P2Y 1 receptor modulation of Ih in the preBötzinger inspiratory rhythm generator may underlie the ATP‐mediated attenuation of the hypoxic respiratory depression

Abstract

Premature infants often stop breathing briefly (apnea) because the brainstem respiratory network is still immature (apnea of prematurity, AOP). During these apneas, oxygen levels fall (hypoxia) triggering a biphasic hypoxic ventilatory response comprising an initial increase in ventilation followed by a centrally mediated secondary depression that can be life-threatening. Caffeine is used to stimulate breathing in AOP. However, ~20% of these infants do not respond to caffeine. Thus, alternate therapies are needed. Recent data suggest that during hypoxia, ATP is released in the preBötzinger Complex (preBötC, inspiratory rhythm generator) where it acts via P2Y1 receptors (Rs) to excite inspiratory neurons, increase ventilation and attenuate the secondary hypoxic depression. A mechanistic understanding of this P2Y1R excitation is lacking. Preliminary whole-cell analysis of inspiratory preBötC neurons in vitro revealed P2Y1R currents that reverse near -40 mV and increase in amplitude with neuron hyperpolarization. Based on these data, we tested the hypotheses that the increase in inspiratory frequency evoked by ATP in the preBötC in vitro and the ATP-mediated increase in ventilation during hypoxia in vivo reflects P2Y1R potentiation of the hyperpolarization-activated inward current, Ih (carried by cyclic nucleotide–gated, HCN, channels). Application of MRS2365 (P2Y1R agonist, 100 µM) to Ih-expressing inspiratory preBötC neurons in rhythmic brainstem slices from neonatal rat potentiated Ih by 32±6% (at -100 mV, n=18) and depolarized voltage-dependence of Ih activation by ~10 mV. The MRS2365 inward current and its potentiation of Ih were blocked by the Ih blocker, ZD7288 (100 μM). ZD7288 also attenuated the MRS2365-evoked frequency increase by 60-90%. Ih currents in preBötC neurons were sensitive to ZD7288 (100 µM) but not ivabradine (30 µM), suggesting HCN2 but not HCN1,4 subunit involvement. In paralyzed, anesthetized, vagotomized ventilated adult rats, bilateral microinfusion of ZD7288 (250 µM, but not 100 µM) into the preBötC increased the secondary hypoxic (10% O2) depression by 68±23% (n=6) compared to rats that received aCSF microinfusion (n=4). This effect was regionally specific. If the bilateral microinfusions of ZD7288 were not both in the preBötC (e.g., 1 in preBötC and the other in BötC or rVRG), ZD7288 had no effect on the hypoxic ventilatory response. Finally, in anesthetized, vagotomized, spontaneously-breathing adult rats, unilateral reverse microdialysis of ZD7288 (100 µM) in the preBötC area (n=13) increased the hypoxic frequency depression by 20±6% (compared to aCSF, n=6). These data suggest that the P2Y1R excitation of the preBötC network is produced, in part, via potentiation of Ih in a subgroup of inspiratory neurons, and that potentiation of Ih in the preBötC attenuates the hypoxic ventilatory depression in adult rats. Data also identify the HCN2 subunit as a potential target for new strategies to counteract respiratory depression in AOP.