Hindbrain interneurons and axon guidance signaling critical for breathing : Nature Neuroscience : Nature Publishing Group

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Hindbrain interneurons and axon guidance signaling critical for breathing
Julien Bouvier,Muriel Thoby-Brisson,Nicolas Renier,Véronique Dubreuil,Johan Ericson,Jean Champagnat,Alessandra Pierani,Alain Chédotal& Gilles Fortin

Corresponding authors Journal name: Nature Neuroscience
Year published:
(2010)
DOI:
doi:10.1038/nn.2622
Received02 June 2010
Accepted22 July 2010
Published online02 August 2010

Abstract
Breathing is a bilaterally synchronous behavior that relies on a respiratory rhythm generator located in the brainstem. An essential component of this generator is the preBötzinger complex (preBötC), which paces inspirations. Little is known about the developmental origin of the interneuronal populations forming the preBötC oscillator network. We found that the homeobox gene Dbx1 controls the fate of glutamatergic interneurons required for preBötC rhythm generation in the mouse embryo. We also found that a conditional inactivation in Dbx1-derived cells of the roundabout homolog 3 (Robo3) gene, which is necessary for axonal midline crossing, resulted in left-right de-synchronization of the preBötC oscillator. Together, these findings identify Dbx1-derived interneurons as the core rhythmogenic elements of the preBötC oscillator and indicate that Robo3-dependent guidance signaling in these cells is required for bilaterally synchronous activity.

Figures at a glance
leftFigure 1: Disrupted breathing and rhythm generation in the preBötC of Dbx1 null mice.
(a,b) Plethysmographic recordings of the ventilation of Dbx1LacZ/+ (a) and Dbx1LacZ/LacZ (b) E18.5 embryos 1 min after surgical delivery. All of the Dbx1LacZ/+ mice initiated respiratory cycles of inspirations (upward deflections) and expirations (downward deflections), whereas none of the Dbx1LacZ/LacZ mice showed any sign of ventilation and all died cyanotic. (c,d) Bilateral rhythmic fluorescence changes (ΔF/F) of the preBötC in E15.5 slice preparations (c, dorsal at top) were absent in the mutant (d). (e,f) Corresponding concurrent electrophysiological (Int) and optical (ΔF/F) recordings. Spontaneous preBötC rhythmic bursts increased in frequency in the presence of substance P (SubP) in Dbx1LacZ/+ preparations (e). The preBötC was inactive in Dbx1LacZ/LacZ preparations and could not be induced by SubP (f). (g,h) Calcium imaging of the ventral surface in E15.5 Dbx1LacZ/+ (g) and Dbx1LacZ/LacZ (h) whole hindbrain preparations. Rhythmic activity of the e-pF oscillator (green outline), partially coupled at this stage to that of facial motor nucleus (nVII, white dotted outline), was maintained in the Dbx1 null mutant. (i,j) Simultaneous optical recordings of the e-pF (green) and of the nVII (black) and electrophysiological recording of the fourth cervical spinal root (C4) in Dbx1LacZ/+ (i) and Dbx1LacZ/LacZ (j) preparations. The rhythmic activity of the e-pF was unaffected in Dbx1LacZ/LacZ preparations, albeit in the absence of any activity of the motor outputs. All scale bars in images represent 500 μm.

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Figure 2: Dbx1-derived cells in the preBötC are rhythmically active.
(a) Expression of Dbx1 protein in a Dbx1LacZ/+ E10.5 transverse hindbrain section. (b) β-Gal protein expression. (c) β-Gal protein expression (green) in half of a E15.5 transverse section (midline at right) and Islet1,2 (red) expressed by the hypoglossal nucleus (XIIn) and the nucleus ambiguus (n.a.). (d) Peak fluorescence change (ΔF/F) during one burst of the preBötC in half of a physiological slice (midline at left). (e) Expression of Dbx1 in a Dbx1iresGFP E10.5 transverse hindbrain section. (f) GFP protein expression. (g) GFP expression in a E15.5 preBötC slice preparation during an electrophysiological recording session. (h) Same field (blue rectangle in g) showing GFP expression (red) and Calcium Green-1 AM–loaded cells (CaG, green). A merged image is shown on the right at a higher magnification and was used to derive the individual rhythmic activity of 11 double-labeled (yellow) preBötC cells. (i) Corresponding fluorescence changes of individual cells (black) and averaged preBötC signal (blue). (j) GFP expression (green) and differential interference contrast (DIC) image used to position the patch micropipette (arrows). (k) Membrane potential changes (top trace) of a GFP-positive preBötC neuron showing spontaneous rhythmic burst discharges of action potential phased to the integrated (Int) preBötC population bursting activity. (l) Membrane current changes in the same neuron showing rhythmic bursting volleys of synaptic currents phased to the activity of the preBötC. Scale bars represent 100 μm (a,b,e,f), 500 μm (c,d), 50 μm (h) and 15 μm (j). E10.5 sections are taken from the level of rhombomere 7. XIIn, hypoglossal nucleus.

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Figure 3: Phenotypic profiles of Dbx1-derived cells of the preBötC.

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