094 - The homeodomain protein Dbx1 specifies a vital population of respiratory interneurons in the mouse - 05/12/08

Recent advances in the neurobiology of breathing indicate that the Respiratory Rhythm Generator (RRG) relies on the coupling of two distinct oscillators ventrally located in the brainstem: the para-facial respiratory group (pFRG), comprising pre-inspiratory neurons, and the PreBötzinger complex (PreBötC) playing a dominant role in pacing inspiration. Although vital for life ex utero, the prenatal development of the RRG remains poorly understood. Particularly little is known about the origin in the neural tube of interneurons forming these oscillators. We are addressing this issue considering homeodomain proteins defining distinct progenitor domains along the dorsal ventral (DV) axis of the neural tube. We here report on the role of Dbx1, expressed in the domain of neural progenitors giving rise to the V0 class of interneurons in the mouse. Electrophysiological recordings performed on en bloc and preBötC slice preparations from Dbx1 null (Dbx1LacZ/LacZ) embryos show that the preBötC, but not the pFRG, fails to become active. At birth, Dbx1 null neonates immediately die from a complete failure to breathe. Anatomical analysis in Dbx1LacZ/+ embryos revealed the presence of beta-Gal expressing cells in the preBötC but not the pFRG area. Optical recording of these cells in the Dbx1GFP/GFP line using the calcium indicator Calcium-green- 1 AM ascertained their rhythmic status. The majority of beta-Gal cells in the preBötC expressed the mRNA for the type 2 vesicular glutamate transporter (vGlut2) suggesting their role in excitatory synaptic processing. Indeed, the absence of rhythmic activity in Dbx1LacZ/LacZ embryos was associated to a massive loss of vGlut2 expressing cells in the preBötC Glutamatergic synaptic transmission (i) within the preBötC on one side and (ii) over the midline across the left and right preBötC was found impaired in the mutant. We conclude that Dbx1- derived V0 interneurons constitute an obligatory component of the preBötC oscillator.