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Although a normal respiratory rhythm is vital at birth, little is known about the genetic factors controlling the prenatal maturation of the respiratory network in mammals. In Phox2a mutant mice, which do not express A6 neurons, we previously hypothesized that the release of endogenous norepinephrine by A6 neurons is required for a normal respiratory rhythm to occur at birth. Here we investigated the role of the Ret gene, which encodes a transmembrane tyrosine kinase receptor, in the maturation of norepinephrine and respiratory systems. As Ret-null mutants ( Ret–/–) did not survive after birth, our experiments were performed in wild-type (wt) and Ret–/– fetuses exteriorized from pregnant heterozygous mice at gestational day 18. First, in wt fetuses, quantitative in situ hybridization revealed high levels of Ret transcripts in the pontine A5 and A6 areas. Second, in Ret–/– fetuses, high-pressure liquid chromatography showed significantly reduced norepinephrine contents in the pons but not the medulla. Third, tyrosine hydroxylase immunocytochemistry revealed a significantly reduced number of pontine A5 and A6 neurons but not medullary norepinephrine neurons in Ret–/– fetuses. Finally, electrophysiological and pharmacological experiments performed on brainstem ‘en bloc’ preparations demonstrated impaired resting respiratory activity and abnormal responses to central hypoxia and norepinephrine application in Ret–/– fetuses. To conclude, our results show that Ret gene contributes to the prenatal maturation of A6 and A5 neurons and respiratory system. They support the hypothesis that the normal maturation of the respiratory network requires afferent activity corresponding to the A6 excitatory and A5 inhibitory input balance. [ABSTRACT FROM AUTHOR]

Although respiration is vital to the survival of all mammals from the moment of birth, little is known about the genetic factors controlling the prenatal maturation of this physiological process. Here we investigated the role of the Phox2a gene that encodes for a homeodomain protein involved in the generation of noradrenergic A6 neurons in the maturation of the respiratory network. First, comparisons of the respiratory activity of fetuses delivered surgically from heterozygous Phox2a pregnant mice on gestational day 18 showed that the mutants had impaired in vivo ventilation, in vitro respiratory-like activity, and in vitro respiratory responses to central hypoxia and noradrenaline. Second, pharmacological studies on wild-type neonates showed that endogenous noradrenaline released from pontine A6 neurons potentiates rhythmic respiratory activity via α1 medullary adrenoceptors. Third, transynaptic tracing experiments in which rabies virus was injected into the diaphragm confirmed that A6 neurons were connected to the neonatal respiratory network. Fourth, blocking the α1 adrenoceptors in wild-type dams during late gestation with daffy injections of the α1 adrenoceptor antagonist prazosin induced in vivo and in vitro neonatal respiratory deficits similar to those observed in Phox2a mutants. These results suggest that noradrenaline, A6 neurons, and the Phox2a gene, which is crucial for the generation of A6 neurons, are essential for development of normal respiratory rhythm in neonatal mice. Metabolic noradrenaline disorders occurring during gestation therefore may induce neonatal respiratory deficits, in agreement with the catecholamine anomalies reported in victims of sudden infant death syndrome. [ABSTRACT FROM AUTHOR]

Previous experiments performed in the in vitro newborn rat brainstem-spinal cord preparation reported that the addition of serotonin (5-HT, 30-50 μM) to the bathing medium induced increases in the respiratory frequency and a large tonic discharge on all the cervical ventral roots. The aim of the present work was to define whether the 5-HT-induced tonic discharge involved respiratory or non-respiratory motoneurones. Intracellular recordings demonstrated that cervical (C2) motoneurones ( n = 27) were depolarized by 5-HT but that the 5-HT-induced tonic discharge was mainly due to recruitment of silent motoneurones ( n = 18) which fired permanently (15/18; 17 ±3 Hz) under 5-HT. The respiratory motoneurones ( n = 9) retained a phasic inspiratory discharge (5/9), even if some (4/9) occasionally exhibited a few spikes during expiration. Therefore, it is concluded that the 5-HT-induced tonic discharge is unlikely to have functional significance in respiration. [ABSTRACT FROM AUTHOR]

1. Intracellular recordings were made from 220 Phrenic Motoneurons (PM) in anaesthetized, spontaneously breathing cats, deafferented from C3 to C7, in order to look for somatic events related to the Recurrent Responses (RR) evoked in PM axons by repetitive stimulation of the phrenic nerve. RR appear sporadically at a constant latency, originate from a spinal nicotinic mechanism and can be evoked in a PM without the presence of an antidromic volley in its axon (Khatib et al. 1986). 2. Using stimuli effective for eliciting RR in axons, we failed to observe intracellularly somatic events corresponding to RR after the occurence of an antidromic action potential. RR were observed extracellularly in two cases, but in both cases the recording originated from axons. 3. We attempted to elicit somatic RR without a preceding antidromic action potential, using either parathreshold stimulation of the impaled PM, or suprathreshold stimulation of a phrenic strand which excluded the axon of the impaled PM. In both cases, RR-like events, with very stable latencies, appeared sporadically in 4/142 and 2/15 PMs respectively. 4. Parathreshold stimuli or stimulation of a strand were coupled with averaging of the synaptic noise in order to look for small events temporally related to the stimuli. Short latency small depolarizations, looking-like recurrent EPSPs, were revealed in 22/142 and 5/15 PMs respectively. 5. These results confirm the existence of interrelations between PMs, providing for re-excitation and coupling within the phrenic pool, in addition to centrally imposed synchronization. [ABSTRACT FROM AUTHOR]

1. Interactions between phrenic motoneurons have been analysed in anaesthetized, paralyzed cats after C3 to C7 deafferentation. Effects of electrical stimulation of the C5 phrenic axons have been studied on thin filaments dissected from the stimulated nerve. Repetitive stimulation could elicit, after the primary direct response of the stimulated axons, a secondary response named Recurrent Response, RR. 2. RRs have been obtained in 117/186 phrenic axons. They appear sporadically (mean occurrence: 3.75 RRs elicited by 100 shocks of stimulation) at a constant latency. They originate from a spinal mechanism since they persist after C2 transection and disappear after section of the ventral roots. 3. The mechanism responsible for RR shows spatial and temporal facilitation. The RR probability increases with the number of antidromically invaded motoneurons as revealed by changes either of stimulation intensity or of central respiratory drive. However, RR could be evoked in a motoneuron without an antidromic volley in its axon. 4. Systemic injections of nicotinic blocking drugs such as dihydro-β-erytroidin or mecamylamine decrease or suppress the occurrence of RR; therefore, cholinergic synapses are involved in the RR generating process. 5. RR are assumed to be due to direct excitatory interactions between homonymous motoneurons. Recurrent axon collaterals impinging directly on neighbouring motoneurons would link together the different motoneurons of the phrenic pool. The functional significance of this phenomenon is discussed. [ABSTRACT FROM AUTHOR]

In anaesthetized cats (chloralose-urethan) the effects of brief tetanic electrical stimulation (50 to 100 ms) of the mesencephalic central gray matter and reticular formation on the inspiratory on-switch were studied during the expiratory (E) phase on the gross and unitary activities of phrenic, laryngeal inspiratory and laryngeal expiratory nerves. On the inspiratory laryngeal and phrenic nerves, stimulation elicited a short latency gross response concomitant with the train: the inspiratory Primary Response (Prim.R.) which is followed by an inspiratory Patterned Response (Patt.R.) of longer duration which corresponded to the inspiratory on-switch. The Patt.R. generally appeared from the Prim.R. within a latent period (Silent Phase: Sil.P.) as long as 100 ms. On the expiratory laryngeal nerve, stimulation elicited a brief activation (expiratory Prim.R.) concomitant with the beginning of the inspiratory laryngeal Prim.R. and which rapidly stopped as the latter continued during the stimulus train. The inspiratory Prim.R. corresponded to a simultaneous activation of both early and late (so defined during their spontaneous discharge) inspiratory motoneurones. The laryngeal motoneurones were more strongly activated than the phrenic ones. During the inspiratory Patt.R. all the phrenic motoneurones presented a recruitment delay earlier, compared with the spontaneous one, whereas the recruitment drastically changed from an inspiratory laryngeal motoneurone to another. Thus, the two pools of motoneurones presented different properties of activation. During the inspiratory Sil.P. no concomitant expiratory laryngeal activation was observed when most of the inspiratory motoneurones were inactive. As some inspiratory laryngeal motoneurones did not stop firing, the existence of some central respiratory neurones exhibiting a similar persistent activity and subserving the inspiratory on-switch mechanisms may be hypothesized. [ABSTRACT FROM AUTHOR]