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J Appl Physiol 94: 1204-1212, 2003. First published November 15, 2002; doi:10.1152/japplphysiol.00558.2002
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Vol. 94, Issue 3, 1204-1212, March 2003

Central CO2 chemoreception in developing bullfrogs: anomalous response to acetazolamide

Barbara E. Taylor1, Michael B. Harris1, E. Lee Coates2, Matthew J. Gdovin3, and J. C. Leiter1

1 Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001; 2 Department of Biology, Allegheny College, Meadville, Pennsylvania 16335; and 3 Biology Department, University of Texas at San Antonio, San Antonio, Texas 78249


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Central CO2 chemoreception and the role of carbonic anhydrase were assessed in brain stems from Rana catesbeiana tadpoles and frogs. Buccal and lung rhythms were recorded from cranial nerve VII and spinal nerve II during normocapnia and hypercapnia before and after treatment with 25 µM acetazolamide. The lung response to acetazolamide mimicked the hypercapnic response in early-stage and midstage metamorphic tadpoles and frogs. In late-stage tadpoles, acetazolamide actually inhibited hypercapnic responses. Acetazolamide and hypercapnia decreased the buccal frequency but had no effect on the buccal duty cycle. Carbonic anhydrase activity was present in the brain stem in every developmental stage. Thus more frequent lung ventilation and concomitantly less frequent buccal ventilation comprised the hypercapnic response, but the response to acetazolamide was not consistent during metamorphosis. Therefore, acetazolamide is not a useful tool for central CO2 chemoreceptor studies in this species. The reversal of the effect of acetazolamide in late-stage metamorphosis may reflect reorganization of central chemosensory processes during the final transition from aquatic to aerial respiration.

isolated brain stem preparation; carbonic anhydrase; acetazolamide; hypercapnia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CENTRAL RESPIRATORY CHEMORECEPTOR neurons are sensors for CO2 and/or pH within the brain, and they are essential for respiratory acid-base regulation in air-breathing animals. Evidence for the occurrence of central respiratory chemoreceptors exists for many vertebrates: air-breathing fish (29, 42), amphibians (2, 3, 32), reptiles (17), crocodilians (4), birds (24), and mammals (26). In response to even slight increases in PCO2, populations of CO2 chemosensory neurons in the medulla and pons stimulate ventilation in an effort to return PCO2 to normal levels. The ontogeny of central CO2 chemoreception has been challenging to elucidate in mammals due to the internal development of the fetus. Amphibians develop as free-living organisms, and they provide an appealing alternative vertebrate model in which to study the developmental progression of CO2 chemoreception. Many amphibians also develop from water-breathing larval forms into air-breathing adults, which requires plasticity in form and function of the respiratory system. As a tadpole metamorphoses into a frog, a transition occurs in the primary mode of O2 exchange from cutaneous and gill ventilation in younger tadpoles to pulmonary respiration in older tadpoles and frogs (6). The ontogenetic changes in respiratory mode are associated with the emergence of CO2 as the major source of respiratory drive for lung ventilation (31). During the transition from aquatic to aerial respiration, the blood pH decreases, whereas the bicarbonate (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) concentration and PCO2 increase (19). In the face of these changes, one might anticipate alterations in the form and function of central respiratory CO2 chemoreceptors and the central pattern generators sustaining ventilation. In a comparison of fictive gill and lung ventilation under conditions of normocapnia and hypercapnia among selected developmental stages, Torgerson et al. (37) found that CO2 ventilatory drive switched from increasing gill frequency to increasing lung frequency. This is consistent with the replacement of gills by lungs as the primary O2-uptake organs. Torgerson et al. suggested that respiratory chemosensitivity emerges as a feature of the rostral ventral medulla during development (35); however, their investigation focused on a few early and late developmental stages. Therefore, ontogenetic plasticity in central respiratory CO2 chemoreception warrants further investigation.

Carbonic anhydrase, which catalyzes the reversible hydration of CO2 to H+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, is required for the normal function of various peripheral CO2 chemoreceptors in mammals in the carotid body (15) and the larynx (8) and in intrapulmonary receptors in birds (30). Carbonic anhydrase activity has also been implicated in the function of the central CO2 chemoreceptors of mammals (9, 10) and pulmonate snails (12). Acetazolamide is a membrane-permeable carbonic anhydrase inhibitor, and focal injections of acetazolamide have been used to define regions of chemosensitivity within the mammalian central nervous system. Application of acetazolamide generates a localized region of tissue acidosis akin to respiratory acidosis (27) and stimulates ventilation. Thus acetazolamide may provide a useful pharmacological tool to identify regions of the brain that may possess chemosensory activity, and the responsiveness to acetazolamide may give insight into the chemosensory mechanism, i.e., whether carbonic anhydrase participates in the chemosensory process.

We investigated the response of fictive gill and lung ventilation to hypercapnia and acetazolamide throughout development. It was our hypothesis that acetazolamide would stimulate fictive lung ventilation, and the potency of acetazolamide would increase as metamorphosis progressed and central CO2 chemoreception came to provide greater ventilatory drive. These expectations were not fully corroborated: hypercapnia consistently stimulated fictive lung ventilation in all developmental stages, but acetazolamide, although it mimicked the hypercapnic response in most tadpoles, actually decreased fictive lung ventilation in late metamorphic tadpoles.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies were performed on 32 Rana catesbeiana tadpoles and frogs of either sex purchased from a commercial supplier (Sullivan, Nashville, TN). Tadpoles were assigned to one of four groups: early-stage (forelimbs absent, hindlimbs paddlelike without joints or separated toes), midstage (forelimbs absent, hindlimbs developing joints and toes), and late-stage (forelimbs developing, tail being resorbed) tadpoles and juvenile frogs. These groupings are based primarily on anatomic changes. The division between mid- and late-stage tadpoles was based on the physiological changes in gas-exchange mode and blood PCO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> buffering; pulmonary gas exchange increases (5), and the PCO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations rise dramatically at about stage XVIII (19). For those familiar with the commonly used classification scheme of Taylor and Kollros (34), early stage corresponds with TK I-X, midstage with TK XI-XVII, and late stage with TK XVIII-XXV. All animals were maintained at 25°C in aquaria containing dechlorinated water and were fed tropical fish food (tadpoles) or crickets (frogs). The institutional animal resource committee approved the research and animal use protocols, and the experimental protocols conformed to local and national standards of ethics.

Surgical preparation. Each animal was anesthetized by immersion for 1-2 min in a cold (4°C) 0.2 mM solution of tricaine methanesulfonate (MS222; Sigma Chemical, St. Louis, MO) in dechlorinated water buffered with NaHCO3 to pH 7.4. Under a dissecting microscope, the dorsal cranium was removed, the forebrain rostral to the optic lobes was resected, and the fourth ventricle was exposed by removing the choroid plexus. The brain stem and spinal cord were removed en bloc from the cranium and spinal canal, the dura mater was stripped, and the brain was transected rostral to the optic tectum and caudal to the brachial nerve. During dissection, exposed tissues were superfused with cold (4°C) artificial cerebrospinal fluid (aCSF) composed of (in mM) 104 NaCl, 4 KCl, 1.4 MgCl2, 10 D-glucose, 25 NaHCO3, and 2.4 CaCl2 equilibrated with 100% O2. The aCSF HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration is similar to that of frog plasma but higher than the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration in tadpole plasma. However, to ensure comparability between experiments on animals of different stages, the aCSF composition was kept consistent in all experiments.

The isolated brain stem was transferred to a low-volume (0.5 ml) flow-through recording chamber (36, 37). Each isolated brain stem was supported ventral side up between coarse nylon mesh such that all surfaces were bathed with aCSF flowing from rostral to caudal regions at a rate of 5 ml/min. A supply of aCSF, equilibrated with O2-CO2 mixtures to produce the desired pH, flowed through glass tubing to the chamber. After dissection, the brain stem was allowed to stabilize while superfused with aCSF at 25°C, pH 7.8, and ~9 Torr PCO2 for 1 h. During experiments, the pH of the aCSF was maintained at either pH 7.4 or 7.8 by adjusting the fractional concentrations of O2 and CO2 in the equilibration gas. CO2 was monitored with a carbon dioxide analyzer (Capstar-100, CWE, Ardmore, PA).

Nerve recording. The seventh cranial nerve (CNVII) and the second spinal nerve (SNII) both innervate respiratory muscles. Cranial nerve VII innervates buccal levators (14), and SNII innervates the hypoglossal motoneurons, which innervate both buccal levator and buccal depressor muscles (21). Both nerves produce fictive respiratory activity (14). The nerve roots of CNVII and SNII were drawn into glass suction electrodes pulled from 1-mm-diameter capillary glass to tip diameters of 30-60 µm. Whole-nerve discharge was amplified (100× by DAM-50 amplifier, World Precision Instruments, Sarasota, FL; 1,000× by model 1700 polarographic amplifier, A-M Systems, Carlsborg, WA) and filtered (1-Hz low-pass and 500-Hz high-pass by amplifier) and fed to an integrator with a time constant of 50 ms (MA-821/RSP, CWE). Both raw (amplified) and integrated signals were digitized at 200 Hz per channel, and these neurograms were archived as computer files (DATAPAC, RUN Technologies, Mission Viejo, CA) for subsequent analyses.

Protocol. Baseline nerve activity under normocapnia was first established by superfusing the brain stem with aCSF at pH 7.8 (equilibrated with 1.5% CO2-98.5% O2). A pH of 7.8 is comparable to the arterial pH in intact frogs and tadpoles at room temperature. The ventilatory response to CO2 was assessed by increasing the CO2 concentration of the aCSF superfusate to hypercapnic conditions (aCSF equilibrated with 4.7% CO2-95.3% O2; pH 7.4). Responses to acetazolamide, a carbonic anhydrase inhibitor, were assessed in preparations that exhibited an easily recognized hypercapnic response, defined by an increase in fictive lung ventilatory bursts in response to increased CO2. Acetazolamide (Sigma Chemical) was added to the aCSF at a final concentration of 25 µM. This dose of acetazolamide is ~250 times the inhibition constant of carbonic anhydrase, and, at this dose, 99.99% of the carbonic anhydrase activity should be inhibited. No nonspecific effects of acetazolamide have been reported at this dose (22).

Histological staining for carbonic anhydrase activity. The activity of carbonic anhydrase was detected using a cobalt-phosphate histochemical method (11, 16). Eleven brain stems were isolated from representative individuals of the four developmental groups according to the protocol described above (Surgical preparation). Tissues were fixed for 3 h at 4°C in aCSF containing 4% glutaraldehyde, washed for 1 h at 4°C in aCSF, and frozen in OCT compound (Fischer, Pittsburgh, PA). We analyzed carbonic anhydrase activity in three locations of the medulla: a rostral region at the level of fifth cranial nerve, a caudal region at the level of SNII, and a region midway between the rostral and caudal regions at the level of 9th and 10th cranial nerves. Within each region, we analyzed carbonic anhydrase staining throughout a 100-µm-thick cross section of the medulla. The brain stems were sliced in a cryostat into 20-µm sections, and cross sections of the brain stem were placed on slides that were processed in batches so that sections from all regions and all stages were exposed to identical staining conditions. The sections were incubated in a solution composed of (in mM) 2.90 CoSO4, 17.6 KH2PO4, 156 NaHCO3, and 15.9 H2SO4. A group of slices from similar anatomic areas of the same brain stems was immersed for 3-5 s in an incubation solution that also contained 100 µM acetazolamide; this provided a control for nonspecific staining. The difference in inhibition of carbonic anhydrase between the 25 µM dose used in the isolated brain stem and the 100 µM dose used to stain the tissue is negligible. The slides were removed from the incubation solution, and any remaining incubation solution was suctioned away. The slices were placed in a stream of compressed air for 30 s to dry the tissue. This procedure of immersing and drying the slices was performed four to six times, resulting in a total incubation period of ~10 min. The slices were rinsed in distilled water, immersed in 0.5% ammonium sulfide, and rinsed again. The slides were dehydrated in alcohol, rinsed in xyline, and mounted with a coverslip. Intensity of staining was scored on a scale from zero to four, with zero being no staining and four being maximal staining. The slides were reviewed independently by two investigators, each of whom was ignorant of the metamorphic stage, section within the medulla, and histological treatment of each slide. When scores from the two reviewers were divergent, both investigators, working together, arrived at a consensus score.

Data analysis. Neurograms of fictive ventilation were scored as buccal or lung ventilatory bursts (14, 36). Burst activity patterns were designated as either buccal or lung based on the amplitude of the integrated neurogram and the presence or absence of coincident firing in both CNVII and SNII. Buccal bursts were those of lesser amplitude in CNVII without coincident firing in SNII, whereas lung bursts were of greater amplitude in CNVII with coincident firing in SNII. Each burst event was demarcated with an onset and offset, and events were counted and divided by the time period of measurement to generate frequency data (in bursts per minute). The duty cycle was computed as the average elapsed period (in seconds) between the onset and offset of each burst. These quantifications of buccal and lung ventilation were made over 2-10 consecutive min of recorded fictive ventilation. The information derived from frequency and duty cycle is not equivalent. The duty cycle reflects the intrinsic rhythm of the pattern generator, but the frequency reflects the interaction between pattern generators. For example, when lung frequency increases, buccal frequency must necessarily decrease because the lung breaths now occupy a greater part of every minute, but the duty cycles of lung and buccal pattern generators need not change. In adult frogs, the buccal rhythm may be absent or imperceptible at times (20), and, in the juvenile frogs, the buccal rhythm did occasionally wax and wane. However, disappearance of the buccal rhythm was infrequent and not associated with any particular treatment. Therefore, the buccal and lung rhythms were calculated in the juvenile frogs from data segments only when both rhythms were present. We did not see systematic variation in the amplitude of the buccal or lung bursts over the pH ranges we studied; therefore, we relied solely on the frequency and duty cycle of buccal and lung bursts to quantify the response to treatments.

Statistical analyses. The data on fictive buccal and lung ventilation in tadpoles and frogs were analyzed with a three-way repeated-measures ANOVA in which developmental stage was a between-subject factor and the CO2 level (normocapnia vs. hypercapnia) and acetazolamide (present vs. absent) were within-subject factors. When normalized ventilatory responses within each metamorphic stage were analyzed, each value was expressed as a percentage of the control condition (normocapnia or no acetazolamide present), and normalized values were compared as a function of stage using a one-way ANOVA. When the ANOVA indicated that significant differences existed between the treatment groups, multiple preplanned comparisons were made by using t-values adjusted by the Bonferroni method, and post hoc comparisons were made with Scheffé's method. Values reported in the text represent means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The neuroventilatory responses to normocapnia and hypercapnia are illustrated by the representative neurograms in Fig. 1, which were recorded from the brain stem of a late-stage tadpole. Neural recordings were made from CNVII and SNII. Measurements of fictive ventilatory frequency and duty cycle were made from CNVII neurograms because both buccal and lung bursts were always evident. SNII neurograms always demonstrated lung bursts coincident with those on CNVII, but buccal bursts were frequently absent. Both recorded nerves innervate the buccal musculature, which provides the main ventilatory pump for both buccal (gill) and lung ventilation. As seen in Fig. 1, lung bursts were easily distinguished from buccal bursts by virtue of their greater amplitude.


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  		Fig. 1.   Neuroventilatory response to normocapnia (A) and hypercapnia (B) generated by the isolated brain stem of a late-stage tadpole. Each data panel depicts 30 s of data [a "raw" and integrated (int) signal] from the seventh cranial nerve (CNVII) and the second spinal nerve (SNII). A: response of each nerve to perfusion with normocapnic artificial cerebrospinal fluid (aCSF; pH 7.8 created by equilibrating aCSF with 98.5% O2-1.5% CO2). A steady buccal bursting pattern interrupted by 2 large-amplitude lung bursts is shown. B: response to hypercapnic stimulation with aCSF at pH 7.4 (created by equilibrating aCSF with 95.3% O2-4.7% CO2). Note the marked increase in the number of lung bursts.

Duty cycle of respiratory bursting in response to CO2 and acetazolamide. The average duty cycle of fictive buccal and lung ventilation are presented in Table 1 as a function of stage, CO2 level, and acetazolamide treatment. When the buccal duty cycle was analyzed across all stages, there was a significant linear decline in the duty cycle as metamorphosis progressed (P = 0.02). For example, buccal duty cycle in frogs (0.7 ± 0.2 s) was significantly shorter than in midstage tadpoles (1.1 ± 0.2 s) and early-stage tadpoles (1.1 ± 0.3 s). However, the differences in buccal duty cycle were not statistically significant in the younger metamorphic groups. In addition, treatment with acetazolamide consistently prolonged the buccal duty cycle (P = 0.042) when the data from all metamorphic stages and CO2 concentrations were pooled. However, there were no significant differences in the response to CO2 or acetazolamide within any metamorphic grouping. In contrast, lung duty cycles were consistent and invariant among all metamorphic stages and acetazolamide and CO2 treatments.

                              
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  		Table 1.   Buccal and lung duty cycle responses

Lung frequency responses to CO2 and acetazolamide. The average frequencies of fictive lung ventilation are presented in Fig. 2 as a function of stage, CO2 level, and acetazolamide treatment. Lung burst frequency increased as the tadpoles progressed through metamorphosis (P < 0.001). Hypercapnia consistently increased the lung burst frequency (P < 0.001) when examined across all metamorphic stages and acetazolamide treatment conditions. Also, lung burst frequency during both normocapnia and hypercapnia increased significantly after acetazolamide treatment (P < 0.001). The pattern of response to hypercapnia was not modified by acetazolamide treatment; rather, the effects of the two treatments were additive. Consistent with this observation, the three-way ANOVA indicated that there was no dependence of the CO2 response profile on the presence or absence of acetazolamide or on metamorphic stage.


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  		Fig. 2.   Neuroventilatory responses of lung burst frequency during normocapnia (pH 7.8), hypercapnia (pH 7.4), and with and without 25 µM acetazolamide added to the normocapnic and hypercapnic aCSF. Each line represents the change in mean lung burst frequency ± SD for 4-6 tadpoles or frogs, when CO2 was increased during treatment with (open circle ) and without () 25 µM acetazolamide. * Significant response to hypercapnic treatment within a metamorphic group (P < 0.05). ** Significant response to acetazolamide treatment within a metamorphic group (P < 0.05). §Difference between lung burst frequency with and without acetazolamide present was significantly reduced in the late-metamorphic group compared with all other stages (P < 0.005).

The overall effect of hypercapnia was to increase fictive lung ventilation. The absolute increase in lung burst frequency after hypercapnia did not reach statistical significance in the early-stage and midstage tadpoles. However, fictive lung ventilation increased significantly in late-stage tadpoles (from 8.4 ± 9.3 to 25.8 ± 7.7 lung bursts/min; P < 0.001) and in juvenile frogs (from 12.4 ± 11.1 to 22.5 ± 16.4 lung bursts/min; P < 0.01). Furthermore, the fictive lung burst rate increased over the course of metamorphosis, and lung ventilation under hypercapnia was significantly greater in frogs than in late-stage tadpoles (P < 0.05) and significantly greater in late-stage tadpoles than in early-stage and midstage tadpoles (P < 0.001), although these two youngest stages were statistically indistinguishable. Despite the lack of statistically significant changes in absolute lung frequencies at the earliest stages of metamorphosis, the relative change in lung burst frequency caused by hypercapnia, expressed as a function of the baseline normocapnic activity in each state, was remarkably consistent across all stages. The average increase in lung ventilation frequency was 315 ± 207% across all stages during hypercapnia relative to the normocapnic condition (P < 0.001), and there were no significant differences in the relative change in lung bursting among stages (P > 0.5). Thus, when hypercapnic responses were compared within each metamorphic grouping, a hypercapnic response was apparent in all stages. Even in the early-stage tadpoles, lung frequency increased during hypercapnia in five of five animals, although the absolute changes in frequency were small compared with the changes in lung ventilation in later stages.

The average frequency of fictive lung ventilation as a function of metamorphic stage and acetazolamide treatment is also presented in Fig. 2. The most interesting aspect of the neuroventilatory response to acetazolamide is that, whereas acetazolamide increased lung burst frequency in early-stage and midstage tadpoles and frogs, the effect was opposite in late-stage tadpoles. For example, the frequency of fictive lung ventilation averaged over both pH-CO2 conditions decreased from 17.1 ± 5.1 to 12.1 ± 3.6 bursts/min in response to acetazolamide in late-stage tadpoles (P < 0.01), whereas in frogs it increased from 17.5 ± 6.6 to 30.6 ± 11.5 bursts/min (P < 0.001). The increase in lung burst frequency after acetazolamide treatment was not statistically significant in the early-stage and midstage tadpoles. The situation is similar to the hypercapnic response in that the relative increase in lung ventilation frequency expressed as a function of the baseline normocapnic activity at each stage was actually slightly greater in the early-stage and midstage tadpoles compared with the frogs. We analyzed the change in lung frequency between control and acetazolamide treatment conditions as a function of metamorphic stage, and the decline in lung burst frequency during late metamorphosis was significantly different from the slight to moderate increase in lung burst frequency in all other stages (P < 0.005 for each of the three comparisons with Scheffé's method to adjust P values for post hoc comparisons). Thus acetazolamide treatment increased the lung ventilation frequency in early-stage and midstage tadpoles and frogs but significantly decreased the lung ventilation frequency in the late-metamorphic tadpoles. These data indicate a marked ontogenetic change in either central CO2 chemoreceptor function or in the action of the lung central rhythm generator.

Buccal frequency responses to CO2 and acetazolamide. The average frequencies of fictive buccal ventilation are presented in Fig. 3 as a function of metamorphic stage, CO2 level, and acetazolamide treatment. Buccal frequency was not affected by metamorphic stage. Across all stages, both hypercapnia and acetazolamide consistently reduced the buccal ventilation rate (P = 0.002 and 0.024, respectively), but the effects of the two treatments were simply additive. The average reduction in buccal frequency was ~4 bursts/min after acetazolamide treatment or hypercapnic acidosis (pH 7.4), and, when the treatments were combined, the buccal frequency dropped by ~9 bursts/min. There were no interactions between hypercapnia, acetazolamide, or metamorphic stage. In summary, acetazolamide and hypercapnia decreased fictive buccal frequency, but this may reflect the dependence of buccal frequency on lung frequency, since the intrinsic buccal duty cycle was unaffected by either treatment. The buccal duty cycle did, however, diminish as the tadpoles completed metamorphosis and entered adulthood.


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  		Fig. 3.   Neuroventilatory responses of buccal burst frequency during normocapnia (pH 7.8, 1.5% CO2), hypercapnia (pH 7.4, 4.7% CO2), and with and without 25 µM acetazolamide added to the normocapnic and hypercapnic aCSF. Each line represents the change in mean lung burst frequency ± SD for 4-6 tadpoles or frogs, when CO2 was increased during treatment with (open circle ) and without () 25 µM acetazolamide.

Carbonic anhydrase activity in different metamorphic groups. Hansson staining for the activity of carbonic anhydrase was performed on 350 cross sections of the brain stem from four developmental stages, and the results are summarized in Fig. 4. In Fig. 4a, the ventrolateral segment of a medullary cross section is shown, and individual neurons stained for carbonic anhydrase can be seen. Carbonic anhydrase activity was evident throughout the medulla in all developmental stages (Fig. 4c), this despite the fact that acetazolamide affected metamorphic stages differently. In some animals, the staining was particularly dense in the circumferential ependymal layers along the rostrocaudal extent of the medulla (Fig. 4b; shaded areas located dorsally and ventrally). However, the circumferential ependymal staining did not seem to vary systematically among metamorphic groups, and it was not present consistently in every brain stem in any particular group. Sections from the rostral region of the medulla of late-stage tadpoles and frogs, which may contain emergent CO2 chemosensory regions (35), did not differ with respect to the intensity of carbonic anhydrase activity compared with the presumably nonchemosensory caudal medullary cross sections of those stages, nor were they different from the medullary cross sections of earlier stages. Finally, when 100 µM acetazolamide was included in the incubation medium, we found no staining in any section. Thus the staining we detected was specific for carbonic anhydrase activity.


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  		Fig. 4.   Hansson staining for carbonic anhydrase activity in brain stem slices from frogs and 3 metamorphic stages. The photograph (a) shows the typical pattern of carbonic anhydrase activity at the ventral edge of the medulla, with evenly dispersed staining within individual neurons moving toward the brain stem core. The cross-sectional schematic of the medulla (b) shows the sites of carbonic anhydrase activity as they would be situated anywhere along the medulla in any developmental stage. The table (c) summarizes the Hansson staining scores for 350 slices from the 4 developmental groups.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study of fictive ventilation in the brain stem of developing bullfrogs yielded four important findings. First, there is an ontogenetic increase in lung burst frequency, which is to be expected because the lungs gain importance over the course of development as organs of oxygen uptake. Second, tadpoles of the earliest stages increased fictive lung ventilation in response to increased PCO2, and this response remained consistent throughout ontogeny. Third, the response to acetazolamide was paradoxical in view of the previous trend. In early-stage and midstage tadpoles and frogs, fictive lung ventilation increased in response to both acetazolamide and hypercapnia, and the effects of combined treatment were additive. However, fictive lung ventilation decreased in response to acetazolamide treatment despite an increased lung burst frequency during hypercapnia in late-stage tadpoles. Finally, despite the anomalous response to acetazolamide in late-stage metamorphosis, carbonic anhydrase activity was similarly distributed in the brain stem throughout bullfrog development.

Ontogenetic changes in respiratory pattern. Many ontogenetic changes in anatomy and physiology occur during bullfrog metamorphosis, and these changes facilitate the animal's transition from the aquatic to the aerial environment. Early-stage tadpoles exhibit primarily gill breathing interspersed with sporadic lung breathing, and the frequency of lung breaths gradually increases until adulthood when the frog ventilates its lungs in episodes demarcated by a few buccal pumps that ventilate only the buccal cavity (31, 41). The neural correlates of lung ventilation in the isolated tadpole brain stem parallel this gradual ontogenetic increase as demonstrated by Torgerson et al. (38) and this study. We found that the intrinsic frequency of the buccal pattern generator also increased (shorter duty cycle) over the course of development. An increase in lung frequency would usually come at a price of decreased buccal frequency, and we saw this trend in that buccal frequency decreased during hypercapnia and acetazolamide treatment as lung frequency increased. However, this trend was opposed by the effect of metamorphosis on the duty cycle of buccal ventilation, which tended to increase buccal frequency because each buccal burst was shorter. Thus the buccal frequency in the control condition was more stable over the course of metamorphosis than it would otherwise have been given the reciprocal relationship between lung frequency and buccal frequency.

Neuroventilatory responses to hypercapnia. A hypercapnic response, manifest as increased frequency of lung ventilation, is well established for air-breathing lower vertebrates: turtles (17), toads (2, 32), lungfish (29), and gar (42). CO2-pH sensitivity of isolated brain stems has been shown for mammals (25, 39) and turtles (18) as well as bullfrog tadpoles (37) and adult frogs (20). Reported here is further evidence that the isolated bullfrog brain stem is capable of generating a fictive hypercapnic response in all stages of metamorphosis, and this response is predictable and reliable throughout development. The lung burst frequency and the magnitude of the frequency response to CO2 that we found in all stages were similar to results reported by others only in later metamorphic stages (37). When we expressed lung frequency during hypercapnia (pH 7.4) as a function of the baseline lung frequency (pH 7.8), even the early-stage tadpoles demonstrated a threefold increase in lung frequency. Thus the metamorphic stage at which hypercapnic responses in lung ventilation can be detected was earlier in our study than that previously reported (37). This implies that the same CO2 chemosensory neurons are present in the earliest stages of metamorphosis, even though the animal has a purely aquatic existence.

It is difficult to characterize the buccal component of the hypercapnic ventilatory response. The frequency of gill activity increases during environmental hypercapnia in water-breathing fish, but the mechanisms of this change have not been shown to rely on central CO2 chemosensory mechanisms (23). Earlier investigators reached contradictory conclusions regarding the effect of hypercapnia on fictive buccal ventilation of the tadpole brain stem. Walker et al. (40) demonstrated no change in buccal frequency when the superfusate pH was changed from 7.8 to 7.2, whereas Torgerson et al. (38) reported that buccal burst frequency increased during hypercapnia. However, Torgerson et al. normalized the buccal frequency response to the value at pH 8.0, although the normal arterial pH of frogs and tadpoles is closer to 7.8 and the CSF and intracellular pH are likely to be lower still. There was no difference in buccal burst frequency between pH 7.8 and 7.4 (the range we tested). Thus, to date, the majority of evidence suggests that buccal ventilatory rate is unchanged by hypercapnia. Our results are at odds with the foregoing studies; buccal frequency decreased slightly during hypercapnia and acetazolamide treatment. Part of the decline in buccal frequency occurred as a result of the increase in lung frequency associated with these treatment conditions. It may be significant that the frequency of lung bursts rose more during hypercapnia in our study than in previous studies (37), and therefore the decline in buccal frequency was likely to be greater.

Perhaps more important in terms of chemosensory mechanisms, neither the lung nor the buccal duty cycle was affected by hypercapnic stimulation. Thus we have no evidence that the mechanisms controlling the lung and buccal pattern generators are sensitive to CO2. Hypercapnia seems to increase the occurrence of lung breaths (at the expense of buccal breaths) without altering the temporal pattern of each individual breath.

Effect of acetazolamide on neuroventilatory output. In our attempt to use acetazolamide as a pharmacological means of activating central CO2 chemosensory responses, we identified a remarkable developmental change in central chemosensitivity of bullfrogs. Acetazolamide induces a tissue acidosis akin to respiratory acidosis and has been used to explore chemosensory function in mammalian preparations (26). We believe that a similar acidosis was generated in the bullfrog brain stem, and this was the means by which acetazolamide evoked increased fictive lung ventilation in the present study. Bath application of the drug is a limitation of this study in so far as it leaves some ambiguity as to the site of action of the drug. However, the staining for carbonic anhydrase activity indicated the presence of abundant carbonic anhydrase activity in the brain stem at all metamorphic stages examined and particularly prominent staining of the ventral medullary surface in the older metamorphic groupings, which may provide evidence that central chemoreceptors are located in this area.

It was our assumption that acetazolamide decreased the pH at any given PCO2 level and increased the putative central CO2 chemoreceptor stimulus. The increase in lung burst frequency in the early-stage and midstage tadpoles and juvenile frogs is consistent with this response; application of acetazolamide increased the lung frequency in every animal tested in these stages, and a stimulatory effect of hypercapnia alone was also present. However, the most noteworthy aspect of the present study is that the acetazolamide-induced increase in fictive lung ventilation was absent in the late-stage tadpoles, despite maintenance of responsiveness to hypercapnia that was indistinguishable from the other metamorphic stages. In fact, acetazolamide depressed the normocapnic and hypercapnic lung ventilation frequency at this metamorphic stage. Hypercapnia and acidosis inhibit many neurons and excite others (33). Previous studies of the ventilatory effects of hypercapnia noted its dual excitatory and inhibitory influences (7, 28). The ultimate response to hypercapnia and acid stimuli will depend on the balance of excitatory and inhibitory influences. It is our hypothesis that the greater acidosis during combined hypercapnia and acetazolamide treatment in the late-stage tadpole was associated with greater inhibition of neural activity and reduced lung bursting in the late-stage tadpoles. The excitatory effect of hypercapnia alone on central CO2 chemoreceptors was still apparent in this metamorphic stage, but inhibition, perhaps of nonchemosensory elements in the rhythm-generating apparatus, dominated the lung-bursting response to combined treatment with CO2 and acetazolamide. Thus we believe that the effect of acetazolamide on tissue pH was similar across metamorphic stages, but the respiratory response to the acidosis induced by acetazolamide differed in the late-stage tadpoles. One limitation of our study is that we did not measure the interstitial or intracellular pH associated with hypercapnia and acetazolamide treatment in the different metamorphic stages. However, the direction of pH changes we are suggesting is consistent with previous measurements during similar treatments, and there is no reason to believe that the pattern of pH changes associated with each treatment should have varied among the different metamorphic stages studied.

The paradoxical response to acetazolamide in late-stage tadpoles is especially intriguing because carbonic anhydrase activity was consistently present in the medulla of the bullfrog throughout metamorphosis, and the distribution of staining, within the limits of our ability to detect it, was not different among the stages. It is possible that subtle differences in the staining of specific neurons went undetected; however, evenly dispersed neuronal staining was a consistent finding across development. Although intriguing in itself, the fact that different stages have contradictory responses to acetazolamide negates the usefulness of this drug as a tool for investigating central CO2 chemosensitivity in the bullfrog.

Perspective. The transition from an aquatic to an aerial environment in late metamorphosis is associated with a variety of changes in sensory systems. For example, tadpoles appear to pass through a period of transient "deafness" as the neural structures of the auditory system are modified to match changes in the primary organs that sense acoustic pressure changes (1). A similar reorganization of the circuitry of the visual system has also been described during amphibian metamorphosis (13). Just as the physical natures of visual and acoustic signals change in aquatic and aerial environments, the stimulus for breathing changes from a system dominated by hypoxic ventilatory drive in aquatic animals to one dominated by hypercapnic ventilatory drive in air-breathing animals. It is our hypothesis that the metamorphic reorganization of the neural structures serving ventilation, in which central CO2 chemosensitivity emerges as a much more important ventilatory drive, is associated with a period in which the excitatory effects of acidosis are attenuated or less effectively communicated to the lung pattern generator such that the more profound acidosis associated with acetazolamide treatment caused inhibition, rather than stimulation, of lung burst activity.

In summary, the present study offers the following insights into the ontogeny of central chemoreception and its modulation of ventilatory pattern in the bullfrog. First, the neural correlate of lung burst frequency increases during development despite the fact that the gills cease to exist in fully developed animals. Second, a consistent hypercapnic response can be generated by the brain stem of any stage of tadpole or frog. Finally, there is a transient inhibitory response to acetazolamide among the late-stage tadpoles, although the excitatory response to hypercapnia persists, and carbonic anhydrase activity is present throughout the medulla in all developmental stages. The anomalous response to acetazolamide occurred at a time in metamorphosis when other sensory systems are reorganized in preparation for an aerial existence, and the dramatic change in the response to acetazolamide may reflect similar neuronal reorganization and plasticity of the respiratory control system during which central CO2 chemosensory inputs are reintegrated into a respiratory control system adapted for air breathing.


    ACKNOWLEDGEMENTS

This research was funded by Special Neuroscience Research Project Grant 39409 from the National Institute for Neurological Disease and Stroke.


    FOOTNOTES

Original submission in response to a special call for papers on "Plasticity in Respiratory Motor Control."

Address for reprint requests and other correspondence: B. E. Taylor, Dept. of Physiology, Borwell Bldg., 1 Medical Center Drive, Lebanon, NH 03756-0001 (E-mail address: barbara.e.taylor{at}dartmouth.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.

First published November 15, 2002;10.1152/japplphysiol.00558.2002

Received 26 June 2002; accepted in final form 12 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 94(3):1204-1212
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