Figure 6:

The projection of NMBR⁺ neurons to the caudal ventral respiratory group (cVRG). (A) Representative imaging of brainstem coronal sections showing that NMBR⁺ neurons (green) comprise a highly restricted population in the sneeze-evoking region (red, labeled by WGA-555 applied to mouse nasal cavity). Neurons were marked by NeuN (blue). (B) Representative imaging showing that NMBR⁺ neurons selectively project to cVRG but no other respiratory regions including the rostral ventral respiratory group (rVRG), pre-BÖtzinger complex (preBÖtC) and BÖtzinger complex (BÖtC) as revealed by axonal tracing in Nmbr^eGFP reporter line. Images on the right show higher-magnification view of each boxed region. (C) Representative imaging showing that NMBR-GFP⁺ nerve fibers (green) express the presynaptic marker synaptophysin 1 (red) and synapse with cVRG neurons (marked by NeuN, blue). (D-E) Retrograde axonal tracing by microinjection of CTB-555 into cVRG of Nmbr^eGFP mice shows that CTB labeled NMBR-GFP⁺ neurons (indicated by arrows) are localized within the sneeze-evoking region in the spinal trigeminal nucleus and absent from other brain regions including the pre-BÖtzinger complex (preBÖ) and nucleus tractus solitarius (NTS). All images shown are representatives of three biologically independent mice. Scale bars: 100 μm. Each dot represents an individual mouse (n=3 mice/group). Data are represented as mean ± SEM. ** P≤0.01, *** P≤0.001. See also Figure S4.