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Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908-0735
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The rostral ventrolateral medulla
(RVLM) may play an important role in the sympatholytic and hypotensive
effects of clonidine. The present study examined which type of
presympathetic RVLM neuron is inhibited by clonidine, and whether the
adrenergic presympathetic RVLM neurons are essential for
clonidine-induced sympathoinhibition. In chloralose-anesthetized and
ventilated rats, clonidine (10 µg/kg iv) decreased arterial pressure
(116 ± 6 to 84 ± 2 mmHg) and splanchnic nerve activity
(93 ± 3% from baseline). Extracellular recording and
juxtacellular labeling of barosensitive bulbospinal RVLM neurons
revealed that most cells were inhibited by clonidine (26/28) regardless
of phenotype [tyrosine hydroxylase (TH)-immunoreactive cells: 48 ± 7%; non-TH-immunoreactive cells: 42 ± 5%], although the
inhibition of most neurons was modest compared with the observed sympathoinhibition. Depletion of most bulbospinal catecholaminergic neurons, including 76 ± 5% of the rostral C1 cells, by
microinjection of saporin anti-dopamine 
-hydroxylase into the
thoracic spinal cord (levels T2 and T4, 42 ng · 200 nl
1 · side1) did not alter the
sympatholytic or hypotensive effects of clonidine. These data show that
although clonidine inhibits presympathetic C1 neurons, bulbospinal
catecholaminergic neurons do not appear to be essential for the
sympatholytic and hypotensive effects of systemically administered
clonidine. Instead, the sympatholytic effect of clonidine is likely the
result of a combination of effects on multiple cell types both within
and outside the RVLM.
rostral ventrolateral medulla; anti-dopamine -hydroxylase
saporin; splanchnic nerve activity; tyrosine hydroxylase; phenylethanolamine-N-methyl transferase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CLONIDINE, AN
2-adrenergic receptor agonist,
produces a long-lasting decrease in arterial pressure (AP) by a
centrally mediated inhibition of sympathetic vasomotor tone. Although
multiple sites within the central nervous system are sensitive to
clonidine (10, 27), the rostral ventrolateral medulla
(RVLM) appears to be an important target for clonidine-induced
hypotension (7, 24-26). The decrease in AP produced
by intravenously administered clonidine is mimicked by microinjection
directly into the RVLM and is reversed by microinjection of the
2-adrenergic receptor antagonist idazoxan into the same
area (24).
Some barosensitive bulbospinal (presympathetic) neurons in the RVLM are inhibited by clonidine administered intravenously and by local microiontophoresis (3, 9, 33). Moreover, the inhibition of RVLM neurons by intravenous clonidine can be attenuated by microiontophoresis of idazoxan into the vicinity of the recorded RVLM neuron (3). The presympathetic RVLM neurons most clearly inhibited by clonidine have relatively low rates of discharge and slow axonal conduction velocities (3, 9, 32, 33), suggesting that they include C1 cells (28). However, the RVLM also contains non-C1 cells that are likely to be important for the generation of sympathetic vasomotor tone (15, 28), and whether these neurons are also inhibited by clonidine is not known.
Several lines of evidence suggest that clonidine may not
preferentially target C1 cells in the RVLM. Although bulbospinal C1
cells have postsynaptic 2-adrenergic receptors
(8), which likely contribute to the clonidine-induced
inhibition of these cells, clonidine also affects neuronal activity via
presynaptic 2-adrenergic receptors (10,
35). In fact, in the RVLM, the majority of
2-adrenergic receptors are located in axons and axon terminals (18). Some of these terminals have
synaptic contacts with C1 neurons, but contacts are also made with
noncatecholaminergic neurons within the RVLM. Furthermore, in neonate
rat brain stem slices, stimulation of 2-adrenergic
receptors inhibits bulbospinal RVLM neurons primarily by a presynaptic
inhibition of glutamatergic inputs, which is equally effective in
catecholaminergic and noncatecholaminergic RVLM neurons
(11). However, whether these noncatecholaminergic RVLM
neurons recorded in vitro are related to cardiovascular function could
not be ascertained.
One goal of the present study was to seek definitive evidence that
sympatholytic doses of clonidine inhibit presympathetic C1 neurons. In
addition, we sought to determine whether noncatecholaminergic presympathetic RVLM neurons are also inhibited by clonidine. Finally, we tested whether C1 cells are important for the sympatholytic and
hypotensive effects of clonidine by determining the consequences of
destroying the C1 presympathetic neurons with the use of the immunotoxin anti-dopamine -hydroxylase-saporin (anti-DH-Sap).
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Male Sprague-Dawley rats, weighing 250-350 g (Hilltop Laboratories, Scotsdale, PA), were housed in groups (4 rats/cage) with a 12:12-h light-dark cycle wherein food and water were available ad libitum. All procedures were performed in accordance with National Institutes of Health and University of Virginia Animal Care and Use Guidelines.
Surgical procedures and experimental protocol. Anesthesia was induced with 5% halothane (in 100% O2). Rats were intubated and artificially ventilated with 1.5-1.9% halothane in 100% O2 during surgical procedures. A brachial artery was cannulated to measure AP and heart rate (HR), and a brachial vein was cannulated to administer anesthetic and paralytic agents. A femoral vein was cannulated for administration of clonidine. An inflatable snare was placed around the abdominal aorta just below the diaphragm to permit rapid control of upper body AP (28). After placing the rat in a stereotaxic apparatus, the left splanchnic nerve was isolated as previously described (29) and placed on two Teflon-coated silver wires (250-µm tip bared; A-M Systems, Everett, WA). The wires were embedded in a dental impression material (polyvinylsiloxane; Darby Dental Supply, Westbury, NY), and the wound was closed around the exiting wires. A laminectomy was performed at spinal segment T2 for implantation of a bipolar stimulation electrode into the dorsolateral funiculus to antidromically activate RVLM neurons (28, 36). The mandibular branch of the facial nerve on each side was exposed for implantation of a bipolar stimulation electrode to elicit field potentials in the facial motor nucleus. The skull was exposed, and interparietal plate was removed for insertion of a recording electrode into the RVLM.
The halothane was replaced by-chloralose (31 mg/ml solution in 3%
sodium borate; 70 mg/kg initial bolus and hourly supplements of 20 mg/kg; Fisher Scientific, Pittsburgh, PA) on completion of surgery.
Rats were allowed to stabilize for at least 45 min before baseline
recordings were obtained. Ten minutes before recordings began, rats
were paralyzed with pancuronium bromide (1 mg/kg iv; Elkins-Sinn,
Cherry Hill, NJ) to allow for antidromic activation of RVLM neurons.
Presympathetic RVLM neurons were recorded extracellularly with the use
of glass electrodes filled with 1.5% biotinamide in 0.5 M NaCl as
previously described (28, 36). Units were selected on the basis of the following criteria: 1) location
(rostrocaudally within 500 µm of the caudal pole of the facial
nucleus, ventrally within 300 µm of the bottom of the facial nucleus,
and 1.7-1.9 mm lateral to the midline), 2) spontaneous
activity, 3) time-locked inhibition by increased AP, and
4) antidromic activation from the thoracic spinal cord.
After characterization of a stable presympathetic RVLM unit, the
effects of clonidine were examined.
Baseline parameters were measured for 45 s to 1 min (Fig. 1), and
then clonidine was administered in three doses (1, 1.5, and 7.5 µg/kg
iv) given at 5-min intervals. Measurements were taken during the minute
preceding each dose and at ~10 min after the last dose (Fig. 1). In a
subset of animals, clonidine was administered in a single 10-µg/kg
dose.
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Juxtacellular labeling and phenotypic identification of RVLM units. The recorded RVLM neurons were individually filled with biotinamide as previously described (23, 28, 36). Pulses of positive current (200 ms, 50% duty cycle) were delivered through the recording electrode while the unit activity was monitored with the amplifier in bridge mode. The activity of the cell was entrained to the positive pulses of current (0.5-5.0 nA; 1-5 min). This procedure ejects biotinamide from the recording electrode into the vicinity of the recorded neuron and reliably produces the label of a single neuron that is very likely to be the recorded cell (23, 28).
The animal was maintained in the anesthetized state for at least 30 min to allow for dispersion of biotinamide into the recorded neuron. Then the animal was deeply anesthetized (5% halothane) and perfused transcardially with phosphate-buffered saline (250 ml, pH 7.4) and 500 ml of 4% phosphate-buffered formaldehyde (Fisher Scientific). The brain stem was removed and stored in fixative overnight. Brain stem sections were cut (30 µm) with the use of a Vibratome and stored in a cryoprotectant solution at
20°C.
Biotinamide-labeled neurons were revealed by incubating the sections in
streptavidin-Cy3 conjugate (1:1,000; 3 h; Jackson Immunoresearch
Laboratories, West Grove, PA). Sections were mounted onto uncoated
slides with coverslips applied with the use of a glycerol-based
mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and
examined with the use of a fluorescence microscope. As previously
described (28), the biotinamide-labeled cell was located,
photographed (standard 35-mm camera and 1600 ASA color slide film), and
its structure along with the outline of the section were drawn with the
use of a Neurolucida software (Microbrightfield, Colchester, VT) and a
Ludl motor driven stage. The sections containing the labeled cell
bodies were removed from the slides and processed to reveal tyrosine
hydroxylase (TH) immunoreactivity with the use of a sensitive
peroxidase-antiperoxidase method (28). Sections were
incubated with a mouse monoclonal TH antibody (1:2,000; 24 h at
4°C; Chemicon, Temecula, CA) in 10% blocking serum and 0.1% Triton
X-100, followed by a goat-anti-mouse IgG (1:150; 45 min; Sternberger
Monoclonal, Baltimore, MD) and then mouse ClonoPAP (1:150; 30 min;
Sternberger). Immunoreactivity for TH was revealed by incubation for
5-10 min in a 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0.005% hydrogen peroxide solution. Sections were
mounted onto gelatin-coated slides, cleared in graded alcohols and
xylenes, and coverslipped with DPX (Aldrich, Milwaukee, WI). As
previously described (28), to determine whether the
recorded neuron was catecholaminergic, the Neurolucida drawing of the
biotinamide-labeled cell was superimposed onto the binocular of the
microscope with the use of the Lucivid camera system
(Microbrightfield). The TH-immunoreactive (TH-ir) neurons were
photographed with the use of a 35-mm camera and TMAX-100 black and
white film.
Microinjections of saporin conjugates into the thoracic spinal
cord.
Anesthesia was induced with 5% halothane, and during surgery rats were
maintained with 1.9% halothane inhaled through a nose cone. The rat
was placed in a stereotaxic apparatus, and a dorsal laminectomy was
performed to expose the upper thoracic spinal cord. As previously
described (29), bulbospinal catecholaminergic neurons were
depleted with the use of the ribosomal toxin saporin conjugated to an
antibody for dopamine -hydroxylase (anti-DH-Sap; Chemicon). Rats
received bilateral microinjections of anti-DH-Sap (42 ng · 200 nl1 · injection1)
into the region of the intermediolateral cell column of two levels of
the spinal cord (at T2 and T4). Another group of rats received
bilateral microinjections of saporin conjugated to a mouse IgG
(IgG-Sap; 40 ng · 200 nl1 · injection1; Chemicon) into
the same levels of the thoracic spinal cord. Because IgG-Sap does not
alter the number of catecholaminergic neurons, these rats served as an
operated control group. Rats were allowed to recover 3-5 wk and
then were prepared as described above for measuring the effects of
clonidine on AP, HR, and splanchnic nerve activity (SNA). No RVLM units
were recorded in these animals.
Verification of lesions by anti-DH-Sap.
To determine the extent of depletion of bulbospinal catecholaminergic
neurons in rats treated with anti-DH-Sap, brain stem sections from
rats treated with anti-DH-Sap, rats treated with IgG-Sap, and
untreated control rats were processed to reveal immunoreactivity for
phenylethanolamine-N-methyl transferase (PNMT) and TH. All immunohistochemical procedures were performed with the use of Tris-buffered saline (0.1 M Tris, pH 7.4) at room temperature unless
otherwise noted. To determine the loss of C1 neurons in the RVLM, for
each animal a one in six series of sections were incubated with a
rabbit polyclonal antibody for PNMT (1:2,000 with 10% serum and 0.1%
Triton X-100, overnight at 4°C; DiaSorin, Stillwater, MN) followed by
a biotinylated goat anti-rabbit IgG (1:200; 45 min; Vector
Laboratories). The PNMT-immunoreactive (PNMT-ir) neurons were revealed
by incubation with streptavidin-Cy3 conjugate (1:1,000; 1 h;
Jackson Immunoresearch Laboratories). To determine the loss of A5 cells
in the ventrolateral pons, a separate set of one in six sections were
incubated with a mouse monoclonal antibody for TH (1:2,000 with 10%
serum and 0.1% Triton X-100 overnight at 4°C; Chemicon) followed by
a biotinylated goat anti-mouse IgG (1:200; 45 min; Vector
Laboratories). The TH-ir cells were revealed by incubation with
streptavidin-Cy3 conjugate (1:1,000; 1 h; Jackson Immunoresearch
Laboratories). All sections were mounted onto slides, and coverslips
were applied with Krystalon mounting medium (EM Diagnostic Systems,
Gibbstown, NJ).
11.8 and 11.6 (2 sections/animal). This method reliably estimates the depletion of
bulbospinal neurons, but it may underestimate the depletion of spinally
projecting neurons on average by 12% due to interspersed C1 cells that
project to the hypothalamus and not to the spinal cord (29,
31). To demonstrate consistency of PNMT immunoreactivity among
the groups of rats, PNMT-ir cells were plotted from sections corresponding to A-P levels 13.2 and 13.0. These caudal C1 cells do
not project to the spinal cord (29, 31), and they would not be expected to be affected by intraspinal injections of
anti-DH-Sap (29). To determine the depletion of A5
noradrenergic neurons, TH-ir cells were plotted in the ventral half of
sections ranging from A-P levels 9.6, 9.4, 9.2, and 9.0 (4 sections/animal).
Data analyses and statistics. All physiological measures were monitored on a chart recorder and stored with the use of a video cassette recorder with a digitizer interface (Vetter 3000A, frequency range: DC-22 kHz, Vetter Digital, Rebersberg, VA). Mean AP (MAP), HR, integrated SNA, and integrated rate histograms of RVLM single-unit activity were analyzed with the use of a Metrabyte Dash-16 A/D interface and custom-designed software.
The effects of clonidine on MAP and HR were analyzed by one-way ANOVA with repeated measures. The effects of clonidine on SNA and RVLM unit activity were analyzed by Kruskal-Wallis ANOVA on ranks. The effects of treatment with anti-DH-Sap or IgG-Sap on the number of C1 cells and
A5 cells and responses to clonidine were analyzed by two-way ANOVA.
Student-Newman-Keuls post hoc tests were performed when ANOVA showed a
significant effect. Significance was set at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of intravenous clonidine on mean SNA, MAP, and HR.
Intravenously administered clonidine produced the expected dose-related
biphasic effect on MAP (Fig. 1,
top; 3, 24). At first, clonidine transiently increased AP by
activation of peripheral 2-adrenergic receptors. The
increases in AP were accompanied by brief dose-related inhibitions of
SNA (Fig. 1, middle), attributable to the stimulation of
arterial baroreceptors.
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Which presympathetic RVLM units are sensitive to intravenous clonidine? A presympathetic RVLM unit was recorded in each of the seven animals described above, and an additional 21 presympathetic RVLM units were recorded in 19 rats where SNA was not recorded (in 2 rats, clonidine was given twice separated by 2 h). Although SNA was not recorded in these animals, the responses of the RVLM units and the hypotension produced by clonidine were comparable to those seen in rats with recordings of SNA. With eight of the recorded RVLM units, clonidine was given as a single 10-µg/kg dose. Because this method produced equivalent decreases in AP, HR, and RVLM unit activity compared with the responses seen in rats that received the 10-µg/kg dose in three injections, the data were pooled.
Presympathetic RVLM units have a large range of conduction velocities and heterogeneous phenotypes (15, 21, 28, 31). To determine whether clonidine preferentially affected a subset of these neurons, the data were analyzed with consideration of conduction velocity and catecholaminergic phenotype. The RVLM units were divided into three conduction-velocity ranges on the basis of our previous findings (28): <1 m/s (unmyelinated C1 cells), 1-3 m/s (lightly myelinated mostly C1 cells), and >3 m/s (mostly non-C1 cells). As shown previously (28), the basal firing rates of the presympathetic neurons were related to their conduction velocities, with the slower conducting neurons having a lower discharge rate (Fig. 2E). Clonidine inhibited most of the recorded presympathetic RVLM neurons (26 of 28). However, two presympathetic RVLM cells showed no change in activity, although the clonidine-induced decreases in SNA were substantial in these cases. On average, presympathetic RVLM neurons of all conduction velocities were comparably inhibited by clonidine (Fig. 2E). However, the degree of inhibition of individual RVLM units was highly variable within each category: 0-83% inhibition in cells with a conduction velocity <1 m/s (n = 13), 0-100% inhibition in cells with a conduction velocity in the 1- to 3-m/s range (n = 10), and 18-78% inhibition in cells with a conduction velocity >3 m/s (n = 5). In addition, the percent inhibition of RVLM units in each category was substantially less than the percent inhibition observed in the SNA (Fig. 2E). Most of the recorded RVLM units (19/28) were filled with biotinamide to determine whether they contained TH immunoreactivity, a reliable marker for C1 neurons in this region of the RVLM (31). An example of one recorded RVLM neuron that was filled with biotinamide and found to be TH-ir is shown in Fig. 3. Clonidine inhibited both TH-ir and non-TH-ir presympathetic RVLM neurons equally (Figs. 4 and 5). However, the sensitivity of the cells was variable: 14-83% inhibition in TH-ir cells with conduction velocity <1 m/s (n = 7), 23-98% inhibition in TH-ir cells with a conduction velocity >1 m/s (n = 8), and 30-51% inhibition in non-TH-ir cells (n = 4).
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Does depletion of bulbospinal catecholaminergic neurons alter
responses to clonidine?
As expected (29), microinjection of anti-DH-Sap into
the upper thoracic spinal cord produced massive depletions of the rostral Cl cells (example in Fig. 6). In
these treated rats, the number of PNMT-ir cells in the rostral C1 cell
group was reduced by 76 ± 4.8% (range 59-95%) compared
with control rats (Table 1). In contrast,
the number of PNMT-ir cells in the caudal C1 cell group was unaffected
by treatment with anti-DH-Sap (Table 1), indicating the
immunohistochemical detection of C1 cells was comparable between
groups. Treatment with anti-DH-Sap also reduced the number of TH-ir
A5 neurons in the ventrolateral pons at every level examined (88 ± 3%; range 74-95%). In contrast, treatment with IgG-Sap did
not alter the number of A5 neurons (data not shown) or rostral C1
neurons (Table 1).
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H-Sap (n = 8), and in
rats treated with IgG-Sap (n = 6) with the use of the
same three-dose protocol (1, 1.5, and 7.5 µg/kg separated by 5 min).
In rats treated with anti-DH-Sap, clonidine produced transient
increases in AP and baroreceptor-mediated decreases in SNA and HR (Fig.
7, middle and
bottom). These responses were followed by sustained
decreases in MAP, HR, and SNA (Figs. 7 and
8) that were not different from control
rats or rats treated with IgG-Sap (Fig. 8).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study demonstrates that bulbospinal barosensitive C1
neurons are inhibited by sympatholytic doses of systemically administered clonidine. In addition, barosensitive bulbospinal RVLM
neurons with no detectable catecholaminergic phenotype are equally
inhibited by clonidine. Massive depletion of bulbospinal C1 cells by
anti-DH-Sap, which would spare the non-C1 presympathetic RVLM
neurons, did not alter the sympatholytic or hypotensive effects of
clonidine. These data suggest that although both cell types may
contribute to the sympatholytic effect of clonidine, the presympathetic C1 cells are not essential for this response. Finally, presympathetic RVLM neurons, regardless of conduction velocity or phenotype, were
inhibited to a lesser degree on average than SNA, suggesting that the
RVLM is only one of several important central target sites for clonidine.
Technical considerations.
To determine the necessity of presympathetic C1 neurons in the
physiological responses to clonidine, we depleted bulbospinal C1 cells
with the use of a newly described immunotoxin, saporin conjugated to an
antibody for dopamine -hydroxylase (16, 29, 37). We
have previously shown that microinjection of anti-DH-Sap into the
thoracic spinal cord effectively depletes the rostral C1 cells
(29), and coinjection of the retrograde tracer Fast Blue
into adjacent spinal levels in these animals indicates that the vast
majority of bulbospinal C1 cells is destroyed. Counts of PNMT-ir
neurons alone reliably estimate the percent depletion of bulbospinal C1
cells, although on average they underestimate by 12% due to counts of
interspersed PNMT-ir cells that do not project to the cord. Therefore,
the average depletion of 76 ± 5% of rostral PNMT-ir neurons in
the present study is likely to be a conservative estimate of the
depletion of bulbospinal PNMT-ir neurons.
H-Sap is selective for the depletion of catecholaminergic
neurons because the number of bulbospinal non-C1 cells in the RVLM and
serotonergic neurons in the adjacent raphe is not affected
(29). However, other bulbospinal catecholaminergic neurons
including the A5 group are virtually eliminated by intraspinal injection of anti-DH-Sap (present study, 29). Therefore, although anti-DH-Sap is effective and selective for the depletion of C1 neurons within the RVLM, the additional loss of the pontine
noradrenergic bulbospinal neurons must be considered when evaluating
effects of the lesion.
Effect of clonidine on presympathetic RVLM neurons. Barosensitive bulbospinal RVLM neurons were inhibited by clonidine within a range of doses that produced a decrease in SNA and AP. However, in contrast to our previous studies (3, 33), inhibition by clonidine was not restricted to barosensitive neurons with the slowest conduction velocities. A major difference between the present study and the previous ones is the choice of anesthesia. In the study by Sun and Guyenet (33), rats were anesthetized with halothane, a condition in which the discharge rate of presympathetic RVLM neurons and sympathetic tone are very high. Under these conditions, clonidine is not very efficacious for decreasing SNA (6) or RVLM unit activity (33). The slowly conducting neurons are relatively more inhibited by clonidine under halothane anesthesia, but the responses seen in these neurons are small (18% average inhibition). One speculation of this study was that the apparent preferential inhibition of the slowly conducting cells may be indicative of their catecholaminergic phenotype. Indeed, we have recently shown that the slowly conducting presympathetic RVLM neurons are C1 cells (28), however, the C1 cells are not limited to this range of conduction velocity. Therefore, although the clonidine-responsive cells in the study by Sun and Guyenet (33) were probably C1 cells, many of the unresponsive neurons were also likely to have been C1 cells. In the study by Allen and Guyenet (3), the rats were anesthetized with urethan, and the RVLM neurons displayed a wider range of inhibitory responses to clonidine (45-100% inhibition). However, the classification of cells that responded with 10-35% inhibition as nonresponding and unidentified phenotypes of the recorded RVLM neurons makes it difficult to assess whether inhibition of presympathetic neurons in the RVLM is restricted to the C1 cells. In the present study, we could conclusively demonstrate that both C1 and non-C1 presympathetic neurons were equally likely to be inhibited by clonidine. Therefore, inhibition of presympathetic neurons is not restricted to the C1 cells, and the relative inhibition of C1 and other presympathetic RVLM neurons depends on the state of the animal.
The inhibition of presympathetic RVLM neurons by clonidine presumably has more to do with the nature and strength of their synaptic inputs in a given condition than their phenotype. Indeed, within the RVLM, clonidine exerts its effects by activation of both pre- and postsynaptic receptors (11, 14, 35). Although presympathetic C1 neurons have postsynaptic2-adrenergic receptors (8), the
postsynaptic effects of agonists for these receptors appear to be small
and variable, and non-C1 cells have small postsynaptic responses to
agonists for 2-adrenergic receptors as well (11, 14). Presynaptic 2-adrenergic receptors appear to
play a more important role in the presympathetic RVLM neuron's
response to 2-adrenergic receptor agonists. Analysis by
electron microscopy shows that most 2-adrenergic
receptors in the RVLM are in axons and axonal terminals that contact
both C1 and non-C1 neurons (18). In agreement, patch-clamp
recordings in bulbospinal RVLM neurons of rat brain stem slices show
that stimulation of 2-adrenergic receptors produces
powerful presynaptic inhibition of both glutamatergic and GABAergic
inputs (11). Under conditions when most of the synaptic
input to RVLM bulbospinal neurons is excitatory,
2-adrenergic agonists would be expected to inhibit these
cells strongly. However, the overall effect of clonidine on the
activity of presympathetic RVLM neurons will be contingent on the
balance of excitatory and inhibitory inputs that these neurons receive
under a given condition.
Another factor to be considered in response of the presympathetic RVLM
neurons to systemically administered clonidine is the effects on
neurons in other brain regions that, in turn, affect the activity of
the presympathetic RVLM neurons. Clearly, 2-adrenergic receptors are located in many other areas of the brain that regulate autonomic outflow via the RVLM (34). Clonidine produces a
decrease in AP when microinjected into the nucleus of the solitary
tract (27). Conversely, clonidine microinjected into the
caudal ventrolateral medulla increases AP and renal sympathetic nerve
activity, apparently by inhibition of inhibitory inputs to the RVLM
(30). These effects antecedent to the RVLM will obviously
alter the inputs to the presympathetic neurons and, in turn,
modulate the response to clonidine within the RVLM. Therefore, the
responses of the presympathetic RVLM neurons to clonidine are the
result of a combination of actions on 2-adrenergic
receptors within and outside the RVLM.
Importance of bulbospinal C1 cells for the sympatholytic and
hypotensive effects of clonidine.
The inhibition of bulbospinal barosensitive C1 neurons by clonidine
suggests that these neurons contribute to the inhibition of SNA, but it
does not provide conclusive evidence. The RVLM contains other types of
barosensitive bulbospinal neurons (15, 28), and the
relative contributions of C1 and non-C1 cells to the generation of
sympathetic vasomotor tone are not known and probably vary with the
state of the animal. We have previously demonstrated that depletion of
the vast majority of presympathetic C1 neurons by treatment with
anti-DH-Sap does not chronically alter AP in chloralose-anesthetized
rats (29). In these rats, the RVLM continues to generate
sympathetic vasomotor tone to maintain a normal AP (unpublished
observation), suggesting that the non-C1 presympathetic RVLM neurons
are fully capable of generating SNA. In the present study, counts of
PNMT-ir neurons in the RVLM from two rostral sections showed a 76 ± 5% depletion compared with control rats. This is a conservative
estimate of the depletion of bulbospinal C1 neurons, because counts of
PNMT-ir neurons would also include some C1 neurons that project to the
hypothalamus and not to the spinal cord (29, 31).
Nevertheless, because a few C1 cells remained in the RVLM after
treatment with anti-DH-Sap, the possibility that these C1 neurons
could account for the persistent clonidine responses in the present
study must be entertained. However, on average, the depletion of
rostral C1 neurons was quite substantial, and the SNA and AP responses
to clonidine in an animal with a 95% depletion of rostral C1 cells
were indistinguishable from control rats. Thus the most likely reason
for the clonidine-induced inhibition of SNA in rats treated with
anti-DH-Sap is that bulbospinal C1 neurons are not essential for the
response. In addition, the depletion of bulbospinal A5 noradrenergic
neurons by treatment with anti-DH-Sap also confirms that these cells
are not essential for the sympatholytic and hypotensive effects of
clonidine (5). Because the noncatecholaminergic
presympathetic RVLM neurons are sensitive to clonidine (Fig. 5) and
would not be depleted by intraspinal anti-DH-Sap, their inhibition
likely contributes to the sympathoinhibition and hypotension elicited
by clonidine in rats treated with anti-DH-Sap.
How important is the contribution of RVLM to the sympatholytic
effect of clonidine?
The findings that effects of systemically administered clonidine can be
mimicked by local microinjection into the RVLM and reversed by
microinjection of an 2-adrenergic receptor antagonist into the same site (24) likely overestimate the role of
the RVLM. A comparable sympathoinhibition and hypotension may be
achieved by the partial inhibition of several sites by intravenous
clonidine versus the very effective inhibition of one central site by
the local microinjection of clonidine into the RVLM. For instance, in
the present study, clonidine produced a relatively modest inhibition of
presympathetic RVLM neurons (40% on average) with a dose that nearly
eliminated SNA (10 µg/kg). In contrast, activation of the baroreceptor reflex, which reduces SNA by inhibition of RVLM neurons, produced at least as much inhibition of RVLM unit activity as of SNA
(Figs. 1 and 4A; 33). In addition, because microinjection of
2-adrenergic receptor antagonists into the RVLM
increases AP, SNA, and RVLM unit activity in the absence of clonidine
(12), the apparent reversal of the effects of systemically
administered clonidine does not signify that all the effects of
systemically administered clonidine occur within the RVLM.
2-adrenergic receptors in the
spinal cord.
In summary, the inhibition of barosensitive bulbospinal neurons in the
RVLM by clonidine has been cited as evidence that these cells are
important targets for the sympatholytic effect of this drug (3,
25, 33). The present study demonstrates that in chloralose-anesthetized rats, barosensitive bulbospinal C1 and non-C1
neurons in the RVLM are inhibited by clonidine in doses that inhibit
SNA, suggesting that both cell types may play a role in the decrease in
SNA produced by clonidine. Nevertheless, depletion of most
presympathetic C1 neurons did not alter effects of clonidine on SNA and
AP, suggesting that these responses to clonidine do not require
presympathetic C1 cells. Finally, the modest inhibition of
presympathetic RVLM neurons by a dose of clonidine that nearly eliminated SNA suggests that other central sites contribute to the
inhibitory effects of clonidine. Thus, although central
catecholaminergic systems are a target for antihypertensive drugs such
as clonidine, the sympatholytic and hypotensive effects of these drugs
are likely to be achieved by affecting a variety of cell types via pre-
and postsynaptic mechanisms at multiple sites within the central
nervous system.
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. G. Guyenet, Dept. of Pharmacology, Univ. of Virginia Health System, P.O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735 (E-mail: pgg{at}virginia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 May 2000; accepted in final form 18 July 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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,
Onset of clonidine injections.
, PNMT-ir neuron. Both sections correspond to
anterior-posterior level 
