Activation of 5-hydroxytryptamine-1A receptors suppresses cardiovascular
responses evoked from the paraventricular nucleus
Jouji Horiuchi, Alp Atik, Kamon Iigaya, Lachlan M. McDowall, Suzanne Killinger,
and Roger A. L. Dampney
School of Medical Sciences (Physiology) and Bosch Institute, University of Sydney, New South Wales, Australia
Submitted 18 March 2011; accepted in final form 12 July 2011
Horiuchi J, Atik A, Iigaya K, McDowall LM, Killinger S, Dampney
RAL. Activation of 5-hydroxytryptamine-1A receptors suppresses car-
diovascular responses evoked from the paraventricular nucleus. Am J
Physiol Regul Integr Comp Physiol 301: R1088–R1097, 2011. First
published July 13, 2011; doi:10.1152/ajpregu.00144.2011.—Activation
of central 5-hydroxytryptamine-1A (5-HT1A) receptors powerfully
inhibits stress-evoked cardiovascular responses mediated by the dor-
somedial hypothalamus (DMH), as well as responses evoked by direct
activation of neurons within the DMH. The hypothalamic paraven-
tricular nucleus (PVN) also has a crucial role in cardiovascular
regulation and is believed to regulate heart rate and renal sympathetic
activity via pathways that are independent of the DMH. In this study,
we determined whether cardiovascular responses evoked from the
PVN are also modulated by activation of central 5-HT1Areceptors. In
anesthetized rats, the increases in heart rate and renal sympathetic
nerve activity evoked by bicuculline injection into the PVN were
greatly reduced (by 54% and 61%, respectively) by intravenous
administration of (?)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-
DPAT), an agonist of 5-HT1A receptors, but were then completely
restored by subsequent administration of WAY-100635, a selective
antagonist of 5-HT1A receptors. Microinjection of 8-OH-DPAT di-
rectly into the PVN did not significantly affect the responses to
bicuculline injection into the PVN, nor did systemic administration of
WAY-100635 alone. In control experiments, a large renal sympatho-
excitatory response was evoked from both the PVN and DMH but not
from the intermediate region in between; thus the evoked responses
from the PVN were not due to activation of neurons in the DMH. The
results indicate that activation of central 5-HT1A receptors located
outside the PVN powerfully inhibits the tachycardia and renal sym-
pathoexcitation evoked by stimulation of neurons in the PVN.
dorsomedial hypothalamus; renal sympathetic activity; heart rate;
IT IS NOW WELL ESTABLISHED that activation of 5-hydroxytrypta-
mine-1A (5-HT1A) receptors in the brain can modulate physi-
ological and behavioral responses to a wide range of stressors.
In particular, systemic administration of (?)8-hydroxy-2-(di-
n-propylamino)tetralin (8-OH-DPAT; an agonist of 5-HT1A
receptors) has been shown to reduce, in a dose-dependent
manner, the increases in heart rate and arterial blood pres-
sure evoked by acute psychological stressors (33–35, 48,
49). Systemic administration of 8-OH-DPAT also reduces
the cutaneous vasoconstriction evoked by physical stressors
such as cold exposure or fever (34, 37, 38). In several of
these studies the effects of 8-OH-DPAT were shown to be
selective for the 5-HT1Areceptors, because they were abol-
ished by the selective 5-HT1A receptor antagonist WAY-
100635 (33, 35, 37, 38, 49). In addition, the effects of
8-OH-DPAT can be attributed to a central action of the drug
because it is known that systemically administered 8-OH-
DPAT affects sympathetic activity by an action on the
central nervous system (18, 29).
Microinjection of 8-OH-DPAT into the medullary raphé
reduces the tachycardia evoked by psychological stress or
fever (33, 35), and microinjection of WAY-100635 into this
region reverses the inhibition of cold-evoked cutaneous
vasoconstriction induced by systemic administration of
8-OH-DPAT (38). Thus, the medullary raphé is one site at
which 5-HT1Areceptors can suppress stress-evoked cardiac
or cutaneous vasoconstrictor responses (33, 35, 38). At the
same time, activation of 5-HT1Areceptors located in higher
brain regions, such as limbic regions, may also reduce
stress-evoked cardiovascular responses via an anxiolytic
effect rather than via a direct effect on brainstem cardiovas-
cular neurons (49).
Apart from the fact that they are inhibited by activation of
central 5-HT1Areceptors, cardiovascular responses evoked by
acute psychological stress, cold exposure, or fever are also all
mediated to a significant extent by neurons within the dorso-
medial hypothalamus (DMH) (9–11, 32). Furthermore, the
cardiovascular responses evoked by disinhibition of neurons in
the DMH of anesthetized rats are also powerfully inhibited by
systemic administration of 8-OH-DPAT, and these effects are
also completely reversed by subsequent administration of
WAY-100635 (24). This raises the question as to whether
cardiovascular responses mediated by brain regions that are
independent of the DMH are also modulated by activation of
central 5-HT1Areceptors. In particular, the hypothalamic para-
ventricular nucleus (PVN) contains neurons that regulate the
sympathetic outflow via descending pathways that do not
include synapses in the DMH [e.g., direct projections to sym-
pathetic preganglionic neurons and an indirect projection via
sympathetic premotor neurons in the rostral ventrolateral me-
dulla (RVLM)] (7, 44, 46).
The PVN has long been known to have a crucial role in
cardiovascular regulation, particularly in regard to the mainte-
nance of fluid balance (6, 14). The PVN may also be involved
in the long-term regulation of sympathetic activity and blood
pressure (8), and there is now considerable evidence that it
contributes to the sustained activation of renal and cardiac
sympathetic activity that occurs in heart failure and neurogenic
hypertension (1, 4, 14, 40). It has been proposed that the PVN
is a potential target for treatment of sympathetic dysfunction in
these conditions (14), and so it is important to identify the
mechanisms that modify the regulation of sympathetic activity
by the PVN. The aim of this study was to test whether
activation of 5-HT1A receptors can modify the regulation of
Address for reprint requests and other correspondence: R. A. L. Dampney,
School of Medical Sciences (Physiology), F13, The Univ. of Sydney, NSW
2006, Australia (e-mail: email@example.com).
Am J Physiol Regul Integr Comp Physiol 301: R1088–R1097, 2011.
First published July 13, 2011; doi:10.1152/ajpregu.00144.2011.
0363-6119/11 Copyright © 2011 the American Physiological Societyhttp://www.ajpregu.org R1088
arterial pressure, heart rate, and renal sympathetic nerve activ-
ity by the PVN.
MATERIALS AND METHODS
General procedures. Experiments were performed using male
Sprague-Dawley rats (body wt 370–480 g) supplied by University of
Sydney Laboratory Animal Services. All experimental procedures
were approved by the Animal Ethics Committee of the University of
Sydney and were carried out in accordance with the Guidelines for
Animal Experimentation of the National Health and Medical Research
Council of Australia. Anesthesia was initially induced by inhalation of
isoflurane (2–3% in oxygen-enriched air). A thermo-regulated heating
pad was used to maintain body temperature in the range 37–38°C as
measured via a rectal probe. Catheters were placed in a femoral artery
and a femoral vein for the recording of pulsatile arterial pressure and
drug injection, respectively. After the surgery, the isoflurane anesthe-
sia was gradually withdrawn while being replaced by urethane (1.3
g/kg iv with supplementary doses of 0.1 g/kg iv, if required). The
adequacy of anesthesia was verified by the absence of the corneal
reflex and a withdrawal response to nociceptive stimulation of a hind
paw. A tracheotomy was performed to maintain an unobstructed
airway, and the animals were allowed to breathe freely.
The rat was then mounted prone in a stereotaxic frame with the
incisor bar at 19 mm below the interaural line. The dorsal surface of
the cortex was exposed for approaching the PVN and DMH. The renal
sympathetic nerve on the left side was isolated from surrounding
connective tissue and its activity was recorded as described previously
(16). The signal from the electrodes was amplified, passed through a
band pass filter (100–1,000 Hz), and then rectified and integrated over
successive 10-s intervals. At the end of the experiments, the baseline
noise level of renal sympathetic nerve activity (RSNA) was deter-
mined by application of 2% lignocaine to the proximal end of the
nerve. The mean arterial pressure (MAP) and heart rate (HR) signals
were derived from the pulsatile pressure signal via a low-pass filter
and rate meter, respectively. All signals were recorded on a computer
using a PowerLab system (AD Instruments).
Microinjections were made using a glass micropipette held in a
micromanipulator at an angle of 28 degrees (tip caudal). The com-
pound injected was bicuculline methochloride (20 pmol or 10 pmol in
20 nl; Tocris Bioscience, Ellisville, MO). The vehicle solution was
artificial cerebrospinal fluid adjusted to pH 7.4, and the drug solution
contained 1% fluorescent microspheres to allow later histological
determination of the injection sites. The tip of the micropipette was
positioned stereotaxically (1.8 mm caudal to the bregma, 0.5–0.6 mm
lateral to the midline, and 7.5–7.6 mm from the surface of the cortex).
In some experiments, the tip of the micropipette was positioned in
the DMH (3.1 mm caudal to bregma, 0.5–0.6 mm lateral to the
midline, and 8.5–8.6 mm ventral to the surface of the cortex) or at a
site in between the PVN and DMH (2.5 mm caudal to bregma,
0.5–0.6 mm lateral to the midline, and 7.5–7.6 mm ventral to the
surface of the cortex). Microinjections were made by pressure, and the
volume injected was measured by the displacement of the meniscus in
the pipette with respect to a horizontal grid viewed through an
Experimental procedures. In six rats, an initial microinjection of
bicuculline (20 pmol in 20 nl) was made into the PVN. After a waiting
period of 30–50 min, to allow for all cardiovascular variables to
stabilize again, the selective 5-HT1A receptor agonist 8-OH-DPAT
(Tocris Bioscience) was administered (100 ?g/kg iv). After a further
5–10 min, a second microinjection of bicuculline was made into the
same PVN site, and there was then a further waiting period of 30–50
min. The selective 5-HT1Areceptor antagonist WAY-100635 (Sigma)
was then administered (100 ?g/kg iv) after which there was a further
waiting time of 5–10 min followed by a third and final microinjection
of bicuculline into the same site in the PVN.
In a second series of experiments in eight rats, the effects of
WAY-100635 alone on the responses was also tested. In these
experiments, a microinjection of bicuculline [either 20 pmol (n ?
4) or 10 pmol (n ? 4)] was first made into the PVN. After a waiting
time of 30–60 min, WAY-100635 was administered (100 ?g/kg
iv) after which there was a further waiting time of 5–10 min
followed by a second microinjection of bicuculline into the same
site in the PVN.
In a third series of experiments in seven rats, the effect of blockade
of 5-HT1Areceptors within the PVN itself on the responses evoked by
microinjection of bicuculline into the PVN was also tested. In these
experiments, the procedure was the same as in the first series of
experiments, except that instead of a systemic injection of 8-OH-
DPAT, a microinjection of 8-OH-DPAT (1 nmol in 100 nl solution)
was made at the same coordinates in the PVN as the site at which
bicuculline was injected.
In a fourth series of experiments in 10 rats, the responses evoked by
microinjections of bicuculline (20 pmol in 20 nl) in the PVN were
compared with those evoked by microinjections into the DMH or into
an intermediate region in between in the same experiment. In these
experiments there was also a waiting period of 30–50 min between
At the end of each experiment, the rat was euthanized with an
overdose of pentobarbital sodium, the brain was removed, and after
fixation in 4% paraformaldehyde solution, coronal sections (50
?m) were cut on a freezing microtome and mounted onto glass
slides. The labeled microinjection sites were identified by exam-
ining the sections under a fluorescence microscope. In the third
series of experiments, in which microinjections of bicuculline and
of 8-OH-DPAT were made into the same coordinates in the PVN,
each injectate contained microspheres with different colored fluo-
rescent labels, so the injection sites could be distinguished. Injec-
tion sites were determined using a fluorescence microscope and
mapped onto standard sections of the atlas by Paxinos and
Data analysis. The baseline MAP, HR, and RSNA were measured
as the average values of these variables over the 2-min period
preceding each microinjection into the PVN, DMH, or intermediate
region. The maximum changes in MAP, HR, and integrated RSNA
compared with their respective preinjection baseline levels were
determined following each microinjection of bicuculline. One-factor
ANOVA was used to compare the bicuculline-evoked peak changes in
MAP, HR, and RSNA before and after 8-OH-DPAT administration,
and after subsequent WAY-100635 administration, followed by
paired comparisons using the Student’s t-test with application of the
Holm step down procedure for multiple comparisons as appropriate
(45). The same procedure was used to compare the increases in MAP,
HR, and RSNA evoked by bicuculline microinjections into the PVN,
DMH, and intermediate region. The time courses of the changes in
MAP, HR, and RSNA following bicuculline microinjection under
different conditions were compared using two-factor ANOVA,
where the factors were treatment (control, after 8-OH-DPAT
administration, or after subsequent WAY-100635 administration)
and time (after injection). For the experiments in which the effects
of WAY-100635 alone was tested on the peak responses evoked by
microinjections of bicuculline (10 or 20 pmol) into the PVN,
two-factor ANOVA was also used, where the factors were treat-
ment (control or WAY-100635 administration) and dose of bicu-
culline. A value of P ? 0.05 was taken as statistically significant.
All values are presented as means ? SE.
As previously reported (20, 24, 36), intravenous injection of the
5-HT1A receptor agonist 8-OH-DPAT (100 ?g/kg) resulted in
decreases in MAP and HR, reaching new stable levels by 5–10
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