it is well established that vertebrates display respiratory responses to changes in the arterial blood gases, and the underlying control mechanisms are very similar among different taxa. The transition of amphibians to air breathing was accompanied by an increase in the sensitivity to CO₂/H⁺ (45). Interestingly, the ontogenetic changes of anuran amphibians involve modifications of the control system from being almost entirely oxygen driven to a combination of acid-base and oxygen driven (11).

Central respiratory CO₂ chemoreceptors have been clearly established in adult anuran amphibians (8, 46), and these receptors are probably distributed throughout the rostral medulla, surrounding the fourth ventricle (50). In mammals, these receptors were once thought to be located only close to the surface of the ventral medulla, but it is now clear that they are widespread within the brainstem (12). Recently, sites have been identified in the ventrolateral medulla, nucleus of the solitary tract, ventral respiratory group, locus coeruleus (LC), caudal medullary raphe, and fastigial nucleus of the cerebellum (for a review, see Ref. 35). However, it remains unclear whether chemoreceptors are also widely distributed in amphibians. An evaluation of specific contributions of these chemosensitive sites can be achieved by CO₂ challenge, focal stimulation, or focal disruption. Focal acidification of a single central chemosensitive site increases ventilation, whereas disruption decreases ventilation (3, 28, 37, 47). According to Nattie (35), the presence of widespread central chemoreceptors may be related to the increased demands of a CO₂-sensitive control system that occurred in ancestral amphibians during the transition from water- to air-breathing.

In mammals, LC is a noradrenergic nucleus located in the dorsomedial region of the pons, close to the floor of the fourth ventricle. The LC neurons are of particular interest in CO₂ challenge because >80% of neurons are found to be chemosensitive, responding to hypercapnia with an increased firing rate (13, 37, 38). Moreover, CO₂ stimulation increases c-fos expression in the LC (20, 49). Recently, Li and Nattie (28) lesioned the catecholaminergic (CA) neurons of the rat brain stem and found that the ventilatory response to 7% CO₂ was significantly decreased in sleep and wakefulness, suggesting that brain stem CA neurons participate in central chemoreception in vivo during both non-rapid eye movement sleep and wakefulness. In their study ∼84% of LC-CA neurons were eliminated, indicating that LC is an important site for hypercapnic ventilatory response. In amphibians, LC is placed in the isthmus region, which lies at the rostral end of the hindbrain and is considered to be homologous to the LC of mammals. This homology is based on its position, noradrenergic content, and projections to both the telencephalon and spinal cord (17, 18, 32). No data are available on the role of LC in relation to the CO₂ drive to breathing in amphibians. Therefore, we decided to test the hypothesis that LC is a chemosensitive site in toads (Bufo schneideri). To this end, we first used Fos immunoreactivity to verify whether LC is activated by hypercarbia (5% CO₂ in air). In addition, we studied the role of noradrenergic LC neurons on cardio-respiratory responses to hypercarbia by using 6-hydroxydopamine (6-OHDA) lesion. We further explored the role of LC in central chemosensitivity by microinjecting solutions with different pH values (7.2, 7.4, 7.6, 7.8, and 8.0) into this nucleus.

METHODS

Animals

B. schneideri (Werner, 1894; formerly Bufo paracnemis: Lutz, 1925) of either sex weighing 150–300 g were collected in the vicinity of Ribeirão Preto, São Paulo, Brazil, during the rainy summer months. Animals were captured and transported in agreement with the Instituto Brasileiro do Meio Ambiente e dos Recursos Renováveis (Animal license No. 025/2005). The toads were maintained in containers with free access to water and a basking area. Food was withheld for 1 wk before the experiment. Each toad was used in only one experiment, and all experiments were performed between 8:00 AM and 5:00 PM. The study was conducted in compliance with the guidelines of the American Physiological Society (2) and with the approval of the University of São Paulo Animal Care and Use Committee (Protocol # 04.1.740.53.0).

Surgery

Animals were anesthetized in an aqueous solution of 3-aminobenzoic acid ethyl ester (MS-222; 0.3% Sigma, St. Louis, MO) and implanted with a Silastic tube segment, 1.5 mm in outer diameter, into the right femoral artery. The head was then fixed to a Kopf stereotaxic apparatus (Model 900 Small Animal Stereotaxic, Tujunga, CA), and the skin covering the skull was removed using a bone scraper. The skull was opened above the midbrain region by means of a small diameter drill (Foredom Electric; Bethel, CT).

For the chemical lesions, the tip of a glass micropipette was inserted into the LC (1.5–2.0 mm below the level of the cerebellum and 0.5 mm from the midline), according to the coordinates of the stereotaxic atlas for B. schneideri (23), and 6-OHDA (Sigma) was injected (0.8 μg in 0.2 μl of vehicle over 5 min). Sham-operated animals were injected with vehicle (0.2 μg ascorbic acid in 0.2 μl of saline over 5 min). Dose and methods of administration were chosen on the basis of pilot experiments and previous studies (1, 15).

For microinjection, a guide cannula prepared from a hypodermic needle segment 14 mm in length and 0.5 mm in outer diameter was attached to the tower of the stereotaxic apparatus and placed unilaterally in contact with the exposed surface of the midbrain inside the LC region. The cannula was attached to the bone with stainless-steel screws and acrylic cement. A tight-fitting stylet was kept inside to prevent occlusion. The experiments were initiated 5 days after brain surgery.

Measurements of Ventilation

Measurements of pulmonary ventilation (V_E) were performed by the pneumotachographic method (16), based on the Poiseuille principle that a laminar flow of a gas is proportional to the pressure gradient across a tube. A lightweight transparent face mask provided an air-tight connection between the nostrils and a Fleisch tube. Inspiratory and expiratory gas flows were monitored with a differential pressure transducer (model MP45–14-871, Validyne, Northridge, CA). Signals from the air differential transducer were collected by a differential pressure signal conditioner connected to a physiograph (Gould Instrument Systems, model TA 240, Valley View, OH), passed through an analog-to-digital converter (CAD-12/36; Lynx Tecnologia Eletrônica, São Paulo, Brazil) digitized in a microcomputer equipped with data-acquisition software (AQ DADOS, data acquisition system, Lynx Tecnologia Eletrônica), and then integrated and analyzed with data-analysis software (WINDAQ). Calibration for volume was obtained during each experiment by injecting the face mask with known amounts of air (1, 2, and 3 ml) using a graduated syringe.

Measurements of Arterial Blood Pressure and Heart Rate

Arterial blood pressure was measured by connecting the arterial catheter to a Gould pressure transducer (Gould Instrument Systems, Valley View, OH), and the signals from the transducers were recorded on paper (Gould Instrument Systems, Valley View, OH). Heart rate (f_H) was determined by counting pressure pulses.

Experimental Protocols

Experiment 1.

This experiment evaluated whether the LC is activated by hypercarbia (5% CO₂ in air, AGA, Sertãozinho, SP, Brazil). Basal c-fos expression was assessed in normocarbic toads, under the same ambient conditions (n = 3). The stimulated animals were submitted to a hypercarbic gas mixture (n = 3) for 3 h. This duration was selected on the basis of previous reports in mammals (4, 49) and pilot experiments. To minimize unspecific c-fos expression, the animals were installed in the chamber used for stimulation the day before the experiment. The chamber was constantly ventilated with humidified air before the stimulation period. Ambient temperature was controlled and maintained at 25°C throughout the experiments. The stimulations were routinely delivered between 8:00 AM and 1:00 PM. At the end of the stimulation or of the control period, the toads were deeply anesthetized in an aqueous solution of 3-aminobenzoic acid ethyl ester (MS-222; 0.3%) and transcardially perfused with 0.01 M phosphate buffer saline, followed by 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4).

Experiment 2.

This experiment evaluated the effects of LC lesions on hypercarbic drive to ventilation. The animals were divided into four groups: 1) control group (n = 9): toads without brain surgery; 2) vehicle group (n = 9), 3) 6-OHDA-lesioned toads (6-OHDA, n = 7), and 4) peri-LC group: considered lesions located in nuclei surrounding the LC (n = 6). The toads were placed in a plastic chamber at the experimental temperature of 25°C, while the animal chamber was continuously flushed with humidified air (1.5 l/min). Once conditions were stable under normocarbia (∼1 h), a hypercarbic gas mixture (5% inspired CO₂, AGA, Sertaozinho, SP, Brazil) was applied for 30 min at the same flow (1.5 l/min). Blood pressure and V_E were recorded continuously during these exposures.

Experiment 3.

This procedure served to test whether LC neurons are chemoreceptors by focal acidification. Microinjections of mock cerebrospinal fluid (CSF) with different pH values: 7.2 (n = 6), 7.4 (n = 10), 7.6 (n = 6), 7.8 (n = 6) and 8.0 (n = 9) were performed into the LC, and ventilation was measured. The group injected with the pH of 7.8 was considered to be the control, since this value is expected from the arterial pH in intact toads at 25°C (8, 9). The composition of mock CSF was based on a previous study by Wilson et al. (55; in mM): 104 NaCl, 4 KCl, 1.4 MgCl₂, 10 d-glucose, 25 NaHCO₃, 2.4 CaCl₂. Bicarbonate and chloride concentrations were adjusted to the desired pH values for each solution. The solution was constantly equilibrated with 2% CO₂, and the pH was determined using a Micronal pH analyzer (model B374, São Paulo, SP, Brazil). The pH meter was calibrated by means of high-precision buffer solutions (S1500 and S1510, Radiometer).

The experiments were initiated 5 days after surgery. Equipped with a face mask, the toad was placed into the experiment chamber for at least 4 h before measurements. The chamber was continuously flushed with humidified air. After stable ventilation in room air (1 h), the animal received microinjections (0.1 μl) of mock CSF solutions into the LC, and ventilation was measured for 10 min. Each animal received two microinjections (1-h interval between them) of different pH value solutions in a random order. Intra-LC injections were performed using a thin dental needle introduced until its tip was 2 mm below the cannula end. A volume of 0.1 μl was injected over a period of 30 s using a Hamilton microsyringe. Drug flow was confirmed by the movement of an air bubble inside the polyethylene (PE)-10 tubing connecting the microsyringe to a dental needle.

Fos Immunohistochemistry

At the end of the experiments (experiment 1), the animals were anesthetized with MS-222 (0.3%) and perfused transcardially (right atrium cut) with PBS (0.01 M, pH 7.4) followed by 4% paraformaldehyde in PBS. The brains were removed, postfixed with 4% paraformaldehyde for 4 h, then immersed in a 20% sucrose solution for 24 h, and then immersed in a 30% sucrose solution for a further 24 h. Serial sections (40 μm) of the brain stem were made using a cryostat microtome (Leica, model CM1850, Nussloch, Germany). Free-floating sections were washed in PBS (0.01 M, pH 7.4) and then incubated in PBS containing 1% hydrogen peroxide for 10 min to deactivate endogenous peroxidase activity. After several rinses in PBS for 30 min, the sections were placed in 5% normal goat serum (Vector, Burlingame, CA) for 45 min and then incubated for 24–48 h at 4°C with polyclonal anti-c-fos serum generated in rabbits (Santa Cruz Biotechnology). The serum was diluted (1:1,000) in PBS containing 1% bovine serum albumin and 0.5% triton X-100. After being rinsed in PBS, the sections were incubated for 1.5 h at room temperature with biotinylated goat anti-rabbit IgG (1:400; Vector Laboratories). Subsequently, they were washed in PBS and placed for 30 min in avidin-biotin peroxidase complex (ABC kit, Vectastain, Vector, Burlingame, CA). Labeled neurons were visualized by a 5 to 10 min incubation with 0.05% 3,3′ diaminobenzidine tetrachloride and 0.1% hydrogen peroxide. Sections were mounted on gelatin-coated glass slides, dehydrated through an ascending ethanol series, xylene-cleared, and coverslipped with Entellan. Because the antibody used in this study recognizes c-fos and Fos-related proteins, immunoreactive neurons are described as Fos-like immunoreactive (Fos-IR). In negative control of the immunohistochemistry, the first antibody was omitted (polyclonal anti-c-fos serum generated in rabbits; Santa Cruz Biotechnology).

Assessment of 6-OHDA Chemical Lesion Effectiveness and Placement

Serial sections (40 μm) of the brain stem were made using a cryostat microtome (Leica, Model CM1850) and mounted on gelatin-coated glass slides. The first and second series of frontal sections were stained by cresyl violet and tyrosine hydroxylase (TH), respectively. To verify the correct placement and effectiveness of the chemical lesions, TH immunoreactivity was assessed using a marker of catecholaminergic neurons (56). To this purpose, the sections were incubated for 48 h with a rabbit polyclonal anti-TH antibody (1:1,000; Calbiochem, San Diego, CA) followed by a 2-h incubation with a biotinylated goat polyclonal anti-rabbit IgG antibody (1:400; Vector Laboratories). The biotinylated antibody was complexed with avidin DH-biotinylated horseradish peroxidase (Vector, code PK-4001), and the complex was developed by the addition of the peroxidase substrate 3,3′-diaminobenzidine tetrahydrochloride, according to manufacturer's instructions (Sigma). The selection of acceptable lesions was made on the basis of anatomical criteria.

Histological Procedures for Experiment 3

At the end of the experiment 3, the animals were anesthetized with MS-222 (0.3%, Sigma) and perfused through the heart with Ringer solution followed by 10% formalin solution. A dental needle was inserted through the guide cannula, a 0.1-μl microinjection of Evan's blue was performed, and the heads were removed and fixed in 10% formalin solution. On the following day, the brains were removed from the skull, immersed in paraffin, sectioned on a microtome, and stained with hematoxylin-eosin for light microscopy determination of the region reached by the microinjection needle.

Data Processing and Analysis

All values are reported as means ± SE.

Experiment 1.

The sections were analyzed by light microscopy, and labeled neurons were registered with the use of an image analysis system (Keiss KS 300). For quantification, one brain section of each nucleus was selected for unilateral counts from each toad. Images were captured and analyses were performed using a computerized image analysis system (U.S. National Institutes of Health System, Image J, available on the Internet at http://www.rsb.info.nih.gov/nih-image/). Fos-IR cells in each section were counted by setting a size range for cellular nuclei (in pixels) and a threshold level for staining intensity. Representative sections in control and experimental groups were acquired at exactly the same level, with the aid of the Adobe Photoshop Image Analysis Program, 5.5 version. The counting was done in three animals for each condition and was repeated at least twice on each section analyzed, which ensured that the number of profiles obtained was similar. Number of Fos-IR neurons was compared by t-test, with P < 0.05, indicating a significant difference.

Experiment 2.

Calculations of mean arterial blood pressure (MAP), heart rate (f_H), and V_E were based on 10-min recording periods. Respiratory frequency (f) was quantified by analyzing the number of respiratory events (lung breaths) per minute. Tidal volume (V_T) was obtained from the integrated area of the inspired flow signal. Buccal ventilations were identified by small positive and negative pressures, whereas lung ventilations involved larger pressure changes (24). V_E (V_E = V_T × f) was expressed as ml STPD·kg⁻¹·min⁻¹, where STPD is standard temperature, standard pressure, deg. Duration of each nonventilatory period (apnea) and number of breaths per episode were calculated. A breathing episode consists of inhalations that are not separated by a nonventilatory period longer than the length of two ventilation cycles. Duration of nonventilatory period was defined according to the criteria of Sanders and Milsom (43), consisting of a period between two fictive breaths, with a duration of at least two fictive buccal oscillations. Apnea duration was measured from the end of the last breath of an episode to the onset of the first breath of the subsequent episode. Instantaneous breathing frequency was determined according to the criteria of Kinkead and Milsom (26) by calculating the inverse of the period between two successive, uninterrupted breaths within an episode and multiplying by 60. Heart rate was determined by counting pressure pulses, and MAP was estimated using the following formula: mean pressure = diastolic pressure + 1/3 (systolic pressure minus diastolic pressure) (29). The effects of hypercarbia on ventilatory and cardiovascular variables were evaluated by two-way ANOVA followed by a point-by-point one-way ANOVA and paired t-test, respectively, to assess differences between groups. The Tukey-Kramer multiple comparisons test was applied as a post hoc test. A P < 0.05 was considered significant.

The TH-immunoreactive cells (TH-IR) were counted using the same procedure of experiment 1. Number of TH-IR neurons was compared by unpaired t-test, with P < 0.05 indicating a significant difference.

Experiment 3.

V_E was calculated on 10-min recording periods for the control period. For analysis of the pH effect, the calculations were based on the basis of 3-min recording periods after the microinjection. V_E, V_T, and f were determined as in the experiment 2. The effects on ventilation of microinjections of different pH values into the LC on ventilation were evaluated by one-way ANOVA followed by the Tukey-Kramer multiple-comparisons test. A P < 0.05 was considered significant.

RESULTS

Experiment 1

Within the LC of normocarbic animals, only a few immunoreactive neurons were found, which indicates a low level of basal expression of c-fos (Fig. 1A). Hypercarbia significantly increased the number of immunoreactive neurons of the LC (Fig. 1, A and B) when compared with normocarbia (P = 0.0035, unpaired t-test). Slices processed as negative controls, that is, which were not incubated with the primary antibody, presented no staining (Fig. 1A).

Experiment 2

The immunohistochemistry for TH shows the effectiveness of chemical lesions of the LC with 6-OHDA (Fig. 2). The LC of control animals was intact and typically appeared as a compact cluster of intensely stained cells. Successful chemical (6-OHDA) lesions of the LC were revealed mostly by the disappearance of TH-positive cells. The number of TH immunoreactive cells was dramatically reduced or even eliminated after 6-OHDA lesions (P < 0.001; paired t-test).

Figure 3 shows breathing traces obtained from the control, vehicle, peri, and 6-OHDA groups during room air and hypercarbia (5% CO₂), illustrating the patterns of breathing present under these conditions. During normocarbia, all animals breathed episodically and showed buccal ventilations (small positive and negative pressure changes). Table 1 presents the corresponding effects of each condition on breathing pattern. Under normocarbia, none of the experiment's conditions had any significant effect on breathing pattern in any group. Exposure to hypercarbia transformed the breathing pattern from episodic to continuous in some animals (three out of nine of the control group, six out of nine of the vehicle group, four out of six of the peri group, and one out of seven of the 6-OHDA toads); thus we did not perform statistical analysis for this parameter. Hypercarbia decreased significantly the nonventilatory period of the vehicle (P = 0.0010, paired t-test), peri (P = 0.0157, paired t-test), and the 6-OHDA group (P = 0.0302, P = 0.0302, paired t-test, but not of the control group). Hypercarbia had no effect on instantaneous breathing frequency in any group. No buccal ventilations were observed during hypercarbic challenge.

Figure 4 shows the effect of hypercarbia on ventilation. Hypercarbia caused an increase in pulmonary ventilation in all groups (P = 0.006 for control group, P = 0.0043 for vehicle group, P = 0.0048 for peri group, and P = 0.0031 for the 6-OHDA, paired t-test), which resulted from increases of f and V_T in control, vehicle, and peri groups. This difference was due to the absence of an increase in V_T in 6-OHDA toads. Figure 4 also shows the effect of 6-OHDA lesions of the LC on hypercarbia. The hypercarbic ventilatory response was significantly decreased in 6-OHDA toads compared with control, vehicle, and peri groups (P < 0.05, one-way ANOVA). This difference was due to a decreased V_T in 6-OHDA toads (P < 0.05; one-way ANOVA).

Table 2 shows the effects of the chemical lesions in the LC on cardiovascular parameters. None of the experimental conditions had any significant effect on MAP and f_H.

Experiment 3

Figure 5 shows the effects on V_T, f, and V_E of the microinjection of mock CSF with different pH values (7.2, 7.4, 7.6, 7.8, and 8.0) into the LC. The microinjection of mock CSF of pH 7.8 and 8.0 had no effect on V_E compared with the value before the injection (no injection group, NG). V_T and f were not significantly different between groups, but their product (V_E) increased with microinjection of mock CSF of pH 7.6 (P < 0.05, one-way ANOVA), 7.4 (P < 0.01, one-way ANOVA), and 7.2 (P < 0.001, one-way ANOVA) compared with NG, pH 7.8 and pH 8.0 groups.

Peri-LC microinjections were used as negative controls for each one of the different mock CSF pH values. In the NG group, V_E was 41.7 ± 14.4 ml STPD·kg⁻¹·min⁻¹, and no significant difference was found in relation to other groups (8.0 = 27.4 ± 15.1 ml STPD·kg⁻¹·min⁻¹; 7.8 = 38.8 ± 18.4 ml STPD·kg⁻¹·min⁻¹, 7.6 = 39.5 ± 16.1 ml STPD·kg⁻¹·min⁻¹, 7.4 = 39.5 ± 2.0 ml STPD·kg⁻¹·min⁻¹). We have no peri-LC data for pH value of 7.2 because all of the microinjections at this pH successfully reached LC.

DISCUSSION

In the present study, we have used selective chemical lesion of LC catecholaminergic neurons to verify the possible involvement of this nucleus in the cardio-respiratory responses to hypercarbia. Our data show that chemical lesions of the LC with 6-OHDA resulted in an attenuation of hypercarbia-induced hyperventilation, but they had no cardiovascular effect. In addition, we provided morphologic evidence, that is, the expression of c-fos in neurons of the LC after hypercarbic challenge. This finding associated with the fact that the isthmic catecholaminergic cell group of amphibians (where LC is placed) does not contain dopaminergic or adrenergic cell bodies (18) strongly suggests that noradrenergic LC neurons are involved in processing or modulating central chemoreceptor information in amphibians. Next, we investigated whether LC neurons are intrinsically pH-sensitive. To test this hypothesis, we performed local acidification by microinjecting mock CSF with different pH values. Our data indicate that the LC neurons are pH sensitive, as microinjection of acid mock CSF solutions (pH 7.6, 7.4, and 7.2) increased pulmonary ventilation.

It has traditionally been suggested that central CO₂/H⁺ chemoreceptors exist in the adults of all tetrapod vertebrates, that is, amphibians (8, 9, 46), reptiles (21), crocodilians (10), birds (34), and mammals (30, 44). More recently, it has been reported that central chemosensitvity to CO₂/H⁺ is already present in the lungfish Lepidosiren paradoxa, the sister group of Tetrapoda (42). According to Milsom (33), once central chemoreceptors appear, they take on the predominant role of providing chemosensory drive under steady-state conditions. In amphibians, this dominant role of central chemoreceptors appears at metamorphosis, when these receptors assume the generation of the respiratory drive (48, 51). The present study adds information specifically related to the LC as a chemosensitive site in the central nervous system (CNS) of adult toads to this scenario. Actually, as far as we are concerned, this is the first report documenting that a nonmammalian vertebrate may possess multiple sites of central chemosensitivity, as already reported for mammals (35, 19, 41). These chemosensitive sites include the LC, where noradrenergic neurons compose the largest percentage (>80%), excited by high CO₂/H⁺ of the CNS (13, 37, 38). Because LC is a region associated with pain sensation, attention, learning, and anxiety, it is possible that the primary function of LC CO₂/H⁺-sensitive neurons is to produce an aversive or anxiety response to elevated CO₂ levels (39).

According to Richerson (40) and Putnam et al. (39), there are two essential criteria that a central chemoreceptor neuron must possess: 1) it must respond to changes in CO₂ that occur under nonpathological conditions in vivo, and this response must be due to mechanisms intrinsic to the specific cell; and 2) it must have the capability of increasing respiratory output in response to an increase in CO₂, which could be accomplished if the neuron is part of the respiratory network or projects to respiratory neurons. Our results fit the first criterion since lesions of the LC resulted in an attenuation of the hypercarbia-induced hyperventilation, and local tissue acidosis increased the ventilation of the toads. Regarding the second criterion, it is well known that the LC of mammals projects to respiratory neurons, such as ventral medullary and solitary tract nuclei (52, 53). There is neuroanatomical evidence suggesting that the LC of amphibians is homologous to the LC of mammals primarily on the basis of its position, noradrenergic content, and projections to brain stem structures (17, 18, 32). Furthermore, under in vivo conditions, in mammals, LC neurons appear to be activated by systemic hypercapnia, as judged by increased expression of c-fos gene product (20). The c-fos expression technique has been extensively used as a marker of neuronal activity, induced by a number of stimuli, including hypercarbia. We found that an increased inspired CO₂ concentration (5% CO₂) induces Fos-like immunoreactivity in the LC of toads (Fig. 5), reinforcing the idea that the LC of amphibians is homologous to the LC of mammals. Further investigation is needed to understand the specific functional connections of the LC with the neuronal circuitry involved in the control of respiration in amphibians, but we can speculate that the LC of amphibians might be a central chemoreceptor site.

Mock CSF perfusion is a well-established method for studying the central chemoreceptor drive to breathe (8, 22, 42). We performed local acidification of the LC by microinjecting mock CSF with different pH values. Interestingly, pulmonary ventilation increased after local reduction of the pH (mock CSF of 7.2, 7.4, and 7.6), which suggests that LC is a chemosensitive site in the CNS of amphibians.

Hypercarbia is a powerful stimulus for the anuran respiratory control system (8, 9, 26, 31, 46, 50, 54). In intact unanesthetized toads, the central chemoreceptors contribute about 80% of the hypercarbic drive to ventilation (8). Furthermore, the ventilatory response of toads to both hypercapnia and hypoxia is greater than in mammals (cf. Ref. 8 and 27), which is in agreement with the present data, in which a seven-fold increase was measured in response to under 5% CO₂. Moreover, we found that an LC lesion reduced the ventilatory response to CO₂ by 75%, and acidification of this site increased ventilation to up four- to fivefold (Fig. 5). The reduced ventilatory responses after lesion, as well as the increased responses to acidification, contrast with relatively modest responses in mammals (35). This may relate to different modes of gas exchange and respiratory mechanics (for instance, toads have no rib cage and use the skin as a gas exchange site) and to specific characteristics of breathing control (different receptors have been reported for amphibians that are not present in mammals; for a review, see Ref. 33).

The toad Bufo schneideri is widely distributed over the South American continent, including Central Brazil, the Argentinean Chaco, and Jujuy Province. From there, it is distributed through the Pantanal of Mato Grosso to the northern extreme of Cordilheira and is commonly found in the central plateau of the continent (23). In the São Paulo state, Brazil, where our experiments were performed, the winter is the dry season (36). Bufo schneideri is known to estivate, burrowing into the soil without forming a cocoon during winter (6), whereas it is active during rainy seasons (spring and summer). In fact, this species has already been found in an Ecological Station in Itirapina, São Paulo, in burrows down to 1.0 and 1.5 m below the surface (C. A. Brasileiro, personal communication). Although measurements of O₂ and CO₂ levels in these microhabitats have not been reported, it is possible that these burrows also become hypoxic and hypercarbic. Consistently, Boutilier et al. (7) reported elevated arterial Pco₂ levels in burrowing cane toads, whose nostrils were open to the air while the skin was surrounded by sand at 25°C.

In agreement with previous studies on anuran species, hypercarbia (5, 14) had no effect on cardiovascular responses. Moreover, the present results showed that LC plays no role in cardiovascular control.

In conclusion, our results indicate that LC noradrenergic neurons of toads are a site of central chemoreception. On the basis of these data, we can suggest that the presence of widespread central chemoreceptor sites evolved early, as they are present in amphibians. Very likely, the transition from water to air breathing was associated with demands for a more flexible and sensitive CO₂ control system, which brought the development of multiple central sites for CO₂/H⁺ detection. Our findings further emphasize the similarities between anuran and mammalian LC and support the proposed homology of this nucleus in both groups.

Perspectives

Considerable advances have been recently achieved in the understanding of the neurorespiratory control mechanisms in amphibians. However, the presence of widespread central CO₂/pH chemosensitive sites has been less investigated in nonmammalian species. The present study is the first to demonstrate that the surface of the ventral medulla does not seem to be the exclusive site of central chemoreceptors in amphibians. Future research will be necessary to specify the stimulus or stimuli responsible for LC activation (intracellular pH, extracellular pH, and/or molecular CO₂) by using electrophysiological recordings. Additionally, it will be interesting to investigate the role of LC in chemosensitivity during ontological development of amphibians and also the role of other brain stem structures, considered chemoreceptive sites in mammals, such as raphe, nucleus tractus solitarius, and retrotrapezoid nucleus.

GRANTS

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico. Carolina R. Noronha-de-Souza was the recipient of a FAPESP undergraduate scholarship (04/10454–0).

FOOTNOTES

• 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.