Figure 4. Brainstem Neuropathologies of Nkx2.2^Cre; Phox2b^Δ8 mice are accompanied by weakened inspiratory drive from the preBötC.

A and B. No apparent defect in the Locus coeruleus (TH+) in Nkx2.2^Cre, Phox2b^Δ8 mice compared to controls noted. C and D. VII nucleus (ChAT in red) and RTN with PHOX2B-positive neurons (green) at the ventral medullary surface. E-H. Significant dysgenesis of dorsal motor nucleus of vagus nerve and nucleus ambiguus at the level of the post-inspiratory complex (PiCo) respiratory oscillator (PICO is delineated in higher magnification in panels g and h for control and mutant mice, respectively). Panels I-J Dorsal motor nucleus of the vagus nerve, nucleus ambiguus and a decreased intensity of NK1R-immunoreactive cells noted. K-P. Box plots show solid black line to represent the median, the box represents in the interquartile range, and the whiskers represent 1.5 times the interquartile range. P-values of no significance are shown in the box plots in black font, with corrected P-values of < 0.05 shown in red. In box plots the parameter tested is labeled on Y-axis corresponding to either the number of ChAT-positive cells/section (K, L, O, P), PHOX2B positive cells/section (M), or NK1R Mean gray Value (N). Genotypes are delineated on X-axis. TH = Tyrosine Hydroxylase, ChAT = Choline Acetyltransferase, NK1R = Neurokinin 1 Receptor, VGLUT2 = vesicular glutamate transporter-2. Nkx2.2^Cre, Phox2b^Δ8 n = 10 images obtained from three pups, control n = 5 images obtained from two pups. Q-W. Representative image of the integrated inspiratory rhythm from isolated preBötC left, and the mean inspiratory burst (thick gray trace) of seven individual inspiratory bursts (thin gray traces) right, from a control preBötC isolated in a brainstem slice (Q); representative image of the integrated inspiratory rhythm from isolated preBötC left, and the mean inspiratory burst (thick green trace) of seven individual inspiratory bursts (thin green traces) right from a preBötC isolated in a brainstem slice from a Nkx2.2^Cre, Phox2b^Δ8 animal (R). While no differences were observed when comparing the irregularity score (IrS) of amplitude (S, P = 0.92) and period (T, P = 0.62) between the preBötC from control (n = 8) and Nkx2.2^Cre, Phox2b^Δ8 (n = 7) animals, halfwidth of preBötC burst from Nkx2.2^Cre, Phox2b^Δ8 animals has a significant shorter duration (U, P = 0.011). No difference in the mean IrS of period observed. Variance in IrS period was different between the two groups (F = 6.538, P-value for F test to compare variance = 0.026). Positive relationship existed between IrS of period and period in the control group (r² = 0.62, P = 0.021) (V). In the Nkx2.2^Cre, Phox2b^Δ8 group, the relationship was flat (r² = 0.004, P = 0.886) (W).