Vol. 273, Issue 6, H2899-H2909, December 1997
SPECIAL COMMUNICATION
Extracellular serotonin changes in VLM during muscle
contraction: effects of
5-HT1A-receptor
activation
Gudbjorn
Asmundsson,
Daryl
Caringi,
David J.
Mokler,
Toshio
Kobayashi,
Takeshi
Ishide, and
Ahmmed
Ally
Departments of Pharmacology and Biochemistry, College of Osteopathic
Medicine, University of New England, Biddeford, Maine 04005; and
The Health Science Center, Tokyo University of Mercantile Marine,
Tokyo 135, Japan
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ABSTRACT |
This study determined whether muscle
contraction causes an increase in extracellular levels of serotonin
(5-HT) in the rostral (rVLM) or caudal ventrolateral medulla (cVLM) in
anesthetized rats. Muscle contraction, evoked by tibial nerve
stimulation, increased mean arterial blood pressure (MAP) by 27 ± 4 mmHg (n = 8). In addition, 5-HT levels
in the rVLM were elevated by 65 ± 9% during the contraction
(n = 8). Results were similar over two
repeated contractions. In contrast, muscle contraction increased MAP,
but not 5-HT, levels in the cVLM (n = 6). Tibial nerve stimulation after muscle paralysis had no effect on
either MAP or 5-HT levels in both rVLM and cVLM. Microdialysis of a
5-HT1A agonist, 8-OH-DPAT (10 mM),
into the rVLM for 30 min (n = 6)
blunted the MAP change and reduced 5-HT release during contraction.
Administration of NAN-190, a
5-HT1A antagonist, into the rVLM
had no effect on 5-HT release and cardiovascular responses during
muscle contraction and blocked the changes in 5-HT, MAP, and heart rate
to static contraction after subsequent microdialysis of 8-OH-DPAT.
Results demonstrate that 5-HT levels in the rVLM increase during muscle contraction and that
5-HT1A-receptor activation in the
rVLM blunts MAP response to muscle contraction via a decrease in the
extracellular concentration of 5-HT.
arterial blood pressure; heart rate; exercise pressor reflex; rostral ventrolateral medulla; caudal ventrolateral medulla; rat
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INTRODUCTION |
THE VENTROLATERAL MEDULLA (VLM) is divided into caudal
(cVLM) and rostral (rVLM) portions, and these regions have been
suggested to be opposing in nature with regard to regulation
and/or integration of cardiovascular responses (8). For
example, the rVLM elicits pressor effects in response to electrical and
chemical stimulation, whereas the cVLM evokes hypotension (8, 34, 43).
The VLM has also been implicated in mediating increases in mean
arterial blood pressure (MAP) and heart rate (HR) during static muscle contraction, commonly known as the exercise pressor reflex, in anesthetized cats (4, 11, 21, 22, 24, 29) and rats (2). The pressor
response during muscle contraction was abolished after bilateral
electrolytic lesioning of an area within the VLM (21). Also, c-Fos
expression (24) and radioactive glucose (22) studies have highlighted
regions within the medulla, including the rVLM and cVLM, that are
active during static muscle contraction.
Serotonin (5-HT) within the rVLM and adjacent reticular formation
contributes to descending control of autonomic functions and regulates
sympathetic outflow (26, 27, 40). Release of 5-HT has been implicated
in the interaction between antinociceptive/cardiovascular control and
specific descending medullary neurons projecting into sympathetic or
somatomotor regulatory regions in the spinal cord (28, 44). Studies
using 5-HT1A agonists demonstrate
5-HT1A-mediated cardiovascular
effects through the rVLM, including the raphe pallidus and the C1
region of the rVLM (14, 15). Stimulation of
5-HT1A receptors in the rVLM
evokes a decrease in sympathetic activity, resulting in hypotension and
bradycardia (15, 19, 40). Recently, Ally et al. (2) found evidence that
activation of 5-HT1A receptors within the rVLM, but not the cVLM, inhibits cardiovascular responses elicited during static muscle contraction and suggested that this attenuation is possibly mediated through changes in 5-HT release.
Therefore, the purpose of the present study was to determine whether
rVLM or cVLM changes in extracellular 5-HT concentration are associated
with cardiovascular responses during static muscle contraction and
whether 5-HT1A-receptor activation
within the rVLM attenuates increases in MAP and HR during muscle
contraction via a change in 5-HT release. We quantified the rVLM and
cVLM extracellular fluid 5-HT in response to muscle contraction using microdialysis techniques. Also, we examined the effects of
8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; RBI, Natick, MA), a
5-HT1A-receptor agonist
administered into the rVLM, on the exercise pressor reflex with
concomitant measurement of 5-HT release. Receptor specificity was
further confirmed by prior administration of
1-[2-methoxyphenyl]-4-[4-(2-phthalimido)butyl]piperazine (NAN-190; RBI), a 5-HT1A-receptor
antagonist, followed by subsequent microdialysis of 8-OH-DPAT into the
rVLM.
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METHODS |
Surgery
Male Sprague-Dawley rats (300-350 g) were initially anesthetized
with 25 mg/kg pentobarbital sodium (Sigma Chemical, St. Louis, MO) and
75 mg/kg chloral hydrate (Sigma Chemical). The rats were maintained at
37-38°C with the use of a heating pad and an infrared heat
lamp. Additional doses of chloral hydrate were given based on
appearance of a corneal reflex, changes in blood pressure during surgical manipulation, and/or a response to a noxious stimulus, i.e., paw or tail pinch. One common carotid artery was catheterized and
coupled with a pressure transducer (model P23 ID; Statham, Oxnard, CA)
to allow measurement of arterial pressure using a physiological chart
recorder (model 79D; Grass Instruments, Quincy, MA). MAP and HR were
obtained by integrating the arterial pressure signal with a time
constant of 2 s. The animal was allowed to breathe spontaneously after
cannulation of the trachea. However, during experiments involving
neuromuscular blockade with intravenous administration of pancuronium
bromide through a cannula inserted into a jugular vein, a respirator
(model 681; Harvard Apparatus, South Natick, MA) was used for
artificial ventilation (room air, 60 strokes/min, 1 ml/100 g body wt).
Arterial blood gases and pH were periodically checked (ABL-3;
Radiometer, Copenhagen, Denmark) and were maintained within normal
limits by providing supplemental oxygen, inflating the lungs using a
ventilator, and/or injecting sodium bicarbonate intravenously.
After the left tibial nerve was isolated, the nerve was placed on a
bipolar platinum hook electrode connected to a stimulator (model S88,
Grass) via a stimulus isolation unit (model SIU5C, Grass). The hip and
left knee were secured to prevent movement during contractions. The
triceps sura muscle was exposed and kept moist with mineral oil over
wet gauze. Muscle tensions generated by tibial nerve stimulation were
measured by a force transducer (model FT03, Grass) attached to the
corresponding Achilles tendon.
Microdialysis
The head of the rat was fixed in a stereotaxic frame (Kopf Instruments,
Tujunga, CA), and a static muscle contraction was induced by
stimulating the tibial nerve (3× motor threshold, 40 Hz, 0.1 ms)
while monitoring arterial pressure, MAP, HR, and developed tension. The
dorsal medulla was exposed after retraction of the dorsal neck muscles,
the caudal half of the cerebellum, and the dura, thereby revealing the
floor of the fourth ventricle rostral to the caudal aspect of the
inferior cerebellar peduncle. Two microdialysis probes (model CMA-11;
CMA, Acton, MA) with a 1-mm membrane tip (0.24 mm outer diameter) were
placed bilaterally into either the rVLM (2.0 mm rostral to the caudal
tip of the area postrema, 1.9 mm lateral to midline, and 2.4 mm ventral
to the floor of the fourth ventricle) or the cVLM (0.5 mm rostral to
the caudal tip of area postrema) based on the rat atlas (33). With the
use of a microdialysis pump (CMA/100, CMA), the probes were
continuously perfused at 1 µl/min with artificial cerebrospinal fluid
(CSF: 125 mM NaCl, 1.26 mM CaCl2,
2.5 mM KCl, 1.18 mM MgCl2) at pH
7.4 and osmolality of ~309 mosmol/kg. This artificial fluid served as
the delivery system for the drug used in the experiments.
After setup, verification of proper placement of microdialysis probes
was performed before each experiment by perfusing 1 nM
L-glutamate (RBI, Natick, MA)
for 5 min into either the rVLM or the cVLM. If the probes were in the
rVLM, an increase in MAP was noted after subsequent
L-glutamate administration.
Conversely, L-glutamate dialysis
into the cVLM exhibited a decrease in MAP. After correct placement of
probes was functionally assessed, a static muscle contraction was
evoked by stimulating the tibial nerve at parameters similar to those
described above. Arterial pressure, MAP, HR, and developed tension were
recorded and compared with those recorded before insertion of the
probes. This step was performed to determine whether insertion of the
probes disrupted the functional integrity of the rVLM or the cVLM. New
probes were used for each experiment.
Protocols
Release of 5-HT in rVLM and cVLM.
Perfusion of artificial CSF continued at 1 µl/min, and nine 10-min
collection periods were performed so that a stable baseline 5-HT
release (control) was achieved over a 90-min period (Table 1). A 10-min collection was necessary in
this protocol for the dialysate volume to be 20 µl (bilateral
collection) to run neurochemical assays for 5-HT. Subsequently, a 2-min
static contraction was evoked by stimulating the tibial nerve (3×
motor threshold, 40 Hz, 0.1 ms) during a 10-min collection period. The
animal was then allowed to recover for 60 min with six 10-min
collections, followed by another 2-min stimulation-evoked muscle
contraction (10-min collection). This was performed to determine
whether repeated muscle contractions elicit similar 5-HT release
patterns. Artificial CSF was then dialyzed for another 60 min (six
10-min collections) to establish a recovery of 5-HT. Lastly, the animal
was paralyzed with 200 µg pancuronium bromide intravenously
(Elkins-Sinn, Cherry Hill, NJ) to allow observation of whether
identical stimulation of the tibial nerve using prior parameters
produced a change in 5-HT or cardiovascular variables following
neuromuscular blockade. This protocol was implemented in separate rats,
and samples were collected from both the rVLM
(n = 8) and the cVLM
(n = 6). Muscle tension, HR, and MAP
were continuously monitored and documented throughout the protocol.
Samples were immediately stored at 
80°C. The 5-HT
concentrations of the perfusates were measured using high-performance
liquid chromatography with electrochemical detection (HPLC-EC; see
Biochemical Assay of 5-HT). Analyses
were done without bias, because samples were coded and the person
performing the assays was unaware of the code or the protocol.
Effect of a 5-HT1A-receptor agonist
(8-OH-DPAT) microdialyzed into rVLM.
Six rats were used to determine the effects of the
5-HT1A-receptor agonist 8-OH-DPAT
on cardiovascular responses and changes in extracellular 5-HT
concentration during muscle contraction by dialyzing the drug into the
rVLM. The cVLM did not warrant further investigation because 5-HT
concentrations in this area did not change significantly after muscle
contractions despite increases in MAP and HR (see
RESULTS). Furthermore, 8-OH-DPAT was
not microdialyzed into the cVLM, because a recent study (2) demonstrated that such administration had no effect on cardiovascular responses during muscle contraction.
After the surgical setup, nine 10-min control collections were
performed for 90 min (Table 2). A 2-min
muscle contraction was then elicited during another 10-min collection
period (Table 2). The rat was allowed to recover for 60 min (six 10-min
collections). After control cardiovascular responses were measured and
the dialysates were collected, 8-OH-DPAT (10 mM) was microdialyzed for
30 min during three 10-min collection periods. The muscle contraction was then repeated as described, and the dialysate was collected for 10 min. Artificial CSF was dialyzed through the probes for an additional
60 min with six 10-min collection periods to determine whether 5-HT
recovers to precontraction levels. The dialysate samples were stored at
80°C, and 5-HT levels were measured by HPLC-EC (see
Biochemical Assay of 5-HT). Tibial
nerve stimulation was conducted using prior stimulation parameters
after muscle paralysis with pancuronium, and the dialysate was
collected for 10 min. The dose of 8-OH-DPAT used in this study was 10 mM, because a recent dose-response experiment (2) determined this to be the effective dose.
Effect of a 5-HT1A-receptor antagonist
(NAN-190) microdialyzed into rVLM.
The effects of the 5-HT1A-receptor
antagonist NAN-190 on cardiovascular responses and changes in
extracellular 5-HT concentration during muscle contraction were
investigated by dialyzing the drug into the rVLM
(n = 5). The effects on 5-HT, MAP, and
HR during a static muscle contraction after a subsequent administration of 8-OH-DPAT into the rVLM were then determined.
In this protocol, nine 10-min control collections were performed for 90 min. A 2-min muscle contraction was then elicited during another 10-min
collection period. The rat was allowed to recover for 60 min (six
10-min collections). After control cardiovascular responses were
measured and the dialysates were collected, NAN-190 (10 mM) was
microdialyzed for 30 min during three 10-min collection periods. The
muscle contraction was then repeated as described, and the dialysate
was collected for 10 min. Thereafter, 8-OH-DPAT (10 mM) was
administered into the rVLM for 30 additional min and a contraction was
repeated. The dialysate samples were stored at 80°C, and
5-HT levels were measured by HPLC-EC (see Biochemical Assay of 5-HT). Tibial nerve stimulation was
conducted using prior stimulation parameters after muscle paralysis
with pancuronium, and the dialysate was collected for 10 min. The dose
of NAN-190 was 10 mM, because a previous study (2) determined this dose to be effective in blocking the attenuating effects on cardiovascular responses during muscle contraction after a subsequent administration of 8-OH-DPAT into the rVLM.
Biochemical Assay of 5-HT
Analysis of 5-HT was done using HPLC-EC. A reversed phase
C18 3-µm column (Rainin
Instruments, Woburn, MA) was used with a mobile phase consisting of 75 mM monobasic sodium phosphate, 1.4 mM sodium octanyl sulfonate, 100 µM EDTA, and 12% acetonitrile brought to pH 5.6 with NaOH. Analysis
of the eluent was by electrochemical detection (ESA Coulochem II,
Bedford, MA). The area under the curve was compared with standards of
5-HT (Sigma Chemical) injected onto the column at the beginning of each
run. Standards were prepared daily from a
10
6 M stock standard that
was stored at 80°C. Dilutions of
109, 5 × 1010,
1010, and
1011 M were analyzed to
establish a standard curve. The use of Justice Innovations ChromPerfect
(Palo Alto, CA) software allowed determination of regression for the
standard. A minimum correlation coefficient of 0.95 was used for all
standard curves. Routinely, voltammograms were generated to verify
retention times of standards and to maximize the sensitivity of the
system for 5-HT. Voltammograms of samples were also done to compare the
retention time and oxidation-reduction ratios with authentic standards.
Histology
Methylene blue (10 mM) was dialyzed for 30 min at 1 µl/min through
the microdialysis probes at the completion of every experiment. The
animal was perfused transcardially with 0.9% saline and then with 10%
phosphate-buffered Formalin. The medulla was removed, fixed in 10%
phosphate-buffered Formalin, and then stored at 4°C. The
locations of microdialysis probes were later determined by mounting the
medulla on the stage of a model Pelco 101 vibratome (Ted Pella,
Redding, CA), taking 50-µm transverse sections, and examining under a
microscope (Zeiss). The spread of the dye rostrocaudally as well as
laterally was measured to compare diffusion of the drug. Only animals
in which the probes were centered at the target sites (cVLM or rVLM)
were included in the final data analyses.
Statistical Analyses
All data are expressed as means ± SE. Normality of the data was
tested so that appropriate parametric and/or nonparametric statistics could be performed. Baseline and peak values of MAP, HR,
tension, and percent 5-HT release elicited by two repeated muscle
contractions were analyzed using a one-way analysis of variance (ANOVA)
with repeated measures. Baseline value was the average of the 2-min
period before a manipulation. Peak changes in MAP, HR, developed
tension, and 5-HT were defined as the maximum values obtained during
the contraction periods. The one-way repeated-measures ANOVA was also
used to compare the hemodynamic, tension, and 5-HT data before and
after administration of 8-OH-DPAT. Also, the one-way repeated-measures
ANOVA was used to compare MAP, HR, tension, and 5-HT data before and
after administration of NAN-190 and after administration of 8-OH-DPAT.
Post hoc analyses for the ANOVAs were performed using
Student-Newman-Keuls tests, and for all statistical evaluations
P < 0.05 was considered significant.
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RESULTS |
Confirmation of Proper Probe Placements and Functional Integrity
Bilateral insertion of the dialysis probes into either the rVLM or the
cVLM had no effects on changes in arterial pressure, MAP, HR, and
developed tension generated by contraction of the triceps sura muscle
in response to tibial nerve stimulation. In all rats
(n = 20), MAP, HR, and tension rose by
28 ± 3 mmHg, 28 ± 4 beats/min, and 410 ± 23 g,
respectively, before insertion of the probes. After the probes were
placed, a similar contraction increased MAP, HR, and tension by 27 ± 3 mmHg, 26 ± 4 beats/min, and 423 ± 29 g, respectively.
If the probes were within the rVLM or the cVLM,
L-glutamate (1 nM)
administration elicited pressor or depressor responses, respectively.
Microdialysis of L-glutamate into the rVLM revealed a significant increase in MAP of 40 ± 4 mmHg
within 1 min, and such administration into the cVLM produced a
significant depressor response of 43 ± 5 mmHg.
Postexperimental methylene blue dialysis followed by histological
analysis confirmed successful implantation of probes in targeted areas.
Release of 5-HT During Muscle Contraction
For the experiments with the rVLM, after a 90-min collection period to
achieve stable basal values of extracellular 5-HT, a tibial nerve
stimulation-evoked static muscle contraction was performed. Basal
levels of 5-HT were achieved at the second collection period, i.e.,
after a 20-min stabilization period. The novel finding was that
extracellular fluid concentrations of 5-HT in the rVLM significantly
increased from 0.7 ± 0.3 to 1.2 ± 0.5 fmol/20 µl dialysate
(an increase of 65 ± 9%; P < 0.05) following muscle contraction and paralleling a pressor response,
as shown in Fig. 1. MAP and HR also
increased by 27 ± 4 mmHg and 29 ± 4 beats/min, respectively, in
response to a developed tension of 433 ± 30 g during the
contraction (n = 8; Fig.
2). Percent change of extracellular 5-HT in
the rVLM and cardiovascular changes during a second contraction were
consistent with the first contraction in all rats (Figs. 1 and 2).
During the second contraction, MAP, HR, and developed tension increased
by 25 ± 5 mmHg, 27 ± 5 beats/min, and 423 ± 32 g,
respectively. The 5-HT level returned to baseline value within 20 min
after each contraction. Stimulation of the tibial nerve after
neuromuscular blockade resulted in constant HR and MAP and a baseline
extracellular level of 5-HT (Fig. 1). Hemodynamic, tension, and
5-HT-level data are summarized in Table 3.
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Fig. 1.
Percent extracellular fluid serotonin concentration sampled from
rostral ventrolateral medulla (rVLM) during two 2-min tibial nerve
stimulation-evoked static muscle contractions repeated at an interval
of 60 min and during a tibial nerve stimulation following neuromuscular
blockade (muscle paralysis arrow). Values are means ± SE
(n = 8).
* P < 0.05 compared with
respective controls (values before each contraction).
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Fig. 2.
Average peak changes in mean arterial pressure (MAP), heart rate (HR),
and developed tension during two 2-min stimulations of tibial nerve
repeated at an interval of 60 min in experiments in which microdialysis
probes were placed within rVLM. Values are means ± SE
(n = 8).
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Table 3.
Hemodynamic, tension, and 5-HT levels during two 2-min tibial nerve
stimulation-evoked muscle contractions before and after neuromuscular
blockade in experiments in which microdialysis probes were placed
into either rVLM or cVLM
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For the protocol in which dialysate samples were collected from the
cVLM, two repeated muscle contractions revealed no significant changes
among baseline, contraction, and postcontraction values in
extracellular fluid concentration of 5-HT (Fig.
3). However, during the two contractions,
MAP, HR, and tension increased similarly (Fig.
4). Basal and peak hemodynamic, tension,
and 5-HT data are summarized in Table 3.
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Fig. 3.
Percent extracellular fluid serotonin concentration sampled from caudal
ventrolateral medulla (cVLM) during two 2-min tibial nerve
stimulation-evoked static muscle contractions repeated after 60 min and
during a tibial nerve stimulation following muscle paralysis. Values
are means ± SE (n = 6). Control
values are levels before respective contractions.
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Fig. 4.
Average peak changes in MAP, HR, and developed tension during two 2-min
stimulations of tibial nerve at an interval of 60 min in experiments in
which microdialysis probes were inserted into cVLM. Values are means ± SE (n = 6).
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Effect of 5-HT1A-Receptor Activation on
Extracellular 5-HT Levels in rVLM and Concomitant Cardiovascular
Responses During Muscle Contraction
The effects of a 30-min perfusion with 10 mM 8-OH-DPAT on the rVLM
concentrations of 5-HT before and after muscle contractions and after
neuromuscular blockade are shown in Fig. 5.
The dose (10 mM) and site of administration of this drug were selected on the basis of a recent study (2) that used a similar dose and site to
affect cardiovascular responses during muscle contraction. Before
8-OH-DPAT was administered, MAP and HR increased by 26 ± 3 mmHg and
28 ± 4 beats/min, respectively, with a developed tension of 422 ± 19 g (n = 6; Fig.
6). After the drug was administered, increases were noted in MAP and HR during the contraction, but the
elevations were significantly diminished (
MAP = 13 ± 2 mmHg and
HR = 15 ± 3 beats/min; P < 0.05) (Fig. 6). Muscle tension developed to 429 ± 25 g and was
similar to that before the drug.
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Fig. 5.
Percent extracellular fluid serotonin concentration sampled from rVLM
during 2-min tibial nerve stimulation-evoked static muscle contractions
before and 30 min after microdialysis of 10 mM 8-OH-DPAT and during a
tibial nerve stimulation following muscle paralysis. Values are means ± SE (n = 6).
* P < 0.05 compared with
respective controls (values before each contraction).
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Fig. 6.
Average peak changes in MAP, HR, and developed tension during a 2-min
tibial nerve stimulation-evoked static muscle contraction before
(control) and after 30 min of microdialysis of 10 mM 8-OH-DPAT into
rVLM. Values are means ± SE (n = 6). * P < 0.05 compared with
control.
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Of novel importance was the percent change in extracellular fluid
levels of 5-HT in the rVLM, which was significantly inhibited (42 ± 6%; P < 0.05)
during the muscle contraction after the 30-min perfusion of 8-OH-DPAT
(Fig. 5). In addition, there was an ~10-fold increase in 5-HT
baseline level after the 30-min 8-OH-DPAT microdialysis. Lastly, by
paralyzing the animal, the percent change in extracellular 5-HT
concentration, MAP, and HR did not deviate from baseline after a tibial
nerve stimulation.
Effect of 5-HT1A-Receptor Antagonism on
Extracellular 5-HT Levels in rVLM and Subsequent Cardiovascular
Responses During Muscle Contraction Before and After 8-OH-DPAT
Administration
The effects of a 30-min perfusion with 10 mM NAN-190 on the rVLM
concentrations of 5-HT and on MAP and HR before and after muscle
contractions and after neuromuscular blockade are shown in Table
4. Before NAN-190 was administered, muscle
contraction increased MAP and HR by 24 ± 3 mmHg and 25 ± 4 beats/min, respectively, with a developed tension of 434 ± 25 g (n = 5). The
extracellular concentration of 5-HT increased from 0.7 ± 0.2 to 1.5 ± 0.3 fmol/20 µl fluid (5-HT = 0.8 ± 0.3 fmol/20 µl
fluid) during a muscle contraction. After NAN-190 was administered for
30 min, MAP and HR increased by 22 ± 4 mmHg and 28 ± 5 beats/min, respectively, in response to another muscle contraction. In
addition, microdialysis of NAN-190 into the rVLM resulted in no
significant change in extracellular 5-HT concentration during the
muscle contraction (Table 4). Developed muscle tension was 440 ± 29 g and was similar to that before the drug. Furthermore, prior
administration of NAN-190 blocked the attenuating effects on
extracellular 5-HT level and cardiovascular responses after subsequent
8-OH-DPAT perfusion into the rVLM (MAP = 25 ± 5 mmHg,
HR = 26 ± 5 beats/min, 5-HT = 0.8 ± 0.3 fmol/20
µl fluid, and developed tension = 430 ± 30 g) (Table 4).
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Table 4.
Hemodynamic, tension, and 5-HT levels during 2-min tibial nerve
stimulation-evoked muscle contractions before and after administration
of 8-OH-DPAT into rVLM followed by neuromuscular blockade and
during muscle contractions before and after microdialysis of
NAN-190 and after perfusion of 8-OH-DPAT into rVLM
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Histology and Diffusion of Methylene Blue
Histological sections of the medullary region showed that the membranes
of the probes were within the rVLM and the cVLM as described by the rat
brain atlas of Paxinos and Watson (33). Also, after histological
sections were visualized, the spread of methylene blue dye for 30 min
in each region was measured to be ~600 µm (Fig.
7), both rostrocaudally and laterally. This
diffusion was similar in all sections, and the distribution pattern
corresponds to a recent study (2) that used similar techniques.
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Fig. 7.
Schematic diagram of a transverse section of medulla showing tract made
by a microdialysis probe (MP) inserted into rVLM. An estimate of
approximate lateral diffusion of methylene blue (MB; ~600 µm) is
shown by transverse lines. A similar spread was seen in rostrocaudal
plane and experiments with cVLM. MLF, medial longitudinal fasciculus;
NTS, nucleus tractus solitarii; STN, spinal trigeminal nucleus.
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DISCUSSION |
This study is the first to demonstrate that an increase in
extracellular 5-HT concentration within the rVLM is associated with
cardiovascular responses during static muscle contraction. Furthermore,
our results have determined that administration of a
5-HT1A agonist into the rVLM
attenuates increases in MAP and HR in response to muscle contraction
mediated via a decrease in extracellular concentrations of 5-HT. This
finding adds significant conceptual and mechanistic information to the
rVLM 5-HT1A-mediated system
involved in static exercise. This research also showed a lack of 5-HT
change within the cVLM during muscle contraction, implying that changes
in 5-HT in the cVLM have no possible role in the exercise pressor
reflex. The cardiovascular and neurotransmitter responses were due to
contraction-evoked activation of muscle afferents, because stimulation
of the tibial nerve after neuromuscular blockade evoked no changes in
MAP, HR, or 5-HT concentration.
The VLM is a functionally identified area within the reticular
formation of the medulla oblongata and has been implicated in various
autonomic regulatory roles (8, 12). Furthermore, the rostral and caudal
portions of the VLM, i.e., the rVLM and the cVLM, respectively, have
been shown to play opposing roles in regulating and integrating
cardiovascular adjustments resulting from local or peripheral
stimulation (17, 42). The rVLM is critical in central regulation of
sympathetic nerve discharge because it projects directly to
preganglionic neurons in the intermediolateral cell column of the
spinal cord (3, 17). In contrast, the cVLM neurons send inhibitory
projections to the rVLM neurons that appear to tonically inhibit
rVLM-neuronal activity (17, 43). Both the rVLM and cVLM have been
demonstrated to be involved in integration of blood pressure and HR
responses during static muscle contraction. For example, lesioning the
rVLM (5) or locally administering kynurenic acid, an excitatory amino
acid antagonist (4), abolishes the pressor response during muscle
contraction. In addition, radioactive glucose (22) and c-Fos expression
(24) studies have identified both the rVLM and cVLM as important areas active during the exercise pressor reflex. Thus the role of the rVLM
and cVLM in integrating cardiovascular responses during static exercise
has become a focus of sustained interest.
Histofluorescence and neuroanatomic studies have described the
existence of several neurotransmitters in the VLM, including catecholamines and other neuropeptides, contributing to regulation of
cardiovascular functions (for reviews, see Refs. 8 and 35). In
addition, clustered groups of 5-HT cell bodies/neurons have been
identified in the VLM of rats (6, 20, 36). The term "B1/3 cell
group" for the 5-HT-containing neurons located within the rVLM was
introduced by Jacobs et al. (23). Furthermore, the role of 5-HT in the
VLM in evoking centrally mediated cardiovascular effects has been well
documented by numerous investigators (10, 15, 19, 27, 28). However, a
recent review of the involvement of 5-HT in the central regulation of
cardiovascular homeostasis notes that "findings regarding release of
endogenous 5-HT itself in the VLM are scarce" (35). Also, there is
no literature with respect to release of 5-HT in the extracellular
space within either the rVLM or the cVLM. The present study is the
first determination of such release of 5-HT from both the rVLM and cVLM
using microdialysis techniques that allow a site-specific approach to
the measurement of release of neurotransmitter(s). We have demonstrated
that extracellular 5-HT in the rVLM increases during static muscle
contraction, whereas 5-HT levels in the cVLM do not change during
muscle manipulation. These results suggest that release of 5-HT in the
rVLM appears to play a role in mediating cardiovascular responses
during static exercise. It is noteworthy that plasma levels of 5-HT
have been shown to increase during exercise in humans (31). Static
muscle contraction increased 5-HT by ~75% (0.9 fmol). Although this
femtomolar concentration seems quite small, it may represent a
functionally important increase. A change of 0.9 fmol represents 15 × 106 molecules of 5-HT.
Depending on the number of synapses that the probe sampled, this change
may have a physiological significance on the neurochemistry of rVLM
regulation during cardiovascular responses elicited by muscle
contraction. The relation between this release of 5-HT and a
cardiovascular response is shown in the present study. However, the
potential functional implication of the 15 × 106 molecules of 5-HT increase
cannot be fully explained by our study.
The VLM neurons contain a high density of 5-HT binding sites,
particularly the 5-HT1A subtype
(38). These 5-HT1A receptors are
present in the C1 area of the rVLM, the raphe pallidus, the parapyramidal region, the ventromedial medulla, and scattered locations
throughout the VLM (38, 39). These receptors play a role in mediating
cardiovascular effects, because local or iontophoretic application of
the 5-HT1A-receptor agonist
8-OH-DPAT in the rVLM inhibits neuronal activity of the rVLM and
elicits a decrease in MAP and HR by reducing efferent sympathetic
outflow (19, 41). Furthermore, activation of
5-HT1A receptors in the rVLM, but
not in the cVLM, results in attenuation of cardiovascular responses
evoked during static muscle contraction in anesthetized rats (2), which
is confirmed by the present study. In addition, the present study
measures changes in extracellular fluid 5-HT levels during muscle
contraction before and after administration of 8-OH-DPAT and supports
the concept put forward by Ally and colleagues (2) that
5-HT1A-receptor activation
attenuates MAP and HR changes during muscle contraction via an
inhibition of presynaptic 5-HT release. Presumably, 8-OH-DPAT is
selective for 5-HT1A
receptors, because previous studies have shown that prior administration of the
5-HT1A-receptor antagonist NAN-190
blocked cardiovascular effects of the drug (2, 14, 15). Furthermore, because prior administration of NAN-190 blocked the attenuating effects
of 8-OH-DPAT on 5-HT, MAP, and HR observed in the experiments in which
8-OH-DPAT was microdialyzed alone, it can be suggested that the
selectivity for 5-HT1A receptors
was functionally specific. However, the 2 mM dose of 8-OH-DPAT (final
extracellular concentration of the drug as determined by in vitro
recovery experiments; see below) may affect other receptors besides
5-HT1A, including
5-HT1B, 5-HT2, and
2-adrenergic receptors (28,
30). Therefore, we cannot exclude the possibility that 8-OH-DPAT
attenuated the cardiovascular responses and 5-HT concentration during
muscle contraction via these other receptors. Along the same line,
Adell et al. (1) suggested that 10 mM 8-OH-DPAT, administered into the
raphe nucleus, may produce nonspecific effects. Similarly, it has been
shown that a 2 mM dose of NAN-190 binds with
5-HT1B,
5-HT2, and
2-adrenergic receptors in
addition to 5-HT1A receptors (16).
However, Nóbrega et al. (32) demonstrated that the specific
5-HT1B-receptor agonist 1-[3-(trifluoromethyl)phenyl]piperazine failed to modify
cardiovascular responses to muscle contraction, suggesting
that 5-HT1B receptors have no
possible role in regulating blood pressure and HR responses during
static contraction. In addition, it has been shown that the
5-HT2-receptor antagonist
ketanserin and the
2-adrenoceptor blocker idazoxan
failed to inhibit cardiovascular effects of 8-OH-DPAT, thereby
suggesting that 8-OH-DPAT effects are not mediated via 5-HT2 or
2-adrenergic receptors (14,
28). Nevertheless, the present study using the 2 mM concentration of
8-OH-DPAT and NAN-190 cannot clearly rule out the possibility of an
effect of the drugs on other receptors besides
5-HT1A. Our findings of increased
5-HT release and successful pharmacological manipulation with a
5-HT1A agonist add to the scheme
of central cardiovascular regulation via neurotransmitters and provide
additional insights into the neural control of blood pressure during
static exercise.
Microdialysis of 8-OH-DPAT into the rVLM resulted in a 10-fold increase
in basal extracellular 5-HT levels. During the contraction period,
8-OH-DPAT was continuously dialyzed for 10 min. This procedure strengthens our finding that, despite a 10-fold increase in 5-HT while
8-OH-DPAT was administered during rest, muscle contraction indeed
attenuated 5-HT release in the rVLM, further suggesting that 8-OH-DPAT
attenuates cardiovascular responses during muscle contraction via a
decrease in extracellular 5-HT concentration. However, because prior
administration of NAN-190 blocked the 10-fold increase in 5-HT after
administration of 8-OH-DPAT during rest and inhibited the increase in
5-HT during muscle contraction, it can be assumed that 8-OH-DPAT
mediated its effects via 5-HT release. Using in vivo brain
microdialysis, studies have measured the somatodendritic release of
5-HT in the raphe nuclei of the rat (1, 14). Perfusion of 1 µM
8-OH-DPAT into the raphe did not produce any significant effect on 5-HT
release; however, at doses of 10 mM and higher, 8-OH-DPAT
dose-dependently increased dialysate 5-HT within the raphe (1). That
study (1) suggests that in vivo 5-HT release does not depend on nerve
impulses of serotonergic neurons and that somatodendritic release of
5-HT is independent of local autoreceptor activation. Furthermore, Adell and co-workers (1) have also postulated that the increase in
extracellular 5-HT concentration after local dialysis of 8-OH-DPAT is
possibly due to a blockade of the 5-HT transport, because the drug
inhibits the 5-HT reuptake process. Because our results revealed an
increased baseline level of 5-HT in the rVLM after microdialysis of 10 mM 8-OH-DPAT, it is possible that a serotonergic mechanism similar to
the raphe is present in the rVLM. However, this study cannot clearly
explain potential mechanisms by which 8-OH-DPAT decreases 5-HT levels
to less than control levels during muscle contraction but not during
rest. To the best of our knowledge, this study is the first to present
this data on the basis of measurement of 5-HT in the rVLM. However, it
has been shown that 8-OH-DPAT administration into the ventromedial
medulla (a region ~1 mm medial to the rVLM) decreases extracellular
concentrations of 5-HT in the ventromedial medulla, and it has been
suggested that this decrease in 5-HT may play a role in modulation of
nociception via 5-HT1A-receptor
activation (37). It may be possible that a mechanism similar to that in
the ventromedial medulla exists in the rVLM. This may also explain the
decrease in 5-HT during muscle contraction after perfusion of
8-OH-DPAT. However, other potential mechanisms for the action of
8-OH-DPAT exist in addition to the effects on 5-HT release. For
example, a recent study has shown that 8-OH-DPAT induces release of
norepinephrine in the hippocampal formation using in vivo methods,
suggesting a noradrenergic-serotonergic mechanism (18). Furthermore,
8-OH-DPAT has also been shown to facilitate acetylcholine release in
the rat frontal cortex (9). These effects of 8-OH-DPAT raise the
possibility of other potential mechanisms for its action. However, the
exact mechanism of the increased 5-HT level in the rVLM after
administration of 8-OH-DPAT cannot be explained by the present study.
Doses of 8-OH-DPAT <10 mM have shown no attenuating effects on
cardiovascular responses during contraction (2). Nevertheless, it is
possible that the increased baseline 5-HT level after microdialysis of
8-OH-DPAT might have contributed to the attenuation of 5-HT levels
following muscle contraction. However, this seems unlikely given the
fact that a negative percent change in 5-HT concentration occurred during the contraction following the drug, strongly suggesting that the
higher baseline may not have been the causative factor.
The 10-fold increase in basal 5-HT following 8-OH-DPAT (10 mM)
administration did not elicit a shift in baseline blood pressure or HR.
The concentration of 5-HT in the rVLM after 8-OH-DPAT perfusion was
increased to 7 fmol/20 µl of extracellular fluid. Thus far, no
microdialysis studies have been done in which 5-HT is administered into
the rVLM along with the recording of concomitant changes in blood
pressure. However, intracerebroventricular administration or local
application of 5-HT into the nucleus tractus solitarii has been
performed in previous studies. For example, in one study (7) in which
5-HT was locally injected into the nucleus tractus solitarii, the dose
of 5-HT was in the picomolar range to elicit a change in blood
pressure. That picomolar dose (7) is higher than the femtomolar
concentration of 5-HT measured following 8-OH-DPAT administration in
our experiments, and hence it appears that the concentration of 5-HT in
the femtomolar range is not sufficient to evoke a change in blood
pressure. In another study (13) using intracerebroventricular
administration of 5-HT, a dose of 100 nmol was shown to evoke an
increase in blood pressure, a concentration much higher than that
measured in the rVLM after 8-OH-DPAT perfusion in our study.
Furthermore, the prolonged administration and the low rate of perfusion
of 8-OH-DPAT also attribute to the lack of effect on baseline
cardiovascular parameters. In addition, on the basis of in vitro
recovery studies, it has been determined that ~20% of 8-OH-DPAT
crossed the microdialysis membrane, a concentration much less than that
required to evoke a change in blood pressure as shown in a previous
study (25). In our experiments, 5-HT was sampled site-specifically by
the microdialysis probes from the rVLM or cVLM. We determined correct
placement of the probes within the rVLM or cVLM by perfusing
L-glutamate and observing a
pressor or depressor response, respectively. Furthermore, analyses of
the diffusion of methylene blue dye, microdialyzed for the same
duration and at the same rate as the drug, showed a spread of ~600
µm, suggesting that the dialysate was collected from within the
respective area. Obviously, the possibility of collecting 5-HT from
distant areas and a potentially wider diffusion of 8-OH-DPAT to
structures other than the rVLM exists. However, our results demonstrated an increase in 5-HT during muscle contraction measured from the rVLM and no change when collected from the cVLM. The rVLM and
cVLM in the rat are separated by ~1.5 mm. If 5-HT was sampled from an
area that was 1.5 mm away or farther, then analyses of 5-HT collected
from the cVLM region would have shown an increase during muscle
contraction. Therefore, our experimental method proves that the
sampling was site specific. Furthermore, Ally and co-workers (2) have
shown that administration of 10 mM 8-OH-DPAT into the cVLM did not
attenuate the cardiovascular responses during muscle contraction. In
our study, the drug and dose used were the same as those used by Ally
et al. (2), and dye diffusion studies indicate that 8-OH-DPAT did not
spread to an area that is 1.5 mm away. This suggests that the drug
possibly diffused similar to the dye and that 5-HT was sampled from
within the rVLM or the cVLM.
In conclusion, the present study demonstrated that an increase in
extracellular fluid concentration of 5-HT in the rVLM, but not in the
cVLM, is associated with cardiovascular responses elicited during
static muscle contraction. Furthermore, activation of
5-HT1A receptors within the rVLM
inhibits increases in MAP and HR during muscle contraction mediated via
a reduction in extracellular concentration of 5-HT. The present study
provides conceptual information regarding the mechanism of
5-HT1A modulation of the exercise
pressor reflex and sheds further light on the neurochemical basis of
circulatory adjustments during static exercise.
| |
ACKNOWLEDGEMENTS |
D. Caringi was supported by a fellowship from the American Heart
Association (Maine Affiliate) and the University of New England Dean's
Summer Research Grant of 1996. G. Asmundsson also received the 1996 University of New England Dean's Summer Research Grant.
| |
FOOTNOTES |
A part of this study has been previously presented in abstract form
(FASEB J. 11: A53, 1997).
Present address of T. Ishide: 3rd Dept. of Internal Medicine, Chiba
University School of Medicine, Inohana 1-8-1, Chiba 260, Japan.
Address for reprint requests: A. Ally, Depts. of Pharmacology and
Biochemistry, College of Osteopathic Medicine, Univ. of New England, 11 Hills Beach Rd., Biddeford, ME 04005.
Received 24 April 1997; accepted in final form 2 September 1997.
| |
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Abstract
Introduction
