Defective exercise-related expiratory muscle recruitment in patients with PHOX2B mutations: A clue to neural determinants of the congenital central hypoventilation syndrome.

INTRODUCTION AND OBJECTIVES: The human congenital central hypoventilation syndrome (CCHS) is caused by mutations in the PHOX2B (paired-like homeobox 2B) gene. Genetically engineered PHOX2B rodents exhibit defective development of the brainstem retrotrapezoid nucleus (RTN), a carbon dioxide sensitive structure that critically controls expiratory muscle recruitment. This has been linked to a blunted exercise ventilatory response. Whether this can be extrapolated to human CCHS is unknown and represents the objective of this study. MATERIALS AND METHODS: Thirteen adult CCHS patients and 13 healthy participants performed an incremental symptom-limited cycle cardiopulmonary exercise test. Responses were analyzed using guideline approaches (ventilation V'E, tidal volume VT, breathing frequency, oxygen consumption, carbon dioxide production) complemented by a breathing pattern analysis (i.e. expiratory and inspiratory reserve volume, ERV and IRV). RESULTS: A ventilatory response occurred in both study groups, as follows: V'E and VT increased in CCHS patients until 40 W and then decreased, which was not observed in the healthy participants (p<0.001). In the latter, exercise-related ERV and IRV decreases attested to concomitant expiratory and inspiratory recruitment. In the CCHS patients, inspiratory recruitment occurred but there was no evidence of expiratory recruitment (absence of any ERV decrease, p<0.001). CONCLUSIONS: Assuming a similar organization of respiratory rhythmogenesis in humans and rodents, the lack of exercise-related expiratory recruitment observed in our CCHS patients is compatible with a PHOX2B-related defect of a neural structure that would be analogous to the rodents' RTN. Provided corroboration, ERV recruitment could serve as a physiological outcome in studies aiming at correcting breathing control in CCHS.

[Alone or combined with serotoninergic agents, the progestins of the gonane family may represent a therapeutic alternative for Ondine's curse syndrome]. / Les progestatifs de la famille des gonanes, seuls ou associés à des agents sérotoninergiques, une éventuelle piste thérapeutique pour le syndrome d'Ondine.

Central hypoventilation syndromes such as Ondine's curse are rare neurorespiratory diseases associated with dysregulation of the vegetative (or autonomous) nervous system. They are characterized by a defect in the automatic control of ventilation, with a severe reduction or even the absence of CO2/H+ chemosensitivity. As no effective pharmacotherapy currently exists, lifelong mechanical ventilation is the only available treatment for patients suffering from these syndromes. In this context, clinical observation and studies in animal models suggest that gonane progestins, alone or in combination with serotonergic agents, could constitute a therapeutic alternative.

Growth restriction induced by chronic prenatal hypoxia affects breathing rhythm and its pontine catecholaminergic modulation.

Impaired transplacental supply of oxygen leads to intrauterine growth restriction, one of the most important causes of perinatal mortality and respiratory morbidity. Breathing rhythm depends on the central respiratory network modulated by catecholamines. We investigated the impact of growth restriction, using prenatal hypoxia, on respiratory frequency, on central respiratory-like rhythm, and on its catecholaminergic modulation after birth. At birth, respiratory frequency was increased and confirmed in en bloc medullary preparations, where the frequency of the fourth cervical (C4) ventral root discharge was increased, and in slice preparations containing the pre-Bötzinger complex with an increased inspiratory rhythm. The inhibition of C4 burst discharge observed in pontomedullary preparations was stronger in the growth-restricted group. These results cannot be directly linked by the tyrosine hydroxylase activity increase of A1/C1 and A2/C2 cell groups in the medulla since blockade of α1- and α2-adrenergic receptors did not abolish the difference between both groups. However, in pontomedullary preparations, the stronger inhibition of C4 burst discharge is probably supported by an increased inhibition of A5, a respiratory rhythm inhibitor pontine group of neurons displaying increased tyrosine hydroxylase activity, because blockade of α2-adrenergic receptors abolished the difference between the two groups. Taken together, these results indicate that growth restriction leads to a perturbation of the breathing frequency, which finds, at least in part, its origin in the modification of catecholaminergic modulation of the central breathing network.

Anandamide centrally depresses the respiratory rhythm generator of neonatal mice.

Endogenous cannabinoid receptors are widely distributed throughout the CNS, including the brainstem, and modulate a variety of functions, including breathing. In adult rats, activation of cannabinoid 1 receptors has been shown to depress breathing. Here in neonatal mice, we used in vitro electrophysiology, pharmacology, and immunohistochemistry to analyse the central effects of the endocannabinoid anandamide (AEA) on the activity of the medullary respiratory rhythm generator (RRG). First of all, in vitro electrophysiology on medullary preparations has revealed that bath application of AEA (30 µM, 15 min) significantly depressed respiratory activity. Secondly, applying pre-treatments with alpha-1 (Prazosin, 5 µM, 10 min) and alpha-2 (Yohimbine, 5 µM, 10 min) adrenoceptor antagonists prior to AEA application abolished the AEA-induced depression of the RRG. Finally, immunostaining revealed a dense network of fibres positive for the cannabinoid 1 receptor in the ventrolateral medulla (VLM), a region known to contain both the RRG and the modulatory A1/C1 catecholaminergic group. Moreover, cannabinoid 1 receptor positive fibres were found in close apposition with A1/C1 catecholaminergic cells, identified by the presence of tyrosine hydroxylase. In regard of our electrophysiological, pharmacological and immunostaining results, we conclude that AEA has a central depressive effect on the neonatal RRG, probably via the medullary A1/C1 catecholaminergic neurons which are already known to modulate the respiratory rhythm generator.

Antenatal environmental stress and maturation of the breathing control, experimental data.

The nervous respiratory system undergoes postnatal maturation and yet still must be functional at birth. Any antenatal suboptimal environment could upset either its building prenatally and/or its maturation after birth. Here, we would like to briefly summarize some of the major stresses leading to clinical postnatal respiratory dysfunction that can occur during pregnancy, we then relate them to experimental models that have been developed in order to better understand the underlying mechanisms implicated in the respiratory dysfunctions observed in neonatal care units. Four sections are aimed to review our current knowledge based on experimental data. The first will deal with the metabolic factors such as oxygen and glucose, the second with consumption of psychotropic substances (nicotine, cocaine, alcohol, morphine, cannabis and caffeine), the third with psychoactive molecules commonly consumed by pregnant women within a therapeutic context and/or delivered to premature neonates in critical care units (benzodiazepine, caffeine). In the fourth section, we take into account care protocols involving extended maternal-infant separation due to isolation in incubators. The effects of this stress potentially adds to those previously described.

Shortened seasonal photoperiodic cycles accelerate aging of the diurnal and circadian locomotor activity rhythms in a primate.

The gray mouse lemur (Microcebus murinus), a prosimian primate, exhibits seasonal rhythms strictly controlled by photoperiodic variations. Previous studies indicated that longevity can be altered by long-term acceleration of seasonal rhythms, providing a model for assessing various aspects of aging. To assess the effect of aging and accelerated aging on the circadian system of this primate, we compared the circadian rhythm of the locomotor activity in adult mouse lemurs (2-4.5 years, n = 9), aged mouse lemurs (5-9 years, n = 10), and adult mouse lemurs that had been exposed from birth to a shortened seasonal photoperiodic cycle (2-4.5 years, n = 7). Compared to adult animals, aged mouse lemurs showed a significant increase in intradaily variability and an advanced activity onset. Aging was characterized by a decrease in amplitude, with both a decrease in nocturnal activity and an increase in daytime activity. When maintained in constant dim red light, aged animals exhibited a shortening of the free-running period (22.8 +/- 0.1 h) compared to adult animals (23.5 +/- 0.1 h). A 3- to 5-year exposure to an accelerated seasonal photoperiodic rhythm ("annual" duration of 5 months) in accelerated mouse lemurs produced disturbances of the locomotor activity rhythm that resembled those of aged mouse lemurs, whether animals were studied in entrained or in free-running conditions. The present study demonstrated a weakened and fragmented locomotor activity rhythm during normal aging in this primate. Increasing the number of expressed seasonal cycles accelerated aging of parameters related to circadian rhythmicity in adult animals.

Redistribution of CB1 cannabinoid receptors during evolution of cholinergic basal forebrain territories and their cortical projection areas: a comparison between the gray mouse lemur (Microcebus murinus, primates) and rat.

Endocannabinoid signaling, mediated by presynaptic CB1 cannabinoid receptors on neurons, is fundamental for the maintenance of synaptic plasticity by modulating neurotransmitter release from axon terminals. In the rodent basal forebrain, CB1 cannabinoid receptor-like immunoreactivity is only harbored by a subpopulation of cholinergic projection neurons. However, endocannabinoid control of cholinergic output from the substantia innominata, coincident target innervation of cholinergic and CB1 cannabinoid receptor-containing afferents, and cholinergic regulation of endocannabinoid synthesis in the hippocampus suggest a significant cholinergic-endocannabinergic interplay. Given the functional importance of the cholinergic modulation of endocannabinoid signaling, here we studied CB1 cannabinoid receptor distribution in cholinergic basal forebrain territories and their cortical projection areas in a prosimian primate, the gray mouse lemur. Perisomatic CB1 cannabinoid receptor immunoreactivity was unequivocally present in non-cholinergic neurons of the olfactory tubercule, and in cholecystokinin-containing interneurons in layers 2/3 of the neocortex. Significantly, CB1 cannabinoid receptor-like immunoreactivity was localized to cholinergic perikarya in the magnocellular basal nucleus. However, cortical cholinergic terminals lacked detectable CB1 cannabinoid receptor levels. A dichotomy of CB1 cannabinoid receptor distribution in frontal (suprasylvian) and parietotemporal (subsylvian) cortices was apparent. In the frontal cortex, CB1 cannabinoid receptor-containing axons concentrated in layers 2/3 and layer 6, while layer 4 and layer 5 were essentially devoid of CB1 cannabinoid receptor immunoreactivity. In contrast, CB1 cannabinoid receptors decorated axons in all layers of the parietotemporal cortex with peak densities in layer 2 and layer 4. In the hippocampus, CB1 cannabinoid receptor-containing terminals concentrated around pyramidal cell somata and proximal dendrites in the CA1-CA3 areas, and granule cell dendrites in the molecular layer of the dentate gyrus. CB1 cannabinoid receptors frequently localized to inhibitory GABAergic terminals while leaving glutamatergic boutons unlabeled. Aging did not affect either the density or layer-specific distribution of CB1 cannabinoid receptor-immunoreactive processes. We concluded that organizing principles of CB1 cannabinoid receptor-containing neurons and their terminal fields within the basal forebrain are evolutionarily conserved between rodents and prosimian primates. In contrast, the areal expansion and cytoarchitectonic differentiation of neocortical subfields in primates is associated with differential cortical patterning of CB1 cannabinoid receptor-containing subcortical and intracortical afferents.

Fos study of ponto-medullary areas involved in the in vitro hypoxic respiratory depression.

In this study, the brainstem-spinal cord preparation isolated from newborn rats, an established model for the study of the hypoxic respiratory depression (HRD), has been used. The comparison of Fos expression in ponto-medullary areas in these preparations placed either in normoxic or hypoxic conditions suggests that only the retrotrapezoid nucleus (RTN) and the ventrolateral medulla (VLM) are involved in the in vitro HRD. Hypoxic preparations exhibit a Fos expression enhanced in the RTN, suggesting that the RTN might play a crucial role in the HRD. As well as this, VLM neurons presented a decrease in Fos expression that could be related to the decline of the respiratory output induced by hypoxia.

5-HT(2A/2C) receptor-mediated hypopnea in the newborn rat: relationship to Fos immunoreactivity.

Previous data derived from anesthetized, decerebrate, or in vitro preparations suggested that 5-HT(2) receptor activation might be responsible for respiratory dysfunction. Such a mechanism has not yet been documented in the intact animal, but might be of clinical relevance to the apneic spells of the premature infant. In the present investigation on conscious newborn rats we analyzed the respiratory response to the activation of 5-HT(2A/2C) receptors by the agonist 1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and we delineated central structures possibly involved in this response, using Fos expression as a marker of neuronal activation. We demonstrated that intraperitoneal injection of 5 mg/kg DOI produced a long-lasting decrease in respiratory frequency and tidal volume, which could be blocked by the antagonist ritanserin. Fos immunohistochemistry suggested that the rostral ventrolateral medulla and the lateral paragigantocellular nucleus might have a key role in the respiratory response to 5-HT(2) receptor activation. In addition, double immunostaining for Fos and tyrosine hydroxylase suggested that the contribution of catecholaminergic neurons to this response might be modest and indirect.

5-HT acting on 5-HT(1/2) receptors does not participate in the in vitro hypoxic respiratory depression.

The involvement of serotoninergic mechanisms in the central respiratory depression produced by hypoxia was studied in the newborn rat brainstem-spinal cord preparation. The respiratory frequency measured by the C4 ventral root activity was recorded. 5-HT (30 microM) superfusion elicited a rapid increase in respiratory frequency, prevented by a treatment with methysergide (a 5-HT(1/2) receptor antagonist) (40 microM). To investigate the possible participation of 5-HT in hypoxic respiratory depression, this concentration of methysergide was added to the bathing medium during hypoxia. Methysergide did not modify the decrease in respiratory frequency produced by hypoxia. In order to ensure that other 5-HT subtype receptors were not involved in hypoxic respiratory depression, 5-HT was added to the bath during hypoxic-methysergide tests; no effect on respiratory frequency was observed. These results suggest that in the newborn rat brainstem-spinal cord preparation, serotoninergic mechanisms are not involved in the elaboration of the in vitro respiratory response to hypoxia.

Possible role of retrotrapezoid nucleus and parapyramidal area in the respiratory response to anoxia: an in vitro study in neonatal rat.

The brainstem-spinal cord preparation from neonatal rat has been used in several reports to evaluate the central effect of low oxygen level on the respiratory network. We demonstrate that bilateral lesion of retrotrapezoid nucleus and parapyramidal area unmasks an early reinforcement of the respiratory output in response to anoxia. This suggests that neurons in both areas might trigger or relay a central depressive influence of hypoxia on the respiratory network.

Postnatal changes in Fos-like immunoreactivity evoked by hypoxia in the rat brainstem and hypothalamus.

We have recently used Fos expression in adult rats to map neuronal populations activated in the brainstem and hypothalamus during the acute ventilatory response to moderate hypoxia (O(2) 11%). Although present at birth, this response evolves postnatally. The present investigation aimed at a better understanding of these maturational processes by delineating structures that might functionally develop after birth. The developmental pattern Fos expression evoked by hypoxia was analysed in rats aged from 0 to 26 postnatal days. The numbers of Fos positive neurons markedly increased with the age in the medullary areas related to respiratory control during the 2 first postnatal weeks. Thereafter, the response plateaued in the nucleus tractus solitarius and attenuated in the ventral medulla. In the upper brainstem (parabrachial area, central grey) and the hypothalamus (posterior and dorsomedial nuclei, ventral zone), Fos response to hypoxia was absent or weak at birth and increased until late development. The significance of the development of evoked Fos expression in these rostral sites is discussed together with their possible contribution to the maturation of O(2)-sensitive chemoreflex pathways.