Role of inspiratory pacemaker neurons in mediating the hypoxic response of the respiratory network in vitro

In severe hypoxia the breathing frequency is modulated in a biphasic manner: an initial increase (augmentation) is followed by a depression and cessation of breathing (apnea). Using a mouse slice preparation that contains the functional respiratory network, we aimed at identifying the neurons responsible for this frequency modulation. Whole-cell patch recordings revealed that expiratory neurons become tonically active during anoxia, indicating that these neurons cannot be responsible for the respiratory frequency modulation. Inspiratory neurons tended to depolarize (by 6.9 mV; n = 9), and the frequency of rhythmic activity was significantly increased during anoxia (from 0.16 to 0.4 Hz; n = 9). After the blockade of network activity with 6-cyano-7-nitroquinoxaline-2,3-dione, most inspiratory neurons became tonically active (72%; n = 25, non-pacemaker). In anoxia, the membrane potential of these non-pacemaker neurons did not change (-0.26 mV; n = 6), and their tonic activity ceased. Only a subpopulation of inspiratory neurons remained rhythmically active in the absence of network activity (pacemaker neurons, 28%, 7 of 25 inspiratory neurons). In anoxia two subgroups of pacemaker neurons were differentiated; one group showed a transient increase in the bursting activity, followed by a decrease and cessation of rhythmic activity. These neurons tended to depolarize (by 10.3 mV) during anoxia. The second group remained rhythmic during the entire anoxic exposure and exhibited no depolarization. The time course of the frequency modulation in all pacemaker neurons resembled that of the intact network. We conclude that pacemaker neurons are primarily responsible for the frequency modulation in anoxia and that in the respiratory network there is a strict separation between rhythm- and pattern-generating mechanisms.