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CARDIAC, RENAL, AND RESPIRATORY INTEGRATION
1Department of Biology, St. Lawrence University, Canton, New York 13615; and 2Departments of Physiology and Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756
Submitted 6 May 2003 ; accepted in final form 27 September 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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or Na+ from the perfusate acidified the glial cells, but the acidification after Na+ removal was greater in the RTN than in the NTS. Treatment of the slice with 5-(N-ethyl-N-isopropyl)amiloride (100 µM) in saline containing acidified the cells in both nuclei, but the acidification was greater in the NTS. Restoration of extracellular Cl- after Cl- depletion during the control condition acidified the cells. Immunohistochemical studies of glial fibrillary acid protein demonstrated much denser staining in the RTN compared with the NTS. We conclude that there is evidence of 
cotransport and Na+/H+ exchange in glia in the RTN and NTS, but the distribution of glia and the distribution of these pH-regulatory functions are not identical in the NTS and RTN. The differential strength of glial pH regulatory function in the RTN and NTS may also alter CO2 chemosensory neuronal function at these two chemosensitive sites in the brain stem. astrocytes; central chemosensitivity; respiratory control
The foregoing comments emphasize the relation of pHe and pHi to neuronal function, but the neurons are surrounded by glia. The activity of glial cells can modify pHe, but glia have received relatively little attention with respect to central CO2 chemosensitivity. Manipulation of pHe in the region of chemosensory neurons by disrupting glial function alters ventilation. Fluorocitrate is a selective and reversible glial toxin that depolarizes glia in vitro and has no apparent effect on the intrinsic biophysical properties of neurons (29, 36). Focal perfusion of fluorocitrate in the retrotrapezoid nucleus (RTN) of the anesthetized rat was associated with a significant decline in pHe and significant stimulation of ventilation (20). Perfusion of 1 mM fluorocitrate unilaterally into the RTN in conscious rats also stimulated ventilation (26). These studies indicate that glial activity may modify neuronal function in central CO2 chemosensory areas in part by changing pHe.
Cortical glia express a welter of exchange mechanisms, including NHE, Na+-independent and Na+-dependent 
exchange, and electrogenic cotransport (NBC) mechanisms (16), and the activity of any one or all of these processes may modify glial pHi and interstitial pHe. We have been particularly interested in CO2 chemosensory function in the brain stem in the nucleus tractus solitarius (NTS) and the ventral medulla, including the RTN. The nuclei differ anatomically: the NTS is a compact nucleus with few glial cells, and the RTN is loosely aggregated with many more glia (37). Furthermore, the ventilatory responses to fluorocitrate differed between the two nuclei: fluorocitrate applied focally within the RTN stimulated ventilation in conscious rats (26), but the same treatment applied focally within the NTS failed to stimulate ventilation during either normocapnia or hypercapnia (Erlichman, unpublished observations). Therefore, we tested the hypothesis that glial pH-regulatory mechanisms active under steady-state conditions would differ between the RTN and the NTS. To the extent that different glial pH-regulatory mechanisms are present in chemosensory areas, the ventilatory stimulus, as reflected by pHe, may differ among the areas even when the acidic stress, hypercapnia for example, may be identical.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Solutions. The control aCSF contained (in mM) 124 NaCl, 5 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.24 KH2PO4, 26 NaHCO3, and 10 glucose. Control aCSF was equilibrated with 95% O2-5% CO2 to maintain a pH of 7.48 at 37°C. We used a variety of substituted saline solutions to test the function of various acid-base transport mechanisms. We used a CO2-free aCSF in which 
was replaced by Na+ HEPES buffer and titrated to pH 7.48 at 37°C. In Na+-free solutions, NaCl was replaced with N-methyl-D-glucamine chloride (NMDG-Cl), NaHCO3 was omitted, and the solution was gassed with 5% CO2 for 30 min and titrated to pH 7.48 with NMDG. We made Cl--free solutions by substituting gluconate for Cl-, and the concentration of Ca2+ was increased to compensate for the binding of Ca2+ to the anion substitutes (50). As a result, the Cl--free aCSF contained (in mM) 124 Na-gluconate, 5 K-gluconate, 12 Ca-gluconate, 1.3 MgSO4, 1.24 KH2PO4, 26 NaHCO3, and 10 glucose. High-K+ solutions were prepared by increasing the KCl concentration from 5 to 20 mM (total K+ 6.24 to 21.24 mM). In some high-K+ experiments, valinomycin (10 µM) and bumetanide (15 µM) were added to the aCSF. Valinomycin is a K+ ionophore, and bumetanide inhibits Na+-K+-Cl- cotransport. Activity of the Na+-K+-Cl- cotransporter can alter cell volume (41), which may in turn alter NHE and affect pHi (12, 21). Before delivery to the tissue, all solutions were loaded into 60-ml glass syringes and preheated to 37°C using a servocontrolled syringe heater block (model SW-707; Warner Instruments, New Haven, CT) and equilibrated with the appropriate gas mixture.
We used a modified aCSF solution for brain sectioning. Cell death is often associated with increased intracellular Na+ and Ca2+ and with glutamatergic excitation, which also enhances Ca2+ loading. Therefore, we used a slicing solution that contained no Na+ (choline was substituted for Na+), reduced Ca2+, and elevated Mg2+, and added kynurenic acid, a glutamate antagonist. We also included ascorbic acid as an antioxidant. Thus the choline-substituted slicing solution contained (in mM) 135 choline chloride, 1 KCl, 0.5 CaCl2, 20 MgCl2, 1.4 NaH2PO4, 24 choline bicarbonate, 20 kynurenic acid, 5 ascorbic acid, and 10 glucose. The choline-substituted slicing solution was equilibrated with 95% O2-5% CO2 and chilled to 4°C. The pH of the choline-substituted slicing solution was 
6.9. Although this solution is quite acidic compared with aCSF, the tissue viability of slices sectioned in the presence of ascorbic acid and low pHe was better than sections perfused with aCSF alone without ascorbic acid. The improved health of the sections was probably the result of both the antioxidant properties of the ascorbic acid and the decrease in solution pH, which inhibits the inward movement of cations through NMDA channels.
The nigericin calibration solution contained (in mM) 104 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.24 KH2PO4, 10 glucose, 25 NMDG-HEPES, 25 K-HEPES, and 1.6 x10-2 nigericin. A calibration curve was constructed by measuring the fluorescence of cells exposed to nigericin-containing calibration solutions of known pH ranging from 5.8 to 8.6 pH units at 37 °C. Nigericin-containing solutions, titrated to 7.3 at 37 °C (8), were used to perform a one-point calibration at the end of each experiment. Each experimental value was transformed into normalized fluorescence (Nfl) using the fluorescence of each cell after exposure to the nigericin calibration solution at pH 7.3. pHi was calculated from the following calibration equation: pHi = 6.864 + log[(Nfl - 0.485)/(1.283 - Nfl)]. The calibration curve had a correlation coefficient (r2) of 0.98. After each calibration, the entire perfusion apparatus was soaked and washed three times in 70% ethanol in water and subsequently rinsed repeatedly in distilled water to remove any retained nigericin.
Specific inhibitors of NHE and exchange were also used. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) was dissolved in DMSO and used at a final concentration of 100 µM (the final DMSO concentration in the perfusate was <0.1%). DIDS was dissolved in the solutions at a final concentration of 0.5 mM. All reagents were purchased from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
Superfusion chamber. Single brain slices, loaded with 2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein (BCECF; see below), were immobilized in a 500-µl perfusion chamber using a platinum wire and nylon grid (17). The chamber was continuously superfused with control or test solutions at a rate of 1 ml/min. Solutions entering the perfusion chamber were heated to 37°C using a servocontrolled in-line heater (model SF-28; Warner Instrument, New Haven, CT), and a continuous stream of 95% O2-5% CO2 or 100% O2, as appropriate for the particular perfusate, was directed over the air-fluid interface of the chamber. The pH of superfused solutions was monitored using a pH electrode (Beetrode, WPI, Sarasota, FL).
Measurement of pHi. Slices were loaded with the pH-sensitive fluoroprobe BCECF (Molecular Probes, Eugene, OR) by incubating them in a 20 µM solution of the acetoxymethyl ester of BCECF in control saline bubbled with 95% O2-5% CO2 for 45 min at room temperature. To create brain slices enriched with glia, kainic acid was added to the BCECF-loading solution at a final concentration of 5 mM. BCECF-loaded, kainic acid-treated slices were transferred to a holding chamber containing control saline (no dye) bubbled with 95% O2-5% CO2 at room temperature and maintained in this solution in the dark for 45 min. The slices were washed in control aCSF and held at room temperature for a further 30-45 min to allow cells to recover from the kainic acid exposure before experimentation.
pHi was measured in single, BCECF-loaded slices placed in a chamber mounted on the stage of an upright Nikon E600FN microscope (Nikon, Melville, NY) and continuously superfused with control saline. The pHi of individual cells was measured within discrete areas (0.042 mm2) of the medulla by alternately exciting the area with light from a 75-W Xe lamp (Chiu Industries, Kings Park, NY) at 500 ± 10 and 440 ± 10 nm wavelengths for 0.3-0.8 s. The excitation filters were switched by a computer-controlled filter wheel (Lambda 10-2, Sutter Instruments, Novato, CA). The emitted fluorescence from the intracellular BCECF was filtered using a 515-nm dichroic mirror and a 535 ± 25-nm emission filter (Chroma Technology, Bennington, VT) and intensified and measured with a cooled CCD camera (Photometrics, Tucson, AZ). The digital images were acquired and processed with Compix software (C Imaging Systems, Cranberry Township, PA).
Protocol. We studied pHi regulation in glia in two regions: the NTS and the RTN. The NTS was identified grossly by its relation to the floor of the fourth ventricle and microscopically by the location of the NTS and proximity of adjacent nuclei, such as the spinal trigeminal tract, and hypoglossal and medial vestibular nuclei. The RTN was identified grossly by the shape of the ventral medulla and microscopically by the presence of the facial nucleus, pyramids, and spinal trigeminal tract. The cells recorded from the RTN in this study were located from -9.68 to -11.80 mm from bregma. Cells recorded from the NTS were located -11.30 to -14.03 mm from bregma (37).
Each study began with a period of 5-10 min of perfusion with the control solution during which pHi stabilized. Next, the slice was exposed to a test solution, and pHi was measured when steady pHi values were reached (8-15 min). In the final condition, the perfusate was usually restored to the initial control condition, and pHi was measured when it stabilized (8-15 min). Steady-state pHi values were obtained by averaging at least three contiguous values at the conclusion of each treatment condition.
Cell viability after kainic acid treatment. The rate of BCECF leakage is a sensitive measure of cell viability (3), and a rate of leakage <5%/min correlates well with cell viability. However, the rates of BCECF leakage reported by Bevensee et al. (3) were measured over short durations (2 min). In the current study, we measured the average rate for BCECF loss from the rate of fall of the pH-insensitive BCECF fluorescence during excitation at 440 nm, but our experiments often lasted 20-30 min. Therefore, we used linear regression to calculate the rate of decline of fluorescence at 440 nm as a function of time from the start to the conclusion of each experiment. The average rate of decline among the cells we studied was 0.5%/min, and we excluded data from cells in which the rate of BCECF leakage exceeded 1%/min. Cells with leakage exceeding 1%/min consistently 1) had more acidic pHi in aCSF, 2) had more pronounced photobleaching, and 3) could not be calibrated reliably. Consequently, we used this more conservative rate of decline since it seemed better suited to our relatively long experiments.
Immunocytochemistry. Rats were deeply anesthetized with ketamine-xylazine and perfused transcardially with fixative (STF, Strek Laboratories, La Vista, NE), and the brain was removed for histological analysis. Regions of the brain containing the NTS and RTN were blocked, mounted (Tissue-Tek, Fisher Scientific), and sectioned on a cryostat (40 µm) maintained at -17°C (Reichert-Jung, Buffalo, NY). Sections were washed two times with PBS (pH = 7.4) plus 0.15% Triton X-100 for 1 h (this step and subsequent steps were performed at room temperature). The slices were removed from the PBS-Triton solution and incubated in a solution containing 10% normal goat serum (NGS)-PBS-Triton for 1 h. A mouse monoclonal anti-glial fibrillary acidic protein (GFAP, clone G-A-5, Sigma Immunochemicals) was added to NGS-PBS-Triton solution at a dilution of 1:60, and sections were incubated overnight. Subsequently, slices were washed with PBS-Triton three times for 1 h. Secondary labeling of GFAP was performed using a goat anti-mouse antibody conjugated to FITC at a 1:50 dilution (Alexa Fluor 488, Molecular Probes). Sections were incubated in the dark for 3 h and washed two times for 1 h using the PBS-Triton solution and mounted. In some experiments, sections were also labeled with 1,5-bis{[2-(methylamino)ethyl]amino}-4,8-dihydroxy anthracene-9,10-dione (DRAQ-5) to stain neuronal and glial nuclei (Biostatus, Leicestershire, UK). Processed sections were viewed with either a Nikon E600FN microscope equipped with epifluorescence and a combination FITC/DAPI filter (Chroma Technology, Brattleboro, VT) or a Leica DMIRE2 scanning laser confocal microscope equipped with argon (488 nm; Alexa Fluor) and HeNe (633 nm; DRAQ-5) lasers.
Data analysis and statistics. Statistical comparisons were made using a two-way repeated-measures ANOVA in which the experimental conditions were a repeated within-subjects factor and the nucleus (RTN vs. NTS) was a between-subjects factor (Systat 9.0, SPSS Science, Chicago, IL). When the results of the ANOVA indicated that significant differences existed among treatment conditions or between nuclei, specific preplanned comparisons were made using P values adjusted by the Bonferroni method. A P value 
0.05 was considered statistically significant. All values are reported as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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pHi responses to depolarization. NBC is well represented in glial cells. The stoichiometry of NBC is probably 1:2 in glia, and NBC transport is, therefore, electrogenic (16). The direction of transport will vary as a function of the membrane potential (Vm), and when Vm is depolarized above the equilibrium potential for the NBC, and Na+ enter the cell and alkalinize pHi (11, 35, 46). We examined the activity of NBC in the NTS and RTN by elevating extracellular K+ from 6.24 to 21.24 mM to depolarize the glia in kainic acid-treated slices. In cultured astrocytes, this change in extracellular K+ raised membrane potential 20 to 40 mV (35). The results are summarized in Fig. 2, which shows the responses of two typical cells, a summary of the initial dpH/dt in each nucleus during the hyperkalemic treatment, and the average steady-state pHi responses to each experimental condition. In the examples, note that the average stable pHi values were calculated during the times that are shaded on each example figure. The linear regression of pHi against time is also shown during the test condition. The average baseline pHi values during perfusion with aCSF did not differ significantly in the RTN (n = 16) and NTS (n = 27). However, in the presence of elevated K+, pHi rose significantly compared with the baseline control value in the RTN (P < 0.001), and pHi fell below the initial control pHi when the control aCSF was restored (P < 0.002). The increase in pHi during exposure to elevated K+ compared with the initial aCSF exposure was not significant in the NTS (P = 0.064). However, after returning to the control aCSF perfusate, pHi remained elevated and was significantly greater than the initial control aCSF pHi (P = 0.014). The initial rate of alkalinization was significantly greater in the RTN than in the NTS (P < 0.001), and the final average increase in pHi during exposure to elevated K+ was significantly greater in the RTN than in the NTS (P < 0.001). Thus depolarization of Vm alkalinized glia in the RTN but failed to change pHi in glia in the NTS.
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The stilbene derivative DIDS inhibits NBC (7, 35). Therefore, we tested the hypothesis that DIDS would prevent the depolarization-induced alkalinization of glial cells during exposure to elevated K+ in kainic acid-treated slices. Examples of the effect of DIDS on pHi during treatment with increased extracellular K+ (6.24 to 21.24 mM) and the steady-state average pHi values during each condition are shown in Fig. 3. In this set of experiments, the initial pHi values in the NTS were significantly more acidic than in the RTN (P = 0.002). However, within each nucleus, the glial cells showed no change in pHi during the period of depolarization or in the recovery period when the perfusate was returned to control aCSF (the fall in pHi in the RTN during DIDS treatment did not reach statistical significance, P = 0.12). When the baseline pHi is more alkaline, depolarization-induced alkalosis may be greater (35). To determine whether the difference in initial pHi values affected the response to elevated K+ and DIDS, we analyzed the cells from both nuclei in which the initial pHi values were similar (pHi = 7.15-7.35), and in this subset of cells, DIDS also blocked the alkalinization of pHi during treatment with high K+ in the RTN and in the NTS. The response of this subset of cells in the NTS was identical to the response of all cells from the NTS. Thus the initial pHi values did not seem to modify the response to elevated K+ and DIDS treatment.
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When studying voltage-dependent alkalinization, some authors include valinomycin and bumetanide in the high-K+ solutions (39). Valinomycin is a K+ ionophore, and when intercalated into the cell membrane, the cell rapidly attains the membrane potential appropriate for the K+ equilibrium potential (28). However, when external K+ is elevated, K+ entry may alter the activity of volume-regulatory processes (i.e., Na-K-Cl cotransport) in response to the increased osmolarity, which in turn can alter proton transport independent of changes in pH (41). Bumetanide inhibits Na-K-Cl cotransport, but bumetanide does not seem to modify pH regulation in glia in the absence of cell volume regulation mediated by Na-K-Cl cotransport (13). Therefore, we assessed the effect of elevated extracellular K+ on pHi in the presence of valinomycin (10 µM) and bumetanide (15 µM) in kainite-treated slices. The results of these studies are shown in Fig. 4. We were not able to obtain reliable calibrations in the presence of bumetanide and valinomycin; the cells were healthy and stable during each of the experimental conditions, but the rate of loss of BCECF fluorescence at 440 nm increased dramatically when the calibration solution was added at the conclusion of each experiment, and some cells actually lysed. Therefore, results from these experiments are presented as the uncalibrated ratios of fluorescence at 500 and 440 nm. In both nuclei, exposure to elevated extracellular K+ alkalinized the cells significantly (P < 0.001 for both nuclei), and the relative alkalinization reflected by the increased ratios was not different between the two nuclei. pHi returned to the same value present during the initial control period once the perfusate was returned to control aCSF.
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In a final experiment examining the effects of membrane depolarization on pHi, we studied slices in which kainic acid was not used as a pretreatment. These slices contain a mixed population of neurons and glia. The responses of individual cells and the average responses of all cells are shown in Fig. 5. Based on the number of cells in each slice, we believe most of the cells were actually neurons; these slices were much more densely populated with viable cells than the kainate-treated slices (this was particularly true in the compact portions of the NTS). The average pHi of these cells was 7.13 ± 0.12 in the NTS and 7.22 ± 0.10 in the RTN; both values are more acidic, and closer to neuronal pHi values, than the average pHi values measured in glia after kainic acid treatment of the brain stem slices. Cells in the NTS were more acidic under baseline conditions than were cells in the RTN, but this difference did not reach the level of statistical significance (P = 0.056). There was no significant alkalinization in cells from either nucleus during the high-K+ treatment. The NTS remained at a stable pHi when returned to control aCSF, but the pHi of cells in the RTN fell significantly when perfusion with aCSF was restored (P = 0.002). Thus the depolarization-induced alkalosis seen in the RTN in kainate-treated slices was no longer apparent in slices in which neurons were present.
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Response to CO2- or Cl--free perfusate. To assess the role of -dependent transport processes in acid-base regulation in glia from the RTN and NTS in kainic acid-treated slices, we removed CO2 and from the perfusate. When slices were perfused with CO2-free Na-HEPES-buffered saline (NHB, pHe = 7.48), the cells promptly acidified (Fig. 6). Removing extracellular enhances the electrochemical gradient favoring outward flux of , and the acidification, which occurred when extracellular was removed, revealed the activity of a significant exchange process. pHi did not differ in the two nuclei during the initial control aCSF perfusion. When was removed, the fall in pHi was significant in both nuclei compared with control conditions (P = 0.003), and the reduction in pHi was not different between cells in the two nuclei. When aCSF containing was restored in the perfusate, pHi returned to a value that was not significantly different from the initial control value in the RTN.
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Both Na+-dependent and Na+-independent anion exchangers that swap for Cl- are well represented in glia (13, 30, 46). Therefore, the dependence of pHi regulation revealed in the foregoing studies might reflect exchange. We evaluated this by incubating the slices in Cl--free media. Thus the control condition was perfusion of the slices with Cl--free solutions, and the test solution contained Cl- in the standard aCSF. We chose this protocol since we could be certain that intracellular Cl- was very low in the control condition. If the order of testing were reversed, it would be difficult to know exactly what the intracellular Cl- was during the initial exposure to Cl--free perfusate. The time necessary to deplete cells of Cl- is variable, but in cultured astrocytes Cl- fell to low levels in 11 min (1). Therefore, we placed the slices in Cl--free media immediately after they were sectioned. The cells were treated with kainic acid in Cl--free saline (>45 min) and stabilized in the perfusion chamber before the experiment in Cl--free saline. Thus the cells were in Cl--free media for at least 90 min before the experiment began. In this set of experiments, the pHi of the RTN was consistently more acidic than the NTS by 0.1 pH units in all treatment conditions (P < 0.001; Fig. 7). However, this was a main effect apparent only when pH values from all three test conditions were pooled, and the comparisons of pHi between nuclei within each treatment condition were not significantly different. Regardless of the starting pHi values, the pattern of responses was identical in the nuclei. pHi fell significantly in both nuclei when Cl- was introduced into the perfusate (P < 0.001), and the magnitude of the pHi change was similar in the two nuclei. pHi recovered partially in cells in both nuclei when Cl- was removed from the perfusate but remained significantly more acidic than the initial Cl--free condition (P = 0.005). Thus these data provide evidence for the activity of a exchange mechanism that was not different in glia between the two nuclei.
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Response to Na+-free perfusate. There are two exchange processes known to require Na+: Na+-driven exchange (NDCBE) and NBC. Therefore, we examined the role of Na+ in acid-base regulation by exposing brain slices to Na+-free aCSF. This treatment will also affect NHE mechanisms to the extent they are present. In the absence of extracellular Na+, NHE, NDCBE, and NBC may all run backward and acidify the cell. To try to separate the activity of NHE from the Na+- and -dependent processes, we followed the Na+-free treatment with Na+-free HEPES buffered saline, which was also free. If Na+-independent exchange were active, we would have expected some further intracellular acidification beyond what we saw with the Na+-free aCSF alone. In this protocol, we were unable to complete the calibration in all cells tested, so ratios only are reported. As in the valinomycin/bumetanide treatment, the cells appeared healthy until we added the calibration solution, at which point the fluorescence at 440 nm dropped more rapidly and some cells lysed. There was a significant fall in pHi when Na+ was removed from the perfusate (Fig. 8; P < 0.001 for cells in both nuclei), and the fall in pHi was greater in the RTN than in the NTS (P < 0.001). Removing and CO2 by perfusing the Na-free HEPES-buffered saline reduced pHi in the NTS and elevated pHi in the RTN, but neither of these changes was statistically significant. These data indicate that Na-dependent processes play an important role in pHi regulation, but they provide no support for the activity of any Na+-independent exchange under these conditions.
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In the final set of experiments, we examined the effect of EIPA, an inhibitor of NHE. The initial pHi values were not different in the two nuclei, and pHi fell significantly in both nuclei when 100 µM EIPA was added to the perfusate (Fig. 9; P < 0.001 in both nuclei). The decline in pHi after administration of EIPA was much greater in cells within the NTS compared with cells within the RTN (P = 0.002). After removal of EIPA from the perfusate, pHi remained unchanged in both nuclei; there was no evidence of pHi recovery in either location. These results indicate that a NHE mechanism is active under normal conditions in both nuclei.
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Anatomic distribution of glia within the RTN and NTS. We assessed the number of glial elements in the RTN and NTS by examining the distribution of GFAP, a protein that is ubiquitously expressed in astrocytes. Examples of these immunohistochemical studies are shown in Fig. 10. The RTN (Fig. 10, A-D) is found between the ventral medullary surface and the facial nucleus (VII). The RTN was stained intensely by the antibody directed against GFAP as is apparent in the region just ventral to the compact facial nucleus, which is relatively lucent (Fig. 10B). The NTS is also a compact nucleus, and it appears as a relatively lucent area in the dorsal medulla (Fig. 10F). Higher-magnification views also demonstrated greater density of GFAP in the RTN (Fig. 10, C and D). Glial elements, which form part of the blood-brain barrier, surrounded the microvessels in each area. In the compact portion of the nucleus, virtually the only glia in the NTS were adjacent to vessels (Fig. 10, G and H). We stained the nuclei of cells in the NTS with DRAQ-5 and confirmed that the large GFAP-free areas in the NTS nonetheless contained many cells (Fig. 10G, blue nuclei).
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DISCUSSION |
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Acid-base regulatory function at physiological pHe: NBC. We identified three transport processes in the NTS and RTN: NBC, NDCBE, and NHE. Although mRNA for the NBC has been isolated in neurons (45), only glia are thought to express NBC activity (16). Electrogenic NBC is identified by its dependence on extracellular Na+, lack of dependence on Cl-, activation by depolarization, and inhibition by stilbene derivatives (7). No single test can provide convincing evidence of NBC, and we studied multiple conditions to assess the activity of NBC. The alkalinization of pHi that occurred in the RTN after depolarization of the cells by raising extracellular K+ (Fig. 2), which was inhibited by DIDS (Fig. 3), suggests that NBC was present in these glia. We cannot exclude the activity of other anionic transporters (e.g., lactate), but these processes are unlikely to account for the DIDS-inhibitable component of depolarization-induced alkalosis that we found. Furthermore, once DIDS was present, there was little evidence of any non-NBC electrogenic transport.
The activity of NBC was greater in the RTN than in the NTS. The simplest hypothesis is that there was greater expression of NBC in the RTN than in the NTS. However, the apparent activity of NBC may vary if the resting membrane potential of glia differed in the RTN and NTS. Glial cells are usually hyperpolarized compared with neurons; the average resting membrane potential of glia is approximately -70 mV (34), and the reversal potential for NBC is approximately -85 to -90 mV assuming 2:1 :Na+ cotransport. If the membrane potential were less negative in the RTN or if the intracellular Na+ concentration were greater in the NTS than in the RTN, the apparent activity of NBC might have been less in the NTS compared with the RTN even if the number of transport proteins were similar. The concentration was probably similar in the two nuclei and unlikely to contribute to the observed differences in NBC activity since glial pHi was similar in both nuclei and differences in intrinsic buffer capacity have not been described. A wide range of glial membrane potentials has been described in cultured rat hippocampal astrocytes (34) although we are not aware of any systematic differences in membrane potential based on the neuroanatomic location of the glia. Differences in glial membrane potential may originate from differences among the Na+/K+ permeability ratios in different cells (47), which may reflect the particular K+ channels expressed in any group of astrocytes (10). In this respect, it is interesting to note that when glia were depolarized by elevating the extracellular K+ in the presence of valinomycin, a K+ ionophore that would be expected to enhance K+ permeability and increase the depolarizing effect of elevated extracellular K+, the differences between the RTN and the NTS were reduced and the apparent activity of NBC was increased in the NTS. Thus the hypothesis that differences in membrane potential and/or patterns of K+ channel expression may have contributed to the observed differences in NBC activity between the RTN and NTS may receive some support from the response to depolarization in the presence of valinomycin.
Whatever the explanation for the divergent pHi responses to depolarization in the RTN and the NTS (differential expression of the transporter, different electrochemical gradients, or different K+ permeabilities in the two nuclei), the RTN behaves as if NBC is more active. Thus any elevation in extracellular K+ associated with neuronal activity will lead to greater alkalinization of glial pHi and a greater fall in pHe in the RTN compared with the NTS.
Na+-driven 
exchange. The response to depolarization-induced alkalosis indicates that NBC was present in these nuclei, and the effect on pHi of perfusion with Cl--containing media after depletion of intracellular Cl- indicates that exchange was present as well. However, it is difficult to differentiate Na+-dependent and Na+-independent exchange mechanisms based on the limited experiments we did. There is ample evidence in previous studies of glia that NDCBE participates in acid-base regulation under control conditions (46). However, the role of NDCBE in the regulation of resting pHi in our studies is hard to assess. The zero Cl- pretreatment had little effect on pHi; the pHi values were quite similar to pHi values during perfusion with CO2-containing aCSF. If a Cl--dependent processes were active, we should have observed an acid shift of baseline pHi during perfusion with Cl--free saline. Thus one interpretation of the Cl--free treatment is that NDCBE exists in glia in both the RTN and NTS, but this exchange mechanism is not active under resting conditions. If this is the case, the interpretation of the response to Na+-free perfusion may change slightly. Unfortunately, we could not calibrate all of the Na+-free treatment experiments, but the change in the ratios was greater in the RTN than in the NTS. If there is no significant NDCBE active at resting pH values, then the greater response in the RTN may be attributed to greater activity of NBC. Greater activity of NBC in the RTN is also consistent with the response to depolarization seen in the elevated K+ experiments. In the zero Na+ experiments, we observed no additional acidification when was also removed from the perfusate. This implies that Na+-independent exchange was also not particularly active in these cells. NBC cannot account for all pHi regulation in these cells since the application of EIPA also decreased pHi. Taken together, these data suggest (but do not prove) that steady-state pHi is dependent on the activity of both NHE and NBC with only a minimal contribution of NDCBE. However, clearly defining the role of Na+-dependent and Na+-independent exchange processes will require further experiments. There is one further caveat: it is possible that NDCBE remained active even during the zero Cl- pretreatment. Bevensee et al. (1) found that Cl--dependent exchange remained active even when the Cl- concentration was extremely low; thus it could still contribute to regulation of pHi although that contribution does not appear to be marked.
We failed to identify any evidence of Na+-independent exchange although this transport mechanism has been identified previously in glia (13, 30), but unlike the first three mechanisms, which have the effect of extruding protons, Na+-independent exchange is active at more alkaline pHi values (46) and loads protons into the intracellular space. We did not examine pHi under alkaline conditions, and we were, therefore, unlikely to detect Na+-independent exchange in the experiments we did.
Na+/proton exchange. The Na+-dependent processes that we identified probably represent NBC, NHE, and possibly some element of NDCBE. The presence of an EIPA-inhibitable process, which is consistent with the activity of NHE, provides additional evidence for the activity of NHE in glia in the RTN and NTS. Furthermore, Na+/proton exchange has been described previously in glia in the cortex (31, 46). The activity of NHE seemed to differ between chemosensory sites: NHE activity was greater in the NTS than in the RTN.
Glial anatomy of the RTN and the NTS. Not only were there functional differences between the RTN and NTS, but also the anatomic relationships between neurons and glia seemed to differ in the two nuclei. The NTS is a compact nucleus, and the neurons are concentrated in a densely populated region that was relatively devoid of glial elements except adjacent to small vessels (Fig. 10, F-H). In the RTN, the glia were spread throughout this more diffusely organized nucleus (Fig. 10, B-D), and there seemed to be a closer relationship between the glia and neurons in the RTN. One might worry that GFAP labeling is also heterogeneous and, therefore, not representative of the actual distribution of glia in the brain stem. Patterns of GFAP labeling among glia have not been studied extensively, but 80% of the brain stem glia are GFAP positive (22). Also, our immunohistochemical studies show that staining was heavy in both nuclei around the microvasculature. Thus GFAP staining was present in both nuclei at the blood-brain barrier (where one would expect it), and there is, therefore, no evidence that we selectively missed GFAP elements in the NTS. As a result of the distribution of glia in these nuclei, neuronal behavior seems less likely to be modulated by glial processes in the NTS compared with the RTN. This speculation, based on the anatomy of the nuclei, is consistent with the functional responses to the glial toxin fluorocitrate; fluorocitrate stimulated ventilation when administered in the RTN (26) but not when administered focally within the NTS (Erlichman, unpublished observations).
Limitations of the methods. Glial pH regulation has been studied in the past in transformed cell lines or primary cell cultures from the cortex or hippocampus (4, 31, 44, 46). Thus acute studies of glial pH regulation in brain slices are unusual. Furthermore, previous studies have described the distribution of pH-regulatory mechanisms (as we have as well), but direct comparisons among or between regions have not been described. Because we used an unusual preparation, it behooves us to examine the potential problems with this preparation. A major consideration is that we removed neurons and obtained a brain slice dominated by glia by killing the neurons with kainic acid. Kainic acid induces an excitotoxic cell death mediated principally by non-NMDA receptors. The neurons are acidified, Na+ and Ca2+ enter the cell, and the cells are killed, possibly by necrosis rather than apoptosis (18, 27, 49). Glia are relatively resistant to the effects of kainic acid. After exposure to kainic acid, glia manifest a biphasic pH response: the cells acidify briefly, but alkalinization follows quickly. These pH changes have been attributed to inward movement of Na+ through non-NMDA receptors, and the associated changes in membrane potential activate inward or outward movement of through the NBC mechanism (44). The glial K+ concentration probably declines as K+ moves down its electrochemical gradient through the nonspecific cation channel activated by kainate binding. The effects of kainic acid on glia are transient, and pHi recovers normally once kainic acid is removed from the bath. The kainic acid-mediated killing of neurons is incomplete, and it is likely that the population of glia is enriched relative to the number of neurons, but some neurons almost certainly persisted in the brain slices we studied. Two findings indicate that we are studying primarily glia and the cells were healthy. First, the average pHi measured in the cells we studied was virtually identical to pHi values measured in primary cultures of glia (46). Furthermore, the cells appeared to represent a similar and homogeneous group of glia (see Fig. 1). Second, when we did not expose the brain slices to kainic acid, we obtained resting pHi measurements similar to published values of pHi in neurons. The pHi values that we measured in brain slices exposed to kainic acid were stable over time, and the rate of BCECF dye loss from the cells was not excessive. Thus acute treatment of brain slices appears to be an effective mechanism of isolating healthy glia in vitro for pH studies. A similar strategy to our own has been used in which brain slices enriched in glial elements were prepared from kainate-lesioned animals (25).
We were not able to calibrate pHi measurements in all conditions. The glia lost cell membrane integrity when the calibration solution was introduced. Other investigators studying glia have had similar problems (5, 6), and they attributed the loss of viability to the combination of ion shifts, low pHi, and hypoxia. We were not studying hypoxia, but loss of cell viability was increased in longer protocols associated with significant ion fluxes and marked reductions in pHi. Nonetheless, the direction of changes in pHi reflected by the fluorescence ratios still accurately represents the pattern, if not the absolute value, of pHi changes under the conditions studied (5).
Perspectives
The acid-base regulatory mechanisms described above have all been described previously in astrocytes. The interesting aspect of the study is the heterogeneous distribution of these exchange processes between two nuclei that share at least one common function: both the RTN and NTS are thought to contain CO2 chemosensory neurons (14). Moreover, there are differences in the number of glia present in the two nuclei: glia are more abundant in the diffusely organized RTN than in the more compact NTS. To the extent that glial pHi regulation modifies pHe (and glia are thought to play a major role conditioning the extracellular fluid), these qualitative and quantitative differences in glia in the RTN and NTS could lead to differential regulation of pHe in the RTN and NTS. For any given chemosensory stimulus, pHe is less likely to be modified by glial mechanisms in the NTS. The major pH-regulatory mechanism likely to modify pHe is NBC, and this is both less well represented in glia in the NTS, and there are fewer glia in the NTS. On the other hand, pHe in the RTN is more likely to be affected by glial mechanisms: activity of NBC was more abundant in glia in the RTN, and more glia exist in the RTN.
Ransom (40) put forth the hypothesis that NBC by glia might participate in a negative-feedback loop in which increased neuronal activity during seizures would increase extracellular K+, depolarize glia, activate NBC, alkalinize glial pHi, but acidify pHe, and the fall in pHe might inhibit neuronal activity and limit the extent of seizure activity. This remains aviable hypothesis for neurons that are inhibited by acidosis, but not all neurons are inhibited by acidosis. The activity of CO2 chemosensory neurons increases when the neurons are acidified. Thus, in CO2 chemosensory regions, neuronal activity associated with hypercapnic stimulation might increase extracellular K+, activate NBC, and enhance the fall in pHe, and rather than blunting neuronal activity, this action of NBC might actually amplify the stimulus for CO2 chemosensory cells. Furthermore, this amplification mechanism may differ among CO2 chemosensory regions to the extent that the number of glia and the activity of the NBC differ among CO2 chemosensory sites. Thus we would predict that even if the CO2 chemosensory mechanisms were similar in the NTS and RTN, the RTN would nonetheless express greater neuronal sensitivity for any level of hypercapnia by virtue of the amplifying effect of glial activity on pHe. We conclude that glial function may play an important role in information processing in CO2 chemosensory regions of the brain by modifying one of the putative chemosensory stimuli, pHe. However, this modulatory function of glia is probably heterogeneously distributed among CO2 chemosensory sites within the brain.
The foregoing analysis is based on work in brain slices, and we have ignored the effects of blood flow on the make up of extracellular fluid. It seems likely to us that blood flow may blunt the differences in extracellular fluid buffering that originate from the heterogeneous patterns of glial function and anatomy, but blood flow will not completely abrogate these differences. Fluorocitrate is a specific glial toxin that depolarizes glia, increases extracellular K+ concentrations, and reduces pHe (20, 29). Fluorocitrate stimulates ventilation in awake and anesthetized animals when microperfused into the RTN (20, 26). The respiratory effects were correlated with an extracellular acid shift that stimulated this chemosensory region (20). Fluorocitrate is an ineffective stimulus when injected into the NTS (Erlichman, unpublished observations) even though injection of acidic stimuli into the NTS is a potent respiratory stimulus (14). It is our hypothesis that there are few glia in the NTS and those glia present in the NTS do not express significant NBC activity. Therefore, there is little effect of the fluorocitrate-induced depolarization on pHe in the NTS. Thus the heterogeneous distribution of glial function may alter neural processing of chemosensory information even in the presence of intact blood flow.
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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.
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  N. A. Ritucci, J. S. Erlichman, J. C. Leiter, and R. W. Putnam Response of membrane potential and intracellular pH to hypercapnia in neurons and astrocytes from rat retrotrapezoid nucleus Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2005; 289(3): R851 - R861. [Abstract] [Full Text] [PDF]
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 M. R. Hodges, P. Martino, S. Davis, C. Opansky, L. G. Pan, and H. V. Forster Effects on breathing of focal acidosis at multiple medullary raphe sites in awake goats J Appl Physiol, December 1, 2004; 97(6): 2303 - 2309. [Abstract] [Full Text] [PDF]
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A. Hewitt, R. Barrie, M. Graham, K. Bogus, J. C. Leiter, and J. S. Erlichman Ventilatory effects of gap junction blockade in the RTN in awake rats Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2004; 287(6): R1407 - R1418. [Abstract] [Full Text] [PDF]
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