A brainstem peptide system activated at birth protects postnatal breathing.

Among numerous challenges encountered at the beginning of extrauterine life, the most celebrated is the first breath that initiates a life-sustaining motor activity1. The neural systems that regulate breathing are fragile early in development, and it is not clear how they adjust to support breathing at birth. Here we identify a neuropeptide system that becomes activated immediately after birth and supports breathing. Mice that lack PACAP selectively in neurons of the retrotrapezoid nucleus (RTN) displayed increased apnoeas and blunted CO2-stimulated breathing; re-expression of PACAP in RTN neurons corrected these breathing deficits. Deletion of the PACAP receptor PAC1 from the pre-Bötzinger complex-an RTN target region responsible for generating the respiratory rhythm-phenocopied the breathing deficits observed after RTN deletion of PACAP, and suppressed PACAP-evoked respiratory stimulation in the pre-Bötzinger complex. Notably, a postnatal burst of PACAP expression occurred in RTN neurons precisely at the time of birth, coinciding with exposure to the external environment. Neonatal mice with deletion of PACAP in RTN neurons displayed increased apnoeas that were further exacerbated by changes in ambient temperature. Our findings demonstrate that well-timed PACAP expression by RTN neurons provides an important supplementary respiratory drive immediately after birth and reveal key molecular components of a peptidergic neural circuit that supports breathing at a particularly vulnerable period in life.

The Purinome and the preBötzinger Complex - A Ménage of Unexplored Mechanisms That May Modulate/Shape the Hypoxic Ventilatory Response.

Exploration of purinergic signaling in brainstem homeostatic control processes is challenging the traditional view that the biphasic hypoxic ventilatory response, which comprises a rapid initial increase in breathing followed by a slower secondary depression, reflects the interaction between peripheral chemoreceptor-mediated excitation and central inhibition. While controversial, accumulating evidence supports that in addition to peripheral excitation, interactions between central excitatory and inhibitory purinergic mechanisms shape this key homeostatic reflex. The objective of this review is to present our working model of how purinergic signaling modulates the glutamatergic inspiratory synapse in the preBötzinger Complex (key site of inspiratory rhythm generation) to shape the hypoxic ventilatory response. It is based on the perspective that has emerged from decades of analysis of glutamatergic synapses in the hippocampus, where the actions of extracellular ATP are determined by a complex signaling system, the purinome. The purinome involves not only the actions of ATP and adenosine at P2 and P1 receptors, respectively, but diverse families of enzymes and transporters that collectively determine the rate of ATP degradation, adenosine accumulation and adenosine clearance. We summarize current knowledge of the roles played by these different purinergic elements in the hypoxic ventilatory response, often drawing on examples from other brain regions, and look ahead to many unanswered questions and remaining challenges.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism.

KEY POINTS: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure. ABSTRACT: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

Excitatory Modulation of the preBötzinger Complex Inspiratory Rhythm Generating Network by Endogenous Hydrogen Sulfide.

Hydrogen Sulfide (H2S) is one of three gasotransmitters that modulate excitability in the CNS. Global application of H2S donors or inhibitors of H2S synthesis to the respiratory network has suggested that inspiratory rhythm is modulated by exogenous and endogenous H2S. However, effects have been variable, which may reflect that the RTN/pFRG (retrotrapezoid nucleus, parafacial respiratory group) and the preBötzinger Complex (preBötC, critical for inspiratory rhythm generation) are differentially modulated by exogenous H2S. Importantly, site-specific modulation of respiratory nuclei by H2S means that targeted, rather than global, manipulation of respiratory nuclei is required to understand the role of H2S signaling in respiratory control. Thus, our aim was to test whether endogenous H2S, which is produced by cystathionine-ß-synthase (CBS) in the CNS, acts specifically within the preBötC to modulate inspiratory activity under basal (in vitro/in vivo) and hypoxic conditions (in vivo). Inhibition of endogenous H2S production by bath application of the CBS inhibitor, aminooxyacetic acid (AOAA, 0.1-1.0 mM) to rhythmic brainstem spinal cord (BSSC) and medullary slice preparations from newborn rats, or local application of AOAA into the preBötC (slices only) caused a dose-dependent decrease in burst frequency. Unilateral injection of AOAA into the preBötC of anesthetized, paralyzed adult rats decreased basal inspiratory burst frequency, amplitude and ventilatory output. AOAA in vivo did not affect the initial hypoxia-induced (10% O2, 5 min) increase in ventilatory output, but enhanced the secondary hypoxic respiratory depression. These data suggest that the preBötC inspiratory network receives tonic excitatory modulation from the CBS-H2S system, and that endogenous H2S attenuates the secondary hypoxic respiratory depression.

Laser ablation of Dbx1 neurons in the pre-Bötzinger complex stops inspiratory rhythm and impairs output in neonatal mice.

To understand the neural origins of rhythmic behavior one must characterize the central pattern generator circuit and quantify the population size needed to sustain functionality. Breathing-related interneurons of the brainstem pre-Bötzinger complex (preBötC) that putatively comprise the core respiratory rhythm generator in mammals are derived from Dbx1-expressing precursors. Here, we show that selective photonic destruction of Dbx1 preBötC neurons in neonatal mouse slices impairs respiratory rhythm but surprisingly also the magnitude of motor output; respiratory hypoglossal nerve discharge decreased and its frequency steadily diminished until rhythm stopped irreversibly after 85±20 (mean ± SEM) cellular ablations, which corresponds to ∼15% of the estimated population. These results demonstrate that a single canonical interneuron class generates respiratory rhythm and contributes in a premotor capacity, whereas these functions are normally attributed to discrete populations. We also establish quantitative cellular parameters that govern network viability, which may have ramifications for respiratory pathology in disease states.

Neuromodulation: purinergic signaling in respiratory control.

The main functions of the respiratory neural network are to produce a coordinated, efficient, rhythmic motor behavior and maintain homeostatic control over blood oxygen and CO2/pH levels. Purinergic (ATP) signaling features prominently in these homeostatic reflexes. The signaling actions of ATP are produced through its binding to a diversity of ionotropic P2X and metabotropic P2Y receptors. However, its net effect on neuronal and network excitability is determined by the interaction between the three limbs of a complex system comprising the signaling actions of ATP at P2Rs, the distribution of multiple ectonucleotidases that differentially metabolize ATP into ADP, AMP, and adenosine (ADO), and the signaling actions of ATP metabolites, especially ADP at P2YRs and ADO at P1Rs. Understanding the significance of purinergic signaling is further complicated by the fact that neurons, glia, and the vasculature differentially express P2 and P1Rs, and that both neurons and glia release ATP. This article reviews at cellular, synaptic, and network levels, current understanding and emerging concepts about the diverse roles played by this three-part signaling system in: mediating the chemosensitivity of respiratory networks to hypoxia and CO2/pH; modulating the activity of rhythm generating networks and inspiratory motoneurons, and; controlling blood flow through the cerebral vasculature.

Glia contribute to the purinergic modulation of inspiratory rhythm-generating networks.

Glia modulate neuronal activity by releasing transmitters in a process called gliotransmission. The role of this process in controlling the activity of neuronal networks underlying motor behavior is unknown. ATP features prominently in gliotransmission; it also contributes to the homeostatic ventilatory response evoked by low oxygen through mechanisms that likely include excitation of preBötzinger complex (preBötC) neural networks, brainstem centers critical for breathing. We therefore inhibited glial function in rhythmically active inspiratory networks in vitro to determine whether glia contribute to preBötC ATP sensitivity. Glial toxins markedly reduced preBötC responses to ATP, but not other modulators. Furthermore, since preBötC glia responded to ATP with increased intracellular Ca(2+) and glutamate release, we conclude that glia contribute to the ATP sensitivity of preBötC networks, and possibly the hypoxic ventilatory response. Data reveal a role for glia in signal processing within brainstem motor networks that may be relevant to similar networks throughout the neuraxis.

Tripartite purinergic modulation of central respiratory networks during perinatal development: the influence of ATP, ectonucleotidases, and ATP metabolites.

ATP released during hypoxia from the ventrolateral medulla activates purinergic receptors (P2Rs) to attenuate the secondary hypoxic depression of breathing by a mechanism that likely involves a P2Y(1)R-mediated excitation of preBötzinger complex (preBötC) inspiratory rhythm-generating networks. In this study, we used rhythmically active in vitro preparations from embryonic and postnatal rats and ATP microinjection into the rostral ventral respiratory group (rVRG)/preBötC to reveal that these networks are sensitive to ATP when rhythm emerges at embryonic day 17 (E17). The peak frequency elicited by ATP at E19 and postnatally was the same ( approximately 45 bursts/min), but relative sensitivity was threefold greater at E19, reflecting a lower baseline frequency (5.6 +/- 0.9 vs 19.0 +/- 1.3 bursts/min). Combining microinjection techniques with ATP biosensors revealed that ATP concentration in the rVRG/preBötC falls rapidly as a result of active processes and closely correlates with inspiratory frequency. A phosphate assay established that preBötC-containing tissue punches degrade ATP at rates that increase perinatally. Thus, the agonist profile [ATP/ADP/adenosine (ADO)] produced after ATP release in the rVRG/preBötC will change perinatally. Electrophysiology further established that the ATP metabolite ADP is excitatory and that, in fetal but not postnatal animals, ADO at A(1) receptors exerts a tonic depressive action on rhythm, whereas A(1) antagonists extend the excitatory action of ATP on inspiratory rhythm. These data demonstrate that ATP is a potent excitatory modulator of the rVRG/preBötC inspiratory network from the time it becomes active and that ATP actions are determined by a dynamic interaction between the actions of ATP at P2 receptors, ectonucleotidases that degrade ATP, and ATP metabolites on P2Y and P1 receptors.

P2Y1 receptor modulation of the pre-Bötzinger 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-Bötzinger complex (preBötC) (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 preBötC based on histology, overlap of injection sites with NK1R immunolabeling, and potentiation or inhibition of respiratory frequency by SP ([Sar9-Met(O2)11]-substance P) or DAMGO ([D-Ala2,N-MePhe4,Gly-ol5]-enkephalin), respectively. The relative potency of P2R agonists [2MeSADP (2-methylthioadenosine 5'-diphosphate) approximately = 2MeSATP (2-methylthioadenosine 5'-triphosphate) approximately = ATPgammas (adenosine 5'-[gamma-thio]triphosphate tetralithium salt) approximately = ATP >> UTP approximately = alphabeta meATP (alpha,beta-methylene-adenosine 5'-triphosphate)] and attenuation of the ATP response by MRS2179 (2'-deoxy-N6-methyladenosine-3',5'-bisphosphate) (P2Y1 antagonist) indicate that the excitation is mediated by P2Y1Rs. The post-ATP inhibition, which was never observed in response to ATPgammas, is dependent on ATP hydrolysis. These data establish in neonatal rats that respiratory rhythm generating networks in the preBötC are exquisitely sensitive to P2Y1R activation, and suggest a role for P2Y1Rs in respiratory motor control, particularly in the P2R excitation of rhythm that occurs during hypoxia.

Developmental downregulation of P2X3 receptors in motoneurons of the compact formation of the nucleus ambiguus.

Motoneurons of the compact division of the nucleus ambiguus (cNA) are the final output neurons of the swallowing pattern generator. Thus, their normal function is critical to neonatal survival. To explore the role of purinergic signaling in modulating the excitability of these motoneurons during development, immunohistochemical and whole-cell recording techniques were used to characterize expression patterns of ionotropic P2X receptors and the effects of ATP on cNA motoneurons. Medullary slices containing the cNA were prepared from neonatal (P0-4) and juvenile (P15-21) rats. In neonatal cNA motoneurons, local application of 1 mM ATP produced a large (-133 +/- 17 pA; n = 78), desensitizing, inward current that was mimicked by 1 mM alpha,beta meATP and 2meSATP, and inhibited by the P2 antagonist, PPADS (5 microM), and the P2X3 antagonist, A-317481 (0.1-1 mM). In juvenile cNA motoneurons, 1 mM ATP produced negligible currents, while 10 mM ATP produced small (-59 +/- 14 pA; n = 42), primarily non-desensitizing currents. Immunohistochemistry demonstrated that in the neonate, the expression of P2X3 was robust, P2X2 and P2X5 moderate, P2X4 and P2X6 weak, and P2X1 absent. In the juvenile cNA, only low levels of P2X5 and P2X6 labeling were detected. These data indicate that P2X receptors in cNA motoneurons are profoundly downregulated during the first two postnatal weeks, and suggest a role for the purinoceptor system, particularly P2X3 receptors, in the control of esophageal motor networks during early postnatal periods.

Modulation of phrenic motoneuron excitability by ATP: consequences for respiratory-related output in vitro.

On the basis of the high level of P2X receptor expression found in phrenic motoneurons (MN) in rats (Kanjhan et al., J Comp Neurol 407: 11-32, 1999) and potentiation of hypoglossal MN inspiratory activity by ATP (Funk et al., J Neurosci 17: 6325-6337, 1997), we tested the hypothesis that ATP receptor activation also modulates phrenic MN activity. This question was examined in rhythmically active brain stem-spinal cord preparations from neonatal rats by monitoring effects of ATP on the activity of spinal C4 nerve roots and phrenic MNs. ATP produced a rapid-onset, dose-dependent, suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium-sensitive increase in C4 root tonic discharge and a 22 +/- 7% potentiation of inspiratory burst amplitude. This was followed by a slower, 10 +/- 5% reduction in burst amplitude. ATPgammaS, the hydrolysis-resistant analog, evoked only the excitatory response. ATP induced inward currents (57 +/- 39 pA) and increased repetitive firing of phrenic MNs. These data, combined with persistence of ATP currents in TTX and immunolabeling for P2X2 receptors in Fluoro-Gold-labeled C4 MNs, implicate postsynaptic P2 receptors in the excitation. Inspiratory synaptic currents, however, were inhibited by ATP. This inhibition differed from that seen in root recordings; it did not follow an excitation, had a faster onset, and was induced by ATPgammaS. Thus ATP inhibited activity through at least two mechanisms: 1) a rapid P2 receptor-mediated inhibition and 2) a delayed P1 receptor-mediated inhibition associated with hydrolysis of ATP to adenosine. The complex effects of ATP on phrenic MNs highlight the importance of ATP as a modulator of central motor outflows.

Prenatal nicotine exposure increases apnoea and reduces nicotinic potentiation of hypoglossal inspiratory output in mice.

We examined the effects of in utero nicotine exposure on postnatal development of breathing pattern and ventilatory responses to hypoxia (7.4 % O2) using whole-body plethysmography in mice at postnatal day 0 (P0), P3, P9, P19 and P42. Nicotine delayed early postnatal changes in breathing pattern. During normoxia, control and nicotine-exposed P0 mice exhibited a high frequency of apnoea (f(A)) which declined by P3 in control animals (from 6.7 +/- 0.7 to 2.2 +/- 0.7 min(-1)) but persisted in P3 nicotine-exposed animals (5.4 +/- 1.3 min(-1)). Hypoxia induced a rapid and sustained reduction in f(A) except in P0 nicotine-exposed animals where it fell initially and then increased throughout the hypoxic period. During recovery, f(A) increased above control levels in both groups at P0. By P3 this increase was reduced in control but persisted in nicotine-exposed animals. To examine the origin of differences in respiratory behaviour, we compared the activity of hypoglossal (XII) nerves and motoneurons in medullary slice preparations. The frequency and variability of the respiratory rhythm and the envelope of inspiratory activity in XII nerves and motoneurons were indistinguishable between control and nicotine-exposed animals. Activation of postsynaptic nicotine receptors caused an inward current in XII motoneurons that potentiated XII nerve burst amplitude by 25 +/- 5 % in control but only 14 +/- 3 % in nicotine-exposed animals. Increased apnoea following nicotine exposure does not appear to reflect changes in basal activity of rhythm or pattern-generating networks, but may result, in part, from reduced nicotinic modulation of XII motoneurons.