Central Respiratory Rhythmogenesis Is Abnormal inLbx1- Deficient Mice

Abstract

Lbx1 is a transcription factor that determines neuronal cell fate and identity in the developing medulla and spinal cord. Newborn Lbx1 mutant mice die of respiratory distress during the early postnatal period. Using in vitro brainstem-spinal cord preparations we tested the hypothesis that Lbx1 is necessary for the inception, development and modulation of central respiratory rhythmogenesis. The inception of respiratory rhythmogenesis at embryonic day 15 (E15) was not perturbed in Lbx1 mutant mice. However, the typical age-dependent increase in respiratory frequency observed in wild-type from E15 to P0 was not observed in Lbx1 mutant mice. The slow respiratory rhythms in E18.5 Lbx1 mutant preparations were increased to wild-type frequencies by application of substance P, thyrotropin releasing hormone, serotonin, noradrenaline, or the ampakine drug 1-(1,4-benzodioxan-6-yl-carbonyl) piperidine. Those data suggest that respiratory rhythm generation within the pre-Bötzinger complex (preBötC) is presumably functional in Lbx1 mutant mice with additional neurochemical drive. This was supported by anatomical data showing that the gross structure of the preBötC was normal, although there were major defects in neuronal populations that provide important modulatory drive to the preBötC including the retrotrapezoid nucleus, catecholaminergic brainstem nuclei, nucleus of the solitary tract, and populations of inhibitory neurons in the ventrolateral and dorsomedial medullary nuclei. Finally, we determined that those defects were caused by abnormalities of neuronal specification early in development or subsequent neuronal migration.

Defective Respiratory Rhythmogenesis and Loss of Central Chemosensitivity in Phox2b Mutants Targeting Retrotrapezoid Nucleus Neurons

“…Here, we strengthen the case for the RTN as principal chemosensor by showing that central chemosensitivity is lost in two additional mutations that affect the RTN but leave intact other candidate chemosensors. The CO 2 /pH responsiveness of other mutants affecting the RTN was either not explored in Lbx1 mutants (Pagliardini et al, 2008) or found to be preserved in vivo in Egr2 mutants (Jacquin et al, 1996). The latter result contrasts with the complete lack of chemosensitivity observed here in brainstem preparations of Egr2 cre ;Phox2b lox/lox embryos.…”

“…By genetic tracing, partnering either the Lbx1 cre or the Egr2 cre allele with the Tau GFPnLacZ reporter, we first confirmed published evidence (Pagliardini et al, 2008;Thoby-Brisson et al, 2009) that the majority of RTN neurons arise from the Lbx1 cre -expressing domain of the alar plate (Sieber et al, 2007), in the Egr2 creexpressing r3 or r5 (Voiculescu et al, 2001) (Fig. 3A-J (Fig.…”

“…RTN neurons have recently been found to be depleted in Lbx1 Ϫ/ Ϫ (Pagliardini et al, 2008) and Egr2 ) neonatal lethality and a slowed-down respiratory rhythm at birth (Jacquin et al, 1996;Pagliardini et al, 2008), again consistent with a role of the RTN in setting the appropriate respiratory pace. However, the massive reorganization of the hindbrain in these mutants may also contribute to the phenotype.…”

“…Hence, at least in vitro, a number of other candidate chemosensitive sites (Nattie and Li, 2009) are unable to compensate for the loss of RTN neurons. The candidate chemosensors de facto ruled out by our study include those that do not depend on Phox2b-5-HT neurons (Pattyn et al, 2003) and the preBötC (this paper)-and those that do not derive from Lbx1 ϩ precursors (and thus do not lose Phox2b expression in Phox2b lox/lox ;Lbx1 cre/ϩ mutants)-noradrenergic cell groups, including the locus ceruleus, and the nucleus of the solitary tract (Sieber et al, 2007;Pagliardini et al, 2008). Our results thus argue in favor of an eminent role for the small number of Phox2b ϩ sites, including the RTN, which are affected by both conditional mutations.…”

“…Rather, RTN neurons are a sparse collection of small-to mediumsized neurons that occupy an area with ill defined borders, ventral and immediately lateral and caudal to nVII (Cream et al, 2002;Takakura et al, 2008). Therefore, RTN neurons are best defined at this time by their functional and molecular properties: they are CO 2 -responsive, glutamatergic (Vglut2 ϩ ), TH-negative, Phox2b-expressing neurons, located parafacially near the medullary surface, that also express NK1R Stornetta et al, 2006;Dubreuil et al, 2008;Onimaru et al, 2008;Pagliardini et al, 2008;Takakura et al, 2008). This definition leaves out probably only a minority of neurons in the same location since Ͼ90% of the neurons under the lateral two-thirds of nVII appear to express Phox2b in the adult rat (Takakura et al, 2008).…”