between the dorsal cap and the lateral margins of the OVLT. These results are consistent with previous studies that demonstrate FosB staining requires at least 8 h to return to baseline after osmotic stimulation (30). In the SON, 24-h and 22-h water deprivation, followed by 2 h access to water did not significantly increase FosB staining. FosB staining in the SON was significantly increased after 48 h of water deprivation. After 46 h of water deprivation and 2 h of access to water, FosB staining in the SON was significantly greater than the control group but was significantly attenuated compared with 48 h of water deprivation. Because FosB has a longer expression than c-Fos (19, 33) and has been shown to accumulate with chronic stimulation (33), it might be expected that FosB staining in the SON did not return to control levels following rehydration after 46-h water deprivation and was not significantly increased at 24 h of water deprivation. Overall, these results show that 24-h water deprivation was not sufficient to significantly increase FosB staining in the SON, although it did increase staining in the MnPO and OVLT. Furthermore, rehydration did lead to diminished FosB staining in the SON, but not the MnPO or OVLT. Thus, as observed with c-Fos staining, water deprivation and rehydration produced clearly distinctive patterns of FosB staining among these three regions.

Electrophysiological studies have shown that the discharge of vasopressin and oxytocin magnocellular neurons in the SON to osmotic challenges is linearly related to the degree of hypertonicity, although the hormonal response of oxytocin may be altered by stimulus-secretion coupling in the neural lobe (22). This linear relationship between osmolality and magnocellular single unit activity requires the coactivation of excitatory and inhibitory synaptic inputs to the SON (22). Given that the MnPO and OVLT have each been shown to have excitatory and inhibitory influences on SON neurons (34, 47), c-Fos, and FosB staining in the MnPO and OVLT of rehydrated rats may be related to an activation of inhibitory neurons, whereas excitatory neurons that are stimulated by water deprivation may no longer express these inducible transcription factors.

Another interesting observation is that in the SON and MnPO, c-Fos staining appears to increase with the duration of water deprivation, i.e., the number of c-Fos-positive cells after 48 h of water deprivation is greater than the number of c-Fos cells after 24 h of water deprivation. These results are consistent with the findings of Morien et al. (32). A similar trend also is present in the OVLT, although it fails to achieve statistical significance. When regions of the OVLT were analyzed separately, it was found that cells in the lateral margins did show a graded response to water deprivation, whereas cells in the dorsal cap did not. The lateral margins of the OVLT have been shown to contain cells that express c-Fos after peripheral administration of ANG II (28, 35). Cells in the dorsal cap of the OVLT have been shown to project to the SON and are reported to be responsive to osmolality and relaxin (27, 33). FosB staining also appears to be graded in proportion to the duration of water deprivation in the SON but not the OVLT or_MnPO. In the present study, plasma osmolality was not significantly different between 24 and 48 h of water deprivation. On the other hand, on the basis of hematocrit and plasma protein measurements, the degree of hypovolemia was graded with the duration of water deprivation. This suggests that the circulating levels of ANG II may also have been graded with the degree of hypovolemia. These factors could have contributed to the regional differences that we observed in c-Fos and FosB staining related to the duration of water deprivation. Alternatively, the function of c-Fos and FosB in these regions of the central nervous system has not been determined. It could be that the cellular function and metabolism of c-Fos and FosB in these different areas have contributed to the regional differences in staining. These regional and temporal differences in staining also could suggest that c-Fos and FosB expression may be activated by different transduction mechanisms and/or different populations of neurons that might respond at different time points during water deprivation. Because we performed the c-Fos and FosB staining in different sets of serial sections from the same brains, we cannot determine whether they were expressed in the same cells. These issues will be addressed in future studies.

Perspectives

Fos immunocytochemistry has been widely used to map regions of the central nervous system that are activated by acute cardiovascular-related homeostatic changes (11, 12, 16). Given its time course of expression, the use of Fos immunocytochemistry to map the nervous system after chronic or progressive physiological challenges has been controversial. FosB and other Fos-related antigens have been shown to have longer time courses of expression that might make them more useful in these situations (19, 33). The results of the present study show that in the specific case of water deprivation, c-Fos and FosB staining produce generally similar results. However, there were important regional differences.

Although it might be assumed that the appearance of immunocytochemically detectable levels of c-Fos and FosB is related to synaptic activation of neurons, the activation of inducible transcription factors represents a distinct biological response (16). Therefore, the results indicate that the cellular processes, which are regulated by c-Fos and FosB, are likely to be different in the SON, OVLT, and MnPO. In the SON, genes that are regulated by c-Fos containing AP-1 dimers are affected by water deprivation, but these effects would be reduced dramatically once the animals are allowed to rehydrate. The regulatory effects of FosB in the SON might not be apparent unless the water deprivation is >24 h and could continue for at least 2 h after rehydration. In the MnPO and OVLT, both c-Fos and FosB would be activated by water deprivation and their influences on gene expression could continue after rehydration.

These findings indicate that the transcriptional regulation of gene expression by c-fos and fosB is distinct with respect to brain region and the duration of water deprivation. Maintenance of c-Fos and FosB expression after rehydration raises the interesting possibility that plasticity among body fluid regulatory pathways might also persist well after the acute perturbation, possibly leading to altered responses (facilitated or depressed) to subsequent disturbances to body fluid homeostasis.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grants R01-HL-0756834 (to G. M. Toney) and R01-HL-55692, and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-57822 (to J. T. Cunningham).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. T. Cunningham, Dept. of Pharmacology, University of Texas Health Science Center at San Antonio, 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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