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Hypertension. 1996;27:433-441

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(Hypertension. 1996;27:433-441.)
© 1996 American Heart Association, Inc.

Articles

Altered c-fos in Rostral Medulla and Spinal Cord of Spontaneously Hypertensive Rats

Jane Minson; Leonard Arnolda; Ida Llewellyn-Smith; Paul Pilowsky; John Chalmers

From the Department of Medicine and Centre for Neuroscience, School of Medicine, Flinders University of South Australia, Adelaide.


*    Abstract
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 *Abstract
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Abstract Neurons immunoreactive for Fos, the protein product of the immediate early gene c-fos, have been compared in the rostral ventral medulla and spinal cord of conscious normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) after baroreceptor unloading. Hypotension induced by a 60-minute intravenous infusion of sodium nitroprusside reduced baroreceptor activity; controls received intravenous saline. In WKY, 474±56 (n=6) Fos-positive neurons were identified in the rostral ventral medulla after nitroprusside infusion, a fivefold increase from controls; 50% of the tyrosine hydroxylase–containing neurons in the rostral ventral medulla were activated by this hypotension. Sympathetic preganglionic neurons, mainly sympathoadrenal neurons, were Fos positive after nitroprusside, but Fos-positive sympathetic preganglionic neurons were not observed in control WKY. In SHR, Fos immunoreactivity in the rostral ventral medulla was elevated in the control group compared with the WKY controls (236±31 and 93±15, respectively, n=6 for both). Nitroprusside hypotension did not further increase Fos immunoreactivity in the rostral ventral medulla, although the number of Fos-positive spinal sympathetic neurons increased. Our results have identified different neuronal activities between WKY and SHR in sites that are critical to sympathetic outflow. In WKY, nitroprusside effects are consistent with an activation of rostral ventral medulla neurons, including bulbospinal neurons, that are normally inhibited by baroreceptor activity. In SHR, basal nerve activity is increased, so even at rest, rostral ventral medulla neurons and sympathetic preganglionic neurons, mainly sympathoadrenal neurons, are Fos immunoreactive. These activated neurons are likely to contribute to the elevated blood pressure in this rat strain.


Key Words: blood pressure • immunohistochemistry • genes • retrograde tracing • rostral ventral medulla • rats, inbred SHR


*    Introduction
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 *Introduction
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The major source of descending drive to the sympathetic preganglionic neurons of the spinal cord is the spinally projecting neurons in the RVM.1 2 3 The activity of these neurons determines arterial BP, in that stimulation of the RVM increases BP and sympathetic vasomotor tone, and inhibition reduces BP to spinal levels.3 The activity of neurons in the RVM is modulated by excitatory and inhibitory inputs arising from different sites, including the baroreceptor afferents. Increases in baroreceptor activity slow the firing rate of the RVM neurons, and decreases in baroreceptor afferent activity increase their activity. These baroreceptor effects are mediated by a tonically active inhibitory input to the RVM that arises in the caudal ventrolateral medulla and uses GABA as a neurotransmitter.4 5 In SHR, it is likely that tonic activity is reduced in this inhibitory pathway from the caudal to rostral medulla.6 7 Such a change in activity would be consistent with the increased activity in bulbospinal sympathoexcitatory pathways in SHR, observed in pharmacological8 and electrophysiological9 studies, as well as the overall increase in sympathetic nerve activity in this rat strain.10 11 12 13

The immediate early gene c-fos is transiently expressed after a variety of stimuli and is considered to be a useful marker of neuronal activity in different sites,14 15 including those important in BP control.16 17 18 19 20 21 22 23 24 Within the RVM, c-fos expression is likely to be critical for BP control; our own studies have shown that the blockade of c-fos expression in this region, by local injection of an antisense oligonucleotide to c-fos mRNA, attenuates resting and stimulated BP levels.25 Recently, we reported that the GABA receptor agonist muscimol, injected into the caudal ventrolateral medulla to inhibit local neuronal activity, acutely increased both BP and immunoreactivity to Fos, the protein product of the c-fos gene, in bulbospinal neurons in the RVM.17 18 This result was consistent with a disinhibition of RVM neurons.

In the present study, we used Fos immunohistochemistry to identify the neurons in the RVM and spinal cord activated during isovolemic hypotension by intravenous infusion of sodium nitroprusside for 60 minutes. The stimulus was applied in conscious normotensive WKY and genetically hypertensive SHR. We suggest that the neurons in the RVM and spinal cord activated by this stimulus, which unloads the arterial baroreceptors, are those neurons that subserve the arterial baroreceptor reflex. Since catecholamine and serotonin neurons in the RVM are likely to be involved in BP regulation,26 we used double-labeling immunohistochemistry to determine whether these two chemically distinct neuron groups are activated by this stimulus. Retrograde neuronal tracing was used to identify the targets of the sympathetic preganglionic neurons that were stimulated by nitroprusside infusion.


*    Methods
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 *Methods
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Animal Preparation
Male, age-matched (16 to 18 weeks old, 300 to 400 g) WKY and SHR were used. The procedures followed were in accordance with the National Health and Medical Research Council Code of Practice and were approved by the institutional Animal Welfare Committee. Rats were anesthetized with sodium pentobarbital (60 mg/kg IP), and superficial branches of the left femoral artery and vein were implanted with vinyl catheters (SV10, Dural Plastics). The catheters were filled with heparinized saline (200 IU/mL), plugged, tunneled subcutaneously, and exteriorized dorsally at the scapulae. Rats were placed on a warming pad and, after regaining consciousness, were housed alone, with food and water available ad libitum. At 24 hours after recovery, and while remaining in their home cages, the rats were connected to a MacLab System 8 Recorder (ADInstruments) via the arterial catheter. An infusion line from a multichannel peristaltic pump (IPS-4, Ismatec SA) was connected to the venous catheter. The rats were kept undisturbed for the duration of the experiment. Resting arterial BP, MAP, and heart rate were calculated over a 30-minute period before infusion. Two rats were infused simultaneously; one received sodium nitroprusside (David Bull Laboratories, 3.4 mmol/L in saline) and the other saline only. Nitroprusside was infused at a rate to reduce and maintain MAP at 60% of the calculated resting MAP. BP was constantly monitored, and the infusion rate (range, 2 to 20 µL/min) was modified as appropriate in response to fluctuations in MAP. A second line from the multichannel pump provided the saline infusion such that the control rat was subject to the same variations in the infusion rate over the course of the experiment. Infusions continued for 60 minutes. The rats rested for another 60 minutes and BP was monitored intermittently. At 120 minutes after the start of the infusion, rats were anesthetized (sodium pentobarbital, 100 mg/kg IP) and perfused through the heart with 200 mL oxygenated tissue culture medium (D-8900, Sigma Chemical Co), followed by 1 L of 4% formaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. Brain stem and spinal cord tissues were postfixed overnight in the same fixative solution.

Immunohistochemistry
Vibratome sections (50 µm) were cut transversely through the brain stem and horizontally through the spinal cord. Immunohistochemistry was used to reveal Fos, TH, and serotonin immunoreactivities in the brain stem; Fos and ChAT immunoreactivities were revealed in the spinal cord. GFAP immunoreactivity was revealed in some brain stem and spinal cord sections. Tissue sections were washed in several changes of 10 mmol/L sodium phosphate buffer containing 10 mmol/L Tris, 0.9% NaCl, 0.05% thimerosal (Sigma), and 0.3% Triton X-100 (TPBS-Triton) and incubated in 10% NHS diluted in TPBS-Triton for 30 minutes. Alternate brain stem sections were incubated in sheep anti-Fos antiserum (1:20 000) and either mouse anti-TH antibody (1:100, Boehringer Mannheim Australia) or rabbit anti-serotonin antiserum (1:10 000, Arnel Products Co) in TPBS-Triton containing 10% NHS for 48 hours at room temperature with gentle agitation. Spinal cord sections were incubated in the same manner in sheep anti-Fos antiserum (1:15 000) and rabbit anti-ChAT antiserum (1:2000, Chemicon International). Some brain stem and spinal cord sections were incubated in sheep anti-Fos antiserum and rabbit anti-GFAP antibody (1:1000, Dako Corp). The antibody to Fos17 18 25 was raised in sheep after immunization with the synthetic peptide (Auspep) corresponding to residues 4 through 17 of the N-terminal domain of human Fos conjugated to bovine thyroglobulin using ethyl carbodiimide. Fos 4-17 has little homology with known Fos-related proteins. After washing, the sections were incubated overnight in biotinylated donkey anti-goat immunoglobulin (Jackson ImmunoResearch Laboratories) diluted 1:1000 in TPBS-Triton containing 1% NHS, followed by 4 hours in a 1:1500 dilution of ExtrAvidin conjugated to horseradish peroxidase (Sigma) in TPBS-Triton. Sections were washed in three changes of TPBS-Triton after each incubation. Gray-black Fos-immunoreactive nuclei were revealed by a nickel-intensified diaminobenzidine reaction with peroxide being generated by glucose oxidase.27 Subsequently, tissue sections were incubated overnight in biotinylated donkey anti-mouse immunoglobulin (Jackson ImmunoResearch; for TH) or biotinylated donkey anti-rabbit immunoglobulin (Jackson ImmunoResearch; for serotonin, ChAT, or GFAP) diluted 1:1000 in TPBS-Triton containing 1% NHS and in ExtrAvidin (1:1500, 4 hours). An imidazole-intensified diaminobenzidine reaction revealed this second immunoreactive product as an amber-colored deposit.27 Fos-immunoreactive brain stem neurons were counted in alternate 50-µm sections extending caudally 600 µm from the caudal tip of the facial nucleus. The area of RVM counted was bordered laterally by a line extending from compact formation of the nucleus ambiguus to the ventral pole of the spinal trigeminal tract and medially by a line extending from the nucleus ambiguus to bisect the pyramidal tract. This area, defined as RVM, encompassed the rostral ventrolateral medulla, including the catecholamine-containing neurons, and the rostral ventromedial medulla, including the serotonin-containing neurons in the parapyramidal area. Immunoreactive spinal cord neurons were counted in the intermediolateral cell column. The spinal cord was marked according to the 13 thoracic vertebrae so that the spinal tissue examined extended caudally from thoracic segment T1 to lumbar segment L4.28

The preabsorption of anti-Fos antiserum with synthetic Fos peptide abolished Fos staining in medulla and spinal cord.17 Some sections were incubated in TPBS-Triton containing 10% NHS without primary antiserum; there was no specific staining in these control experiments.

Retrograde Labeling of Sympathetic Preganglionic Neurons
Three to 4 weeks before experiment, WKY (n=3) and SHR (n=6) were injected with the retrograde tracer cholera toxin B subunit conjugated to 7 nm gold (CTB-gold)29 into the adrenal medulla and superior cervical ganglion. The rats were anesthetized (sodium pentobarbital, 60 mg/kg IP); 10 µL of tracer was injected into the left adrenal medulla via a flank incision with a 50-µm-tip glass pipette, and 5 µL of tracer was injected into the right superior cervical ganglion via a midline ventral incision. Incisions were sutured and the rats allowed to recover. Nitroprusside or saline infusions were as outlined above. Retrogradely labeled sympathetic preganglionic neurons were revealed before immunohistochemical processing with a Silver Enhancement Kit (Sigma). Immunoreactive spinal cord neurons and immunoreactive spinal cord neurons containing retrogradely transported gold particles were counted in the intermediolateral cell column.

Controls for Fos Immunoreactivity
Two groups of rats were used as controls for Fos expression. The ambient control group (WKY, n=3; SHR, n=3) included rats housed identically to the treatment rats but not subjected to surgery or other intervention. These rats were anesthetized immediately before perfusion. The catheter control group rats (WKY, n=2; SHR, n=2) were implanted with arterial and venous catheters but were not handled further; on the day of experiment, the catheters were not connected. These rats were perfused at times after cannulation that matched those of infused rats.

Statistical Analysis
Results are expressed as mean±SEM. Two-way ANOVAs were used to compare the effects on Fos immunoreactivity of the different interventions in SHR and WKY. Pairwise post hoc comparisons were made with Student's t tests. Bonferroni adjustments were used to reduce the risk of type 1 error.30 A probability level of .05 or less was considered to be statistically significant.


*    Results
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 up arrowMethods
*Results
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Nitroprusside infusion induced an immediate fall in BP in WKY and SHR as well as tachycardia (Fig 1Down, TableDown). Nitroprusside reduced MAP in WKY by 48±2 mm Hg (n=6, 59±0.7% of resting levels) and in SHR by 65±3 mm Hg (n=6, 59±1.4% of resting levels); infusion rates were the same in both strains. BP immediately recovered toward resting levels when the nitroprusside infusion was stopped (Fig 1Down, TableDown). Saline infusion did not affect MAP and heart rate in WKY and SHR (TableDown). Two-way ANOVAs comparing strains (WKY, SHR) and treatment (nitroprusside, saline, catheter, ambient) revealed a significant strainxtreatment interaction (F3,26=11.6) on Fos immunoreactivity in the RVM and significant treatment (F1,14=14.6) and strain (F1,14=8.4) effects on Fos immunoreactivity in the spinal cord.



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  		Figure 1. Effects of nitroprusside infusion on arterial BP and heart rate (HR) in conscious WKY (top) and SHR (bottom). Nitroprusside was infused during the period marked by the bar. Rats rested for 60 minutes after infusion before being anesthetized and perfused.


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  		Table 1. Blood Pressure and Heart Rate Changes During Nitroprusside or Saline Infusion in Conscious WKY and SHR

Fos Immunoreactivity in WKY
Fos in RVM
Fos immunoreactivity, confined to nuclei and stained gray to black, was observed in the RVM (Fig 2Down). In nitroprusside-treated WKY, 474±56 (n=6) Fos-positive RVM neurons were counted in each rat, a significant increase from the saline group (93±15 [n=6], P<.01). Almost half of the RVM TH neurons were Fos positive after nitroprusside (Fig 3ADown and 3BDown), but only a small number of these neurons were Fos positive in the saline group (P<.001, Fig 4Down). TH-immunoreactive neurons made up 30±2% (n=6) of all the Fos-positive RVM neurons after nitroprusside. Nitroprusside increased the number of Fos-positive serotonin-immunoreactive RVM neurons compared with the saline group (P<.02); these serotonin neurons were medial to the TH neurons, clustering near the lateral edges of the pyramidal tracts. The increase in the incidence of Fos immunoreactivity in the serotonin neurons was smaller than that in the TH population, and serotonin-immunoreactive neurons made up 3±1% (n=6) of all the RVM Fos neurons after nitroprusside. Fos immunoreactivity was not seen in glial profiles (Fig 3CDown).



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  		Figure 2. Fos-positive neurons (examples labeled with arrow) in RVM of WKY after 60-minute infusion of nitroprusside (A) or saline (B). Each photomicrograph shows the left side of the RVM, 300 µm caudal to the caudal tip of the facial nucleus. NA indicates the compact area of the nucleus ambiguus; V, the ventral surface of the medulla. Scale bar=100 µm.



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  		Figure 3. Photomicrographs show immunoreactivity in RVM of nitroprusside-infused WKY. The sections are at the same rostrocaudal level as in Fig 2Up. A, Fos-positive neurons (examples labeled with arrowheads) and Fos-positive TH-immunoreactive neurons (arrows) are visible. Only two of the TH neurons in this field do not contain Fos (double arrowheads). B, Enlargement of the field labeled with the asterisk in A. Fos-positive neurons and Fos-positive TH-immunoreactive neurons are visible. C, Fos-positive nuclei (arrowheads) in RVM are not associated with stained glial profiles; GFAP-positive cells are labeled (arrows). Scale bar=50 µm (A), 25 µm (B and C).



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  		Figure 4. Bar graphs show Fos immunoreactivity in TH- or serotonin-positive (5 HT) RVM neurons in WKY and SHR after nitroprusside (NP) or saline infusion. Numbers of Fos-positive immunoreactive neurons are expressed as percentages of the whole population of TH or serotonin neurons in the RVM. Results are mean±SEM, n=6 in each group. Numbers of TH-immunoreactive RVM neurons or serotonin-immunoreactive RVM neurons were similar in WKY and SHR (TH: WKY, 183±16, SHR, 214±9; serotonin: WKY, 110±16, SHR 80±7; each group n=12; P>.05).

Fos immunoreactivity in the RVM was examined in two control groups. In ambient controls (no surgery or handling), 112±12 (n=3) Fos-positive RVM neurons were counted; in catheter controls (catheters implanted, no infusion), 110±3 (n=2) Fos-positive RVM neurons were counted. These numbers were similar to those observed in the saline-infusion group (P>.05). The control tissues were processed simultaneously with the experimental groups, but the Fos-immunoreactive signal in these groups was always paler than the reaction product seen in the treatment groups (Fig 5ADown through 5C).



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  		Figure 5. Photomicrographs show Fos-positive RVM neurons in WKY (A through C) and SHR (D through F) at the same RVM level as in Figs 2Up and 3Up. Rats were infused with nitroprusside (A and D) or saline (B and E) or were perfused without experiment (ambient controls, C and F). Fos immunoreactivity was stained gray to black and confined to the nuclei. In WKY controls (B and C), this immunoreactivity was pale; these nuclei are labeled with arrows. The sections were also processed to reveal serotonin immunoreactivity, and varicose fibers are visible in each field; the relationship of these fibers to the Fos nuclei was not determined. Scale bar=25 µm and applies to all panels.

Fos in Spinal Cord
Fos-positive ChAT-immunoreactive sympathetic preganglionic neurons were not seen in the saline-infused or control WKY groups, but these neurons were found in the thoracic and upper lumbar spinal cord after nitroprusside infusion (Fig 6ADown and Fig 7Down); in each nitroprusside-infused WKY, 263±56 (n=6) Fos-positive sympathetic preganglionic neurons were counted. Fos-positive neurons were observed in the dorsal horn of rats receiving either nitroprusside or saline. The activation of sympathetic preganglionic neurons observed after nitroprusside was widespread. Fos-positive sympathetic preganglionic neurons were found in T2 through L2 segments and were most densely localized in the vertebral levels that contained T7 through T11 (Fig 7Down).



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  		Figure 6. Photomicrographs show sympathetic preganglionic neurons in the intermediolateral cell column in WKY (A and C) and SHR (B) after nitroprusside infusion. A, WKY segment T10; ChAT-immunoreactive sympathetic preganglionic neurons lie in the intermediolateral cell column. Some of these neurons are Fos positive (examples labeled with arrows). B, SHR segment T7; many ChAT-immunoreactive sympathetic preganglionic neurons in the intermediolateral cell column are visible. Some of these neurons are Fos positive (examples labeled with arrows). C, WKY segment T7; a single ChAT-immunoreactive sympathetic preganglionic neuron is shown. The black punctate deposits in the cytoplasm are silver-intensified CTB-gold, retrogradely transported from the adrenal medulla. This sympathoadrenal neuron has Fos immunoreactivity in the nucleus. Scale bar=50 µm (A), 100 µm (B), 10 µm (C).



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  		Figure 7. Bar graphs show Fos immunoreactivity in ChAT-positive sympathetic preganglionic neurons in WKY (top) and SHR (bottom) after nitroprusside (NP) or saline infusion. Spinal cords were marked according to the 13 thoracic vertebrae; thoracic and lumbar segments are indicated at the bottom of the graph. No Fos-positive sympathetic preganglionic neurons were identified in the saline-infused WKY. Results are mean±SEM; nitroprusside, n=6 in each group; saline, n=3.

Fos in Retrogradely Labeled Sympathetic Preganglionic Neurons
Retrogradely labeled sympathetic preganglionic neurons were observed on the left side of the spinal cord after injection of CTB-gold into the left adrenal medulla. These sympathoadrenal neurons extended from T2 to T13 but mostly were localized in the lower thoracic cord. The numbers of sympathetic preganglionic neurons containing CTB-gold alone were not counted in this study. Fos-positive sympathoadrenal neurons were found after nitroprusside (Fig 6CUp, Fig 8Down). Sympathoadrenal neurons accounted for 71±2% (n=3) of all the activated ChAT-positive sympathetic preganglionic neurons and were most densely distributed in segments T8 and T9 (Fig 8Down). After CTB-gold injection into the right superior cervical ganglion, retrogradely labeled sympathetic preganglionic neurons were identified on the right side of the cord from T1 to T5; these neurons were never identified as Fos positive after nitroprusside.



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  		Figure 8. Bar graphs show Fos immunoreactivity in sympathetic preganglionic neurons on the left side of the spinal cord in WKY after nitroprusside (NP) infusion (top) and in SHR after nitroprusside or saline infusion (middle and bottom). Rats were previously injected with CTB-gold into the left adrenal medulla. Fos-positive sympathoadrenal neurons (speckled bars) and Fos-positive sympathetic preganglionic neurons not retrogradely labeled (shaded bars) were counted. Spinal cord dissection was as in Fig 7Up legend. Results are mean±SEM, n=3 in each group. SPN indicates sympathetic preganglionic neuron.

Fos Immunoreactivity in SHR
Fos in RVM
In SHR after nitroprusside infusion, 269±33 (n=6) Fos-positive neurons were counted in the RVM. This incidence was not different from that found in the saline group (236±31 [n=6], P>.05). Fos-positive RVM neurons immunoreactive for TH or serotonin were observed in the RVM, but nitroprusside infusion did not increase the incidence of Fos immunoreactivity in these neurons (Fig 4Up, P>.05).

The incidence of Fos-positive RVM neurons in the saline-treated SHR was significantly increased from the number in the saline-treated WKY (P<.02). Fos-immunoreactive RVM neurons were also found in the control SHR groups (Fig 5DUp through 5F). In ambient control SHR (no surgery or handling), 260±25 (n=3) Fos-positive RVM neurons were counted; in catheter control SHR (catheters implanted, no infusion), 189±21 (n=2) Fos-positive RVM neurons were counted. This incidence was similar to that seen in the saline-infused SHR group (P>.05).

Fos in Spinal Cord
Fos-positive ChAT-immunoreactive sympathetic preganglionic neurons were found in the thoracic and upper lumbar spinal cord in the saline SHR group (167±50, n=3) and in the noninfused controls. Nitroprusside infusion significantly increased the number of these neurons (707±130, n=6; P<.02). As in WKY, Fos-positive neurons were found in the dorsal horn of SHR after either nitroprusside or saline infusion. In the nitroprusside-treated SHR, Fos-positive sympathetic preganglionic neurons were identified from T1 through L2 segments, localized mainly in the middle to lower thoracic cord (Fig 6BUp, Fig 7Up). These neurons were also identified in the middle to lower thoracic cord in the saline-treated SHR (Fig 7Up).

Fos in Retrogradely Labeled Sympathetic Preganglionic Neurons
The distribution of retrogradely labeled sympathetic preganglionic neurons in SHR, after CTB-gold injection into either the adrenal medulla or superior cervical ganglion, was similar to that observed in WKY. Fos-positive sympathoadrenal neurons were identified in the middle to lower thoracic cord in saline-infused SHR (Fig 8Up); these sympathoadrenal neurons represented 66±6% (n=3) of all the Fos-positive sympathetic preganglionic neurons. Nitroprusside treatment increased the number of Fos-positive sympathoadrenal neurons. These neurons were localized mainly in segments T7 through T11 (Fig 8Up) and accounted for 39±12% (n=3) of the Fos-positive sympathetic preganglionic neurons in SHR after nitroprusside. Rare Fos-positive and retrogradely labeled sympathetic preganglionic neurons projecting to the superior cervical ganglion were identified in SHR (eight neurons in two nitroprusside-infused SHR, no neurons in the third SHR). A single labeled Fos-positive retrogradely labeled neuron was found in one of the three saline-infused SHR.


*    Discussion
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*Discussion
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Fos immunoreactivity in the RVM and intermediolateral cell column, compared in normotensive and hypertensive rats, showed strain differences and changes after nitroprusside-induced hypotension. First, nitroprusside infusion in WKY evoked a fivefold increase in the number of Fos-positive neurons in RVM, and many of these activated neurons were catecholaminergic (containing TH). Second, an activation of sympathetic preganglionic neurons was observed after nitroprusside in WKY, and this spinal activation was almost exclusively confined to the sympathoadrenal neurons. Third, the basal incidence of Fos-immunoreactive neurons was higher in SHR than in WKY. Fos-positive RVM neurons, including TH-containing neurons, and Fos-positive sympathetic preganglionic neurons, including sympathoadrenal neurons, were observed in control SHR. Fourth, nitroprusside treatment failed to increase the number of Fos-positive RVM neurons in SHR, although Fos in the spinal cord was increased. The results in WKY are consistent with an activation of RVM neurons normally inhibited by baroreceptor activity. The activation of half of the RVM catecholaminergic neurons suggests that these neurons probably play an integral, though not exclusive, role in mediating baroreflex changes in BP. The activation of sympathetic preganglionic neurons most likely follows as a consequence of the activation of RVM neurons. In SHR the increased basal activity of RVM and spinal sympathetic neurons is consistent with a disinhibition of the RVM.

We used Fos immunoreactivity in this study as a marker of activation in the brain stem and spinal cord. Fos immunoreactivity was not associated with glial profiles, so the patterns of Fos staining we have observed are likely to reflect neuronal activity within these central areas. We suggest that many of the neurons immunoreactive for Fos after arterial baroreceptor unloading are the vasomotor neurons that mediate the baroreceptor reflex. We avoided anesthesia effects on c-fos expression31 ; and using different controls, we determined that Fos immunoreactivity in the RVM and intermediolateral cell column was not a consequence of infusion without BP change or of surgical intervention and handling. In contrast, the Fos immunoreactivity observed in the dorsal horn in both saline- and nitroprusside-infused rats was likely to be evoked by handling, consistent with other studies.32 Since Fos is a transcription regulating factor,15 c-fos expression in RVM and spinal neurons is likely to promote changes in the expression of other genes, and these changes might underlie long-term alterations in the activity of these neurons. One target gene regulated by c-fos is the TH gene, and c-fos has been linked with the transsynaptic induction of this gene.33 In the RVM neurons revealed as Fos positive in this study, Fos could promote TH gene transcription to replenish intracellular catecholamine stores depleted during RVM disinhibition.

Fos Immunoreactivity in Normotensive WKY
Hypotension in WKY evoked a fivefold increase in the number of Fos-positive neurons in the area of the RVM, which is the major source of descending drive to the sympathetic preganglionic neurons of the spinal cord.1 2 3 Fos immunoreactivity in these neurons is consistent with disinhibition of the RVM neurons, hypotension decreasing baroreceptor input and, via a multisynaptic pathway, reducing the inhibitory drive to the RVM. Nucleus tractus solitarius to caudal ventrolateral medulla and caudal ventrolateral medulla to RVM projections are probably involved.3 34 The caudal ventrolateral medulla to RVM pathway provides a tonic inhibitory GABA input to RVM4 5 35 and mediates the baroreflex control of sympathetic activity.36 37 38 The increase in Fos immunoreactivity in WKY after nitroprusside is consistent with our earlier studies, in which reduced neuronal activity in the caudal ventrolateral medulla by direct GABA agonist injection evoked a short hypertensive response and increased Fos-positive neurons in the RVM.17 18 Other studies in rat, cat, and rabbit have demonstrated Fos immunoreactivity in the RVM after reductions in baroreceptor activity by hemorrhage19 20 21 22 or hypotensive drug infusion.22 23 24

A basal incidence of Fos-immunoreactive RVM neurons was observed in normotensive rats. This expression might reflect ongoing, repetitive neuronal activation related to the generation of sympathetic tone and is consistent with the lowering of BP after an RVM injection of antisense oligonucleotide to c-fos mRNA.25 This incidence of Fos-positive neurons was similar in untreated, catheterized but not infused, and saline-infused WKY, so surgical preparation and infusion without BP change were not stimuli for c-fos expression in RVM neurons. The immunoreactive product of this constitutive Fos was pale compared with the product observed in the nitroprusside group and is consistent with low Fos protein levels at rest.

The widespread activation of catecholaminergic neurons in the RVM after nitroprusside, 50% of TH-immunoreactive neurons expressing Fos, supports an integral role for these neurons in the baroreflex control of BP. Other c-fos studies have also reported an activation of RVM catecholaminergic neurons in association with cardiovascular stimuli.18 21 22 23 In contrast, there was only a small increase in the number of Fos-positive serotonin neurons in the RVM. This result suggests that although these neurons have an important pressor role,26 39 40 41 their activity might not be essential for the mediation of the baroreceptor reflex. Previously we observed Fos immunoreactivity in RVM serotonin neurons after muscimol injection in the caudal ventrolateral medulla.18 Such expression would be consistent with an interruption of inhibitory pathways from the caudal ventrolateral medulla, which project to the RVM but have no role in the baroreflex arc.42

Sympathetic preganglionic neurons, identified by immunoreactivity for ChAT and localized in the intermediolateral cell column, were identified from T1 to L3 spinal segments, in accord with other studies.43 In WKY there was no constitutive Fos immunoreactivity in these neurons, but after nitroprusside treatment, Fos-positive sympathetic preganglionic neurons were identified in the middle and lower thoracic segments, consistent with an activation of sympathoexcitatory bulbospinal RVM neurons during hypotension. Neuronal activation in the spinal cord was selective, Fos-positive neurons being mainly sympathoadrenal neurons. These neurons were most densely distributed in the T8 to T9 level, the spinal level that is the major source of input to the adrenal gland.43 44 This increase in Fos immunoreactivity in the sympathoadrenal neurons suggests that adrenal catecholamines are likely to be secreted in response to the hypotension. We have identified a significant elevation in plasma epinephrine concentration associated with a pronounced activation of sympathoadrenal neurons, as revealed by Fos immunoreactivity, in another study.16 Sympathetic outflow to the heart, and to the head and thoracic organs, arises from the upper thoracic cord,43 but there was no evidence of activation of sympathetic preganglionic neurons at these spinal levels, including those sympathetic preganglionic neurons retrogradely labeled from the superior cervical ganglion. The absence of Fos-positive sympathetic neurons in the upper thoracic cord suggests that changes in vagal tone or hormonal effectors underlie the nitroprusside-induced tachycardia, although it is possible that the activation of cardiac sympathetic preganglionic neurons might not involve c-fos expression.

Fos Immunoreactivity in Hypertensive Rats
This study reveals a pattern of neuronal disinhibition in the RVM of SHR, such that Fos immunoreactivity in the RVM and spinal cord in SHR shows similarities with that in the baroreceptor-unloaded WKY. The basal incidence of Fos-positive RVM neurons in SHR (observed in untreated, catheterized but not infused, and saline-infused SHR) was twofold higher than in WKY, suggesting that the constitutive c-fos activity in RVM neurons, and consequently in sympathetic preganglionic neurons, was increased in SHR. This neuronal activation in SHR is likely to contribute to the higher resting sympathetic tone in this strain12 and is consistent with electrophysiological9 and pharmacological8 evidence of bulbospinal sympathoexcitatory pathway activation in SHR. Impaired inhibitory inputs to the RVM from the caudal ventrolateral medulla7 are likely to contribute to this neuronal activation such that the sympathoexcitatory RVM neurons might be chronically disinhibited. TH-positive neurons were identified among the disinhibited (ie, Fos-positive) RVM neurons in control SHR, so the hypertension of this strain might result, at least in part, from the increased activity of these catecholaminergic neurons. In WKY, the TH neurons were revealed as an integral neuronal link in the baroreflex control of BP, and the changed activity of the TH neurons associated with chronic hypertension again points to a critical role of these neurons in the central control of BP. Neuronal activation in the RVM was selective, and the incidence of serotonin-positive Fos neurons did not increase between control SHR and WKY. Fos-positive sympathetic preganglionic neurons were observed in the middle and lower thoracic cord in control SHR. This activation, a likely consequence of the increased constitutive activity of the RVM neurons, is consistent with the increased renal12 and splanchnic13 nerve activity in this strain. Fos-positive sympathoadrenal neurons point to an activation of these neurons in SHR such that an increased activity of the sympathoadrenal axis contributes to the hypertension in this strain.

Nitroprusside infusion in SHR failed to evoke an increase in the incidence of Fos-immunoreactive neurons in the RVM. This failure to alter c-fos expression demonstrates an impaired baroreceptor reflex in SHR such that acute hypotension cannot stimulate the withdrawal of the inhibitory input to the RVM. It is also possible that the cellular mechanisms of the RVM neurons in SHR might have been altered by the long-term alteration in the activity, so an acute nitroprusside stimulus might not promote c-fos gene transcription. Numbers of Fos-positive sympathetic preganglionic neurons were increased in SHR after nitroprusside infusion. The restricted spinal distribution of these neurons suggests a stimulation of selective afferent pathway(s) to the spinal cord, but clearly this does not involve a c-fos–mediated activation of RVM neurons. The source of the sympathetic preganglionic neuron stimulation in SHR is unclear. The activation of the sympathetic preganglionic neurons could reflect differences between SHR and WKY in the innervation of these neurons or even differences within the sympathetic preganglionic neurons themselves. One possibility is an alteration in N-methyl-D-aspartate (NMDA) excitatory amino acid receptors in the sympathetic preganglionic neurons of SHR. Expression of c-fos is directly linked to NMDA receptor activation,15 45 and we have demonstrated a sensitivity of SHR sympathetic preganglionic neurons to specific NMDA antagonism that was not observed in WKY.8

In conclusion, we applied a hypotensive drug infusion, to unload the arterial baroreceptors, in conscious normotensive WKY and genetically hypertensive SHR and used Fos immunohistochemistry to identify the ensuing neuronal activation in the RVM and spinal cord. In WKY the powerful inhibitory drive to the RVM is removed by hypotension, and many bulbospinal, presumed vasomotor, neurons, including catecholamine neurons, are activated. Sympathetic preganglionic neurons, principally those innervating the adrenal medulla, are also activated. Resting SHR displayed many of the characteristics of WKY with RVM disinhibition, with a high incidence of Fos immunoreactivity in RVM and spinal sympathetic neurons. These data provide the first anatomic evidence that the hypertension in SHR is associated with an impaired inhibitory drive to the RVM. Few of the connections from the caudal ventrolateral medulla to the RVM are likely to be functional in SHR because baroreceptor unloading fails to increase Fos immunoreactivity in the RVM. The differences in the activation of RVM neurons in SHR and WKY, and the apparent differences in the influence of RVM bulbospinal neurons on the activity of the sympathetic preganglionic neurons in the two strains, suggest that there may be fundamental differences in the central mechanisms regulating sympathetic activity in the SHR.


*    Selected Abbreviations and Acronyms
 
BP = blood pressure
ChAT = choline acetyltransferase
GABA = {gamma}-aminobutyric acid
GFAP = glial fibrillary acidic protein
MAP = mean arterial pressure
NHS = normal horse serum
RVM = rostral ventral medulla
SHR = spontaneously hypertensive rat(s)
TH = tyrosine hydroxylase
WKY = Wistar-Kyoto rat(s)


*    Acknowledgments
 
This work was supported by a Programme Grant from the National Health and Medical Research Council of Australia and a Grant-in-Aid from the National Heart Foundation of Australia. We thank Anne Shephard, Michelle Anderson, and Claire Nicholls for their expert technical assistance. We thank Dr John Oliver, Department of Medicine, Flinders Medical Centre, for his kind gift of Fos antiserum.


*    Footnotes
 
Reprint requests to Dr J.B. Minson, Department of Medicine, Flinders Medical Centre, Bedford Park SA 5042, Australia.

Portions of this work have been presented in abstract form (Proc Aust Neurosci Soc. 1995;6:108 and Proceedings of the High Blood Pressure Research Council of Australia. 1994;16:13).

Received June 13, 1995; first decision August 10, 1995; accepted December 7, 1995.


*    References
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*References
 

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