Journal of Applied Physiology Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 90: 1539-1547, 2001;
8750-7587/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Holleran, J.
Right arrow Articles by Erlichman, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Holleran, J.
Right arrow Articles by Erlichman, J. S.
Vol. 90, Issue 4, 1539-1547, April 2001

Ventilatory effects of impaired glial function in a brain stem chemoreceptor region in the conscious rat

Julianne Holleran1, Melissa Babbie2, and Joseph S. Erlichman2

1 Department of Biology, Allegheny College, Meadville, Pennsylvania 16335; and 2 Department of Biology, St. Lawrence University, Canton, New York 13617


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glia are thought to regulate ion homeostasis, including extracellular pH; however, their role in modulating central CO2 chemosensitivity is unclear. Using a push-pull cannula in chronically instrumented and conscious rats, we administered a glial toxin, fluorocitrate (FC; 1 mM) into the retrotrapezoid nucleus (RTN), a putative chemosensitive site, during normocapnia and hypercapnia. FC exposure significantly increased expired minute ventilation (VE) to a value 38% above the control level during normocapnia. During hypercapnia, FC also significantly increased both breathing frequency and expired VE. During FC administration, maximal ventilation was achieved at ~4% CO2, compared with 8-10% CO2 during control hypercapnic trials. RTN perfusion of control solutions had little effect on any ventilatory measures (VE, tidal volume, or breathing frequency) during normocapnic or hypercapnic conditions. We conclude that unilateral impairment of glial function in the RTN of the conscious rat results in stimulation of respiratory output.

brain stem pH regulation; central chemoreception; control of breathing; retrotrapezoid nucleus; brain stem glia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE INTERPLAY BETWEEN NEURONS and glia in the central nervous system is more complicated than previously envisioned. Glial cells have been implicated in diverse cellular functions, including the metabolic support of neurons and extracellular ion homeostasis (1, 14, 17, 27, 32). For example, there is increasing evidence suggesting that glia regulate the extracellular concentrations of K+, amino acids, and H+ (9, 11, 17), thereby potentially altering the activity of neurons (18, 32).

Decreases in extracellular pH (pHo) that normally occur during respiratory and metabolic acidosis also affect neuronal activity. The increase in ventilation associated with hypercapnia and other acidic stimuli is well documented and appears to originate from central CO2 chemoreceptor cells located at sites within the medulla oblongata (6, 22). Although the unique stimulus to these cells is not known, a central feature in many hypotheses involves the effects of pH on neuronal excitability (10, 22). Changes in intracellular pH (pHi) or pHo and modulation of synaptic transmission by pH have all been implicated in the chemotransduction pathway (10, 22). Therefore, factors that influence pH in or around sites of chemosensitivity may affect ventilatory responsiveness to acidic stimuli.

The retrotrapezoid nucleus (RTN) is located within a few hundred micrometers of the rostral ventral lateral surface of the medulla. The RTN region in the rat extends from the rostral border of the nucleus ambiguus to the rostral border of the facial nucleus, bounded laterally by the spinotrigeminal tract and medially by the pyramidal tract. Anatomic tracing of neurons in the RTN has shown extensive projections to both the dorsal and ventral respiratory groups in the brain stem (30, 38). RTN neurons discharge with the respiratory rhythm and increase their firing during hypercapnia (7, 23). Focal acidosis in the region of the RTN increases phrenic nerve output, whereas neuronal ablation at this site decreases CO2 sensitivity, indicating the presence of CO2 chemoreceptors within this region (20, 24, 25).

Previously, our laboratory showed that selective impairment of glial function using fluorocitrate in situ in the RTN of the anesthetized rat resulted in extracellular acidification and increased phrenic nerve activity during normocapnia (11). Although these data suggest that glia may influence the microenvironment surrounding chemoreceptor cells in the RTN, it is not known what role glia may play in ventilatory control in the conscious rat or the effects of glial impairment on CO2 sensitivity. Therefore, in this study, we focally delivered fluorocitrate into the RTN of chronically instrumented conscious rats while measuring ventilation using a whole body plethysmograph. To examine the ventilatory effects of glial impairment on CO2 sensitivity, we generated a CO2 response curve by varying the CO2 in the plethysmograph from ~0.5 to 10%. We show that unilateral perfusion of fluorocitrate into the RTN increases submaximal ventilation at all CO2 levels compared with control. We also show that maximal ventilation was not different between fluorocitrate and control experiments, although the maximal ventilation attained by the animal was observed at a lower CO2 level during fluorocitrate perfusion. Our findings support our hypothesis that glia in the region of the RTN play an important role in respiratory control in the conscious rat.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The care of the animals and the experimental protocol used in this study were approved by the institution's Animal Review Committee. Adult Sprague-Dawley rats of either sex, weighing between 350 and 450 g, were used for this study. Animals were kept on a 12:12-h light/dark cycle and provided food and water ad libitum.

Rats were initially anesthetized with an intraperitoneal injection of a mixture of ketamine · HCl (70 mg/kg) and xylazine (6 mg/kg). Before surgery, the depth of anesthesia was determined by the lack of a pinch reflex in the hind paw. In the event that an animal needed additional anesthesia during the course of the surgery, one-fourth to one-third of the original dose of ketamine-xylazine was administered.

The scalp was shaved, and the animal was secured in a stereotaxic apparatus (Kopf Instruments). A midline incision was made, and a hole was drilled in the skull 2 mm caudal to lambda and 1.5 mm lateral to midline. Three additional holes were drilled into the skull for placement of anchor screws. A push-pull guide cannula (0.29 mm ID, 0.56 mm OD; Plastics One, Roanoke, VA) was placed with the tip dorsal to the RTN and ~500 µm from the ventral surface of the brain (Fig. 1A). The guide cannula and the anchor screws were attached to the skull with cranioplast (Plastics One), and care was taken to ensure that the exposed end of the guide cannula was free of the acrylic cement. Rough spots in the cement were leveled, and the scalp was sutured up to the border of the cement cap. A dummy cannula was screwed into the guide cannula to maintain patency of the lumen during the recovery period and between experiments. The dummy cannula extended ~0.3 mm beyond the end of the guide cannula.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 1.   Inner delivery cannula (A) and outer guide cannula (B). The inner delivery cannula has an inner diameter of 0.1 mm and an outer diameter of 0.2 mm. The outer guide cannula has an inner diameter of 0.29 mm and an outer diameter of 0.56 mm. Solutions were delivered through the cannula using a push-pull syringe pump.

After surgery, animals were examined for signs of infection, such as redness, swelling, and discharge. In one instance, an animal developed signs of infection and was given oral antibiotics. This animal and two others who pulled out their cannulas during the recovery period were excluded from the study. Approximately 3-4 wk after surgery, ventilatory measurements were made on the remaining six animals.

Solutions. A 1 mM fluorocitrate solution was prepared as previously described (11). Briefly, the barium salt of fluorocitric acid was dissolved in 0.1 M HCl, and the barium was precipitated by the addition of Na2SO4 (0.1 M) to the solution. This solution was then buffered with Na2PO4 (0.1 M) and centrifuged at 800 g for 10 min. The supernatant was removed and added to artificial cerebral spinal fluid (aCSF).

aCSF was composed of the following salts (in mM): 124 NaCl, 5 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.24 KH2PO4, 26 NaHCO3, and 10 glucose. Before delivery, aCSF was equilibrated with 95% O2 and 5% CO2, warmed to 37°C, and loaded into 3-ml plastic syringes. The pH of all solutions was 7.48. For citrate control experiments, citrate was dissolved directly into aCSF at a final concentration of 1 mM.

Solution delivery. Solutions were delivered to the RTN by using a push-pull syringe pump (World Precision Instruments, Sarasota, FL). Flow was directed through polyethylene tubing that was connected to an inner delivery cannula (Plastics One; 0.1 mm ID, 0.2 mm OD). Before perfusate delivery, the line and inner cannula were flushed with either aCSF, aCSF + citrate, or aCSF + fluorocitrate. The inner cannula was then screwed into the guide cannula, the return vacuum line was connected (Fig. 1B), and the animal was placed in a whole body plethysmograph. Like the dummy cannula, the inner delivery cannula extended 0.3 mm beyond the end of the guide cannula. The flow through the cannula was 0.06 ml/h. Test solutions were continuously perfused throughout the experiment.

Experimental design. Baseline, presurgical ventilatory measurements were made in each rat before cannulation during normocapnic conditions. Animals were placed in the plethysmograph and allowed to accommodate to the chamber for 30 min before the chamber CO2 was elevated. In cannulated animals, perfusion was initiated at the beginning of the 30-min accommodation period in the plethysmograph and was terminated after the animal was exposed to incremental hypercapnia. Cannulated animals were perfused with either aCSF alone, aCSF + citrate, or aCSF + fluorocitrate, or no perfusate was delivered. All rats were exposed to each experimental treatment. No animal was exposed to more than one perfusate per day, and the order that the perfusates were delivered was randomized. Body weight and barometric pressure were determined before each experiment. After the completion of all experimental conditions, fast green was added to aCSF, and this solution was perfused into the RTN for a similar amount of time as the experimental trials (~1.5 h) to evaluate the distribution of perfusate within the medulla.

Ventilatory responses to CO2. The rats were exposed to increasing levels of inspired CO2 in the plethysmograph, beginning with room air and progressing in 2% increments to a final concentration of ~10% CO2. Before delivery into the chamber, the compressed air and CO2 were mixed and humidified by bubbling of the air through a water column. Animals were maintained at each level of CO2 for ~10 min after the stabilization of CO2 levels within the plethysmograph chamber. The chamber had an approximate volume of 5 liters, and the rate of air flow through the box was 1.5-2 l/min. Stable CO2 values within the chamber were achieved in 3-4 min.

Changes in chamber CO2 (CD3A, Applied Electrochemistry) and pressure (PT5, Grass Instruments) were measured continuously, digitized, and stored on a personal computer (Biopac, World Precision Instruments). Both the chamber and rat body temperature were measured at the end of each step change in CO2 concentration. Body temperature was measured by using an infrared sensor (C1600, Linear Laboratories). For tidal volume (VT) calculations, this value was corrected to rectal temperature based on preliminary experiments in which both body temperature, as determined by infrared emissivity, and rectal temperature were measured simultaneously.

Data analysis. Values used to calculate VT were digitized at 20 Hz, low-pass filtered at 10 Hz, and smoothed by using a moving-average transformation (Biopac, World Precision Instruments). An average pressure reflecting the magnitude of the VT was determined from 120 breaths during the last 2 min of each period (i.e., CO2 level). The frequency of breathing was measured during the last minute of each period. VT was calculated by using the method described by Pappenheimer (28) and the following equation
V<SC>t</SC><IT>=</IT><FR><NU><IT>&Dgr;</IT>P<SUB>chamber</SUB></NU><DE><IT>&Dgr;</IT>P<SUB>ml</SUB></DE></FR><IT>×</IT><FENCE><FR><NU><IT>1</IT></NU><DE><IT>1−</IT><FR><NU>(P<SUB>B</SUB><IT>−</IT>P<SC>h</SC><SUB>2</SUB><SC>o</SC><SUB>b</SUB>)</NU><DE>(P<SC>b</SC><IT>−</IT>P<SC>h</SC><SUB>2</SUB><SC>o</SC><SUB>chamber</SUB>)</DE></FR><IT>×</IT><FR><NU>T<SUB>chamber</SUB></NU><DE>T<SUB>b</SUB></DE></FR></DE></FR></FENCE>
where VT is in milliliters BTPS, Delta Pchamber is change in plethysmograph chamber pressure (mmHg), Delta P1 ml is change in plethysmograph chamber pressure with the addition of 1 ml of air (mmHg), PB is barometric pressure (mmHg), PH2ob is vapor pressure of water determined by body temperature (mmHg), PH2ochamber is vapor pressure of water at the plethysmograph chamber temperature (mmHg), Tchamber is plethysmograph chamber temperature (K), and Tb is body temperature (K). Typical pressure tracings using this method are shown in Fig. 2.


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 2.   Typical pressure measurements collected using the whole body plethysmograph. A: plots utilizing raw pressure values. B: plots of the same data at a higher gain that have been smoothed using a moving average transformation. Bar = 5 s.

Histology. After the final perfusate was tested, the rat was anesthetized and transcardially perfused through the left ventricle of the heart with 0.1 M PBS, pH 7.3, containing 4% paraformaldehyde. After fixation, the brain was removed, flash frozen (Histofreeze, Fisher Scientific), and sectioned (50-µm thickness) with a cryostat (Histostat, Reichert) maintained at -20°C. Tissue sections were mounted on glass slides, stained with neutral red, and viewed under bright field. Serial images of the sections were captured by using a digital camera (Sensys 1400, Photometrics). The serial images were compared with photographic plates in a stereotaxic atlas (Ref. 28a, plates 59-64) to identify the location of sections. Placement of the cannula was determined by identifying the section in which the center of the cannula was located (i.e., identifying the section with the largest cross-sectional lesion). Distances of the lesion from traditional anatomic landmarks (i.e., pyramids, facial nucleus, ventral medullary surface) were determined from digitized images of the sections. Distance, in pixels, was measured by using software (UTSA Image) and converted to millimeters by using a stage micrometer.

Statistical analysis. The main effects of the type of perfusate delivered (i.e., aCSF, aCSF + citrate, aCSF + fluorocitrate) and CO2 were analyzed by using a two-factor repeated-measures ANOVA design. The main effects of the surgical procedure and perfusion (i.e., no perfusion vs. aCSF and aCSF + citrate perfusion) were determined using separate, one-factor repeated-measures ANOVA designs. Significant main effects and interactions were evaluated by using simple, repeated, and difference contrasts. All significance values were corrected for sphericity with the use of the Greenhouse-Geisser epsilon. Values reported are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Histology. Examination of the histological sections revealed that the placement of the terminal portion of the cannula was either in the RTN or in the superficial margin of neighboring nuclei (Fig. 3). Tissue staining with Fast Green outside the site of the lesion was minimal and present only on the lateral margins of the lesion, suggesting that the mobility of the perfusate was restricted to the tissue adjacent to the cannula.


View larger version (68K):
[in this window]
[in a new window]
  		Fig. 3.   Location of cannula placement in the retrotrapezoid nucleus of the 6 rats studied (A-F). Solid rectangles represent the approximate width of the center of the guide cannula and its placement within the medulla. The figure was made by comparing serial images with the photographic plates in the stereotaxic atlas of Paxinos and Watson (plates 59-64; Ref. 28a), and the corresponding reference sections were identified. Placement of the cannula along the rostral-caudal axis was determined by counting the number of sections (50 µm each); the center of the guide cannula was located from the reference sections identified in the stereotaxic atlas. Distances of the lesion within the section from traditional anatomic landmarks [i.e., pyramids (PYR), facial nucleus (VII), ventral medullary surface] were determined using software (UTSA Image). LPGi, lateral paragigantocellular nucleus; SPT, spinotrigeminal tract. Figures are modified from Paxinos and Watson with permission. G and H: transverse sections of the medulla showing the lesion size resulting from the cannula. Fast green was only slightly visible on the margins of lesion at high power. Bar = 1 mm for G and H.

Effects of surgery on ventilation. The presurgical values for VT per kilogram, VE per kilogram, and breathing frequency were not significantly different among the no perfusion, aCSF, or aCSF + citrate conditions during normocapnia (P >=  0.24). The mean CO2 concentration in the plethysmograph, while room air was delivered to the chamber at a rate of 1.5-2 l/min, was 0.58 ± 0.08% and was likely the result of incomplete washout of expired air from the chamber. VT per kilogram, VE per kilogram, and breathing frequency were not different during unilateral perfusion in the RTN among the no perfusion, aCSF perfusion, or aCSF + citrate conditions across CO2 levels (main effect of perfusion, P >=  0.10; CO2, P <=  0.02; interaction of flow and CO2, P >=  0.38). For control conditions (i.e., no flow, aCSF alone, aCSF + citrate), the increase in maximal VE during hypercapnia was the result of an ~50% increase in frequency and a 50% increase in VT.

Effects of perfusate type on ventilation. Although hypercapnia increased VE in all experimental conditions, VE was significantly greater during aCSF + fluorocitrate treatment compared with other conditions (P = 0.007; Fig. 4). In addition, there was no statistical difference in VE across CO2 levels for any of the conditions (P > 0.08). Post hoc analysis revealed that expired VE during the fluorocitrate condition was significantly higher compared with control conditions at all CO2 levels tested (P <=  0.025), and the control conditions were not significantly different from each other (P = 0.51). During the fluorocitrate trial, all animals were capable of tolerating elevations in CO2 up to ~4% (Fig. 5). In three of six rats, the experiment was terminated at the end of the 4% CO2 condition (Fig. 5, C, E, and F) due to apparent respiratory distress experienced by the animal. This discomfort was manifest as one of two behaviors: either the rat showed labored breathing while on its side and was incapable of righting itself (2 of 3 rats) or the rat attempted to chew through the rubber stoppers used as sampling ports in the side of the plethysmograph (1 of 3 rats). All three of these animals recovered when the level of hypercapnia was reduced. None of these behaviors was observed during any of the other trials (i.e., no flow, aCSF, or aCSF + citrate), implicating fluorocitrate as the probable cause. Statistical comparisons of VE per kilogram during fluorocitrate exposure at 4 vs. ~10% CO2 during control conditions (i.e., aCSF and aCSF + citrate) revealed that VE was not statistically different between these conditions, suggesting that animals had reached their maximal ventilation (P = 0.84). Although three of six rats tolerated higher levels of CO2, none of the animals could achieve the same maximal level of CO2 used during perfusions with aCSF or aCSF + citrate, and little change in ventilation was evident at CO2 concentrations >4% (Fig. 5, A, B, and D).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4.   Effects of perfusate on minute ventilation (VE) during normocapnia and hypercapnia. Exposure to fluorocitrate resulted in a significant increase in VE at all CO2 levels up to ~4% compared with artificial cerebral spinal fluid (aCSF) alone or aCSF + citrate controls (* P < 0.025). Values are means ± SE. Nos. above each data point indicate no. of animals completing all 4 trials at each level of hypercapnia.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of perfusate on VE during normocapnia and hypercapnia in individual animals (A-F).

Because VE is a product of both frequency of breathing and VT, both factors could account for the increase in VE per kilogram during fluorocitrate perfusion. Statistical analysis showed that the increase in VE was due principally to an increase in breathing frequency evident at all CO2 concentrations during fluorocitrate perfusion (P = 0.01; Fig. 6). There was no significant change in frequency across CO2 levels in any of the groups (P = 0.87). The breathing frequencies associated with control conditions were significantly lower than those of the fluorocitrate trials (P <=  0.04). The aCSF + citrate resulted in higher breathing frequencies that approached significance compared with trials with no perfusate or aCSF alone (P = 0.08). Although fluorocitrate resulted in an increase in both VE per kilogram and breathing frequency, it had less of an effect on VT per kilogram. Hypercapnia resulted in an increased VT per kilogram; however, this change was not statistically significant and there was no statistical effect of the type of perfusate or the interaction between these terms (P >=  0.10; Fig. 7). Body temperature decreased slightly during hypercapnia in all groups; however, this decrease was not statistically significant (P = 0.24) nor were there statistical differences among treatments (P = 0.13). The effect of hypercapnia on body temperature for all animals and conditions is shown in Fig. 8.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6.   Effects of perfusate on breathing frequency. Exposure to fluorocitrate significantly increased breathing frequency at all CO2 levels up to 4%, compared with aCSF alone or aCSF + citrate (* P = 0.01). Values are means ± SE. Nos. above each data point indicate no. of animals completing all 4 trials at each level of hypercapnia.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 7.   Effects of perfusate on tidal volume (VT). Hypercapnia resulted in a slight, but not significant, increase in VT in all trials and was most evident at CO2 levels >4% (P > 0.1). Values are means ± SE. Nos. above each data point indicate no. of animals completing all 4 trials at each level of hypercapnia.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 8.   Effect of hypercapnia on body temperature for all rats during all trials. Hypercapnia resulted in a progressive decrease in body temperature; however, this decrease was not significant (P = 0.13) and did not differ among trials (P = 0.24). Values are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Role of glia in central chemoreception. The ventilatory effects that we observed with fluorocitrate perfusion into the RTN were marked. At all levels of CO2, VE and breathing frequency were statistically higher compared with other conditions. Our observation that maximal VE was not different between the fluorocitrate trials during 4% CO2 exposure compared with control trials (aCSF, aCSF + citrate) utilizing 8-10% CO2 indicates that the rats attained the same maximal ventilation albeit at a lower CO2 during fluorocitrate perfusion. Although one-half of the animals in this study could tolerate higher levels of CO2 >4%, the small, additional increase in VE was not significant, indicating that maximal ventilation had been reached.

Similar to previous reports, maximal ventilation in all trials except fluorocitrate was observed between 8 and 10% CO2 (2, 4, 16). Our data showed a significant increase in VE per kilogram during hypercapnia for all groups that was due principally to an increase in breathing frequency. VT also increased during hypercapnia; however, this was most evident at CO2 levels >4%. The change in VT was incremental and more variable during mild hypercapnia (2 and 4% CO2), and the change in VT was not statistically significant, even though resting and maximal VT values were different from each other and similar to those reported by others (2, 16, 28).

The precise mechanism underlying the selective nature of fluorocitrate impairing glial function is not known. Previous studies in vitro have shown that fluorocitrate rapidly decreases ATP and glutamine levels by inhibiting the tricarboxylic acid cycle enzyme aconitase (13). In both the hippocampus and the medulla, changes in neuronal morphology and function during fluorocitrate perfusion in situ are minimal when tissue is exposed to this agent for <4 h at 1 mM concentrations (11, 17). Here we have shown that the ventilatory effects of this compound are reversible, suggesting that the ability of fluorocitrate to impair glial function is likely restricted to the time during exposure. This is supported by our findings that the functional effects of fluorocitrate impairment are not carried over from one trial to another.

There is increasing evidence suggesting that glia can influence neuronal function by at least three mechanisms. First, neuronal excitability can be altered by glial uptake of glutamate from the synaptic cleft (3). Second, the bidirectional, Ca2+-dependent communication reported between glia and neurons appears to play a role in synaptic plasticity (39). Third, glia appear to regulate the extracellular ionic milieu surrounding neurons (11, 17, 18, 27). In the case of central chemoreceptors, changes in accumulation of neurotransmitters, K+, or protons could all potentially alter chemoreceptor function, thereby altering ventilatory control. Regarding the role of pH, we showed previously that transient impairment of glial function in the RTN in the anesthetized rat led to a rapid and reversible acidification of the extracellular space that correlated well with increased phrenic nerve activity (11). Although the extent to which changes in pHi or pHo mediate central chemoreceptor activity is unknown, it has been shown that the application of other acidifying stimuli in the region of the RTN increases ventilation during normocapnia (6, 22). Hence, it is intriguing to consider the role glia may play in pHo regulation. Previously, Deitmer (8) showed that pHo buffering in the leech neuropile was dependent on the availability of bicarbonate and linked to the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter present in the glial membrane. Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport has been described in many vertebrate glial cell types; however, this transporter has not been identified in neurons (35). An essential feature of this proton transporter is that it is electrogenic, having a stoichiometry of 1 (Na+):2 or 3 (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>), depending on the cell type. The reversal potential for this transporter is near the resting membrane potential of most glia. Therefore, membrane hyperpolarization in RTN glia may result in intracellular acidification, whereas membrane depolarization may lead to intracellular alkalinization. Thus the linkage of glial membrane potential and pHi may enable the glia to influence pHo by transporting acid equivalents between the extracellular and intracellular compartments (9, 32). Recently, Largo et al. (18) showed that glial cells depolarize in the hippocampus in the presence of fluorocitrate in a time-dependent manner. In light of this finding and our previous observations, this suggests that the metabolic inhibition generated by fluorocitrate exposure may lead to a decrease in membrane potential in glia in the RTN, resulting in the movement of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> from the extracellular space into glia, thus leading to extracellular acidification via Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. Using medullary brain slices pretreated with kainic acid to generate slices devoid of neurons (24), we are presently examining the proton transport mechanisms present in the RTN glia in vitro and have preliminary data supporting the presence of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport. Determining the role of glia in brain stem pH regulation is crucial to our understanding of ventilatory control, given the important role of pHo and pHi in chemoreceptor function (10, 11, 22, 34).

The findings in this study are consistent with an increase in the gain of the respiratory system rather than an increase in CO2 sensitivity. Such a change would be expected if there were an acid shift in the extracellular space, consequent to fluorocitrate exposure, that was additive of the acidification associated with CO2. Previously, our laboratory (11) showed, in the anesthetized rat in situ, that unilateral perfusion of fluorocitrate resulted in an acidification of the extracellular fluid and an increase in phrenic nerve activity of ~43%. Consistent with these findings, we observed a 38% increase in VE during normocapnia with the perfusion of the fluorocitrate into the RTN in the conscious rat in this study. Using a microdialysis probe, Li and Nattie (19) found that focal acidification with CO2 in the RTN resulted in a 24% increase in VE above baseline in the anesthetized rat. Using electrodes to measure pHo, they showed that this method resulted in acidification that was largely restricted to the RTN. Here we report a slightly greater increase in resting ventilation with fluorocitrate perfusion. Assuming that the increase in VE is associated with acidification of the extracellular space, resulting from glial impairment, we can speculate either that fluorocitrate results in greater acidification of the RTN or more tissue is acidified, thus affecting additional chemosensitive sites compared with focal hypercapnia. The extent of acidification that our laboratory measured in our previous study (11) using fluorocitrate in the anesthetized rat in situ is similar to the values reported by Li and Nattie (19), suggesting that the effects of fluorocitrate may have extended beyond the RTN rather than result in greater acidification. Although the Fast Green we added to the perfusate was restricted to the tissue directly adjacent to the perfusion pipette, it is unlikely that this stain was reflective of the true mobility of fluorocitrate. Once taken up by glia, fluorocitrate could diffuse to distant sites via intracellular coupling of glia by gap junctions. Labeling of up to 100 astrocytes has been reported after injection of the fluorescent dye Lucifer Yellow into a single glial cell (31). Alternatively, the clearance of fluorocitrate by blood flow may have transported this compound to other areas of the brain stem. Thus it is likely that we have underestimated the region of tissue affected by fluorocitrate in this study.

An important finding of this study was our observation that unilateral impairment of glial function in the RTN of the conscious rat is sufficient to stimulate ventilation. The depressant effects of anesthesia and the state-dependent nature of ventilatory control make it difficult to extend many of the previous findings to the conscious animal (19, 26). In anesthetized and decerebrate animals, destruction of the RTN markedly reduces ventilatory responses to hypercapnia. In contrast, bilateral cooling of rostral regions of the ventral lateral medulla surface in goats results in a sustained apnea under anesthesia but has only modest effects on breathing in the unanesthetized animal. Similarly, unilateral lesions in the RTN attenuate the ventilatory responses to CO2 much less compared with anesthetized animals (2). Recently, Li and Nattie (19) showed that focal CO2 stimulation increases ventilation in anesthetized, decerebrate, and conscious, but not sleeping, animals.

Technical considerations. The whole body plethysmography method used in this study permits noninvasive and unrestricted ventilatory measurements to be made in the conscious rat. Our presurgical values for VT per kilogram of 5.78 ml/kg during room-air breathing are similar to those reported previously by Pappenheimer (28) of 5.66 ml/kg in awake rats using a similar technique. In addition, our frequency data of 102 breaths/min are similar to the values reported by both Akilesh et al. (2) and Lai et al. (16). Our observation that maximal VE during hypercapnia was the result of an approximately equal increase in both breathing frequency and VT is also consistent with previous studies in vivo in rats (2, 19). The decrease in body temperature that we observed in the present study was not statistically significant; however, the magnitude of the decrease was similar to values reported in other studies (19, 21). Although most studies in the rat report a hypothermic response during CO2 exposure, Saiki and Mortola (36) showed in the rat that decreases in body temperature during hypercapnia are dependent on ambient temperature, suggesting that some of the variability among studies may be attributed to this factor. In their study, maintaining an ambient temperature of 25°C eliminated decreases in body temperature during hypercapnia. In the present study, the ambient temperature range was 25-29°C.

The effects of cannulation alone on the ventilatory measurements in this study were minimal. Cannulation resulted in a slight decrease in both VT (5.78 vs. 5.63 ml/kg) and frequency (103 vs. 101 breaths/min) in baseline conditions; however, neither of these decreases was statistically significant. In addition, the ventilatory effects of perfusion of the vehicle alone into the RTN, compared with the no perfusion or presurgical values, were not significant. Hence both the vehicle (aCSF) and inactive analog citrate had minimal effects on ventilation during normocapnia. The small increase in breathing frequency that we observed during citrate perfusion may have been due to increased neuronal excitability of cells exposed to this compound. Previous studies have shown that citrate is selectively sequestered, produced, and released by astrocytes (39, 41). Citrate can chelate extracellular zinc and other normally occurring divalent ions, resulting in enhanced N-methyl-D-aspartate receptor activity and increased neuronal firing (40, 42). Thus the addition of exogenous citrate may have resulted in increased chemoreceptor activity, leading to a slight increase in ventilation. In contrast, the application of fluorocitrate has little effect on biophysical properties of neurons and no known effect on glutamate receptor activity (5, 15, 17, 18).

Summary. Our data suggest that glia in the RTN can dramatically affect the ventilatory responses arising from this chemosensitive site. The increase in ventilation was likely due to a decrease in pHo associated with impaired glial function; an observation that our laboratory has previously reported (11). We hypothesize that glia may regulate pHo, possibly through the activation of Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransport, to maintain a stable milieu surrounding central chemoreceptors. Our data support the findings of others (2, 19) that the RTN is an important site in ventilatory control during wakefulness and anesthesia. Together, these findings raise the possibility that the contribution that a given chemoreceptor site may have on shaping central ventilatory control may critically depend on arousal state.


    ACKNOWLEDGEMENTS

We thank Donald Bartlett, James C. Leiter, and Gene Nattie for thoughtful review of this manuscript and Aihua Li for technical assistance.


    FOOTNOTES

This work was supported by National Science Foundation Grant IBN-98-10809 (to J. S. Erlichman), the Phelps Trust (to M. Babbie), the Alden Trust (to J. Holleran), and a grant from Merck-American Association for the Advancement of Science (to M. Babbie).

Address for reprint requests and other correspondence: J. S. Erlichman, Dept. of Biology, St. Lawrence Univ., Canton, NY 13617 (E-mail: jerlichman{at}stlawu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 August 2000; accepted in final form 22 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adelman, WJ, and Fitzhugh R. Solutions of the Hodgkin-Huxley equations modifies for potassium in periaxonal spaces. Fed Proc 34: 1322-1329, 1975[Web of Science][Medline].

2.   Akilesh, M, Kamper M, Li A, and Nattie EE. Effects of unilateral lesions of the retrotrapezoid nucleus on breathing in awake rats. J Appl Physiol 83: 469-479, 1997[Web of Science].

3.   Aoki, C, Milner TA, Sheau KFR, Blass JP, and Pickel VM. Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate transmission. J Neurosci 7: 2214-2231, 1987[Abstract].

4.   Bartlett, D, Jr, and Tenney SM. Control of breathing in experimental anemia. Respir Physiol 10: 384-395, 1970[Web of Science][Medline].

5.   Berg-Johnson, J, Paulsen RE, Fronnum F, and Langmoen IA. Changes in evoked potentials and amino acid content during fluorocitrate action studied in rat hippocampal cortex. Exp Brain Res 96: 241-246, 1993[Web of Science][Medline].

6.   Coates, EL, Li A, and Nattie EE. Widespread sites of brain stem ventilatory chemoreceptors. J Appl Physiol 75: 5-14, 1993[Abstract/Free Full Text].

7.   Connelly, CA, Ellenberger HH, and Feldman JL. Respiratory activity in the retrotrapezoid nucleus in the cat. Am J Physiol Lung Cell Mol Physiol 258: L33-L44, 1990[Abstract/Free Full Text].

8.   Deitmer, JW. Evidence of glial control of extracellular pH in the leech central nervous system. Glia 5: 43-47, 1992[Web of Science][Medline].

9.   Deitmer, JW, and Rose CR. pH regulation and proton signaling by glial cells. Prog Neurobiol 48: 73-103, 1996[Web of Science][Medline].

10.   Erlichman, JS, and Leiter JC. Comparative aspects of central CO2 chemoreception. Respir Physiol 110: 177-185, 1997[Web of Science][Medline].

11.   Erlichman, JS, Li A, and Nattie EE. Ventilatory effects of glial dysfunction in a rat brain stem chemoreceptor region. J Appl Physiol 85: 1599-1604, 1998[Abstract/Free Full Text].

12.   Forster, HV, Ohtake PJ, Pan LG, Lowry TF, Korducki MJ, Aaron EA, and Forster AL. Effects of breathing of ventrolateral medullary cooling in awake goats. J Appl Physiol 78: 258-265, 1995[Abstract/Free Full Text].

13.   Hassel, B, Sonnewald U, Unsgard G, and Fonnum F. NMR spectroscopy of cultured astrocytes: effects of glutamine and the gliotoxin fluorocitrate. J Neurochem 62: 2187-2194, 1994[Web of Science][Medline].

14.   Heinemann, U, and Lux HD. Ceiling of stimulus induced rises in extracellular potassium concentration in the cerebral cortex of the cat. Brain Res 120: 231-249, 1977[Web of Science][Medline].

15.   Keyser, DO, and Pellmar TC. Synaptic transmission in the hippocampus: critical role for glial cells. Glia 10: 237-243, 1994[Web of Science][Medline].

16.   Lai, YL, Tsuya Y, and Hildebrandt J. Ventilatory responses to acute CO2 exposure in the rat. J Appl Physiol 45: 611-618, 1978[Free Full Text].

17.   Largo, C, Cuevas P, Somjen GG, Martin del Rio R, and Herreras O. The effect of depressing glial function in rat brain in situ on ion homeostasis, synaptic transmission, and neuron survival. J Neurosci 16: 1219-1229, 1996[Abstract/Free Full Text].

18.   Largo, C, Ibarz JM, and Herreras O. Effects of fluorocitrate on spreading depression and glial membrane potential in rat brain in situ. J Neurophysiol 78: 295-307, 1997[Abstract/Free Full Text].

19.   Li, A, and Nattie EE. CO2 microdialysis in the retrotrapezoid nucleus of the rat increases breathing in wakefulness but not sleep. J Appl Physiol 87: 910-919, 1999[Abstract/Free Full Text].

20.   Li, A, and Nattie EE. Focal central chemoreceptor sensitivity in the retrotrapezoid nucleus studied with a CO2 diffusion pipette. J Appl Physiol 83: 420-428, 1997[Abstract/Free Full Text].

21.   Mortola, JP, and Gautier H. Interaction between metabolism and ventilation: effects of respiratory gases and temperature. In: Regulation of Breathing (2nd ed.), edited by Dempsey JA, and Pack AI.. New York: Dekker, 1994, p. 1011-1064.

22.   Nattie, EE. Central chemoreception. In: Regulation of Breathing (2nd ed.), edited by Dempsey JA, and Pack AI.. New York: Dekker, 1994, p. 473-501.

23.   Nattie, EE, Fung ML, Li A, and St. John W. Responses of respiratory modulated and tonic units in the retrotrapezoid nucleus to CO2. Respir Physiol 94: 35-50, 1993[Web of Science][Medline].

24.   Nattie, EE, and Li A. Retrotrapezoid nucleus lesions decrease phrenic activity and CO2 sensitivity in rats. Respir Physiol 97: 63-77, 1994[Web of Science][Medline].

25.   Nattie, EE, and Li A. Central chemoreception in the region of the ventral respiratory group of the rat. J Appl Physiol 81: 1987-1995, 1996[Abstract/Free Full Text].

26.   Ohtake, PJ, Forster HV, Pan LG, Lowry TF, Korducki MJ, Aaron EA, and Weiss EM. Ventilatory responses to cooling the ventral medullary surface of awake and anesthetized goats. J Appl Physiol 78: 247-257, 1995[Abstract/Free Full Text].

27.   Orkand, RK, and Opava S. Glial function in homeostasis of the neuronal microenvironment. News Physiol Sci 9: 265-267, 1994[Abstract/Free Full Text].

28.   Pappenheimer, JR. Sleep and respiration of rats during hypoxia. J Physiol (Lond) 266: 191-207, 1977[Abstract/Free Full Text].

28a.   Paxinos, G, and Watson C. The Rat Brain in Stereotaxic Coordinates (4th ed.). San Diego, CA: Academic, 1998, p. 59-64.

29.   Paulsen, RE, Constestabile A, Villani L, and Fronnum F. An in vivo model for studying function of brain tissue temporarily devoid of glial cell metabolism: the use of fluorocitrate. J Neurochem 48: 1377-1385, 1987[Web of Science][Medline].

30.   Pearce, RA, Stornetta RL, and Guyenet P. Retrotrapezoid nucleus in the rat. Neurosci Lett 101: 138-142, 1998.

31.   Ransom, B. Gap junctions. In: Neuroglia, edited by Kettenmann H, and Ransom BR.. New York: Oxford University Press, 1995, p. 299-319.

32.   Ransom, B. Glial modulation of neural excitability mediated by extracellular pH: a hypothesis. Prog Brain Res 94: 37-46, 1992[Web of Science][Medline].

33.   Richerson, G. Responses to CO2 of neurons in the rostral ventral medulla in vitro. J Neurophysiol 73: 933-943, 1995[Abstract/Free Full Text].

34.   Ritucci, NA, Dean JB, and Putnam RW. Intracellular pH responses to hypercapnia in neurons from chemosensitive areas of the medulla. Am J Physiol Regulatory Integrative Comp Physiol 273: R433-R441, 1997[Abstract/Free Full Text].

35.   Romero, MF, and Boron WF. Electrogenic Na+/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters: cloning and physiology. Annu Rev Physiol 61: 699-723, 1999[Web of Science][Medline].

36.   Saiki, C, and Mortola JP. Effect of CO2 on the metabolic and ventilatory responses to ambient temperatures in conscious adult and newborn rats. J Physiol (Lond) 491: 261-269, 1996[Abstract/Free Full Text].

37.   Schousboe, A, Westergaard N, Waagepetersen HS, Larsson OM, Baaken IJ, and Sonnewald U. Trafficking between glia and neurons of TCA cycle intermediates and related metabolites. Glia 21: 99-105, 1997[Web of Science][Medline].

38.   Smith, JC, Morison DF, Ellenberger HH, Otto MR, and Feldman JL. Brain stem projections to the major respiratory neuron populations in the medulla of the cat. J Comp Neurol 281: 69-96, 1989[Web of Science][Medline].

39.   Vernadakis, A. Glia-neuron intercommunications and synaptic plasticity. Prog Neurobiol 49: 185-214, 1996[Web of Science][Medline].

40.   Westbrook, GL, and Mayer ML. Micromolar concentrations of Zn2+ antagonize NMDA and GABA responses of hippocampal neurons. Nature 328: 640-643, 1987[Medline].

41.   Westergaard, N, Sonnewald U, Unsgard G, Peng L, Hertz L, and Schousboe A. Uptake, release and metabolism of citrate in neurons and astrocytes in primary cultures. J Neurochem 62: 1727-1733, 1994[Web of Science][Medline].

42.   Westergaard, N, Banke T, Wahl P, Sonnewald U, and Schousboe A. Citrate modulates the regulation of Zn2+ of NMDA receptor mediated channel current neurotransmitter release. Proc Natl Acad Sci USA 92: 3367-3370, 1995[Abstract/Free Full Text].

J APPL PHYSIOL 90(4):1539-1547
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Neurosci.Home page
J. S. Erlichman, A. Hewitt, T. L. Damon, M. Hart, J. Kurascz, A. Li, and J. C. Leiter
Inhibition of Monocarboxylate Transporter 2 in the Retrotrapezoid Nucleus in Rats: A Test of the Astrocyte-Neuron Lactate-Shuttle Hypothesis
J. Neurosci., May 7, 2008; 28(19): 4888 - 4896.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
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]

Home page
 Am. J. Physiol. Cell Physiol.Home page
R. W. Putnam, J. A. Filosa, and N. A. Ritucci
Cellular mechanisms involved in CO2 and acid signaling in chemosensitive neurons
Am J Physiol Cell Physiol, December 1, 2004; 287(6): C1493 - C1526.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
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]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
J. S. Erlichman, A. Cook, M. C. Schwab, T. W. Budd, and J. C. Leiter
Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2004; 286(2): R289 - R302.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Holleran, J.
Right arrow Articles by Erlichman, J. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Holleran, J.
Right arrow Articles by Erlichman, J. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online