Viemari J C, Maussion G, Bévengut M, Burnet H, Pequignot J M, Népote V, Pachnis V, Simonneau M, Hilaire G
FRE CNRS 2722, 280 Boulevard Sainte Marguerite, 13009 Marseille, France.
Eur J Neurosci. 2005 Nov;22(10):2403-12. doi: 10.1111/j.1460-9568.2005.04441.x.
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.
尽管正常的呼吸节律在出生时至关重要，但对于控制哺乳动物呼吸网络产前成熟的遗传因素却知之甚少。在不表达A6神经元的Phox2a突变小鼠中，我们之前推测A6神经元释放内源性去甲肾上腺素是出生时出现正常呼吸节律所必需的。在此，我们研究了编码跨膜酪氨酸激酶受体的Ret基因在去甲肾上腺素和呼吸系统成熟过程中的作用。由于Ret基因敲除突变体（Ret-/-）出生后无法存活，我们的实验在妊娠第18天从怀孕的杂合小鼠体内取出的野生型（wt）和Ret-/-胎儿中进行。首先，在野生型胎儿中，定量原位杂交显示脑桥A5和A6区域有高水平的Ret转录本。其次，在Ret-/-胎儿中，高压液相色谱显示脑桥中的去甲肾上腺素含量显著降低，但延髓中的含量未降低。第三，酪氨酸羟化酶免疫细胞化学显示，Ret-/-胎儿中脑桥A5和A6神经元的数量显著减少，但延髓去甲肾上腺素能神经元数量未减少。最后，对脑干“整体”标本进行的电生理和药理学实验表明，Ret-/-胎儿的静息呼吸活动受损，对中枢性缺氧和去甲肾上腺素应用的反应异常。总之，我们的结果表明Ret基因有助于A6和A5神经元以及呼吸系统的产前成熟。这些结果支持了这样一种假说，即呼吸网络的正常成熟需要与A6兴奋性和A5抑制性输入平衡相对应的传入活动。
