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Third Department of Internal Medicine, University of the Ryukyus School of Medicine, Okinawa 903 - 0215, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We examined the effects
of clonidine injected unilaterally into the rostral ventrolateral
medulla (RVLM) of conscious, unrestrained rats. We also examined
whether the local 
2-adrenoceptor mechanism contributed
to the action of clonidine injected into the RVLM. Injection of
clonidine but not vehicle solution significantly decreased the mean
arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve
activity (RSNA) in conscious, unrestrained rats as well as in
propofol-anesthetized rats. The frequency of natural behavior was
significantly lower after clonidine injection than after vehicle
injection. The depressor and sympathoinhibitory responses were
significantly larger in the propofol-anesthetized rats than in the
conscious rats. Coinjection of a selective
2-adrenoceptor antagonist, 2-methoxyidazoxan, with
clonidine into the RVLM significantly attenuated the depressor,
bradycardiac, sympathoinhibitory, and sedative effects of clonidine
injected alone. In conclusion, clonidine injected into the RVLM
decreased MAP, HR, and RSNA and caused sedation in conscious,
unrestrained rats. The action of clonidine in the RVLM was at least
partly mediated by 2-adrenoceptor mechanisms.
blood pressure; 2-adrenoceptor; renal sympathetic
nerve activity; natural behavior
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CLONIDINE elicits
sympatholytic and sedative effects by acting on the central nervous
system (29). Intravenous administration of clonidine
transiently increases blood pressure and subsequently decreases
peripheral sympathetic tone and blood pressure (29, 35).
The sympathoinhibitory effect of clonidine is believed to result from
action on imidazoline receptors and/or 2-adrenoceptors in the medullary cardiovascular nuclei. Systemic administration of
clonidine also causes sedation. Catecholaminergic neurons in the locus
ceruleus have been reported to be involved in the sedative effect of
systemically administered clonidine (8), probably by
stimulating 2-adrenoceptors (38).
The rostral ventrolateral medulla (RVLM) is a vasomotor center where
cardiovascular sympathetic premotor neurons are located (7,
34). The neurons in the RVLM directly innervate sympathetic preganglionic neurons in the intermediolateral cell column of the
spinal cord and also innervate to supramedullary brain regions (7). The neurons generate sympathetic tone and also
participate in reflex control of the cardiovascular system. The RVLM
neuron has been regarded as the main site of the hypotensive action of clonidine (34). Radioligand binding studies revealed
clonidine-binding sites in the RVLM (13). Imidazoline
receptors and 2-adrenoceptors exist on the RVLM neurons
(13, 28). Microinjection of clonidine into the RVLM of
anesthetized animals causes long-lasting hypotensive and
sympathoinhibitory effects (9, 13, 23). Previous studies suggested that the sympathoinhibitory action of clonidine in the RVLM
was mediated by the local 2-adrenoceptors
(16) or novel I1-imidazoline receptors
(14) or both (19). The functional predominance of either 2-adrenoceptors or
I1-imidazoline receptors remains to be elucidated
(27).
Cardiovascular, sympathetic, and behavioral responses to various
drugs are influenced by anesthetics. For example, urethane attenuates
cardiovascular responses of central 2-adrenoceptor stimulation (2), and pentobarbital sodium anesthesia
enhances the cardiovascular effects of rilmenidine and
clonidine in rats (31). In fact, in pentobarbital
sodium-anesthetized rats, intracerebroventricular administration of
clonidine decreased arterial pressure, whereas in conscious rats,
intracerebroventricular administration of clonidine increased
arterial pressure (21). To the best of our knowledge, all
previous microinjection studies of clonidine in the RVLM were conducted
on anesthetized animals.
We examined the cardiovascular, sympathetic, and behavioral effects of
clonidine microinjected unilaterally into the RVLM of conscious,
unrestrained rats. We also examined whether the local
2-adrenoceptors contributed to the action of clonidine microinjected into the RVLM.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Male Sprague-Dawley rats (340-550 g) were purchased from Charles River Japan and fed standard laboratory rat chow and tap water ad libitum. The rats were kept in a room maintained at constant temperature (24 ± 2°C) and humidity (55 ± 10%) under a 12-h light period between 0800 and 2000. After 7 days of adaptation to these conditions, the experimental procedures were performed. All procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Use Committee, University of the Ryukyus.
Implantation of guide cannula. Each rat was anesthetized with an intraperitoneal injection of 50 mg/kg of pentobarbital sodium, and the rats were placed on a stereotaxic frame (Narishige Scientific Instruments, Tokyo, Japan) in a prone position. An incisor bar was set at 4 mm below the interaural line. The skin overlying the midline of the skull was incised, and a small hole was drilled to the dorsal surface of the cranium according to the following coordinates: 2.0 mm lateral to the midline and 3.5 mm posterior to the lambdoid suture. Two screws were fixed to the cranium beside the hole. A 25-gauge stainless steel guide cannula was lowered 6 mm vertically from the skull surface, and the tip was placed 4 mm above the left RVLM according to the coordinates of Paxinos and Watson (25). The guide cannula was fixed to the skull with screws, and the screws were hardened to the skull using cyanoacrylate adhesive (Aron Alpha: Toa Gosei Chemical Industries, Tokyo, Japan) and secured with dental cement. Before and after surgery, each rat received an intramuscular injection of 20,000 U/kg body wt of penicillin G for prophylaxis.
Implantation of arterial and venous catheters and the renal nerve electrode. At least 7 days after implantation of the guide cannula, rats were reanesthetized with pentobarbital sodium, and vascular catheters (PE-10 fused with PE-50) were inserted through the right femoral artery and vein for blood pressure recording and drug administration, respectively. The left renal nerves were exposed through a retroperitoneal approach. A branch of the nerves was separated from surrounding connective tissue, and a bipolar silver wire electrode (no. 7855; A-M Systems, Carlsborg, WA) was placed under the nerve branch. When an optimal neurogram was obtained, the nerve and electrode were embedded in silicone gel (Semicosil 932; Wacker, Munich, Germany) and allowed to harden. Catheters and lead wires from the electrode were exteriorized at the interscapular region through a subcutaneous tunnel and fixed to the skin. After surgery, each rat received an intramuscular injection of 40,000 U/kg body wt of penicillin G for prophylaxis.
Microinjection procedure. Drugs were microinjected unilaterally into the RVLM. The injection volume was always 200 nl on the basis of previous studies of conscious rats (15, 30). A 33-gauge stainless steel injector needle glued with a 27-gauge stainless steel connector was used in the experiment. The injector was connected via a polyethylene tube (both ends of PE-10 were fused with PE-20) to a Hamilton microsyringe (5 µl). The injections were delivered by hand, and the injection volume was measured by observing the movement of the fluid meniscus in the PE-10 tube marked with a scale of every 200-nl volume.
Unilateral injection of clonidine into the RVLM of conscious, unrestrained rats. At least 24 h after the implantation of the arterial and venous catheters and the renal nerve electrode, the rat was placed in an 18-cm-diameter plastic bowl and was allowed to move freely. During the recording period, acoustic disturbances were avoided, and the room was kept at constant temperature and with a moderate degree of illumination. After a stabilization period of at least 30 min, arterial catheter and lead wires from the electrode were connected to a pressure transducer (P10EZ; Spectramed, Tokyo, Japan) and biophysical amplifier (DPA-100E; Dia Medical System, Tokyo, Japan), respectively. The original renal nerve signals were amplified and filtered between 100 and 1,000 Hz. The amplified nerve pulses were counted with a spike counter (DSE-325A; Dia Medical System). The number of nerve spikes per second was continuously displayed on a chart recorder (RJG-4128; Nihon Kohden, Tokyo, Japan) together with pulsatile pressure, mean arterial pressure (MAP), and heart rate (HR), which was derived from blood pressure signals. The number of spikes per second after intravenous administration of 40 mg/kg of hexamethonium was determined as background noise level. Changes in renal sympathetic nerve activity (RSNA) were expressed as percent changes from baseline spike counts. After recording resting MAP, HR, and RSNA for at least 30 min, a 33-gauge stainless steel injector needle was inserted through the guide cannula previously fixed to the skull of the rat. The injector needle was extended 4 mm beyond the tip of the guide cannula. Blood pressure, HR, and RSNA were then allowed to stabilize for at least 20 min before the first injection was made. The RVLM was identified by the response to a unilateral injection of 2 nmol of L-glutamate on the basis of the modified criteria outlined previously (37): 1) the latency of the onset of changes in MAP was no more than 5 s, 2) the response plateau occurred within 20 s, and 3) the change in MAP was at least 25 mmHg. After identification of the RVLM, the injector needle was replaced by another containing clonidine solution or the vehicle solution. After a 20-min period for restabilization, 3 nmol of clonidine or vehicle solution was then injected unilaterally into the RVLM of the conscious, unrestrained rat. The injector needle was removed 3 min after the injection. MAP, HR, and RSNA were continuously recorded for 60 min. During the recording period, the onset and the end of each natural behavior, grooming and exploring, that lasted more than 30 s were marked on the chart to examine frequency and duration of the behavior. Grooming was defined as the animal scratching, licking, or rubbing any part of its body, and exploring was defined as the animal sniffing and standing on its hindlimbs to look outside the bowl (36). The preparation and microinjection procedures of conscious, unrestrained rats have been described in detail elsewhere (30).
Unilateral injection of clonidine into the RVLM of
propofol-anesthetized rats.
To compare the cardiovascular and sympathetic effects of clonidine
injected into the RVLM between the conscious rat and the anesthetized
rat, a separate group of rats with the guide cannula fixed to the skull
7 days previously was anesthetized with propofol. In these experiments,
anesthesia was induced with an intraperitoneal injection of
pentobarbital sodium. Right femoral artery and bilateral femoral veins
were cannulated for monitoring arterial pressure and drug
administration, respectively, and the electrode was placed under the
renal nerve branch. As the effects of the pentobarbital sodium began to
disappear, propofol infusion was started. After the surgical
procedures, continuous infusion of propofol was reduced at a rate of 20 mg · kg
1 · h1, and a
stabilization period of at least 30 min was taken. Baseline MAP, HR,
and RSNA were then recorded. Drugs were unilaterally injected into the
RVLM by using a 33-gauge stainless steel injector needle. The surgical
preparation and microinjection procedures were the same as in the
conscious rats. The RVLM was identified by injection of 2 nmol of
L-glutamate on the basis of the same criteria listed above.
MAP, HR, and RSNA were continuously recorded for 60 min after injection
of 3 nmol of clonidine or vehicle.
Coinjection of clonidine and 2-methoxyidazoxan unilaterally into
the RVLM of conscious, unrestrained rats.
To clarify whether the effects of clonidine injected into the RVLM of
conscious, unrestrained rats were mediated by the local 2-adrenoceptors, a selective
2-adrenoceptor antagonist was coinjected with clonidine
into the RVLM. The dose of each drug was chosen on the basis of
previous studies (18, 20). After identification of the
RVLM, 2 nmol of clonidine alone or 2 nmol of 2-methoxyidazoxan together
with 2 nmol of clonidine was injected unilaterally into the RVLM of
conscious, unrestrained rats. MAP, HR, and RSNA were continuously
recorded for 60 min. Onset and end of natural behaviors were marked on
the chart.
Unilateral blockade of 2-adrenoceptors in the RVLM
of conscious, unrestrained rats.
To clarify whether the 2-adrenoceptors in the RVLM
contributed to the ongoing levels of MAP, HR, RSNA, and natural
behaviors of conscious, unrestrained rats, 2-methoxyidazoxan was
injected into the RVLM. After identification of the RVLM, 2 nmol of
2-methoxyidazoxan or vehicle was injected unilaterally into the RVLM of
conscious, unrestrained rats. MAP, HR, and RSNA were continuously
recorded for 60 min. The onset and the end of natural behaviors were
marked on the chart.
Drugs. Drugs were dissolved in artificial cerebrospinal fluid (aCSF; 133.3 mol/l sodium chloride, 3.4 mmol/l potassium chloride, 1.3 mmol/l calcium chloride, 1.2 mmol/l magnesium chloride, 0.6 mmol/l sodium dihydrogen orthophosphate, 32.0 mmol/l sodium bicarbonate, and 3.4 mmol/l glucose). Concerning the coinjection of 2-methoxyidazoxan and clonidine and the injection of 2-methoxyidazoxan alone, 5% glucose solution was used as the vehicle. Mixed drugs for coinjection were always prepared just before the microinjection. Clonidine was purchased from Research Biochemicals International (Natick, MA). 2-Methoxyidazoxan was purchased from Sigma Chemical (St. Louis, MO).
Histological examinations. At the end of the experiments, rats were anesthetized with pentobarbital sodium and received an injection of 200 nl of Alcian blue dye to mark the injection site. Rats were then perfused transcardially with 50 ml of 0.9% sodium chloride followed by 100 ml of 10% phosphate-buffered formalin. The brain stem was removed and stored in 10% phosphate-buffered formalin. The day before sectioning of the brain tissue, the brain stem was transferred to a fixative containing 20% sucrose. Frozen brain tissues were sectioned in the coronal plane (50 µm) and stained with neutral red. Microinjection sites were identified by the deposition of Alcian blue dye and referred to standard anatomic structures of the rat medulla oblongata according to the atlas of Paxinos and Watson (25). We excluded those findings from further analysis when the Alcian blue dye penetrated to the ventral surface or apparent bleeding was observed.
Statistical analysis. Values are expressed as means ± SE. Differences among the groups were tested by two-way ANOVA with or without repeated measures. Subsequent analysis for significant difference was performed using Scheffé's F test. Differences between two groups were tested by unpaired Student's t-test. A value of P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of clonidine injected unilaterally into the RVLM of
conscious, unrestrained rats.
The microinjection of 3 nmol of clonidine into the RVLM was performed
in eight conscious, unrestrained rats. After insertion of the 33-gauge
injector needle toward the RVLM, slight pressor and bradycardiac
changes were typically observed. Mostly within 20 min, the rat became
undisturbed and calm inside the bowl, and both MAP and HR became
stable. Preinjection values of MAP and HR and maximal changes in values
of MAP, HR, and RSNA after injection of the drugs are shown in Table
1. Typical traces of pulsatile arterial
pressure, MAP, HR, and RSNA recorded for 60 min after clonidine
injection into the RVLM of a conscious rat and the marked periods of
natural behaviors that occurred after clonidine injection are shown in
Fig. 1. Those traces and marked periods
of natural behavior after aCSF injection are shown in Fig.
2. Unilateral injection of 3 nmol of
clonidine but not aCSF into the RVLM gradually decreased MAP, HR, and
RSNA in conscious, unrestrained rats. The depressor response peaked at
17 ± 3 min and returned to preinjection values within 45-60
min in five rats, and the response of the other three rats continued
for >60 min. The time course of the MAP, HR, and RSNA responses to
clonidine injection is shown in Fig. 3.
The frequency of natural behaviors was significantly (P < 0.01) lower after clonidine injection than after vehicle injection. The duration of each behavior and changes of MAP, HR, and RSNA in response to natural behaviors were similar between
clonidine-injected rats and vehicle-injected rats (Table
2).
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Effects of clonidine microinjected unilaterally into the RVLM of propofol-anesthetized rats. The microinjection of 3 nmol of clonidine into the RVLM was performed in five propofol-anesthetized rats. Preinjection MAP was significantly (P < 0.05) lower in the propofol-anesthetized rats than that in the conscious rats, whereas HR was similar in the propofol-anesthetized rats and the conscious rats (Table 1). The unilateral injection of 3 nmol of clonidine into the RVLM decreased MAP, HR, and RSNA in the propofol-anesthetized rats. The depressor response peaked at 18 ± 2 min and returned to preinjection levels within 55 min in one rat. The depressor response continued for more than 60 min in four rats. The time course of the MAP, HR, and RSNA responses to clonidine injection is shown in Fig. 3. The depressor and sympathoinhibitory responses to clonidine injection were significantly larger in the propofol-anesthetized rats than in the conscious rats, whereas the bradycardiac response was similar in the conscious rats and the propofol-anesthetized rats (Table 1).
Effects of coinjection of clonidine and 2-methoxyidazoxan
unilaterally into the RVLM of conscious, unrestrained rats.
To examine whether local 2-adrenoceptors contribute to
the action of clonidine in the RVLM, coinjection of
2-methoxyidazoxan, a selective 2-adrenoceptor
antagonist, with clonidine into the RVLM was performed in six
conscious, unrestrained rats. Preinjection values of MAP and HR;
changes in values of MAP, HR, and RSNA; and the frequency of natural
behavior after injection of the drugs are shown in Table
3. The unilateral injection of 2 nmol of
clonidine alone into the RVLM decreased MAP, HR, and RSNA.
Coinjection of 2 nmol of 2-methoxyidazoxan with 2 nmol of clonidine
into the RVLM of conscious rats significantly (P < 0.05) attenuated the depressor, bradycardiac, and sympathoinhibitory
effects of clonidine injected alone. The frequency of the natural
behaviors was significantly (P < 0.01) higher after
coinjection of 2-methoxyidazoxan with clonidine injection than after
clonidine injection alone.
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Effects of unilateral blockade of 2-adrenoceptors in
the RVLM of conscious, unrestrained rats.
To clarify whether the 2-adrenoceptors in the RVLM
contributed to the ongoing levels of MAP, HR, RSNA, and natural
behaviors, microinjection of 2 nmol of 2-methoxyidazoxan into the RVLM
was performed in six conscious, unrestrained rats. Preinjection values of MAP and HR; changes in values of MAP, HR, and RSNA; and the frequency of natural behavior after injection of the drugs are shown in
Table 3. The unilateral injection of 2 nmol of 2-methoxyidazoxan or 5%
glucose solution into the RVLM caused no significant change in MAP, HR,
and RSNA. The frequency of the natural behaviors was similar after
2-methoxyidazoxan injection and after vehicle injection.
Histological analysis.
A typical Alcian blue dye injection site of conscious rats is shown in
Fig. 4, right. The injection
sites were restricted to an area encompassing the dorsolateral aspect
of the lateral paragigantocellular nucleus (LPGi) and the region
dorsolateral to the LPGi. This area lies at the caudal end of the
facial nucleus, which is known as the pressor area of the RVLM.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The principal observations in the present study were that
clonidine injected unilaterally into the RVLM decreased MAP, HR, and
RSNA in conscious, unrestrained rats as well as in
propofol-anesthetized rats. Clonidine injection into the RVLM decreased
the frequency of natural behaviors such as grooming and exploring. The
actions of clonidine were attenuated by coinjection of
2-methoxyidazoxan, a selective 2-adrenoceptor antagonist.
Clonidine, an imidazoline receptor and 2-adrenoceptor
agonist, elicits hypotensive and sedative effects by acting on the central nervous system. Intravenous administration of clonidine increases blood pressure transiently and then gradually decreases peripheral sympathetic tone and blood pressure (29, 35).
The sympathoinhibitory and depressor effects of clonidine were mediated by the action within the central nervous system (29). The
RVLM has been nominated as the main site of action of clonidine in the
central nervous system (13, 29, 34). Blockade of
2-adrenoceptors/imidazoline receptors in the RVLM
eliminates the hypotensive effects of systemic clonidine
(17). Radioligand binding studies revealed clonidine binding sites in the RVLM (13). Imidazoline receptors and
2-adrenoceptors exist on the RVLM neurons (13,
28). 2-Adrenoceptors have been detected in the
RVLM by membrane binding studies (13), immunohistochemistry (28), in situ hybridization
(39), and electrophysiology (1).
Microinjection of clonidine and other 2- and/or
imidazoline receptor agonists into the RVLM of anesthetized animals
either unilaterally or bilaterally caused long-lasting sympathoinhibitory, hypotensive, and bradycardiac effects (9, 13,
23). In the present study, we injected 3 nmol of clonidine unilaterally into the RVLM of conscious, unrestrained rats, a dose
based on the findings of previous studies performed on anesthetized rats (9, 13). In conscious rats, the unilateral injection of 3 nmol of clonidine into the RVLM also caused sympathoinhibitory, hypotensive, and bradycardiac effects. The depressor and
sympathoinhibitory responses to clonidine injection were significantly
larger in the propofol-anesthetized rats than in the conscious rats.
These findings suggested that propofol anesthesia enhanced the
depressor and sympathoinhibitory action of clonidine injected into the
RVLM of rats. We recently reported that urethane anesthesia reduced the
magnitude of sympathetic responses to glutamate and glycine injected into the RVLM (30). Taken together, anesthetic
agents may variously influence the sympathetic responses evoked by
neuroexcitatory or inhibitory drugs injected into the RVLM.
Clonidine acts on the RVLM. Phenotypically identified catecholaminergic
and noncatecholaminergic neurons in the RVLM were equally
inhibited by systemically administered clonidine (32). Either 2-adrenoceptors (16) or novel
I1-imidazoline receptors (14) or both
(19) may be responsible for the sympathoinhibitory action
of clonidine in the RVLM. The central administration of an imidazoline
receptor antagonist, either intracerebroventricularly (6)
or by microinjection into the RVLM (26), blocked the antihypertensive actions of systemically administered clonidine and/or
rilmenidine or moxonidine. A number of selective
2-antagonists appeared to have weak or no blocking
effects on the action of peripherally administered clonidine or its
related compounds (24). These studies (6, 24,
26) suggested the functional dominance of the imidazoline
receptors. However, discharges of neurons in the RVLM that expressed
2-adrenoceptors were inhibited by systemic and/or
iontophoretic application of either catecholamines or clonidine (1). Transgenic mice expressing mutated
2A-adrenoceptor, with intact 2B- and
2C-adrenoceptor subtypes of
2-adrenoceptor, were reported to lack hypotensive
responses to imidazoline analogs (22). In the present
study, we used 2-methoxyidazoxan as a selective 2-adrenoceptor antagonist. This drug reported an
~200-fold higher affinity for 2-adrenoceptor than
imidazoline receptor (12) and was used as the
2-adrenoceptor antagonist (18, 19). The sympatholytic, hypotensive, bradycardiac, and sedative effects of
clonidine microinjected into the RVLM were attenuated by coinjection of
2-methoxyidazoxan in conscious, unrestrained rats. The results of the
present study are quite different from those of Ernsberger et al.
(13), who reported that in anesthetized rats the drug SKF-86466, a selective 2-adrenoceptor antagonist, had no
effect on the hypotensive response to clonidine in the RVLM. Compared with SKF-86466, 2-methoxyidazoxan has 15 times higher affinity to the
2-adrenoceptor (12). We used 2 nmol of
2-methoxyidazoxane, whereas Ernsberger et al. (13) used an
even smaller dose, namely 1 nmol, of SKF-86466. Furthermore, they used
urethane as the anesthetic. Urethane is reported to attenuate the
central 2-adrenoceptor-mediated response
(2). We suppose that in the study of Ernsberger et al.
(13), the use of SKF-86466 in a smaller dose in
urethane-anesthetized rats masked the effect of the
2-adrenoceptor antagonist on the hypotensive response to
clonidine in the RVLM. The findings of the present and previous studies
(1, 22) support the notion that
2-adrenoceptors in the RVLM, at least in part,
contribute to the action of clonidine in the RVLM. Head et al.
(19) proposed a hypothesis that imidazoline receptors are
presynaptic on noradrenergic terminals, whereas
2-adrenoceptors lie downstream on cell bodies or other
nonadrenergic terminals.
The magnitude of the depressor response evoked by clonidine in both the conscious and propofol-anesthetized rats was less than in previous studies in which similar doses of clonidine were injected into the RVLM of anesthetized rats (9, 13). Drolet et al. (9) used spontaneously hypertensive rats, whereas we used normotensive Sprague-Dawley rats in the present study. The difference in the baseline blood pressure levels could have affected the magnitude of the depressor responses evoked by clonidine. We performed unilateral injection of clonidine into the RVLM, whereas Ernsberger et al. (13) performed bilateral injection. Compared with unilateral injection, bilateral injection of clonidine is expected to cause more profound sympathoinhibition that yields a larger depressor response.
We also examined whether the action of the
2-adrenoceptors in the RVLM contributed to the ongoing
levels of MAP, HR, RSNA, and natural behaviors. The injection of 2 nmol
of 2-methoxyidazoxan alone into the RVLM caused negligible effects on
MAP, HR, RSNA, and natural behavior in conscious, unrestrained rats.
These findings do not support the significant role of the local
2-adrenoceptors in maintaining the ongoing activity of
the RVLM neurons. In the present study, however, we only used one dose
of the 2-antagonist. We could not exclude the
possibility that higher doses of 2-methoxyidazoxan blocked the ongoing
activity of the RVLM neurons, resulting in cardiovascular and
sympathetic responses. In urethane-anesthetized spontaneously
hypertensive rats, microinjection of 4 nmol of efaroxan, an
2-antagonist with relatively higher affinity for
I1-imidazoline receptors, into the bilateral RVLM increased
MAP and HR, whereas 10 nmol of SKF-86466, a selective
2-antagonist, did not change baseline MAP and HR
(17). In the caudal ventrolateral medulla, microinjection
of 2 nmol of SKF-86466 decreased MAP and RSNA in urethane-anesthetized
rats (33). These previous studies (17, 33)
suggested that the imidazoline receptor with or without the
2-adrenoceptor mechanism in the RVLM and in the caudal
ventrolateral medulla tonically regulated blood pressure in
urethane-anesthetized rats.
Another important observation of the present study was the attenuation
of the natural behaviors after the clonidine injection into the RVLM.
The suppression of the frequency of natural behaviors was blocked by
coinjection of 2-adrenoceptor antagonist. We do not have
a clear explanation for the mechanism of this observation. Decreasing
arterial pressure in a normotensive animal might reduce the occurrence
of natural behaviors. In our previous study (30), however,
the frequency of natural behaviors of normotensive rats did not
decrease during the depressor response by the 
16 ± 3 mmHg
evoked by microinjection of glycine into the RVLM. Thus the decrease of
natural behaviors in the present study was probably not caused by the
blood pressure reduction. Furthermore, Takishita et al.
(36) reported that in spontaneously hypertensive rats, grooming behavior was equally observed both before and after the intravenous administration of manidipine, which decreased MAP by
13 ± 1 mmHg. Another possibility was the involvement of the locus
ceruleus. Sedation is related to reduction of noradrenergic input to
the thalamus and cerebral cortex (29). Noradrenergic neurons in the locus ceruleus send major efferent fibers to the thalamus and the cerebral cortex (5). These noradrenergic
neurons in the locus ceruleus receive major afferent input from the
RVLM (4). In most cases, excitation of the RVLM neurons by
electrical stimulation mostly excites the activity of locus ceruleus
neurons (11). The excitatory input caused by electrical
stimulation of the RVLM neurons to the locus ceruleus neurons appears
to be mediated by the excitatory amino acid pathway because the
excitatory response was blocked by kynurenic acid and
-D-glutamylglycine (10). Injection of
kynurenic acid into the locus ceruleus significantly decreased the
spontaneous discharge rate of the locus ceruleus neurons
(3). Taken together, it is possible that injection of
clonidine into the RVLM caused inhibitory effects on the RVLM neurons,
resulting in a decrease in the excitatory input to the locus ceruleus,
which sends noradrenergic fibers to the forebrain sites. Further
studies are definitely needed to confirm this hypothesis.
In summary, clonidine injected unilaterally into the RVLM decreased
MAP, HR, and RSNA in conscious, unrestrained rats, as well as in
propofol-anesthetized rats. Clonidine injection into the RVLM decreased
the frequency of natural behaviors. The actions of clonidine were
attenuated by coinjection of an 2-adrenoceptor antagonist, 2-methoxyidazoxan. These findings suggest that clonidine injected into the RVLM decreases MAP, HR, and RSNA in conscious, unrestrained rats, as well as in anesthetized rats, and causes sedation. The action of clonidine in the RVLM is at least partly mediated by the local 2-adrenoceptors.
Perspectives
There are various substances taking charge of neural control of blood pressure in the RVLM. In the present study, the depressor and sympathoinhibitory responses to clonidine injection into the RVLM were significantly larger in the propofol-anesthetized rats than in the conscious rats. These findings clearly demonstrated the influence of propofol anesthesia on the cardiovascular and sympathetic responses to2-adrenoceptor stimulation in the RVLM. To know whether
a substance endogenous to the RVLM contributes to the ongoing levels of
sympathetic tone and blood pressure, microinjection of the receptor
antagonist, or the antisense of the substance, into the RVLM would be
an appropriate method. However, microinjection of
2-antagonist into the RVLM yielded results of an
inconclusive nature about whether the 2-adrenoceptor in the RVLM contributed to the ongoing levels of blood pressure and sympathetic tone. To address this issue, bilateral injection of 2-antagonist into the RVLM would be helpful.
Establishment of the technique of bilateral injection into the RVLM of
conscious animals is the next step to be achieved in this field of
study. Furthermore, the use of conscious animals enables us to observe the change in naturally occurring behavior. In the present study, microinjection of clonidine into the RVLM caused sedation of the rats.
The use of naturally behaving conscious animals allowed us to have more
insight into the role of neuroacting substances in the RVLM.
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ACKNOWLEDGEMENTS |
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This study was, in part, supported by Ministry of Health and Welfare Research Grant 9C-1.
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FOOTNOTES |
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Parts of this study were presented at the 17th and 18th Scientific Meeting of the International Society of Hypertension and at the 2000 American Physiological Society Conference, Baroreceptor and Cardiopulmonary Receptor Reflexes.
Address for reprint requests and other correspondence: M. Yamazato, Third Dept. of Internal Medicine, Univ. of the Ryukyus School of Medicine, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan (E-mail: sannai{at}med.u-ryukyu.ac.jp).
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 29 December 2000; accepted in final form 7 August 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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|---|
1.
Allen, AM,
and
Guyenet PG.
2-Adrenoceptor-mediated inhibition of bulbospinal barosensitive cells of rat rostral medulla.
Am J Physiol Regulatory Integrative Comp Physiol
265:
R1065-R1075,
1993
2.
Armstrong, JM,
Lefevre-Borg F,
Scatton B,
and
Cavero I.
Urethane inhibits cardiovascular responses mediated by the stimulation of -2 adrenoceptors in the rat.
J Pharmacol Exp Ther
223:
524-535,
1982
3.
Aston-Jones, G,
Astier B,
and
Ennis M.
Inhibition of noradrenergic locus coeruleus neurons by C1 adrenergic cells in the rostral ventral medulla.
Neuroscience
48:
371-381,
1992[ISI][Medline].
4.
Aston-Jones, G,
Ennis M,
Pieribone VA,
Nickell WT,
and
Shipley MT.
The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network.
Science
234:
734-737,
1986
5.
Aston-Jones, G,
Shipley MT,
and
Brzanna R.
The locus coeruleus, A5 and A7 noradrenergic cell groups.
In: The Rat Nervous System (2nd ed.), edited by Paxinos G.. New York: Academic, 1995, p. 183-213.
6.
Chan, CK,
Sannajust F,
and
Head GA.
Role of imidazoline receptors in the cardiovascular actions of moxonidine, rilmenidine and clonidine in conscious rabbits.
J Pharmacol Exp Ther
276:
411-420,
1996
7.
Dampney, RA.
Functional organization of central pathways regulating the cardiovascular system.
Physiol Rev
74:
323-364,
1994
8.
De Sarro, GB,
Ascioti C,
Froio F,
Libri V,
and
Nistico G.
Evidence that locus coeruleus is the site where clonidine and drugs acting at -1 and -2 adrenoceptors affect sleep and arousal mechanisms.
Br J Pharmacol
90:
675-685,
1987[ISI][Medline].
9.
Drolet, G,
Aslanian V,
Minson J,
Morris M,
and
Chalmers J.
Differences in the central hypotensive actions of -methyldopa and clonidine in the spontaneously hypertensive rat: contribution of neurons arising from the B3 and the C1 areas of the rostral ventrolateral medulla.
J Cardiovasc Pharmacol
15:
118-123,
1990[ISI][Medline].
10.
Ennis, M,
and
Aston-Jones G.
Activation of locus coeruleus from nucleus paragigantocellularis: a new excitatory amino acid pathway in brain.
J Neurosci
8:
3644-3657,
1988[Abstract].
11.
Ennis, M,
and
Aston-Jones G.
A potent excitatory input to the nucleus locus coeruleus from the ventrolateral medulla.
Neurosci Lett
71:
299-305,
1986[ISI][Medline].
12.
Ernsberger, P,
Friedman JE,
and
Koletsky RJ.
The I1-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease.
J Hypertens Suppl
15:
S9-S23,
1997[Medline].
13.
Ernsberger, P,
Giuliano R,
Willette RN,
and
Reis DJ.
Role of imidazole receptors in the vasodepressor response to clonidine analogs in the rostral ventrolateral medulla.
J Pharmacol Exp Ther
253:
408-418,
1990
14.
Ernsberger, P,
and
Haxhiu MA.
The I1-imidazoline-binding site is a functional receptor mediating vasodepression via the ventral medulla.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R1572-R1579,
1997.
15.
Fontes, MA,
Pinge MC,
Naves V,
Campagnole-Santos MJ,
Lopes OU,
Khosla MC,
and
Santos RA.
Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats.
Brain Res
750:
305-310,
1997[ISI][Medline].
16.
Guyenet, PG.
Is the hypotensive effect of clonidine and related drugs due to imidazoline binding sites?
Am J Physiol Regulatory Integrative Comp Physiol
273:
R1580-R1584,
1997
17.
Haxhiu, MA,
Dreshaj I,
Schafer SG,
and
Ernsberger P.
Selective antihypertensive action of moxonidine is mediated mainly by I1-imidazoline receptors in the rostral ventrolateral medulla.
J Cardiovasc Pharmacol
24:
S1-S8,
1994.
18.
Head, GA,
Burke SL,
and
Chan CK.
Central imidazoline receptors and centrally acting anti-hypertensive agents.
Clin Exp Hypertens
19:
591-605,
1997.
19.
Head, GA,
Chan CK,
and
Burke SL.
Relationship between imidazoline and 2-adrenoceptors involved in the sympatho-inhibitory action of centrally acting antihypertensive agents.
J Auton Nerv Syst
72:
163-169,
1998[ISI][Medline].
20.
Head, GA,
Godwin SJ,
and
Sannajust F.
Differential receptors involved in the cardiovascular effects of clonidine and rilmenidine in conscious rabbits.
J Hypertens
11, Suppl5:
S322-S323,
1993.
21.
Kawasaki, H,
Araki H,
Futagami K,
and
Gomita Y.
Age-related decrease in centrally-mediated pressor response to clonidine in conscious rats.
Brain Res
782:
333-336,
1998[ISI][Medline].
22.
MacMillan, LB,
Hein L,
Smith MS,
Piascik MT,
and
Limbird LE.
Central hypotensive effects of the 2a-adrenergic receptor subtype.
Science
273:
801-803,
1996[Abstract].
23.
McAuley, MA,
Macrae IM,
and
Reid JL.
The cardiovascular actions of clonidine and neuropeptide-Y in the ventrolateral medulla of the rat.
Br J Pharmacol
97:
1067-1074,
1989[ISI][Medline].
24.
Nosjean, A,
and
Guyenet PG.
Role of ventrolateral medulla in sympatholytic effect of 8-OHDPAT in rats.
Am J Physiol Regulatory Integrative Comp Physiol
260:
R600-R609,
1991
25.
Paxinos, G,
and
Watson C.
The Rat Brain in Stereotaxic Coordinates (2nd ed.). New York: Academic, 1986.
26.
Punnen, S,
Urbanski R,
Krieger AJ,
and
Sapru HN.
Ventrolateral medullary pressor area: site of hypotensive action of clonidine.
Brain Res
422:
336-346,
1987[ISI][Medline].
27.
Reis, DJ,
and
Piletz JE.
The imidazoline receptor in control of blood pressure by clonidine and allied drugs.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R1569-R1571,
1997
28.
Rosin, DL,
Zeng D,
Stornetta RL,
Norton FR,
Riley T,
Okusa MD,
Guyenet PG,
and
Lynch KR.
Immunohistochemical localization of 2A-adrenergic receptors in catecholaminergic and other brainstem neurons in the rat.
Neuroscience
56:
139-155,
1993[ISI][Medline].
29.
Ruffolo, RR, Jr,
Nichols AJ,
Stadel JM,
and
Hieble JP.
Pharmacologic and therapeutic applications of 2-adrenoceptor subtypes.
Annu Rev Pharmacol Toxicol
33:
243-279,
1993[ISI][Medline].
30.
Sakima, A,
Yamazato M,
Sesoko S,
Muratani H,
and
Fukiyama K.
Cardiovascular and sympathetic effects of L-glutamate and glycine injected into the rostral ventrolateral medulla of conscious rats.
Hypertens Res
23:
633-641,
2000[ISI][Medline].
31.
Sannajust, F,
Cerutti C,
Koenig-Berard E,
and
Sassard J.
Influence of anaesthesia on the cardiovascular effects of rilmenidine and clonidine in spontaneously hypertensive rats.
Br J Pharmacol
105:
542-548,
1992[ISI][Medline].
32.
Schreihofer, AM,
and
Guyenet PG.
Role of presympathetic C1 neurons in the sympatholytic and hypotensive effects of clonidine in rats.
Am J Physiol Regulatory Integrative Comp Physiol
279:
R1753-R1762,
2000
33.
Sesoko, S,
Muratani H,
Yamazato M,
Teruya H,
Takishita S,
and
Fukiyama K.
Contribution of 2-adrenoceptors in caudal ventrolateral medulla to cardiovascular regulation in rat.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R1119-R1124,
1998
34.
Sun, MK.
Pharmacology of reticulospinal vasomotor neurons in cardiovascular regulation.
Pharmacol Rev
48:
465-494,
1996[ISI][Medline].
35.
Takishita, S,
Muratani H,
Kawazoe N,
Kimura Y,
Tozawa M,
and
Fukiyama K.
Neural effects of renal blood flow during acute hypotension vary with antihypertensive drugs.
Hypertension
23:
I97-I101,
1994.
36.
Takishita, S,
Muratani H,
Teruya H,
Sesoko S,
Kawano Y,
and
Fukiyama K.
Neural effects of natural behavior on renal blood flow in spontaneously hypertensive rats.
Clin Exp Nephrol
2:
234-239,
1998.
37.
Teruya, H,
Yamazato M,
Muratani H,
Sakima A,
Takishita S,
Terano Y,
and
Fukiyama K.
Role of ouabain-like compound in the rostral ventrolateral medulla in rats.
J Clin Invest
99:
2791-2798,
1997[ISI][Medline].
38.
Tibirica, E,
Feldman J,
Mermet C,
Gonon F,
and
Bousquet P.
An imidazoline-specific mechanism for the hypotensive effect of clonidine: a study with yohimbine and idazoxan.
J Pharmacol Exp Ther
256:
606-613,
1991
39.
Travares, A,
Handy DE,
Bogdanova NN,
Rosene DL,
and
Gavras H.
Localization of 2A- and 2B-adrenergic receptor subtypes in brain.
Hypertension
27:
449-455,
1996
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, injection sites of conscious rats; 
,
injection sites of propofol-anesthetized rats. Amb, nucleus ambiguus;
NTS, nucleus of the solitary tract.