Innervation of the rat trachea by bilateral cholinergic projections from the nucleus ambiguus and direct motor fibers from the cervical spinal cord: a retrograde and anterograde tracer study

A tract-tracer method was employed to examine the innervation of the rat trachea. Cholera toxin β subunit (CTB) was injected into the following locations in separate groups of rats: (1) ventral trachea, (2) lateral trachea, (3) ventral trachea after the excision of the nodose ganglion, and (4) ventral trachea after the transection of C1-C2 spinal nerves. CTB injection in the ventral trachea showed bilateral labeling of neurons in the nucleus ambiguus (NA), medial subnucleus of the nucleus of the solitary nucleus, dorsal motor nucleus of the vagus (DMV), and lamina IX of C1-C6. CTB injection in the lateral trachea showed significant ipsilateral predominance of neuronal labeling in the NA and lamina IX of C1-C2 segments. CTB injection in rats after the excision of the nodose ganglion revealed no labeling in the ipsilateral DMV and NA and a significant reduction of neuronal labeling in C1. CTB injection in rats after the transection of C1-C2 spinal nerves showed a significant decrease in the number of labeled neurons in ipsilateral NA, C1, and C2 and no labeling of fibers in C1-C2. The combination of retrograde fluorogold labeling and choline acetyltransferase (ChAT) immunostaining revealed that all fluorogold-labeled neurons in the NA and lamina IX of C1-C2 colocalized with ChAT. The injection of biotinylated dextran amine in NA produced labeling in axonal terminals on postganglionic neurons, but not in other regions of the trachea. Our findings indicate that the rat trachea is innervated bilaterally by cholinergic motor neurons in NA and C1-C2, while those traveling through the spinal nerves project directly to the trachea.