We examined the role of the hyperpolarization-activated current (I h ) in the generation of the respiratory rhythm using a spontaneously active brainstem slice of mice. This preparation contains the hypoglossus (XII) nucleus, which is activated in-phase with inspiration and the pre-Bo ¨tzinger complex (PBC), the presumed site for respiratory rhythm generation. Voltage-clamp recordings (n ϭ 90) indicate that cesium (Cs) (5 mM) blocked 77.2% of the I h current, and ZD 7288 (100 M) blocked 85.8%

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... the I h current. This blockade increased the respiratory frequency by 161% in Cs and by 150% in ZD 7288 and increased the amplitude of integrated population activity in the XII by 97% in Cs and by 162% in ZD 7288, but not in the PBC (Cs, by 19%; ZD 7288, by Ϫ4.56%). All inspiratory PBC neurons (n ϭ 44) recorded in current clamp within the active network revealed a significantly decreased frequency of action potentials during the interburst interval and an earlier onset of inspiratory bursts after I h current blockade. However, hyperpolarizing current pulses evoked only in a small proportion of inspiratory neurons (0% of type I; 29% of type II neurons) a depolarizing sag. Most of the neurons expressing an I h current (86%) were pacemaker neurons, which continued to generate rhythmic bursts after inactivating the respiratory network pharmacologically with CNQX alone or with CNQX, AP-5, strychnine, bicuculline, and carbenoxolone. Cs and ZD 7288 increased the frequency of pacemaker bursts and decreased the frequency of action potentials between pacemaker bursts. Our findings suggest that the I h current plays an important role in modulating respiratory frequency, which is presumably mediated by pacemaker neurons.