Serotonergic neurons in the nucleus raphé obscurus are not involved in the ventilatory and thermoregulatory responses to hypoxia in adult rats
Introduction
Ventilatory responses to hypoxia involve the peripheral chemoreceptors which sense hypoxia and a central network where the sensory inputs are integrated. The peripheral chemoreceptor afferents make their first synapses in the nucleus tractus solitarii (NTS), and additional brainstem regions are involved in the full expression of the ventilatory responses to hypoxia (Gargaglioni et al., 2003, Ross et al., 1985, Takakura et al., 2006, Teppema et al., 1997).
The medullary raphé (MR) is connected with the NTS (Thor and Helke, 1987) as well as other regions of the respiratory control network (Hodges and Richerson, 2008, Richerson, 2004). Serotoninergic neurons (5-HT) are found in the brainstem in distinct cell groups classified as the raphe (Hilaire et al., 2010), which is anatomically subdivided into three nuclei in the MR: raphé magnus (RMg; rostral portion of MR), raphé pallidus (RPa; ventral portion of MR) and raphé obscurus (ROb; caudal portion of MR). Recent studies have suggested that these nuclei may be functionally distinct regarding their involvement in respiratory control (Besnard et al., 2009, Cao et al., 2006, da Silva et al., 2011, da Silva et al., 2012, Dias et al., 2008, Dias et al., 2007, Gargaglioni et al., 2003, Li et al., 2006). For instance, the RMg contributes more to the hypercapnic ventilatory response compared to the ROb (da Silva et al., 2011, da Silva et al., 2012, Dias et al., 2007, Li et al., 2006). Gargalioni and colleagues (Gargaglioni et al., 2003) demonstrated that chemically lesioning the RMg by ibotenic acid injection provoked an increased ventilatory response to hypoxia (7% O2), suggesting an inhibitory modulation of RMg neurons under this condition. The authors did not, however, assess the specific role of RMg 5-HT neurons. Furthermore, the role of the ROb region in hypoxic ventilatory responses has not yet been established, nor is the function of medullary 5-HT neurons clear during this response (Hodges et al., 2008, Li and Nattie, 2008, Nattie, 2008, Taylor et al., 2005b, Taylor et al., 2005a).
As well as increasing pulmonary ventilation, hypoxia also elicits a thermal response, named anapyrexia. This regulated drop in body temperature (Tb) (for detailed review see Steiner and Branco, 2002) is thought to be beneficial because it reduces oxygen demand when oxygen supply is limited. Brain serotonin has been implicated in the regulation of Tb (Feldberg and Myers, 1963, Myers, 1981, Ray et al., 2011, Steiner and Branco, 2002, Steiner et al., 2002). In this context, Gargaglioni et al. (2005) reported that 5-HT receptors (5-HT1A and 5-HT7) in the anteroventral preoptical region of hypothalamus, an important site for thermoregulation, modulate the hypoxia-induced drop in Tb. Other recent evidence points to the contribution of 5-HT neurons in thermoregulation (Hodges et al., 2008, Ray et al., 2011) and specifically those located in the medullary raphé (Berthoud et al., 2005, Cao et al., 2004, Martin-Cora et al., 2000, Morrison, 2004b).
Despite all of the evidence of ROb involvement in respiratory control (da Silva et al., 2011, Depuy et al., 2011, Hilaire et al., 2010, Hodges et al., 2008, Ptak et al., 2009, Ray et al., 2011, Taylor et al., 2005b, Zanella et al., 2008) and thermoregulation (Berthoud et al., 2005, Cao et al., 2004, Hodges et al., 2008, Morrison, 2004b) there are no reports available on the role of the ROb and their 5-HT neurons in hypoxia-induced hyperventilation and anapyrexia. Therefore, the objective of the present study was to evaluate to role of ROb and, more specifically, 5-HT neurons confined in the ROb on ventilatory and thermoregulatory responses to hypoxia. We did so by inducing either a non-specific (ibotenic acid) or a 5-HT specific lesion (anti-SERT-SAP) of the ROb and assessed pulmonary ventilation and Tb during normoxia and hypoxia exposure.
Section snippets
Animals
Animals (adult male Wistar rats; 275–300 g) were kept in the animal facility in a 12 h dark-light cycle (light on at 7am) and had free access to water and food. Animal care and all procedures were carried out in compliance with the guidelines set by SBCAL (Sociedade Brasileira de Ciência em Animais de Laboratório/Brazilian Society of Animal Science) and approved by the University of São Paulo Animal Care and Use Committee (Protocol # 040/2007).
Stereotaxic surgery and microinjection
Animals were submitted to general anesthesia
Nonspecific neuronal lesion (ibotenic acid)
Representative photomicrographs of the nonspecific lesions are shown in Fig. 1. In the IBO group, cresyl violet sections revealed typical aspects of ibotenic acid-induced neuronal degeneration, that is neuronal body rarefaction, edema, and gliosis in damaged areas. The mean distance (from bregma) for injection needle tips was −12.45 ± 0.55 mm (mean ± S.E.) (IBO group, n = 8) and −12.25 ± 0.62 mm (vehicle group, n = 7)
Specific 5-HT neuronal lesion:
The immunohistochemistry for 5-HT showed anti-SERT-SAP chemical lesions of the ROb 5-HT
Discussion
In the present study we assessed the contribution of the ROb in hypoxic ventilatory and thermoregulatory responses. Our data suggest that ROb, and 5-HT neurons within ROb, are not involved in the ventilatory and thermoregulatory responses to hypoxia. Given the difference that may exist comparing the rostral (RMg) to caudal (ROb) MR regions (Besnard et al., 2009, Cao et al., 2006, da Silva et al., 2012, Li et al., 2006), it is important to assess these MR areas separately in order to understand
Conclusion
Under our experimental conditions, the present study suggests that the ROb and, 5-HT neurons within, are not involved in the ventilatory response to hypoxia in conscious adult rats. In addition, we demonstrated that the ROb is not involved in the regulation of body temperature during normoxia and hypoxia. It remains possible, however, that a larger ROb lesion could elicit different responses.
Acknowledgments
We would like to thank Rubens F. de Melo for the excellent technical assistance in the histological procedures. We also would like to thank Dr. Ann Revill who kindly suggested English corrections to the manuscript. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: #07/51581-2 and #06/60696-5) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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