The airway function is dominated by the parasympathetic nervous system.A subpopulation of the airway-related preganglionic parasympathetic neurons (APPNs) located in the external formation of the nucleus ambiguus (eNA) play critical roles in the physiological and pathological control of tracheobronchial smooth muscle.However, the firing properties of these APPNs are not clear.In the present study, APPNs in the eNA were retrogradely labeled with a fluorescent tracer applied to the extrathoracic trachea and identified in rhythmically firing brainstem slices, and their electrophysiological properties were examined using patch-clamp technique.The results showed that inspiratory and expiratory APPNs in the eNA were relatively dissociated anatomically.Activation of the sodium channels in both types of neurons showed L-shaped current-voltage relationship curve.In both types of neurons depolarization evoked a transient outward potassium current that was sensitive to 4-aminopyridine (4-AP), and a lasting outward potassium current that was partially sensitive to 4-AP in inspiratory APPNs and insensitive to 4-AP in expiratory APPNs.Both types of neurons have an afterhyperpolarization (AHP) after each action potential, which was mediated by small-conductance Ca2+-activated K+ channel (SKCa) in inspiratory APPNs and by large-conductance Ca2+-activated K+ channel (LKca) in expiratory APPNs.These findings have led to better understanding of the physiological properties of the APPNs controlling the tracheal or tracheobronchial muscles.