(Hypertension. 1996;27:287-296.)
© 1996 American Heart Association, Inc.
Articles |
Medullary Neurons Activated by Angiotensin II in the Conscious Rabbit
From the Department of Physiology and Sydney Institute for Biomedical Research, University of Sydney, New South Wales (Y.H., P.D.P., R.A.L.D.), and the Baker Medical Research Institute, Prahran, Victoria (G.A.H., S.J.G., R.D.B.), Australia.
Correspondence to Dr Roger A.L. Dampney, Department of Physiology (F13), University of Sydney, New South Wales 2006, Australia. E-mail rogerd@physiol.su.oz.au.
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Abstract Previous studies have shown that angiotensin II (Ang II) can activate cardiovascular neurons within the medulla oblongata via an action on specific receptors. The purpose of this study was to determine the distribution of neurons within the medulla activated by infusion of Ang II into the fourth ventricle of conscious rabbits, using the expression of Fos, the protein product of the immediate early gene c-fos as a marker of neuronal activation. Experiments were done in both intact and barodenervated animals. In comparison with a control group infused with Ringer's solution alone, in both intact and barodenervated animals, fourth ventricular infusion of Ang II (4 to 8 pmol/min) induced a significant increase in the number of Fos-positive neurons in the nucleus of the solitary tract and in the rostral, intermediate, and caudal parts of the ventrolateral medulla. Double-labeling for Fos and tyrosine hydroxylase immunoreactivity showed that 50% to 75% of Fos-positive cells in the rostral, intermediate, and caudal ventrolateral medulla and 30% to 40% of Fos-positive cells in the nucleus of the solitary tract were also positive for tyrosine hydroxylase in both intact and barodenervated animals. The distribution of Fos-positive neurons corresponded very closely to the location of Ang II receptor binding sites as previously determined in the rabbit. The results indicate that medullary neurons activated by Ang II are located in discrete regions within the nucleus of the solitary tract and ventrolateral medulla and include, in all of these regions, both catecholamine and noncatecholamine neurons.
Key Words: angiotensin II • blood pressure • brain • immunohistochemistry • pressoreceptors • rabbits
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Introduction |
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There is much evidence that central Ang II plays an important role in the regulation of blood pressure and blood volume in both normotensive and hypertensive animals by an action on neurons at all levels of the brain (for review, see Reference 1). Autoradiographic binding studies in several mammalian species, including humans, have demonstrated Ang II receptor binding sites within discrete regions in the forebrain and brain stem.2 3 4 5 In particular, there is a very high density of Ang II receptor binding sites within the medulla oblongata in the NTS and in the caudal and rostral parts of the VLM.2 3 4 6 Neurons within all these regions are known to be essential components of the central pathways that regulate blood pressure.7 8 9
Physiological studies have demonstrated that Ang II can affect blood pressure by actions within all these medullary regions.6 10 11 12 13 14 15 16 In particular, several recent studies have focused on the actions of Ang II in the rostral VLM, since this region contains a group of bulbospinal sympathoexcitatory neurons that are a major source of the tonic excitatory input to sympathetic preganglionic vasomotor neurons, as well as a site of convergence of central pathways mediating cardiovascular responses elicited from peripheral receptors or supramedullary nuclei.8 9 These studies have demonstrated that rostral VLM sympathoexcitatory neurons are excited by exogenously applied Ang II.6 14 15 16 17 Furthermore, there is evidence that endogenous Ang II helps to maintain the resting activity of rostral VLM sympathoexcitatory neurons, as well as facilitate their response to excitatory synaptic inputs.15 16 18 19
Previous studies have indicated that not all
sympathoexcitatory neurons in the rostral VLM
are activated by Ang II.18 20 In particular,
electrophysiological studies in vitro have
shown that in the rat medullary slice preparation Ang II excites most
rostral VLM neurons that have a slow and irregular spontaneous firing
rate,20 but it has no effect on neurons with rapidly
firing pacemakerlike activity.20 21 Furthermore,
nearly
all Ang-sensitive neurons, but none of the Ang-insensitive
neurons, were inhibited by 
2-adrenergic receptor
agonists.21 Because 2-adrenergic receptors
are believed to be associated with catecholamine neurons in
the rostral VLM,22 these observations suggest that Ang II
may act selectively on catecholamine neurons in the rostral
VLM and raise the possibility that Ang-sensitive neurons in other
regions of the medulla (caudal VLM and NTS) may also synthesize
catecholamines.
The main aim of the present study was to identify the population of neurons in the medulla oblongata that is activated by Ang II. For this purpose, we used the expression of the immediate early gene c-fos as a marker of individual neurons that are activated by Ang II. Expression of c-fos, as indicated by immunohistochemical labeling of its protein product Fos, occurs in a wide variety of central neurons in response to activation of neurons by many different stimuli; provided careful controls are carried out to minimize the possibility of nonspecific effects, Fos expression is thought to be an effective means of identifying neurons that are activated by specific physiological or pharmacological stimuli.23 24 25 26 Previous studies have used the method of Fos expression to identify neurons within the forebrain that respond to intracerebroventricular administration of Ang II,27 28 29 as well as neurons in circumventricular organs that respond to intravenous administration of Ang II,30 31 but have not examined in detail the effect of Ang II on Fos expression in the medulla.
In the present study, therefore, we mapped the distribution of neurons in the medulla oblongata that express Fos in response to Ang II injected directly into the fourth ventricle of conscious rabbits. A previous study in the conscious rabbit has indicated that, in contrast to intracerebroventricular administration, injection of Ang II into the fourth ventricle acts principally on neurons within the lower brain stem.13 Second, in view of the above-stated hypothesis that Ang II may act selectively on catecholamine neurons, we also combined Fos labeling with immunohistochemical labeling of the catecholamine-synthesizing enzyme TH. Experiments were performed in rabbits with denervated carotid sinus and aortic baroreceptors, as well as in intact rabbits, because we have previously observed a greater sensitivity to fourth ventricular administration of Ang II in barodenervated rabbits.32 In addition, this allowed the direct effects of Ang II to be observed in the absence of compensatory effects from arterial baroreflexes.
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Surgical Procedures
Experiments were performed on crossbred male and female rabbits with an average body weight of 2.9 kg (range, 2.6 to 3.3 kg) supplied by the Baker Medical Research Institute. In a preliminary operation, a polyvinyl catheter was implanted into the fourth ventricle as described previously.33 In brief, the rabbits were anesthetized with halothane (ICI) after induction with a mixture of alfaxalone and alfadolone (Saffan, Pitman-Moore, total steroids 5 mg/kg IV). The atlanto-occipital membrane was exposed through a midline skin incision, and a hole was made with a 25-gauge needle at the rostral midline border of the membrane with the occipital bone. The vinyl catheter (SV10, 0.28-mm ID, 0.61-mm OD, Dural Plastics) was passed 8 mm through the hole so that it lay in the fourth ventricle. Cerebrospinal fluid flow under gentle hydrostatic pressure was used as confirmation of catheter placement. The catheter was then stitched to the atlanto-occipital membrane with a 6-0 atraumatic needle and suture.
In some animals, bilateral denervation of the carotid sinus and aortic baroreceptors was performed in a separate operation with animals under halothane anesthesia, according to the procedure as described previously.34 The aortic nerve was identified as it entered the vagus nerve close to the origin of the superior laryngeal nerve, and a 1-cm section of the aortic nerve was then removed. The carotid sinus was exposed and elevated, and the carotid sinus nerve was identified and sectioned. The outer layers of all major and minor arterial branches within 1 cm of the carotid bifurcation were then stripped completely to ensure total denervation of the carotid sinus. Animals were then monitored closely after surgical procedures, and there was a waiting period of at least 6 days before further procedures were carried out. All experiments were carried out in accordance with the guidelines for animal experimentation of the National Health and Medical Research Council of Australia.
Experimental Procedures
On the day of the experiment, the
central ear artery was
cannulated transcutaneously with a 22-gauge
polytetrafluoroethylene (Teflon) catheter,
and the end of the fourth ventricular catheter was
retrieved from under the skin after injection of a local anesthetic.
The fourth ventricular catheter was flushed with 50 µL
sterile Ringer's solution at room temperature. There was then a
resting period of at least 1 hour after these procedures before the
commencement of the experiment. Arterial pressure was
measured with a pressure transducer, and mean arterial
pressure and heart rate were derived from the pulsatile pressure signal
with a low-pass filter and ratemeter, respectively. All
cardiovascular variables were displayed on a chart
recorder.
The effectiveness of surgical denervation of sinoaortic baroreceptors was assessed during the resting period before the commencement of the experiment. Normally, this is done by testing the heart rate response to an intravenous injection of phenylephrine. In the present study, this procedure was not appropriate because it may have induced Fos expression independent of any effect of Ang II. The effectiveness of barodenervation, however, was indicated by the fact that the resting heart rate (range, 232 to 316 bpm) in these animals was much higher than in all barointact rabbits (range, 152 to 204 bpm). Furthermore, the lability of resting arterial pressure (measured by the coefficient of variance of mean arterial pressure) in all but one of the barodenervated rabbits was much higher (range, 13.5% to 24.3%) than in all animals in the barointact group (range, 3.1% to 8.7%). In the remaining animal subjected to sinoaortic denervation, the coefficient of variance was 7.6% (in the upper part of the range for barointact animals), but the resting mean arterial pressure (103 mm Hg) and heart rate (232 bpm) were both much higher than the corresponding variables for all animals in the barointact group.
After the recovery period, Ang II (human, Penninsula Laboratories) was infused into the fourth ventricle at a rate of 4 to 8 pmol/min (2 to 4 µL/min) for 60 minutes in 6 rabbits (3 intact and 3 with denervated baroreceptors). The dose of Ang II was chosen to elicit a sustained increase in mean arterial pressure of approximately 10 mm Hg in barointact animals. In a separate group of 6 rabbits (4 intact and 2 barodenervated), the vehicle Ringer's solution was infused into the fourth ventricle at a rate of 4 µL/min for 60 minutes. After infusion, there was a waiting period of 30 minutes. Rabbits were then deeply anesthetized with sodium pentobarbital (50 mg/kg IV) and perfused transcardially with 500 mL saline, followed by 2 L of 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline. The brain was removed and immersed in 20% sucrose in phosphate-buffered saline. Five series of coronal sections (40 µm) were cut on a freezing microtome, after which they were washed in 50% ethanol for 1 hour and stored in 0.1 mol/L TBS containing 0.1% sodium azide at 4°C.
Staining Procedure for Fos and TH Immunoreactivity
To
visualize the Fos protein, an immunohistochemical
avidin-biotin-peroxidase staining procedure was used. The
sections were incubated in 15% normal horse serum in 0.1 mol/L TBS for
60 minutes and then in polyclonal sheep anti-Fos antibody (Cambridge
Research Biochemicals) diluted 1:4000 in 1% normal horse serum–TBS at
4°C for 2 days. Sections were then rinsed three times (10 minutes
each time) in 1% normal horse serum–TBS and incubated in biotinylated
anti-sheep IgG (Vector, 1:400 in 1% normal horse serum–TBS) for 2
hours at room temperature. The tissue was washed again and then
incubated in ExtrAvidin peroxidase conjugate (Sigma, 1:400 in 1%
normal horse serum–TBS) for 1 hour. The sections were then
preincubated for 10 minutes in a TBS solution containing 0.05%
3,3'-diaminobenzidine hydrochloride and 0.1% nickel ammonium sulfate
before being reacted in the same solution with the addition of 0.005%
hydrogen peroxide for 3 to 5 minutes. We have shown previously that no
specific immunoreactivity is observed if this procedure is carried out
with the omission of the Fos antibody.24
One of the series of sections was also stained for TH immunoreactivity. After the Fos immunohistochemical procedures were completed, these sections were washed again in TBS and then incubated in mouse monoclonal anti-TH antibody (1:4000, Incstar) for 24 hours. The sections were washed and incubated in biotinylated anti-mouse antibody for 2 hours. After being washed again, they were incubated in 0.05% 3,3'-diaminobenzidine hydrochloride solution containing 0.005% hydrogen peroxide in TBS. Consequently, Fos-positive cell nuclei were stained black, whereas TH-positive cell bodies and processes were brown, as described previously.24
After completion of the reactions, the sections were mounted on slides coated with gelatin–chromium potassium sulfate, dried, and placed under coverslips.
Microscopy and Quantification
Sections were examined with an
Olympus BH2 microscope. Labeled
cells were mapped and quantified using the Magellan Image
Analysis Program35 and a 486DX-33 IBM-compatible
computer. In the medulla, where double-labeled neurons extended
over considerable rostrocaudal distances, several sections at different
levels (approximately 0.4 mm apart) were mapped. In addition, the
distribution of labeled cells in representative
sections through the pons, midbrain, and hypothalamus were also mapped.
Anatomic structures in each section were identified with reference to
the atlas of Meesen and Olszewski.36
In each animal, the mean number per section of labeled cells of each type (Fos-positive, TH-positive, Fos/TH-positive) was calculated for each region (eg, NTS, caudal VLM, etc). This was done by counting bilaterally, for each region in each experiment, the number of labeled cells of each type in sections approximately 0.4 mm apart over the rostrocaudal extent of that region. Depending on the region examined, the number of sections counted per region varied between three and six. For each experiment, the mean number of labeled cells of each type in each region was then calculated; these mean values were then pooled with corresponding values from other experiments and used to calculate the overall mean value for the entire group of experiments of the same type. Except in the case of the area postrema, which is a midline structure, these numbers were then divided by 2 so that the final values presented in "Results" represent the means per section for one side.
Statistical Analysis
Data are given as the mean±SEM.
The
cardiovascular data were analyzed by a
two-factor repeated measures ANOVA. Measurements of mean
arterial pressure and heart rate during the periods of Ang
II infusion were further analyzed to determine any trend with
time, using orthogonal partitioning.37 For the
histological data, a two-factor repeated measures
ANOVA was first applied to test whether there was a significant
difference in overall cell counts in all regions between the
experimental groups (intact versus barodenervated and control versus
Ang II infusion). Where a significant difference was found, the
unpaired t test was then used to compare the number of
Fos-positive cells within each region between the experimental
groups. Statistical significance was set at a value of
P<.05.
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Results |
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Effects of Fourth Ventricular Infusion of Ang II on Mean Arterial Pressure and Heart Rate
Before Ang II infusion, the resting values for mean arterial pressure and heart rate were 78±2 mm Hg and 161±9 bpm in barointact rabbits and 92±8 mm Hg and 249±15 bpm in barodenervated rabbits, respectively. Fourth ventricular infusion of Ang II resulted in an average increase in mean arterial pressure above the preinfusion level of 9±2 mm Hg in barointact rabbits and 22±6 mm Hg in barodenervated rabbits. In barointact animals, the initial increase in mean arterial pressure at the onset of Ang II infusion was followed by a gradual but significant (P<.05) decline back toward the preinfusion level (Fig 1
). By contrast, the mean
arterial pressure remained elevated throughout the infusion
period in barodenervated animals (Fig 1). The larger response
in
barodenervated rabbits confirms previous observations that
barodenervation increases the pressor response to centrally
administered Ang II.32 Ang II infusion also resulted in an
increase in heart rate that was of similar magnitude (approximately 15
to 25 bpm) in barointact and barodenervated rabbits (Fig 1). In
barointact rabbits infused only with the vehicle Ringer's solution,
there was a slight but significant (P<.05) fall in both
mean arterial pressure and heart rate during the infusion
period. No significant change in mean arterial pressure and
heart rate was observed in barodenervated rabbits infused only with the
vehicle Ringer's solution.
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) or Ringer's
solution (
) in barointact and barodenervated rabbits. Error bars
indicate average SEM.
Distribution of Fos-Positive Cells
In both barointact and
barodenervated rabbits infused with
Ringer's solution alone, there was very little Fos expression in all
regions examined (Table 1). By contrast, fourth
ventricular infusion of Ang II resulted in a significant
increase, compared with infusion of Ringer's solution, in the number
of Fos-positive neurons in the NTS and caudal, intermediate, and
rostral parts of the VLM in both barointact and barodenervated rabbits
(Figs 2 and 3, Table 1). There
was also a
small increase in the number of Fos-positive cells in the area
postrema in both the barointact and barodenervated groups, although in
the latter group this was not statistically significant. Although there
tended to be more Fos-positive neurons in the barointact group
compared with the barodenervated group, particularly in the NTS and
intermediate VLM (Table 1), this difference in overall cell
counts
failed to achieve statistical significance (ANOVA, P=.079),
and the pattern of labeling was very similar in the two groups.
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In the
NTS, Fos-positive cells were distributed throughout its
entire rostrocaudal extent, with the majority located within the medial
and commissural portions of the nucleus (Figs 2
and
3). In the caudal
VLM (defined as the region between the obex and the level 2.0 mm more
caudal), Fos-positive neurons were concentrated in a circumscribed
area between the nucleus ambiguus and the lateral reticular nucleus
(Figs 2A, 2B, 3A, and
3B). More rostrally, in the intermediate VLM
(defined as the region extending from the level of the obex to the
level 1.8 mm more rostral), the Fos-positive cell group shifted
closer to the ventral medullary surface after the disappearance of the
lateral reticular nucleus (Figs 2C and 3C). In
the rostral VLM (defined
as the region between the level 1.8 mm rostral to the obex and the
level of the caudal pole of the facial nucleus), a significant number
of Fos-positive cells were seen in the region ventrolateral to the
retrofacial nucleus (Figs 2D and 3D). There were
also some
Fos-positive cells located in a band extending from the NTS to the
nucleus ambiguus or retrofacial nucleus (eg, Figs 2C and
3D). Very few
Fos-positive neurons were found within the nucleus ambiguus or
retrofacial nucleus.
In the pons, an increased number of Fos-positive cells was observed in the A5 area and locus ceruleus after fourth ventricular administration of Ang II compared with the control group of animals. Scattered Fos-positive cells were also identified in the parabrachial region, but the degree of Fos expression in this region varied among animals. There were also Fos-positive cells in the midbrain periaqueductal gray matter and in the hypothalamus, but in these regions the degree of Fos expression was similar to that observed in the control group of animals.
Double-Labeling for Fos and TH Immunoreactivity
In both
barointact and barodenervated rabbits, approximately one
third of the Fos-positive cells in the NTS were also immunoreactive
for TH (Table 2) and were therefore
catecholamine-synthesizing neurons of the A2/C2 group.
These cells had a more restricted distribution than the entire group of
Fos-positive cells in the NTS because they were principally
confined to the caudal part of the NTS, in the region just dorsal and
lateral to the dorsal motor nucleus of the vagus (Fig 4). At
all levels of the VLM, the majority of
Fos-positive cells in both barointact and barodenervated animals
were also TH-positive (Table 2). The double-labeled cells in
the
VLM had a distribution similar to that of the entire group of
Fos-positive cells within this region except that very few were
found dorsal to the nucleus ambiguus and retrofacial nucleus (Figs
2, 3, and 5).
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In all these
regions, the proportions of Fos-positive cells that
were also TH-positive were similar in both barointact and
barodenervated animals except that a higher proportion of
double-labeled cells was found in the caudal and intermediate VLM
in the barodenervated group compared with the barointact group (Table
2). Overall, however, these differences in proportions just
failed to
achieve statistical significance (ANOVA, P=.054).
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This study shows that fourth ventricular administration of Ang II results in a highly distinctive pattern of Fos expression in the medulla of the conscious rabbit. Although several previous studies in rats have reported that intracerebroventricular administration of Ang II results in Fos expression in the forebrain,27 28 29 this is the first study in which Fos expression in the medulla has been studied after injection of Ang II into the fourth ventricle in close proximity to the medulla. Since Fos expression is believed to be a marker of neuronal activation,23 25 26 these results therefore indicate for the first time the distribution of the population of neurons within the medulla that are activated by Ang II. In particular, they show that such activated neurons are located principally within the NTS and the caudal, intermediate, and rostral VLM and include both catecholamine and noncatecholamine neurons within all these regions.
Methodological Considerations
An important advantage of the
experimental approach used in this
study is that the Ang II was administered in normal conscious animals,
thus avoiding the confounding effects of anesthesia on
central neurons. A possible disadvantage of using a conscious
preparation, however, is that arousal or stress associated with
environmental factors may induce Fos expression.23 38
In
the present study, we took great care to minimize arousal and
stress associated with handling, surgical preparation, and drug
infusion. The baseline level of Fos expression in control animals
infused with the vehicle solution alone was very low, indicating that
the Fos expression in the experimental group of animals was a
consequence of administration of Ang II rather than nonspecific factors
associated with the experimental procedure.
Since Ang II infusion resulted in an increase in pressure, the question arises as to whether the Fos expression may have been a secondary effect resulting from stimulation of peripheral baroreceptors. Our results showed, however, that the pattern of Fos expression that followed Ang II administration was very similar in both barointact and barodenervated animals. Furthermore, the increase in mean arterial pressure (average, 9 mm Hg) produced by Ang II administration in barointact animals was rather modest. In a previous study in our laboratory, we found that an increase in arterial pressure of this magnitude produced by intravenous infusion of phenylephrine in conscious rabbits did not result in significant Fos expression.24 With regard to barodenervated rabbits in which the increase in mean arterial pressure was greater (average, 22 mm Hg), we have recently found that in such animals a period of phenylephrine-induced hypertension of the same magnitude (20 to 25 mm Hg) and duration (60 minutes) as in the present experiments resulted in a very low level of Fos expression in the NTS and the caudal, intermediate, and rostral VLM, which was not significantly different from the baseline level of Fos expression observed in control animals (P.D. Potts and R.A.L. Dampney, unpublished observations, 1994). Thus, we conclude that the Fos expression observed after fourth ventricular administration of Ang II in both barointact and barodenervated rabbits is due mainly to the effects of Ang II on medullary neurons rather than being a secondary effect arising from the increase in arterial pressure.
A further general consideration that needs to be taken into account when interpreting the results is that the Fos-positive neurons could include both neurons that are directly activated by Ang II and those that are indirectly activated as a consequence of stimulation of Ang II–sensitive neurons located elsewhere. As discussed in detail below, the distribution of Fos-positive neurons in the NTS and the caudal, intermediate, and rostral VLM corresponded very closely to the distribution of Ang II receptor binding sites in the same regions, as determined by in vitro autoradiography in the same species.2 4 The simplest explanation for this correlation is that Fos expression occurred mainly as a consequence of direct activation of neurons mediated via specific Ang II receptors. At the same time, we cannot exclude the possibility that at least some Fos-positive neurons in the present study were not directly sensitive to Ang II but instead were part of multisynaptic pathways activated by antecedent Ang II–sensitive neurons.
Distribution of Fos-Positive Neurons in Medullary
Regions
NTS and Area Postrema
The NTS in the rabbit is
known to contain a high density of Ang II
receptor binding sites,4 which are predominantly of the
AT1 subtype,2 as in humans.39
There is evidence that these binding sites are located on afferent
fibers as well as on cell bodies in the NTS,40 41
indicating that Ang II may act both presynaptically and
postsynaptically. The present results show that, regardless of the
precise site of its action, Ang II activates many neurons
within the NTS in the conscious rabbit. Furthermore, the distribution
of the Ang II–activated neurons in the NTS is very similar to
that of Ang II binding sites.4 In particular, Mendelsohn
et al4 noted that in the rabbit there is a particularly
high concentration of Ang II binding sites just dorsal to the dorsal
motor nucleus of the vagus. Our results showed that this region also
contained a high concentration of Fos-positive cells. Furthermore,
many of these activated cells were immunoreactive for TH and
thus are part of the A2/C2 group of catecholamine
neurons.
In the anesthetized rat, microinjection of Ang II in the NTS can inhibit the baroreceptor reflex.10 42 It is therefore possible that some of the neurons within the NTS activated by Ang II are intrinsic interneurons that modulate the transmission of baroreceptor signals through the NTS. On the other hand, Dorward and Rudd43 found that in conscious rabbits the baroreflex control of heart rate was not significantly altered by continuous infusion of Ang II into the fourth ventricle at a rate very similar to that used in the present study. It therefore follows that under the experimental conditions of the present study Ang II is unlikely to have significantly modulated the baroreceptor reflex by an action within the NTS. It is possible, however, that Ang II may have had actions on NTS neurons that are independent of the baroreceptor reflex, as has been described previously.12 Consistent with this, our results show that Ang II produced a significant degree of Fos expression in the NTS in barodenervated as well as in barointact rabbits. Thus, the large number of neurons that are activated by Ang II may reflect the fact that the peptide has multiple actions in regulating cardiovascular neurons within the NTS (for review, see Reference 44).
Overall, the number of Fos-positive cells in barodenervated rabbits was less than in barointact animals, although this difference did not quite achieve statistical significance (P=.079). This difference was most marked in the NTS, where the number of Fos-positive cells was approximately half that observed in barointact rabbits. The reduced number of Fos-positive cells in the NTS of barodenervated rabbits could reflect a loss of Ang II receptors after section of the carotid sinus and aortic nerves, in the same way that vagotomy has been shown to reduce Ang II receptor binding in the NTS of rats.40 In particular, axotomy is believed to cause a loss of presynaptic receptors.40
Unlike the NTS, the area postrema has a relatively low density of Ang II receptors in rabbits4 ; this was matched by the lower density of Fos-positive neurons found in this area, although it was higher than in control experiments. These observations therefore contrast with the view that the area postrema plays an important role in mediating the cardiovascular effects produced by Ang II, which is based largely on studies in dogs and rats.45 46 It should be noted, however, that there are major species differences in the density of Ang II binding sites in the area postrema. For example, in contrast to rabbits, there is a high density of binding sites in the area postrema in dogs and rats.5 47 In this respect, the rabbit is more similar to humans, in whom Ang II receptor binding is absent in the area postrema.3
VLM
The distribution of
Fos-positive cells in the VLM was also
remarkably similar to the distribution of Ang II receptor binding sites
in this region in the rabbit,4 which are also
predominantly of the AT1 subtype,2 as in
humans.39 It has been shown previously that Ang II
receptor binding sites in the VLM of the rabbit are closely associated
with neuronal cell bodies within this region.4 In
particular, the distribution of Ang II binding sites corresponds
closely to the location of catecholamine neurons in the
rostral, intermediate, and caudal parts of the VLM, although the
functional relationship between the binding sites and the
catecholamine neurons has not previously been determined.
Our results demonstrate for the first time that the majority of VLM
neurons activated by Ang II are catecholamine
neurons and include both C1 neurons in the rostral VLM and A1 neurons
in the intermediate and caudal VLM.48
The rostral VLM
contains a group of
sympathoexcitatory spinally projecting
neurons that play a critical role in the tonic and reflex regulation of
blood pressure (for reviews, see References 8 and 9). Some of these
neurons are part of the C1 group of catecholamine neurons,
whereas others are noncatecholamine
cells.8 9 Microinjection of Ang II into the rostral
VLM
causes a rise in sympathetic activity and arterial
pressure,6 14 15 and an
electrophysiological study has identified
neurons within the rostral VLM that are excited by iontophoretic
application of Ang II.17 Furthermore, Ang II in the
rostral VLM appears to be selective for vasomotor
sympathoexcitatory neurons, since it has no
effect on respiratory activity.49 There is some evidence,
however, that Ang II does not excite all
sympathoexcitatory neurons within the rostral
VLM. In particular, previous studies using the medullary slice
preparation have shown that direct application of Ang II to rostral VLM
neurons in vitro has no effect on putative
sympathoexcitatory neurons that have a
pacemakerlike discharge.20 21 Since an earlier
study50 reported that rostral VLM neurons with a
pacemakerlike discharge are not immunoreactive for phenylethanolamine
N-methyl transferase (a marker of C1
catecholamine neurons), it has been suggested that Ang II
does not affect non-C1 cells in the rostral VLM.21 As
mentioned above, this hypothesis is also supported by the finding that
nearly all Ang II–sensitive neurons, but none of the Ang
II–insensitive neurons, are inhibited by agonists of
2-adrenergic receptors,20 which have been
shown to be associated with catecholamine but not
noncatecholamine neurons in the rostral
VLM.22
In the present study, however, we found that approximately 40% of neurons in the rostral VLM that expressed Fos after fourth ventricular infusion of Ang II were not catecholamine cells. It is possible that these noncatecholamine Fos-positive cells were not activated directly by Ang II but instead were excited by inputs from Ang II–sensitive neurons in the caudal or intermediate VLM or in the NTS. Microinjection of Ang II into the caudal or intermediate VLM in rabbits, however, causes a depressor and sympathoinhibitory response15 51 that is probably mediated by an inhibitory input to rostral VLM sympathoexcitatory neurons.52 In the NTS, Ang II produces either pressor or depressor responses, depending on the dose injected. With low doses of 2.5 pmol or less, however, no response or a depressor response is elicited in rats11 53 and rabbits (G.A. Head and N.S. Williams, unpublished data, 1990), indicating that low doses of Ang II in the NTS do not result in excitation of rostral VLM sympathoexcitatory neurons, at least in anesthetized animals. In the present study, Ang II was infused at a low rate (4 to 8 pmol/min in 2 to 4 µL/min) into the fourth ventricle. Taking into account the fact that Ang II would have been considerably diluted in the cerebrospinal fluid, the amount of the peptide diffusing to NTS cells would be equivalent to a low dose of Ang II directly injected into the NTS. Thus, although it is possible that in conscious rabbits application of even low doses of Ang II in the NTS could lead to activation of rostral VLM neurons, the available evidence from previous studies in anesthetized animals does not support this hypothesis.
An alternative explanation for the apparent discrepancy between our results and those of previous electrophysiological studies in vitro is that Ang II could exert its effects on rostral VLM sympathoexcitatory neurons by a presynaptic action, which may not be observed in vitro. The precise site of Ang II receptors within the rostral VLM is not known, although they do appear to be closely associated with cell bodies, as mentioned above.4 In any case, our results indicate that at least in conscious rabbits Ang II acts on both catecholamine and noncatecholamine neurons in the rostral VLM.
Microinjection of Ang II directly into the caudal or intermediate VLM of rabbits elicits, as mentioned above, a depressor and sympathoinhibitory response15 51 and also an increase in the levels of circulating vasopressin.51 The depressor and sympathoinhibitory effect is believed to be mediated by noncatecholamine cells that project to the rostral VLM, whereas the vasopressin-releasing effect is mediated predominantly by catecholamine (A1) neurons that project directly to the supraoptic nucleus in the hypothalamus (for review, see Reference 7). Thus, our finding that both catecholamine and noncatecholamine neurons in the caudal and intermediate parts of the VLM expressed Fos after fourth ventricular infusion of Ang II into the medulla is entirely consistent with these previous observations.
Other Medullary Regions
In rabbits, Ang II receptor binding sites have been found
previously in the region between the VLM and NTS,2 4
which
corresponds to the intermediate reticular nucleus as defined in
rats.54 Ang II receptor binding sites, which are
predominantly of the AT1 subtype, are also located in this
region in humans.3 39 It has been suggested
previously4 that these binding sites may represent
Ang II receptors being transported in axons that connect the two
regions.55 56 The present results show, however, that
Ang II activates neurons within this region, indicating that at
least some binding sites in this intermediate region are associated
with neuronal cell bodies.
Functional Significance of Fos Expression by Ang
II–Sensitive Neurons
In the central nervous system, Fos and
other products of
immediate early genes are believed to act as transcription factors that
play a role in neuronal plasticity.25 It is therefore
possible that endogenous Ang II could induce the expression
of transcription factors that in turn have long-term effects on
medullary cardiovascular regulatory mechanisms in
addition to its short-term effects on the activity of medullary
cardiovascular
neurons.6 10 11 12 13 14 15 16 17 18 19 20
Further
studies will be required to determine the precise nature and mechanisms
of such long-term effects.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
|---|
This study was supported by the National Heart Foundation of Australia, the National Health and Medical Research Council of Australia, and the Ramaciotti Foundations. We are very grateful to Jaimie Polson for helpful discussions.
Received May 19, 1995; first decision August 7, 1995; accepted October 3, 1995.
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References |
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