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J Appl Physiol 103: 1780-1788, 2007. First published September 6, 2007; doi:10.1152/japplphysiol.00424.2007
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Raphe magnus nucleus is involved in ventilatory but not hypothermic response to CO2

Mirela B. Dias,1 Tatiane B. Nucci,1 Lisandra O. Margatho,1 José Antunes-Rodrigues,1 Luciane H. Gargaglioni,2 and Luiz G. S. Branco3

1Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo; 2Department of Animal Morphology and Physiology, UNESP-São Paulo State University; and 3Department of Physiology, Dental School of Ribeirão Preto, University of São Paulo, São Paulo, Brazil

Submitted 18 April 2007 ; accepted in final form 27 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
There is evidence that serotonin [5-hydroxytryptamine (5-HT)] is involved in the physiological responses to hypercapnia. Serotonergic neurons represent the major cell type (comprising 15–20% of the neurons) in raphe magnus nucleus (RMg), which is a medullary raphe nucleus. In the present study, we tested the hypothesis 1) that RMg plays a role in the ventilatory and thermal responses to hypercapnia, and 2) that RMg serotonergic neurons are involved in these responses. To this end, we microinjected 1) ibotenic acid to promote nonspecific lesioning of neurons in the RMg, or 2) anti-SERT-SAP (an immunotoxin that utilizes a monoclonal antibody to the third extracellular domain of the serotonin reuptake transporter) to specifically kill the serotonergic neurons in the RMg. Hypercapnia caused hyperventilation and hypothermia in all groups. RMg nonspecific lesions elicited a significant reduction of the ventilatory response to hypercapnia due to lower tidal volume (VT) and respiratory frequency. Rats submitted to specific killing of RMg serotonergic neurons showed no consistent difference in ventilation during air breathing but had a decreased ventilatory response to CO2 due to lower VT. The hypercapnia-induced hypothermia was not affected by specific or nonspecific lesions of RMg serotonergic neurons. These data suggest that RMg serotonergic neurons do not participate in the tonic maintenance of ventilation during air breathing but contribute to the ventilatory response to CO2. Ultimately, this nucleus may not be involved in the thermal responses to CO2.

hypercapnia; ventilation; serotonin

HYPERCAPNIA elicits a number of compensatory responses in a wide variety of animal species, such as an increased ventilatory response and a reduced body temperature (Tb) (7, 41). Central chemoreceptors detect changes in CO2/pH providing regulation of breathing and are located primarily in the brain stem, close to the surface of the ventral medulla (35, 37, 55). However, it is now clear that central respiratory CO2 chemoreceptors are widespread within the brain stem (10, 31, 41, 43), including the medullary raphe region (MRR; raphe magnus, pallidus, and obscurus).

The raphe magnus nucleus (RMg) is of particular interest in CO2 challenge since it contains a very large percentage of serotonergic neurons (15–20%) (17), and there is physiological and anatomic evidence for its role in the control of breathing (13, 14, 15, 19, 22, 23, 24). We have recently reported that nonspecific chemical lesions of the RMg cause an increased ventilatory response to hypoxia, a result that suggests that this nucleus exerts an inhibitory modulation of ventilation in rats (18). Moreover, Teppema et al. (59) have shown that hypercapnia induces c-fos expression in the RMg, indicating that RMg is probably part of the neuronal pathway activated during hypercapnia. However, the specific involvement of RMg neurons mediating the ventilatory response to hypercapnia has never been assessed until now. Moreover, despite the fact MRR has a fairly heterogeneous function with respect to chemoreception and breathing (32), studies have assessed this brain area as a whole (57), and thus in the present study RMg nucleus has been assessed separately, adding specificity to the scenario.

It has long been recognized that the central serotonergic system may also modulate thermoregulatory mechanisms in many species, including rodents and humans (11, 25, 29, 33, 34, 62). For instance, it is known that MRR neurons participate in the regulation of Tb by means of effects on sympathetic outflow to brown adipose tissue (9). Additionally, it has been demonstrated that RMg serotonergic neurons modulate the relationship between output of thermointegrative centers and thermoregulatory effector responses (4). On the other hand, rats submitted to killing of serotonergic neurons of MRR have not shown any change in the thermal response to hypercapnia (44). However, no data exist about the specific involvement of RMg in thermoregulation during hypercapnia.

In view of these considerations, the goal of the present study was to investigate the participation of RMg in the ventilatory and thermal responses to hypercapnia and to test the hypothesis that RMg serotonergic neurons are specifically involved in these responses. Thus we microinjected 1) ibotenic acid to cause nonspecific lesioning of neurons in the RMg, or 2) anti-SERT-SAP to kill serotonergic neurons in the RMg.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals

Experiments were performed on adult male Wistar rats weighing 280–310 g. The animals had free access to water and food and were housed in a temperature-controlled chamber at 24–26°C (model ALE 9902001; Alesco, Monte Mor, SP, Brazil) with a 12:12-h light-dark cycle (lights on at 6:00 AM). All procedures were in accordance with Colégio Brasileiro de Experimentação Animal (COBEA) and were approved by the University of São Paulo Animal Care and Use Committee (protocol no. 214/2005).

Surgery

Animals were submitted to general anesthesia by intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). The head and a portion of the abdomen were shaved, and the skin was sterilized with betadine solution and alcohol. Rats were fixed in a Kopf stereotaxic frame and received two microinjections into the RMg region. The coordinates for the placement were 10.52 and 11.3 mm from the bregma in the midline, and 10.4 mm below the dorsal surface of the skull (47). The microinjections were made by using a 0.5-µl Hamilton microsyringe with a 28-gauge dental needle and were performed with a microinjector machine (model 310, Stoelting, IL). Each microinjection lasted 4 min, and the needle remained in position for another 5 min before removal. The wound was then sutured. Additionally, animals of all groups were submitted to paramedian laparotomy for the insertion of a biotelemetry capsule (model VM-FH, Mini-Mitter, Sunriver, OR) into the peritoneal cavity. The abdominal wound was then closed, and the implanted capsule was used for body temperature (Tb) measurement. At the end of surgery, rats received 0.2 ml (1,200,000 U) of benzyl-penicillin administered intramuscularly. The surgical procedures were performed over a period of ~40 min.

Determination of Ventilation

Measurements of ventilation (VE) were performed by the body plethysmograph method (2). Freely moving rats were kept in a 5-liter plexiglas chamber and allowed to move about freely while the chamber was ventilated with room air or with a humidified normoxic hypercapnic gas mixture of 7% CO2 (AGA). During VE measurements, the flow was interrupted and the chamber was sealed for 1 min (which is enough to evaluate 100–170 respiratory cycles analyzed to obtain the respiratory variables); the pressure oscillations caused by breathing were monitored with the use of a differential pressure transducer (model MP45–14-871, Validyne, Northridge, CA). The signals were fed into a differential pressure signal conditioner, passed through an analog-to-digital converter, and digitized in a microcomputer equipped with data-acquisition software (Gold Instrument Systems, Valley View, OH; Acquire 6600 Data Acquisition System). The results were analyzed with the data-analysis software Windaq (v1.32 data acquisition system, DI-720, DATAQ Instruments). Calibration for volume was obtained during each experiment by injecting the animal chamber with a known amount of air (1 ml). Respiratory variables such as respiratory frequency (f) and tidal volume (VT) were calculated with the appropriate formula of Malan (36):

Formula
where PT is the pressure deflection associated with each VT, PK is the pressure deflection associated with injection of the calibration volume (VK), Tb is the body core temperature, TA is the air temperature in the animal chamber, PB is the barometric pressure, PR is the vapor pressure of water at Tb, PC is the vapor pressure of water vapor in the animal chamber, and TR is the room temperature. VE and VT are presented at the ambient barometric pressure and at body core temperature and saturated with water vapor at this temperature (BTPS). Tb was monitored by biotelemetry (model ER-3000, Mini-Mitter), and the air temperature in the animal chamber was monitored with the use of a thermoprobe (model 8502-10, Cole Parmer, Chicago, IL). According to Malan (36), TR may be slightly lower than TA, because of the heat production of the animal in the chamber. The PC (vapor pressure of water vapor in the animal chamber) was calculated indirectly by using an appropriate table (12).

Determination of Tb

Tb was measured by biotelemetry, and the animal chamber was placed on the RLA 3000 telemetry receiver (Mini-Mitter). The output of the receiver displayed the pulse frequency of the transmitter capsule and the corresponding Tb on the screen of a microcomputer containing appropriate software (Vital View).

Experimental Protocol

Nonspecific chemical lesion into the RMg on ventilatory and thermal response to hypercapnia.   The chemical lesions were made using ibotenic acid obtained from Sigma (St. Louis, MO). This acid is an excitotoxin that destroys neuronal bodies but spares fibers of passage (1). Each rat received two injections of 0.2 µg/0.1 µl of ibotenic acid, dissolved in phosphate-buffered saline, pH 7.4. Rats in the vehicle group underwent the same procedures except that vehicle (phosphate-buffered saline, pH 7.4.) was microinjected instead of the ibotenic acid. The experiments were performed 6 days after brain surgery (18). Dose and methods of administration were chosen on the basis of pilot experiments and previous studies (8, 18).

Six days after surgery, each animal was individually placed in a plexiglas chamber (5 liter) kept at the experimental temperature of 25°C, and allowed to move about freely while the chamber was flushed with humidified air. After the animals remained calm (at least 30 min), control Tb and VE were measured. Subsequently, a normoxic hypercapnic gas mixture of 7% CO2 was flushed through the chamber for 60 min. VE was measured 5, 20, 35, 50, and 70 min after the start of hypercapnia. Finally, rats were returned to a 60-min period of normocapnia exposure when VE was measured again.

Hypercapnic levels and the period of time for hypercapnic exposure before measurements, were chosen on the basis of pilot experiments and previous studies (44, 58).

Specific lesion of RMg serotonergic neurons on ventilatory and thermal response to hypercapnia.   The specific lesions were made using anti-SERT-SAP (Advanced Targeting Systems, San Diego, CA). The anti-SERT is bound to the saporin (SAP), which is a ribosome-inactivating protein. This complex utilizes a monoclonal antibody to the third extracellular domain of the serotonin reuptake transporter (SERT). Each rat received two injections of 1 µM in a final volume of 0.1 µl each, dissolved in artificial cerebrospinal fluid, pH 7.4. Rats in the control group underwent the same procedures except that IgG-SAP was microinjected instead of the anti-SERT-SAP. The experiments were performed 9–10 days after brain surgery. Dose and methods of administration were chosen on the basis of pilot experiments and previous studies (44).

Nine days after surgery, each animal was individually placed in a plexiglas chamber (5 liters) kept at the experimental temperature of 25°C and was submitted to the same procedures described in the ibotenic acid protocol.

Histology

On completion of the experiments of the nonspecific lesion protocol (see Nonspecific chemical lesion in to the RMg on ventilatory and thermal response to hypercapnia), the animals were deeply anesthetized with 2,2,2-tribromoethanol and perfused intracardially with saline followed by 10% formalin solution. The brain was removed and stored in 10% formalin for at least 2 days. After fixation, the brain stem was embedded in paraffin, sectioned on a microtome (30-µm-thick coronal sections), and stained by the Nissl method for light microscopy determination of the region reached by the microinjection according to the atlas of Paxinos and Watson (47). Cresyl violet sections revealed gliosis in the sites of ibotenic acid injections. Only the rats with a positive site of microinjection into the RMg were considered. The estimation of the lesion was based on the fact that inside the core of microinjection sites there is moderate edema surrounding many damaged neuronal perikarya and picnotic nuclei strictly situated in the RMg. Whenever these alterations were observed outside RMg, rats were considered as negative control of misplaced lesions ("peri-RMg").

Immunohistochemistry

On completion of the experiments of the specific lesion protocol (see Specific lesion of RMg serotonergic neurons on ventilatory and thermal response to hypercapnia), the animals were deeply anesthetized with 2,2,2-tribromoethanol and then transcardially perfused with 300 ml saline followed by 300 ml of chilled 4% paraformaldehyde [4% in 0.1 M phosphate buffer (PB), pH 7.4]. The brain was removed and postfixed overnight in 4% paraformaldehyde at 4°C and then cryoprotected for 48 h in 30% saccharose. The brains were sectioned at 30-µm thickness on a cryostat (Leica, CM 1850). The free-floating sections were washed in PB (0.1M, pH 7.4) and incubated in PB containing 10% hydrogen peroxide and 10% methanol for 60 min to inactivate endogenous peroxidase activity. After three rinses in PB for 15 min, the sections were placed in 10% normal goat serum (Vector, Burlingame, CA) and 5% bovine serum albumin for 60 min. Next, the sections were incubated with a rabbit polyclonal antibody against 5-HT (1:1,000, Sigma) for 48 h at 4°C. After being rinsed in PB, the sections were incubated for 1.5 h at room temperature with biotinylated goat anti-rabbit IgG (1:400, Vector). They were subsequently washed in PB and placed for 30 min in avidin-biotin peroxidase complex (ABC kit, Vectastain, Vector). The labeled neurons were revealed by a 5- to 10-min incubation with 0.05% 3,3'-diaminobenzidine tetrachloride-0.1% hydrogen peroxide. The sections were mounted on gelatin-coated slides, dehydrated through an ascending ethanol series, cleared with xylene, and coverslipped with Entellan.

Cell Counting

To estimate the total number of 5-HT-immunoreactive (5-HT-ir) neurons of RMg and RPa, we averaged the counts of somatic profiles obtained at levels –9.30 to –9.80 mm, –10.04 to –10.30 mm, –10.52 mm, and –11.30 mm from bregma. The rats used for the physiological experiments were the same as the rats used for subsequent anatomy experiments. Numbers of 5-HT-ir neurons are given bilaterally and were quantified using a computerized system that includes a Zeiss microscope equipped with a DC 200 Leica digital camera attached to a contrast enhancement device.

Data and Statistical Analysis

Values are reported as means ± SE. In the analysis of ventilatory data, we used two approaches. First, our study design included a control and a lesioned group, with measurements made before CO2 exposure, at 5, 20, 35, 50, and 60 min during hypercapnia, and after CO2 exposure. We performed a two-way ANOVA on these data with treatment and time as factors and using a Duncan's test for post hoc comparisons. In analysis of Tb data, we used two-way ANOVA with treatment and time as factors followed by Duncan's test for post hoc comparisons. Counts of somatic cell profiles of 5-HT-ir neurons were compared using Student's t-test. Values of P < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Anatomy

Nonspecific lesions.   Representative photomicrographs of the nonspecific lesions are represented in Fig. 1. In the ibotenic acid group, cresyl violet sections revealed gliosis in the sites of microinjections. The selection of acceptable chemical lesions was made based on anatomic criteria. In all accepted rats, at least 50% of the RMg was damaged bilaterally. Figure 2 shows serial sections from the injection site, indicating that lesion is relatively restricted to ~400 µm from the injection site (black lined circle).


Figure 1
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  		Fig. 1. Nonspecific lesioning of neurons in the raphe magnus nucleus (RMg). Photomicrographs of coronal brain sections (30 µm) of a representative rat with a control rat (A and B) and ibotenic acid lesion of the RMg (C and D). B and D are higher magnifications (x20) of the sectors indicated by a dotted line in A and C, respectively. Note the presence of glia, degenerative debris, and decreased identifiable neurons in D.

 

Figure 2
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  		Fig. 2. Serial sections from the injection site. Note that fusiform neurons are present in B and C but not in A, D, E, and F where they are absent, indicating that lesion is relatively restricted to ~400 µm from the injection site (black lined circle).

 
Specific lesions.   The immunohistochemistry for 5-HT showed the effectiveness of chemical lesions of the RMg serotonergic cells with anti-SERT-SAP, which was revealed mostly by the reduction of 5-HT-positive cells (Fig. 3). Substantial heterogeneity in somatic shape and dendritic branching pattern of serotonergic RMg cells exists (16). The morphology of 5-HT-ir cells in this study was similar to that of serotonergic cells previously described (16, 17). The number of 5-HT-ir cells was dramatically reduced (at levels –10.52 mm and –11.30 mm from bregma) after anti-SERT-SAP lesions compared with the IgG-SAP control group (Fig. 4; P < 0.05; unpaired t-test).


Figure 3
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  		Fig. 3. Specific killing of serotonergic neurons in the RMg. Immunoreactivity (5-HT) in the RMg of anti-SERT-SAP-injected (A–C; G–I) and IgG-SAP-injected (D–F; J–L) animals. anti-SERT-SAP is an immunotoxin that utilizes a monoclonal antibody to the third extracellular domain of the serotonin reuptake transporter (SERT). A–F: representative sections of the more rostral level (–10.52 mm from bregma). G–L: representative sections of the caudal level (–11.3 mm from bregma). B, E, H, and K are higher magnifications (x20) of the sectors indicated by dotted line in plates A, D, G, and J, respectively. C, F, I, and L are higher magnifications (x40) of cells indicated by arrows in B, E, H, and K, respectively. Note loss of 5-HT-immunoreactivity in the RMg area of D and J. Scale bar, 100 µm. RPa, nucleus raphe pallidus; Py, pyramidal tract.

 

Figure 4
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  		Fig. 4. Comparison of the 5-HT-immunoreactive (5-HT-ir) neuron counting of anti-SERT-SAP-treated and IgG-SAP-treated rats. A: data of average number of 5-HT-ir neurons at levels –9.30 to –9.80 mm, –10.04 to –10.30 mm, –10.52 mm, and –11.30 mm of RMg. B: data of average number of 5-HT-ir neurons at levels –9.30 to –9.80 mm, –10.04 to –10.30 mm, –10.52 mm, and –11.30 mm of RPa. Values are means ± SE; n = 6 for both groups. *Significant difference between anti-SERT-SAP and IgG-SAP groups (P < 0.05, Student's t-test).

 
Nonspecific Chemical Lesion into the RMg on VE and Tb

Chemical lesion of RMg with ibotenic acid caused no change in VE during air breathing (Fig. 5). Figure 5 also shows that the ventilatory response to hypercapnia was significantly lower in the RMg lesioned group compared with both the peri-RMg and vehicle groups. The difference between the groups was due to a decreased VT and f in RMg lesioned rats.


Figure 5
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  		Fig. 5. Effects of ibotenic acid lesion of RMg and vehicle injection on tidal volume (VT), respiratory frequency (f), and ventilation (VE) of rats. Ventilatory values are shown for normocapnia (room air) and hypercapnia (7% CO2) exposure. Values are expressed as means ± SE. *Significant difference between lesioned group and control group during hypercapnia (P < 0.05, 2-way ANOVA, Duncan's post hoc test). No. of animals in each group is shown in parentheses.

 
Typical hypercapnia-induced hypothermia was observed in both groups, with no difference between groups (see Fig. 7A).


Figure 7
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  		Fig. 7. Nonspecific lesioning and specific serotonergic neuron killing of RMg do not change the hypercapnia-induced hypothermia. A: effects of ibotenic acid lesion of RMg and vehicle injection on the body temperature (Tb) of rats during normocapnic and hypercapnic conditions (7% CO2). B: effects of anti-SERT-SAP and IgG-SAP injections on Tb of rats during normocapnic and hypercapnic conditions (7% CO2).Values are means ± SE. No. of animals in each group is shown in parentheses.

 
Specific Lesion of RMg Serotonergic Neurons on VE and Tb

Figure 6 shows the effect of anti-SERT-SAP injection on ventilation. As observed, under normocapnia, no consistent difference could be observed among the experimental groups. The ventilatory response to hypercapnia was significantly lower in the anti-SERT-SAP group compared with the peri-RMg and vehicle groups. The difference between the groups was entirely due to a decreased VT in anti-SERT-SAP-injected rats.


Figure 6
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  		Fig. 6. Effects of anti-SERT-SAP and IgG-SAP injections on VT, f, and VE of rats. Ventilatory values are shown for normocapnia (room air) and hypercapnia (7% CO2) exposure. Values are expressed as means ± SE. *Significant difference between lesioned group and control group during normocapnia and hypercapnia (P < 0.001, 2-way ANOVA; P < 0.05, Duncan's post hoc test). No. of animals in each group is shown in parentheses.

 
Typical hypercapnia-induced hypothermia was observed in both groups, with no statistical difference between groups (Fig. 7B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The present data provide evidence that RMg serotoninergic neurons specifically play a key role in the ventilatory response to CO2. This finding adds to previous studies reporting that medullary raphe as a whole is involved in this response. Actually, specific lesion of the RMg serotoninergic neurons caused a 31% reduction in the ventilatory response, whereas medullary raphe as a whole causes ~16% reduction in the ventilatory response to the same amount of CO2 (44), indicating a major role of RMg among medullary raphe. It will be of interest to compare and contrast the role of other raphe nuclei in the hypercapnic ventilatory response when data are available.

In the present study we have used ibotenic acid lesion as a preliminary approach to produce unspecific ablation and provide the basis for more refined lesion. We also used anti-SERT-SAP to identify whether the serotoninergic neurons or other cell types in the RMg account for the changes in the respiratory responses. This compound, designed to kill neurons that express SERT, has been used successfully by others (44).

The major findings of the present study were the following: 1) RMg plays an excitatory role in the ventilatory but not in the thermal response to hypercapnia, and 2) RMg serotonergic neurons participate in the ventilatory response to CO2. Supporting these findings, injection of both ibotenic acid or anti-SERT-SAP caused no change in thermal responses to CO2 but reduced the hypercapnic ventilatory response.

Tonic Drive Breathing

As shown in Figs. 5 and 6, neither nonspecific lesion nor the serotonergic cell killing within the RMg caused any consistent change in the VE during air breathing. This result indicates that RMg serotonergic neurons play no role in the tonic maintenance breathing.

Role of RMg on Ventilatory Response to Hypercapnia

The MRR has been extensively studied because it has been implicated in chemoreception (3, 42, 60). As to RMg, Teppema et al. (59) demonstrated that the neuronal pathway activated during hypercapnia includes the RMg by showing that this area presents increased c-fos staining during high levels of CO2 exposure. In the present study, we demonstrated that the nonspecific cell killing in the RMg neurons caused a decreased ventilatory response to hypercapnia (Fig. 1). This response was due to a lower VT and f. These results are consistent with the notion that RMg has a role in chemoreception. However, the phenotype of neurons responsible for this response is unknown. The neurons within the RMg are heterogeneous; however, the principal cell type is serotonergic, comprising 15–20% of RMg neurons (17). Much attention has been given to the serotonergic raphe neurons because abnormalities in this system have been implicated in sudden infant death syndrome (26, 27, 28, 45, 46, 54).

To investigate the hypothesis that RMg serotonergic neurons are involved in ventilatory response to hypercapnia, we microinjected anti-SERT-SAP within this nucleus. We observed that a reduction of ~35% of serotonergic neurons was associated with a reduction in the response to CO2 by ~24%. These data provide evidence that the role of RMg in the ventilatory response to hypercapnia may arise mainly from serotonergic neurons of this nucleus.

Animals injected with ibotenic acid had a decrease in both VT and f, while the anti-SERT-SAP group had no effect on the f. On the basis of these observations, we may speculate that other cell types, besides the serotonergic neurons, may have different projections to other areas responsible for the control of breathing within the central nervous system. Further studies are needed to clarify this matter.

Recently a number of different reports attempt to characterize the physiological role of the serotonergic neurons of MRR, and it has been suggested that these neurons are chemosensitive (50, 51, 52, 61). In vivo studies have shown that MRR serotonergic neurons have an important role in the CO2 response, as demonstrated by microdialysis-induced focal acidification and by selective lesioning of serotonergic neurons in goats and rats (20, 21, 31, 42, 44). In a previous study, Nattie et al. (44) demonstrated that the loss of medullary serotonergic neurons by anti-SERT-SAP injections reduced slightly the ventilatory response to CO2. In the present study, the specific lesion of RMg serotoninergic neurons caused a major decrease in the hypercapnic ventilatory response (31%), indicating that this nucleus exerts an important contribution to CO2 drive to breathing. It is important to mention that as shown in Fig. 4, the lesions in the present study took place approximately at the level of –10.52 mm and 11.30 mm from bregma, i.e., they affected mostly the caudal portion of the RMg.

Recently, Penatti et al (48) reported that 5-HT neurons may affect CO2 sensitivity in male newborn piglets during NREM sleep and the frequency response to hypoxia in both sexes, indicating that the 5-HT system plays an important role in the control of breathing during development. However, the same study also documented that lesions of the same neurons have no impact on the CO2 response of piglets. Reconciling the existing data, one can come to the conclusion that the role of 5-HT neurons of RMg depends on development.

Thermoregulatory Response to Hypercapnia

A decrease in body temperature during hypercapnia has often been observed in several species, ranging from amphibians (7) to mammals (56). Lai et al. (30) showed that in rats, even when O2 consumption increased during hypercapnia, body temperature consistently decreased by 1–1.5°C, a phenomenon which probably reflects the heat loss of hyperpnea and vasodilatation. According to Saiki and Mortola (53), hypercapnia may interfere with thermoregulation largely as a result of the increase in heat dissipation, whereas the effect of CO2 on heat production may be indirect, via the decrease in body temperature.

Studies have shown that MRR can be a sympathetic premotor region involved in thermoregulation (40). Stimulation of MRR increases the sympathetic activity of the thermoregulatory effector organs: brown adipose tissue (38, 39), tail artery of rats (6, 49) and ear pinna blood vessels of rabbits (5). Our results showed that either ibotenic acid or anti-SERT-SAP microinjections within the RMg does not affect hypercapnic hypothermia, indicating that the RMg plays no role in the hypothermic response to hypercapnia. This is in agreement with a previous study of our laboratory that demonstrated that RMg do not participate in the hypoxia-induced hypothermia (18). It is possible that other raphe nuclei participate in this response, such as raphe pallidus.

In summary, the present study provides evidence that RMg plays a role in the ventilatory but not in the thermal response to hypercapnia, suggesting a major role of RMg among medullary raphe in CO2 drive to breathing.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Programa de Apoio para grupos de excelência (PRONEX). M. B. Dias was the recipient of a FAPESP scholarship.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Gustavo Michel Batista de Souza and Lidiane C. Anastacio for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. G. S. Branco, Faculdade de Odontologia de Ribeirão Preto, USP, 14040-904 Ribeirão Preto, SP, Brazil (e-mail: branco{at}forp.usp.br)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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