P2Y1 receptor modulation of the pre-Botzinger complex inspiratory rhythm generating network in vitro

ATP is released during hypoxia from the ventrolateral medulla (VLM) and activates purinergic P2 receptors (P2Rs) at unknown loci to offset the secondary hypoxic depression of breathing. In this study, we used rhythmically active medullary slices from neonatal rat to map, in relation to anatomical and molecular markers of the pre-Botzinger complex (preBotC) ( a proposed site of rhythm generation), the effects of ATP on respiratory rhythm and identify the P2R subtypes responsible for these actions. Unilateral microinjections of ATP in a three-dimensional grid within the VLM revealed a "hotspot" where ATP (0.1 mM) evoked a rapid 2.2 +/- 0.1-fold increase in inspiratory frequency followed by a brief reduction to 0.83 +/- 0.02 of baseline. The hotspot was identified as the preBotC based on histology, overlap of injection sites with NK1R immunolabeling, and potentiation or inhibition of respiratory frequency by SP ([Sar(9)-Met(O-2)(11)]-substance P) or DAMGO ([D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin), respectively. The relative potency of P2R agonists [2MeSADP (2-methylthioadenosine 5'-diphosphate) approximate to 2MeSATP (2-methylthioadenosine 5'-triphosphate) approximate to ATP gamma s ( adenosine 5'-[gamma-thio] triphosphate tetralithium salt) approximate to ATP >> UTP approximate to alpha beta meATP (alpha, beta-methylene-adenosine 5'-triphosphate)] and attenuation of the ATP response by MRS2179 (2'-deoxy-N6-methyladenosine-3', 5'-bisphosphate) (P2Y(1) antagonist) indicate that the excitation is mediated by P2Y(1)Rs. The post-ATP inhibition, which was never observed in response to ATP gamma s, is dependent on ATP hydrolysis. These data establish in neonatal rats that respiratory rhythm generating networks in the preBotC are exquisitely sensitive to P2Y(1)R activation, and suggest a role for P2Y(1)Rs in respiratory motor control, particularly in the P2R excitation of rhythm that occurs during hypoxia.