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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]

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A-deficient (Tg8) strains, in which levels of endogenous serotonin (5-HT) were drastically increased, we investigated how 5-HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5-HT(2A) receptor (5-HT(2A)-R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5-HT(1B) receptor (5-HT(1B)-R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5-HT(2A)-R and 5-HT(1B)-R during maturation by treating pregnant C3H mice with a 5-HT(2A)-R agonist (2,5-dimethoxy-4-iodoamphetamine hydrochloride). This pharmacological overactivation of 5-HT(2A)-R induced a somatic expression of 5-HT(1B)-R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5-HT(2A)-R antagonist (ketanserin) decreased the 5-HT(1B)-R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5-dimethoxy-4-iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H- to a Tg8-like pattern. These results show that a prenatal 5-HT excess affects, via the overactivation of 5-HT(2A)-R, the expression of 5-HT(1B)-R in PhMns and the organization of their premotor network.

In vivo (plethysmography) and in vitro (en bloc preparations) experiments were performed from embryonic day 16 (E16) to postnatal day 9 (P9) in order to analyse the perinatal maturation of the respiratory rhythm-generator in mice. At E16, delivered foetuses did not ventilate and survive but at E18 they breathed at about 110 cycles/min with respiratory cycles of variable individual duration. From E18 to P0-P2, the respiratory cycles stabilised without changes in the breathing parameters. However, these increased several-fold during the next days. Hypoxia increased breathing frequency from E18-P5 and only significantly affected ventilation from P3 onwards. At E16, in vitro medullary preparations (pons resection) produced rhythmic phrenic bursts at a low frequency (about 5 cycles/min) with variable cycle duration. At E18, their frequency doubled but cycle duration remained variable. After birth, the frequency did not change although cycle duration stabilised. At E18 and P0-P2, the in vitro frequency decreased by around 50% under hypoxia, increased by 40-50% under noradrenaline or substance P and was permanently depressed by the pontine A5 areas. At E16 however, hypoxia had no effects, both noradrenaline and substance P drastically increased the frequency and area A5 inhibition was not expressed at this time. At E18 and P0-P2, electrical stimulation and electrolytic lesion of the rostral ventrolateral medulla affected the in vitro rhythm but failed to induce convincing effects at E16. Thus, a major maturational step in respiratory rhythmogenesis occurs between E16-E18, in agreement with the concept of multiple rhythmogenic mechanisms.

Substance P and neurokinin-1 receptors (NK1) modulate the respiratory activity and are expressed early during development. We tested the hypothesis that NK1 receptors are involved in prenatal development of the respiratory network by comparing the resting respiratory activity and the respiratory response to hypoxia of control mice and mutant mice lacking the NK1 receptor (NK1-/-). In vitro and in vivo experiments were conducted on neonatal, young and adult mice from wild-type and NK1-/- strains. In the wild strain, immunohistological, pharmacological and electrophysiological studies showed that NK1 receptors were expressed within medullary respiratory areas prior to birth and that their activation at birth modulated central respiratory activity and the membrane properties of phrenic motoneurons. Both the membrane properties of phrenic motoneurons and the respiratory activity generated in vitro by brainstem-spinal cord preparation from NK1-/- neonate mice were similar to that from the wild strain. In addition, in vivo ventilation recordings by plethysmography did not reveal interstrain differences in resting breathing parameters. The facilitation of ventilation by short-lasting hypoxia was similar in wild and NK1-/- neonates but was significantly weaker in adult NK1-/- mice. Results demonstrate that NK1 receptors do appear to be necessary for a normal respiratory response to short-lasting hypoxia in the adult. However, NK1 receptors are not obligatory for the prenatal development of the respiratory network, for the production of the rhythm, or for the regulation of breathing by short-lasting hypoxia in neonates.

The hyperpolarization-activated cationic current (I(h)) was characterized and its maturation studied on phrenic motoneurons (PMNs), from reduced preparations of foetal (E18 and E21) and newborn (P0-P3) rats, using the whole-cell patch-clamp technique. In voltage-clamp mode, 2-s hyperpolarizing steps (5-mV, -50 to -110 mV) elicited a noninactivating inward current, blocked by external application of Cs+ or ZD 7288. At -110 mV, Ih current density averaged 0.67 +/- 0.41 pA/pF at E18, reached a transient peak at E21 (1.38 +/- 0.11 pA/pF) and decreased at P0-P3 (0.77 +/- 0.22 pA/pF). V1/2 was similar at E18 and E21 (-79 mV) but was significantly hyperpolarized at P0-P3 (-90 mV). The time constant of activation was voltage-dependent, and significantly faster at E21. Reversal potential was similar at all ages when estimated by extrapolation or tail current procedures. It was positively shifted by 25 +/- 6 mV when external potassium was raised from 3 to 10 m M, suggesting a similar sensitivity to K+ from E18 to P0-3. Cs(+) or ZD 7288 applications on PMNs at rest in current-clamp mode, in a partitioned chamber, induced a 10 +/- 2 mV hyperpolarization at E18 and E21, and an 8 +/- 2 mV hyperpolarization at P0-3. The area of the central respiratory drive potential or current was increased by 33 and 31%, respectively, at E21, but was not significantly modified at E18 and P0-3. Our data suggest a critical period during the perinatal maturation of Ih during which it is transiently upregulated and attenuates the influence of the central respiratory drive on PMNs just prior to birth.

Experiments were carried out on the in vitro brainstem-spinal cord preparation of the newborn rat to analyse the effects of substance P (SP) on phrenic motoneuron (PMN) activity. In current-clamp mode, SP significantly depolarized PMNs, increased their input resistance, decreased the rheobase current and shifted the firing frequency-intensity relationships leftwards, but did not affect spike frequency adaptation or single spike configuration. The neurokinin receptor agonist NK1 had SP-mimetic effects, whereas the NK3 and NK2 receptor agonists were less effective and ineffective, respectively. In a tetrodotoxin-containing aCSF, only SP or the NK1 receptor agonist were still active. No depolarization was observed when the NK1 receptor agonist was applied in the presence of muscarine. In voltage-clamp mode, SP or the NK1 receptor agonist produced an inward current (ISP) which was not significantly reduced by extracellular application of tetraethylammonium, Co2+, 4-aminopyridine or Cs+. In aCSF containing tetrodotoxin, Co2+ and Cs+, ISP was blocked by muscarine. No PMN displayed any M-type potassium current but only a current showing no voltage sensitivity over the range -100 to 0 mV, reversing near the expected EK +, hence consistent with a leak current. SP application to the spinal cord only (using a partitioned chamber) significantly increased the phrenic activity. Pretreatment with the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) decreased the C4 discharge duration and blocked the effect of SP, thus exhibiting an NMDA potentiation by SP. In conclusion, SP modulates postsynaptically the response of phrenic motoneurons to the inspiratory drive through the reduction of a leak conductance and the potentiation of the NMDA component of the synaptic input.