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Hypercapnia-induced activation of brainstem GABAergic neurons during early development
Respiratory Physiology & Neurobiology 136 (2003) 25 /37 www.elsevier.com/locate/resphysiol
Hypercapnia-induced activation of brainstem GABAergic neurons during early development L. Zhang a, C.G. Wilson a,*, S. Liu a, M.A. Haxhiu a,b,c,d, R.J. Martin a a
Department of Pediatrics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA b Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA c Anatomy, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA d Department of Physiology and Biophysics, Howard University, College of Medicine, Washington, DC 20059, USA Accepted 24 January 2003
Abstract During early development, GABAergic mechanisms contribute to the regulation of respiratory timing in response to CO2. In 5 /7 day old piglets, a double labeling technique was used to determine whether GABA-containing neurons are activated by normoxic hypercapnia (10% CO2, 21% O2, and 69% N2). The c-Fos gene encoded protein (c-Fos) was employed to localize CO2 activated cells within the piglet medulla oblongata. Parvalbumin was used as a marker for GABAergic neurons. In animals breathing room air, only scant c-Fos-like immunoreactive neurons were observed. A marked increase in c-Fos positive cells was induced after a 60 min exposure to hypercapnia. Colocalization studies revealed that hypercapnia significantly increased c-Fos expression in GABA-containing neurons in the medulla oblongata, especially in the ventral aspect of the medulla, within the Bo¨tzinger region, the gigantocellular reticular nucleus, and the caudal raphe nuclei. Only a few double-labeled cells were observed within the nucleus tractus solitarius. Therefore, brainstem GABAergic neurons are part of the neural networks that respond to CO2 and may contribute to respiratory frequency responses to hypercapnia during early development. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Carbon dioxided, respiratory timing; Control of breathing, respiratory timing; Development, GABAergic mechanisms; Gene, c-Fos; Mammals, piglet; Pattern of breathing, GABA
1. Introduction
* Corresponding author. Address: Division of Neonatology, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106-6010, USA. Tel.: /1-216-3683757; fax: /1-216-844-3380. E-mail address: [email protected] (C.G. Wilson).
Chemical regulation of breathing and ventilatory responses to hypercapnia have been well characterized during development. Studies in various species and human infants have consistently demonstrated strong evidence for immaturity of hypercapnic ventilatory responses during early postnatal life in both unanesthetized premature
1569-9048/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1569-9048(03)00041-7
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non-primates, and in preterm human infants (Abu-Shaweesh et al., 1999; Rigatto et al., 1975; Noble et al., 1987). In preterm human infants, hypercapnia induces a sustained increase in tidal volume, but unlike in adults, frequency begins to fall after 1 min of hypercapnic exposure, primarily due to an increase in expiratory duration (Martin et al., 1985). This is consistent with recent findings in piglets and rat pups that hypercapnia induces centrally mediated prolongation of expiratory time (Dreshaj et al., 1999; Abu-Shaweesh et al., 1999). In addition to the excitation of respiratory neural output elicited by hypercapnia, an increase in CO2/H concentration also appears to enhance GABA-(g-aminobutyric acid) ergic inhibition of respiratory timing in newborn animals (AbuShaweesh et al., 1999; Dreshaj et al., 1999). In fact, there is evidence for tonic GABAergic inhibition of respiratory drive (Dreshaj et al., 1999; Tonkovic-Capin et al., 2001; Nattie et al., 2001). Hence, the present study was designed to test the hypothesis that a subpopulation of GABA-containing neurons in the medulla oblongata is activated by hypercapnic loading. We assume that their activation may modulate ventilatory and autonomic nervous system responses to an increased concentration of CO2/H via local projections, and/or axons that innervate the respiratory-related rhythm generating network, inspiratory bulbospinal cells, and/or spinal motor neurons. To define whether hypercapnia activates GABA-containing neurons, we evaluated the expression of c-Fos protein (c-Fos), the product of an immediate-early gene c-Fos in normocapnic and hypercapnic challenged animals. C-Fos protein has been used to identify activated neurons within the central nervous system (CNS). This gene is rapidly and transiently expressed following cell activation by different stimuli (Morgan et al., 1987; Bullit, 1990), including hypercapnic stimulation in adults and during development (Sato et al., 1992; Haxhiu et al., 1996; Belegu et al., 1999). In the present study immunohistochemistry for the calcium binding protein parvalbumin (PV) was used to identify GABAergic neurons, as PV is selectively expressed in GABA-containing cells in the CNS (Amadeo et al., 2001; McDonald and
Betette, 2001). The results of this study show that hypercapnia activates brainstem GABAergic neurons, which are part of the neural networks that respond to changes in chemical drive, and may play an important role in regulating expiratory duration during early development.
2. Methods 2.1. Animals In these studies, awake, nonsedated, 5/7 day old piglets were used. The experimental protocol was approved by the Case Western Reserve University School of Medicine Institutional Animal Care and Use Committee. The methods employed have been described earlier (Haxhiu et al., 1996). Briefly, we placed individual animals in a plexiglass chamber and either exposed them to room air for 60 min (three piglets; control group), or to a gas mixture containing 10% CO2, 21% O2, and 69% N2 (six piglets; experimental group). Immediately after exposure, each piglet was deeply anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and perfused through the ascending aorta with Ca2 free Tyrode solution followed by 4% paraformaldehyde prepared in 0.1 M sodium phosphate buffer (pH 7.4). The brains were removed, stored in 4% paraformaldehyde for 48 h, and then transferred to a 30% sucrose solution for 2/3 days. Transverse sections of the brain were cut at 50 m. 2.2. Immunohistochemistry We characterized the responses of brainstem neurons to hypercapnic loading, using c-Fos gene encoded protein (Fos), as a marker of neuronal activity. To visualize c-Fos expression, the freefloating sections were washed in PBS containing 0.3% Triton-X, and then 1-in-5 series sections were exposed to PBS-Triton solution containing 3% normal rabbit serum, to block non-specific binding sites for 30 min. After a further wash, the tissue was placed overnight at room temperature in a primary polyclonal antibody solution (1:10 000 dilution of rabbit anti-Fos in PBS; oncogene).
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The sections were rinsed, incubated with biotinylated goat anti-rabbit secondary antiserum (1:300) and further processed using the standard biotinand avidin-peroxidase kit (Vector, ABC-elite kit). The dilution factor of the ABC complex was 1:50. The immunoreaction was visualized by incubating the sections with 0.02% 3,3?-diaminobenzidine (DAB) containing 0.01% hydrogen peroxide for 6 min. A purple /black reaction product was obtained by adding nickel chloride to the peroxidase reaction (40 ml of 8% NiCl2 solution per 100 mL of DAB solution). Subsequently, the sections were washed twice in PBS. After the sections were stained for c-Fos and washed, they were incubated overnight with primary mouse anti-PV antibody (1:2000; Sigma-Aldrich), then washed and reacted with 1:100 secondary FITC conjugated goat antimouse antibody (Jackson ImmunoResearch Laboratories) for 3 h. All sections were washed and mounted on gelatin-coated glass slides, and the slides were coverslipped with FluoromountTM for microscopy. Control experiments were performed to determine whether the primary or the secondary antibodies produced false /positive results. Omission of primary or secondary antibodies resulted in the absence of labeling, demonstrating that no false / positive results were obtained with these reagents. Stained sections were mounted on glass slides, dried, and counter-stained with neutral red. The labeling pattern was carefully analyzed from the pontomedullary border to the decussation of the pyramids. Drawings presented in this report are based on the drawings of brainstem coronal sections, at the level of the obex, and 1.5, 3.0 and 4.5 mm rostral to the obex. For better orientation, findings of Fos-positive neurons in the rostro-caudal plane, at four defined rostrocaudal levels, are presented in parallel with corresponding drawings showing brain structures in reference to the obex. 2.3. Microscopy Slides were viewed with a fluorescence microscope (Leica DM LB) equipped with appropriate filter cubes to observe the green FITC and red TRITC fluorescence.
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2.4. Quantification A minimum of three immunofluorescent sections at each of the four rostrocaudal levels (obex, /1.5, /3.0, and /4.5 mm rostral), were used for quantification in each animal. The cell counts from all of the piglets studied were combined into one data set and then comparisons were made between control and hypercapnia exposed piglets. Since our preliminary studies showed no major differences between c-Fos expression in PV containing neurons and in GABA immunoreactive neurons, coexpression of c-Fos and PV is presented. All cells determined to be PV positive were labeled with a fluorescence intensity greater than 2 / the level of background fluorescence intensity for a given section (determined using IMAGEJ, http://rsb.info.nih.gov/ij/). We counted the total number of cells at the level of the obex, /1.5, /3.0, and /4.5 mm rostral to the obex for three different groups: (1) those that expressed only c-Fos, (2) those that expressed only PV, and (3) the number of neurons that co-expressed c-Fos and PV. We calculated the percentage of GABA-containing neurons activated by hypercapnic stress by dividing the total number of double labeled neurons by the total number of PV labeled neurons (sum of only PV labeled neurons and c-Fos expressing PV cells) and multiplying by 100. Data are presented as mean9/S.D. for each data set analyzed at the ventral, midline and dorsal aspect of the medulla oblongata. We used the student t -test to compare differences in cFos expression of PV containing neurons between animals exposed to room air and normoxic hypercapnia.
3. Results 3.1. c-Fos expression in the medulla oblongata We characterized the responses of brainstem neurons to hypercapnic loading, using c-Fos protein (c-Fos), as a marker of CO2 activated neurons. Within the medulla oblongata of control unanesthetized piglets, only a few scattered c-Fos immunoreactive nuclei were observed. In any of the studied piglets, exposure to 10% CO2 for 1 h
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produced a consistent increase in the number of activated neurons within the ventral, midline, and the dorsal aspects of the brainstem, as summarized in Figs. 2, 4 and 5. In this study, we chose to analyze c-Fos expression in the rostral and caudal medulla oblongata, which are known sites of GABA-containing neurons within chemosensory regions (Ellenberger, 1999; Blessing, 1990). The rostral ventrolateral medulla was defined as an area extending from the rostral border of the area postrema to the rostral border of the nucleus ambiguous, extending laterally to the spinal trigeminal tract, and medially to the pyramidal tract. This region in rats includes the pre-Bo¨tzinger complex of rhythm generating neurons (Smith et al., 1991) and the parapyramidal area. Our definition of RVLM differs from that published in a recent piglet study from Eugene Nattie’s lab at Dartmouth. They considered RVM to be a region ‘within 2 mm of the ventral surface, more than 0.5 mm from the midline, and less than 2 mm rostral to the rostral pole of the facial nucleus (VII), less than 2 mm caudal to the caudal pole of VII, and less than 1 mm lateral to the border of VII’ (Darnall et al., 2001). The caudal medulla was considered to be the region that extends from the obex to the most rostral border of the area postrema, bounded laterally by the spinal trigeminal tract and medially by the pyramidal tract. This cytoarchitectonic cell group includes the lateral reticular nucleus. In the ventrolateral aspect of the medulla oblongata, c-Fos positive cells were observed extending from the pontomedullary border to the decussation of the pyramids. c-Fos positive cells were additionally observed in the lateral paragigantocellular and gigantocellular reticular nuclei and in the medullary midline complex (raphe pallidus, raphe obscurus, and raphe magnus). In the dorsal aspect of the medulla oblongata, c-Fos positive neurons were observed along the nucleus tractus solitarius (nTS), and area postrema. These findings indicate that neurons activated by increases in CO2/H concentrations appear to be well developed in the first days of postnatal life in the piglet.
3.2. Distribution of GABAergic neurons in the chemosensory regions of the medulla oblongata In the piglets, a majority of the GABA-, or PVimmunoreactive neurons were located just lateral to the midline in the medial reticular formation, with the highest population observed in the ventral reticular nucleus. The midline raphe nuclei of the medulla oblongata (pallidus, obscurus, and magnus) contained PV-immunoreactive neurons. In the ventrolateral medulla, the majority of PVimmunoreactive neurons were located just dorsal to the lateral reticular nucleus, with fewer labeled neurons distributed dorsomedially. Individual PVimmunoreactive neurons were seen within and dorsolateral to the ventral respiratory group. Furthermore, individual as well as clusters of PV-immunoreactive cells were found in caudal regions of the ventrolateral medulla and within the lateral reticular nucleus, including the parvicellular subnucleus. In the dorsal aspect of medulla oblongata, GABA-immunoreactive neurons were observed within the nTS subnuclei. As summarized in Figs. 2, 4 and 5 hypercapnic exposure had no consistent effect on the number of PV labeled neurons at the various sites analyzed. 3.3. Hypercapnia-induced c-Fos expression in GABAergic neurons of the medulla oblongata Following exposure to hypercapnic stress, we observed c-Fos expression in subsets of PV-immunoreactive neurons localized in the caudal and the rostral ventrolateral aspect of the medulla oblongata. A subpopulation of PV-immunoreactive neurons in the ventral aspect of the medulla oblongata was activated by hypercapnia (c-Fos positive cells) and formed a network extending from the pontomedullary border to the decussation of the pyramidal tracts. In Fig. 1 we show an example of c-Fos expression in GABA-containing neurons of Bo¨tzinger complex, within the rostral ventrolateral aspect of the medulla oblongata of a 5 day old piglet, in Fig. 2 we present the average number of neurons (mean9/S.D.), at four different levels of the medulla oblongata that were labeled with c-Fos alone, PV alone, and PV colocalized with c-Fos. The counts from all four levels in each
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Fig. 1. (A) Schematic diagram of the medulla oblongata of a piglet showing location of CO2-induced c-Fos expression in GABAcontaining neurons of the rostral ventrolateral medulla. (B) Representative fluorescent photomicrograph showing PV expressing neurons (green) and a PV expressing neuron showing c-Fos expression (arrow). (C) Brightfield micrograph of nuclei of cells that expressed c-Fos. The arrow indicates CO2-induced c-Fos expression in the PV-immunoreactive neuron indicated in (B).
animal were combined into one data set. In normocapnic controls (n /3) we counted 70 cells that expressed c-Fos alone, 489 that expressed PV alone and calculated that 49/1% of PV labeled cells expressed c-Fos. Following exposure to hypercapnia, (n /6) we counted 458 cells that expressed c-Fos alone, 824 neurons that expressed PV alone, and observed c-Fos expression in 169/ 2% of PV positive cells (4 versus 16%, P B/0.001). Hypercapnia also induced c-Fos expression in a subpopulation of PV-immunoreactive neurons in the medial reticular formation, including GABAergic neurons within the raphe pallidus, raphe obscurus, and raphe magnus. An example of a PV expressing neuron in the raphe obscurus is presented in Fig. 3, and average results are shown in Fig. 4. When cell counts at four levels were again combined from all normocapnic controls (n /3) we counted 20 cells expressing c-Fos alone, and 84 expressing PV alone, while 69/2% of PV positive cells expressed c-Fos. Following exposure to hypercapnia (n/6), we counted 122 cells that expressed c-Fos alone, 178 neurons that contained PV alone, while 189/2% of PV positive cells expressed c-Fos (6 versus 18%, P B/0.01).
In the dorsal aspect of the medulla oblongata, following exposure to CO2, only a few of GABAergic cells expressed c-Fos like immunoreactivity. In Fig. 5 we present transverse sections through the medulla oblongata, indicating the distribution of c-Fos-positive neurons, PV containing neurons, and PV positive neurons that express c-Fos at four levels. When neurons in nTS subnuclei at the dorsal medulla were counted from all four levels, during normocapnia 78 expressed cFos, 93 expressed PV and there was no coexpression of c-Fos and PV. Following hypercapnia, 508 cells expressed c-Fos, 217 expressed PV and 59/3% of PV positive cells expressed c-Fos (0 versus 5%, NS) (Fig. 6). A summary of percent co-localization, based on c-Fos expression in PV containing neurons at ventrolateral, midline and dorsal (nTS) medullary sites is presented in Fig. 6.
4. Discussion The results of these studies extend previous findings, by demonstrating for the first time that in the developing pig, as in newborn, juvenile and
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Fig. 2. The average (mean9/S.D.) number of c-Fos positive, PV positive, and double labeled neurons in the ventrolateral medulla of control (unfilled) and CO2 exposed (hatched) piglets at different rostro-caudal levels. Levels are shown in regard to camera lucida drawings of piglet medulla oblongata.
adult rats, exposure to CO2 results in expression of c-Fos gene encoded protein (Fos protein) in subsets of neurons within previously described chemosensitive regions of the ventrolateral medulla (Mitchell et al., 1963; Loeschcke et al., 1970;
Schla¨fke and Loeschcke, 1967; Ballantyne and Scheid, 2000). Activated cells were also demonstrated in other regions located outside putative chemosensory fields, such as neurons found within the nTS, area postrema (Haxhiu et al., 1996; Sica
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Fig. 3. (A) Schematic diagram of the midline nuclei of a piglet medulla oblongata showing location of CO2-induced c-Fos expression in GABA-containing neurons of the raphe obscurus. (B) Representative fluorescent photomicrograph showing PV expressing neurons. Arrow indicates PV-expressing neuron with c-Fos expression. (C) Nuclei of neurons that expressed c-Fos. The arrow indicates CO2induced c-Fos expression in PV-immunoreactive neuron; see in (B). Scale bar/50 mm.
et al., 1999), raphe nuclei (Bernard et al., 1996; Dreshaj et al., 1998; Richerson et al., 2001; Pete et al., 2002), lateral paragigantocellular, and gigantocellular nuclei (Belegu et al., 1999; Haxhiu et al., 1996; Teppema et al., 1997). Our observations regarding the distribution of neurons activated by CO2 and identified by Fos expression, are consistent with functional magnetic resonance imaging studies (Gozal et al., 1994). Furthermore, chemosensitive regions defined by hypercapniainduced c-Fos-expression in the brainstem of piglets, correspond well with the chemosensitive sites identified by local changes in CO2 concentrations and are analogous to sites where, under in vitro experimental conditions, decrease in pH activates medullary neurons. This observation is also supported by physiologic studies that acidification by topical application of acetazolamide causes an increase in respiratory output (Coates et al., 1993; Bernard et al., 1996). The finding of hypercapnia-induced c-Fos expression in a subset of neurons within these medullary regions does not prove that these neurons are chemosensitive, since they can be activated synaptically. However, our data indicate
that these cells are part of the chemosensory network that is involved in responses to hypercapnia. An increase in CO2/H concentration may either hyperpolarize or depolarize the neurons in organotypic cultures of the fetal rat medulla (Wellner-Kienitz and Shams, 1998), and medullary raphe neurons (Wang et al., 1998). Therefore, the results of the present study do not exclude the possibility that an increase in CO2/H concentration did not inhibit a subset of GABA-containing neurons. As inhibited neurons do not express cFos, only activated cells are visualized by this technique. We assume that metabolic acidosis could also activate GABA-containing cells, since changes in pH are detected by chemosensory cells (Loeschcke, 1982; Nattie, 1999). In this study, we determined the distribution of medullary GABAergic neurons activated by hypercapnia in piglets, using immunocytochemistry for the calcium binding protein PV. This protein is preferentially associated with the more active (electrically and metabolically) neurons in a given functional system (Celio, 1990), and GABAergic neurons from different brain regions contain high levels of PV both in their soma and in their
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Fig. 4. The average (mean9/S.D.) number of c-Fos positive, PV positive, and double labeled neurons in the midline nuclei of control (unfilled) and CO2 exposed piglets (hatched) at different rostro-caudal levels of medulla oblongata.
processes. Previous work has shown that at all developmental ages, GABA-containing neuronal cell bodies, dendrites and axonal terminals are PVimmunoreactive (Amadeo et al., 2001). Hence, PV expression can be used to define the organization
of GABAergic inhibitory circuits in the brain (McDonald and Betette, 2001). We did not observe differences in PV expression between normocapnic and hypercapnic conditions. The present study showed that within the medulla oblongata,
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Fig. 5. The average (mean9/S.D.) number of c-Fos positive, PV positive, and double labeled neurons in the nTS of control (unfilled) and CO2 exposed piglets (hatched) at different rostro-caudal levels.
subsets of PV-containing neurons are located within the same medullary regions as chemosensory cells, and GABA synthesizing neurons in adult rats (Ellenberger, 1999). We have demonstrated for the first time that hypercapnia activates a subpopulation of GABA inhibitory cells, which
can be regarded as part of a broader ventral medullary network involved in modulation of the basic rhythm and pattern of breathing in response to hypercapnic loading in early life. Recently, using pharmacological tools we investigated mechanisms involved in hypercapnia-
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Fig. 6. The percent (mean9/S.D.) of PV-immunoreactive neurons within different regions of the medulla oblongata expressing c-Fos in control (unfilled) and CO2 exposed piglets (hatched). Asterisks indicate statistically significant difference (P B/0.05).
induced slowing of breathing frequency in the piglet (Dreshaj et al., 1999). Our present findings extend these results, showing that hypercapnia activates GABAergic neurons, which in turn may modulate respiratory drive via projections to spinal motoneurons, bulbospinal inspiratory-related cells, or the inspiratory rhythm-generating network. We assume that bulbospinal inhibitory inputs to phrenic motoneurons during the expiratory phase arise from GABAergic neurons of the rostral medulla oblongata, located within the Bo¨tzinger region. This assumption is based on findings that the Bo¨tzinger neurons synthesize GABA (Livingston and Berger, 1989), which monosynaptically inhibits phrenic motoneurons (Merrill and Fedorko, 1984; Tian et al., 1998) as well as ventral (Fedorko et al., 1989) and dorsal bulbospinal inspiratory neurons (Merrill et al., 1983). In this
study, we did observe the presence of GABAergic neurons along the ventrolateral aspect of the medulla, including a subset of neurons within the most rostral part of the ventral respiratory group, known as the Bo¨tzinger complex. Furthermore, we found that a number of GABAergic neurons of the ventromedial medulla are also activated by hypercapnic loading. These neurons are observed within the caudal raphe complex (raphe obscurus, raphe pallidus and raphe magnus), and within the nearby parapyramidal nucleus, the paragigantocellular and gigantocellular reticular nuclei. This is consistent with our earlier physiologic studies showing that medullary midline neurons are required for full expression of hypercapnic ventilatory responses in the piglet (Dreshaj et al., 1998). These ventromedial medullary sites synchronize multiple autonomic functions, and innervate phrenic motor nuclei (Holtman et al., 1984; Ellenberger, 1999) and project to motoneurons of the cranial nerves (Haxhiu et al., 1993; Waldbaum et al., 2001). A subpopulation of the midline bulbospinal neurons contain serotonin, often co-expressed with neuropeptides (i.e., thyrotropin-releasing hormone, substance P, and met-enkephalin) (Ho¨kfelt et al., 2000), and/or GABA (Millhorn et al., 1988), probably to a less extent that originally thought. It would appear that midline neurons expressing cFos contain a variety of transmitters, both excitatory and inhibitory, and the level of neuronal stimulation and maturation may affect the balance of release of these substances. The present findings suggest that GABAergic neurons activated by hypercapnic loading innervate rhythm generating cells of the pre-Bo¨tzinger
Fig. 7. Schematic model indicating the effect of increasing CO2 on the response of the neural network regulating inspiratory drive and respiratory timing, (GLU/glutamate).
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complex, and/or project to inspiratory bulbospinal, or inspiratory spinal motoneurons. Previous findings showed that GABA terminal varicosities innervate respiratory neurons within the dorsal respiratory group (Lipski et al., 1990) and phrenic motoneurons in the spinal cord (Ellenberger, 1999), Furthermore, exogenous application of GABA inhibits medullary respiratory neurons via activation of GABAA receptors (Haji et al., 1990), and hypercapnia-induced prolongation of expiratory duration is blocked by prior GABAA receptor blockade in both piglets and rat pups (Dreshaj et al., 1999; Abu-Shaweesh et al., 1999). In addition, in maturing pigs, blockade of GABAA receptors by bicuculline reduces hypoxic respiratory depression caused by single or intermittent oxygen deprivation (Huang et al., 1994; Miller et al., 2000), and/or stimulation of the superior laryngeal nerves (Abu-Shaweesh et al., 2001). The latter is consistent with a role for GABA in the prolongation of expiration, activation of post-inspiratory neurons and inhibition of inspiratory neurons described by Lawson in newborn piglets exposed to laryngeal stimuli (Lawson et al., 1999). These findings are also consistent with earlier studies of the ontogeny and distribution of GABAA receptors, demonstrating a relatively high GABAA receptor binding density in the brainstem of rat pups in early postnatal life (Xia and Haddad, 1992). Analysis of nTS neurons activated by hypercapnia showed that GABA-containing neurons within the nTS rarely express c-Fos. Although the sensitivity and widespread applicability of c-Fos as a marker of cell activity are well documented, it is not likely that all GABAergic neurons are equally sensitive to changes in CO2/H. Hence, the number of activated GABA neurons may not represent all GABA-containing neurons that respond to hypercapnia. Additionally, a large population of nTS and ventral medullary neurons activated by hypercapnia, based on c-Fos expression, were not GABAergic cells. These neurons may express excitatory neurotransmitters such as substance P (Pete et al., 2002) and may play a role in transmission of excitatory inputs generated by hypercapnia, partly via cholinergic mechanisms,
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and partly through glutamate release (Nattie, 1999). In Fig. 7 we provide a diagrammatic overview of the respiratory circuitry affected by an increase in CO2. We propose that GABAergic neurons in the network regulate ventilatory responses to hypercapnia, CO2 (acting directly or through interconnected glutamatergic neurons) stimulates respiration in parallel with GABAergic neurons that may shape the discharge pattern of bulbar respiratory neurons (Haji et al., 1990; Schmid et al., 1996; Tonkovic-Capin et al., 2001; Nattie et al., 2001). In our model, the affected glutamatergic neurons may be part of the inspiratory rhythm generating network (indicated by direct excitatory connections from CO2) and thus may be excitatorily coupled (Butera et al., 1999) as we have shown by broken arrows. This is consistent with a prominent role for GABA in prolonging expiratory duration in piglets exposed not only to hypercapnia, but also laryngeal stimulation (Abu-Shaweesh et al., 2001). These and future neuroanatomic observations, when correlated with physiologic data will allow us to define the role of specific neurotransmitter pathways in mediating maturational changes in respiratory control. Grant support HL 62526 and NS 39407 (NINDS, NCRR).
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L. Zhang et al. / Respiratory Physiology & Neurobiology 136 (2003) 25 /37 Nattie, E., 1999. Brainstem chemoreceptors and breathing. Prog. Neurobiol. 59, 299 /331. Nattie, E., Shi, J., Li, A., 2001. Bicuculline dialysis in the retrotrapezoid nucleus (RTN). Respir. Physiol. 124, 179 / 193. Noble, L.M., Carlo, W.A., Miller, M.J., DiFiore, J.M., Martin, R.J., 1987. Transient changes in expiratory time during hypercapnia in premature infants. J. Appl. Physiol. 62, 1010 /1013. Pete, G., Mack, S.O., Haxhiu, M.A., Walbaum, S., Gauda, E.B., 2002. CO2-induced c-Fos expression in brainstem preprotachykinin mRNA containing neurons. Respir. Physiol. Neurobiol. 130, 265 /274. Richerson, G.B., Wang, W., Tiwari, J., Bradley, S.R., 2001. Chemosensitivity of serotonergic neurons in the rostral ventral medulla. Respir. Physiol. 129, 175 /189. Rigatto, H., Brady, J.P., Verduzco, R., 1975. Chemoreceptor reflexes in preterm infants: II. The effect of gestational and postnatal age on the ventilatory response to inhaled carbon dioxide. Pediatrics 55, 614 /620. Sato, M., Severinghaus, J.W., Basbaum, A.I., 1992. Medullary CO2 chemoreceptor neuron identification by c-Fos immunocytochemistry. J. Appl. Physiol. 73, 96 /100. Schla¨fke, M.E., Loeschcke, H.H., 1967. Lokalisation eines an der regulation von Atmung und Kreislauf beteiligten Gebietes an der ventralen Oberfla¨che der Medulla oblongata durch Ka¨lteblockade. Pflu¨gers Arch. 297, 201 /220. Schmid, K., Foutz, A.S., Denavit-Saubie, M., 1996. Inhibitions mediated by glycine and GABAA receptors shape the discharge pattern of bulbar respiratory neurons. Brain Res. 710, 150 /160. Sica, A.L., Gootman, P.M., Ruggiero, D.A., 1999. CO2induced expression of c-Fos in the nucleus of the solitary
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tract and the area postrema of developing swine. Brain Res. 837, 106 /116. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W., Feldman, J.L., 1991. Pre-Bo¨tzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726 /729. Teppema, L.J., Veening, J.G., Kranenburg, A., Dahan, A., Berkenbosch, A., Olievier, C., 1997. Expression of c-Fos in the rat brainstem after exposure to hypoxia and to normoxic and hyperoxic hypercapnia. J. Comp. Neurol. 388, 169 / 190. Tian, G.F., Peever, J.H., Duffin, J., 1998. Bo¨tzinger-complex expiratory neurons monosynaptically inhibit phrenic motoneurons in the decerebrate rat. Exp. Brain Res. 122, 149 / 156. Tonkovic-Capin, V., Stucke, A.G., Stuth, E.A., TonkovicCapin, M.D., Krolo, M., Hopp, F.A., McCrimmon, D.R., Zuperku, E.J., 2001. Differential modulation of respiratory neuronal discharge patterns by GABA(A) receptor and apamin-sensitive K(/) channel antagonism. J. Neurophysiol. 86, 2363 /2373. Waldbaum, S., Hadziefendic, S., Erokwu, B., Zaidi, S.I., Haxhiu, M.A., 2001. CNS innervation of posterior cricoarytenoid muscles: a transneuronal labeling study. Respir. Physiol. 126, 113 /125. Wang, W., Pizzonia, J.H., Richerson, G.B., 1998. Chemosensitivity of rat medullary raphe neurones in primary tissue culture. J. Physiol. (Lond.) 511, 433 /450. Wellner-Kienitz, M.-C., Shams, H., 1998. CO2-sensitive neurons in organotypic cultures of the fetal rat medulla. Respir. Physiol. 111, 137 /151. Xia, Y., Haddad, G.G., 1992. Ontogeny and distribution of GABAA receptors in rat brainstem and rostral brain regions. Neuroscience 49, 973 /989.
Hypercapnia-induced activation of brainstem GABAergic neurons during early development L. Zhang a, C.G. Wilson a,*, S. Liu a, M.A. Haxhiu a,b,c,d, R.J. Martin a a
Department of Pediatrics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA b Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA c Anatomy, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA d Department of Physiology and Biophysics, Howard University, College of Medicine, Washington, DC 20059, USA Accepted 24 January 2003
Abstract During early development, GABAergic mechanisms contribute to the regulation of respiratory timing in response to CO2. In 5 /7 day old piglets, a double labeling technique was used to determine whether GABA-containing neurons are activated by normoxic hypercapnia (10% CO2, 21% O2, and 69% N2). The c-Fos gene encoded protein (c-Fos) was employed to localize CO2 activated cells within the piglet medulla oblongata. Parvalbumin was used as a marker for GABAergic neurons. In animals breathing room air, only scant c-Fos-like immunoreactive neurons were observed. A marked increase in c-Fos positive cells was induced after a 60 min exposure to hypercapnia. Colocalization studies revealed that hypercapnia significantly increased c-Fos expression in GABA-containing neurons in the medulla oblongata, especially in the ventral aspect of the medulla, within the Bo¨tzinger region, the gigantocellular reticular nucleus, and the caudal raphe nuclei. Only a few double-labeled cells were observed within the nucleus tractus solitarius. Therefore, brainstem GABAergic neurons are part of the neural networks that respond to CO2 and may contribute to respiratory frequency responses to hypercapnia during early development. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Carbon dioxided, respiratory timing; Control of breathing, respiratory timing; Development, GABAergic mechanisms; Gene, c-Fos; Mammals, piglet; Pattern of breathing, GABA
1. Introduction
* Corresponding author. Address: Division of Neonatology, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106-6010, USA. Tel.: /1-216-3683757; fax: /1-216-844-3380. E-mail address: [email protected] (C.G. Wilson).
Chemical regulation of breathing and ventilatory responses to hypercapnia have been well characterized during development. Studies in various species and human infants have consistently demonstrated strong evidence for immaturity of hypercapnic ventilatory responses during early postnatal life in both unanesthetized premature
1569-9048/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1569-9048(03)00041-7
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non-primates, and in preterm human infants (Abu-Shaweesh et al., 1999; Rigatto et al., 1975; Noble et al., 1987). In preterm human infants, hypercapnia induces a sustained increase in tidal volume, but unlike in adults, frequency begins to fall after 1 min of hypercapnic exposure, primarily due to an increase in expiratory duration (Martin et al., 1985). This is consistent with recent findings in piglets and rat pups that hypercapnia induces centrally mediated prolongation of expiratory time (Dreshaj et al., 1999; Abu-Shaweesh et al., 1999). In addition to the excitation of respiratory neural output elicited by hypercapnia, an increase in CO2/H concentration also appears to enhance GABA-(g-aminobutyric acid) ergic inhibition of respiratory timing in newborn animals (AbuShaweesh et al., 1999; Dreshaj et al., 1999). In fact, there is evidence for tonic GABAergic inhibition of respiratory drive (Dreshaj et al., 1999; Tonkovic-Capin et al., 2001; Nattie et al., 2001). Hence, the present study was designed to test the hypothesis that a subpopulation of GABA-containing neurons in the medulla oblongata is activated by hypercapnic loading. We assume that their activation may modulate ventilatory and autonomic nervous system responses to an increased concentration of CO2/H via local projections, and/or axons that innervate the respiratory-related rhythm generating network, inspiratory bulbospinal cells, and/or spinal motor neurons. To define whether hypercapnia activates GABA-containing neurons, we evaluated the expression of c-Fos protein (c-Fos), the product of an immediate-early gene c-Fos in normocapnic and hypercapnic challenged animals. C-Fos protein has been used to identify activated neurons within the central nervous system (CNS). This gene is rapidly and transiently expressed following cell activation by different stimuli (Morgan et al., 1987; Bullit, 1990), including hypercapnic stimulation in adults and during development (Sato et al., 1992; Haxhiu et al., 1996; Belegu et al., 1999). In the present study immunohistochemistry for the calcium binding protein parvalbumin (PV) was used to identify GABAergic neurons, as PV is selectively expressed in GABA-containing cells in the CNS (Amadeo et al., 2001; McDonald and
Betette, 2001). The results of this study show that hypercapnia activates brainstem GABAergic neurons, which are part of the neural networks that respond to changes in chemical drive, and may play an important role in regulating expiratory duration during early development.
2. Methods 2.1. Animals In these studies, awake, nonsedated, 5/7 day old piglets were used. The experimental protocol was approved by the Case Western Reserve University School of Medicine Institutional Animal Care and Use Committee. The methods employed have been described earlier (Haxhiu et al., 1996). Briefly, we placed individual animals in a plexiglass chamber and either exposed them to room air for 60 min (three piglets; control group), or to a gas mixture containing 10% CO2, 21% O2, and 69% N2 (six piglets; experimental group). Immediately after exposure, each piglet was deeply anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and perfused through the ascending aorta with Ca2 free Tyrode solution followed by 4% paraformaldehyde prepared in 0.1 M sodium phosphate buffer (pH 7.4). The brains were removed, stored in 4% paraformaldehyde for 48 h, and then transferred to a 30% sucrose solution for 2/3 days. Transverse sections of the brain were cut at 50 m. 2.2. Immunohistochemistry We characterized the responses of brainstem neurons to hypercapnic loading, using c-Fos gene encoded protein (Fos), as a marker of neuronal activity. To visualize c-Fos expression, the freefloating sections were washed in PBS containing 0.3% Triton-X, and then 1-in-5 series sections were exposed to PBS-Triton solution containing 3% normal rabbit serum, to block non-specific binding sites for 30 min. After a further wash, the tissue was placed overnight at room temperature in a primary polyclonal antibody solution (1:10 000 dilution of rabbit anti-Fos in PBS; oncogene).
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The sections were rinsed, incubated with biotinylated goat anti-rabbit secondary antiserum (1:300) and further processed using the standard biotinand avidin-peroxidase kit (Vector, ABC-elite kit). The dilution factor of the ABC complex was 1:50. The immunoreaction was visualized by incubating the sections with 0.02% 3,3?-diaminobenzidine (DAB) containing 0.01% hydrogen peroxide for 6 min. A purple /black reaction product was obtained by adding nickel chloride to the peroxidase reaction (40 ml of 8% NiCl2 solution per 100 mL of DAB solution). Subsequently, the sections were washed twice in PBS. After the sections were stained for c-Fos and washed, they were incubated overnight with primary mouse anti-PV antibody (1:2000; Sigma-Aldrich), then washed and reacted with 1:100 secondary FITC conjugated goat antimouse antibody (Jackson ImmunoResearch Laboratories) for 3 h. All sections were washed and mounted on gelatin-coated glass slides, and the slides were coverslipped with FluoromountTM for microscopy. Control experiments were performed to determine whether the primary or the secondary antibodies produced false /positive results. Omission of primary or secondary antibodies resulted in the absence of labeling, demonstrating that no false / positive results were obtained with these reagents. Stained sections were mounted on glass slides, dried, and counter-stained with neutral red. The labeling pattern was carefully analyzed from the pontomedullary border to the decussation of the pyramids. Drawings presented in this report are based on the drawings of brainstem coronal sections, at the level of the obex, and 1.5, 3.0 and 4.5 mm rostral to the obex. For better orientation, findings of Fos-positive neurons in the rostro-caudal plane, at four defined rostrocaudal levels, are presented in parallel with corresponding drawings showing brain structures in reference to the obex. 2.3. Microscopy Slides were viewed with a fluorescence microscope (Leica DM LB) equipped with appropriate filter cubes to observe the green FITC and red TRITC fluorescence.
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2.4. Quantification A minimum of three immunofluorescent sections at each of the four rostrocaudal levels (obex, /1.5, /3.0, and /4.5 mm rostral), were used for quantification in each animal. The cell counts from all of the piglets studied were combined into one data set and then comparisons were made between control and hypercapnia exposed piglets. Since our preliminary studies showed no major differences between c-Fos expression in PV containing neurons and in GABA immunoreactive neurons, coexpression of c-Fos and PV is presented. All cells determined to be PV positive were labeled with a fluorescence intensity greater than 2 / the level of background fluorescence intensity for a given section (determined using IMAGEJ, http://rsb.info.nih.gov/ij/). We counted the total number of cells at the level of the obex, /1.5, /3.0, and /4.5 mm rostral to the obex for three different groups: (1) those that expressed only c-Fos, (2) those that expressed only PV, and (3) the number of neurons that co-expressed c-Fos and PV. We calculated the percentage of GABA-containing neurons activated by hypercapnic stress by dividing the total number of double labeled neurons by the total number of PV labeled neurons (sum of only PV labeled neurons and c-Fos expressing PV cells) and multiplying by 100. Data are presented as mean9/S.D. for each data set analyzed at the ventral, midline and dorsal aspect of the medulla oblongata. We used the student t -test to compare differences in cFos expression of PV containing neurons between animals exposed to room air and normoxic hypercapnia.
3. Results 3.1. c-Fos expression in the medulla oblongata We characterized the responses of brainstem neurons to hypercapnic loading, using c-Fos protein (c-Fos), as a marker of CO2 activated neurons. Within the medulla oblongata of control unanesthetized piglets, only a few scattered c-Fos immunoreactive nuclei were observed. In any of the studied piglets, exposure to 10% CO2 for 1 h
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produced a consistent increase in the number of activated neurons within the ventral, midline, and the dorsal aspects of the brainstem, as summarized in Figs. 2, 4 and 5. In this study, we chose to analyze c-Fos expression in the rostral and caudal medulla oblongata, which are known sites of GABA-containing neurons within chemosensory regions (Ellenberger, 1999; Blessing, 1990). The rostral ventrolateral medulla was defined as an area extending from the rostral border of the area postrema to the rostral border of the nucleus ambiguous, extending laterally to the spinal trigeminal tract, and medially to the pyramidal tract. This region in rats includes the pre-Bo¨tzinger complex of rhythm generating neurons (Smith et al., 1991) and the parapyramidal area. Our definition of RVLM differs from that published in a recent piglet study from Eugene Nattie’s lab at Dartmouth. They considered RVM to be a region ‘within 2 mm of the ventral surface, more than 0.5 mm from the midline, and less than 2 mm rostral to the rostral pole of the facial nucleus (VII), less than 2 mm caudal to the caudal pole of VII, and less than 1 mm lateral to the border of VII’ (Darnall et al., 2001). The caudal medulla was considered to be the region that extends from the obex to the most rostral border of the area postrema, bounded laterally by the spinal trigeminal tract and medially by the pyramidal tract. This cytoarchitectonic cell group includes the lateral reticular nucleus. In the ventrolateral aspect of the medulla oblongata, c-Fos positive cells were observed extending from the pontomedullary border to the decussation of the pyramids. c-Fos positive cells were additionally observed in the lateral paragigantocellular and gigantocellular reticular nuclei and in the medullary midline complex (raphe pallidus, raphe obscurus, and raphe magnus). In the dorsal aspect of the medulla oblongata, c-Fos positive neurons were observed along the nucleus tractus solitarius (nTS), and area postrema. These findings indicate that neurons activated by increases in CO2/H concentrations appear to be well developed in the first days of postnatal life in the piglet.
3.2. Distribution of GABAergic neurons in the chemosensory regions of the medulla oblongata In the piglets, a majority of the GABA-, or PVimmunoreactive neurons were located just lateral to the midline in the medial reticular formation, with the highest population observed in the ventral reticular nucleus. The midline raphe nuclei of the medulla oblongata (pallidus, obscurus, and magnus) contained PV-immunoreactive neurons. In the ventrolateral medulla, the majority of PVimmunoreactive neurons were located just dorsal to the lateral reticular nucleus, with fewer labeled neurons distributed dorsomedially. Individual PVimmunoreactive neurons were seen within and dorsolateral to the ventral respiratory group. Furthermore, individual as well as clusters of PV-immunoreactive cells were found in caudal regions of the ventrolateral medulla and within the lateral reticular nucleus, including the parvicellular subnucleus. In the dorsal aspect of medulla oblongata, GABA-immunoreactive neurons were observed within the nTS subnuclei. As summarized in Figs. 2, 4 and 5 hypercapnic exposure had no consistent effect on the number of PV labeled neurons at the various sites analyzed. 3.3. Hypercapnia-induced c-Fos expression in GABAergic neurons of the medulla oblongata Following exposure to hypercapnic stress, we observed c-Fos expression in subsets of PV-immunoreactive neurons localized in the caudal and the rostral ventrolateral aspect of the medulla oblongata. A subpopulation of PV-immunoreactive neurons in the ventral aspect of the medulla oblongata was activated by hypercapnia (c-Fos positive cells) and formed a network extending from the pontomedullary border to the decussation of the pyramidal tracts. In Fig. 1 we show an example of c-Fos expression in GABA-containing neurons of Bo¨tzinger complex, within the rostral ventrolateral aspect of the medulla oblongata of a 5 day old piglet, in Fig. 2 we present the average number of neurons (mean9/S.D.), at four different levels of the medulla oblongata that were labeled with c-Fos alone, PV alone, and PV colocalized with c-Fos. The counts from all four levels in each
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Fig. 1. (A) Schematic diagram of the medulla oblongata of a piglet showing location of CO2-induced c-Fos expression in GABAcontaining neurons of the rostral ventrolateral medulla. (B) Representative fluorescent photomicrograph showing PV expressing neurons (green) and a PV expressing neuron showing c-Fos expression (arrow). (C) Brightfield micrograph of nuclei of cells that expressed c-Fos. The arrow indicates CO2-induced c-Fos expression in the PV-immunoreactive neuron indicated in (B).
animal were combined into one data set. In normocapnic controls (n /3) we counted 70 cells that expressed c-Fos alone, 489 that expressed PV alone and calculated that 49/1% of PV labeled cells expressed c-Fos. Following exposure to hypercapnia, (n /6) we counted 458 cells that expressed c-Fos alone, 824 neurons that expressed PV alone, and observed c-Fos expression in 169/ 2% of PV positive cells (4 versus 16%, P B/0.001). Hypercapnia also induced c-Fos expression in a subpopulation of PV-immunoreactive neurons in the medial reticular formation, including GABAergic neurons within the raphe pallidus, raphe obscurus, and raphe magnus. An example of a PV expressing neuron in the raphe obscurus is presented in Fig. 3, and average results are shown in Fig. 4. When cell counts at four levels were again combined from all normocapnic controls (n /3) we counted 20 cells expressing c-Fos alone, and 84 expressing PV alone, while 69/2% of PV positive cells expressed c-Fos. Following exposure to hypercapnia (n/6), we counted 122 cells that expressed c-Fos alone, 178 neurons that contained PV alone, while 189/2% of PV positive cells expressed c-Fos (6 versus 18%, P B/0.01).
In the dorsal aspect of the medulla oblongata, following exposure to CO2, only a few of GABAergic cells expressed c-Fos like immunoreactivity. In Fig. 5 we present transverse sections through the medulla oblongata, indicating the distribution of c-Fos-positive neurons, PV containing neurons, and PV positive neurons that express c-Fos at four levels. When neurons in nTS subnuclei at the dorsal medulla were counted from all four levels, during normocapnia 78 expressed cFos, 93 expressed PV and there was no coexpression of c-Fos and PV. Following hypercapnia, 508 cells expressed c-Fos, 217 expressed PV and 59/3% of PV positive cells expressed c-Fos (0 versus 5%, NS) (Fig. 6). A summary of percent co-localization, based on c-Fos expression in PV containing neurons at ventrolateral, midline and dorsal (nTS) medullary sites is presented in Fig. 6.
4. Discussion The results of these studies extend previous findings, by demonstrating for the first time that in the developing pig, as in newborn, juvenile and
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Fig. 2. The average (mean9/S.D.) number of c-Fos positive, PV positive, and double labeled neurons in the ventrolateral medulla of control (unfilled) and CO2 exposed (hatched) piglets at different rostro-caudal levels. Levels are shown in regard to camera lucida drawings of piglet medulla oblongata.
adult rats, exposure to CO2 results in expression of c-Fos gene encoded protein (Fos protein) in subsets of neurons within previously described chemosensitive regions of the ventrolateral medulla (Mitchell et al., 1963; Loeschcke et al., 1970;
Schla¨fke and Loeschcke, 1967; Ballantyne and Scheid, 2000). Activated cells were also demonstrated in other regions located outside putative chemosensory fields, such as neurons found within the nTS, area postrema (Haxhiu et al., 1996; Sica
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Fig. 3. (A) Schematic diagram of the midline nuclei of a piglet medulla oblongata showing location of CO2-induced c-Fos expression in GABA-containing neurons of the raphe obscurus. (B) Representative fluorescent photomicrograph showing PV expressing neurons. Arrow indicates PV-expressing neuron with c-Fos expression. (C) Nuclei of neurons that expressed c-Fos. The arrow indicates CO2induced c-Fos expression in PV-immunoreactive neuron; see in (B). Scale bar/50 mm.
et al., 1999), raphe nuclei (Bernard et al., 1996; Dreshaj et al., 1998; Richerson et al., 2001; Pete et al., 2002), lateral paragigantocellular, and gigantocellular nuclei (Belegu et al., 1999; Haxhiu et al., 1996; Teppema et al., 1997). Our observations regarding the distribution of neurons activated by CO2 and identified by Fos expression, are consistent with functional magnetic resonance imaging studies (Gozal et al., 1994). Furthermore, chemosensitive regions defined by hypercapniainduced c-Fos-expression in the brainstem of piglets, correspond well with the chemosensitive sites identified by local changes in CO2 concentrations and are analogous to sites where, under in vitro experimental conditions, decrease in pH activates medullary neurons. This observation is also supported by physiologic studies that acidification by topical application of acetazolamide causes an increase in respiratory output (Coates et al., 1993; Bernard et al., 1996). The finding of hypercapnia-induced c-Fos expression in a subset of neurons within these medullary regions does not prove that these neurons are chemosensitive, since they can be activated synaptically. However, our data indicate
that these cells are part of the chemosensory network that is involved in responses to hypercapnia. An increase in CO2/H concentration may either hyperpolarize or depolarize the neurons in organotypic cultures of the fetal rat medulla (Wellner-Kienitz and Shams, 1998), and medullary raphe neurons (Wang et al., 1998). Therefore, the results of the present study do not exclude the possibility that an increase in CO2/H concentration did not inhibit a subset of GABA-containing neurons. As inhibited neurons do not express cFos, only activated cells are visualized by this technique. We assume that metabolic acidosis could also activate GABA-containing cells, since changes in pH are detected by chemosensory cells (Loeschcke, 1982; Nattie, 1999). In this study, we determined the distribution of medullary GABAergic neurons activated by hypercapnia in piglets, using immunocytochemistry for the calcium binding protein PV. This protein is preferentially associated with the more active (electrically and metabolically) neurons in a given functional system (Celio, 1990), and GABAergic neurons from different brain regions contain high levels of PV both in their soma and in their
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L. Zhang et al. / Respiratory Physiology & Neurobiology 136 (2003) 25 /37
Fig. 4. The average (mean9/S.D.) number of c-Fos positive, PV positive, and double labeled neurons in the midline nuclei of control (unfilled) and CO2 exposed piglets (hatched) at different rostro-caudal levels of medulla oblongata.
processes. Previous work has shown that at all developmental ages, GABA-containing neuronal cell bodies, dendrites and axonal terminals are PVimmunoreactive (Amadeo et al., 2001). Hence, PV expression can be used to define the organization
of GABAergic inhibitory circuits in the brain (McDonald and Betette, 2001). We did not observe differences in PV expression between normocapnic and hypercapnic conditions. The present study showed that within the medulla oblongata,
L. Zhang et al. / Respiratory Physiology & Neurobiology 136 (2003) 25 /37
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Fig. 5. The average (mean9/S.D.) number of c-Fos positive, PV positive, and double labeled neurons in the nTS of control (unfilled) and CO2 exposed piglets (hatched) at different rostro-caudal levels.
subsets of PV-containing neurons are located within the same medullary regions as chemosensory cells, and GABA synthesizing neurons in adult rats (Ellenberger, 1999). We have demonstrated for the first time that hypercapnia activates a subpopulation of GABA inhibitory cells, which
can be regarded as part of a broader ventral medullary network involved in modulation of the basic rhythm and pattern of breathing in response to hypercapnic loading in early life. Recently, using pharmacological tools we investigated mechanisms involved in hypercapnia-
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L. Zhang et al. / Respiratory Physiology & Neurobiology 136 (2003) 25 /37
Fig. 6. The percent (mean9/S.D.) of PV-immunoreactive neurons within different regions of the medulla oblongata expressing c-Fos in control (unfilled) and CO2 exposed piglets (hatched). Asterisks indicate statistically significant difference (P B/0.05).
induced slowing of breathing frequency in the piglet (Dreshaj et al., 1999). Our present findings extend these results, showing that hypercapnia activates GABAergic neurons, which in turn may modulate respiratory drive via projections to spinal motoneurons, bulbospinal inspiratory-related cells, or the inspiratory rhythm-generating network. We assume that bulbospinal inhibitory inputs to phrenic motoneurons during the expiratory phase arise from GABAergic neurons of the rostral medulla oblongata, located within the Bo¨tzinger region. This assumption is based on findings that the Bo¨tzinger neurons synthesize GABA (Livingston and Berger, 1989), which monosynaptically inhibits phrenic motoneurons (Merrill and Fedorko, 1984; Tian et al., 1998) as well as ventral (Fedorko et al., 1989) and dorsal bulbospinal inspiratory neurons (Merrill et al., 1983). In this
study, we did observe the presence of GABAergic neurons along the ventrolateral aspect of the medulla, including a subset of neurons within the most rostral part of the ventral respiratory group, known as the Bo¨tzinger complex. Furthermore, we found that a number of GABAergic neurons of the ventromedial medulla are also activated by hypercapnic loading. These neurons are observed within the caudal raphe complex (raphe obscurus, raphe pallidus and raphe magnus), and within the nearby parapyramidal nucleus, the paragigantocellular and gigantocellular reticular nuclei. This is consistent with our earlier physiologic studies showing that medullary midline neurons are required for full expression of hypercapnic ventilatory responses in the piglet (Dreshaj et al., 1998). These ventromedial medullary sites synchronize multiple autonomic functions, and innervate phrenic motor nuclei (Holtman et al., 1984; Ellenberger, 1999) and project to motoneurons of the cranial nerves (Haxhiu et al., 1993; Waldbaum et al., 2001). A subpopulation of the midline bulbospinal neurons contain serotonin, often co-expressed with neuropeptides (i.e., thyrotropin-releasing hormone, substance P, and met-enkephalin) (Ho¨kfelt et al., 2000), and/or GABA (Millhorn et al., 1988), probably to a less extent that originally thought. It would appear that midline neurons expressing cFos contain a variety of transmitters, both excitatory and inhibitory, and the level of neuronal stimulation and maturation may affect the balance of release of these substances. The present findings suggest that GABAergic neurons activated by hypercapnic loading innervate rhythm generating cells of the pre-Bo¨tzinger
Fig. 7. Schematic model indicating the effect of increasing CO2 on the response of the neural network regulating inspiratory drive and respiratory timing, (GLU/glutamate).
L. Zhang et al. / Respiratory Physiology & Neurobiology 136 (2003) 25 /37
complex, and/or project to inspiratory bulbospinal, or inspiratory spinal motoneurons. Previous findings showed that GABA terminal varicosities innervate respiratory neurons within the dorsal respiratory group (Lipski et al., 1990) and phrenic motoneurons in the spinal cord (Ellenberger, 1999), Furthermore, exogenous application of GABA inhibits medullary respiratory neurons via activation of GABAA receptors (Haji et al., 1990), and hypercapnia-induced prolongation of expiratory duration is blocked by prior GABAA receptor blockade in both piglets and rat pups (Dreshaj et al., 1999; Abu-Shaweesh et al., 1999). In addition, in maturing pigs, blockade of GABAA receptors by bicuculline reduces hypoxic respiratory depression caused by single or intermittent oxygen deprivation (Huang et al., 1994; Miller et al., 2000), and/or stimulation of the superior laryngeal nerves (Abu-Shaweesh et al., 2001). The latter is consistent with a role for GABA in the prolongation of expiration, activation of post-inspiratory neurons and inhibition of inspiratory neurons described by Lawson in newborn piglets exposed to laryngeal stimuli (Lawson et al., 1999). These findings are also consistent with earlier studies of the ontogeny and distribution of GABAA receptors, demonstrating a relatively high GABAA receptor binding density in the brainstem of rat pups in early postnatal life (Xia and Haddad, 1992). Analysis of nTS neurons activated by hypercapnia showed that GABA-containing neurons within the nTS rarely express c-Fos. Although the sensitivity and widespread applicability of c-Fos as a marker of cell activity are well documented, it is not likely that all GABAergic neurons are equally sensitive to changes in CO2/H. Hence, the number of activated GABA neurons may not represent all GABA-containing neurons that respond to hypercapnia. Additionally, a large population of nTS and ventral medullary neurons activated by hypercapnia, based on c-Fos expression, were not GABAergic cells. These neurons may express excitatory neurotransmitters such as substance P (Pete et al., 2002) and may play a role in transmission of excitatory inputs generated by hypercapnia, partly via cholinergic mechanisms,
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and partly through glutamate release (Nattie, 1999). In Fig. 7 we provide a diagrammatic overview of the respiratory circuitry affected by an increase in CO2. We propose that GABAergic neurons in the network regulate ventilatory responses to hypercapnia, CO2 (acting directly or through interconnected glutamatergic neurons) stimulates respiration in parallel with GABAergic neurons that may shape the discharge pattern of bulbar respiratory neurons (Haji et al., 1990; Schmid et al., 1996; Tonkovic-Capin et al., 2001; Nattie et al., 2001). In our model, the affected glutamatergic neurons may be part of the inspiratory rhythm generating network (indicated by direct excitatory connections from CO2) and thus may be excitatorily coupled (Butera et al., 1999) as we have shown by broken arrows. This is consistent with a prominent role for GABA in prolonging expiratory duration in piglets exposed not only to hypercapnia, but also laryngeal stimulation (Abu-Shaweesh et al., 2001). These and future neuroanatomic observations, when correlated with physiologic data will allow us to define the role of specific neurotransmitter pathways in mediating maturational changes in respiratory control. Grant support HL 62526 and NS 39407 (NINDS, NCRR).
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