5-HT neurons of the medullary raphe contribute to respiratory control in toads

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Highlights

  • 5-HT immunoreactive neurons were found clustered at the midline of the brainstem of the toad Rhinella diptycha

  • The serotoninergic neurons of the medullary raphe nuclei of toads do not exert a tonic role in the respiratory control.

  • 5-HT specific chemical lesions of the medullary raphe promoted an attenuation of the respiratory chemoreflex responses to hypercarbia, similar to mammals.

  • Different from mammals, the serotoninergic neurons of the medullary raphe nuclei of toads play a excitatory role in the hypoxic ventilatory response.

Abstract

Air-breathing vertebrates undergo respiratory adjustments when faced with disturbances in the gas composition of the environment. In mammals, the medullary raphe nuclei are involved in the neuronal pathway that mediates the ventilatory responses to hypoxia and hypercarbia. We investigate whether the serotoninergic neurons of the medullary raphe nuclei of toads (Rhinella diptycha) play a functional role in respiratory control during resting conditions (room air), hypercarbia (5% CO2), and hypoxia (5% O2). The raphe nuclei were located and identified based on the location of the serotoninergic neurons in the brainstem. We then lesioned the medullary raphe (raphe pallidus, obscurus and magnus) with anti-SERT-SAP and measured ventilation in both control and lesioned groups and we observed that serotonin (5-HT) specific chemical lesions of the medullary raphe caused reduced respiratory responses to both hypercarbia and hypoxia. In summary, we report that the serotoninergic neurons of the medullary raphe of the cururu toad Rhinella diptycha participate in the chemoreflex responses during hypercarbia and hypoxia, but not during resting conditions. This current evidence in anurans, together with the available data in mammals, brings insights to the evolution of brain sites, such as the medullary raphe, involved in the ventilatory chemoreflex in vertebrates.

Introduction

In adult anurans, ventilatory adjustments ensure adequate oxygen (O2) uptake and carbon dioxide (CO2) removal when metabolic demands increase or the gas composition of the environment is altered (Gargaglioni and Milsom, 2007; Wang et al., 1999;). Environmental hypoxia or hypercarbia induces hyperpnea (Gamperl et al., 1999; Gargaglioni and Branco, 2003; Gargaglioni et al., 2002; Kruhøffer et al., 1987; Noronha-de-Souza et al., 2006). The system controlling the adequate respiratory output depends on the integration of sensory information concerning mechanical and gas exchange performance in the central nervous system (CNS) (Gargaglioni and Milsom, 2007).

The sensory pathways involved in the mediation of the respiratory responses to environmental hypoxia and hypercarbia have been extensively studied in adult anurans. During hypoxia, decreases in the O2 levels in the blood (Wang et al., 1994) are sensed by chemoreceptors of the systemic and possibly the pulmonary circulation (Ishii and Kusakabe, 1985; Van Vliet and West, 1992; Wang et al., 2004). In contrast, hypercarbia can be sensed not only by the chemoreceptors of the systemic circulation but also by CO2-sensitive chemoreceptors present in the airways and in the CNS (Branco et al., 1992; Coates and Ballam, 1990; Kinkead and Milsom, 1996; Noronha-de-Souza et al., 2006; Van Vliet and West, 1992; Santin and Hartzler, 2013). Additionally, pulmonary stretch receptors, that monitor the degree of inflation and deflation of the lung, are also sensitive to CO2 (Milsom and Jones, 1977).

Previous research suggests indirectly that the modulation of ventilation is a result of an integration of the sensory information from chemo- and mechanoreceptors in the CNS (reviewed by Reid, 2006, and Wang et al., 2004). However, few studies have directly addressed this issue. Some areas of the amphibian’s brainstem, such as the nucleus isthmi (NI) and the locus coeruleus (LC), have been considered essential for mediating the CO2 drive for breathing (Gargaglioni et al., 2002; Noronha-de-Souza et al., 2006). The NI also participates in the ventilatory response to hypoxia (Gargaglioni et al., 2002). Additionally, orexins contribute to the ventilatory response to hypoxia and hypercarbia in adult toads (Fonseca et al., 2016) and pre-metamorphic bullfrog tadpoles (Fonseca et al., 2021).

In mammals, the major sites involved in respiratory control receive serotoninergic input through projections from the medullary raphe nuclei (MR), which includes the raphe magnus, pallidus and obscurus (reviewed by Richerson, 2004). Furthermore, electrical stimulation of MR neurons in anesthetized rats can either inhibit or stimulate ventilatory movements, depending on the specific site stimulated (Cao et al., 2006). Studies examining c-fos expression suggest that the MR nuclei are not activated during resting situations but are involved in the ventilatory response to hypoxia and hypercapnia (Erickson e Millhorn, 1994; Larnicol et al., 1994; Teppema et al., 1997). Consistently, the serotoninergic neurons from the MR increase their firing rate during hypercapnia in awake cats (Veasey et al., 1995). Moreover, chemical lesion of the raphe magnus increases the ventilatory response to hypoxia, suggesting that this site exerts an inhibitory influence over ventilation during hypoxia (Gargaglioni et al., 2003). In contrast, chemical lesion of the raphe magnus attenuates the ventilatory response to hypercapnia, suggesting that this site has an excitatory influence during CO2 exposure (Dias et al., 2007).

The raphe nuclei of amphibians are possibly homologous to those of mammals (Adli et al., 1999), making them an ideal candidate for cardiorespiratory studies. In the literature, only one study showed that chemical and electrical stimulation of the MR influences ventilation in pre-metamorphic tadpoles using a reduced preparation of the brainstem of Lithobates catesbeianus, but no effect of this stimulation was found in adults (Belzile et al., 2007). To evaluate the participation of serotoninergic neurons of the medullary raphe in respiratory control in adult toads (Rhinella diptycha) during resting conditions and hypoxia or hypercarbia, we first located the raphe nuclei based on the location of serotoninergic neurons in the brainstem. Ventilation was then measured in non-anesthetized animals during normoxia, hypoxia, and hypercarbia after lesioning the 5-HT-neurons of the raphe pallidus, obscurus and magnus with anti-SERT-SAP.

Section snippets

Animals

Cururu toads Rhinella diptycha (Cope, 1862; previously known as Rhinella schneideri; cf. Lavilla and Brusquetti, 2018) of either sex, weighing 120-240 g, were collected in the vicinity of Jaboticabal, São Paulo, Brazil, during the rainy summer months from 2016 to 2019. The toads were captured and transported to the laboratory in agreement with the SISBIO-ICMBio (license #13243-1) and maintained at 25 °C and natural light-dark cycle in containers with free access to water and a basking area.

Location and identification of the raphe nuclei in the brainstem

At all four transverse levels analyzed (Fig. 1), 5-HT immunoreactive neurons were found clustered at the midline. At the rhombencephalic levels (Fig. 2 - Levels A, B, and C), the cells were found exclusively in a single cluster at the midline. At the mesencephalic level (Fig. 2 - Level D), immunoreactive cells were also arrayed laterally from the midline, whereas the cells at the midline formed two bilateral clusters. Adli et al. (1999) performed a complete analysis of the reticular formation

Discussion

In our study, we located the raphe nuclei of Rhinella diptycha based on the immunohistochemistry labeling of serotoninergic neurons in the brainstem and named them as described previously by Adli et al. (1999). Furthermore, we showed that 5-HT specific chemical lesions of the medullary raphe promoted an attenuation of the respiratory chemoreflex responses to hypercarbia and hypoxia. To our knowledge, this is the first study to specifically assess the role of the 5-HT medullary raphe neurons in

Author contribution

EMF, CNS and LHG designed the research. EMF and CNS performed the experiments and analyzed the data. EMF, CNS, LGSB, KCB and LHG interpreted the data. All authors provided critical and intellectual input during the preparation of the manuscript and approved the final version.

Declaration of Competing Interest

The authors report no declarations of interest.

Funding

This work received financial support from the Sao Paulo Research Foundation (FAPESP; 2019/09469-8 and 2016/17681-9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - 407490/2018-3).

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