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1 Department of Medicine, Keio University, Tsukigase Rehabilitation Center, Shizuoka-ken 410-3293; 4 Department of Physiology, School of Medicine, Teikyo University, Tokyo 173-8605, Japan; 2 Department of Cell and Molecular Physiology, and 3 Lineberger Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The anatomical structure of central respiratory chemoreceptors in the superficial ventral medulla of rats was studied by using hypercapnia-induced c-fos labeling to identify cells directly stimulated by extracellular pH or PCO2. The distribution of c-fos-positive cells was found to be predominantly perivascular to surface vessels. In the superficial ventral medullary midline, parapyramidal, and ventrolateral regions where c-fos-positive cells were concentrated, we found a common, characteristic, anatomical architecture. The medullary surface showed an indentation covered by a surface vessel, and the marginal glial layer was thickened. We classified c-fos-positive cells into two types. One (type I cell) was small, was located inside the marginal glial layer and close to the medullary surface, and surrounded fine vessels. The other (type II cell) was large and located dorsal to the marginal glial layer. c-fos Expression under synaptic blockade suggested that type I cells are intrinsically chemosensitive. The chemosensitivity of surface cells (possible type I cells) surrounding vessels was confirmed electrophysiologically in slice preparations. We suggest that this characteristic anatomical structure may be the central chemoreceptor.
respiratory control; chemoreceptor; central chemosensitivity; carbon dioxide; synaptic blocker; c-fos
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE RESPIRATORY CONTROL SYSTEM maintains a stable arterial PCO2 level by continuously adjusting ventilation via regulatory inputs from peripheral and central chemoreceptors. In 1963, Mitchell et al. (18) identified the classical rostral chemosensitive area in the cat ventral medullary surface; this was followed by the discovery of the classical caudal chemosensitive area [see the review by Loeschcke (17)]. In 1979, it was reported that coagulation of the ventral medullary surface eliminates central chemosensitivity (31). In 1992, Sato et al. (30) reported the use of hypercapnia-induced c-fos labeling technique to locate the chemoreceptor cells in the superficial ventral medulla. In addition, several recent experiments have indicated a widespread distribution of possible chemoreception sites within the central nervous system, including the nucleus tractus solitarius (3), hypothalamus (4), midline raphe (1), locus coeruleus (26), and ventral respiratory group region (21). However, the exact location of primary chemoreceptor cells and the specific anatomic structure of central chemoreceptors in the ventral medulla and in other brain areas are still controversial [see the review by Okada et al. (23)]. We hypothesize that the ventral medullary chemoreceptor cells are preferentially located adjacent to small vessels. The purpose of the present study is to elucidate the anatomic structure of central chemoreceptors by focusing on the anatomic relationship between hypercapnia-activated cells and blood vessels in the ventral medulla. For this purpose, we examined quantitatively the overall distribution patterns of hypercapnia-induced c-fos expression in the superficial layer of the ventral medulla and compared these patterns with those of surface vessels. Next, we explored the anatomic arrangement of hypercapnia-activated cells in the superficial ventral medulla. We also conducted perforated patch recordings in slice preparations to confirm the CO2 responsiveness of cells in the surface regions of the ventral medulla.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Preparations for c-fos Immunohistochemistry
Conscious animal experiments. In each experiment, a pair of conscious, freely moving adult rats (Sprague-Dawley; a total of 12 pairs) was placed overnight in separate, small chambers (one for hypercapnic stimulation, the other as a control) in an identical dark and quiet environment. The next morning, the still darkened chamber of one rat was closed and flushed for 3 h with a humidified hypercapnic gas mixture (10% CO2-21% O2 in N2). CO2 level inside the chamber was monitored with an infrared CO2 analyzer and kept between 70 and 78 Torr. The control rat was allowed to breathe normal air for the same period of time. After 3 h under these conditions, the two animals were immediately killed with an excessive amount of pentobarbital sodium (50 mg/kg ip) or diethyl ether (inhalation) and immediately perfused transcardially with standard Tyrode followed by a fixative solution containing paraformaldehyde (4%) and sucrose (5%) in 0.1 M phosphate buffer (pH 7.4). The lower brain stem was removed, kept in the same fixative at 4°C for 60-90 min, and then placed in a solution with 30% sucrose and 0.1 M phosphate buffer (pH 7.4).
Anesthetized animal experiments. Adult rats (n = 7) were anesthetized first with diethyl ether or methoxyflurane. After the trachea had been cannulated, the femoral vein and femoral artery were catheterized for infusion and blood pressure measurement, respectively. A mixture of urethane (500 mg/kg) and chloralose (10 mg/kg) was administered intravenously. The animals were artificially ventilated, and end-tidal PCO2 was continuously monitored with an infrared CO2 analyzer. The animals were divided into three groups. In the first group (n = 2), eucapnia (end-tidal PCO2 of 32-36 Torr) was maintained throughout the experiment. In the second group (n = 2), hypocapnia (end-tidal PCO2 of 10-13 Torr) was induced by increasing both tidal volume and respiratory frequency. In the third group (n = 3), the animals were ventilated with the same hypercapnic gas mixture used in the conscious animal experiments to maintain end-tidal PCO2 between 70 and 80 Torr. After 3 h of these exposures, the animals were immediately killed and perfused transcardially, and the brain stems were fixed, as in the conscious animal experiments described above.
In situ transarterial perfusion experiments. To determine whether c-fos-positive cells in the superficial ventral medulla are activated directly by CO2/H+ or indirectly through transsynaptic mechanisms, we performed an in situ transarterial perfusion study (11) in young rats (10-14 days old; n = 12). The animals were anesthetized with pentobarbital (35 mg/kg ip). After the chest was opened and the right atrium was incised, a catheter was inserted into the left ventricle, and transarterial perfusion was performed with a normal mock cerebrospinal fluid (CSF) [(in mM) 125 NaCl, 5.0 KCl, 1.0 NaH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, 15 glucose] or a mock CSF for synaptic blockade, i.e., a mock CSF with a composition low in calcium and high in magnesium [(in mM) 125 NaCl, 5.0 KCl, 1.0 NaH2PO4, 0.2 CaCl2, 6 MgSO4, 26 NaHCO3, 15 glucose] or a mock CSF containing TTX (1.0 µM), all at 26°C. The perfusion pressure was adjusted to 50-60 Torr. In preliminary experiments, to confirm the viability of our in situ preparations, the respiratory movements of the thorax were observed for >4 h during perfusion with a normal mock CSF. Animals were divided into four groups (n = 3 in each group): 1) perfused with a hypercapnic (saturated with 8% CO2 and 92% O2) normal mock CSF alone, 2) perfused with a hypercapnic mock CSF with added synaptic blocker, 3) perfused with hypocapnic (saturated with 100% O2) mock CSF alone, and 4) perfused with hypocapnic mock CSF with added synaptic blocker. After an animal had been perfused, the brain stem was isolated, and the brain tissue was fixed with 4% paraformaldehyde.
In vitro whole brain stem experiments. To observe the distribution pattern of c-fos expression on the superficial ventral medulla, we performed immunohistochemical procedures on the whole brain stem tissues of neonatal rats (n = 10). Each rat (4 days old) was anesthetized with diethyl ether. Then the brain stem was isolated, placed in a chamber, and superfused with a mock CSF, as in the experiments with the isolated brain stem-spinal cord preparation (24, 25). Hypercapnic or hypocapnic mock CSF (for composition, see In situ transarterial perfusion experiments above) was used to superfuse the tissue. In four preparations, TTX (1.0 µM) was added to the mock CSF to block synaptic transmission. At the end of 1 h of superfusion, the preparation was fixed with 4% paraformaldehyde.
Tissue Preparation and Immunohistochemistry
Sectioning with a cryostat. The frozen brain tissue was transversely sectioned with a cryostat. To obtain a detailed distribution map of c-fos-positive cells in the entire ventral medullary surface, serial sections (thickness of 60 µm) were floated in 0.1 M phosphate buffer (pH 7.4) and processed for Fos-like protein immunohistochemistry. A sheep polyclonal antibody to Fos (Genosys) was used as the first antibody. Tissues from control and CO2-exposed animals were processed with the same procedure. The sections were placed in a 0.1 M phosphate buffer, containing 2% normal rabbit serum and 0.3% Triton X-100 detergent, and then incubated for 24-48 h at 4°C with Fos antiserum. After the sections were rinsed with phosphate buffer containing normal rabbit serum, they were incubated with biotinylated anti-sheep IgG as the secondary antibody (Kit PK-4006, Vector), followed by avidin-biotin complex reagent, and allowed to react in Ni-diaminobenzidine solution. Then the sections were floated onto gelatin-coated slides, air dried, and covered. Control immunohistochemical experiments were also conducted by omitting primary antiserum.
Sectioning with a paraffin-embedding technique. To preserve the surface vessels of the medulla, the fixed brain stem was embedded in paraffin. Sectioning of the paraffin-embedded brain tissue was carried out with a microtome. Serial sections (5-10 µm thick and 50-60 µm apart) were floated onto gelatin-coated slides and air dried. The sections were dewaxed and processed in the same immunohistochemical manner as described above.
Whole brain stem preparation. Whole brain stem preparations were processed for c-fos immunohistochemistry in the same immunohistochemical manner as described above but without sectioning. After being allowed to react with Ni-diaminobenzidine, the wet brain stem preparation was examined directly under a microscope. The nuclei of c-fos-positive cells with dark brown color labeling on the surface of the ventral brain stem were clearly recognized through the pia mater.
Perforated Patch Recording of Superficial Ventral Medullary Neurons
Responses of superficial medullary neurons to CO2 were analyzed in slice preparations. Medullary slice preparations (300-500 µm thick) were made from 25 rats (Wistar, 3-7 days old) by using a brain slicer (Microslicer DTK-1000, DSK, Kyoto, Japan). Slices obtained from the rostral medulla were placed in a recording chamber (volume = 1.0 ml) and continuously superfused (4-6 ml/min) with an oxygenated mock CSF (for composition, see In situ transarterial perfusion experiments above). For visualization of neurons in slices, we used a fixed-stage upright microscope (Nikon Measurescope UM-2, Tokyo, Japan) with a long working distance objective lens (×40 CFWI, Nikon) after we modified the microscope as follows: an illuminating system with optic fiber, halogen lump (ELI-100S, Mitsubishi Rayon, Tokyo, Japan), and red colored glass filter (MC R1, Kenko, Tokyo, Japan) was used instead of the original lighting system. Neurons were visualized on a videomonitor (PC-TV354, NEC, Tokyo, Japan) by using a high-sensitivity charge-coupled device video camera (WAT-308A, Watec, Tokyo, Japan). Discordance of the lighting axis produced an image similar to an infrared differential interference contrast image. Thirty-nine spontaneously firing surface neurons (distance from the ventral medullary surface of <100 µm; mostly <80 µm) were selected, and the membrane potentials and firing activities of these neurons were analyzed with a perforated patch recording technique. The recording was under direct neuronal visualization but was primarily based on a blind patch recording technique described previously (13). Briefly, a glass pipette (GC100-TF-10, Clark) was pulled with a horizontal puller (PA-91, Narishige, Tokyo, Japan) to a tip size of ~2 µm. Electrode resistance was 12 M
when filled with a solution containing (in mM) 140 potassium gluconate,
3.0 KCl, 10 EGTA, 10 HEPES, 1.0 CaCl2, 1.0 MgCl2, and 100 µg/ml nystatin (pH adjusted to
7.2-7.3 with KOH). A micropipette with positive pressure
(10-20 cmH2O) applied inside was placed above a target
neuron and downed vertically by using a manual hydraulic
micromanipulator. When the tip of the micropipette touched a neuron,
gentle suction was applied to form a cell-attached patch. Membrane
potentials were recorded with a whole cell patch amplifier (CEZ 3100, Nihon Kohden, Tokyo, Japan). After a gigaohm seal was obtained, the
recorded membrane potential became gradually negative and was
stabilized in ~10 min. Membrane potential was presented without
correcting a liquid junction potential. Responses of these neurons to
CO2 were examined by switching the superfusate
CO2 fraction from 2 to 8%. Neurons were classified as
CO2 excitable when neuronal firing frequency was increased
by >20% by hypercapnic (i.e., 8% CO2) mock CSF.
Data Analysis
Camera lucida drawings and photographs of immunostained materials were made to view the precise anatomic location and structure of the medullary regions where dense populations of c-fos-positive cells were found. The coordinate brain sites were determined according to the rat brain atlas (27). Quantitative evaluation of c-fos expression was made in two ways. 1) c-fos-Positive cells in the ventral medulla were consecutively counted in serial sections (thickness of 60 µm); each counting area was 160 µm in width and 50 µm in depth from the ventral surface. In each counting area, all c-fos-positive cells were counted through the entire section thickness (60 µm) by gradually and carefully changing focal planes. In the data presentation, the effect of tissue shrinkage caused by fixation was not corrected. The numbers of c-fos-positive cells in each experimental group were averaged and used to construct a distribution map showing c-fos-positive cells within the superficial ventral medulla. The average number of c-fos-positive cells at selected brain stem levels were plotted. 2) c-fos-Positive cells within the superficial ventral medulla were divided into two groups according to the size of their nuclei (nuclei diameter <9 µm or >10 µm; see Classification of hypercapnia-induced c-fos-positive cells below). The diameters of cell nuclei were measured under a microscope scale. In cells with oval-shaped nuclei, the long axis diameter was measured. Statistical analyses were conducted by using one way analysis of variance followed by a t-test. P < 0.05 was assumed to be significant.| |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypercapnia-Induced c-fos-Positive Cells in the Ventral Medulla
In the ventral medulla of both conscious and anesthetized adult rats, an increase in c-fos expression was found in several ventral medullary regions processed with a cryostat sectioning technique after the rats were exposed to CO2 for 3 h. Clusters of c-fos-positive cells were located in the nucleus retrotrapezoideus, the nucleus reticularis lateralis, the nucleus paragigantocellularis lateralis, the nucleus retroambiguous, the superficial parapyramidal region, a region ventrolateral to the nucleus olivaris inferioris, and the nucleus raphe pallidus. These observations are largely consistent with those reported by other investigators who used hypercapnic/acidic stimulation (5, 10, 14, 19, 30, 34). We found hypercapnia-induced c-fos expression in a large number of cells in the most superficial layer of the ventral medulla (i.e., in the marginal glial layer) in both conscious, spontaneously breathing rats (Figs. 1A and 2) and anesthetized, artificially ventilated rats (Fig. 3A). Figure 1A shows the distribution map of hypercapnia-induced c-fos-positive cells in the superficial ventral medullary areas (from the surface up to 50 µm deep; average data of 8 rats) prepared with a cryostat sectioning technique. There were four major groups of c-fos-positive cells. A narrow cell column (Fig. 1A, area 1) in the ventral medullary midline area occupied the most ventral portion of the nucleus raphe pallidus, which had an uneven distribution pattern with several high-density peaks rostrocaudally. Three lateral groups of c-fos-positive cells were found; the first one is distributed in the rostral parapyramidal area 0.8-1.1 mm lateral to the ventral medullary midline, the second one in the rostral area 2.3-3.2 mm lateral to the ventral medullary midline, and the third one in the caudal area 0.7-1.5 mm lateral to the ventral medullary midline (surroundings of the hypoglossal nerve rootlets). In most parts of the ventral medulla, c-fos-positive cells were arranged in a single layer, but multilayer clusters of c-fos-positive cells were found in the thickened marginal glial layer of several regions that corresponded to the high-density c-fos-positive regions shown in Fig. 1A. The highest density subpopulation was located along the lateral border of the pyramidal tract, corresponding to the nucleus parapyramidalis superficialis (Fig. 1A, area 2) (9), and in the surroundings of the hypoglossal nerve rootlets (Fig. 1A, area 5). Areas with a medium density of c-fos-positive cells were observed widely along the more lateral portions of the ventral medulla, which included the areas ventrolateral to the lateral superior olive, the caudal periolivary nucleus and the retrotrapezoid nucleus (Fig. 1A, area 3), ventrolateral to the paragigantocellular nucleus (Fig. 1A, area 4), and ventrolateral to the ventrolateral reticular nucleus (Fig. 1A, area 6). In conscious rats, increases of c-fos expression were remarkable in the superficial raphe,and in the parapyramidal and retrotrapezoid regions at the mid- to rostral medullary levels (Fig. 2; stars indicate significant differences between the hypercapnic and eucapnic groups). In the anesthetized hyperventilated rats, only a small number of c-fos-positive cells were found in the ventral medullary midline, parapyramidal and ventrolateral regions (Fig. 3B). When evaluating c-fos expression in both conscious and anesthetized animals on the basis of PCO2 levels, our results demonstrated dose-dependent increases of c-fos-positive cells in the superficial ventral medulla (Figs. 2 and 3, A and B). Hypercapnia induced a further increase of c-fos expression in both ventral medullary midline and ventrolateral medullary regions.
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Hypercapnia-Induced c-fos-Positive Cells Intimately Related to Surface Vessels
One of the most important findings in the present study is an intimate anatomic relationship between hypercapnia-induced c-fos-positive cells and surface vessels. There was a high degree of coincidence between the distribution of c-fos-positive cells and the location of surface vessels (Figs. 1A and 3, A and C). For example, the ventral medullary midline group lies under the basilar artery. The parapyramidal region, which includes the highest density of c-fos-positive cells, lies under the inferior petrosal sinus. In the rostrolateral and caudolateral medulla, c-fos-positive cells were scattered over areas that were perfused through the anastomoses of the anterior and posterior inferior cerebellar and paraolivary arteries. In transverse brain sections, the ventral medullary surface where high concentrations of c-fos-positive cells were found consistently displayed a concave shape, a surface indentation associated with surface vessels (Fig. 4, A-C). Concentrations of c-fos-positive cells were often found surrounding fine vessels. Figure 4C shows clusters of cells that surround fine vessels (apparently fine artery and vein) in the most superficial region of the nucleus raphe pallidus that is covered by the basilar artery. Figure 4D demonstrates a penetrating branch of the basilar artery (the median medullary artery) that was surrounded by c-fos-positive cells. Most of these vessel-surrounding c-fos-positive cells were located only superficially (mostly within 200 µm of the ventral medullary surface; many are within 100 µm).
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Because in a cryostat sectioning technique surface vessels are usually
detached and lost, we used a paraffin-embedding technique that
permitted the preservation of surface vessels in immunohistochemical processes. Using this technique, we investigated the spatial
relationship between c-fos-positive cells and adjacent
vessels in the superficial ventral medulla. We observed characteristic
arrangements of c-fos-positive cells and surface vessels in
thickened parts of the marginal glial layer (Fig.
5). In the ventral medullary midline
areas, c-fos-positive cells were concentrated in a small
region dorsal to the basilar artery (Fig. 4, A and
B). This region occupied the superficial portion of the
nucleus raphe pallidus. In the rostral medulla, this region exhibited
an arrowhead-shaped profile and was separated from the other brain
tissue by a thin capsule-like layer (Fig. 4B). Several
arterioles, venules, and capillaries were often found to be closely
surrounded by or mingled with c-fos-positive cells. When
sections were made containing the median medullary artery (Fig.
4D), clusters of c-fos-positive nuclei were
observed closely surrounding the arterial wall along the superficial
portion of the penetrating artery. In addition, in the parapyramidal
and ventrolateral medullary regions, similar arrangements of
c-fos-positive cells with blood vessels were found. Figure
4C shows that c-fos-positive cells and a
capillary are embedded in the marginal glial layer in the region of the
nucleus parapyramidalis superficialis (9). In this region,
the medullary surface was indented, and the region was further covered
by surface vessels. The distance between the fine vessels and the
vessel-surrounding c-fos-positive cells was mostly <20
µm (usually <10 µm). In other parts of the ventral medulla,
c-fos-positive cells were scattered within a thin part of
the marginal glial layer; however, three to five
c-fos-positive cells were often found surrounding a
capillary.
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Classification of Hypercapnia-Induced c-fos- Positive Cells
There was a diversity of sizes and shapes among the c-fos-positive cell nuclei in the superficial ventral medulla. We examined the size of c-fos-positive cell nuclei located in the superficial ventral medulla (from surface to 50 µm deep) to see whether the difference reflected heterogeneous groups of cells. In 50 brain stem sections prepared with a cryostat sectioning technique from 10 conscious hypercapnic rats, we found that in the ventral medullary midline and in the parapyramidal and ventrolateral regions, the diameters of most cell nuclei ranged from 5 to 16 µm (Fig. 6). There were two distribution peaks in the cell nuclei diameters; one was at 7-8 µm and the other at 12 µm. On the basis of these findings in conjunction with the location of the cells and the shape of cell nuclei, we classified the c-fos-positive cells into two types. Three-fourths of the cells had small round- or oval-shaped nuclei 6-9 µm in diameter; we termed these type I cells. Most type I cells were embedded in the marginal glial layer adjacent to the ventral medullary surface. Some type I cells were found surrounding penetrating arterioles and venules. The other type, which we termed type II cells, were characterized by variously shaped, relatively large nuclei (nuclear diameter of >10 µm; mostly 11-14 µm). Some type II cells were found in the marginal glial layer intermingled with type I cells, and the rest were located immediately dorsal to the marginal glial layer. Classification of these two types of cells was also supported by studies with synaptic blockers (see below).
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Hypercapnia-Induced c-fos Expression Under Synaptic Blockade
To determine whether the c-fos-positive cells being studied were activated directly by an increase of CO2 or indirectly through transsynaptic mechanisms, we conducted studies using synaptic blockers (mock CSF containing low calcium and high magnesium or mock CSF containing TTX). The results for the three ventral medullary regions (midline, parapyramidal, and ventrolateral regions) are presented. In the normal (control) hypercapnic perfusate group containing normal concentration of calcium, a large number of cells in the midline, parapyramidal, and ventrolateral regions of the superficial ventral medulla expressed c-fos (Fig. 7). Both type I (nuclear diameter of 6-9 µm) and type II (nuclear diameter of >10 µm) c-fos-positive cells were observed. In the second group of animals that received a hypercapnic perfusate with low calcium and high magnesium composition, the number of type II c-fos-positive cells was greatly reduced (compared with that of the normal hypercapnic perfusate group, P < 0.01). However, the number of type I c-fos-positive cells was only slightly less than that of the normal hypercapnic perfusate group. In the group that received a hypocapnic perfusate causing synaptic blockade, only a few c-fos-positive cells were found (data not shown). The absence of c-fos expression in the animals perfused with this synaptic blockade solution could be caused by low calcium (20). To avoid this complication, we performed studies with another type of synaptic blocker, i.e., TTX (Fig. 8). We found that the number of type II cells in each of the three regions of the superficial ventral medulla was markedly reduced with hypercapnic perfusate containing TTX compared with those perfused with normal hypercapnic solution (P < 0.01). No c-fos-positive cell with a nuclear diameter >14 µm was observed in the presence of TTX. However, c-fos expression in type I cells was not inhibited by TTX but rather very slightly augmented (statistically not significant).
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We also examined the whole brain stem preparation without sectioning to
analyze the effect of TTX. Surface c-fos-positive cells
could be clearly visualized from the surface through the semitransparent pia mater. Figure 9 shows
examples of camera lucida drawings of c-fos-positive nuclei
on the rat ventral medullary surface. We found similar distribution
patterns of c-fos-positive cells in the two groups that were
superfused with normal hypercapnic (Fig. 9A) and
TTX-containing hypercapnic mock CSF (Fig. 9B), respectively, and these distribution patterns of c-fos-positive cells were
similar to those observed in the in vivo hypercapnic conscious adult
rats (see Fig. 1A). Only a few c-fos-positive
cells were observed in the TTX-treated hypocapnic brain stem (Fig.
9C).
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Perforated Patch Recording of Superficial Ventral Medullary Neurons
We conducted perforated patch recording of superficial ventral medullary neurons under direct visualization and analyzed CO2 responsiveness in neurons of the superficial ventral medulla. Of the 39 recorded neurons, 17 were located in the ventrolateral medullary regions and 22 were in the ventral medullary midline. Twenty-nine percent (5 of 17) of the ventrolateral neurons were excited by hypercapnic CSF, and the rest were either insensitive to or inhibited by hypercapnic CSF. Of the ventral midline neurons, 27% (6 of 22) were excited by hypercapnic CSF. An example of a surface neuron of the ventral medullary midline is shown (Fig. 10, A-C). This neuron exhibited excitation in response to hypercapnia. In addition, we succeeded in recording CO2-excitable neurons that had close contact with fine vessels in the superficial ventral medulla. Figure 10, D and E, demonstrates a CO2-excitable neuron that was in close contact with a penetrating vessel in the superficial region of the ventral medullary midline.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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c-fos Expression of neurons in hypercapnic rats indicated that those neurons were being activated under experimental conditions, but we could not determine the source of the stimulus, whether due to CO2/H+ or another source (e.g., input from mechanoreceptors in the chest or abdomen or from nociceptors). The c-fos-positive neurons that we observed in the superficial ventral medulla may represent the result of any number of various stimuli. To analyze the source of the stimuli, we performed group experiments with hypocapnic, eucapnic, and hypercapnic rats under conscious and anesthetized conditions. Furthermore, we obtained principally same data in the experiments by using the isolated whole brain stem preparation that lacks all the peripheral input. It should be noted that anesthesia itself may induce c-fos expression in the ventrolateral medulla. Takayama et al. (33) claimed that urethane alone, but not a chloralose-urethane mixture, caused an increase in c-fos expression in the ventrolateral medulla. We therefore used chloralose-urethane as an anesthetic. In hyperventilated anesthetized rats, end-tidal PCO2 was reduced to a level below the phrenic threshold. We found that c-fos expression disappeared almost completely in the superficial ventral medulla, which indicated that there was minimal background neural activity in the hypocapnic condition and that chloralose-urethane alone does not induce c-fos expression (Fig. 3B). However, moderate c-fos expression was observed in the superficial ventral medulla in eucapnic rats, with similar distribution patterns in both anesthetized and conscious rats. Thus c-fos expression in the superficial ventral medulla under eucapnic conditions can be associated with a physiological level of CO2.
In the evaluation of cell depths from the medullary surface, the effect of tissue shrinkage induced by fixation must be noted. We conducted mapping of c-fos-positive cells in paraformaldehyde fixed brain stem tissue. We counted c-fos-positive cells from the ventral medullary surface up to 50 µm and presented the data without correcting for the influence of tissue shrinkage. Therefore, it must be noted that the investigated regions correspond to those up to slightly deeper than 50 µm of raw (prefixed) tissue.
We observed a hypercapnia-induced increase of c-fos-positive cells in various superficial ventral medullary regions, including traditionally recognized and recently reported areas (1, 6, 8, 9, 17, 18, 29). Many c-fos-positive cells are embedded in the marginal glial layer, i.e., the most superficial layer of the medulla, and this finding is well consistent with the study of Sato et al. (30), who reported that 67% of CO2-induced c-fos-positive cells were identified within 50 µm below the ventral medullary surface. We confirmed CO2-excitability of surface cells that are embedded in the ventral medullary marginal glial layer by perforated patch recording (Fig. 10). This finding supports our idea that surface cells embedded in the ventral medullary marginal glial layer, including those in contact with fine vessels, play a role in central chemoreception.
The different patterns of hypercapnia-induced c-fos expression in the conscious and anesthetized rats were interesting. When we compared the density of c-fos-positive cells in the hypercapnic group with that in the eucapnic group of the conscious rats, we found that a hypercapnia-induced increase of c-fos-positive cells that was statistically significant was found only in some restricted areas (see Fig. 2). On the other hand, in the anesthetized rats, hypercapnia-induced c-fos expression was found in broad areas of the superficial ventral medulla (especially in the rostral ventrolateral areas) (Fig. 3A). The observed difference of c-fos expression in awake and anesthetized rats may seem unusual because anesthesia is usually a ventilatory depressant. This may be due to awake inhibition of chemoreceptor activity or partly due to anesthetic-induced c-fos expression (33). Assuming that the hypercapnia-induced c-fos-positive cells that we studied represent a part of the neural substrate of central chemoreceptors in the superficial ventral medulla, our results show that sensitivity of ventral medullary chemoreceptors to CO2 stimulation is state dependent. This may be one of the possible causes of the different effects of surface ventral medullary chemoreceptors on control of breathing under conscious and anesthetized conditions (7, 28). The state dependency of central chemosensitivity has also been studied recently by Nattie's group (16, 22), and the contribution to central chemoreception of the retrotrapezoid nucleus in the awake state and that of the ventral medullary midline (raphe) in the sleeping state has been described. These reports together with our present results will contribute to the further study on the state-dependency of central chemoreception in each brain stem region.
It is important to recognize that c-fos expression induced by CO2 exposure does not distinguish primary chemosensitive cells from second-order neurons. The diversity in sizes and shapes of c-fos-positive cells in the superficial ventral medulla has encouraged us to classify them according to their function. By measuring the sizes of c-fos-positive cell nuclei in the superficial ventral medulla, we found two distribution peaks in diameters of cell nuclei (Fig. 6). Three-fourths of the cells within the marginal glial layer had small nuclei with round or oval shapes. The cells with larger nuclei exhibited various shapes and were more deeply located. We therefore classified hypercapnia-induced c-fos-positive cells into two types (type I and type II). Our classification of cells appears to coincide with that of previously reported superficial cell typing; Leibstein et al. (15) reported that superficial ventral medullary neurons can be classified into two types on the basis of cell body sizes and cell depths from the ventral medullary surface, although they did not address the chemosensitivity of these cells. Their report supports the validity of our cell type classification. Our present study is the first to show that there are (at least) two types of CO2-activated cells in the superficial ventral medulla, and we expect that this finding will contribute to future studies that seek to identify the cellular substrate for central chemoreceptor complex.
The classification of c-fos-positive cells is further supported by studies with synaptic blockades. We found that in situ transarterial perfusion of preparations with a solution containing a synaptic blocker (low calcium and high magnesium or TTX), hypercapnia-induced c-fos expression was blocked in the cells with large cell nuclei (type II cells) but not in cells with small nuclei (type I cells) (Figs. 7 and 8). In addition, we observed hypercapnia-induced c-fos expression in the whole brain stem preparation in which only surface cells are visualized. In the whole brain stem experiment, c-fos expression in surface cells was retained even under synaptic blockade (Fig. 9), although statistical analysis was not conducted in this observation. In the whole brain stem experiment, only very superficially located cells (surface cells) could be observed, and this finding also supports the idea that CO2 responsiveness of surface cells (many are expected to be type I cells but not type II cells) is TTX resistant. These observations support our hypothesis that type I cells within the marginal glial layer of the ventral medulla are intrinsically CO2 sensitive and that the CO2 excitability of type II cells is synaptically transmitted. This is one of the most important issues in the study of central chemosensitivity and must be further studied, for example, by using electrophysiological techniques.
Previous studies have identified a characteristic morphological profile of neuronal elements in the ventrolateral surface of the medulla; neuronal somata with numerous synapses were found scattered among the processes of marginal glia (35). However, on the basis of these purely morphological studies, it was impossible to determine which neurons were functionally related to chemoreception. In the present study, hypercapnia-induced c-fos expression under a synaptic blockade was used as a tool to visualize intrinsically chemosensitive cells. By applying c-fos immunocytochemistry, we found an intimate relationship between CO2-sensitive cells and vessels in the superficial ventral medulla. In pictures of the ventral medullary surface, either reconstructed from a series of transverse sections or processed in whole brain stem preparations, we found that the distribution patterns of CO2-sensitive cells corresponded well to surface arteries and veins. The most important finding of the present study is the cellular and vascular architecture in the thickened marginal glial layer in the ventral medullary midline (superficial portion of the nucleus raphe pallidus), parapyramidal, and ventrolateral medullary regions. These regions are morphologically characterized by 1) the concave shape of the medullary surface with a thickened marginal glial layer covered by a surface vessel, 2) the existence of penetrating vessels, 3) clusters of hypercapnia-induced c-fos-positive cells, including intrinsic chemosensitive cells, 4) the anatomic separation of these regions from other brain parts, and 5) an intimate relationship between hypercapnia-induced c-fos-positive cells and vessels; i.e., type I cells surround the fine vessels in the thickened marginal glial layer (Fig. 5). Because surface vessels are usually detached from the brain tissue during a cryostat sectioning process, we applied a paraffin-embedding technique and found an anatomic relationship between surface vessels and hypercapnia-induced c-fos-positive superficial cells. This characteristic anatomic structure appears to be ideal for a central chemoreceptor complex. In the present study, our perforated patch recording experiments also revealed CO2 excitability of superficial ventral medullary cells that were in close contact with penetrating vessels (Fig. 10, D and E). Consistent with the idea that changes of PCO2 in arterial blood are much more effective in stimulating respiration than equivalent changes of PCO2 in CSF (32), we suggest that this characteristic structure composed of surface cells and vessels is effective for the rapid sensing of local pH or PCO2 changes within the perivascular interstitial space. We therefore propose that this structure represents the neural substrate for central chemoreceptors in the ventral medulla. It must be emphasized that the hypercapnia-induced c-fos-positive cells (type I cells) surrounding penetrating vessels are distributed only at the very superficial part of the vessels. The physiological significance of this distribution may be that such an arrangement is efficient in detecting perivascular PCO2/pH changes even before the blood reaches deeper brain stem regions.
It is interesting that Kawai et al. (12) demonstrated dendritic projection of chemosensitive neurons to the concave part of the ventral medullary surface (see Fig. 7, E and F, of Ref. 12). Although they did not conduct simultaneous staining of superficial medullary cells, we hypothesize that dendrites terminating near the ventral medullary surface receive information from the surface chemoreceptor cells (type I cells). Fine morphological studies on the possible synaptic formation between surface-projecting dendrites from deeper neurons and type I cells must be done in the future.
Recently, a growing body of evidence has shown that in addition to the traditionally known chemosensitive areas in the ventrolateral medulla, several other sites in the central nervous system (i.e., the nucleus tractus solitarius, the ventral respiratory neuron group, and the locus coeruleus) are sensitive to hypercapnia (1-5, 9, 10, 21, 26, 29). Our findings regarding the putative neural substrate for central chemoreceptors in the ventral medulla will be useful to identify the structure and physiological roles of the intracranial chemoreceptors in other brain sites.
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ACKNOWLEDGEMENTS |
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The authors thank Kirk McNaughton for assistance in histological processing.
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FOOTNOTES |
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This study was supported by the National Heart, Lung, and Blood Institute Merit Award Grant HL-17689 (to F. L. Eldridge), and the Research Grant for Specific Diseases from the Japanese Ministry of Health and Welfare and the Grant-in-Aid for Exploratory Research from the Japan Society for the Promotion of Science (both to Y. Okada).
Present address of Z. Chen: Division of Biology, GlaxoSmithKline, Five Moore Dr., Research Triangle Park, NC 27709.
Address for reprint requests and other correspondence: Y. Okada, Dept. of Medicine, Keio Univ., Tsukigase Rehabilitation Center, Tsukigase 380-2, Amagiyugashima-cho, Tagata-gun, Shizuoka-ken 410-3293, Japan (E-mail: yasumasaokada{at}1979.jukuin.keio.ac.jp).
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.
March 15, 2002;10.1152/japplphysiol.00620.2000
Received 26 June 2000; accepted in final form 6 March 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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, Hypercapnic rats exposed to 10%
CO2; 
, eucapnic rats breathing room air.
*Significant difference (P < 0.05).
