Endogenous 5-HT_1/2 systems and the newborn rat respiratory control

Abstract

Consequences of 5-HT_1/2 systems blockade by methysergide on newborn rats respiratory drive were evaluated in vivo with unrestrained animals and in vitro using brainstem-spinal cord preparations. A decrease in respiratory frequency until a plateau level was observed under both in vivo (82.8±0.6% of control values) and in vitro (76.8±0.8% of control values) conditions whereas an increase in inspiratory amplitude (135.1±2.1% of control values) was only retrieved in vivo. By the use of the c-fos expression analysis, we correlated these effects with neuronal activity changes, particularly, in vivo in two key structures between the respiratory ponto-medullary network and the peripheral or suprapontine afferences, namely the commissural subnucleus of the nucleus of the solitary tract and the lateral parabrachial nucleus. Thus, peripheral and suprapontine inputs seem to be of a primeval importance in the respiratory influence of endogenous 5-HT. Besides, as 5-HT is involved in the respiratory perturbations that occur in sudden infant death syndrome (SIDS), our results suggest a participation of peripheral and suprapontine inputs in these disorders.

Introduction

The central serotoninergic systems consist of clusters of neurons containing serotonin (5-hydroxytryptamine; 5-HT) involved in several autonomic regulations such as the respiratory control (Halliday et al., 1995, Hilaire and Duron, 1999). Serotoninergic systems have been implicated in the occurrence of respiratory perturbations that happen in sudden infant death syndrome (SIDS) (Filiano et al., 1990, Caroff et al., 1992, Panigraphy et al., 2000, Kinney et al., 2001). Although serotoninergic mechanisms linked to SIDS are still misread, clinical studies suggested two leads. Cerebrospinal fluid levels of 5-HT metabolites such as the 5-hydroxyindoleacetic acid were measured in 102 infants during the first year of the life and compared with 53 victims of SIDS. The concentration of 5-HT metabolites was significantly higher in the SIDS group than in the control group (Caroff et al., 1992, Cann-Moisan et al., 1999). This suggests that an increase in 5-HT level is involved in the occurrence of SIDS. During the last decade, animal studies in vivo as in vitro have shown that an exogenous supply of 5-HT induced changes of the newborn respiratory control (Lindsay and Feldman, 1993, Khater-Boidin et al., 1996, Onimaru et al., 1998, Khater-Boidin et al., 1999, Schwarzacher et al., 2002), supporting the hypothesis that a high level of 5-HT participates in the occurrence of respiratory perturbations. On the other hand, it has been proposed that SIDS is due to a developmental abnormality in medullary serotoninergic neurons including the arcuate nucleus (putative human homologue of the parapyramidal group and the nucleus raphe pallidus of the rat). This abnormality results in a failure of protective response to life-threatening stresses (e.g. asphyxia, hypoxia, hypercapnia) during sleep as infants pass through a critical period in homeostatic control called the medullary serotoninergic network deficiency (Filiano et al., 1990, Kinney et al., 2001, Matturri et al., 2002). These anatomical perturbations confirmed by recent clinical data (Okado et al., 2002) indicate a decrease in the endogenous 5-HT level that could in turn participate in the occurrence of SIDS. A better understanding of the influence of the endogenous 5-HT on the respiratory control would permit to surround the possible dysfunction occurring during SIDS. Unfortunately, only few in vitro studies are available. These latter demonstrate that (i) endogenous 5-HT increases the respiratory frequency in newborn (Monteau et al., 1990, Morin et al., 1991, Cayetanot et al., 2001a, Pena and Ramirez, 2002) as during the foetal period (Di Pasquale et al., 1994), and ii) a depletion of 5-HT affects the capacity of respiratory centers to develop a stable respiratory activity at birth (Bou-Flores et al., 2000). In this context, the present work aims at clarifying the influence of the endogenous 5-HT on the respiratory control of the newborn rat. A blockade of the serotoninergic systems was performed with methysergide, an antagonist of the 5-HT_1/2 receptors that has been shown to be responsible for the 5-HT influence on the respiratory control (Morin et al., 1991, Hilaire and Duron, 1999, Onimaru et al., 1998). The use of methysergide as 5-HT antagonist is therefore justified by its lack of specificity upon type 1 or 2 of receptors. In order to evaluate the importance of peripheral and suprapontine inputs on the endogenous 5-HT influence on the respiratory control, we examined the consequences of the 5-HT_1/2 systems blockade on in vivo and in vitro preparations of newborn rats. First of all, these consequences on respiratory parameters and c-fos expression were evaluated in vivo using unrestrained animals as well as in vitro with brainstem-spinal cord preparations. The immunohistochemical revelation of the Fos protein was then used in order to identify areas that presented modifications of neuronal activity.