Title: Disparate purinergic modulation of respiration in rats and mice
Abstract: Chemosensitive reflexes are a key integrative property of the central circuits that generate and control ventilation. Purine nucleotides play an important role in the reflex pathways that regulate O2, CO2 and pH, and the central mechanisms that underlie purinergic modulation of breathing have been a topic of great interest in recent years because they may augment the drive to breathe in perinatal and preterm infants. In this issue, Zwicker et al. (2011) show surprising new data that reveals a disparity in the purinergic modulation of respiration between neonatal rats and mice, two prominent animal models in respiratory neurobiology. Their analysis focuses on the preBötzinger complex (preBötC) of the ventral medulla, which controls inspiratory breathing movements. Unravelling the mechanisms of purinergic modulation is an ongoing challenge. This tripartite system is composed of adenosine triphosphate (ATP) and adenosine (ADO), which generally exert opposing effects on the breathing rhythms, as well as ectonucleotidases that govern the hydrolysis of ATP to ADO. ATP acts at ionotropic (P2X) and metabotropic (P2Y) purinergic receptor subtypes. Metabolites of ATP such as adenosine diphosphate (ADP) act at P2 receptors too. However, ADO acts at P1 purinergic receptors. While P2 receptors are generally excitatory, P1 receptors like the A1 subtype generally inhibit central respiratory circuits. The balance of purine nucleotides is governed by the expression of particular ectonucleotidases. Thus the net effect of extracellular ATP will depend on which receptors are present and their relative expression, as well as the complement of ectonucleotidases. ATP generally enhances ventilation. This has been measured in anaesthetized adult rats in vivo and in fictive respiration in neonatal rat preparations in vitro. Hypoxia causes ATP release from sites throughout the ventral medulla (Gourine et al. 2005a,b). ATP ameliorates the long-lasting depression that normally follows. ADO acting at A1 receptors generally exerts an inhibitory effect on central mechanisms of respiration in adult as well as fetal mammals. The long-lasting depression of respiratory activity in hypoxia may depend on ADO signalling pathways, but the sites and mechanisms of its actions have remained obscure, and highly developmentally regulated. In some cases, ADO has been reported to be without respiratory effects in rat models in vitro. To better understand the sites and mechanisms of ATP modulation in the neonatal preBötC, Zwicker et al. use transverse slice preparations from newborn mice and rats in which the preBötC is located at the rostral face. Rat slices serve as a control, since a great deal more is already known about purinergic modulation in rats. Neuroanatomical criteria were used in conjunction with a mapping strategy to locate the core of the preBötC. ATP microinjection into the preBötC transiently enhanced the frequency of fictive respiration in rats but not mice. While ATP microinjection into the mouse preBötC failed to stimulate fictive respiration, microinjection of a specific P2Y1 receptor agonist caused a dramatic increase in respiratory frequency. This suggested that the lack of effect of ATP might be attributed to the degradation of ATP to ADO. If so, the co-recruitment of excitatory P2Y1 receptors as well as inhibitory A1 receptors could nullify each other. So the authors pre-applied an A1 receptor antagonist in the perfusate. Afterwards, ATP microinjections into the preBötC dramatically increased respiratory frequency, which could be completely eliminated if the perfusate also contained a P2Y1 receptor antagonist. The authors hypothesized that the differential effects of ATP in the rat and mouse preBötC are attributable to disparate expression of ectonucleotidases. They used real-time polymerase chain reaction (PCR) to quantify the expression of ectonucleotidases in the preBötC. Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) hydrolyse ATP to AMP; isoform ENTPDase2 has particularly high affinity for ATP and thus leads to ADP accumulation. Tissue-non-specific alkaline phosphatase (TNAP) is responsible for hydrolysis of ATP to ADO. The natural prediction is that ectonucleotidase expression in rat should favour ENTPDase2, whereas mouse should favour TNAP. And this is exactly what the authors found. In mice, TNAP rapidly degrades ATP to ADO and thus P2Y1 receptor- and A1 receptor-mediated effects appear to cancel one another out. In rats, by contrast, ENTPDase2 degrades ATP to ADP, and both purine nucleotides act at excitatory P2Y1 receptors. The disparity between the effects of ATP on rat and mouse models is important to understand because these two are the dominant animal models used in studies of respiratory neurobiology; their comparative neurophysiology pertains to the neural control of breathing in mammals in general. Zwicker et al. focus on purinergic modulation of the preBötC in neonatal stages in mice. Equipped with the knowledge of the key role played by TNAP in the hydrolysis of ATP to ADO, it will be important to determine the cellular-level mechanisms in greater detail. For example, to what extent do P2Y1 receptors evoke non-specific mixed cationic current or close K+ channels in preBötC neurons, or does P2Y1 receptor activation affect another type of current such as persistent Na+ current (INaP) (Lorier et al. 2008)? Another important issue is the role of glia, which have been shown to be chemosensitive in respiratory circuits of the brainstem including, but not limited to, the preBötC and contribute to hypoxic respiratory reflex responses via gliotransmission (Gourine et al. 2010; Huxtable et al. 2010). Hypoxic ventilatory depression and the role of purinergic modulation of respiration is a clinically important issue that affects premature infants. Understanding the purinergic signalling mechanisms may be applicable to development of therapies that modify the relative levels of ATP and ADO in ways that favour the stimulatory actions of ATP at P2Y receptors and diminish the inhibitory effects of ADO.