Multicolor labeling of airway neurons and analysis of parasympathetic heterogeneity

We report subpopulations of airway parasympathetic neurons expressing substance P, neuronal nitric oxide synthase, and tyrosine hydroxylase, highlighting unexplored heterogeneity in this population. These neurotransmitter-specific subpopulations did not form intraganglionic interneurons, but rather, extended outside the ganglia, into the airways, to distant innervation targets. Our experiments demonstrate the utility of multicolor labeling to characterize airway innervation, allowing us to confirm the extensive heterogeneity of postganglionic parasympathetic neurons. These methods will facilitate future investigations of neurophysiology and neural contributions to airway disease.