Cellular and ionic mechanisms underlying the P2Y1receptor‐mediated excitation of thepreBötzinger Complex inspiratory network in vitro

Abstract

A host of adaptive responses have evolved to protect brain oxygen levels. Prominent among these is the biphasic hypoxic ventilatory response (HVR), in which a fall in arterial oxygen, detected at the peripheral chemoreceptors triggers a rapid increase in breathing that is followed by a centrally‐mediated secondary hypoxic respiratory depression (HRD). The HRD is most pronounced in premature infants and can be life‐threatening. Our recent data challenge dogma that the only contribution of the CNS to the HVR is this secondary depression. Specifically, in vitro and in vivo data from rat suggest that astrocytes in the pre‐Bötzinger complex (pre‐BötC, critical site for inspiratory rhythm generation) release ATP in response to hypoxia, which acts via P2Y1 receptors (P2Y1Rs) on inspiratory neurons to increase ventilation and attenuate the HRD. In other parts of the brains, P2Y1Rs signal via the Gq second messenger pathway, but the signalling cascade(s) and ion channel(s) via which P2Y1Rs excite inspiratory neurons to increase inspiratory frequency remain unknown. Previous efforts from our lab to address these questions tested the effects of second messenger and ion channel blockers on ATP currents, but data were confounded by the finding that while P2Y1Rs account for the entire network frequency effect, they account for only a fraction (~30%) of the total ATP current. Thus, the first objective of this study was to revisit this question by examining the effects of Gq and ion channel blockers on currents evoked in inspiratory preBötC neurons by the P2Y1R agonist, MRS 2365. Our second goal was to determine whether the excitatory effect of P2Y1Rs on the preBötC network is mediated through activation of excitatory (glutamatergic) or inhibitory GABA/glycinergic) preBötC neurons.Analysis of whole‐cell recordings from inspiratory preBötC neurons in rhythmic medullary slices from neonatal (P0‐4) SD rats revealed that inward currents evoked by MRS 2365 (30 sec, 100 mM)immediately after obtaining whole‐cell configuration and again after 15 min of dialysis (via the pipette solution) with the protein kinase C (PKC) inhibitor (Chelerethryne, 10 mM, n = 5), the phospholipase C (PLC) inhibitor (U73122 4 mM, n = 6) or the BKCa channel blocker (paxilline 1 or 4 mM, n = 7), were reduced between 30–40%. To test whether P2Y1R‐sensitive, inspiratory neurons are excitatory or inhibitory, we prepared rhythmic slices from transgenic mice (P0‐4) in which glutamatergic (VGLUT2‐TdTomato) or GABAergic (GAD67‐YFP) neurons are fluorescently labeled. Local application of MRS 2365 evoked inward currents in zero of seven VGLUT2‐TdTomato inspiratory neurons. Analysis of GAD67‐YFP inspiratory neurons is ongoing. We are also applying RNAScope analysis to determine the transmitter phenotype of P2Y1R‐sensitive inspiratory neurons.These data suggest that P2Y1Rs acton non‐glutamatergic, inspiratory neurons, in part via the Gq signalling pathway and BKCa channels, to excite the preBötC inspiratory rhythm generating network.Support or Funding InformationSupported by CIHR, NSERC, CFI, WCHRI and NMHI.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.