Vol. 283, Issue 5, H1975-H1984, November 2002
c-Fos expression in the midbrain periaqueductal gray after
chemoreceptor and baroreceptor activation
Linda F.
Hayward and
Marcela
Von
Reitzenstein
Department of Physiological Sciences, University of
Florida College of Veterinary Medicine, Gainesville, Florida 32601
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ABSTRACT |
The pattern of Fos-like
immunoreactivity (FLI) in the periaqueductal gray (PAG) associated with
activation of arterial chemoreceptors versus baroreceptor afferents was
examined in urethane-anesthetized rats. Chemoreflex responses elicited
by repeat intravenous injections of potassium cyanide (KCN; 90 µg/kg)
significantly increased FLI in all columns of the PAG relative to
saline-injected animals. Pressor responses elicited by intravenous
phenylephrine (PE) produced a similar pattern of increased FLI
throughout the PAG except in the dorsomedial and lateral columns of the
caudal PAG, where FLI was minimal. Chemoreflex responses were unaltered
by blockade of excitatory amino acid receptors in the dorsomedial PAG,
and <10% of the neurons of the caudal PAG that expressed FLI after KCN stimulation were retrogradely labeled from the A5 region of the
caudal ventrolateral pons. These results indicate that integration of
chemoreceptor inputs occurs primarily in the dorsal and lateral columns
of the caudal PAG, but these neurons have little direct descending
influence over lower brain stem regions integral to the central
arterial chemoreflex arc.
midbrain central gray; A5; potassium cyanide; baroreceptors
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INTRODUCTION |
IN MANY
ANIMALS, brief stimulation of arterial chemoreceptors with
cyanide evokes a stereotypic pattern of hyperventilation coupled with
vasodilation in muscle vascular beds and vasoconstriction in the renal
and mesenteric beds (19, 30). In conscious animals, cardiorespiratory adjustments are coupled with behavioral responses that range from mild arousal to running or escape-like behavior (13). Both cardiorespiratory and arousal responses are
eliminated by selective denervation of the carotid sinus nerve
(12). This suggests these responses are mediated through
selective activation of arterial chemoreceptors in the carotid body. On
the basis of similarities between the response to peripheral
chemoreceptor activation and the alerting response evoked by
threatening stimuli, it has been hypothesized that activation of
"defense areas" in the brain, including the midbrain periaqueductal
gray (PAG) (3, 4, 6, 10, 27), must be an integral
component of the central arterial chemoreflex arc (19,
29-31). This hypothesis has been substantiated by numerous
studies demonstrating that c-Fos protein expression increases within
different columns of the PAG after prolonged stimulation of arterial
chemoreceptors (7, 11, 21, 22, 41).
Yet, our understanding of the role of the PAG in the central arterial
chemoreflex pathway remains relatively rudimentary. It remains to be
determined whether lesion of the PAG has any influence on
cardiorespiratory or behavioral responses to chemoreceptor activation.
Furthermore, none of the previous studies examining the effects of
chemoreceptor stimulation on c-Fos expression in the PAG has controlled
for the influence of baroreceptor afferents. Exposure to systemic
hypoxia or direct chemoreceptor afferent nerve stimulation can produce
profound changes in blood pressure and heart rate (HR) (13, 18,
20). Associated fluctuations in baroreceptor inputs may
independently trigger increased c-Fos expression in the PAG (25,
33, 36, 38). The importance of taking into account the influence
of baroreceptor-mediated changes was recently highlighted in a study
examining the c-Fos expression in the PAG after muscle afferent
stimulation (26). In that study, sustained muscle
contractions were shown to significantly increase c-Fos
expression throughout the middle and caudal regions of the PAG. Yet
when the same experimental conditions were applied in
baroreceptor-denervated animals, c-Fos expression declined by 50% or
more. This suggested that changes in baroreceptor input during
experimental manipulation of cardiorespiratory reflexes could play
a significant role in c-Fos expression patterns in the PAG.
The present study was undertaken to examine the contribution of
baroreceptor inputs to c-Fos expression in the PAG after prolonged arterial chemoreceptor activation. On the basis of previous studies, it
was hypothesized that approximately one-half of the c-Fos expression in
the PAG observed after chemoreceptor afferent stimulation was likely to
be associated with reflex-mediated changes in baroreceptor afferent
input. The second objective of the present study was to identify
whether PAG neurons activated by chemoreceptor input play a significant
role in modulating arterial chemoreflex responses in the rat. It was
hypothesized that chemoreceptor activation of PAG neurons plays a
significant role in the arterial chemoreflex response of the rat.
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METHODS |
All experiments were carried out on adult male Sprague-Dawley
rats (weight, 350-420 g) housed in the university animal care facility and exposed to a normal 12-h light (6 AM to 6 PM) to 12-h dark
(6 PM to 6 AM) cycle. All experimental procedures were preapproved by
the University of Florida Institutional Animal Care and Use Committee.
Retrograde labeling.
A small subset of rats underwent placement of a retrograde tracer into
the ventrolateral pons 1 wk before experimentation. Each animal was
deeply anesthetized with a mixture of ketamine-acepromazine (100:10
mg/kg ip). With the use of sterile procedures, the animal was placed in
a stereotaxic head holder (Kopf; Tujunga, CA), and a small hole was
drilled in the skull. A small-diameter glass microinjection pipette
(tip diameter, 15-30 µm) was filled with 2.5% Fluoro-Gold
(Fluorochrome; Denver, CO) diluted in physiological saline. The
microinjection pipette was attached to a microinjection pressure system
(model PPS-2, Medical Systems; Greenvale, NY) and lowered into the left
side of the brain using a micropositioner (Kopf MP660). Coordinates for
microinjection into the ventrolateral pons/A5 cellular region were 
10
to 10.3 mm caudal to the bregma, 2-2.3 mm lateral from midline,
and 8.0 mm ventral to the surface of the brain [coordinates based on
Paxinos and Watson's Rat Brain in Stereotaxic Coordinates
(37)]. The caudal ventrolateral pons was chosen as a
target site based on previous work suggesting that the PAG has more
descending projections to the caudal versus rostral ventrolateral pons
(1, 8). The retrograde tracer was then pressure injected
into the left side of the brain over the course of 30-60 s. The
volume injected was determined by carefully monitoring the movement of
the meniscus with a calibrated ×40 monocular (Titan Tools; Buffalo,
NY). The pipette remained in position for 3 min after fluid ejection
and was then retracted. The wound was sutured (Ethilon, 4-0), and an
antibiotic ointment was applied to the skin. Supplemental doses of
anesthesia were administered intraperitoneally during surgery as
needed. Body temperature was continuously monitored with a rectal probe
and maintained at 38 ± 1°C with a heating pad. To help relieve
postoperative pain, all animals received a subcutaneous injection of
buprenorphine (0.1-0.2 mg/kg). Animals were closely monitored
during recovery for any signs of discomfort or infection. All animals
recovered well from surgery, maintaining steady weight gain and water consumption.
General preparations.
At the time of the terminal experiment, all animals were anesthetized
with urethane (1.3-1.5 gm/kg ip). After the induction of
anesthesia, animals were placed in the supine position and instrumented
with a right carotid arterial catheter (polyethylene-50 tubing) to
record arterial pressure and a right jugular venous catheter for
intravenous fluid administration. A tracheotomy was performed, and the
animals were intubated. A small incision was made in the skin
subcostally. Two small (0.003 mm diameter) Teflon-coated, stainless
steel wires with bared tips were inserted through the abdominal
musculature into the right side of the diaphragm for measurement of
spontaneous inspiratory electromyographic (EMG) activity. Body
temperature was monitored continuously with a rectal temperature probe
(Harvard Apparatus; Holliston, MA) and kept within a normal range
(38 ± 1°C) with a heating blanket. Supplemental anesthesia was
continued (0.05-0.1
g · kg
1 · h1)
thereafter until the experiment was terminated.
The arterial catheter was attached to a calibrated pressure transducer
(Statham) connected to an amplifier (Stoelting; Wood Dale, IL) for
continuous monitoring of arterial pressure. The analog output from the
blood pressure amplifier was connected to a computer data sampling
system [Cambridge Electronics Design (CED) 1401 computer interface;
Cambridge, UK]. The diaphragm EMG (dEMG) wires were connected to a
Grass preamplifier probe (H1P5, Grass Instruments; West Warwick, RI) in
series with a signal amplifier (P511). The dEMG signal was amplified
(5,000-50,0000 times) and bandpass filtered (0.3-3.0 kHz).
The signal was rectified, integrated (Paynter Filter, 50-ms time
constant, BAK Electronics; Rockville, MD), and then sampled using CED
software (Spike2). The baseline dEMG was arbitrarily adjusted to a
value of 1.0-2.0 (arbitrary units) at the beginning of the
experiment. dEMG and arterial pressure were recorded simultaneously.
Experimental procedures for chemoreceptor or baroreceptor
stimulation in c-Fos experiments.
Animals were placed in the supine position after the completion of all
instrumentation. A rest period of 10-15 min was given before drug
administration. Each animal then received 11 bolus intravenous
injections of a single type of solution, repeated every 2 min. This was
followed by a 90-min rest period before euthanization. Chemoreflex
responses were elicited by intravenous injection of potassium cyanide
(KCN; 0.5 mg/ml dissolved in saline). Individual KCN doses were flushed
through the catheter with 150 µl of saline. KCN was chosen as a
stimulus because it provides brief and potent activation of arterial
chemoreceptors and elicits reproducible reflex responses upon repeated
administration (18, 19, 23). Furthermore, the
cardiorespiratory response to KCN in both conscious and anesthetized
rats is dependent on an intact carotid sinus nerve (13, 17,
18). KCN also has little influence on baroreceptor afferents
(12). This suggests that the effects of KCN administration
on c-Fos expression in our protocols would primarily be a function of
changes in peripheral chemoreceptor input and not the direct influence
of KCN on central neurons. The dose range used in the present study was
derived from previous studies that have demonstrated doses 
60 µg/kg
elicit marked cardiorespiratory changes in both conscious and
anesthetized rats (12, 18). In conscious animals,
intravenous injections above 60 µg/kg also elicit arousal and/or
escapelike behavior (12). Because anesthesia is known to
dampen the cardiorespiratory response to KCN (12, 18), 60 µg/kg was chosen as the lowest dose to be tested in the present study.
Baroreflex responses were elicited by intravenous injection of
phenylephrine (PE). PE doses (3-5 µg/kg) were sufficient to raise mean arterial pressure (MAP) ~10 mmHg. Individual doses of PE
were also flushed through the catheter with 150 µl of saline. In
several animals, the effects of repeated bolus injections of 150 µl
of saline alone were tested. During the rest period, all animals
were put on supplemental oxygen.
Experimental procedures for chemical blockade of the PAG.
After the induction of urethane anesthesia, a subset of naïve
animals were instrumented and placed in a stereotaxic head holder. A
small hole was drilled in the region of the skull overlying the PAG,
and a single-barrel glass microinjection pipette was lowered into the
PAG. Arterial chemoreflex responses evoked by 90 µg/kg iv KCN were
tested before and 5 and 45 min after microinjection of kynurenic acid
into the dorsal PAG. Kynurenic acid is a broad-spectrum excitatory
amino acid receptor antagonist (10 mM, Sigma; St. Louis, MO). Each
animal received two central injections (150-200 nl each) into the
left dorsal PAG, including one at 7.5 mm and one at 7.9 mm caudal
to the bregma.
In a separate group of animals, the efficacy of 10 mM kynurenic acid to
block cardiorespiratory responses evoked by excitatory amino acid
receptors was tested. First, 100 nl kynurenic acid (10 mM) was
microinjected into the dorsal PAG (7.8 mm caudal to the bregma,
0.2-0.3 mm lateral from midline, and 4.0 mm ventral from the
surface of the brain). The single-barrel pipette was then withdrawn. A
second pipette containing the GABA-A receptor antagonist bicuculline
methobromide (0.3 mM, Sigma) was immediately repositioned in the dorsal
PAG. Two separate microinjections of 40-60 nl bicuculline were
made into the dorsal PAG, separated in time from the preceding
kynurenic acid injection by 5 and 45 min. Bicuculline, a GABA-A
receptor antagonist, was used to disinhibit PAG neurons and uncover
tonic endogenous excitation (5). All microinjection drugs
were diluted in artificial cerebrospinal fluid (aCSF), which contained
(in mM) 122 NaCl, 3 KCl, 25.7 NaHCO3, and 1 CaCl2, with the pH adjusted to 7.4, mixed with a small
quantity of fluorescent microspheres for subsequent identification of
microinjection sites.
Tissue processing.
At the end of the experiment, animals were given supplemental urethane
(0.2-4 g/kg) and transcardially perfused. Animals were first
perfused with 100-200 ml of ice-cold saline containing 0.9% NaCl,
0.2 g/ml sodium nitroprusside (Sigma), and 100 IU/ml of heparin. This
was followed by 100-200 ml of ice-cold 4% paraformaldehyde (Sigma) in 0.1 M phosphate buffer (pH 7.2). The brains were removed, soaked overnight in 4% paraformaldehyde, and then placed in a 30%
sucrose (in water) solution for 24-48 h. A small cut was made on
the right, ventral surface of the brain to distinguish the right and
left sides. The brains were then frozen and sectioned into 40-µm
transverse slices with a cryostat (15°C, Zeiss HM101). Tissue
sections from those animals that underwent central microinjections of
kynurenic acid or bicuculline were directly mounted on slides, coverslipped with the fluorescent protective agent Antifade (Molecular Probes), and imaged.
Tissue slices to be processed for c-Fos protein expression were soaked
in phosphate-buffered saline (PBS; pH 7.2) for 12-24 h. Tissue
sections were washed in 3% goat serum and 0.4% Triton X (GS-T) in PBS
(3% GS-T-PBS) for 1 h and then incubated for 16-36 h at
4°C in rabbit anti-Fos antibody (1:2,000 dilution, sc52-r, Santa Cruz
Biotechnology; diluted in 1% GS-T-PBS). The tissue was rinsed in 1%
GS-T-PBS for 1 h and incubated in goat anti-rabbit biotinylated
IgG antibody (1:500, diluted in 1% GS-T-PBS, Jackson ImmunoResearch)
for 4 h. The tissue was rinsed again and incubated in an
avidin-biotinylated-peroxidase tertiary molecule solution (ABC
Vectastain Kit, Vector Labs; Burlingame, CA) for 30 min, followed by a
1% GS-T-PBS rinse. The presence of Fos-like immunoreactivity (FLI) in
the tissue slices was visualized as either a brown reaction product
after incubation of the slices for 2-3 min in a chromagen solution
[0.05% diaminobenzidine hydrochloride, 2.5% ammonium sulfate, and
0.033% hydrogen peroxide in 0.05 M Tris · HCl
(DAB), Sigma] or a blue-gray reaction product after incubation for 4 min with Vector SC. Thereafter, the tissue was rinsed three times in
PBS. According to the manufacturer's specifications, this polyclonal antibody recognizes both Fos and Fos-related proteins. Thus the staining that was obtained is described as FLI.
Brain tissue processed for the presence of both FLI (blue-gray reaction
product) and the retrograde tracer Fluoro-Gold was reincubated in 3%
GS-T-PBS for 1 h. Next, the tissue was incubated for 16-36 h
at 4°C in rabbit anti-Fluoro-Gold antibody (1:50,0000 dilution,
Fluorochrome, diluted in 1% GS-T-PBS). The tissue was rinsed in 1%
GS-T-PBS for 1 h and then incubated in goat anti-rabbit biotinylated IgG antibody (1:500, diluted in 1% GS-T-PBS, Jackson ImmunoResearch) for 4 h. Tissue sections were rinsed again and incubated for 30 min in an avidin-biotinylated-peroxidase tertiary molecule solution (ABC Vectastain Kit, Vector Labs). This was followed
by a 1% GS-T-PBS rinse. The presence of Fluoro-Gold was then
visualized as a brown reaction product after incubation for 2-3
min in DAB. Tissue slices were thereafter rinsed three times in PBS and
mounted on slides. The tissue sections were dried overnight, dehydrated, and coverslipped (Vectamount).
To test the specificity of the primary antibodies, tissue sections from
several experiments (two KCN-stimulated animals and two
saline-stimulated animals) were processed in the absence of the primary
antibody (c-Fos antibody or Fluoro-Gold antibodies). After exposure to
the secondary antibody, no FLI or Fluoro-Gold staining was found.
Microscope analysis.
All tissue sections were imaged with a light microscope (Zeiss
Axioskop) was equipped with a videocamera connected to a personal computer with image-capturing software (AIS). For each animal, a total
of six brain sections from the PAG were imaged, including two
representative sections each from the caudal, middle, and rostral PAG.
The criteria for choosing specific sections were based on anatomic
characteristics of the PAG, including 1) the shape of the
central aqueduct; 2) the shape and width of the dorsal and
ventrolateral columns; and 3) the presence of the oculomotor nucleus. The PAG image files were transferred to a second computer program (CorelDraw). Standardized outlines or "masks" of the PAG were superimposed over the images. These standardized masks outlined the boundaries of the different columns of the PAG as described by
Paxinos and Watson (37) and Beitz (6). Images
were magnified, and the numbers of FLI neurons in each PAG column, per
tissue section, were counted. The criterion for the presence of FLI
included the presence of dark black or brown label in a round structure between 7 and 10 µm in diameter, corresponding to the cell nucleus. Cells containing only lightly shaded labeling were not considered activated by the stimulus. Tissue slices that were double labeled for
both c-Fos and Fluoro-Gold were imaged and analyzed in a similar manner. The presence of double labeling, however, was confirmed by
viewing the tissue section directly under the microscope at high
magnification (×40-60).
Brain slices from those animals that only underwent central
microinjections were examined separately with a microscope equipped with epifluorescence (Zeiss). Microinjection sites were recovered and
imaged by identifying the location of the fluorophore.
Data analysis of cardiorespiratory responses.
All data were analyzed off-line using Spike2 software (CED). Peak
changes in MAP, HR, and respiratory rate (RR) during chemoreceptor or
baroreceptor stimulation were calculated from the difference between
the preceding baseline (a 10-s average measured just before each bolus
injection) and the peak deviation from baseline during chemoreceptor or
baroreceptor stimulation (a 3-s average). HR was derived from the
average interval between peak systolic pressure pulses. RR was derived
from the average interval between dEMG bursts. Changes in baseline
activity after central microinjection of bicuculline were calculated
from the difference between a 10-s average taken just before central
microinjection and a 10-s average taken between 70 and 80 s after
onset of central injection.
Statistical analysis.
Preliminary analysis (n = 6) using ANOVA with repeated
measures demonstrated no significant difference in the number of
FLI-positive cells within specific columns of the PAG as a function of
side (right or left). Because retrograde labeling from the
ventrolateral pons was primarily unilateral, a decision was made to
count FLI-positive neurons on only the left side of the PAG.
All statistical comparisons were made between tissue sections in the
same rostral-caudal position of the PAG. The effect of stimulation
condition (KCN, PE, or saline) and PAG column [dorsal (dorsal medial
and dorsal lateral), lateral, and ventrolateral] on FLI was tested
using a two-way ANOVA (effect of stimulation condition and PAG column,
StatView software). Significant differences in cardiorespiratory
responses to KCN, PE, and saline were compared using one-way ANOVA.
Main effects and interactions were examined with Scheffé's post
hoc tests. Significant differences in cardiorespiratory responses to
KCN or bicuculline before and after central microinjections of
kynurenic acid were compared using a paired t-test. Changes were considered significant when P < 0.05. All data
are reported as means ± SE.
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RESULTS |
Cardiorespiratory responses and FLI labeling after exposure to
increasing doses of KCN.
To identify whether a dose-response relationship between FLI labeling
and KCN-evoked chemoreflex responses could be demonstrated in the PAG,
three groups of animals were subjected to repeat injections of KCN (60, 90, or 120 µg/kg KCN). Table 1 shows
the average resting parameters from all three groups. Figure
1A shows a representative cardiorespiratory response to intravenous injection of KCN from a
single animal (90 µg/kg). The chemoreflex response typically included
an increase in RR that began ~5 s after the onset of the intravenous
injection. This was followed by a brief change in both MAP and HR.
Changes in RR and HR were sustained for 15-30 s. All
cardiorespiratory changes associated with KCN stimulation returned to
preinjection levels within 40 s.
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Fig. 1.
Reflex responses evoked by intravenous potassium cyanide
(KCN) injection in spontaneously breathing, urethane-anesthetized rats.
A: cardiorespiratory responses, including arterial pressure
(AP), diaphragm electromyograph (dEMG), heart rate (HR), and
respiratory rate (RR), evoked by intravenous bolus injection of 90 µg/kg KCN from a single animal. Horizontal bar, time of KCN
injection; AU, arbitrary units. B: averaged peak change in
mean AP (MAP), HR, and RR evoked after 60 µg/kg (open bars,
n = 7), 90 µg/kg (solid bars, n = 7),
and 120 µg/kg (hatched bars, n = 7) KCN. bpm, Beats
per minute; brpm, breaths/min. Peak changes were calculated relative to
the preceding baseline activity levels. *Significantly different from
the response evoked by 60 µg/kg KCN (P < 0.05).
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Figure 1B shows the average peak response of all animals
exposed to 60 (n = 7), 90 (n = 7), or
120 µg/kg (n = 7) KCN. Between animals, RR increased
significantly as KCN administration increased. In contrast, the peak
change in MAP and HR induced by 60 µg/kg KCN was significantly
different from that recorded in response to 90 µg/kg KCN but was not
significantly different from responses associated with 120 µg/kg KCN.
This lack of a dose-response-like relationship in the cardiovascular
component of the reflex reflects a more variable response to 120 µg/kg KCN between animals. For instance, 120 µg/kg KCN produced a
decrease in MAP in three of seven animals and a bradycardia in two of
seven animals.
As illustrated in Fig. 2, repeat exposure
to 90 or 120 µg/kg KCN elicited a significant (P < 0.001) increase in FLI in all columns of the PAG relative to 60 µg/kg
KCN, with two exceptions. First, there was no significant difference in
the average number of FLI cells between groups in the dorsal column of
the caudal PAG (both dorsomedial and dorsolateral columns were combined
during initial analysis). Second, there was no significant difference between the number of FLI cells in the lateral column of animals exposed to 60 versus 120 µg/kg KCN in the rostral and caudal PAG. Further comparisons demonstrated that 90 µg/kg KCN consistently induced a greater increase in FLI compared with 120 µg/kg KCN. This
increase in FLI was statistically significant (P < 0.01) in all columns except the dorsal column of the caudal PAG and the
lateral column of the rostral PAG.
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Fig. 2.
Average number of Fos-like immunoreactive (FLI)-labeled
cells observed throughout the rostral-caudal extent of the
periaqueductal gray (PAG) elicited by increasing doses of KCN. Average
FLI labeling evoked by 60 µg/kg (open bars, n = 5),
90 µg/kg (solid bars, n = 5), and 120 µg/kg
(hatched bars, n = 5) KCN is shown for all three PAG
columns. FLI was counted and averaged from six representative sections
per animal, including two sections from the caudal (8.0 to 7.8 mm
caudal to the bregma), 2 sections from the middle (7.4 to 7.2), and
2 sections from the rostral (7.0 to 6.3) PAG. Dorsal includes both
dorsomedial (dm) and dorsolateral (dl) columns combined; lat, lateral;
vlat, ventrolateral. *Significantly different from both 60 and 120 µg/kg KCN, P < 0.001; #significant difference from
60 µg/kg KCN only, P < 0.001.
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c-Fos expression after exposure to chemoreceptor versus
baroreceptor stimulation.
To test the possible role of blood pressure fluctuations and associated
changes in baroreceptor input might play in FLI labeling after
chemoreceptor stimulation, two additional groups of animals were
tested. One group received only repeat bolus injections of saline (the
vehicle of drug administration). The second group underwent repeat
bolus injection of PE. The dose of PE given was sufficient to raise MAP
10-15 mmHg above baseline, similar to the MAP change associated
with bolus injections of 90 µg/kg KCN. The change in blood pressure
evoked by PE was set to match that elicited by 90 and not 120 µg/kg
KCN, based on the more consistent pressor response evoked by 90 µg/kg. The average increases in MAP, HR, and RR evoked in response to
PE, saline, and 90 µg/kg KCN are shown in Table
2. The PE-induced increase in MAP was not
significantly different from that induced by 90 µg/kg KCN. The
PE-induced increase in MAP was, however, significantly different from
the change in MAP associated with saline injections. Bolus injections
of PE also produced small decreases in HR and RR.
Figure 3 shows photomicrographs taken
from animals that received repeat intravenous injections of saline
(left), PE (middle), or KCN (right).
Relative to saline, both PE and KCN increased FLI labeling throughout
the PAG. Comparisons between groups confirmed a trend for PE to induce
increased levels of FLI in all columns of the PAG relative to
saline-injected animals except within the dorsomedial column of the PAG
(see Fig. 4). Yet, PE-induced increases in FLI were only significantly different from saline-injected animals
in the ventrolateral column of the caudal PAG. Relative to
saline-injected animals, repeat exposure to 90 µg/kg KCN induced a
significant increase in FLI labeling throughout all columns of the
rostrocaudal extent of the PAG, with one exception. The increase in FLI
observed in the dorsolateral column in the rostral PAG was not
significantly different between saline- and KCN-injected animals.
Relative to PE-injected animals, however, 90 µg/kg KCN only increased
FLI in select regions of the PAG. These included the dorsomedial column
and the lateral columns of the caudal PAG and in the dorsomedial
columns of the middle and rostral PAG.
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Fig. 3.
FLI labeling in the PAG from urethane-anesthetized rats exposed to
repeated intravenous injections of saline, phenylephrine (PE), or KCN.
Photomicrographs show FLI from representative sections of the PAG from
one animal that received 11 repeated intravenous injections of either
saline (150 µl; left), PE (3-4 µg/kg;
middle), or KCN (90 µg/kg; right). The
approximate location of the representative sections relative to the
bregma are 7.9 mm (top), 7.6 (middle), and
7.2 (bottom). Boundaries of different PAG columns were
defined by Paxinos and Watson's rat stereotaxic atlas
(37).  , Location of the central aqueduct.
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Fig. 4.
Average number of FLI-positive cells throughout the
rostral-caudal extent of the PAG induced by repeat intravenous
injection of KCN, PE, or saline. Average FLI induced by saline (open
bars, n = 5), PE (shaded bars, n = 5),
and 90 µg/kg (solid bars, n = 5) KCN is shown for all
four PAG columns. FLI-labeled cells within the different columns of the
PAG were counted and averaged from six representative sections per
animal, including two sections from the caudal (8.0 to 7.8 mm
caudal to the bregma; left), two sections from the middle
(7.5 to 7.3, middle), and two sections from the rostral
(7.0 to 6.5, right) PAG. *Significant difference from
both saline and PE, P < 0.001; #significant difference
from saline only, P < 0.01.
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c-Fos expression in retrogradely labeled cells of the PAG with
descending projections to the ventrolateral pons.
To test the hypothesis that chemoreceptor-sensitive neurons in the PAG
have direct descending projections to critical brain stem regions
involved in arterial chemoreflex function (16), a third
group of animals was tested. These animals underwent placement of
80-100 nl of the retrograde tracer Fluoro-Gold into the caudal ventrolateral pons. One week after central placement of the retrograde tracer, animals were reanesthetized and exposed to repeated injections of 90 µg/kg KCN. In four of seven animals, the retrograde tracer was
positioned in the caudal ventrolateral pons, just lateral to the exit
of the facial nerve and medial to the inferior olive, near the position
of the A5 noradrenergic cell group (see Fig. 5A). In these animals,
extensive retrograde labeling was observed within the ventrolateral and
lateral columns of the PAG (see Fig. 6).
More moderate retrograde labeling was observed in the dorsomedial columns. Relatively few retrogradely labeled neurons were identified in
the dorsolateral cell column. Table 3
shows the distribution of double-labeled cells in the dorsal and
lateral columns of the caudal and middle PAG (those regions identified
above to primarily contain selective increases in FLI related to
chemoreceptor activation). Relatively few cells were double labeled
with Fluoro-Gold and FLI.
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Fig. 5.
Schematic of reconstructed microinjection sites in the
ventrolateral pons and dorsal PAG. All illustrations were adapted from
Paxinos and Watson (37). A: approximate
location and size of reconstructed of Fluoro-Gold microinjection sites
in the ventrolateral pons. Shaded areas represent injection sites that
resulted in retrogradely labeled neurons in dorsal, lateral, and
ventrolateral columns of the PAG; solid area represents an injection
site that only labeled neurons in the ventrolateral PAG; hatched areas
illustrate reconstructed injections sites that resulted in little or no
retrograde labeling in the PAG. B: centers of injection
sites marked by fluorescent microspheres reconstructed from dorsal PAG
microinjection studies. Shaded ovals illustrate kynurenic acid
injection sites (n = 4); solid ovals represent combined
kynurenic/bicuculline injection sites (n = 3). 3mn,
oculomotor nucleus; 7n, facial nerve; A5, noradrenaline cell group; Dr,
dorsal raphe; g7, genu of facial nerve; LC, locus coeruleus; Py,
pyramidal tract; Sp5, spinal trigeminal tract; scp, superior cerebellar
penducle; Su3, supraoculomotor periaqueductal; , location of the
central aqueduct.
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Fig. 6.
Example of the coexistence of FLI and retrogradely labeled
neurons with projections to the ventrolateral pons in the dorsal and
lateral columns of the PAG after repeat exposure to KCN. Bright-field
images of the middle PAG were taken at ×2.5 (A) and ×10
(B) magnification. Black dots indicate neurons with FLI
labeling in the nucleus; lighter-stained neurons with processes
indicate retrogradely labeled neurons 1 wk after microinjection of
2.5% Fluoro-Gold into the ventrolateral pons. Arrows indicate position
of cells double labeled for both the retrograde tracer and FLI. The
approximate location of the section shown is 8.1 mm caudal to the
bregma. Boundaries of different PAG columns are indicated by
dotted lines and were defined by Paxinos and Watson's rat stereotaxic
atlas (37); , location of the central
aqueduct.
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In two animals, the retrograde tracer was mispositioned lateral and
dorsal to the exit of the facial nerve (see Fig. 6A). In
these two animals, no retrograde labeling was observed in the PAG. In
the third animal, the retrograde tracer was misplaced in the ventral
lateral region of the dorsal pons. Only moderate retrograde tracing,
restricted to the ventral quadrant of the PAG, was observed in this
animal. In these three brains, further examination for double-labeled
cells was not made.
Effect of unilateral excitatory amino acid receptor blockade in the
PAG on chemoreflex function.
The potential role of the PAG in arterial chemoreflex responses was
examined further in four additional animals. These animals underwent
chemoreflex testing before and after central microinjection of
kynurenic acid (10 mM) into the dorsal PAG. Each animal received two
microinjections (150-200 nl each) along the rostrocaudal extent of
the left side of the PAG (one at 7.9 and one at 7.5 mm caudal to
the bregma; see Fig. 5B). Before central microinjection, 90 µg/kg KCN elicited an average increase in MAP, HR, and RR above baseline of 9 ± 3 mmHg, 19 ± 4 beats/min, and 64 ± 7 breaths/min, respectively. Five minutes after microinjection of
kynurenic acid, the average response to 90 µg/kg KCN was unchanged
[7 ± 2 mmHg, 24 ± 6 beats/min, and 67 ± 8 breaths/min (P > 0.4)]. Chemoreflex responses
retested 45 min later remained unchanged (8 ± 4 mmHg, 25 ± 2 beats/min, and 62 ± 7 breaths/min).
In three additional animals, the effectiveness of kynurenic to block
excitatory amino acid receptor function was tested. In these animals,
the cardiorespiratory response to microinjection into the dorsal PAG of
bicuculline, a GABA-A receptor antagonist, was recorded 5 and 45 min
after central microinjection of kynurenic acid (100 nl, 10 mM) into the
same region of the PAG (see Fig. 5B for injection sites).
Microinjection of kynurenic acid alone into the dorsal PAG did not
markedly change baseline MAP, HR, or RR (99 ± 2 vs. 99 ± 4 mmHg, 376 ± 22 vs. 381 ± 15 beats/min, and 105 ± 5 vs. 105 ± 2 breaths/min, before vs. 5 min after kynurenic acid,
respectively). Microinjection of bicuculline into the dorsal PAG 5 min after kynurenic acid microinjections also did not
significantly alter MAP, HR, or RR (3 ± 3 mmHg, 6 ± 1 beats/min, and 2 ± 1 breaths/min). Bicuculline microinjection
into the dorsal PAG at 45 min, however, induced a significant rise in
MAP, HR, and RR (18 ± 6 mmHg, 39 ± 7 beats/min, and 44 ± 11 breaths/min) relative to the response evoked at 5 min
(P < 0.05). This suggested that the dose of kynurenic
acid used was more than sufficient to block increases in excitatory
input. In two animals, the cardiorespiratory response to microinjection
of bicuculline was retested 5 min after central microinjection of aCSF
(the vehicle for bicuculline and kynurenic acid) into the dorsal PAG.
The mean response to bicuculline in these animals was similar before
and after ACSF, suggesting the process of microinjection alone did not
significantly alter cardiorespiratory responses to bicuculline.
| |
DISCUSSION |
In the present study, repeat stimulation of arterial
chemoreceptors evoked a significant increase in FLI throughout the PAG in the anesthetized rat. Across all three doses tested, the increase in
FLI induced by KCN was greatest in the caudal PAG. This pattern of FLI
is similar to that previously reported from conscious rats exposed to
systemic hypoxia (7, 11, 21, 22). Yet, in our study,
further comparisons between groups demonstrated that FLI in the caudal
PAG was greatest in those animals exposed to a dose of KCN that
consistently produced the greatest pressor response, not the highest
dose of KCN. This suggested that a significant percentage of FLI
observed in the PAG after chemoreceptor stimulation was induced
indirectly through reflex-mediated changes in baroreceptor input.
To our knowledge, no previous studies have attempted to identify the
contribution of baroreceptor inputs to c-Fos expression induced by
chemoreceptor activation in the PAG. In our study, repeated exposure to
small increases in blood pressure induced increased levels of FLI in
the middle and rostral PAG. These increases in FLI were similar to
those observed after chemoreceptor stimulation. Identification that
certain regions of the PAG express increased levels of FLI after
increased baroreceptor input is not a new finding. Indeed, the PAG
appears to be one of the more sensitive regions of the brain to
fluctuations in baroreceptor afferent input (33). Previous
studies suggest that hypertension primarily induces increased FLI in
the dorsolateral, lateral (35), and ventrolateral columns
of the caudal and middle PAG (26). Alternatively, hypotension appears to selectively increase FLI in the ventrolateral column of the PAG (25, 35). The results of our study are
in agreement with these findings. Our results also suggest that repeat exposure to increased chemoreceptor afferent input, independent of
changes in baroreceptor input, only moderately increases FLI labeling
in the caudal PAG (~50% above baseline). This finding is in marked
contrast to previous studies that have reported FLI in the PAG
increases 100-200% above baseline after exposure to systemic
hypoxia (7, 22, 41). None of these previous studies, however, assessed the contribution of simultaneous changes in blood
pressure to FLI. Furthermore, the increases in FLI we observed after
chemoreceptor activation were primarily localized to the dorsal and
lateral columns of the caudal PAG. This suggests that increases in
c-Fos expression previously identified as occurring within the
ventrolateral column of the caudal PAG after exposure to hypoxia
(7, 22) may have been primarily related to blood pressure
changes rather than chemoreceptor stimulation or the direct effects of
hypoxia on neurons (7, 22).
To investigate further a potential role for the caudal PAG in the
arterial chemoreflex, we placed a retrograde tracer in the ventrolateral pons. The ventrolateral pons was chosen as a putative target site for chemoresponsive PAG neurons because this region has
been identified as essential for full expression of the sympathetic response to chemoreceptor stimulation in the rat (23, 28). Similar to other reports, 5-7 days after central injection, we observed numerous retrogradely labeled neurons in the lateral and
ventrolateral columns of the PAG (1, 9, 32). Despite extensive retrograde labeling in the PAG, only a small percentage of
cells were double labeled after repeated exposure to KCN. This finding
is in agreement with the results of another study that demonstrated
relatively few double-labeled neurons in the PAG after exposure to
hypoxia in conscious animals (21). In that study, PAG
neurons were retrogradely labeled from the rostroventrolateral medulla,
another region critical for chemoreflex modulation of sympathetic drive
(16).
Next, we examined the effect of the excitatory amino acid blockade in
the dorsal PAG on chemoreflex responses. On the basis of our c-Fos
data, we chose to localize our microinjections sites to the dorsomedial
column of the PAG. Yet, after blockade of the dorsomedial PAG, arterial
chemoreflex responses were not significantly altered. This finding does
not support the hypothesis that the PAG plays an important role in
chemoreflex responses. Still, this finding does corroborate previous
observations that relatively few PAG neurons activated by chemoreceptor
stimulation have direct descending projections to essential components
of central arterial chemoreflex arc. Our results raise the new
possibility that chemoreceptor-related activation of PAG neurons may
play an important role in relaying chemoreceptor-related signals to the
forebrain and in the alerting response to hypoxia. Indeed, excitotoxic
lesions of the dorsal PAG have been shown to significantly alter
baroreflex function (24, 40) without significantly
modulating the cardiovascular response to air jet stress in conscious
rats (24). This suggests that PAG neurons activated by
stressful stimuli, such as hypoxia, may only be indirectly involved in
the cardiorespiratory response to those stimuli.
Methodological considerations.
Although the expression of FLI within neurons can be used as a marker
of cellular excitation, there are limitations to the technique. First,
not all neurons express c-Fos proteins when activated. Thus the lack of
FLI does not necessarily indicate a lack of excitation. Second, the use
of anesthetics can alter c-Fos expression patterns. In the present
study, we used urethane as our anesthetic because it has been shown
have the least effect on chemoreflex responses (13, 18).
Interestingly, the general pattern of FLI that we observed in the PAG
after repeat chemoreceptor stimulation is similar to that reported from
conscious animals. For example, systemic exposure to hypoxia in
conscious animals was recently reported to increase of FLI counts by
40-60 neurons above baseline per PAG section (7). We
observed a similar increase in FLI above baseline in the anesthetized
preparation. This suggests that chemoreceptor stimulation can activate
similar populations of PAG neurons in either anesthetized or conscious animals.
Finally, it might be argued that exposure to moderate hypoxia for
1-3 h in a conscious animal is a more threatening or intense stimulus than repeated injections in KCN for only 20 min. In conscious animals, doses of KCN >60 µg/kg have been shown to be sufficient to
elicit marked cardiorespiratory changes (18) and
escape-like behavior (13, 15). In the present study, 90 µg/kg KCN was shown to elicit similar changes in cardiorespiratory
function to those evoked by 60 µg/kg in conscious animals, minus the
escape-like behaviors (13, 18). This further supports the
notion that stimuli used in the present study were sufficient to
activate PAG neuronal populations responsive to increases chemoreceptor afferent input.
In summary, the results of the present study demonstrate that repeated
stimulation of arterial chemoreceptors with KCN induces significant
increases in FLI throughout the PAG. These increases were greatest in
the caudal PAG. These findings are in strong agreement with previous
studies using systemic hypoxia to activate chemoreflex responses in
conscious animals (7, 22). Yet, when the influence of
reflex-induced changes in baroreceptor input was accounted for,
chemoreceptor-related increases in FLI in the caudal PAG were found to
be relatively modest. Results from additional retrograde labeling and
chemical blockade studies support this finding and suggest for the
first time that the PAG does not play a prominent role in descending
modulation of cardiorespiratory components of the arterial chemoreflex.
The results of the present study raise the possibility that activation
of chemoreceptor-sensitive neurons in the caudal PAG may be more
important in relaying information to the forebrain and/or coordinating
escape behavior associated with the chemoreflex response. Further
investigations are needed to identify the role of the PAG relaying
chemoreceptor information to higher brain centers.
| |
ACKNOWLEDGEMENTS |
We thank Karen Cooper for the hard work on the project and Mabelin
Castellanos for help on the immunohistochemistry.
| |
FOOTNOTES |
This study was supported by National Heart, Lung, and Blood Institute
Grants HL-52607 and HL-63232 (to L. Hayward).
Address for reprint requests and other correspondence:
L. F. Hayward, Univ. of Florida, Dept. of Physiological
Sciences, College of Veterinary Medicine, PO Box 100144, Gainesville,
FL 32610-0144 (E-mail: lindah{at}ufl.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 18, 2002;10.1152/ajpheart.00300.2002
Received 4 April 2002; accepted in final form 11 July 2002.
| |
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ABSTRACT
INTRODUCTION
