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The Journal of Neuroscience, January 1, 1998, 18(1):371-387
Organization and Transmitter Specificity of Medullary Neurons
Activated by Sustained Hypertension: Implications for Understanding
Baroreceptor Reflex Circuitry
Raymond K. W.
Chan and
Paul E.
Sawchenko
Laboratory of Neuronal Structure and Function, The Salk Institute,
La Jolla, California 92037
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ABSTRACT |
In situ expression of c-fos observed
in response to phenylephrine (PE)-induced hypertension provided a basis
for characterizing the organization and neurotransmitter specificity of
neurons at nodal points of medullary baroreflex circuitry. Sustained
hypertension induced by a moderate dose of PE provoked patterns of
c-fos mRNA and protein expression that conformed in the
nucleus of the solitary tract (NTS) to the termination patterns of
primary baroreceptor afferents and in the caudal ventrolateral medulla
(CVLM) to a physiologically defined depressor region. A majority of
barosensitive CVLM neurons concurrently displayed markers for the
GABAergic phenotype; few were glycinergic. Phenylephrine-sensitive
GABAergic neurons that were retrogradely labeled after tracer deposits
in pressor sites of the rostral ventrolateral medulla (RVLM) occupied a
zone extending ~1.4 mm rostrally from the level of the calamus scriptorius, intermingled partly with catecholaminergic neurons of the
A1 and C1 cell groups. By contrast, barosensitive neurons of the NTS
were found to be phenotypically complex, with very few projecting
directly to the RVLM. Extensive colocalization of PE-induced Fos-IR and
markers for the nitric oxide phenotype were seen in a circumscribed,
rostral, portion of the baroreceptor afferent zone of the NTS, whereas
only a small proportion of PE-sensitive neurons in the NTS were found
to be GABAergic. PE treatment parameters have been identified that
provide a basis for defining and characterizing populations of neurons
at the first station in the central processing of primary baroreceptor
input and at a key inhibitory relay in the CVLM.
Key words:
baroreflex; GABA; glycine; nitric oxide; c-fos; nucleus of the solitary tract; ventrolateral
medulla
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INTRODUCTION |
The arterial baroreceptor reflex
provides a rapid negative feedback mechanism that dampens fluctuations
in cardiovascular parameters induced by environmental insults. A rise
in arterial transmural pressure increases the discharge of
high-pressure mechanoreceptors in the carotid sinus and aortic arch
that are sensitive to changes in vessel wall distention. Sensory
information originating from these receptors is conveyed to the medulla
by branches of the glossopharyngeal and vagus nerves to modulate vagal
and sympathetic outflow to the heart and peripheral vasculature,
resulting in bradycardia and peripheral vasodilation, respectively
(Spyer, 1990 ). Despite their essential simplicity, the neural
substrates underlying these responses remain to be fully
elaborated.
The carotid sinus and aortic depressor nerves convey primary
baroreceptor afferent information to partly overlapping regions of the
nucleus of the solitary tract (NTS) (for review, see Loewy, 1990;
Spyer, 1990). Many cells within this barorecipient zone exhibit
powerful, short latency responses to afferent nerve stimulation, supporting the existence of a continuous "baroreceptor strip" region in the NTS (Spyer, 1994). Nevertheless, the complexity of
postsynaptic potentials that may be evoked within the strip region
after baroreceptor activation suggests an involvement of local
microcircuits, even in the early stages of processing (for review, see
Spyer, 1994). The precise location and phenotype of second-order
neurons supplied by baroreceptor afferents, as well as the identity of
subsequent relays within the NTS, have not been rigorously defined.
Output from the NTS modulates the activity of reticulospinal vasomotor
neurons in the rostral ventrolateral medulla (RVLM) that mediate tonic
and reflex adjustments of sympathetic outflow (Morrison et al., 1988;
Haselton and Guyenet, 1989; for review, see Dampney, 1994). The manner
in which baroreceptor information reaches the RVLM remains unsettled.
Although evidence exists of a direct NTS-RVLM projection (Ross et al.,
1985), electrophysiological (Agarwal and Calaresu, 1991; Jeske et al.,
1993) and pharmacological (Willette et al., 1984; Blessing, 1988) data
support the view that baroreceptor information accesses the RVLM
principally via interneurons in a functionally defined "depressor
region" of the caudal ventrolateral medulla (CVLM) (Aicher et al.,
1995; Jeske et al., 1995). A clear anatomic and phenotypic definition
of the CVLM depressor region has not been achieved, owing in part to complications imposed by the fact that depressor neurons may be interdigitated among functionally related catecholaminergic relay (Day
et al., 1983; Aicher et al., 1995), vagal cardiomotor (Bieger and
Hopkins, 1987), and ventral respiratory (Ellenberger et al., 1990;
Pilowsky et al., 1990) cell groups.
Recognition of the ability of certain cellular immediate-early genes
(IEG), notably the c-fos proto-oncogene, to provide
sensitive, inducible, and high resolution markers of extended neural
systems activated by salient environmental events holds potential for functional mapping of cardiovascular regulatory circuitry (for review,
see Morgan and Curran, 1991; Dampney et al., 1995). Using this
technology, we and others have demonstrated that acute or sustained
hypertension provokes c-fos expression in
noncatecholaminergic medullary neurons, the distribution of which in
the NTS conforms to the termination patterns of primary baroreceptor
afferents and in the CVLM overlaps with the caudal depressor region
(e.g., Badoer et al., 1994; Chan and Sawchenko, 1994; Li and Dampney, 1994). In the present study, we sought to characterize further the
organization and neurotransmitter specificity of neurons at these key
points in the baroreflex pathway using approaches that enabled combined
localization of (1) c-fos expression provoked by sustained
phenylephrine-induced hypertension, (2) markers for major inhibitory
neurotransmitter systems, catecholamines, and nitric oxide (NO), and
(3) retrograde labeling after tracer injections into physiologically
defined pressor sites in the RVLM.
Parts of this paper have been published previously in abstract form
(Chan and Sawchenko, 1995b).
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MATERIALS AND METHODS |
Animals and surgery. Adult male Sprague Dawley albino
rats (280-350 gm) were housed individually in a temperature-controlled room on a 12:12 hr light/dark cycle (lights on at 6:00 A.M.), with food
and water available ad libitum, and were adapted to handling for a week before any manipulation. For intravenous infusion, a catheter containing sterile heparin-saline (500 U/ml) was implanted in the external jugular vein under methoxyflurane anesthesia 2 d
before treatment, as described previously (Chan and Sawchenko, 1994).
Cardiovascular challenges. On the day of experimentation,
the jugular catheter was connected to a remote infusion system filled with the vasoconstrictor drug L-phenylephrine hydrochloride
(PE; Sigma, St. Louis, MO) dissolved in 0.9% sterile saline at
moderate (0.6 µg/µl) or higher (0.75-0.8 µg/µl) doses or with
vehicle. Doses of PE were titrated in preliminary experiments to induce a reflex bradycardia and a sustained increase in mean arterial pressure
(MAP) that averaged 24.7 ± 1.1 mm Hg (moderate) or 43.6 ± 1.5 mm Hg (high). Infusions were performed at a constant rate of 10 µl/min for 25 min (i.e., moderate dose, 2.14 µg/kg/min; high dose,
2.85 µg/kg/min). A low infusion rate was used in an attempt to
minimize mechanical stimulation of cardiac volume receptors, as well as
fluid loading (Hainsworth, 1991), whereas the duration was chosen to
provide a sufficiently salient stimulus to evoke comparable
c-fos mRNA and protein responses (cf. Chan and Sawchenko, 1994). Experiments were performed in two separate groups of conscious, freely moving rats (high dose, n = 19 with 3-6/time
point; moderate dose, n = 20 with 3-7/time point)
housed individually in their home cages between 8:30 and 10:30 A.M.
Animals were killed at 0.5, 1, 2, 3, or 4 hr after infusion to permit
analysis of temporal and spatial changes in c-fos expression
in the medulla. Control animals (n = 11) received
similar manipulations and infusions (saline) before perfusion. Animals
were submitted to the experiment in groups of three, in which both
experimental and control groups were represented. All protocols were
approved by the Institutional Animal Care and Use Committee.
Retrograde-tracing studies. To identify PE-responsive
neurons in CVLM that project to RVLM, we anesthetized animals with a ketamine, xylazine, and acepromazine mix (25:5:1 mg/kg, s.c.), and
animals received iontophoretic deposits of the retrogradely transported
fluorescent tracer Fluorogold (2% in saline) (Schmued and Fallon,
1986). The calamus scriptorius, defined as the caudal-most tip of the
area postrema (Ross et al., 1985), was exposed after retraction of the
atlanto-occipital membrane and served as a reference landmark. Deposits
were placed stereotaxically in the rostral aspect of the RVLM
(n = 6/PE dose) at a site identified as one at which
pressure injection (Pneumatic Picopump; WPI) of L-glutamate (injection volume, 12-24 nl; 0.1 M dissolved in 0.1 M sodium phosphate buffer, pH 7.4) yielded pressor
responses of >20 mm Hg (Benarroch et al., 1986). Coordinates used for
tracer injection were 1.9-2.3 mm rostral to the calamus scriptorius,
1.93-1.94 mm lateral to the midline, and 2.94 mm ventral to the dorsal
surface of the medulla. Fluorogold was deposited iontophoretically from
glass micropipettes (10 µm inner diameter; Drummond) by passing 2.5 µA of anodal current for 20-25 min (50% duty cycle) using a
constant current device (Transkinetics).
To allow concurrent localization of a retrograde tracer with Fos-IR and
mRNAs encoding transmitter-related proteins, we pressure injected 10 nl
of colloidal gold-labeled wheat germ agglutinin-horseradish peroxidase
complex (WGA-ApoHRP-Gold; 0.5-1%; EY Laboratories) (Basbaum and
Menétrey, 1987) into pressor sites in rostral RVLM (n = 5-6/dose) via a thick wall glass micropipette.
Silver intensification of WGA-ApoHRP-Gold allowed the retrograde
signals to persist throughout the hybridization protocol. Tracer
deposits in regions of the lateral medullary reticular formation
outside the pressor zone of the RVLM served as controls.
To identify vagal motor neurons, we injected 500-700 nl of 5% true
blue (Dr. Illing GmbH & Co.) suspension in sterile distilled water
beneath the epineurium of the cervical vagus nerve using a microliter
syringe (n = 3/dose). Tissue immediately surrounding the injection site was carefully cleaned to remove any leached tracer.
Animals were allowed to survive for 7 (WGA-ApoHRP-Gold and true blue)
days or 14 (Fluorogold) days before PE challenge and were perfused 2 hr
later, the time at which maximal Fos expression was observed.
Hemodynamic measurements. PE effects on arterial
pressure and heart rate were monitored in separate groups of conscious
animals (n = 4/dose) implanted 48 hr earlier with
femoral arterial (polyethylene-50 tubing) and jugular catheters (see
above) and subjected to PE or vehicle infusions as described above. MAP
was calculated as: 1/3 systolic + 2/3 diastolic pressure. Baroreflex
sensitivity over the course of PE-induced hypertension was determined
as the ratio of the change in heart rate to the change in MAP. Shifting of the arterial pressure-heart rate curve is generally taken as an
index of baroreflex resetting (Chapleau et al., 1988).
Histology. After treatment followed by their respective
survival intervals, rats were deeply anesthetized with chloral hydrate (35 mg/kg, i.p.) and perfused via the ascending aorta with saline followed by 500-700 ml of ice-cold 4% paraformaldehyde in 0.1 M borate buffer, pH 9.5, at 10°C. Brains were post-fixed
for 4 hr and cryoprotected in 20% sucrose in 0.1 M
phosphate buffer overnight at 4°C. Frozen sections were cut in the
coronal plane at 20 µm with a sliding microtome. Six one-in-six
series through the medulla were collected in cold cryoprotectant (Chan
and Sawchenko, 1994) and stored at  20°C until final group sizes
were achieved for histochemical analysis. This enabled all materials
from animals the data of which were to be compared to be processed in
tandem, using common batches of immunological and molecular probes.
Immunohistochemistry. Sections collected from all challenged
animals were processed for immunohistochemical detection of Fos-IR using a polyclonal antiserum raised against a synthetic N-terminal fragment of human Fos (residues 3-16; Santa Cruz Biotechnology, Santa
Cruz, CA), at a dilution of 1:4000. Localization was achieved using a
conventional nickel-enhanced avidin-biotin immunoperoxidase protocol
described previously (Chan and Sawchenko, 1994). The specificity of
immunostaining for Fos was verified by preabsorption controls (50 µM overnight at 4°C) and by substitution of normal for
immune sera; in neither case did this result in discriminable staining
in any brainstem cell group of PE-treated animals.
The distribution of Fos-positive nuclei was plotted onto drawings
prepared from an adjoining series of Nissl-stained sections using a
scanning system coupled to a Macintosh computer. The number of Fos-IR
nuclear profiles within the confines of cell groups of interest was
counted under 250× magnification in complete series of sections;
estimates were corrected for double-counting errors (Abercrombie,
1946). The distribution of barosensitive Fos-IR cells in the CVLM
region was plotted with reference to the rostrocaudal distance from the
level of the calamus scriptorius.
The tracer placements in the RVLM were verified by
counterstaining sections from injected animals for phenylethanolamine
N-methyltransferase (PNMT, a marker for adrenergic neurons)
by indirect immunofluorescence, using a rabbit polyclonal antiserum
raised against rat adrenal PNMT (1:1000 dilution) and kindly supplied
by Dr. M. C. Bohn (see Cunningham et al., 1990). The position of
deposits relative to PNMT-IR C1 neurons was evaluated by fluorescence
(Fluorogold) or polarized epifluorescence (silver-intensified
WGA-ApoHRP-Gold) microscopy.
Choline acetyltransferase (CAT)-IR vagal motor neurons and
catecholaminergic neurons in the VLM were identified using a goat polyclonal antiserum raised against human placental CAT (1:4000 dilution; Chemicon, Temecula, CA) and a rabbit polyclonal antiserum raised against purified rat adrenal tyrosine hydroxylase (TH; 1:2000
dilution; Pel-Freeze Biologicals, Rogers, AR), respectively. Detection
was achieved using the immunoperoxidase protocol. The specificity of
the antisera has been described previously (Shiromani et al., 1990;
Chan and Sawchenko, 1995a).
In situ histochemical hybridization. Hybridization
histochemical localization was performed using antisense
35S-labeled cRNA probes. Protocols for riboprobe synthesis,
hybridization, and autoradiographic localization of mRNA signal were
adapted from Simmons et al. (1989). After immunoperoxidase staining,
tissue sections were mounted onto poly-L-lysine-coated
slides, desiccated under vacuum overnight, fixed, digested by
proteinase K, acetylated, and dehydrated (Chan and Sawchenko, 1994).
After vacuum drying, 100 µl of hybridization mixture
(107 cpm/ml; 50% formamide) was spotted on each
slide, sealed under a coverslip, and incubated at 55°C overnight.
Slides were treated with ribonuclease A and washed in 0.1× SSC for 30 min at 78-82°C. Sections were dehydrated, exposed to x-ray film
( -max; Amersham, Arlington Heights, IL) for 24 hr, defatted in
xylenes, and dipped in NTB2 nuclear emulsion (diluted 1:1 with
distilled water; Eastman Kodak, Rochester, NY). In colocalization
experiments, dehydration steps were abbreviated to minimize dissolution
of diaminobenzidine reaction product (Chan and Sawchenko, 1995a).
Slides were exposed for 10-31 d and developed in D19 developer
(Eastman Kodak) for 4 min at 14°C. All sections were processed in
tandem for the in situ hybridization and autoradiographic
procedures. Grain densities over cell-sized areas exceeding five times
background levels were arbitrarily taken as representing positive
hybridization signals (McCabe and Pfaff, 1989). Similarly labeled
sense-strand runoffs were used as controls. No signal was detected
after pretreatment of sections with ribonuclease or hybridization with
labeled sense-strand probes.
cRNA probes. Antisense and sense (control) cRNA probes were
generated from full-length cDNAs encoding c-fos (2.1 kb; Dr.
T. Curran, St. Jude Children's Hospital; see Morgan and Curran, 1991), NGFI-B (2.4 kb; Dr. J. Milbrandt, Washington University; Milbrandt, 1988), and GAD 65 and 67 (2.0 and 2.7 kb, respectively; Dr. A. J. Tobin, University of California, Los Angeles; Erlander et al., 1991) or
from partial cDNA inserts for the glycine transporter GLYT-2 (3.1 kb;
Dr. N. Nelson, Tel Aviv University; Liu et al., 1993), brain nitric
oxide synthase (bNOS) (2.4 kb; Dr. S. H. Snyder, Johns
Hopkins University; Bredt et al., 1991), TH (1.2 kb; Dr. J. Mallet,
Centre National de la Recherche Scientifique; Grima et al., 1985), and
CAT (2.3 kb; Dr. T. Deguchi, Toyko Metropolitan Institute for
Neurosciences; Ishii et al., 1990). Antisense cRNA probes labeled with
 -35S-UTP and -ATP (NEN) were synthesized using T3 (CAT;
NGFI-B; NOS; TH) or T7 (c-fos; GAD 65; GAD 67; GLYT-2) RNA
polymerases after linearization of the plasmids (pBluescript
SK+) with appropriate restriction enzymes.
Unincorporated nucleotides were removed by Quick Spin columns
(Boehringer Mannheim, Indianapolis, IN). The specific activities of the
probes were approximately 2.3-3 × 109
cpm/µg. Using antisense riboprobes synthesized with two radiolabeled nucleotides (35S-UTP and -ATP) greatly improved the
sensitivity of hybridization histochemical detection of lower abundance
transcripts (Petersen and McCrone, 1994).
Dual labeling. Dual-labeling experiments were performed to
establish the neurochemical specificity and/or connectivity of barosensitive (i.e., Fos-IR) neurons. To identify
neurotransmitter-related molecules expressed by barosensitive neurons,
we coupled avidin-biotin immunoperoxidase localization of Fos either
with hybridization histochemical detection of GAD, GLYT-2, TH, or NOS
mRNAs or with NADPH diaphorase histochemistry. To determine the extent
to which PE-sensitive neurons project to the RVLM, we coupled
immunoperoxidase localization of Fos with fluorescence detection of
retrogradely transported Fluorogold in material from animals bearing
tracer deposits at pressor sites in the RVLM. To probe the topographic relationship between catecholaminergic and GABAergic neurons in the
VLM, we combined immunoperoxidase localization of TH with hybridization
histochemical detection of GAD mRNAs.
In an attempt to identify any vagal motor neurons targeted by the
baroreceptor stimuli, we coupled immunoperoxidase localization of Fos
with hybridization detection of CAT mRNA or with fluorescence detection
of retrogradely transported true blue after injection into the cervical
vagus at the level of the nodose ganglion. Because we failed to detect
PE-induced Fos-IR in the expected complement of vagal motor neurons,
immunoperoxidase localization of CAT was also coupled with
hybridization detection of mRNA encoding a second, independent IEG
marker, NGFI-B. NGFI-B is an orphan steroid hormone receptor
(Milbrandt, 1988) that has been used as an alternative or adjunct to
c-fos in marking activated neurons in brainstem cardiovascular control circuitry (Chan et al., 1993) and has identified sites of situation-specific cellular activation that escaped detection using c-fos expression as a marker (Ericsson et al.,
1995).
Dual localization experiments were performed in material from animals
killed at the times of maximal c-fos induction, that is, at
1 hr after the challenge for c-fos mRNA and at 2 hr for Fos
protein. In all dual-labeling experiments, immunostaining was performed
first, and with the exception of experiments involving fluorescent
tracers (Fluorogold or true blue), nickel enhancement steps were not
used. For combined immuno- and hybridization histochemical localization, the immunostaining methods were modified to avoid RNase
contamination and leaching of reaction product (Watts and Swanson,
1989). These modifications essentially included omission of tissue
pretreatments with hydrogen peroxide and sodium borohydride, as well as
substitution of 2% bovine serum albumin and 5% heparin sulfate for
blocking sera. For NADPH diaphorase histochemistry, sections were
incubated in 0.1 M Tris-HCl, pH 8.0, containing 0.3%
Triton, nitroblue tetrazolium (0.25 mg/ml dissolved in 100 µl/mg of
DMSO), and -NADPH (1 mg/ml) at 37°C for 30-60 min (Vincent and
Kimura, 1992) and then were immunolabeled for nuclear Fos. The number
of doubly-labeled Fos-IR cells in all animals of a given treatment
group was counted visually under 400× magnification in complete series
of coronal sections through cell groups of interest and corrected for
double-counting errors.
Combined retrograde transport (WGA-ApoHRP-Gold),
immunohistochemistry (Fos), and hybridization histochemistry (GAD and
GLYT-2 mRNAs) using color microautoradiography. Animals bearing
WGA-ApoHRP-Gold deposits in the RVLM were challenged with PE, as
described, and killed 2 hr later. Avidin-biotin immunoperoxidase
localization of PE-induced Fos was performed first, followed by silver
enhancement of colloidal gold-based retrograde signal (Basbaum and
Menétrey, 1987) and then hybridization histochemistry. For silver
intensification, sections were rinsed in distilled water for 10 min and
developed in the IntenSEM kit (Amersham) for 15-25 min. After
development, sections were rinsed in distilled water and then in KPBS
for 10 min before mounting onto poly-L-lysine-coated
slides.
In situ hybridization detection of GAD and GLYT-2 mRNAs was
performed with antisense 35S-labeled cRNA probes as
described above. After posthybridization, sections were briefly
dehydrated (three to five rapid dips), exposed to x-ray film for 24 hr,
defatted in xylenes, coated with 3% pyroxylin in ethyl acetate
(Polysciences, Warrington, PA), and dipped in NBT2 nuclear emulsion
(diluted 1:1 with distilled water; Eastman Kodak). After exposure times
of 10 (GLYT-2) days to 31 (GAD 65 and 67) days, the emulsion was
developed, bleached, and coupled to a magenta dye that allowed reduced
silver indicative of a hybridization signal (purple) to be
distinguished from the signal that was representative of colloidal
gold-labeled retrograde tracer (black). The methodology for color
microautoradiography was adapted from Haase et al. (1985). All
procedures before development were performed in a darkroom under
safelight conditions (Wratten filter 2). In brief, slides were
developed in D19 developer (Eastman Kodak) for 4 min at 14°C. Emulsion was stabilized by cross-linking in 0.37% formalin buffered with photographic grade 0.6% sodium carbonate for 3 min. The slides were then washed in distilled water for 2 min, bleached in a solution of 10% potassium ferrocyanide and 5% potassium bromide (photographic grade; Sigma) for 1 min, washed again in distilled water for 2 min, and
developed in freshly prepared dye coupler for 1 min. The dye coupler
was prepared by mixing color developer and magenta dye coupler solution
in a ratio of 9:1 (v/v) immediately before use. The color developer
contained 0.22% CD2
(4-N,N-diethyl-2-methylphenylenediamine monohydrochloride; Acros), 0.22% sodium sulfite (photographic grade;
Sigma), 0.11% potassium bromide, and 2.22% sodium carbonate in
distilled water. The magenta dye coupler solution contained 2% M-38
[1-(2,4,6-trichlorophenyl)3-(p-nitroanilino)2-pyrazolin-5-one; Acros] and 0.2N sodium hydroxide in absolute ethanol. After color development, the slides were washed in distilled water for 2 min, bleached in 10% potassium ferricyanide and 5% potassium bromide for 1 min, washed again in distilled water for 2 min, and fixed in 15%
sodium sulfite solution and 24% sodium thiosulfate (photographic grade; Sigma) for 5 min. The slides were air-dried briefly,
coverslipped in buffered glycerol (50% glycerin in 0.4 M
potassium bicarbonate, pH 8.6), and stored at 20°C.
Statistics. Data presented are mean ± SEM. A one-way
ANOVA followed by the Dunn's test (StatView 4.0) was used to compare MAP changes between treatments. In all analyses, a probability of
p < 0.05 was taken to be statistically
significant.
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RESULTS |
Phenylephrine-induced changes in cardiovascular parameters
We sought to identify PE treatment parameters that would yield a
sustained hypertension that would provoke comparable c-fos mRNA and protein induction at key points along the baroreflex pathway,
to permit characterization of Fos-IR neurons while not seriously
compromising the discreteness of the activation pattern seen in
response to bolus infusions (cf. Chan and Sawchenko, 1994). Continuous
(25 min) infusion of PE at doses of 2.14 and 2.85 µg/kg/min induced a
sustained increase in MAP that averaged 24.7 ± 1.1 mm Hg (+25.8%
from the baseline MAP, 95.6 ± 2.8 mm Hg) and 43.6 ± 1.5 mm
Hg (+45.6%), respectively (Fig. 1).
These responses differed significantly from controls
(p < 0.005) and from one another
(p < 0.01). The elevation in arterial pressure
was also accompanied by a pronounced bradycardia, the magnitude of
which decreased over the course of hypertension. This gradual reduction
in the bradycardiac response was reflected in a decreased  heart
rate (HR)/ MAP ratio and may indicate resetting of baroreceptor
sensitivity in response to sustained hypertension (Chapleau et al.,
1988).
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Figure 1.
Phenylephrine-induced changes in cardiovascular
parameters. Time course of changes in the MAP (top) and
the baroreflex index ( heart rate/ MAP) (bottom)
induced by sustained intravenous infusion of PE. Data are presented as
mean ± SEM; n = 4/dose. PE-induced
hypertensive responses differed significantly from controls
(***p < 0.005) and from one another
(++p < 0.01 compared with the moderate dose). The
solid bar below the curves represents the 25 min
duration of drug or vehicle infusion. The pronounced changes in
baroreflex indices are assumed to be indicative of rapid resetting of
baroreceptor sensitivity in response to a conditioning pressure
ramp.
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Temporal and spatial patterns of PE-induced c-fos
expression in the medulla
In material from control rats, which received prolonged saline
infusion, Fos-IR or c-fos mRNA signals were only
sporadically encountered in the NTS and ventrolateral medullary
reticular formation. The few positively labeled cells that were
detected displayed no suggestion of any preferential regional
distribution or consistent pattern from section to section, within or
between subjects. Although the basal level of Fos-IR in barosensitive
regions of the medulla is low, some degree of constitutive
c-fos expression was displayed consistently in the spinal
trigeminal and lateral reticular nuclei of control, as well as
nonmanipulated, animals.
In contrast to the acute hypertensive model used previously (Chan and
Sawchenko, 1994), sustained PE-induced hypertension provoked
c-fos mRNA and Fos protein expressions that were comparable in strength and distribution in circumscribed regions of the medulla that are generally recognized as nodal points in baroreflex control circuitry (Figs. 2,
3). Fos induction in animals receiving
moderate or higher doses of PE followed similar time courses, with
Fos-IR and c-fos mRNA first appearing within 0.5 hr, the
earliest time point examined. Maximal expression of c-fos
mRNA and Fos-IR were detected at 1 and 2 hr, respectively, after PE
infusion (Figs. 2, 3). These abated gradually thereafter, and by 4 hr
no consistent above-background immuno- or hybridization signals were
detected.
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Figure 2.
Medullary patterns of c-fos mRNA
and Fos protein expression induced by prolonged PE infusion. Dark field
(A, B) and bright field
(A , B, C)
photomicrographs depicting hybridization and avidin-biotin
immunoperoxidase localization of c-fos mRNA and Fos-IR
in the NTS at the levels of the commissural subnucleus (NTSc; A,
A) and the area postrema
(NTSap; B,
B) and in the CVLM (C,
C) at 1 and 2 hr after hypertensive challenge,
respectively. C, Combined immuno- and hybridization
histochemical detection of choline acetyltransferase-IR cells in the
nucleus ambiguus (black cells) and PE-induced
c-fos mRNA (white silver grains), respectively, photographed using polarized epifluorescence
illumination. In contrast to the acute infusion model, sustained
hypertension resulted in comparable strengths and distributions of
c-fos mRNA and protein expression in each region. This
includes robust induction in the dorsal baroreceptor strip of the
commissural NTS (NTSc) that extended into
the dorsal and dorsolateral aspects of NTS at the level of area
postrema (NTSap). Noncatecholaminergic
neurons of the CVLM region were also reliably activated.
AMB, Nucleus ambiguus; AP, area postrema;
cc, central canal; DMX, dorsal motor nucleus of vagus; Gr, gracile nucleus;
IO, inferior olive; NTS, nucleus of
solitary tract; ts, solitary tract; XII,
hypoglossal nucleus. All photomicrographs, 75×.
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Figure 3.
Dose-related patterns of PE-induced Fos-IR
expression in medullary neurons. Line drawings of
coronal sections through the medulla showing the distribution of Fos-IR
nuclei (black dots) at 2 hr after hypertensive
challenges induced by moderate (left) or higher
(right) doses of PE. In addition to a dose-related
increase in Fos-IR neurons in the baroreceptor strip of commissural NTS and the CVLM region, high dose PE also provoked a more-pronounced Fos
induction in the medial subnucleus of NTS and in the lateral reticular
nucleus and medullary raphe nuclei. A1, A1 noradrenergic cell group; AMB, nucleus ambiguus; AP,
area postrema; Cu, cuneate nucleus; dc,
dorsal column; dpy, pyramidal decussation;
EC, external cuneate nucleus; Gr, gracile
nucleus; IO, inferior olive; LRN, lateral
reticular nucleus; MV, medial vestibular nucleus;
NTS, nucleus of solitary tract; PH,
prepositus nucleus; py, pyramidal tract;
RVLM, rostral ventrolateral medulla;
CVLM, caudal depressor region; SpV,
spinal trigeminal nucleus; SV, spinal vestibular nucleus; XII, hypoglossal nucleus.
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Descriptions of c-fos expression patterns are made with
reference to cytoarchitectonic subdivisions described by Loewy and Burton (1978), Kalia and Sullivan (1982), and Altschuler et al. (1989)
for the NTS, by Bieger and Hopkins (1987) for the nucleus ambiguus, and
by Ruggiero et al. (1989) for the VLM. Nomenclature for medullary
catecholaminergic cell groups follows Hökfelt et al. (1984). The
following description pertains to c-fos expression patterns
seen in response to the moderate-dose PE challenge.
Within the NTS, Fos-IR neurons were first detected in circumscribed
regions of the commissural NTS at 1 hr, and their number and staining
intensity peaked at 2 hr. In response to the moderate PE dose, the
total NTS population was estimated at 977 ± 178 neurons in six
animals killed at the 2 hr time point. At levels caudal to the apex of
the calamus scriptorius, the principal grouping of responsive neurons
comprised a horizontal strip at the dorsal margin of the commissural
part of the NTS (NTSc). This was rostrally contiguous with
a bilaterally symmetric, obliquely oriented array that extended into
the dorsal and dorsolateral subnuclei of the NTS, partially enwrapping
medial aspects of the solitary tract at the level of the area postrema
(NTSap) (Figs. 2, 3). The zone of maximal labeling extended
approximately from 120 µm caudal to the level of calamus scriptorius
through the rostral-most extent of the area postrema. Secondary
accumulations of cells displaying PE-induced Fos-IR or c-fos
mRNA were seen reliably in the medial subnucleus of the NTS, which was
rostrocaudally coextensive with induction in the baroreceptor strip
regions highlighted above. In addition, scattered activated neurons
were detected in the dorsal motor nucleus of the vagus and in a
discontinuous ring of cells at the ventral and ventrolateral margins of
the area postrema (Figs. 2, 3). The number of NTS neurons exhibiting
PE-induced c-fos expression tapered sharply at levels
rostral to that of the area postrema, and the lateral division of the
NTS complex contained few activated neurons throughout its rostrocaudal
extent.
In VLM, the bulk of Fos-IR cells (822 ± 122; n = 6) were distributed primarily between the level of the calamus
scriptorius to the midextent of the RVLM, spanning a length of
~1.3-1.4 mm from the apex of the calamus scriptorius and lying
immediately subjacent to the principal column of the ambiguual complex
(Figs. 2, 3). Although these responsive cells were intermingled
extensively with aminergic neurons of the A1 and C1 cell groups, Fos-IR
cells in the CVLM region were overwhelmingly nonaminergic, were
noncholinergic (Fig. 2C), and were not retrogradely labeled
after tracer deposits in the cervical vagus nerve (data not shown),
indicating this PE-activated cell column to be distinct from
functionally allied catecholaminergic cell groups and from vagal
cardiomotor neurons in the ambiguual complex. It is noteworthy that
relatively few Fos-IR neurons were encountered caudal to the level of
the calamus scriptorius, in the so-called caudal subdivision of the
CVLM that is believed not to play a major role in mediating baroreflex
responses (Cravo et al., 1991). At the time of maximal expression, some scattered Fos-IR cells were also detected along the transtegmental tract extending between the NTS and VLM. At the rostral extreme of the
RVLM, in proximity to the facial motor nucleus, Fos-IR cells were
extremely sparse. The moderate-dose PE challenge failed to evoke
reliable c-fos expression in other medullary regions.
Dose response
The higher dose of PE provoked substantially more extensive
c-fos expression in the dorsal commissural strip of
NTSc; the dorsal subnucleus of the
NTSap; the area postrema, proper; the lateral
tegmental field; and the CVLM region (Fig. 3). The total number of
Fos-IR cells counted in the NTS (2416 ± 373) and VLM (2433 ± 297) were elevated 2.5- and 3-fold, respectively, over values
obtained from a comparable sampling of animals treated with the
moderate PE dose (n = 6; animals killed 2 hr after
infusion). The most-pronounced increment of Fos-IR cells was seen in
the medial subnucleus of NTSap, in which the
labeling extended to include somewhat more rostral aspects of NTS (Fig.
3). Also evident in these animals was an increase in the small number
of cells constitutively expressing Fos-IR in the lateral reticular and spinal trigeminal nuclei and novel, although minor, sites of induction in the medial aspects of the gigantocellular reticular nucleus and in
the aspects of the nucleus raphe pallidus.
Phenotypes and connectivities of activated neurons
Neurons activated in response to PE-induced hypertension
were characterized by the labeling for nuclear Fos-IR combining with that of markers for major inhibitory neurotransmitter systems, the
gaseous free radical neurotransmitter nitric oxide, and
catecholaminergic neurons, which comprise important relays for
conveying sensory information from the cardiovascular system to
neuroendocrine and autonomic effectors (Chan and Sawchenko, 1994).
Additional groups of animals received injections of the retrograde
tracer Fluorogold into pressor sites of the RVLM before being subjected
to a PE challenge and subsequent processing for concurrent
demonstration of transported tracer and stimulated Fos-IR.
Tracer deposits in these experiments were 0.8-1.0 mm in diameter and
were centered 0.36-0.54 mm caudal to the caudal pole of the facial
motor nucleus, medial to the compact formation of nucleus ambiguus near
the level of its maximal development, and extended from the base of the
brain to, but not beyond, the principal column of the ambiguual
complex. These gave rise to patterns of retrograde labeling in the
medulla that were comparable with those reported previously using
similar approaches (e.g., Ross et al., 1985; Badoer et al., 1994).
Thus, labeled perikarya in the NTS were concentrated in the caudal
two-thirds of the medial subnucleus. Retrograde labeling in the
baroreceptor strip region was sparse caudal to the level of the calamus
scriptorius and more pronounced rostrally in the dorsal subnucleus at
the level of the area postrema. In the ventrolateral medulla, labeled
cells principally comprised a tight, obliquely oriented column
extending toward the ambiguual complex, which was rostrocaudally
coextensive and overlapping in its ventral aspects with the
distribution of PE-stimulated Fos-IR. Throughout the medulla,
retrogradely labeled cells were distributed bilaterally with a strong
ipsilateral preponderance. Tracer deposits placed outside the pressor
zone of the RVLM resulted in a pronounced diminution in retrograde
labeling of barosensitive neurons in the CVLM. Possible uptake of
Fluorogold by fibers of passage cannot be excluded, but we saw no
evidence to indicate that this may have been pervasive. For example,
deposits in the RVLM region that impinged on internal arcuate fibers
consistently failed to result in retrograde labeling in the dorsal
column nuclei.
GABA
Multiple localization studies were performed to determine
the extent to which PE-responsive neurons in the medulla might express major inhibitory neurotransmitters (GABA and glycine) and/or project to
pressor regions of the RVLM. For GABAergic neurons, the expression patterns of mRNAs encoding both glutamic acid decarboxylase isoforms (GAD 65 and 67) (Erlander and Tobin, 1991; Esclapez et al., 1994) were
analyzed (see Fig. 5). In control animals, GAD 67 mRNAs were found to
be ubiquitously expressed in all major divisions of the NTS and in the
area postrema. In recognized subdivisions of the caudal NTS, neurons
displaying GAD 67 mRNA signals were most abundant in the medial and
interstitial subnuclei, moderate to scattered in the dorsal and
commissural subnuclei (including the baroreceptor strip region), and
very sparse in the subpostrema region and in the dorsal motor nucleus
of the vagus, a pattern consistent with the distribution of GAD-IR
(Blessing et al., 1984; Meeley et al., 1985) or GABA-IR perikarya
(Maley and Newton, 1985; Izzo et al., 1992). Numerous cells exhibiting
hybridization signals for GAD 67 mRNA were also detected throughout the
medullary reticular formation, including the CVLM and RVLM. The
topography of cells labeled for GAD 67 mRNA is similar to that
described based on immunolocalization of GAD in the medulla of
colchicine-treated animals (Meeley et al., 1985; Ruggiero et al.,
1985). Transcripts encoding the GAD 65 isoform were similarly, although
considerably less robustly, expressed when hybridized in material from
the same brains, using probes of comparable size, GC content, and specific activity (data not shown). Similar disparities in the relative
level of constitutive expression of these isoforms have been reported
in other regions of the brain (Esclapez et al., 1994).
Despite the widespread expression of GAD in the NTS, only 19.0 ± 1.1 and 15.8 ± 1.8% of neurons in the baroreceptor strip that
displayed Fos-IR colabeled for GAD mRNA in response to moderate and
higher dose PE challenges, respectively (Figs. 4,
5)
A greater degree (moderate dose, 45.3 ± 6.0%; high dose,
34.5 ± 2.8%) of colocalization was encountered in the medial
subnucleus (Figs. 4, 5), although the bulk of these neurons was not
retrogradely labeled from RVLM at either dose of PE. The handful of
RVLM-projecting neurons that exhibited PE-induced Fos-IR in the medial
subnucleus of NTSap did not display detectable GAD mRNA
signals. By contrast, a substantial fraction (moderate dose, 65.5 ± 3.1%; high dose, 49.3 ± 2.6%) of Fos-IR barosensitive
neurons in the CVLM region displayed GAD transcripts (Figs. 5,
6). Doubly-labeled cells were distributed
uniformly throughout the longitudinal extent of the Fos-IR column. The
higher dose of PE resulted in only a moderate increase in the absolute
number of Fos-positive neurons displaying GAD mRNA in the CVLM, which,
in the face of a more-pronounced increment in the number of Fos-IR
cells, yielded a consistent reduction in the proportion of
barosensitive neurons colabeled for GAD (Fig. 6). This is presumably a
consequence of recruitment to Fos expression of nonintegral components
of baroreflex pathways, such as nearby catecholamine-containing neurons
(see below), in response to more severe hypertensive episodes.
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Figure 4.
Phenotypic characterization of PE-sensitive
neurons in NTS. Top, Schematic drawings of the NTS
depicting the subregions in which PE-induced Fos-IR neurons were
characterized. These include the dorsal aspect of the commissural
division (I) at caudal levels (NTSc; left), the dorsal
and dorsolateral subnuclei (II) at the level of
the area postrema (NTSap;
right), and the medial subnucleus (III) at both levels. Bottom,
Comparison of the proportion of Fos-IR neurons within these aspects of
the NTS that were colabeled for GAD or GLYT-2 mRNAs, TH-IR, or a
retrograde tracer (Fluorogold) after deposits in the RVLM. Data from
animals treated with moderate or higher doses of PE are given.
AP, Area postrema; cc, central canal;
Cu, cuneate nucleus; DMX, dorsal motor
nucleus of vagus; Gr, gracile nucleus;
NTS, nucleus of solitary tract; ts,
solitary tract; XII, hypoglossal nucleus.
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Figure 5.
PE-induced Fos-IR in medullary GABAergic neurons.
A-D, Polarized epifluorescence images showing GAD 67 mRNA signal (aqua grains) and peroxidase staining for
Fos-IR (brown nuclei; A-C) or TH-IR (brown cytoplasm; D) in the
NTSap (A), in the commissural NTS (B), and in the CVLM region (C,
D). Despite robust expression of GAD in the NTS, only a
small subset of neurons in the baroreceptor strip that displayed Fos-IR
in response to a moderate PE dose colabeled for GAD mRNA
(A, B). By contrast, a substantial
fraction of Fos-IR barosensitive neurons in the CVLM region displayed
GAD transcripts (C). Very few TH-IR neurons in
the CVLM region displayed GAD transcripts (D).
Black arrows denote examples of doubly-labeled neurons,
open arrows denote cells displaying GAD mRNA only, and arrowheads indicate cells labeled only for Fos-IR
(A-C) or TH-IR (D).
E, E, Barosensitive GABAergic neurons
projecting to the RVLM; bright-field images of a single field in the
CVLM taken at two focal planes to show PE-induced Fos-IR (brown
nuclei), silver-enhanced retrograde labeling after
WGA-ApoHRP-Gold injection in the RVLM (Retro;
black grains; E), and hybridization
signals for GAD 67 mRNA (magenta grains;
E). The neuron at the bottom right of
E and E displays all three markers. The
cell at the left center of E is
retrogradely labeled only, whereas the one to the upper
right of E is positive only for GAD mRNA.
dc, Dorsal column; ts, solitary tract.
Magnifications: A-D, 150×; E,
E, 375×.
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Figure 6.
Anatomic and phenotypic characterization of
PE-sensitive neurons of the VLM. A, B,
Three-dimensional histograms showing the number of barosensitive
(Fos-IR) neurons seen in response to moderate (A)
and higher (B) doses of PE at regular intervals
through the medulla (orange columns) and the number
colabeled for GAD or GLYT-2 mRNAs, TH-IR, or a retrograde tracer
(FG) after deposits in the RVLM at the level indicated.
Data are presented as mean ± SEM; n = 6-7/dose. Both PE doses induced c-fos expression
concentrated between the level of the calamus scriptorius and that of
the caudal aspect of the RVLM. In material from animals treated with a
moderate PE dose, Fos-IR neurons were found to be predominantly
GABAergic, with a majority projecting to the RVLM. A higher dose of PE
gave rise to more robust Fos expression; a diminished proportion of these colabeled for GAD mRNA or the retrograde tracer, and an increased
fraction displayed the catecholaminergic phenotype.
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Material from rats bearing Fluorogold deposits in the RVLM displayed
retrograde labeling of Fos-IR neurons throughout the longitudinal
extent of the PE-sensitive column of the CVLM. The relative abundance
of these covaried in the rostrocaudal dimension with the barosensitive
GAD-expressing population, although their absolute values were lower
(Fig. 6). To determine whether the activated GAD-expressing and
RVLM-projecting populations overlap, we prepared rats bearing
WGA-ApoHRP-Gold deposits in pressor regions of the RVLM and killed them
2 hr after a PE challenge for concurrent visualization of PE-induced
Fos-IR, silver-intensified tracer, and GAD mRNA, the latter using color
microautoradiography to facilitate discrimination of silver grains
indicative of retrograde tracer from hybridization signals for GAD
transcripts. Examples of triply-labeled cells (Fig.
5E,E) were very prominent within
the ipsilateral CVLM and distributed uniformly throughout the core of
the PE-responsive population. Fewer were seen along the transtegmental
tract extending dorsomedially toward the NTS, and only occasional
examples were detected in the contralateral CVLM. Because of the clear
decrement in the sensitivity of the constituent methods in such
combined applications, no attempt was made to extract quantitative
information from this material.
Glycine
Because a common pathway for glycine biosynthesis is shared by
cells that do and do not use this amino acid as a neurotransmitter, its
biosynthetic enzyme is not a useful phenotypic marker. The neuron-specific glycine transporter GLYT-2 (Liu et al., 1993, Adams et
al., 1995), which serves to terminate synaptic transmission via a
reuptake mechanism (Clark and Amara, 1993), has provided a useful means
of localizing glycinergic neurons.
In the medulla of control animals, neurons expressing GLYT-2 mRNA were
much less abundant than were those hybridized for GAD mRNA, most
notably in the NTS, in which the majority of GLYT-2-positive neurons
were concentrated in the lateral division of the nucleus (Fig. 4). The
few positively hybridized neurons detected in the medial division were
confined principally to the interstitial and dorsal subnuclei (Fig. 4).
A far greater number of neurons expressing the transporter was seen in
the medullary reticular formation, including the CVLM and RVLM, the
distribution of which primarily mirrored that of cells expressing GAD
mRNAs but was less dense. The topography of cells displaying positive
hybridization signals for GLYT-2 mRNA is consistent with the
distribution of glycine-IR perikarya reported in the medulla of
noncolchicine-treated animals (Pourcho et al., 1992; Fort et al.,
1993). In keeping with the paucity of GLYT-2-expressing neurons in the
NTS, examples of dual localization of PE-induced Fos-IR and GLYT-2 mRNA
(0.8-4.0%) were extremely limited throughout the medulla (Fig. 6). A
somewhat higher degree (moderate dose, 7.2 ± 2.2%; higher dose,
9.1 ± 3.0%) of dual labeling was encountered at the rostral end
of the CVLM region (data not shown), and a small fraction of these
cells was retrogradely labeled after tracer deposits in the RVLM.
Nitric oxide
Neurons exhibiting the NO phenotype were detected by NADPH
diaphorase histochemistry and hybridization for mRNA encoding bNOS, which catalyzes NO production from L-arginine (Dawson et
al., 1991; Hope et al., 1991). In the NTS, NADPH- and bNOS-positive neurons displayed indistinguishable distribution patterns (Fig. 7). At
caudal levels, positive labeling for both markers was encountered in
the dorsal aspect of the commissural NTS, in the dorsal subnucleus (with a distribution similar to that of PE-induced Fos-IR), in interstitial and medial aspects of NTSap, and
sporadically in the subpostrema region. A few weakly labeled cells were
occasionally encountered in the area postrema, proper. At rostral
levels, robustly labeled cell clusters were distributed in the central
subnucleus and in medial and ventral aspects of the NTS and were
scattered throughout the dorsal motor nucleus. Labeling in the
medullary reticular formation was considerably less dense than that
obtained using probes for GAD or GLYT-2 mRNAs. The CVLM and RVLM
contained no remarkable accumulations of positive neurons but were
bordered, and sometimes invaded, by moderately dense clusters of
NOS-positive cells associated with adjoining cell groups, such as the
spinal trigeminal complex, the ambiguual complex, and more medial
aspects of the reticular formation. The distribution of NADPH
diaphorase staining and hybridization signals for NOS mRNA encountered
in our material was similar to that of NOS-IR described in the medulla of noncolchicine-treated animals (Dun et al., 1994).
Of particular interest was the finding that a substantial proportion of
the PE-sensitive neurons in the strip region of the NTS displayed
diaphorase activity or NOS mRNA. These were preferentially massed in
the dorsal subnucleus of the NTSap, circumscribing
the dorsomedial aspect of the solitary tract at this level (Fig.
7), where a substantial majority of
activated cells were NOS-positive. The proportion of Fos-IR cells
exhibiting markers for the NO phenotype tapered progressively at levels
caudal to that of the calamus scriptorius. In the dorsal and
dorsolateral subnuclei of the NTSap, NOS mRNA
signals were detected in over 49.1 ± 6.4 and 58.6 ± 4.4% of the Fos-IR neurons seen in response to a moderate and higher PE
doses (Fig. 4). This pattern of double labeling conforms more closely
with the terminal distribution of primary baroreceptor afferents
carried by the aortic depressor, not the carotid sinus, nerve
(Ciriello, 1983; Housley et al., 1987). A substantial fraction (37.8-48.3%) of Fos-IR cells in these regions was also retrogradely labeled from RVLM, and these displayed a topography similar to that of
PE-activated, NOS-expressing neurons.
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Figure 7.
A subset of barosensitive NTS neurons display
markers for the nitric oxide phenotype. Bright-field
(right) and polarized epifluorescence (left) renderings showing peroxidase staining for Fos
(brown nuclei) and either NADPH diaphorase activity
(blue/black cytoplasm; left) or mRNA encoding nitric
oxide synthase (aqua silver grains; right) in the
dorsolateral part of NTS at the level of the area postrema (NTSap). Open arrows, Cells
displaying NADPH diaphorase staining or NOS mRNA; filled
arrowheads, Fos-IR nuclei; filled arrows, cells
displaying both markers. PE-induced Fos-IR was frequently colocalized
with markers for NO in the rostral baroreceptive region of the
NTSap. Cu, Cuneate nucleus. Magnification,
150×.
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Only a very small fraction of Fos-IR neurons in the VLM displayed NADPH
diaphorase activity or positive hybridization signals for NOS mRNA
(both doses, 3.4-3.7%; Fig. 6), and those that did exhibited no
distinctive topography. Few, if any, instances of colocalization were
detected in other regions of the medulla at either dose of PE.
Tyrosine hydroxylase
Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine
biosynthesis, was used as a marker for noradrenergic and adrenergic cell groups in the medulla. In line with our previous findings that
acute hypertension targets preferentially nonaminergic neurons in the
medulla (Chan and Sawchenko, 1994), dual localization of Fos-IR and TH
mRNA in the noradrenergic A1 (10.8 ± 1.6%) and A2 (0%) and the
adrenergic C1 (13.7 ± 2.1%) and C2 (11.5 ± 3.9%) cell
groups was relatively low at the moderate dose of PE. Although intermingled partly with the rostral noradrenergic A1 and the caudal
adrenergic C1 neurons, the bulk of GABAergic barosensitive neurons in
CVLM is situated dorsal to the aminergic cell groups and ventral to the
ambiguual complex (Fig. 8). As has been described (Roland
and Sawchenko, 1993), only small subpopulations of TH-IR neurons
concurrently displaying GAD mRNA were encountered in the VLM
(4.5-5.1%) and the NTS (2.9-3.6%). In contrast, a higher dose of PE
gave rise to much more substantial fractions of the Fos-IR neurons
displaying the aminergic phenotype in the NTS and VLM (A1, 23.1 ± 1.6%; A2, 1.0 ± 0.2%; C1, 30.0 ± 4.1%; C2, 19.9 ± 1.5%) (Figs. 6, 8).
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Figure 8.
Distribution of GABAergic and RVLM-projecting
barosensitive neurons in VLM and their relationship to aminergic cell
groups. A-F, Plots of the distribution of cells
displaying PE-induced Fos-IR and GAD mRNA
(filled circles) or retrogradely labeled after tracer deposits in pressor sites in the RVLM (crosses).
The distribution of medullary aminergic (TH-IR) neurons in the same
animal (open circles) is shown for comparison. The
rostrocaudal distance from the level of the calamus scriptorius is
indicated on the lower right-hand corner
of each panel. The results define a barosensitive CVLM
region extending ~1.3-1.4 mm rostrally from the level of the calamus
scriptorius in which the bulk of PE-sensitive, RVLM-projecting, and/or
GABAergic neurons are found. A1, A1 noradrenergic cell group; Amb, nucleus ambiguus; C1, C1
adrenergic cell group; dpy, pyramidal decussation;
IO, inferior olive; LRN, lateral
reticular nucleus; py, pyramidal tract;
SpV, spinal trigeminal nucleus.
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PE-induced Fos expression in vagal motor neurons
Although PE-induced hypertension was accompanied by a pronounced
bradycardia, cholinergic neurons of the ambiguual complex that were
retrogradely labeled from tracer injections in the cervical vagus nerve
did not display detectable levels of c-fos mRNA or protein
in any experiments (Fig. 2C). This includes cells of the external formation of the complex, the dominant seat of vagal cardiomotor neurons (Bieger and Hopkins, 1987). Use of an independent immediate-early gene marker, NGFI-B, also failed to provide evidence of
PE-induced activation of ambiguual neurons in this region (data not
shown). The somewhat surprising lack of a PE-stimulated activational response at this locus may be indicative of a relative insensitivity of
IEGs to mark activation of motor neurons (see also Menétrey et
al., 1989; Bullit, 1990; Wisden et al., 1990). Nonetheless, a few cells
exhibiting PE-induced Fos-IR and CAT-IR or retrograde tracer were seen
reliably in the dorsal motor nucleus of vagus, aspects of which have
been implicated as participating in the chronotropic regulation of the
heart (Nosaka et al., 1982; Izzo et al., 1993; Standish et al.,
1995).
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DISCUSSION |
PE-induced expression of c-fos has permitted anatomical
and neurochemical characterization of cell groups that, to a first approximation, may be considered the initial station in the central processing of primary baroreceptor input (NTS) and a principal relay
through which the baroreceptor inhibition of sympathetic outflow is
effected (CVLM). A topographically distinct group of PE-sensitive cells
in the NTS displays markers for the NO phenotype, and in contrast to
the remainder of the baroreceptor strip, a substantial fraction of
these project to the RVLM. A more homogeneous arrangement was seen in
the CVLM, where PE-activated neurons comprised a longitudinally
organized column of GABAergic cells, many of which were identified as
projecting to the RVLM (Fig. 9).
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Figure 9.
Summary of the organization of medullary
baroreflex pathways. In the NTS, PE-induced Fos-IR is concentrated in a
continuous strip (dark gray) occupying discrete aspects
of the commissural and dorsal subnuclei at levels 1 and
2, respectively. We provide evidence here that a rostrally
situated subset of barosensitive NTS neurons (level 2)
projects directly to pressor sites in the RVLM (level
4) and/or expresses markers for the NO phenotype. Most
barosensitive neurons of the NTS, however, do not project directly to
the RVLM. The available evidence suggests that they come to influence
sympathetic outflow indirectly, via one or more interneurons in the
medial division of the NTS (light gray), which in turn
projects to the CVLM (dashed lines). Based on criteria of phenotype and connectivity, we define the CVLM as a diffuse, longitudinally organized column of cells, maximally developed at
level 3, and composed primarily of barosensitive neurons
that are GABAergic neurons and project to the RVLM (thick black
line). Drawings of sections through the medulla are modified from
the atlas of Swanson (1992). AMB, Nucleus ambiguus;
AP, area postrema; CVLM, caudal depressor
region; NO, nitric oxide; NTS, nucleus of
solitary tract; RVLM, rostral ventrolateral medulla;
NTSap, NTS at the level of area postrema;
NTSc, commissural NTS.
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PE-induced hypertension as a baroreceptor activation model
PE is a vasoconstrictor agent that acts primarily on
1-adrenoceptors of peripheral vascular beds (Hieble et
al., 1995), also stimulates baroreceptors directly (Kunze, 1981;
Hirooka et al., 1992), and is widely used to assess arterial baroreflex
function (e.g., Imaizumi et al., 1984; Morrison et al., 1988). In a
previous study, bolus injections of PE provoked discrete
c-fos mRNA expression in the barorecipient zone of the NTS
and in nonaminergic neurons of the CVLM, but this was not accompanied
by correspondingly robust Fos protein expression (Chan and Sawchenko,
1994). Here, we have identified PE infusion parameters that reliably
provoke concordant, and comparably discrete, c-fos mRNA and
protein expression patterns throughout the brainstem, providing a basis
for characterizing baroreflex pathways in conscious, freely moving
rats. Similar challenge paradigms have been shown to induce Fos-IR
expression in medullary regions pertinent to the orchestration of
baroreflex responses (Badoer et al., 1994; Li and Dampney, 1994; Polson
et al., 1995). Thus, the extent to which putative second-order NTS neurons exhibit IEG induction seems dependent on the duration, as well
as the magnitude, of the conditioning pressure ramp. This may be
attributed to the nonlinear responsiveness, and differential sensitivity, of NTS neurons to changes in arterial pressure (Rogers et
al., 1993).
A higher PE dose gave rise to less circumscribed Fos induction
patterns, notably including expanded involvement of the medial NTS,
medullary raphe nuclei, and catecholaminergic neurons. Similar observations have been reported after electrical stimulation of baroreceptor nerves (Erickson and Millhorn, 1991; McKitrick et al., 1992; Rutherfurd et al., 1992) and sustained high PE doses (Badoer
et al., 1994). This may reflect activation of nonintegral components of
baroreflex circuitry, such as low-pressure receptors (Li and Dampney,
1994), neurons of the ventral respiratory group (Ellenberger et al.,
1990), or spinally projecting medullary raphe neurons that may modulate
baroreflex functions at the level of the sympathetic preganglionics
(Spyer, 1994). Alternatively, higher doses of PE may recruit neurons
involved in resetting of baroreceptor gain, which is reflected by a
shift of the arterial pressure-heart rate curve over the course of
sustained hypertension (Gonzales et al., 1983). Finally, the activation
of medullary catecholamine neurons in response to more severe levels of
PE-induced hypertension is somewhat paradoxical in view of the
acknowledged involvement of these neurons in stimulating adaptive
autonomic and neuroendocrine responses to hypotensive
stimuli (see, e.g., Chan and Sawchenko, 1994; Li and Dampney, 1994).
Only a very small subset of aminergic neurons was identified as
displaying the GABA phenotype, arguing against any substantial overlap
of the two populations of the VLM. It is worthy of mention that
patients receiving inadvertent overdoses of PE show generalized stress
symptoms (Fraunfelder and Scafidi, 1978), and the recruitment of
medullary catecholamine neurons seen in response to strenuous, but not
milder, PE challenges may help explain such observations. A subset of
these innervates stress-related hypothalamic neurosecretory cell groups
(Cunningham and Sawchenko, 1988; Cunningham et al., 1990), and
distribution of aminergic neurons activated in the high-dose PE
situation more closely approximates that of the hypothalamically
projecting adrenergic population than it does that of the
reticulospinal subset (Tucker et al., 1987). Overall, although the
functional significance of the expanded distribution of IEG expression
seen in response to higher doses of PE is unclear, these seem to
involve nonessential components of the baroreflex pathway. As detailed
below, the moderate dose more discretely activated cells in the
barorecipient zone of the NTS and GABAergic column of the CVLM.
Organization and phenotype of medullary barosensitive neurons
Nucleus of the solitary tract
We identified a contiguous strip of barosensitive neurons situated
in the dorsal part of the commissural NTS and extending rostrally into
circumscribed aspects of the dorsal subnucleus that displayed robust
c-fos expression in response to PE-induced hypertension and
overlaps extensively with terminal fields of the carotid sinus (Housley
et al., 1987) and aortic depressor nerves (Ciriello, 1983). Cells in
these regions have been demonstrated to display short-latency EPSPs to
primary baroreceptor afferent stimulation (Donoghue et al., 1985;
Rogers et al., 1993) and likely conform in large part to second-order
neurons supplied by these nerves. In support of this view, we have
recently found that Fos induction in both the NTS and CVLM in this
paradigm is essentially abolished by complete sinoaortic denervation
(Chan and Sawchenko, 1996) (R. Chan and P. Sawchenko, unpublished
observations). It remains unclear whether neurons comprising a
secondary focus of barosensitive cells in the medial NTS may be
activated directly by baroreceptor afferents or polysynaptically in
response to PE treatment. Unlike the baroreceptor strip, the medial
subnucleus is a major source of projections to the ventrolateral
medulla (Sawchenko and Swanson, 1982; Ross et al., 1985), and the
PE-responsiveness of medial NTS cells may reflect a position as a relay
interposed between second-order neurons and the VLM. On the other hand,
transganglionic tracing experiments have revealed secondary
accumulations of baroreceptor afferent terminals in the medial
subnucleus (Ciriello, 1983; Chan and Sawchenko, 1996), leaving open the
possibility of direct activation by primary afferents.
Despite the great number of GABAergic neurons in the NTS (Blessing et
al., 1984; Izzo et al., 1992), only a small proportion of barosensitive
neurons were found to express GAD mRNA. It is unclear whether those
that do may constitute an organized substrate for inhibitory
interneuronal processing of baroreceptor input within the primary
afferent termination zone. Evidence exists to support a role of
GABAergic interneurons in the modulation of baroreflex function within
the NTS (Sved and Tsukamoto, 1992). In any case, the bulk of GABAergic
barosensitive neurons throughout the NTS do not project to the RVLM,
supporting the notion that NTS is not a major source of inhibitory
influences on cells providing reticulospinal vasomotor outflow (Meeley
et al., 1985; Milner et al., 1987). The extremely limited
representation of GLYT-2-positive barosensitive neurons in the NTS
suggests that glycinergic neurons are unlikely to play a major direct
role in the initial handling of primary baroreceptor input.
By contrast, a subgroup of NO-producing neurons in the NTS are
positioned to play a central role in baroreceptor information processing. We identified a circumscribed group of barosensitive neurons in aspects of the dorsal NTS known to be supplied
preferentially by the aortic depressor nerve (Ciriello, 1983) that
displayed markers for the NO phenotype. A disproportionately high
percentage of these cells could be retrogradely labeled from tracer
deposits in the RVLM, suggesting a direct route by which the
baroreceptor inhibition of sympathetic outflow may be effected. Because
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Abstract
Introduction
