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1 Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 2 Institute of Neurosciences, Fourth Military Medical University, Xi'an 710032, China
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The pre-Bötzinger complex (PBC), thought to be the center of respiratory rhythm generation, is a cell column ventrolateral to the nucleus ambiguus. The present study analyzed its cellular and neurochemical composition in adult rats. PBC neurons were mainly oval, fusiform, or multipolar in shape and small to medium in size. Neurokinin-1 receptor, a marker of the PBC, was present in the plasma membrane of mostly medium and small neurons and their associated processes and boutons. Among neurons immunoreactive for different neurotransmitter or receptor candidates, various numbers were colocalized with neurokinin-1 receptor. The highest ratio was with nitric oxide synthase (52.72%), and the lowest was with glycine receptors (31.93%). Glutamic acid decarboxylase- and glycine transporter 2-immunoreactive boutons, as well as GABAA receptor-immunoreactive plasma membrane processes and boutons, were also identified in the PBC. PBC neurons exhibited different levels of cytochrome oxidase activity, indicating their various energy demands. Our results suggest that synaptic interactions within the PBC of adult rats involve a variety of neurotransmitter and receptor types and that nitric oxide may play an important role in addition to glutamate, GABA, glycine, and neurokinin.
neurokinin-1 receptor; cytochrome oxidase; glutamate; GABA; nitric oxide synthase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE PRE-BÖTZINGER COMPLEX (PBC) in the rostroventrolateral medulla is hypothesized to be the center or kernel of respiratory rhythm generation (10, 19, 33, 34, 37). This proposal is achieved by serially transecting isolated brain stem-spinal cord preparations in neonatal rats (37). Removal of the PBC abolishes rhythm generation, and rhythmicity is maintained only if the PBC is included. Physiologically, the PBC has various types of neurons that presumably are necessary for the generation of respiratory rhythm (10, 19, 33, 34). Although several models of respiratory rhythm generation have been described, it is generally agreed that a hybrid network model exists that is dependent on a complex interaction between pacemaker neurons with intrinsic membrane properties, glutamatergic excitatory transmission, and glycinergic as well as GABAergic synaptic inhibitions (10, 24, 33, 34, 38). The PBC may also contribute to respiratory rhythm generation in adult mammals. In adult cats (6, 36) and rats (39), a transition zone also defined as "pre-Bötzinger complex" has been demonstrated in vivo. It contains a mixed population of respiratory-modulated neurons bearing inspiratory, expiratory, and phase-spanning activities. There is also evidence suggesting that neonatal and adult animals have different neuronal synaptic activities in the PBC. A key discrepancy is that the blockade of chloride-dependent synaptic inhibition with glycine/GABAA receptor (GABAAR) antagonists stops respiratory rhythmogenesis in adult animals (15, 29, 30), but the rhythm remains in the neonate (7, 27, 30). In addition, the blockade of either non-N-methyl-D-aspartate (NMDA) or NMDA glutamate receptors does not abolish respiratory rhythmogenesis in adult animals (1, 9, 32). On the other hand, the blockade of non-NMDA receptors in neonates abolishes rhythm, even though the blockade of NMDA receptors has little effect on respiratory rhythm (11). The discrepancy may reflect the modulatory adjustment of respiration between pacemaker neurons and synaptic interactions in the respiratory network and evolves from a greater reliance on pacemakers in the neonate to synaptic interactions in the adult (3). Regardless of neonatal or adult in vitro or in vivo studies, the central generation of respiratory rhythm relies on chemical neurotransmission to modulate intrinsic conductances underlying pacemaker oscillations and mediating synaptic interactions. Neurotransmitters including excitatory glutamate and inhibitory glycine and GABA putatively play key roles in the generation and modulation of respiratory rhythms (3, 10, 13, 19, 33, 34, 37).
Most previous studies, however, were based on the injection of physiological agonists and antagonists in vivo or in vitro and the recording of rhythmic motor outputs in the hypoglossal or the C5 nerve roots. Very few of these studies could identify neurons that were directly affected by drug applications, or they could only circumscribe the approximate boundaries of the PBC, inasmuch as it lies in the heterogeneous reticular formation of the ventrolateral medulla oblongata. The anatomic structure of the PBC was not known until recently, when the presence of neurokinin-1 receptor (NK1R) immunoreactivity in the ventrolateral medulla of adult rats implied that NK1R-immunoreactive (ir) neurons delineate the precise anatomic location of the PBC (12). Our previous study combined NK1R immunohistochemistry with cytochrome oxidase (CO) histochemistry to analyze the metabolic development of the PBC in postnatal rats (22). The present study again used NK1R as a marker of the PBC and CO as a marker of neuronal activity but in conjunction with a number of neurotransmitter and receptor candidates to investigate the neurochemical organization of the PBC in adult rats. Double labeling using peroxidase immunohistochemistry and immunogold-silver staining (IGSS) methods allowed for colocalization of neurotransmitters and their receptors in the PBC.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Tissue preparation.
Animals were used in accordance with National Institutes of Health and
Medical College of Wisconsin regulations. Experiments were performed on
22 adult Sprague-Dawley rats (250-280 g). The animals were deeply
anesthetized with chloral hydrate (40 mg/100 g body wt ip) and perfused
transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate
buffer (pH 7.4) and 4% sucrose. The brain stems were then removed and
postfixed by immersion in the same fixative for 2 h at 4°C. They
were then cryoprotected in increasing concentrations of sucrose (10, 20, and 30%) in 0.1 M sodium phosphate buffer at 4°C, frozen in dry
ice, and stored at 
80°C until use.
CO histochemistry and optical densitometry. Six animals were used for baseline data of CO and NK1R. Coronal sections of frozen brain stems were cut at 12 µm thickness on a cryostat, and alternate sets of serial sections were mounted on gelatin-coated slides. One set was processed for CO histochemistry (40) and the other for NK1R immunohistochemistry (22). For CO histochemistry, the sections were incubated in 0.1 M sodium phosphate buffer (pH 7.4) containing 25 mg of 3,3'-diaminobenzidine (DAB; Sigma), 10 mg of cytochrome c (type III, Sigma), and 2 g of sucrose per 50 ml of solution. Incubation was carried out at 37°C in the dark for exactly 2 h. Sections were then washed three times in cold 0.1 M sodium phosphate buffer and dehydrated, and coverslips were applied.
After the PBC was located in adjacent NK1R-immunoreacted sections, the intensity of CO reaction product in individual PBC neurons was analyzed with a Zeiss Zonax MPM 03 photometer system through a ×25 objective lens and a 2-µm-diameter measuring spot. White (tungsten) light was used for illumination, and lighting conditions were held constant for all the measurements. Because the white matter exhibited very low levels of CO activity, it was used as an internal standard for our study; hence, the white matter was subtracted as the background and was set to zero over each section examined. Optical densitometry was performed on neuronal perikarya of the PBC, and the optical density of each cell was an average reading of two to four regions of its cytoplasm. A total of 401 neurons were measured in 6 animals (n
60 in each animal). Mean optical densities and
standard deviations were then obtained.
Immunohistochemistry and IGSS. Antibodies used in this study were glutamate receptor subunits 2 and 3 (GluR2/3), GABAA receptor (GABAAR), GABAB receptor (GABABR), glycine receptor (GlyR), and glycine transporter 2 (GlyT2; Chemicon, Temecula, CA); NK1R and glutamate (Sigma); glutamic acid decarboxylase (GAD) (Boehringer Mannheim); nitric oxide synthase (NOS; BD Transduction Labs, San Diego, CA); and NMDA receptor subunit 1 (NMDAR1; BD PharMingen, San Diego, CA). GluR2/3 antibody was used to reveal the location of AMPA receptors in PBC neurons, because these two subunits were reportedly the most prominently labeled AMPA receptor subunits and were widely distributed in the rat medulla (35). GAD was used as a marker for GABAergic neurons or boutons and GlyT2 as a marker for glycinergic neurons or boutons.
Coronal sections of frozen brain stems were cut at 12 µm thickness on a cryostat, and four sets of serial sections were mounted on gelatin-coated slides. One set was used for NK1R immunohistochemistry and the rest for the other antibodies. For single-label immunohistochemistry, slides were first blocked overnight at 4°C in phosphate-buffered saline (PBS; pH 7.4) containing 5% nonfat dry milk, 5% normal goat serum, and 1% Triton X-100. The slides were then incubated in one of the primary antibodies diluted at the appropriate concentration in PBS (1:15,000 for NK1R; 1:1,000 for glutamate, GAD, and NOS; 1:2,000 for Glut2/3; 1:500 for NMDAR1; 1:25,000 for GlyT2; 1:200 for GABAAR; 1:3,500 for GABABR; and 1:300 for GlyR) for 36-48 h at 4°C. This was followed by the secondary antibodies (goat anti-rabbit or goat anti-mouse IgG-horseradish peroxidase) for 4 h at room temperature. The reaction was detected with a DAB solution for 5-10 min and stopped with cold PBS. Slides were then dehydrated, and coverslips were applied. Double labeling of NK1R and one of the other antibodies (glutamate, NOS, GluR2/3, NMDAR1, GABABR, or GlyR) was achieved by performing single-label immunohistochemistry of these antibodies first as described above and then IGSS of NK1R (23). Anti-NK1R antibodies were diluted at 1:15,000 in 5% normal goat serum and applied to slides for 36-48 h at 4°C. After incubation with immunogold (goat anti-rabbit IgG, 5 nm gold conjugate) diluted at 1:100 for 4 h at room temperature, signals were detected by the silver enhancing kit (BB International) for 8-10 min at room temperature in the dark. Between steps, slides were rinsed in PBS three times for 5 min. Before and after silver enhancing, slides were rinsed with distilled water. They were then dehydrated, and coverslips were applied. The sequence of double labeling was tested previously, with the IGSS procedure preceding or following the horseradish peroxidase-DAB reaction, and both proved to yield comparable results (23). Controls in which the primary antibodies were replaced with normal serum or preimmune serum showed no labeling above background (23; present study, data not shown).Cell size measurements.
Neuronal areas of the PBC were measured in NK1R-ir sections by means of
a Leica Quantimat 570C image analyzer. Every third section bearing NK1R
immunoreactivity in the PBC was chosen for neuronal area measurements
under a ×20 objective. Neuronal areas were determined by the image
analysis system, and mean areas and standard deviations were then
obtained. A total of 331 NK1R-ir neurons in the PBC were measured and
analyzed from 6 animals (n 50 in each animal).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NK1R-ir neurons in the PBC.
NK1R immunoreactivity precisely delineated two groups of nuclei in the
ventrolateral medulla oblongata of adult rats: the PBC was localized
ventrally and the nucleus ambiguus (NA) dorsally (Fig.
1A). NK1R immunoreactivity was
expressed mainly along the plasma membrane of cell bodies and processes
of neurons that were dispersed within the ventral reticular formation
(Fig. 1B). At times, the cytoplasm of neurons was also
slightly labeled with NK1R (Fig. 1B). NK1R-ir neurons of the
PBC were oval, fusiform, or multipolar in shape and primarily small to
medium in size; occasionally they were large. Of 331 NK1R-ir neurons in
the PBC, 272 (82.12%) were small [228.5 ± 38.95 (SD)
µm2] and 53 (16.01%) were medium (345.01 ± 34.34 µm2); occasionally, large neurons (>400
µm2) were also found in the PBC. Thin and long processes
of the PBC often traversed among neurons of the PBC, toward the midline
of the brain stem, dorsally to the NA, or ventrally to the surface of
the medulla (Fig. 1B).
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CO-reactive neurons in the PBC.
Two groups of neurons, PBC localized ventrally and NA dorsally, were
also observed in adjacent CO-reacted sections (Fig. 1C). CO-reactive neurons of the PBC were oval, fusiform, pyramidal, or
multipolar in shape and also mainly small to medium in size; occasionally, large neurons were observed. CO reaction product was
present mainly in cell bodies (Fig. 1D), but thin and short processes were also CO reactive in the neuropil. Neurons in the small-
or medium-size categories exhibited dark, moderate, or light
intensities of CO labeling (Fig. 1D), whereas large neurons tended to be intensely labeled for CO. Among 80 medium-sized neurons, 55 (68.75%) had moderate CO activity, and of 224 small-sized neurons, 140 (62.5%) had moderate CO activity. Optical densitometric
measurements of CO reaction product in various-sized neurons of the PBC
are plotted in Fig. 2. CO-reactive
neurons were often surrounded by CO-reactive neuropil.
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Neurotransmitter- and receptor-ir neurons in the PBC.
Immunoreactivities against all the antibodies used in this study were
found in the ventrolateral medulla of adult rats. The labeling in the
PBC (Figs. 3, A and C;
4, A and C;
5, A, C, and E;
and 6, A and
C) could be correlated with that of NK1R in adjacent sections (not shown). Numerous neuronal somata in the PBC were intensely immunoreactive for glutamate (Fig. 3B), NOS (Fig.
3D), NMDAR1 (Fig. 4B), GluR2/3 (Fig.
4D), GABABR (Fig. 6B), or GlyR (Fig.
6D). Short immunoreactive processes were also
observed in the neuropil of the PBC (thin arrows in Figs. 4, B
and D, and 6, B and D).
Immunoreactive neurons were usually oval, fusiform, or pyramidal
in shape and extended longitudinally in a dorsomedial-to-ventrolateral direction. A variety of immunoreactive intensities for different markers were evident in the same cell and in different cells of the
same section. Immunoreactivities against GAD, GlyT2, and
GABAAR presented patterns different from those of the other
neurotransmitter-related markers. GAD and GlyT2 immunoreactivity was
present in boutons, which tended to contact neuronal somata and
outlined the surface of cells (Fig. 5, B and D).
GABAAR immunoreactivity was expressed mainly on neuronal
surfaces, processes, and boutons (Fig. 5F), with little or
no labeling of neuronal somata.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NK1R immunoreactivity is coextensive with the area functionally defined as the PBC in adult rats. Most of the NK1R-ir neurons were small to medium sized (<400 µm2). Neurons in this size category are generally interneurons, and they seldom send long axons down to the spinal cord. Previous retrograde labeling or single-unit extracellular recordings of neurons in the PBC support their propriobulbar characteristic (35, 39). Although lacking bulbospinal projections, processes of NK1R-ir neurons spread toward the ventral surface of the medulla, dorsomedial medulla, midline, or within the PBC. This pattern is consistent with our previous findings in the PBC of postnatal rats (22) and possibly contributes to a complex synaptic network in the brain stem. The superficial medullary projections may be important for central chemosensitivity (17). The midline projection is likely to contribute to the synchronization of the respiratory rhythm between the left and right sides of the brain stem (24). Koshiya and Smith (19) used a calcium-sensitive dye to label neurons of the PBC and found that neurons were retrogradely labeled from the midline, suggesting a cross talk between PBCs of the two sides. The dorsomedial processes are thought to project mainly toward the ipsilateral and/or the contralateral region of the nucleus of the solitary tract (NTS) (28). It is known that neurons in the NTS integrate primary afferent signals from lung sensory receptors (3, 4). Apart from vagal inputs, NTS neurons also integrate primary afferent signals from peripheral carotid chemoreceptors (8), which play an important role in augmenting ventilation during hypoxia (3). Respiratory neurons in the NTS are confined to its ventrolateral part, which contains mostly inspiratory neurons (2, 43). Electrophysiological and anatomic data suggest that GABA may transmit inhibitory inputs from the rostral respiratory group to output neurons in the ventrolateral NTS (21).
All neurotransmitters and receptors examined in the present study were
identified in the PBC of adult rats. Glutamate-ir neurons were found to
colocalize with NK1R. GAD- and GlyT2-ir boutons were intensely labeled
in the PBC. Glutamate, GABA, and glycine are thought to play critical
roles in rhythmogenesis and modulation. Glutamate exerts its function
by activating ionotropic
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA
receptors (3, 13). There is evidence that, in adult
animals, systemic blockade of both AMPA and NMDA receptors causes apnea
or abolishes phrenic nerve activity, but blockade of either receptor
does not evoke apnea, which suggests that the full generation of
inspiratory bursts relies on both AMPA and NMDA receptor contributions
(1, 9, 32). It is known that AMPA and NMDA
receptor-mediated channels have different electrophysiological features
in their activation, inactivation, desensitization, and resensitization
(16, 25). AMPA-mediated channels function rapidly in their
activation, inactivation, desensitization, and resensitization, whereas
NMDA-mediated channels act more slowly. The temporal summation of both
receptors in their dynamic response may be well suited for their
functional demand. Thus glutamate release would first cause a rapid
depolarization through the activation of AMPA receptors, which then
facilitate the removal of the magnesium block from NMDA channels and
activate NMDA receptors. NMDA receptor activation would maintain a
prolonged depolarization (35). In addition, calcium influx
through activated NMDA channels, in turn, upregulates the function of
AMPA channels (20). We found GluR2/3- and NMDAR1-ir
neurons in the PBC that were double-labeled with NK1R. Both receptors
may be colocalized within the same NK1R-ir neuron, a possibility that
remains to be verified by triple-labeling experiments. Although the
relative roles of these receptors in adult animals in in vivo studies
are not as clear-cut as in vitro neonatal studies, AMPA receptors may
be responsible for phasic waves of excitatory postsynaptic potentials
during the active phase of respiratory neurons to shape the pattern,
whereas NMDA receptors may contribute to phase transition from
inspiration to expiration, and both receptors may contribute to tonic
excitatory maintenance (13).
GABA and glycine are essential inhibitory neurotransmitters for generating respiratory rhythm in the mature respiratory network (15, 29, 30). We found GAD- and GlyT2-ir boutons in the PBC. GABA and glycine function mainly through GABAAR- and GlyR-mediated chloride channels, providing phasic waves of inhibitory postsynaptic potentials during the inactive phase of respiratory neuronal function and during the active phase to shape the pattern of respiratory neuronal activation (3, 13). GABABR-ir neurons were also observed in the PBC. The function of GABABR in respiratory regulation is unclear in adult animals, although GABABR-mediated tonic inhibition is present in respiratory neurons (31).
Numerous NOS-ir neurons were found in the PBC and were colocalized with NK1R in the highest ratio among markers examined. As an enzyme for catalyzing the generation of nitric oxide (NO) from L-arginine, NOS is closely coupled to NMDA receptor activity and calcium influx. Glutamate acts on NMDA receptors. Activated NMDA channels cause an increase of calcium influx, which can stimulate constitutive NOS to generate NO (18, 26). NO may diffuse back to the presynaptic terminal, where it stimulates guanylate cyclase and elevates cGMP concentration, leading to a further increase in the release of glutamate and a greater augmentation of synaptic transmission (5, 26). In short-term hypoxia, the NO-cGMP pathway was activated in the central nervous system and contributes to the induction and maintenance of hypoxia-induced increase in respiratory activity (14). The present findings of numerous NOS-ir neurons in the PBC are consistent with NO playing an important role in the regulation of respiration in normal adult rats.
The present study as well as previous data (12, 22) showed that some neurons in the PBC were singly labeled and were not coexpressed with NK1R. One possibility is that the singly labeled neurons may serve functions other than respiration. Because the PBC lies in the heterogeneous reticular formation of the ventrolateral medulla oblongata, neurons there may have a variety of functions besides those related to respiration. Even the preinspiratory neurons in the PBC fire during nonrespiratory activity (44). Given that bursting neurons are embedded within a complex synaptic network, NK1R can serve as a useful marker of respiratory-related neurons (12) and can be correlated with other neurotransmitters and receptors in brain stem respiratory centers. However, the probability remains that singly labeled neurons that do not express NK1R also have respiratory functions.
Neurons in the adult PBC have varying levels of CO activity, just as they do in the neonate (22). As we postulated previously, levels of CO activity in a neuron are closely related to the proportion of excitatory and inhibitory synaptic input received by the neuron. In other words, the level of CO activity in a neuron is most closely correlated with the ion pumping function for the reestablishment of the resting membrane potential after excitatory postsynaptic depolarization (41, 42). Because there are different types of respiratory neurons in the PBC with various depolarization patterns and timing, they are likely to receive different ratios of excitatory and inhibitory inputs, which may place varying demands on their energy metabolism. Indeed, adult PBC neurons in the present study exhibit heterogeneous levels of CO activity, consistent with our previous hypothesis.
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ACKNOWLEDGEMENTS |
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We thank Paulette Jacobs for assistance in reproducing some of the figures.
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FOOTNOTES |
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This study was supported by Children's Hospital Foundation (Milwaukee, WI) and National Natural Science Foundation of China Grant 39870264.
Address for reprint requests and other correspondence: M. T. T. Wong-Riley, Dept. of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: MWR{at}MCW.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 16 January 2001; accepted in final form 16 April 2001.
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TOP
ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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