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Am J Physiol Regul Integr Comp Physiol 280: R913-R920, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 4, R913-R920, April 2001

Sites of respiratory rhythmogenesis during development in the tadpole

C. S. Torgerson1, M. J. Gdovin2, and J. E. Remmers1

1 Respiratory Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1; and 2 Division of Life Sciences, University of Texas at San Antonio, San Antonio, Texas 78249


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During ontogeny, amphibian larvae experience a dramatic alteration in the motor act of breathing as the premetamorphic gill breather develops into the postmetamorphic lung ventilator. We tested the hypothesis that the site of lung rhythmogenesis relocates during metamorphosis by recording fictive lung ventilation before and after transecting the in vitro brain stem of pre- and postmetamorphic Rana catesbeiana into four segments. In premetamorphic tadpoles, the two caudalmost brain stem segments combined proved to be the minimum brain stem configuration necessary and sufficient for lung burst generation. In the postmetamorphic counterpart, this function was supplied by the combination of the two rostralmost brain stem segments. In the postmetamorphic brain stem, a 500-µm segment lying just rostral to cranial nerve IX conveys rhythmogenic capability to neighboring rostral or caudal segments. We conclude that lung rhythmogenic capability translocates rostrally during development as the tadpole shifts from gill to lung ventilation.

central pattern generator; control of breathing; central respiratory chemoreceptor; medulla; ontogeny


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INSIGHT INTO THE NEURAL MECHANISMS controlling amphibian respiration has been facilitated by the development of in vitro tadpole brain stem preparations (5, 26, 27, 29). Correlative studies indicate the two patterns of rhythmic bursting activity recorded from the cranial nerve (CN) roots V, VII, and X and the spinal nerve (SN) root II constitute fictive gill and lung ventilation; the pattern and profile of these bursts resemble those during gill and lung ventilation in the spontaneously breathing, decerebrate tadpole (6, 18). Ontogenic changes in respiratory motor output of the in vitro tadpole brain stem have been previously described: the premetamorphic tadpole brain stem displays predominantly fictive gill ventilation, whereas brain stems from postmetamorphic animals predominantly generate fictive lung bursts (28). Both types of fictive breathing are stimulated by central chemoreceptive input (27).

Whereas both gill and lung oscillators lie within the tadpole brain stem, the exact location and distribution of neurons responsible for the generation of gill and lung motor output at different stages of development are unknown. Early studies in the adult frog by Langendorff (10) demonstrated that breathing continued after medullary transections at the level of CN V and the caudal root of the CN X. Schmidt (22) reported persistence of rhythmic movements of the glottis in the adult frog after transections at CN VIII and the rostral border of SN II. More recently, McLean et al. (12) demonstrated in the isolated adult frog brain stem that injection of excitatory and inhibitory amino acids between CN VIII and CN X influenced both the frequency and amplitude of lung burst activity. Together, these studies suggest that neurons generating lung ventilatory activity in the adult frog lie between CN VIII and CN X.

The goal of the present study was to identify transverse segments of the brain stem critical for lung rhythmogenesis in the developing tadpole. We hypothesize that, as in the neonatal and adult rat and cat (19, 21, 24), the tadpole brain stem contains neural elements in the region of the motor nucleus of CN IX that are necessary for lung ventilatory rhythmogenesis.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General. Larval bullfrog tadpoles (Rana catesbeiana) obtained from a commercial supplier (Charles D. Sullivan, Nashville, TN) were used in all experiments. Animals were housed in aerated, filtered aquariums (19-21°C) and fed fish meal (TetraWerke). Animals were assigned to one of two groups based on the criteria of Taylor and Köllros (25): pre (stages 9-14, n = 14)- and postmetamorphic (stages 24-25, n = 13). The experimental protocol used in these studies was approved by the Animal Care Committee of the University of Calgary, Canada.

Surgical preparation. Tadpoles were anesthetized in tricaine methane sulfonate (1:10,000) and, once unresponsive to tail pinch, decerebrated by transection just rostral to the eyes. This was followed by a caudal transection of the body: just caudal to the level of the opercular slit in premetamorphic animals and just behind the forelimbs in postmetamorphic larvae. A dorsal craniotomy and laminectomy at the first and second spinal segments exposed the brain stem and rostral spinal cord. Throughout the remaining surgical procedures, the brain stem was superfused with artificial cerebrospinal fluid (aCSF) of the following composition (in mM): 104 NaCl, 4 KCl, 1.4 MgCl2, 10 D-glucose, 25 NaHCO3, and 2.4 CaCl2. The superfusate was equilibrated with 98% O2 and 2% CO2 and had a pH of 7.8, corresponding to the plasma pH of larval amphibians (3, 8). With the aid of a dissecting microscope, CNs V, VII, IX, and X and SN II (hypoglossal in the adult) were severed at their ostia, and the dura and arachnoid surrounding the brain stem were carefully removed. After surgery, the brain stem remained loosely attached to the ventral cranium by the unsevered cranial and spinal nerves.

Superfusion-recording chamber. The partially isolated brain stem and ventral cranium were transferred to a superfusion-recording chamber (volume 5 ml) at room temperature. The ventral cranium was pinned to the Sylgard-coated base (Dow Corning) with the dorsal surface of the brain stem upward. During the experiments, the brain stem was superfused with control aCSF (PCO2 = 2.3 kPa; pH 7.8; balance O2) or hypercapnic aCSF (PCO2 = 6.0 kPa; pH 7.4; balance O2). Each of these superfusates was equilibrated in a tonometer at 21-23°C. Superfusate was delivered at a rate of 10 ml/min through an inflow stylus, with the tip positioned near the rostral end of the brain stem, and conducted from the opposite end of the chamber via a paper wick. This partial isolation allowed the ventral cranium to serve as a supportive bed during transection while still allowing superfusate to flow between the ventral surface of the brain and the cranium.

Transection procedures. The brain stem was sectioned serially in the transverse plane (perpendicular to the axis of the brain stem) using a 4-mm segment of a fine-edged razor blade (Feather-S-Blade, Ted Pella). This length approximates the lateral-lateral dimension of the brain stem. The blade was attached to a vertical motorized positioner (Nanostepper, Type B, Scientific Precision Instruments, Oppenheim, Germany) with a resolution of <1 µm. The positioner was mounted on a two-dimensional slide mechanism with the direction of one movement being parallel to the long axis of the brain stem and the other being 90° to this axis. Each slide was positioned by a manual micrometer (Newport, Fountain Valley, CA) with a resolution of 5 µm. The presence of the ventral cranium and intact nerves stabilized the brain stem during sectioning, thereby minimizing displacement and compression. Contact of the blade with the surface of the tissue was determined visually with the aid of a dissecting microscope (×313 magnification). The blade was continuously advanced through the tissue until complete transection was achieved (a process lasting ~2 s).

Figure 1 shows a sagittal projection of the frog brain stem adapted from Senn (23), which depicts the location of transection sites. The initial percutaneous transection of the head and body resulted in brain stem transections x and y, respectively, and created a pretectally decerebrate brain extending from a region just rostral to the optic tectum to 1 mm caudal to SN II. This pretectal brain stem preparation provided baseline data, after which three sequential rostral-to-caudal transections were performed on each brain stem: 1) transection A, located at the rostral margin of the cerebellar bar; 2) transection B, positioned 500 µm rostral to the rostral root of CN IX, or transection C, immediately juxtaposed to the rostral root of CN IX; and 3) transection D, 1 mm caudal to transection C. Our rationale for the position of the transection levels was threefold. First, each brain stem segment (with the exception of segment 2) possessed a nerve root that contained respiratory motor efferents. Second, each transection was topographically reproducible based on consistent external morphology. Third, thinner sections were technically difficult as a result of marked brain stem pliability.


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  		Fig. 1.   Schematic illustration of the tadpole brain stem preparation showing transection sites and resulting segments. Sagittal projection of the tadpole brain stem with serial transection sites x, A, B, C, D, and y creating brain stem segments 0, 1, 2, 3, and 4. Cer., cerebellum; coll. post., colliculus posterior; isth, ganglion isthmi; VIII do, dorsal VIIIth nucleus; VIII ve, ventral VIIIth nucleus; VIII a, nucleus vestibularis anterior; CN, cerebellar nucleus; se, sensory; mo, motor; N. SN, spinal nerves.

As shown in Fig. 1, transection A isolated the brain stem from the tectum and created the tectal segment 0. Transections B or C and D produced four "elemental" brain stem segments (segments 1-4). When two or more elemental segments were contiguous, i.e., before isolation by transection, they formed a "compound" segment. A compound segment was denoted by appropriate segment identification number connected by a dash, e.g., compound segment 2-3-4 for the caudal brain stem after transection B but before transection D. Three of the elemental segments had CN roots for efferent recording, vis, segment 1: CN VII; segment 3: CN X; and segment 4: SN II. Segment 2 was a virtual segment that was not surgically isolated, because no external nerve was available to monitor respiratory activity from this region. In other words, transections B and C were never performed in the same brain stem so that segment 2 was either attached to compound segment 3-4, forming compound segment 2-3-4 after transection B, or attached to segment 1, forming compound segment 1-2 after transection C. The influence of segment 2 on rhythmogenesis was inferred by comparing the behavior of segment 2-3-4 with segment 3-4 and the behavior of segment 1-2 with segment 1.

To precisely locate transections B-D, the dorsal root of CN IX was used as a reference. The blade was positioned at this reference level for transection C. When transection B, rather than transection C, was required, the blade was first positioned at the reference level and then moved 500 µm rostral. To perform transection D, the blade was first positioned at the reference level and then moved 1,000 µm caudal. The elemental segments exhibited similar external morphology and landmarks in brain stems in pre- and postmetamorphic animals. This constancy of segment morphology, independent of development, resulted from two factors. The first derived from the use of the cerebellar bar and CN IX reference levels, and the second stemmed from the relative consistency of the overall rostral-caudal length of the brain stem and distance between adjacent nerve roots in pre- versus postmetamorphic tadpoles.

Recordings. Action potentials were recorded simultaneously from the roots of CNs VII and X and SN II using suction electrodes. The pipettes were constructed from thin-walled borosilicate glass tubing (1 mm OD) pulled to a fine tip with a horizontal micropipette puller (Brown-Flaming, model P80). The tip was broken and beveled (Stähli Läpp-Technik) to achieve various inner-tip diameters ranging from 90 to 350 µm. Action potentials were amplified (AM Systems no. 1700, Tektronix AM 502), filtered (100 Hz to 1 kHz), and recorded on videotape using a pulse-code modulator (Neurodata no. 890). The signals were averaged with a modified Bessel filter (20), displayed on a polygraph (Gould), and digitized and analyzed on a Pentium personal computer using DataPac III software (Run Technologies).

The nerve roots were not intentionally removed from the suction electrode during the sectioning procedure. However, in some experiments, the nerve was inadvertently dislodged from the suction electrode during transection. This raised the possibility that the recorded activity may have been altered by such an event. To evaluate the importance of a possible artifact introduced by such a detachment/reattachment event, preliminary experiments were performed to determine the effect of 11 sequential electrode detachments and reattachments of the root of CN VII. These repeated manipulations indicated that the pattern of the rhythmic lung respiratory activity remained unchanged with reattachment.

Protocol. After transfer to the recording chamber, the pretectally decerebrated brain stem was superfused by control aCSF (pH 7.8) for 30-60 min until stable rhythmic bursting activity was observed from all three neurograms. Baseline respiratory motor output was then recorded for 10 min during normocapnic superfusion followed by a 10-min recording period during superfusion with hypercapnic aCSF. Between these 10-min recording periods, a 5-min equilibration period ensued to allow equalization of tonometer and recording chamber pH and stabilization of the fictive ventilatory response to the new superfusate pH. Three sequential rostral-to-caudal transections were then performed at A, B or C, and D. After each transection, a 30-min recovery period ensued to ensure stabilization of the fictive ventilation. After posttransection stabilization, efferent activity was recorded for 10 min at pH 7.8 and for 10 min at pH 7.4, with a 5-min intervening period between to ensure pH equilibration.

Analysis. In compound segments containing SN II roots, lung burst activity was identified using the SN II amplitude criterion defined by Torgerson et al. (28). Bursts in brain stem segments not containing SN II roots were identified as lung bursts if their amplitude, shape, and duration resembled those recorded under baseline conditions. The effect of transection on fictive gill ventilation was not analyzed, because clear identification of gill bursts was not always possible. For each of the two developmental groups, we analyzed the effects of hypercapnic superfusion on lung ventilatory motor output frequency from CNs VII and X and SN II after each transection. Within each animal, the means of lung burst frequency for all nerves were measured for each 10-min recording period (pH 7.8 and 7.4) after each transection level, and group means ± SE were calculated. The mean value of fictive lung frequency during hypercapnic superfusion (pH 7.4) for each animal was then expressed as the percent change from control superfusion (pH 7.8), and group means ± SE were calculated. The significance of pH effects both within and between developmental groups was examined at each transection level using a one-way ANOVA for repeated measures, with the criterion of statistical significance at P < 0.05. The same test was used to compare lung burst frequency as a function of transection level at each pH. A Student-Newman-Keuls test of pairwise multiple comparisons was used to test significant differences between individuals within treatment groups when statistically significant. Fisher's Exact test was used to compare the prevalence of brain stems displaying lung bursts between pre- and postmetamorphic populations at the same transection level.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adequate neurograms were successfully recorded from the roots of CNs VII and X and SN II in all 27 brain stem preparations. All pre (n = 14)- and postmetamorphic (n = 13) preparations generated robust lung burst activity when superfused with aCSF of pH 7.8 or 7.4 before serial transection. Accordingly, adequate data were derived from all three nerves after all transections. Table 1 provides mean values of lung burst frequency of all elemental and compound segments in pre- and postmetamorphic animals.

                              
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  		Table 1.   Lung burst frequency after sequential transection: response to superfusate pH and development

Premetamorphic segments. The neural substrate sufficient for lung burst generation in premetamorphic tadpoles was localized to compound segment 3-4, as indicated in Table 1. Figure 2 (left) illustrates typical recordings of lung burst activity in SN II during rostral-to-caudal sectioning at A, B, C, and D of premetamorphic brain stems. All compound segments containing segments 3 and 4 in continuity (i.e., segments 1-2-3-4, 2-3-4, and 3-4) showed lung burst activity. By contrast, segments not containing compound segment 3-4 (i.e., segments 1, 2, 1-2, 2-3, 3, or 4) never generated lung bursts. As shown in Table 1, lung burst frequency of compound segments 1-2-3-4, 2-3-4, and 3-4 did not differ significantly from the pretectally decerebrate brain stem (segment 0-1-2-3-4). Thus compound segment 3-4 was the most reduced segment of the premetamorphic brain stem that exhibited a lung rhythm, but neither segment 3 nor segment 4 alone was adequate for the production of lung motor output.


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  		Fig. 2.   Lung burst activity during serial brain stem transection in pre- and postmetamorphic tadpoles. Integrated respiratory motor output simultaneously recorded from cranial nerve (CN) VII, CN X, and SN II. The numbers on the depicted brain stem indicate segments, and the dashed lines demarcate the boundaries of segments 1, 2, 3, and 4. Transection of the brain stem is indicated by discontinuity in the depicted brain stem. In the premetamorphic brain stem, compound segment 3-4 is the most reduced segment generating a lung rhythm. In the postmetamorphic brain stem, compound segment 1-2 is the most reduced segment generating lung bursts.

Table 1 also shows the mean lung burst frequency of various premetamorphic brain stem segments during normocapnic (pH 7.8) and hypercapnic superfusion (pH 7.4). No significant differences in fictive lung ventilatory frequency were observed for any of the segments during the two superfusions, and none of the segments that were silent at pH 7.8 developed a lung rhythm when superfused with hypercapnic aCSF.

Postmetamorphic segments. As shown in Fig. 2 (right) and as indicated in Table 1, two compound segments derived from the postmetamorphic brain stem displayed a lung rhythm, namely, segments 1-2 and 2-3-4. Subdivisions of these compound segments (segments 1, 3, 4, 2-3, and 3-4) never produced a lung rhythm. Thus compound segment 1-2 constitutes the minimal segmental ensemble capable of generating a lung rhythm. Segment 2 appears to be a common element required for rhythmogenesis by compound segments 1-2 and 2-3-4. That segment 2-3 failed to generate a lung rhythm reveals that essential lung rhythmogenic influences may derive from segment 4. The frequency of lung bursts in segments 0-1-2-3-4, 1-2-3-4, and 2-3-4 did not differ, but that for segment 1-2 was significantly lower than the other three rhythmogenic preparations (Table 1), being <10% of the mean values for segments 1-2-3-4 and 2-3-4.

Interestingly, although lung burst frequency in segments 0-1-2-3-4, 1-2-3-4, and 2-3-4 did not differ, a trend toward increased lung burst frequency with the ablation of segment 0 is evident. Segment 0 corresponds to the region of the optic tectum in the tadpole that later develops into the pons. In the neonatal rat, the pons has been shown to display an inhibitory effect on respiratory frequency (15).

Compound segments 0-1-2-3-4, 1-2-3-4, and 1-2 of the postmetamorphic brain stem displayed a statistically significant increase (P < 0.05) in frequency during hypercapnic superfusion (Table 1). The only segment rhythmic during normocapnic superfusion that failed to show a significant response to hypercapnia was segment 2-3-4. All segments that were silent during normocapnic superfusion remained so during superfusion with hypercapnic aCSF.

Comparison of pre- and postmetamorphic segments. As demonstrated in Table 1, the frequency of lung motor output was dependent on developmental stage. Lung burst frequency (pH 7.4) was significantly greater (P < 0.05) in postmetamorphic tadpoles than in premetamorphic brain stems for compound segments 0-1-2-3-4, 1-2-3-4, and 2-3-4. By contrast, the percentage of segment 3-4 preparations displaying a lung rhythm was significantly greater (P < 0.001) for premetamorphic brain stems (100%, 8/8) compared with postmetamorphic animals (0%, 0/6). The percentage of segment 1-2 preparations displaying a lung rhythm was significantly greater (P < 0.001) for postmetamorphic brain stems (100%, 6/6) compared with premetamorphic animals (0%, 0/8).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of the present study suggest that during metamorphosis, the rostral brain stem replaces the caudal medulla in supplying the neural substrate essential for fictive lung rhythmogenesis with development. In premetamorphic larvae, compound segment 3-4 proved to be the minimum segmental ensemble capable of lung rhythmogenesis. By contrast, in the postmetamorphic animals, this compound segment produced no lung burst activity in normocapnic or hypercapnic aCSF. Instead, compound segment 1-2 proved to be the minimal segmental ensemble capable of generating a fictive lung rhythm. However, the fictive lung frequency of segment 1-2 was extremely low (~1/min), whereas that of segment 2-3-4 approximated that of the intact brain stem, indicating that segment 3-4 contributes an important frequency-enhancing action in the postmetamorphic brain stem. Although the intrinsic rhythmogenic capability of segment 2 could not be directly investigated, our results suggest that it plays an important role conveying lung rhythmicity on rostral (segment 1) and caudal (segment 3-4) segments. Results indicating that an isolated or compound segment fails to generate a rhythm do not exclude the possibility that the segment possesses rhythmogenic capability. For instance, a brain stem segment that fails to generate rhythmic motor activity may contain rhythmogenic components that have been separated from essential excitatory input from other brain stem segments.

Our strategy was to isolate segments of the brain stem and infer their contribution to fictive lung ventilation during normocapnia and hypercapnia. Because we positioned the transections with reference to external landmarks (cerebellar bar and dorsal root of CN IX) and because the axial dimensions of the brain stem change little during metamorphosis, the external landmarks of each segment were identical in pre- and postmetamorphic preparations. Unfortunately, the internal anatomy of brain stem structures in the tadpole has not been described, so knowledge of the underlying brain stem structures associated with various segments remains uncertain. With the use of the adult frog as a surrogate (Fig. 1), we suggest that segment 1 contains the nuclei of CNs V, VII, and VIII, segment 2 contains most of CN VII nuclei and rostral portions of CN IX nuclei, segment 3 contains nuclei of CNs VI, IX, and X, and segment 4 contains the hypoglossal motor nucleus (14).

We recognize the need to enhance synaptic input when evaluating the rhythmogenic capability of reduced segments. Our approach to achieving this was to elevate a normal, specific stimulus to the respiratory central pattern generator by using hypercapnia. Whereas other workers have chosen to use supranormal K+ concentration ([K+]) to mimic excitatory input in such experiments, the generalized depolarizing action of a hyperkalemic superfusate introduces the possibility of recruiting nonrespiratory activity.

Lung rhythm generation in premetamorphic tadpoles. The respiratory pattern in premetamorphic, pretectally decerebrate tadpoles consisted predominantly of gill burst activity interrupted occasionally by isolated lung bursts. This behavior resembles the respiratory patterns described for intact premetamorphic tadpoles (2, 7) and confirms previous results from isolated premetamorphic tadpole brain stems (27).

In the premetamorphic brain stem, structures in rostral segments did not influence the frequency of lung breaths generated by the more caudal rhythm generator. Lung burst activity was contingent on the continuity of segments 3 and 4, and this was true even when segment 2 was connected to segment 3. Compound segment 3-4 of premetamorphic tadpoles lies caudal to the location of lung rhythmogenic regions previously reported in the adult frog (10, 12, 22), suggesting that rhythmogenic mechanisms relocate from caudal to rostral brain stem regions with development. Hypercapnia evoked no lung ventilatory response in any of the segments of the premetamorphic brain stem. This finding agrees with previous studies showing that the premetamorphic brain stem lacks a fictive lung ventilatory response to global hypercapnic superfusion (27).

Lung rhythm generation in postmetamorphic tadpoles. Segment 3-4 from postmetamorphic preparations failed to generate fictive lung ventilation. Although we can't exclude the possibility that this caudal brain stem segment possessed rhythmogenic capability, segment 3-4 was capable of generating a rhythm in the premetamorphic brain stem. These results, therefore, suggest that the lung central pattern generator function, initially present in this segment, may have been lost during metamorphosis. In contrast, compound segment 1-2 proved to be the minimal segmental configuration capable of generating a lung rhythm. However, because compound segment 2-3-4 displayed lung rhythmogenesis, we infer that segment 2 of the postmetamorphic brain stem plays an important role, conveying rhythmic capability to segments 3-4 and 1. One possibly important finding is that segment 2-3 did not generate a lung rhythm, which may indicate that segment 2 contains neural substrate that is necessary but not sufficient for lung rhythmogenesis. Another important finding is that the lung rhythm of segment 2-3-4 was high (12.1/min) and that of segment 1-2 was quite low (0.93/min), a difference that points out a major frequency-promoting action of the caudal brain stem.

Segment 2 corresponds to the area between CNs VIII and X in the adult frog brain stem, a region determined to be important for lung burst generation (10, 12, 22). This region also appears to be analogous to the location of the pre-Bötzinger complex in the mammal, just caudal to the glossopharyngeal motor nucleus (see Fig. 1). The pre-Bötzinger complex has been shown to be required for respiratory rhythmogenesis in the isolated brain stem of the neonatal rat and anesthetized adult cat (19, 21, 24). However, within segment 2 of the tadpole brain stem, the precise location and distribution of rhythm-generating neurons needs to be established both rostrocaudally and dorsoventrally.

Whether or not the rostral repositioning of rhythmogenic function with metamorphosis represents a developmental movement of rhythmogenic neurons or a recruitment of new neurons into a rhythmic circuit is unknown. Early developmental studies by Senn (23) demonstrated that differentiation and formation of nerve centers in the growing medulla and spinal cord markedly depend on cellular migration.

The frequency of lung bursts generated in segments 0-1-2-3-4, 1-2-3-4, and 2-3-4 of postmetamorphic tadpoles was significantly greater (P < 0.05) than that produced in corresponding premetamorphic segments. Unlike premetamorphic tadpoles, the frequency of lung bursts generated by compound segments 0-1-2-3-4, 1-2-3-4, and 1-2 of postmetamorphic tadpoles increased in response to pH reduction, a response that is consistent with previous results (27). Such differences agree with previous results in the isolated brain stem preparation (27) and in the intact, freely swimming animal (2, 7, 30). Our results were obtained while superfusing with normocapnic aCSF with normal electrolyte composition. This contrasts with experiments reporting respiratory rhythm generation in the isolated brain stem segments of preparations of the neonatal rat and adult mouse where rhythm generation requires use of a bathing medium having high [K+] to increase neuronal excitability (16, 21, 24).

In the postmetamorphic brain stem, segment 1 appears to play an important role in global chemoreceptive function, because compound segment 1-2-3-4 displayed a response to the hypercapnia, whereas compound segment 2-3-4 did not. Segment 1 may contain chemosensory elements or it may be the location of a circuitous neural pathway originating in chemoreceptors in segment 2-3-4 and traveling through segment 1 to ultimately synapse on target neurons in segment 2. The results reported in the companion paper, showing that chemoreceptive sites are located in regions of the rostral brain stem corresponding to segment 1, support the former possibility (29).

Combining the present results with those of the companion paper reveal an impressive correspondence of rhythmogenic and chemoreceptive regions, indicating that neurons responsible for both functions may be colocalized during amphibian development. These ontogenic findings provide a new type of evidence supporting the notion advanced by Nattie and co-workers that chemoreceptive elements lie in proximity to respiratory neurons (1, 4, 11, 13). Kawai et al. (9) and Oyamada et al. (17) suggested that respiratory neurons are intrinsically chemoreceptive. In the isolated brain stem of the neonatal rat, these investigations provided evidence that "synaptically isolated" respiratory neurons in the ventral respiratory group and locus ceruleus depolarize in response to acid challenge. We conclude that during metamorphosis, the rostral brain stem of the tadpole assumes an essential role in the generation of lung ventilation as it becomes the dominant site for respiratory chemoreception.

Perspectives

The identification of segment 2 as an important area necessary for a lung ventilatory rhythm in postmetamorphic tadpoles fits with recent microinjection studies in similar stage larvae, identifying the presence of a lung oscillator in this region (31). In both studies, the possible homology between the amphibian lung-rhythm generator region and the mammalian pre-Bötzinger is noteworthy. Hence, our results provide suggestive evidence that the pre-Bötzinger region may, indeed, be conserved phylogenetically as the neural substrate for air breathing in bimodal breathers. Furthermore, it establishes the need to conduct more detailed neuroanatomical investigations in this area.


    ACKNOWLEDGEMENTS

This work was supported by the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: J. E. Remmers, Faculty of Medicine, Univ. of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 (E-mail: jeremmer{at}ucalgary.ca).

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 20 April 2000; accepted in final form 3 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bernard, DG, Li A, and Nattie EE. Evidence for central chemoreception in the midline raphe. J Appl Physiol 80: 108-115, 1996[Abstract/Free Full Text].

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6.   Gdovin, MJ, Torgerson CS, and Remmers JE. Neurorespiratory pattern of gill and lung ventilation in the decerebrate spontaneously breathing tadpole. Respir Physiol 113: 135-146, 1998[ISI][Medline].

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Am J Physiol Regul Integr Comp Physiol 280(4):R913-R920
0363-6119/01 $5.00 Copyright © 2001 the American Physiological Society


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