INTRODUCTION

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CENTRAL RESPIRATORY CHEMOSENSITIVITY has been clearly established in amphibians (for reviews, see Refs. 22, 30, and 31). In adult frogs (14-16, 20, 21) and in toads (2, 32), central respiratory chemoreceptors (CRCs) have been shown to regulate lung ventilation. A preliminary report of studies of intact tadpoles by Infantino (9) indicates that larval maturation is accompanied by the emergence of CO₂ chemoreception as a source of respiratory drive for both gill and lung ventilation. Torgerson et al. (35) showed that central chemoreceptors in the tadpole brain stem mediate gill and lung ventilatory output in response to hypercapnia and demonstrate that chemoreceptive influence is transferred from gill to lung regulation. However, the location of CRCs and their transductive mechanisms remain to be established.

In mammals, CRCs have been traditionally described as being located in the superficial layers of the ventral surface of the medulla (19, 23). More recent studies, however, indicate that CRCs are distributed widely within the mammalian brain stem, nested in locations that generally correspond to areas known to have concentrations of respiratory neurons (1, 5, 18, 24, 25). In vivo injection of acetazolimide (AZ) beneath the ventral medullary surface, in the locus ceruleus, nucleus tractus solitarius, caudal medullary raphe, and ventral respiratory group stimulated respiratory output in cats and rats (1, 5, 24). Whereas focal tissue acidosis by AZ injection may be useful in identifying CRC regions, examination of chemoreceptive mechanisms and sensitivity are limited because AZ-induced tissue pH changes are prolonged and of fixed intensity. In contrast, Issa and Remmers (10) and Li and Nattie (18) demonstrated respiratory chemoreceptive areas below the ventral medullary surface in proximity to the location of the ventral respiratory group neurons and in the region of the retrotrapezoid nucleus using hypercapnic artificial cerebrospinal fluid (aCSF). Nevertheless, because the tissue pH in the region of focal acidification was not measured, the CRC fields could not be precisely delineated.

We demonstrate in a companion paper that as ventilatory function shifts from gill to lung, the location of regions essential for lung rhythmogenesis switches from the caudal to the rostral brain stem (37). Assuming that loci of central respiratory chemosensitivity lie in proximity to regions having high density of respiratory neurons and assuming that regions essential for lung rhythmogenesis have a high density of respiratory neurons, it is plausible to hypothesize that sites of chemosensitivity relocate rostrally in the tadpole brain stem during metamorphosis. The aim of the present study was to examine this possibility by localizing regions of the brain stem of pre- and postmetamorphic tadpoles that transduce local changes in PCO₂ and evoke fictive lung ventilation. To describe the distribution of central respiratory chemoreceptive sites, the caudal or the rostral brain stem was systematically and comprehensively explored by microinjecting CO₂-enriched aCSF while monitoring the response of fictive lung ventilation.

	METHODS

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General. Experiments were performed on 24 larval bullfrog tadpoles (Rana catesbeiana) of either sex obtained from a commercial supplier (Charles D. Sullivan, Nashville, TN). Specimens were assigned to one of two developmental groups based on the criteria of Taylor and Köllros (33): premetamorphic (stages 9-12, n = 12,) and postmetamorphic (stages 23-25, n = 12). The pre- and postmetamorphic groups were further subdivided into one group of six for evaluation at the rostral location and one group of six for evaluation at the caudal location for each stage group. During the 5 days preceding experimentation, all tadpoles were housed in aerated, filtered aquariums (19-21°C) and fed fish meal (TetraWerke). The Animal Care Committee of the University of Calgary approved the experimental protocol used in these studies.

Surgical preparation. Tadpoles were anesthetized in tricaine methane sulphonate (1:10,000). Once unresponsive to tail pinch, the dorsal cranium was removed and, with the aid of a dissecting microscope, the cranial and spinal nerves were severed at their ostia. After the dura and arachnoid were removed dorsally and ventrally, the brain stem was transected 1 mm caudal to the root of the second spinal nerve (SN II) and 1 mm rostral to the root of cranial nerve (CN) V. Throughout the dissection, the brain stem was superfused with aCSF having the following composition (in mM): 104 NaCl, 4 KCl, 1.4 MgCl₂, 10 D-glucose, 25 NaHCO₃, and 2.4 CaCl₂. The aCSF was equilibrated with 98% O₂ and 2% CO₂ and had a pH of 7.8, corresponding to the plasma pH of larval amphibians (3, 12).

Recording chamber. The brain stem was transferred to an experimental chamber (volume 0.5 ml) that was at room temperature (21-23°C) and has been described previously (35). Briefly, two superimposed circular discs (diameter 2.5 cm, thickness 1.5 mm) with central elliptical holes (2.0 × 0.5 cm) partitioned the chamber into upper and lower compartments. Fine netting that spanned the holes was attached to the upper surface of the upper and lower discs, thereby creating a brain stem containment reticulum bounded by the upper and lower nets. The brain stem was suspended between the two nets, ventral side up, and was superfused with aCSF equilibrated with 2% CO₂ and 98% O₂ in a tonometer at room temperature. The superfusate was delivered into the chamber through an inflow aperture in the lower compartment (flow rate, 10 ml/min) and was conducted from the upper compartment at the opposite end of the chamber via a paper wick. Thus the superfusate flowed uniformly over the ventral and dorsal surfaces of the suspended brain stem.

Recording. Efferent recordings were obtained from the roots of CN VII and SN II (hypoglossal in the adult) using suction electrodes. The pipettes were fashioned 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 (28), displayed on a polygraph (Gould), and digitized and analyzed on a Pentium personal computer using appropriate software.

CO₂-injection pipette. Four-barrel borosilicate glass tubing [1 mm OD, 0.75 mm ID, Scientific Precision Instruments (SPI), Oppenheim, Germany] was pulled to a fine tip using a vertical pipette puller (Narishige), fractured to produce an outer-tip diameter of 30 µm, and forged. Because the pipette frequently became obstructed during penetration of the brain, two pairs of barrels were filled with identical solutions as follows: hypercapnic aCSF equilibrated with 100% CO₂ (pH 6.0) and control aCSF equilibrated with a PCO₂ of 2.3 kPa (pH 7.8). Each barrel was separately connected to a four-channel picosprizter (General Valve, model 2) for pressure ejection. Short-duration pressure pulses (range 5-300 ms, 125 lbs./in.²) were delivered to individual barrels of the pipette. The volume ejected, calculated from the observed movement of the meniscus, was viewed through a horizontally positioned microscope (Zeiss Opmi-1, ×31.25 magnification) equipped with a fine reticule (maximal resolution, 5 nl).

Characterization of tissue acidification. Preliminary experiments were carried out to ascertain the minimum volume of hypercapnic aCSF required to produce global responses in respiratory motor output. Pilot mapping studies demonstrated that pressure injection of 5.5-11 nl aCSF (0.5-1.0 reticule divisions) equilibrated with 100% CO₂ in the region of CN X often evoked lung burst activity both in CN VII and SN II. However, because injection of 11 nl of hypercapnic aCSF more consistently elicited changes in global respiratory motor output, this volume was used throughout all experiments. To evaluate the reproducibility of the ventilatory response to injection of hypercapnic aCSF, 11 nl of hypercapnic aCSF were repeatedly injected below the ventral brain stem surface at the same site at 2-min intervals while respiratory activity was recorded from CN VII and SN II. These tests revealed that repeated hypercapnic injections at the same site reproducibly elicited lung burst activity with similar amplitude and shape.

Protocol. Once positioned in the recording chamber, the brain stem was superfused with oxygenated aCSF for at least 60 min before recording to allow stabilization. Action potentials were recorded from CN VII and SN II roots. The isolated tadpole brain stem was systematically explored using a three-dimensional 500 × 500 × 500-µm grid, i.e., injection sites were separated by 500 µm in each dimension. This spacing corresponds to the L-L diameter of the tissue-acidification region. With the use of the basilar artery to demarcate the midline and using CN/spinal nerve roots as rostral-caudal markers, eight medial and eight lateral penetration sites were defined, each 500 µm apart, ranging from 500 µm rostral to the level of CN V to 500 µm caudal to the level of SN II (Fig. 4). Thus we define eight transverse segments, each 500 µm thick, delimited by eight paired medial-lateral injection sites. Each transverse segment could be divided into three strata, 500, 1,000, and 1,500 µm below the surface, corresponding to the electrode depth at the time of injection.

Analysis. An injection of aCSF or control solution was considered to produce a positive response if a burst occurred within 2 s after the injection. The use of a brief time-discrimination window (i.e., 2 s after injection) minimized the chance that a positive chemoreceptive response might be the result of the random occurrence of lung burst.

	RESULTS

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Figure 1 demonstrates the spatial-temporal distribution of pH after injection of hypercapnic aCSF, 500 µm below the ventral surface. Figure 1A shows the mean tissue pH change (pH) for five animals as a function of time after injection of 11 nl of hypercapnic aCSF. Tissue pH at 0, 100, 200, and 300 µm lateral to the site of injection and 500 µm below the surface fell to minimal levels 7-12 s after acidic microinjection and returned to baseline (dashed line) after 60 s. The lowest tissue pH was recorded at the site of injection (0 µm), and the magnitude of pH change was progressively smaller at 100 and 200 µm laterally. Little or no tissue pH change was observed 300 µm lateral to the site of injection. Ventral to the injection site, the pH produced by the injection was virtually constant as a function of distance. Mean pH values at 10 s were 0.29, 0.28, and 0.33 pH units at 100, 200, and 300 µm, respectively, ventral to the injection site.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Brain stem tissue pH change in the region of hypercapnic microinjection. Spatial-temporal pH distribution in isolated brain stem of a premetamorphic (stage 11) tadpole during 11-nl microinjection of CO2-enriched artificial cerebrospinal fluid (aCSF). Hypercapnic aCSF was injected 500 µm below the ventral brain stem surface at the level of the cranial nerve (CN) IX root. The dashed line indicates tissue pH, 500 µm below the ventral surface, before injection. A: tissue pH change as a function of time from injection measured at 0, 100, 200, and 300 µm from the microinjection site. B: tissue pH change as a function of distance from injection at 10, 30, and 60 s after microinjection. Plotted are means ± SE, n = 3.

Figure 1B plots mean pH as a function of distance lateral to the microinjection site at 10, 30, and 60 s after injection of hypercapnic aCSF. Ten seconds after hypercapnic aCSF injection, tissue pH at the site of injection decreased 0.23 ± 0.06 (from 7.34 to 7.10), and a steep spatial pH gradient was evident. This gradient was less steep at 30 s and was virtually absent 60 s after acid injection.

In all animals, two patterns of coordinated rhythmic activity were recorded from CN VII and SN II: high-frequency, low-amplitude bursts and low-frequency, high-amplitude bursts. On the basis of correlative studies of spontaneously breathing decerebrate tadpoles, we have previously demonstrated that these bursts represent fictive gill and lung ventilation, respectively (7). Distinction between gill and lung bursts is based on the appearance of a large burst in SN II (36).

Tissue pH was recorded at the site of injection together with an evoked fictive lung ventilatory response in a premetamorphic tadpole (Fig. 2). Injection of 11 nl hypercapnic aCSF produced a transient (60 s) decrease (pH 7.5-7.2) that reached a minimum in 7 s and elicited lung burst activity in CN VII and SN II 2 s after hypercapnic aCSF microinjection, well before minimum tissue pH was reached.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Stimulation of respiratory motor output during hypercapnic microinjection. Simultaneous recordings of CN VII and spinal nerve II (SN II) neurograms showing gill and lung ventilatory motor output and tissue pH from the isolated brain stem of a premetamorphic (stage 14) Rana catesbeiana tadpole superfused with aCSF (PCO2 2.3 kPa; pH 7.8) during 11-nl pressure injection of high-CO2 aCSF (pH 6.0; indicated by the arrow). Hypercapnic aCSF was injected 500 µm below the ventral brain stem surface at the level of CN IX root, and pH was recorded within 25 µm of the injection site. A fictive lung response is indicated by the large burst in SN II, 2 s after injection of hypercapnic aCSF.

Pre- and postmetamorphic brain stems displayed typical positive chemoreceptive responses (Fig. 3). As indicated by the filled arrow, hypercapnic aCSF (11 nl) was injected 500 µm below the ventral surface at the level of SN II in the premetamorphic preparation (Fig. 3A) and 500 µm rostral to the root of CN V in the postmetamorphic brain stem (Fig. 3B). In both cases, injection of 11 nl of hypercapnic aCSF (pH 6.0) evoked both gill and lung bursts in the premetamorphic brain stem (Fig. 3A) but only lung bursts from postmetamorphic brain stems in this time (Fig. 3B). Two minutes after injection of hypercapnic aCSF, control injections of aCSF into the same sites elicited no change in respiratory output.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Typical positive central respiratory chemoreceptor (CRC) responses to 11-nl injection of acidic (filled arrow) and control (open arrow) aCSF in pre (A)- and postmetamorphic (B) brain stem preparations. Ejection of hypercapnic aCSF evoked lung burst activity both in CN VII and SN II 500 µm below the ventral surface at the level of SN II in premetamorphic tadpoles and 500 µm below the ventral surface at a level 500 µm rostral to the CN V in postmetamorphic tadpoles. Ejection of control aCSF in these regions failed to change lung burst frequency in CN VII and SN II.

Comparison of responses to acid and control microinjections revealed that the number of instances when acid, but not control, injection evoked a lung burst far exceeded those when control, but not acid, injection evoked a lung response both for pre- and postmetamorphic preparations (P < 0.001). This indicates that stimulation of lung bursts was strongly related to acid provocation.

Figure 4 summarizes the results of 366 injection trails (each trial = acid + control aCSF injection) in the eight medial and eight lateral penetration sites of pre- and postmetamorphic tadpoles. In premetamorphic larvae, 31 of 126 injection trials produced positive responses, and 21 (68%) of these positive responses lay caudal to CN IX (i.e., in the 4 caudal transverse levels). In postmetamorphic brain stems, 103 of 240 injection trials produced positive chemoreceptive responses, and 81 (79%) of these positive responses were located in areas rostral to CN IX (i.e., in the rostral 4 transverse levels).

View larger version (97K): [in this window] [in a new window]   Fig. 4.   Location of positive and negative CRC responses in pre- and postmetamorphic tadpoles. Topographic ventral surface markings in pre- and postmetamorphic tadpole brain stems showing the 16 medial and lateral penetration sites (large open circles) and the distribution of positive () and negative () chemoreceptive responses. Within each penetration site, the top strata show the number of chemoreceptive responses 500 µm below the ventral surface, the middle 1,000 µm below, and the bottom 1,500 µm below. The black bar indicates that no microinjection occurred. The lines between the 2 brain stem sections delimit the segments formed by transection in the companion manuscript (37).

The distribution of tadpole scores is summarized in Fig. 5. The analysis of variance indicated a statistically significant (P < 0.0001) interaction between stage and rostral/caudal locale, corresponding to the significantly elevated (P < 0.05) chemoreceptive response for the rostral brain stem of postmetamorphic tadpoles compared with the other three groups, i.e., postmetamorphic caudal and premetamorphic rostral and caudal.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Central chemoreception within the brain stem in pre- and postmetamorphic tadpoles. Bottom and top limits of the boxes correspond to lower and upper quartiles, and the "bullet" represents the median. Data points that fall below the lower quartile or above the upper quartile by >1.5 times the interquartile range are plotted separately. The "whiskers" indicate the range of nonoutlying data points.

	DISCUSSION

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The present results indicate that the fundamental ontogenic shift from gill to lung ventilation during metamorphosis is accompanied by the emergence of central chemoreceptor function in the rostral brain stem of the postmetamorphic tadpole. Although not statistically significant, CO₂ chemoreceptive sites tended to be located preferentially in the caudal brain stem of the premetamorphic animal.

The microinjection strategy used in the present experiments produced rapid and transient tissue acidification through convective and diffusive transport of CO₂ from the pipette tip. Although the pH of the unbuffered hypercapnic injectate was low (6.0), this degree of hypercapnia was necessary to elicit a substantial local reduction in the pH of the brain stem tissue because of the action of tissue bicarbonate and tissue buffers in limiting the fall in pH. Measurements of tissue pH indicate that this transport of CO₂ established a region of tissue acidosis with a transverse span of 500 µm, in which tissue pH transiently fell and then returned to baseline. Because the brain stem preparation lacks blood flow, the degree and spread of tissue acidosis may differ from more intact preparations. However, our findings are consistent with calculations described by Nicholson (26), in which pressure injection of 10 nl of a solution into the extracellular space of brain tissue formed a spherical cavity; then the solute diffused away from the cavitation site creating a spherical distribution of solute around the tip of the micropipette. Although the increment in PCO₂ caused by the injection was likely spherically distributed, our tissue pH measurements reveal that the decrement in pH was unevenly distributed around the injection site, being greater in the D-V than in the lateral dimension. We observed that the pH gradient caused by injection of hypercapnic aCSF was steeper in the lateral than in the D-V dimension. This asymmetry is to be expected given the asymmetry of the steady-state PCO₂ gradients existing before injection. As indicated by our previous studies, the lateral PCO₂ gradients are small, and the D-V PCO₂ gradients are steep owing to the rectangular cross-sectional shape of the tadpole brain stem (34). Because of this asymmetrical PCO₂ gradient, an equal increment in PCO₂ at any point equidistant from the injection site will cause a greater pH ventrally than laterally, assuming a constant HCO concentration throughout. The result of this will be a steeper gradient in the lateral than in the D-V dimension.

In the brain stems of premetamorphic larvae, we observed a statistically nonsignificant trend for CO₂ responsiveness to be located within 750 µm of the ventral surface at the level of CN IX and near the root of SN II. Our previous studies using hypercapnic superfusion of tadpole brain stems failed to show a lung ventilatory response in animals less mature than stage 15 (35, 37). Thus the lack of statistically significant CO₂ chemoreception in the caudal brain stem is not surprising.

CO₂-sensitive chemoreceptive fields that activate fictive lung ventilation in postmetamorphic tadpoles were distributed in rostral brain stem areas from CN IX to the rostral limit of the brain stem throughout all tissue layers. These fields correspond to analogous chemoreceptive areas reported by Issa and Remmers (10) and lie within brain stem regions previously shown to contain structures essential for lung burst generation in the adult frog (17, 29). Studies involving brain stem transection in the adult frog have identified critical areas for central respiratory-related activity lying between CN VIII and the caudal border of CN X (17, 29). McLean et al. (21) demonstrated in the isolated frog brain stem that injection of excitatory and inhibitory amino acids between CNs X and VIII influenced both the frequency and amplitude of lung burst activity. In a companion paper, we report that the lung rhythmogenic function of the tadpole brain stem can be localized to rostral segments of the postmetamorphic brain stem (37). Finally, although CO₂-sensitive chemoreceptive fields were located rostrally, we do not exclude the possibility that respiratory chemoreceptors exist elsewhere within the amphibian brain stem.

Locations of rhythmogenic and chemoreceptive sites. The findings of the present study can be correlated with results from our companion study of the location of segments essential for lung rhythmogenic function (35). The caudal brain stem (segment 3-4) of the premetamorphic brain stem was shown to be necessary and sufficient for lung rhythmogenesis, and the trend toward concentration of chemosensory sites in this region is of some interest. The lack of significant CO₂-sensing substrate may relate to the relative immaturity of the lung central pattern generator and reflect the overall lack of chemosensory response of lung ventilation to CO₂. In the postmetamorphic brain stem, the minimum segmental configuration supporting lung rhythmogenesis (segment 1-2) coincides with the chemoreceptive fields in the rostral brain stem identified in the present study. The frequency-enhancing influence of segment 3-4 appears not to depend on intrinsic chemoreceptive function, because no chemoreceptive sites were identified in these segments of the postmetamorphic brain stem. The companion paper reports that of all rhythmic segments of the postmetamorphic brain stem, only compound segment 2-3-4 failed to exhibit a hypercapnic response.

Perspectives

In the mammal, central respiratory chemoreceptors juxtaposed to respiratory neurons are widely distributed within the brain stem, including the pre-Bötzinger area (8). Both pre- and postmetamorphic tadpoles displayed chemosensitive regions in the area between CNs VIII and IX [segment 2 in the companion report (37)]. Thus we speculate that the most primitive site for central respiratory chemoreception lies in this region, which is homologous to the pre-Bötzinger area of the mammal. Further evidence of the possible importance of this region comes from the demonstration that peripheral chemosensory input via CN IX is essential for gill ventilation in premetamorphic larvae and drives lung ventilation in the postmetamorphic tadpole (11). Consequently, the study of respiratory rhythm generation and chemoreception in larval amphibians offers a unique opportunity to gain insights into the origin and evolution of neural respiratory control systems.