P2Y1 receptor-mediated potentiation of inspiratory motor output in neonatal rat in vitro

PreBotzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y(1)R) activation. Despite this, and the fact that inspiratory MNs express P2Y(1)Rs, the role of P2Y(1)Rs in modulating motor output is not known for any MN pool. We used rhythmically active brainstem-spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y(1)R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y(1)R agonist, 0.1mM) potentiated XII inspiratory burst amplitude by 60 +/- 9%; 10-fold higher concentrations potentiated C4 burst amplitude by 25 +/- 7%. In whole-cell voltage-clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca2+-dependent non-selective cation current I-CAN; high intracellular concentrations of BAPTA, which buffers Ca2+ increases necessary for activation of I-CAN; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for I-CAN). Real-time PCR analysis of mRNA extracted from XII punches and laser-microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y(1)R modulation than phrenic MNs and that the P2Y(1)R potentiation of inspiratory output occurs in part via potentiation of TRPM4-mediated ICAN, which amplifies inspiratory inputs.