Introduction

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The increase in BP produced by intravenously infused Ang II has been characterized as having two distinct phases: an acute (fast) phase and a chronic (slow) phase (McCubbin et al., 1965). In the acute phase of Ang II hypertension, the direct contractile action of Ang II on vascular smooth muscle is likely the primary mechanism underlying the increase in BP (Wong et al., 1991). The chronic phase of Ang II hypertension, however, appears to be more complex, and its mechanisms have not been fully characterized. Renal sodium retention, vascular hypertrophy and interactions between Ang II and the CNS and peripheral nervous system have been suggested as possible mechanisms that contribute to chronic Ang II hypertension (Hall, 1986; Lever et al., 1992; Reid, 1992).

The CNS is critical for the chronic hypertensive effects of peripherally infused Ang II (Bickerton and Buckley, 1961; Ueda et al., 1969; Yu and Dickinson, 1971; Fink et al., 1980). Lesions of the area postrema (Fink et al., 1987; Cox and Bishop, 1991), lateral parabrachial nucleus (Fink et al., 1991) and anteroventral region of the third ventricle (the AV3V region) (Fink et al., 1980) prevent or reverse this form of hypertension. Chronic Ang II hypertension is also attenuated by clonidine (Gordea-Opplinger and Fink, 1994), which is reported to have central sympathoinhibitory effects (Van Zwieten, 1973).

The sympathetic nervous system also appears to participate in Ang II hypertension (Brooks and Osborn, 1995; Brooks, 1995; Csiky and Simon, 1997; Fink, 1997). However, whether sympathetic activity is increased during chronic Ang II hypertension remains controversial (Bishop et al., 1995; Brooks and Osborn, 1995; Brooks, 1997; Fink, 1997). We used ganglionic blockers to assay the contribution of the sympathetic nervous system to increases in BP produced by acute and chronic Ang II infusion in rats (Li et al., 1996). The decrease in BP produced by ganglionic blockade during the first few hours of Ang II infusion was smaller than that produced before Ang II infusion. This indicates that during acute Ang II infusion, the sympathetic nervous activity is withdrawn, and the hypertension is initially mediated by Ang II acting on vascular smooth muscle to produce vasoconstriction. After 10-hr Ang II infusion, the decrease in BP produced by ganglionic blockade was larger than the preinfusion decrease, suggesting a greater role for neurogenic mechanisms in chronic Ang II hypertension. Thus, these results support the hypothesis that Ang II hypertension involves two distinct phases, as has been suggested previously (Bickerton and Buckley, 1961). Furthermore, these observations also implicate a change in the response of the sympathetic nervous system during the transition from acute to chronic Ang II hypertension.

In the present study, we investigated the role of medullary regions in the CNS that participate in the control of BP in the transition of Ang II-induced hypertension. Specifically, we investigated the nucleus of the NTS, CVL and RVL. We used immunocytochemistry for the protein product of the early response gene c-fos to determine the effects of acute and chronic Ang II infusion on the CNS. In neurons, Fos, the protein product of c-fos, is involved in initiating genomic effects produced by synaptic activation (Morgan and Curran, 1991). Immunocytochemistry for Fos has been used widely to map regions of the CNS that are sensitive to Ang II (McKinley et al., 1992, 1995) and to changes in BP (Badoer et al., 1994; Li and Dampney, 1994; Graham et al., 1995; Miura et al., 1994; Potts et al., 1997). In some regions of the CNS, Fos is maintained for the duration of the Ang II stimulus (Rowland et al., 1994). Studies in the SHR (Minson et al., 1996) and with c-fos antisense (Suzuki et al., 1994) suggest that sympathoexcitatory neurons in the RVL express c-fos in a manner that is related to BP regulation. All of these observations suggest that Fos can be used to assay neural activity in systems that regulate BP in conscious animals using long-term infusions of Ang II. We examined Fos immunoreactivity in the NTS, CVL and RVL in SAD and intact rats that received an infusion with Ang II for 2 or 18 hr.

	Methods

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Male Sprague-Dawley rats (275-300 g) purchased from Harlan (Indianapolis, IN) were housed individually and allowed access to standard rat chow (Ralston Purina, St. Louis, MO) and tap water ad libitum. The rats were acclimated for1 week to a 12/12-hr light/dark cycle and a stable room temperature of 25°C. The experimental protocol was approved by the Animal Care and Use Committee at the University of Missouri-Columbia.

On the day before the experiment, halothane-anesthetized rats were instrumented with indwelling catheters constructed of Silastic rubber (A-M Systems, Everett, WA) and polyvinyl chloride (Cole-Parmer, Chicago, IL) in the femoral artery and vein for measurement of MAP and for drug delivery, respectively. The distal ends of the catheters were tunneled subcutaneously to exit between the scapulae and filled with a saturated sucrose solution containing 1000 U/ml heparin and 150 µg/ml gentamicin. On the day of the experiment, arterial catheters were connected to a pressure transducer via extension tubing. MAP and HR were recorded continuously. Normal rats were divided randomly into two different groups that received intravenous infusions of either vehicle or Ang II (50 ng/kg/min in 5% dextrose) for 2 hr with use of a syringe pump (Harvard Apparatus, South Natick, MA) at a rate of 0.01 ml/min. MAP and HR were recorded for 30 min before the start of the infusion and during the last 30 min of the Ang II infusion.

The rats receiving vehicle or Ang II (50 ng/kg/min) for 18 hr received osmotic minipumps subcutaneously (flow = 1 µl/hr, model 2001; Alza Corporation, Palo Alto, CA) that were connected via a polyvinyl chloride catheter to the femoral vein on the day before the experiment. Minipumps were filled with vehicle or Ang II solution at a concentration calculated to deliver 50 ng/kg/min. MAP and HR were recorded for 30 min immediately before the animals were killed.

At the end of the infusion period, rats were anesthetized with pentobarbital (60 mg/kg intraperitoneally) and perfused transcardially using a peristaltic pump with PBS followed by cold 4% paraformaldehyde in PBS (250-300 ml in 15-20 min). The brains were then removed, immersed in PBS with 20% sucrose and kept at 4°C for 2 days until they were sectioned.

Baroreceptor denervated rats. Rats were subjected to SAD while under halothane anesthesia according to the method of Krieger (1989). A midline ventral incision was made in the neck to expose the muscle and the neurovascular sheath enclosing the common carotid artery, vagus and sympathetic trunk merging into the superior cervical ganglion. The aortic depressor nerve was severed. A strip of the superior laryngeal nerve and sympathetic trunk including the superior cervical ganglion was resected on both sides. The region of the bifurcation of the carotid artery and all carotid branches were stripped of fibers and connective tissue and painted with 10% phenol in absolute ethanol. The sham operation consisted of exposing the relevant nerve trunks without sectioning the nerves or painting the arteries with phenol.

Fos immunoreactivity. We cut 40-µm coronal sections of the brain using a cryostat and collected them in 0.1 M PBS. The sections were incubated in 0.3% hydrogen peroxide in distilled water at room temperature for 30 min. After a 30-min wash in 0.1 M PBS, the sections were incubated in 3% normal horse serum (Sigma Chemical, St. Louis, MO) in 0.1 M PBS containing 0.25% Triton-100 (PBS diluent) at room temperature for 2 hr. Sheep polyclonal anti-Fos antibody (Genosys, The Woodlands, TX) was diluted 1:1000 in PBS diluent. Sections were incubated with the diluted antibody at 4°C for 2 days. After another 30-min wash in 0.1 M PBS, sections were incubated with a rabbit anti-sheep IgG (1:200 in PBS diluent, Vectastain; Vector Labs, Burlingame, CA) at room temperature for 2 hr and then reacted with an avidin-peroxidase conjugate (ABC kit, Vectastain) and PBS containing 0.04% 3,3-diaminobenzidine hydrochloride, 0.04% nickel ammonium sulfate and 0.025% cobalt. The sections were mounted onto gelatin-coated slides, dried and coverslipped.

Microscopy and quantification. Sections were examined on an Olympus microscope with digital camera connected to an Apple Quadra 800 computer running NIH Image (version I.5). Each section was visually examined by light microscopy. For the quantitative analysis, at least two representative sections were imaged from each brain. The number of stained cells per section in selected brain regions were quantified by observers blinded to experimental conditions. In the NTS, CVL and RVL, the numbers of stained cells were taken from the same rostral-caudal plane of the nucleus, counted bilaterally and averaged for statistical analysis. The RVL was defined according to the criteria used by Badoer et al. (1994). The anterior border of the RVL was defined as the caudal pole of the facial nucleus, and the rostral hypoglossal indicated the posterior border. The CVL was defined posteriorly by the pyramidal decussation and anteriorly by the appearance of the principle nucleus of the inferior olive. For both cell groups, we examined cells that are ventral to the nucleus ambiguous between the pyramidal tracts medially and the spinal nucleus of the trigeminal nerve laterally. The portion of the NTS that was used for the analysis extended from 300 µm caudal to obex (Paxinos and Watson, 1997) to the caudal edge of the area postrema. Previous studies have indicated that this part of the NTS contains neurons that are activated by increases in BP (Badoer et al., 1994; Li and Dampney, 1994; Graham et al., 1995; Miura et al., 1994; Potts et al., 1997).

Statistical analysis. All values are expressed as mean ± S.E.M. MAP and HR data were analyzed using two-way analysis of variance followed by Dunnett's test to determine differences among the groups. Histological data were analyzed using one-way analysis of variance followed by Newman-Keuls test to determine differences among the groups. Significance was determined as P < .05.

	Results

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Effects of Intravenous Infusion of Ang II on MAP and HR

As we reported previously (Li et al., 1996), 2- and 18-hr infusion of Ang II (50 ng/kg/min intravenously) significantly increased arterial pressure in normal, sham and SAD rats (P < .05). There were no significant differences in base-line MAP among any of the groups (table 1, P > .05). In addition, the elevation in MAP in SAD rats after 2-hr Ang II infusion tended to be greater than that in normal and sham rats, but the difference was not statistically significant (table 1). A significant decrease in HR was observed in normal and sham rats in association with the increase in BP (P < .05). However, HR did not change significantly in SAD rats. There also was no significant difference in HR among the groups receiving Ang II infusion for 18 hr (table 1, P = .29).

Fos Immunoreactivity in Medulla

NTS. Intravenous infusion of Ang II in animals with intact baroreflexes resulted in a significant increase in the number of Fos-positive cells compared with rats infused with vehicle (P < .05) This increase was similar at 2 and 18 hr of infusion. Fos-positive cells were located primarily in the medial commissural portion caudal to the obex and in the dorsal and lateral part of the NTS at the level of the area postrema. In SAD rats infused with Ang II, Fos-positive cell numbers in the NTS were not significantly different after Ang II infusion for either 2 and 18 hr compared with vehicle control (table 2, fig. 1). SAD rats infused with vehicle demonstrated no difference in the number of Fos-positive cells from normal rats infused with vehicle.

View larger version (136K): [in this window] [in a new window]   Fig. 1.   Fos-like immunoreactivity in the medial NTS posterior to obex (Paxinos and Watson, 1997) in intact rats infused with vehicle for 2 hr (A) or 18 hr (B), sham rats were infused with Ang II (50 ng/kg/min intravenously) for 2 hr (C) and 18 hr (D) and SAD rats were infused with the same dose of Ang II for 2 hr (E) and 18 hr (F). Note that the 2- and 18-hr infusions of Ang II increase Fos only in the intact rats. The scale bar is 100 µm.

CVL. In normal and sham rats, intravenous infusion of Ang II for 2 hr induced a significant increase in Fos-positive cells in the CVL compared with rats infused with vehicle (table 2, fig. 1, P < 0.05). Fos-positive cells were located predominantly ventrolateral to the retroambiguous nucleus from the level of obex to the level of the area postrema. In SAD rats infused with Ang II for 2 hr, Fos-positive cell numbers were not significantly different from vehicle controls (table 2, fig. 2). In all groups of rats, there were no significant changes in Fos-positive cells in the CVL when Ang II infusion was sustained for 18 hr (table 2, fig. 2).

View larger version (130K): [in this window] [in a new window]   Fig. 2.   Fos immunoreactivity in the CVL in intact rats infused with vehicle for 2 hr (A) or 18 hr (B), intact rats infused with Ang II (50 ng/kg/min intravenously) for 2 hr (C) and 18 hr (D), and SAD rats infused with the same dose of Ang II for 2 hr (E) and 18 hr (F). In this region, only the intact rat infused with Ang II for 2 hr (C) shows an increase in Fos. The scale bar is 100 µm.

RVL. Normal, sham and SAD rats infused with Ang II intravenously for 2 hr had no significant changes in the number of Fos-positive cells in RVL (table 2 and fig. 3). In contrast, intravenous infusion of Ang II for 18 hr induced a significant increase in Fos-positive cells in normal, sham and SAD rats compared with vehicle controls (table 2, fig. 3, P = .005). Fos-positive cells were located primarily ventrolateral to the nucleus ambiguous rostral to area postrema and caudal to the facial nucleus. In SAD rats infused with vehicle for 2 hr, Fos in the RVL was not different from that in normal rats infused with vehicle (table 2).

View larger version (137K): [in this window] [in a new window]   Fig. 3.   Fos immunoreactivity in the RVL in intact rats infused with vehicle for 2 hr (A) or 18 hr (B), intact rats infused with Ang II (50 ng/kg/min intravenously) for 2 hr (C) and 18 hr (D) and SAD rats infused with the same dose of Ang II for 2 hr (E) and 18 hr (F). In this region of the medulla, both the intact and SAD rats infused with Ang II for 18 hr (D and F) demonstrate an increase in Fos. The scale bar is 100 µm.

	Discussion

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The present study is the first to report patterns of Fos immunoreactivity in medullary regions that are associated with reflex control of autonomic outflow during Ang II hypertension. These brainstem regions, all of which have been found to participate in the baroreflex control of arterial pressure, show different patterns of Fos expression after 2 and 18 hr of Ang II infusion.

There was a significant increase in Fos-positive cells in the NTS in normal and sham rats infused with Ang II for 2 and 18 hr. This increase was abolished by SAD, indicating that intact baroreceptors are necessary for increased Fos in the NTS during Ang II infusion. Thus, Fos expression in the NTS appears to be a result of baroreceptor activation and not peripheral Ang II infusion. Others (Badoer et al., 1994; Li and Dampney, 1994; Graham et al., 1995; Potts et al., 1997) have shown that increased arterial pressure produced by intravenous phenylephrine is sufficient to produce increased Fos in the NTS. Badoer et al. (1994) have shown that Ang II infusions produced Fos labeling in the NTS that is similar to the pattern of Fos labeling produced by phenylephrine-induced baroreceptor stimulation. Our data are consistent with these observations and demonstrate that the Fos response of these neurons is maintained for up to 18 hr.

In contrast to the results obtained in the NTS, Fos immunoreactivity in the CVL and RVL changed over the time course of Ang II infusion. In the CVL, 2-hr Ang II infusion resulted in increased Fos in normal and sham rats compared with vehicle-infused controls. This increase was eliminated by SAD, suggesting that the Fos-positive neurons in the CVL responded to increases in baroafferent signals rather than direct effects of peripherally infused Ang II. The Fos-positive cells in the CVL were observed in regions that previously were reported to show increased Fos after baroreceptor activation with either phenylephrine (Badoer et al., 1994; Li and Dampney, 1994; Graham et al., 1995; Potts et al., 1997) or Ang II (Badoer et al., 1994). Electrophysiological studies indicate that this region contains baroreceptor-sensitive neurons that participate in baroreflex control of sympathetic outflow by inhibiting neurons in the RVL (Agarwal and Calaresu, 1991; Masuda et al., 1991). In normal and sham SAD rats infused with Ang II for 18 hr, Fos was no longer elevated in CVL compared with the vehicle control group despite a sustained increase of Fos in the NTS. This change of Fos in the CVL over the time course of Ang II infusion suggests that the synaptic input to these cells is altered so it is no longer sufficient to sustain Fos activation or that these neurons have adapted to the stimulus. Others have shown that circulating Ang II can alter baroreflex function (Guo and Abboud, 1984; Brooks and Reid, 1986; Matsukawa et al., 1989; Brooks and Hatton, 1997). Based on these observations, we postulate that the change in Fos immunoreactivity in the CVL observed between 2- and 18-hr Ang II infusion could represent a change in synaptic input to the CVL neurons. A change in synaptic input to the CVL could then be related to a withdrawal of sympathoinhibition, which apparently occurs during the transition from the acute to the chronic phase of Ang II hypertension.

In the RVL, the number of Fos-positive cells was not significantly increased after 2-hr Ang II infusion in normal and sham rats compared with vehicle-infused control rats. Similarly, 2-hr Ang II infusion in SAD rats was not associated with a significant increase in Fos in the RVL. These data are consistent with the hypothesis that the sympathetic nervous system is not activated during acute infusion of Ang II. However, after 18-hr Ang II infusion, we observed a significant increase in Fos-positive cells in the RVL in normal, sham and SAD rats compared with the vehicle control group. These results indicate that in contrast to the NTS and CVL, the increase in Fos-positive cells in the RVL required a longer period of Ang II infusion because it was observed only after 18 hr. Furthermore, the Fos increases in the RVL after the 18-hr Ang II infusion do not require intact baroreceptors because it was still observed in SAD rats.

The RVL contains bulbospinal neurons that send primarily excitatory projections to sympathetic preganglionic neurons (Calaresu and Yardley, 1988; Morrison et al., 1988; Chalmers and Pilowsky, 1991). Previous studies indicate that many neurons in the RVL are inhibited by baroreceptor stimulation and activated by hypotension or unloading of baroreceptors (Morrison et al., 1988; Agarwal and Calaresu, 1991; Masuda et al., 1991). A number of studies have used Fos in the RVL as an index of sympathetic nervous system activation. In the SHR, Minson et al. (1996) observed that base-line expression of Fos in medullary regions that control sympathetic outflow was elevated compared with the control strain. Furthermore, they observed that Fos in the RVL of SHR was not elevated when baroreceptors were unloaded. This base-line elevation of Fos in the RVL of SHR and lack of a hypotension-induced increase were interpreted as evidence for increased basal sympathetic outflow. The increased Fos in the RVL after 18-hr Ang II infusion is consistent with earlier observations showed that chronic Ang II hypertension has a major neurogenic component (Li et al., 1996). The time course of enhanced Fos expression in the RVL can be correlated with our earlier observations in which enhanced depressor effects of ganglionic blockade were evident after 10-hr Ang II infusion (Li et al., 1996). This suggests there is a centrally mediated increase in sympathetic outflow that is associated only with chronic Ang II infusion. However, it should be noted that we have no direct evidence demonstrating that these RVL cells are bulbospinal neurons. Mechanisms underlying delayed activation of RVL to increase sympathetic outflow also remain to be studied. Several studies suggest that the density of Ang II receptors is increased by manipulations that are associated with increased circulating levels of Ang II (Hwang et al., 1986; Nazarali et al., 1987). It is possible that the delay in the activation of the RVL by chronic Ang II involves a similar mechanism.

Our data suggest that the fast and slow pressor responses to Ang II are associated with different patterns of Fos activation in medullary regions associated with reflex control of BP. The pattern of Fos observed after 2-hr Ang II infusion indicates baroreceptor activation and sympathoinhibition. This is consistent with the traditional description of the mechanism of the fast Ang II pressor response (i.e., peripherally mediated vasoconstriction and baroreflex inhibition of sympathetic outflow). After 18-hr Ang II infusion, the medullary pattern of Fos expression is different. Although baroreceptor-dependent Fos is observed in the NTS, the CVL no longer shows significant Fos activation after 18-hr Ang II infusion. This finding indicates that after 18-hr Ang II infusion, there is no longer a centrally mediated sympathoinhibition even though baroreceptor-related Fos activation persists in the NTS. It has been suggested that Ang II participates in pressure-independent baroreflex resetting (Heesch et al., 1996; Brooks, 1997), and this could account for the change in Fos activation in the CVL.

The 18-hr Ang II infusion increased Fos in the RVL in each group of rats. The activation of neurons in the RVL could be a result of disinhibition associated with baroreceptor resetting. However, results from the SAD experiments do not support this interpretation. Even in the absence of baroreflex inhibition, Ang II infusion for 2 hr did not increase Fos in the RVL, and disinhibition alone was not sufficient for the activation of Fos in the RVL. Because the RVL is within the blood-brain barrier, activation of neurons in the RVL in the present study cannot be a direct effect of intravenous Ang II and could be related to peripheral Ang II acting through a circumventricular organ. The RVL receives projections from the area postrema (Shapiro and Miselis, 1985; Blessing et al., 1987) and is also innervated by regions of the CNS that are connected to circumventricular organs such as the parabrachial nucleus and paraventricular nucleus (Luiten et al., 1985; Herbert et al., 1990). Electrophysiological studies indicate that chemical and electrical stimulation of the area postrema influences the excitability of bulbospinal neurons in the RVL (Sun and Spyer, 1991; Wilson and Bonham, 1994). The area postrema contains neurons that are sensitive to Ang II (Ferguson and Baines, 1997) and appears to be involved in baroreflex modulation (Hasser et al., 1997). It has been suggested that Ang II may produce sympathoexcitation by acting at the area postrema (Fink, 1997). Although we do not present Fos data from the area postrema in the current study, we have preliminary evidence that lesions of the area postrema block the Fos activation in the RVL associated with chronic Ang II (Curtis et al., 1997). This issue will be addressed in subsequent experiments.

The result of RVL activation by 18-hr Ang II could be a centrally mediated sympathoexcitation that contributes to an increase in the neurogenic component as suggested by the ganglionic blockade studies. Thus, 18-hr Ang II infusion is associated with a pattern of Fos activation indicating baroreceptor resetting and sympathoexcitation. Both of these mechanisms could contribute to the high BP and the slow pressor response to Ang II.

The role of the sympathetic nervous system in Ang II-induced hypertension remains controversial (Reid, 1992; Bishop et al., 1995; Brooks and Osborn, 1995; Fink, 1997). Because Ang II has been reported to have peripheral effects on sympathetic nervous transmission, a centrally mediated increase in sympathetic outflow may not be required for the development of Ang II hypertension (Weber et al., 1985). Several groups have produced evidence that sympathetic outflow is increased during Ang II hypertension, and it has been suggested that this increase could be centrally mediated (Fink, 1997). On the other hand, Cox and Bishop (1991) found that renal sympathetic nerve activity decreased in rabbits during acute Ang II infusion, although chronically the level of activity began to approach control levels. Matsukawa et al. (1991) conducted a study on the effects of Ang II on human muscle sympathetic nerve activity. They observed that acute infusions of Ang II alone caused a decrease in muscle sympathetic nerve activity, but when Ang II was infused with nitroprusside, muscle sympathetic nerve activity significantly increased. These data suggest that during the acute stage of Ang II hypertension, the peripheral contractile effects of Ang II activate baroreceptors and inhibit sympathetic outflow. However, if the baroreceptor-mediated effects are compensated for either by infusion nitroprusside (see Matsukawa et al., 1991) or through resetting, Ang II can increase the activity of the sympathetic nervous system. Another possible explanation of the discrepant results is that Ang II does not produce uniform activation of the sympathetic nervous system. Experimental evidence indicates that differential activation of the sympathetic nervous system is possible, although it has not been demonstrated under physiological conditions (Jaenig and McLachlan, 1992).

In summary, we observed different patterns of Fos expression associated with acute vs. chronic administration of Ang II. The pattern of Fos immunostaining in the medulla after 2 hr of Ang II was qualitatively similar to labeling produced by phenylephrine-induced increases in BP. In fact, increases in Fos observed in both the NTS and CVL after 2-hr Ang II infusion were blocked by SAD, and we suggest that the initial response within these areas is consistent with baroreceptor-mediated sympathoinhibition. The pattern of Fos expression changes when Ang II infusion is extended for 18 hr. Although Fos returns to control levels in the CVL at 18 hr, Fos in the NTS remains increased. Furthermore, the RVL, a region associated with sympathoexcitation, shows baroreceptor-independent increases of Fos only after 18-hr Ang II infusion.