Vol. 90, Issue 2, 475-485, February 2001
Decreased CSF pH at ventral brain stem induces widespread
c-Fos immunoreactivity in rat brain neurons
R. M.
Douglas1,
C. O.
Trouth1,
S. D.
James1,
L. M.
Sexcius1,
P.
Kc1,
O.
Dehkordi1,
E. R.
Valladares1, and
J. C.
McKenzie2
Departments of 1 Physiology and Biophysics and
2 Anatomy, College of Medicine, Howard University,
Washington, District of Columbia 20059
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ABSTRACT |
Physiological evidence
has indicated that central respiratory chemosensitivity may be ascribed
to neurons located at the ventral medullary surface (VMS); however, in
recent years, multiple sites have been proposed. Because c-Fos
immunoreactivity is presumed to identify primary cells as well as
second- and third-order cells that are activated by a particular
stimulus, we hypothesized that activation of VMS cells using a known
adequate respiratory stimulus, H+, would induce production
of c-Fos in cells that participate in the central pH-sensitive
respiratory chemoreflex loop. In this study, stimulation of
rostral and caudal VMS respiratory chemosensitive sites in
chloralose-urethane-anesthetized rats with acidic (pH 7.2) mock
cerebrospinal fluid induced c-Fos protein immunoreactivity in
widespread brain sites, such as VMS, ventral pontine surface, retrotrapezoid, medial and lateral parabrachial, lateral reticular nuclei, cranial nerves VII and X nuclei, A1 and
C1 areas, area postrema, locus coeruleus, and
paragigantocellular nuclei. At the hypothalamus, the c-Fos reaction
product was seen in the dorsomedial, lateral hypothalamic, supraoptic,
and periventricular nuclei. These results suggest that 1)
multiple c-Fos-positive brain stem and hypothalamic structures may
represent part of a neuronal network responsive to cerebrospinal fluid
pH changes at the VMS, and 2) VMS pH-sensitive neurons project to
widespread regions in the brain stem and hypothalamus that include
respiratory and cardiovascular control sites.
chemosensitivity; immunocytochemistry
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INTRODUCTION |
EXPERIMENTAL
INVESTIGATIONS have implicated neurons located at the
ventrolateral medullary surface (VMS) in the central regulation of both
cardiovascular and respiratory activity (2, 19, 20, 35). Some of these neurons are believed to be involved
in the central CO2 pH chemoreceptor drive to respiration in
mammals (20, 31). Lesioning or blockade of
discrete areas at the VMS in cats abolishes the respiratory sensitivity
to inspired CO2 (31). Earlier, Berndt et al.
(2) reported that electrical stimulation of the caudal
chemosensitive area [caudal VMS (cVMS)] in peripheral chemoreceptor-denervated cats caused hyperventilation. However, when
procaine was applied topically to the cVMS during electrical stimulation, apnea resulted, and this was only reversed when direct respiratory center stimulation was applied. It was, therefore, postulated that the integrity of the VMS chemosensory neuronal elements
was essential to the respiratory center drive in the absence of
peripheral chemoreceptor afferents. Recent hypotheses concerning
central respiratory chemoreception have been expanded to implicate not
only the VMS but also the retrofacial nucleus, portions of the nucleus
tractus solitarius (NTS), midline raphé structures, and the
retrotrapezoid nucleus (RTN) as potential sites of central respiratory
chemosensitivity (3, 6, 24, 25, 26).
The precise location of the central chemosensitive neurons that
modulate respiratory activity is still being sought, and various techniques have been utilized to identify the exact sites and characteristics of the specific morphological substrates involved. Ideally, a specific marker expressed by neurons that respond to adequate respiratory stimuli could serve to identify the chemoreceptor element and, perhaps, other neurons synaptically coupled to them. In
fact, stimulation of neurons within the central nervous system (CNS) is
known to initiate cascading biochemical reactions, which may activate
immediate early genes. The c-fos gene, which is a transcription modulator, is a DNA binding protein that initiates biochemical long-term adaptive changes in neurons (23,
29). The c-fos and other immediate early genes are
believed to act as third messengers in signal transduction by altering
gene expression in response to neuronal excitation. Expression of the
c-Fos protein, which is translated from the protooncogene
c-fos, is a sign of cell activation and has, therefore, been
utilized immunocytochemically as a metabolic marker and tracer of
activities in the CNS (29).
The present study utilizes the immunocytochemical expression of
c-fos as a marker of increased activity in an attempt to
localize neuronal elements within the rat brain that are activated in
response to stimulation of the VMS with cerebrospinal fluid (CSF) pH
changes. These neurons may represent part of the neuronal network
involved in the central pH chemosensory drive to breathing.
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METHODS |
Adult Sprague-Dawley rats (n = 21; weight
250-350 g) were anesthetized with chloralose-urethane (40 mg/kg
-chloralose and 200 mg/kg urethane ip), and a tracheal cannula was
routinely inserted via tracheotomy. In spontaneously breathing animals,
the VMS was exposed from the lower pons to about spinal cord
C1-C2 level and ~2-3 mm lateral from the
midline. Rectal temperature was maintained at 37 ± 1°C.
Chemical Stimulation
Effect of pH changes.
In a series of studies, pledgets soaked in mock CSF (mCSF) pH 7.2 (acidic) and 7.4 (control) were applied unilaterally to either the
rostral VMS (rVMS) or the cVMS of rats at sites (Fig. 1) analogous to the classic
chemosensitive regions described in the cat (20). The
composition of the mCSF was as follows (in mM): 121 NaCl, 1.14 CaCl2, 24 HCO3
, and 5 KCl (pH 7.4; the
solution was bubbled with 5% CO2 in air and maintained at
37°C). For mCSF pH 7.2, the pH of mCSF was adjusted with HCl while
the solution was bubbled with 5% CO2 in air and maintained
at 37°C. cVMS and rVMS sites may be identified in the vicinity of the
exit of the rootlets of cranial nerves XII and VI, respectively. Both
regions extend lateral (1-2 mm) to the pyramids in a region that
we believe contains the parapyramidal cell group (PPR). In the rat,
cVMS chemosensitive sites appear to extend from the rostral to the
caudal extent of the hypoglossal rootlets (~3-5 mm
rostrocaudally) as they exit the brain stem, whereas rVMS
chemosensitive sites extend 2-3 mm below the pontomedullary border. The pledgets were replaced every 10 min for 2 h to allow for the translation of the gene product, the c-Fos protein. Excess CSF
was blotted from the control and acidic mCSF-soaked pledgets before
application to the VMS to avoid spillage to surrounding regions. In
addition, cottonoid wicks applied bilaterally, at both the rostral and
caudal extent of the exposed medulla, served to absorb
endogenously generated CSF that might mix with and, therefore,
spread the stimulus to adjacent areas.
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Fig. 1.
Diagrammatic representation of the ventral surface of the
medulla oblongata of the rat. The 2 respiratory chemosensitive regions
[rostral (R) and caudal (C)] analogous to the classical
chemosensitive regions described in the cat (20) are
shown. The C1 and A1 areas on the
right represent cardiovascular sites at the ventral medulla
oblongata. V-XII indicate the cranial nerves.
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In this study, we performed three types of control experiments:
1) anesthesia controls, which were perfused immediately on the animal's succumbing to the anesthetic; 2) surgical
controls, which were perfused immediately after the VMS was exposed;
and 3) pH 7.4 controls, wherein pledgets containing mCSF at
pH 7.4 were applied to the VMS for a 2-h period utilizing the same
protocol as for experimental animals. The extent of c-Fos labeling,
both in terms of intensity of staining and number of cells stained, was
much greater in the experimental animals than in controls (i.e.,
anesthesia, surgery, and pH 7.4). The number of animals utilized in
each of the experimental protocols was as follows: surgery controls,
n = 2; pH 7.4 controls at the cVMS, n = 5; pH 7.2 at the cVMS experiments, n = 10; pH 7.4 controls at the rVMS, n = 5; and pH 7.2 at the rVMS
experiments, n = 5. The effect of decreased pH was
assessed by the immunocytochemical methods described below.
Perfusion and Fixation
The animals were rapidly perfused transcardially with 0.9%
saline in 0.1 M phosphate buffer (pH 7.3) and 4% paraformaldehyde in
0.1 M phosphate buffer (pH 7.3). The fixed tissue was dissected out and
placed in fixative for 24 h, followed by cryoprotection in a
glycerol-phosphate buffer for 48 h. Subsequently, 40-µm frozen sections were prepared from the entire brain on an American Optical Cryo-Cut II microtome, placed in PBS, and processed for c-Fos immunocytochemistry, according to the procedure of Uemura et al. (36).
c-Fos Immunocytochemistry
The tissues were washed with 3.0-25.0 ml of PBS three times
for 10 min each. They were then incubated with 0.3% hydrogen peroxide for 30 min at room temperature. The tissues were washed again with PBS
with 0.1% Triton X-100 (TX-100) and incubated with 3.0-10.0 ml
PBS containing 0.3% TX-100 and 5% normal goat serum for 30 min at
room temperature. They were then transferred to 3.0-5.0 ml of
diluted c-Fos primary antibody (1:5,000) (Santa Cruz Biotechnology, Santa Cruz, CA) and incubated 24 h or overnight at 4°C. The
antibody utilized in this study is an affinity-purified polyclonal
antibody raised against a peptide corresponding to amino acids
3-16 of the c-Fos protein. This region maps to the amino terminus
of the human and mouse c-Fos protein and, therefore, does not
cross-react with Fos-related antigens such as Fos B, Fra 1, or Fra 2 (27). After they were washed with PBS containing 0.1%
TX-100 three times for 10 min each, the tissues were incubated with
3.0-5.0 ml of biotinylated anti-goat immunoglobulin G (1:200)
(Vector Laboratories, Burlingame, CA) for 2 h at room temperature.
The tissues were again washed with PBS containing 0.1% TX-100 three
times for 10 min each and incubated with avidin-biotin complex (Vector
Laboratories) reagent for 2 h at room temperature. They were then
washed with PBS three times for 10 min each and incubated in
diaminobenzidine hydrochloride (DAB Kit, Vector Laboratories) with
0.3% H2O2 for 5-10 min. Finally, the
sections were rinsed with tap water, mounted onto gelatin- and
alum-coated slides, dehydrated, coverslipped, and appraised using light
microscopy. Some sections were counterstained with cresyl violet.
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RESULTS |
Expression of c-Fos in Response to pH Changes at the VMS
Response to mCSF pH 7.4 (control).
Figure 1 is a schematic representation of the ventral surface of the
medulla oblongata (VMS) of the rat. Topical application of mCSF pH 7.4 (control) to the cVMS and rVMS over a 2-h period to allow sufficient
time for the translation of the c-Fos protein from the induced
c-fos mRNA produced a light c-Fos reaction at the sites of
mCSF application. There was only scant or no c-Fos immunoreactivity
throughout the rest of the neuraxis, similar to the basal levels
observed in unstimulated tissues and in studies in which the animals
were subjected to anesthesia and/or surgery alone (controls) (Figs.
2 and 3).
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Fig. 2.
Light micrographs showing area
postrema (AP) in the control animal in which pledgets containing mock
cerebrospinal fluid (mCSF) at pH 7.4 were applied to the ventrolateral
medullary surface (VMS) for a 2-h period (top) and in the
experimental animal in which the VMS was stimulated with mCSF pH 7.2 (bottom). Note the amount of stained cells in the
experimental animal compared with the control animal. D,
dorsal.
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Fig. 3.
Light micrographs showing the supraoptic nucleus (SON;
top) and the arcuate nucleus (ARC; bottom) in
animals in which the pledgets containing mCSF at pH 7.4 (control) were
applied on the VMS. OT, optic tract.
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Basal levels of c-Fos were routinely detected at various brain stem
sites, inclusive of the medial vestibular nucleus, ventral and dorsal
cochlear nuclei, and the interpenduncular and red nuclei in the
pons-midbrain.
Response to decreased mCSF pH.
In response to topical application of mCSF at pH 7.2 (acidic) to the
VMS over a 2-h period, the c-Fos protein reaction product was
identified in several brain stem sites and in the hypothalamus.
At the ventral brain stem, the c-Fos reaction product was seen in the
vicinity of the inferior olives (IO) bilaterally, in the lateral
reticular nucleus (LRN) unilaterally, and in the parvocellular region
of the LRN bilaterally (Table 1).
c-Fos-positive cells were also detected in known vasoactive sites such
as the noradrenaline-containing A1 vasodepressor region of
the caudal ventrolateral medulla (Fig. 4). Small c-Fos-positive bipolar and
multipolar neurons were observed embedded within or just beneath the
marginal glia (MG) at the VMS and bilaterally just ventrolateral to the
pyramids (Figs. 4 and 5). A few scattered
c-Fos-positive cells were also noted within the raphé pallidus
(RP) and magnus (RM) (Fig. 5, Table 1). When the decreased mCSF pH
solutions were confined to the rostral brain stem chemosensitive area
(rVMS), no c-Fos-positive cells were noted in the vicinity of the IO.
However, c-Fos immunoreactivity was noted in the vicinity of the facial
nucleus, the nucleus of the trapezoid body (NTZ), as well as the
lateral and medial superior olives and the nucleus
paragigantocellularis lateralis (PGCL) (Figs.
6 and 7 and
Table 1). At the pontomedullary border, c-Fos immunoreactivity was
detectable in the NTZ, in the ventral pontine nuclei, and in the
rostral periolivary region. An intriguing cluster of c-Fos-positive
cells also exists at the ventral pontine surface (VPS) in contiguity
with the cells detected at rVMS levels.
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Table 1.
Topical mock CSF pH 7.2 application to the cVMS and rVMS
induces c-Fos protein expression at multiple brain sites
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Fig. 4.
Light micrograph showing the immunocytochemical Fos-positive
reaction product in neurons at the AP (top left), the caudal
VMS (cVMS; bottom right), in the vicinity of nXII rootlets
as they exit the brain stem (top right), and the
A1 vasomotor area (bottom left) in
response to stimulation of the VMS with mCSF pH 7.2 (acidic). Top
right: D, dorsal.
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Fig. 5.
Light micrograph showing c-Fos-positive cells in the
raphé pallidus (RP) below the basilar artery and in a few
scattered cells about the pyramids (P; top) and in the
rostral VMS (rVMS; bottom).
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Fig. 6.
Distribution of c-Fos-positive reaction product in neurons in
response to stimulation at the brain stem surface with acidic mCSF pH
7.2 at the rostral (top) and caudal (bottom)
medulla. cVRG, caudal ventral respiratory group; DM, dorsomedial
hypothalamus; IO, inferior olives; LC, locus coeruleus; LHB, lateral
habenula; LPB, lateral parabrachial nucleus; NTS, nucleus tractus
solitarius; PGCL, paragigantocellularis lateralis; Pre-BötC,
pre-Bötzinger complex; RTN, retrotrapezoid complex; 7, facial
nerves; VMH, ventromedial hypothalamus; V, ventral; D,
dorsal.
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Fig. 7.
Diagrammatic representation of c-Fos immunoreactivity in the brain
stem of the rat in response to the unilateral topical application of
acidic mCSF (pH 7.2) to the rVMS (left) and to the cVMS
(right). Each dot represents 3 neurons. c-Fos reaction
product can be seen along the VMS, in the ventrolateral IO, and within
the AP. A1, noradrenaline cells; CG, central gray;
C1, adrenaline cells; CVL, caudoventrolateral reticular
nucleus; KF, Kölliker-Fuse nucleus; LRN, lateral reticular
nucleus; LT, lateral trigeminal nucleus; MV, medial vestibular nucleus;
NA, nucleus ambiguus; PBL, lateral parabrachial nucleus; PE,
periventricular hypothalamic nucleus; RVL, rostroventrolateral
reticular nucleus; SON, supraoptic nucleus; cSP5, cervical spinal
trigeminal tract; TZ, nucleus of trapezoid body; VII, facial nerve;
XII, hypoglossal nerve.
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At dorsal regions, immunoreactivity of c-Fos was also detected within
the area postrema (AP) and the ventrolateral portion of the NTS
throughout its rostrocaudal extent (Figs. 4 and 7). c-Fos reaction
product was also noted bilaterally in the locus coeruleus (LC), nuclei
parabrachialis lateralis (PBL), and Kölliker-Fuse (Fig. 7). There
was also a slight response in the cerebellum in the lateral dentate and
interpositus nuclei. At midbrain regions, light staining was noted at
the dorsal aspects of the superior and inferior colliculi. At the level
of the hypothalamus, c-Fos-positive cells were detected in the
dorsomedial, lateral, and periventricular nuclei (Fig.
8). c-Fos-positive cells were also
strongly expressed in the supraoptic nucleus (Figs. 8 and
9). At cortical levels, lightly staining
c-Fos-positive cells were demonstrated in the piriform cortex. Taken
together, these studies demonstrate that decreased pH of mCSF applied
to the VMS induces c-Fos expression in a widespread network of nuclei
within the brain stem and hypothalamus (Table 1).
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Fig. 8.
Light micrograph showing the immunocytochemical localization of
c-Fos-positive reaction product in neurons at the hypothalamus in
response to stimulation of the caudal medulla with acidic mCSF (pH
7.2). a: c-Fos-positive neurons are seen clustered within
the DM, PE, and SON of the hypothalamus. b: c-Fos-positive
neurons are seen clustered within the DM and PE nuclei. c:
c-Fos-positive neurons are seen clustered within the SON of the
hypothalamus.
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Fig. 9.
Light micrograph showing the
immunocytochemical localization of c-Fos in neurons clustered within
the SON of the hypothalamus. Note the intranuclear localization of the
c-Fos reaction product (arrows). Magnification, ×100.
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In general, acidic stimulation of either the cVMS or the rVMS (Table 1)
induced bilateral expression of c-Fos in cells at the cVMS, rVMS, and
caudal VPS. This would indicate the possibility of reciprocal
connections occurring between these superficial brain stem sites. cVMS
stimulation induced consistent c-Fos responses at the A1
vasodepressor area, the AP, and the ventrolateral IO (Fig. 7, Table 1).
rVMS activation, however, induced c-Fos immunoreactivity in the
A5 area, RP, RM, cranial nerve VII, and at rostral
medullary regions that appeared to be insensitive to cVMS stimulation.
An intriguing response to rVMS stimulation occurred in the caudal RP
that did not occur in response to cVMS stimulation. It is known that
connections exist between the rostral ventrolateral medulla (RVLM) and
the caudal RP; therefore, this response could have been due to
descending projections from rostral regions. A finding difficult to
interpret was that rVMS stimulation induced a more extensive c-Fos
response at the cervical spinal cord than did cVMS stimulation. Whereas
at the spinal cord level cVMS stimulation induced c-Fos responsiveness
at dorsal horn cells only, rVMS stimulation additionally caused the
production of c-Fos protein in cells located around the central canal
(Fig. 7).
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DISCUSSION |
The exclusive localization of pH-sensitive sensing elements at the
VMS that modulate respiratory activity has been a controversial issue,
which has been recently compounded by reports that multiple sites of
respiratory chemosensitivity exist throughout the brain stem (24,
29). In an attempt to glean information on the neuroanatomic network involved in responses to brain stem CSF pH changes,
determination of the distribution of c-Fos protein expression in the
rat brain was investigated in response to stimulation of the known
respiratory pH-sensitive regions (19, 20, 22, 35) at the
VMS with acidic mCSF (pH 7.2). The protooncogene c-fos
and its concomitant protein are transiently, rapidly, and
polysynaptically expressed within the cell nucleus in response to cell
activation by a variety of stimuli. Expression of the c-Fos protein in
response to CSF pH changes is, therefore, assumed to identify sites of
cell activation that might contain the central respiratory
chemoreceptor elements as well as neuronal elements synaptically
coupled to them.
One should not, however, overlook the limitations of the c-Fos
technique (7). Traditionally, 2-deoxyglucose (2-DG) uptake has been utilized as a marker of increased metabolic activity within
the CNS; however, mismatches between 2-DG uptake and c-Fos activation
do occur in some brain regions. These mismatches are suggested to occur
because 2-DG can detect axonal and dendritic activity, whereas c-Fos
stains the nucleus and is assumed, therefore, only to detect somatic
changes. Additionally, some brain regions demonstrate a lack of c-Fos
responsiveness, regardless of the stimulus paradigm employed.
Therefore, negative results cannot be absolutely interpreted to
indicate that a particular structure has not been activated. Another
concern of the c-Fos technique is that basal c-Fos expression may
sometimes mask changes related to the activation of specific pathways.
Positive results must also be carefully dissected out, because
activity, handling, and stress of the animal, such as can occur during
surgery, may affect c-Fos levels. To avoid confounding the data with
nonspecific stimuli, we did not monitor respiration or blood pressure
because VMS-modulated effects on these parameters have been well
characterized (19, 22). Conversely, one must ensure that
the stimulus utilized was potent enough to induce a c-Fos response. It
has been recognized that neurons require strong stimulation to generate
c-Fos activation and that anesthetics such as ketamine and morphine can
suppress c-Fos induction. The most critical aspect of c-Fos
biochemistry is the time course of c-Fos activation and decay, which
varies with the brain region, the type of inducing agent, and the route of application of the agent (7, 23). Additionally, it must be noted that some neurons increase c-Fos staining without an increase
in firing, whereas other neurons do not increase c-Fos staining,
despite an increase in firing rate (23). Careful control experiments should also be conducted to ensure that one can separate basal- from stimulus-induced c-Fos activation. We have, therefore, conducted rigorous control experiments, which included anesthesia, surgery, and neutral mCSF application controls, and determined that our
experimental stimulus, acidic mCSF (pH 7.2), was able to induce c-Fos
activation above basal levels in several brain regions.
Responses at Brain Stem Sites to Low-CSF pH
In this study, topical application of mCSF pH 7.4 (control) to the
VMS produced little or no c-Fos immunoreactivity in the brain stem or
throughout the rest of the neuraxis (Figs. 2 and 3). Topical
application of mCSF pH 7.2 to the VMS induced a consistent and clearly
identifiable distribution of c-Fos immunoreactivity throughout the rat
brain. The significance of these findings becomes evident when one
reflects on the known afferent and efferent connections and functions
of a number of these sites.
Ventral brain stem cell groups.
c-Fos responsiveness to low pH at the VMS was observed in neurons
within the MG ventrolateral to the pyramids and within the superficial
regions of the RP. This finding corresponds well with the report of
Sato et al. (29), who described cells along the VMS that
were c-Fos responsive to 1 h of hypercapnia and that reside
ventral to the LRN and the nucleus PGCL. These superficial cell groups
in the rat are reminiscent of the type I neurons in the cat
(19) and the arcuate nucleus in the human
(11), which have been implicated in sudden infant death
syndrome (11, 17) and Ondine's curse (primary alveolar
hypoventilation syndrome) (12).
In our laboratory, we have also detected neuronal cells by light and
electron microscopy at the caudal chemosensitive area (area L) of cats
within the walls of branches of the basilar artery that penetrate the
ventral brain stem surface deep into the neuropil. Scheibel et al.
(30) described similar specialized neurovascular relationships in various subnuclei of the raphé complex and
suggested that this neurovascular system might subserve either a
neurosecretor, chemosensor, mechanoreceptor, or vasomotor function. The
existence of type I cells and neurovascular elements, as well as the
unique properties of the MG in the caudal chemosensitive zone, have
been confirmed by electron microscopy (35) and have been
referred to as surface neuropil by Filiano et al. (11).
The relationship of the nucleus RP, arcuate nucleus (in cat and human),
and VMS cells is an intimate one, whereby the topographic proximity and
the developmental, chemical, and functional similarities argue for a
common function. These regions appear to be ontogenically derived from
neuronal precursors, which migrate ventromedially across the medulla
from the rhombic lip (9). They subsequently differentiate
into the medullary arcuate nucleus, inferior olivary complex, griseum
pontis, and the serotonergic cells of the nucleus RP and nucleus PGCL.
Regardless of their nomenclature, this nuclear group appears to be
functionally and topographically associated with central pH-sensitive
chemosensory elements at the cVMS.
The existence of neurons, as opposed to glia, in the most
superficial aspects of the ventral medulla has been questioned for several years (32). In 1964, Dahlström and Fuxe
(4) described serotonergic neurons in the medulla
oblongata that were localized to the raphé nuclei and in sites
adjacent to the pyramids and that projected to the spinal cord. The
region lateral to the pyramids and ventral to the IO was designated as
the B3 region. Hökfelt et al. (15)
observed dopamine decarboxylase, substance P, and serotonin
[5-hydroxytryptamine (5-HT)] immunoreactivity in raphé nuclei
and in the superficial cells of the arcuate nucleus located medial
(ventral to the RP), ventral, and lateral to the pyramidal tract in the
so-called "subpyramidal" or "arcuate" regions of the medulla.
Ljungdahl et al. (18) found that substance P-containing neurons within the medulla had a similar distribution pattern to that
of 5-HT neurons and that they also projected to the spinal cord. Thor
and Helke (34) demonstrated that cells in the nucleus reticularis pontis and at the ventral surface of the pyramids had a
high intensity of 5-HT immunofluorescence and they projected to the
NTS. Many substance P immunofluorescent neurons located lateral to the
pyramids and along the VMS also projected to the NTS (36),
and double-labeling of cells with 5-HT and substance P
(34) was detected among the arcuate fibers lateral to the pyramids, in the region of the paraolivary nucleus of Hökfelt et
al. (15). In the caudal medulla, double-labeling of cells coincided with the location of the nucleus intrafascicularis
hypoglossi. Sasek and Helke (28) have also demonstrated
superficial medullary enkephalinergic neurons that project to the
intermediolateral cell column (IML). However, electron microscopic
analysis of serotonergic cells at the VMS of the rat (13)
demonstrated that these cells have the usual characteristics of neurons
with observable synapses on somatic and dendritic surfaces. We have,
therefore, adopted the term PPR, as described by Sasek and Helke
(28), to designate all superficially located, medullary
neurons in the vicinity of the pyramids, inclusive of the
B3 region, the paraolivary region, nucleus
intrafascicularis hypoglossi, arcuate nucleus, and the traditional VMS.
In this study, c-Fos-positive cells were also identified ventral to the
facial motor nucleus and in a region functionally defined as the RTN,
which has extensive efferent projections to both the dorsal and ventral
(33) respiratory groups. In the rat, however, the RVLM
appears to contain the rostral chemosensitive zone described in the
cat, which is sensitive to the acid-base changes in the CSF. The RTN
could, therefore, serve as the anatomic substrate for respiratory
chemosensitivity at the RVLM in the rat. At the level of the RVLM,
c-Fos reaction product was also identified in neurons of the
pre-Bötzinger complex [which is considered to be the rostral
component of the ventral respiratory group (33)], the
NTZ, ventral tegmental pontine nuclei, the rostral periolivary region,
and cerebellar nuclei and within the RP and RM. Intriguingly, Richerson
(26), utilizing perforated patch-clamp recordings in rat
medullary slices, has shown that neurons in the rostral raphé
demonstrate CO2 sensitivity and similar
electrophysiological properties as previously described chemoreceptor elements.
c-Fos immunoreactivity was detected in the A1 noradrenaline
area, which contains sympathoinhibitory neurons and appears to act via
inhibitory connections to the RVLM or C1 area. The
A1 vasodepressor site has no spinal projection
(21) but does project to the NTS (34), the
parabrachial complex, and the hypothalamus. Expression of c-Fos was
also noted at the caudal midline raphé complex, which is a major
source of descending input to the IML (21) that projects
via sympathetic premotor neurons to the adrenal medulla and sympathetic
ganglia. The RP and possibly raphé obscurus innervate the IML
(21) and project to catecholaminergic neurons in the RVLM,
implicating the caudal raphé in influencing sympathetic cells via
a relay station in the RVLM. Thus detection of c-Fos immunoreactivity
in the A1 area and in the caudal raphé nuclei (raphé obscurus and RM) may indicate that neuronal connectivity exists between cells at the VMS respiratory chemosensitive areas and
structures involved in the regulation of blood pressure and cardiovascular activity. However, it is difficult to distinguish c-Fos
activation because of VMS-stimulated blood pressure changes from that
which is due to generalized stress-induced blood pressure changes.
Similarly, this paradigm does not permit the precise identification of
neurons that are synaptically coupled within a CNS network.
It should be noted that there was a lack of c-Fos activation in several
respiratory output neuronal groups, such as the nucleus ambiguus,
nucleus para-ambigualis, and the hypoglossal and phrenic nuclei. There
may, therefore, be regions that are responsive to VMS stimulation but
do not increase their c-Fos staining. Conversely, there was an increase
in c-Fos staining in regions without an obvious link to
cardiorespiratory control mechanisms, such as the IO and superior
olives, the facial motor nucleus, and the cervical dorsal horn, which
serves to further complicate the interpretation of the data. However,
the VMS pH-sensitive region may represent a site of central
coinnervation, where one common receptor element might impinge on both
cardiorespiratory and other presently unknown autonomic control mechanisms.
Dorsal cell groups.
The NTS is the primary site of termination of cardiovascular and
peripheral respiratory afferent fibers. It appears to receive projections from the rostral and caudal (21) ventrolateral
brain stem and is densely innervated by catecholaminergic fibers
(4), which are believed to arise from A1 and
C1 catecholaminergic cell groups within the VLM. c-Fos was
expressed within the NTS throughout its rostrocaudal extent and
appeared to be predominantly localized to the ventrolateral portion of
the nucleus. The ventrolateral NTS is known to be the repository of
lung afferents.
In response to decreased CSF pH, c-Fos immunoreactivity was identified
in AP neurons, which are known to project to brain regions involved in
cardiovascular regulation. The AP also receives afferent input from the
periventricular hypothalamic nucleus, the PBL nucleus, the mediocaudal
NTS, and the vagus nerve (32). AP projection targets
include mediocaudal NTS, the PBL, C1 area, dorsal motor
nucleus of the vagus, and the nucleus ambiguus (32), which
may be activated in response to CSF pH changes. c-Fos-positive cells
were identified in the LC in response to decreased CSF pH at the cVMS.
LC or the A6 group of noradrenaline neurons may be involved
in determining the level of vigilance within an animal (1)
and is considered to be a site of convergence of multiple afferent
inputs. At the dorsal pons, moderately stained c-Fos-positive cells
were consistently noted in the nuclei PBL, parabrachialis medialis, and
Kölliker-Fuse. These nuclear groups are presumed to send
descending inhibitory projections to inspiratory neurons within the
respiratory centers. These observations suggest the possible
involvement of multiple regions of the brain stem in the responses to
CSF pH changes.
Hypothalamus.
Indeed, the identification of c-Fos immunoreactivity in the supraoptic,
periventricular, and other hypothalamic nuclei appears to indicate a
linkage between brain stem pH chemosensitive regions and regions
involved in the regulation of water flux and possibly the antidiuretic
hormone, as well as other autonomic and hypophyseotropic hormonal
functions via the norepinephrine-containing monoaminergic system that
projects from the LC and lateral tegmental systems throughout the
spinal cord, brain stem, and hypothalamus. Sympathoexcitatory neurons
scattered throughout the lateral and posterior hypothalamus project to
the RVLM (5).
Summary
Focal, topical application of an acidic
stimulus, mCSF (pH 7.2) to the VMS, both caudal (in the
region of the hypoglossal rootlets) and rostral (in the region of the
facial nucleus and exit of the abducens nerve), induces a nearly
identical c-Fos activation pattern in the brain stem and hypothalamus
of rats. The VMS or PPR is apparently the repository of several
neurotransmitters and neuromodulators or their receptors and also
projects to and receives projections from several CNS regions involved
in autonomic control functions. Intriguingly, this study also indicates
that there is a differential sensitivity between the cVMS and rVMS to
an acidic stimulus, in that some structures activated by caudal stimulation are not c-Fos responsive with rostral stimulation and visa
versa. Previous work and the present study, therefore, suggest that the
VMS, and possibly the VPS, are potentially critical sites, within a
wide and complex network of pH- and CO2-sensitive brain
regions, in the central chemosensory control of respiration and other
autonomic functions.
| |
ACKNOWLEDGEMENTS |
We are grateful to Antoinette Coverton for secretarial assistance
and proofreading.
| |
FOOTNOTES |
This work was supported by Office of Naval Research Grant
N00014-94-0523 (to C. O. Trouth).
Address for reprint requests and other correspondence: C. O. Trouth, Dept. of Physiology and Biophysics, Howard Univ. College of
Medicine, 520 W St., N. W., Washington D. C. 20059 (E-mail: ctrouth{at}fac.howard.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.
Received 6 October 1998; accepted in final form 25 August 2000.
| |
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