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.

Optogenetic excitation of preBötzinger complex neurons potently drives inspiratory activity in vivo.

KEY POINTS: This study investigates the effects on ventilation of an excitatory stimulus delivered in a spatially and temporally precise manner to the inspiratory oscillator, the preBötzinger complex (preBötC). We used an adeno-associated virus expressing channelrhodopsin driven by the synapsin promoter to target the region of the preBötC. Unilateral optogenetic stimulation of preBötC increased respiratory rate, minute ventilation and increased inspiratory modulated genioglossus muscle activity. Unilateral optogenetic stimulation of preBötC consistently entrained respiratory rate up to 180 breaths min(-1) both in presence of ongoing respiratory activity and in absence of inspiratory activity. Unilateral optogenetic stimulation of preBötC induced a strong phase-independent Type 0 respiratory reset, with a short delay in the response of 100 ms. We identified a refractory period of ∼200 ms where unilateral preBötC optogenetic stimulation is not able to initiate the next respiratory event. ABSTRACT: Understanding the sites and mechanisms underlying respiratory rhythmogenesis is of fundamental interest in the field of respiratory neurophysiology. Previous studies demonstrated the necessary and sufficient role of preBötzinger complex (preBötC) in generating inspiratory rhythms in vitro and in vivo. However, the influence of timed activation of the preBötC network in vivo is as yet unknown given the experimental approaches previously used. By unilaterally infecting preBötC neurons using an adeno-associated virus expressing channelrhodopsin we photo-activated the network in order to assess how excitation delivered in a spatially and temporally precise manner to the inspiratory oscillator influences ongoing breathing rhythms and related muscular activity in urethane-anaesthetized rats. We hypothesized that if an excitatory drive is necessary for rhythmogenesis and burst initiation, photo-activation of preBötC not only will increase respiratory rate, but also entrain it over a wide range of frequencies with fast onset, and have little effect on ongoing respiratory rhythm if a stimulus is delivered during inspiration. Stimulation of preBötC neurons consistently increased respiratory rate and entrained respiration up to fourfold baseline conditions. Furthermore, brief pulses of photostimulation delivered at random phases between inspiratory events robustly and consistently induced phase-independent (Type 0) respiratory reset and recruited inspiratory muscle activity at very short delays (∼100 ms). A 200 ms refractory period following inspiration was also identified. These data provide strong evidence for a fine control of inspiratory activity in the preBötC and provide further evidence that the preBötC network constitutes the fundamental oscillator of inspiratory rhythms.

Projections of preBötzinger complex neurons in adult rats.

The preBötzinger Complex (preBötC) contains neural microcircuitry essential for normal respiratory rhythm generation in rodents. A subpopulation of preBötC neurons expresses somatostatin, a neuropeptide with a modulatory action on breathing. Acute silencing of a subpopulation of preBötC neurons transfected by a virus driving protein expression under the somatostatin promoter results in persistent apnea in awake adult rats. Given the profound effect of silencing these neurons, their projections are of interest. We used an adeno-associated virus to overexpress enhanced green fluorescent protein driven by the somatostatin promoter in preBötC neurons to label their axons and terminal fields. These neurons send brainstem projections to: 1) contralateral preBötC; 2) ipsi- and contralateral Bötzinger Complex; 3) ventral respiratory column caudal to preBötC; 4) parafacial respiratory group/retrotrapezoid nucleus; 5) parahypoglossal nucleus/nucleus of the solitary tract; 6) parabrachial/Kölliker-Fuse nuclei; and 7) periaqueductal gray. We did not find major projections to either cerebellum or spinal cord. We conclude that there are widespread projections from preBötC somatostatin-expressing neurons specifically targeted to brainstem regions implicated in control of breathing, and provide a network basis for the profound effects and the essential role of the preBötC in breathing.

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.

Characterization of the null murine sodium/myo-inositol cotransporter 1 (Smit1 or Slc5a3) phenotype: myo-inositol rescue is independent of expression of its cognate mitochondrial ribosomal protein subunit 6 (Mrps6) gene and of phosphatidylinositol levels in neonatal brain.

Ablation of the murine Slc5a3 gene results in severe myo-inositol (Ins) deficiency and congenital central apnea due to abnormal respiratory rhythmogenesis. The lethal knockout phenotype may be rescued by supplementing the maternal drinking water with 1% Ins. In order to test the hypothesis that Ins deficiency leads to inositide deficiencies, which are corrected by prenatal treatment, we measured the effects of Ins rescue on Ins, phosphatidylinositol (PtdIns) and myo-inositol polyphosphate levels in brains of E18.5 knockout fetuses. As the Slc5a3 gene structure is unique in the sodium/solute cotransporter (SLC5) family, and exon 1 is shared with the mitochondrial ribosomal protein subunit 6 (Mrps6) gene, we also sought to determine whether expression of its cognate Mrps6 gene is abnormal in knockout fetuses. The mean level of Ins was increased by 92% in brains of rescued Slc5a3 knockout fetuses (0.48 versus 0.25 nmol/mg), but was still greatly reduced in comparison to wildtype (6.97 nmol/mg). The PtdIns, InsP(5) and InsP(6) levels were normal without treatment. Mrps6 gene expression was unaffected in the E18.5 knockout fetuses. This enigmatic model is not associated with neonatal PtdIns deficiency and rescue of the phenotype may be accomplished without restoration of Ins. The biochemical mechanism that both uniformly leads to death and allows for Ins rescue remains unknown. In conclusion, in neonatal brain tissue, Mrps6 gene expression may not be contingent on function of its embedded Slc5a3 gene, while inositide deficiency may not be the mechanism of lethal apnea in null Slc5a3 mice.

The genesis of epileptogenic cerebral heterotopia: clues from experimental models.

The pre-natal administration of methylazoxymethanol acetate (MAM) in rats is able to induce cerebral heterotopia that share striking similarities with those observed in human periventricular nodular heterotopia, a cerebral dysgenesis frequently associated with drug-resistant focal seizures. In the present study, we investigated the mode of neurogenesis in cerebral heterotopia of MAM-treated rats, by analyzing post-natal cytoarchitectural features and time of neurogenesis using bromodeoxyuridine immunocytochemistry. The cytoarchitectural analysis demonstrated the existence, in the early post-natal period, of white matter cellular bands in close anatomical relationship with the heterotopia, which most likely serve as a reservoir of young, migrating neurons for the newly forming heterotopia. The birth dating analysis demonstrated that the period of generation of neurons within the heterotopia and adjacent white matter bands, was extended in comparison to corticogenesis in normal rat brains. In addition, it demonstrated that the heterotopia were formed through a rather precise outside-in (for cortical and periventricular heterotopia) and dorso-ventral (for intra-hippocampal heterotopia) neurogenetic pattern. We hypothesize that the MAM-induced ablation of an early wave of cortical neurons is sufficient to alter per se the migration and differentiation of subsequently generated neurons, which in turn set the base for the formation of the different types of heterotopia. On this basis, we suggest a neurogenetic scheme for MAM-induced heterotopia that can also explain the origin and intrinsic epileptogenicity of periventricular nodular heterotopia in humans.

The NMDA receptor complex is altered in an animal model of human cerebral heterotopia.

Double intraperitoneal injections of methylazoxymethanol (MAM) in pregnant rats induce developmental brain dysgenesis with nodular heterotopia similar to human periventricular nodular heterotopia (PNH) and composed of hyperexcitable neurons. Here we analyzed the NMDA receptor complex and associated proteins in the heterotopic neurons of 2- to 3-month-old MAM-treated rats by means of a combined immunocytochemical/molecular approach. Our data demonstrated a clear reduction of p286-active form of alphaCaMKII and a selective impairment of both the targeting and the CaMKII-dependent phosphorylation of NR2A/B subunits in the postsynaptic membranes of the MAM-induced heterotopia. The reduced NR2A/B immunofluorescence of the cellular membrane was not due to reduced expression since it was decreased only in postsynaptic fractions but not in the homogenate. NMDA-NR1 and AMPA-GluR2/3 subunits, as well as PSD-95 and total alphaCaMKII protein levels, were not affected in MAM-treated rats, thus revealing that the overall composition of the postsynaptic fraction was not altered. These data clearly suggest that the molecular organization of the NMDA/alphaCaMKII complex is selectively altered in the postsynaptic compartment of heterotopic neurons. This alteration can play a role in determining the hyperexcitability of brain heterotopia in MAM rats as well as in human patients affected by PNH.

Neurogenesis in cerebral heterotopia induced in rats by prenatal methylazoxymethanol treatment.

We have previously demonstrated that the antiproliferative agent methylazoxymethanol acetate (MAM) is able to induce in rats cerebral heterotopia that share striking similarities with those observed in human periventricular nodular heterotopia (PNH), a cerebral dysgenesis frequently observed in human patients affected by drug-resistant focal epilepsy. In this study, we investigated the time-course of neurogenesis in the cerebral heterotopia of MAM-treated rats, with the idea of understanding why PNH develop in human patients. For these goals, we analyzed the cytoarchitectural features, the time of neurogenesis and the cellular phenotype of the heterotopia, by means of BrdU immunocytochemistry and confocal immunofluorescence experiments. Our data demonstrate that the different types of heterotopia in MAM-treated rats are formed through the same altered neurogenetic process, which follows quite organized neurogenetic gradients. The MAM-induced ablation of an early wave of cortical neurons is sufficient to alter per se the migration and differentiation of subsequently generated neurons, which in turn set the base for the formation of the different heterotopic structures. The neurogenesis of MAM-induced heterotopia may explain the origin and intrinsic epileptogenicity of periventricular nodular heterotopia in human patients.

AlphaCaMKII and NMDA-receptor subunit expression in epileptogenic cortex from human periventricular nodular heterotopia.

PURPOSE: Periventricular nodular heterotopia (PNH) is the most common human brain dysgenesis, very frequently characterized by focal drug-resistant epilepsy. To understand the cellular mechanisms underlying its intrinsic hyperexcitability, we investigated the expression of glutamate-receptor subunits and related proteins in four human patients affected by PNH. METHODS: PNH was diagnosed by means of magnetic resonance imaging. The epileptogenic area was revealed by depth electrode recordings and removed during epilepsy surgery. Sections from the removed cerebral tissue were analyzed by means of immunocytochemistry (ICC), with antibodies directed against N-methyl-d-aspartate (NMDA)-receptor subunits, the alpha subunit of the Ca2+/calmodulin-dependent kinase II (alphaCaMKII), and its active phosphorylated form. RESULTS: The ICC data demonstrated that the subcortical heterotopic nodules were consistently characterized by lower expression of alphaCaMKII and its activated form. In more pronounced cases (i.e., when the extension of the nodules to the neocortex determined clear layering abnormalities), the heterotopic tissue also was characterized by a decreased expression of NMDA-receptor subunits, which was particularly evident in the dendritic compartment. CONCLUSIONS: These data suggest the existence of an alteration of alphaCaMKII and the NMDA-receptor complex in the epileptogenic brain tissue of human PNH, which may play a role in the basic mechanisms of hyperexcitability associated with this brain dysgenesis.