Neuronal Mediators of the Frequency Increase Evoked by Activation of P2Y 1 Receptors in the preBötzinger Complex Inspiratory Rhythm Generating Network in Vitro

Abstract

The ventilatory response to acute hypoxia (HVR) comprises an initial, carotid body-mediated increase in breathing followed by a centrally-mediated secondary respiratory depression when ventilation falls to a new steady state. Recent evidence suggests that ATP is released from astrocytes in the preBötzinger Complex (preBötC; site of inspiratory rhythm generation) during the secondary phase where it acts via P2Y1 receptors (Rs) to excite preBötC neurons, increase breathing frequency and attenuate the secondary respiratory depression. The preBötC network comprises a heterogenous population of excitatory and inhibitory inspiratory neurons that can be further classified based on expression of transcription factors, receptors and transmitters, discharge pattern and membrane properties. An important goal is to establish the neuronal phenotype(s) that underlie the P2Y1R frequency increase. Perhaps surprisingly, preBötC frequency increases in response to optogenetic stimulation of excitatory OR inhibitory preBötC neurons. Thus, the objective of this study was to determine if the P2Y1R-mediated frequency increase results from activation of excitatory (glutamatergic) or inhibitory (GABA/glycinergic) preBötC inspiratory neurons. Analysis of P2Y1R and somatostatin (SST) immunolabeling in the medulla of neonatal vGLUT2TdTomato and GLYT2EGFP reporter mice showed that P2Y1R immunolabeled neurons are concentrated in a discrete region of the ventral respiratory column (VRC) that features strong SST immunolabeling (a preBötC marker). P2Y1R+ neurons were sparse in all other regions of the VRC. In addition, 95% of P2Y1R+ neurons were glutamatergic (VGLUT2+), but <10% of glutamatergic neurons were P2Y1R+. None of the GLYT2+ (glycinergic) preBötC neurons were P2Y1R+. Whole-cell analyses of neurons in rhythmic slices from vGLUT2 mice showed that all VGLUT2+ inspiratory preBötC neurons excited by the P2Y1R agonist MRS2365 (100 µM) were glutamatergic, but only1/3 of VGLUT2+ neurons were P2Y1R sensitive. In contrast, calcium imaging of rhythmic slices identified subpopulations (4/16) of glutamatergic and non glutamatergic inspiratory neurons that in TTX were P2Y1R sensitive. Finally, we demonstrated that the frequency increase evoked by MRS2365 (100 µM) in the preBötC was unaffected by block of inhibitory synaptic transmission via GABAA (bicuculline, 20 µM) and glycine (strychnine, 20 µM) receptor antagonists. In summary, we found that P2Y1R-expressing neurons are concentrated in the preBötC and are primarily glutamatergic. P2Y1R-sensitive neurons are primarily glutamatergic, but a subpopulation is nonglutamatergic. These data, combined with the observation that the P2Y1R-evoked excitation of the preBötC does not depend on synaptic inhibition, suggest that the P2Y1R-mediated excitation of the preBötC is primarily achieved through activation of excitatory, glutamatergic neurons.