Adrianne G. Huxtable

Hypoxia‐induced phrenic long‐term facilitation: emergent properties

As in other neural systems, plasticity is a hallmark of the neural system controlling breathing. One spinal mechanism of respiratory plasticity is phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia. Although cellular mechanisms giving rise to pLTF occur within the phrenic motor nucleus, different signaling cascades elicit pLTF under different conditions. These cascades, referred to as Q and S pathways to phrenic motor facilitation (pMF), interact via cross‐talk inhibition. Whereas the Q pathway dominates pLTF after mild to moderate hypoxic episodes, the S pathway dominates after severe hypoxic episodes. The biological significance of multiple pathways to pMF is unknown. This review will discuss the possibility that interactions between pathways confer emergent properties to pLTF, including pattern sensitivity and metaplasticity. Understanding these mechanisms and their interactions may enable us to optimize intermittent hypoxia‐induced plasticity as a treatment for patients that suffer from ventilatory impairment or other motor deficits.

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 Ca2+ 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.

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) ≈ 2MeSATP (2-methylthioadenosine 5′-triphosphate) ≈ ATPγs (adenosine 5′-[γ-thio]triphosphate tetralithium salt) ≈ ATP ≫ UTP ≈ αβmeATP (α,β-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 ATPγs, 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.

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 P2Y1R-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 (∼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 A1 receptors exerts a tonic depressive action on rhythm, whereas A1 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.

Systemic inflammation impairs respiratory chemoreflexes and plasticity

Many lung and central nervous system disorders require robust and appropriate physiological responses to assure adequate breathing. Factors undermining the efficacy of ventilatory control will diminish the ability to compensate for pathology, threatening life itself. Although most of these same disorders are associated with systemic and/or neuroinflammation, and inflammation affects neural function, we are only beginning to understand interactions between inflammation and any aspect of ventilatory control (e.g. sensory receptors, rhythm generation, chemoreflexes, plasticity). Here we review available evidence, and present limited new data suggesting that systemic (or neural) inflammation impairs two key elements of ventilatory control: chemoreflexes and respiratory motor (versus sensory) plasticity. Achieving an understanding of mechanisms whereby inflammation undermines ventilatory control is fundamental since inflammation may diminish the capacity for natural, compensatory responses during pathological states, and the ability to harness respiratory plasticity as a therapeutic strategy in the treatment of devastating breathing disorders, such as during cervical spinal injury or motor neuron disease.

ATP in central respiratory control: A three-part signaling system

The landmark demonstrations in 2005 that ATP released centrally during hypoxia and hypercapnia contributes to the respective ventilatory responses validated a decade-old hypothesis and ignited interest in the potential significance of P2 receptor signaling in central respiratory control. Our objective in this review is to provide a non-specialist overview of ATP signaling from the perspective that it is a three-part system where the net effects are determined by an interaction between the signaling actions of ATP and adenosine at P2 and P1 receptors, respectively, and a family of enzymes (ectonucleotidases) that breakdown ATP into adenosine. We review the rationale for the original interest in P2 signaling in respiratory control, the evolution of this hypothesis, and the mechanisms by which ATP might affect respiratory behaviour. The potential significance of P2 receptor, P1 receptor and ectonucleotidase diversity for the different compartments of the respiratory control system is also considered. We conclude with a look to future questions and technical challenges.

Intermittent Hypoxia-Induced Spinal Inflammation Impairs Respiratory Motor Plasticity by a Spinal p38 MAP Kinase-Dependent Mechanism

Inflammation is characteristic of most clinical disorders that challenge the neural control of breathing. Since inflammation modulates neuroplasticity, we studied the impact of inflammation caused by prolonged intermittent hypoxia on an important form of respiratory plasticity, acute intermittent hypoxia (three, 5 min hypoxic episodes, 5 min normoxic intervals) induced phrenic long-term facilitation (pLTF). Because chronic intermittent hypoxia elicits neuroinflammation and pLTF is undermined by lipopolysaccharide-induced systemic inflammation, we hypothesized that one night of intermittent hypoxia (IH-1) elicits spinal inflammation, thereby impairing pLTF by a p38 MAP kinase-dependent mechanism. pLTF and spinal inflammation were assessed in anesthetized rats pretreated with IH-1 (2 min hypoxia, 2 min normoxia; 8 h) or sham normoxia and allowed 16 h for recovery. IH-1 (1) transiently increased IL-6 (1.5 ± 0.2-fold; p = 0.02) and inducible nitric oxide synthase (iNOS) (2.4 ± 0.4-fold; p = 0.01) mRNA in cervical spinal homogenates, (2) elicited a sustained increase in IL-1β mRNA (2.4 ± 0.2-fold; p < 0.001) in isolated cervical spinal microglia, and (3) abolished pLTF (−1 ± 5% vs 56 ± 10% in controls; p < 0.001). pLTF was restored after IH-1 by systemic NSAID administration (ketoprofen; 55 ± 9%; p < 0.001) or spinal p38 MAP kinase inhibition (58 ± 2%; p < 0.001). IH-1 increased phosphorylated (activated) p38 MAP kinase immunofluorescence in identified phrenic motoneurons and adjacent microglia. In conclusion, IH-1 elicits spinal inflammation and impairs pLTF by a spinal p38 MAP kinase-dependent mechanism. By targeting inflammation, we may develop strategies to manipulate respiratory motor plasticity for therapeutic advantage when the respiratory control system is compromised (e.g., sleep apnea, apnea of prematurity, spinal injury, or motor neuron disease).

Phrenic Long-Term Facilitation Requires PKCθ Activity within Phrenic Motor Neurons

Acute intermittent hypoxia (AIH) induces a form of spinal motor plasticity known as phrenic long-term facilitation (pLTF); pLTF is a prolonged increase in phrenic motor output after AIH has ended. In anesthetized rats, we demonstrate that pLTF requires activity of the novel PKC isoform, PKCθ, and that the relevant PKCθ is within phrenic motor neurons. Whereas spinal PKCθ inhibitors block pLTF, inhibitors targeting other PKC isoforms do not. PKCθ is highly expressed in phrenic motor neurons, and PKCθ knockdown with intrapleural siRNAs abolishes pLTF. Intrapleural siRNAs targeting PKCζ, an atypical PKC isoform expressed in phrenic motor neurons that underlies a distinct form of phrenic motor plasticity, does not affect pLTF. Thus, PKCθ plays a critical role in spinal AIH-induced respiratory motor plasticity, and the relevant PKCθ is localized within phrenic motor neurons. Intrapleural siRNA delivery has considerable potential as a therapeutic tool to selectively manipulate plasticity in vital respiratory motor neurons.

Interpreting deactivations in neuroimaging

Functional magnetic resonance imaging (fMRI) is a relatively young tool for understanding how the brains activities contribute to physical and psychological processes. In the roughly 20 years since its introduction, fMRI has developed at an astounding rate regarding advances in both the acquisition of images, as well as improved post-acquisition analysis (Bandettini, 2011). Blood oxygenation level dependent (BOLD) activity is its key measure and most studies rely on contrasts between two conditions of interest (e.g., BOLD signal during exposure to painful stimuli > signal during exposure to non-painful stimuli) to identify regions of functional significance. Alternately, electrophysiological techniques provide direct measures of neural activity in comparison to neuroimaging techniques. Deciphering the precise activity of the brain, in normal and pathological conditions, is of paramount importance; both neuroimaging and electrophysiological techniques will likely be crucial in this regard.

Adrenergic α1 receptor activation is sufficient, but not necessary for phrenic long-term facilitation

Acute intermittent hypoxia (AIH; three 5-min hypoxic episodes) causes a form of phrenic motor facilitation (pMF) known as phrenic long-term facilitation (pLTF); pLTF is initiated by spinal activation of Gq protein-coupled 5-HT2 receptors. Because α1 adrenergic receptors are expressed in the phrenic motor nucleus and are also Gq protein-coupled, we hypothesized that α1 receptors are sufficient, but not necessary for AIH-induced pLTF. In anesthetized, paralyzed, and ventilated rats, episodic spinal application of the α1 receptor agonist phenylephrine (PE) elicited dose-dependent pMF (10 and 100 μM, P < 0.05; but not 1 μM). PE-induced pMF was blocked by the α1 receptor antagonist prazosin (1 mM; -20 ± 20% at 60 min, -5 ± 21% at 90 min; n = 6). Although α1 receptor activation is sufficient to induce pMF, it was not necessary for AIH-induced pLTF because intrathecal prazosin (1 mM) did not alter AIH-induced pLTF (56 ± 9% at 60 min, 78 ± 12% at 90 min; n = 9). Intravenous (iv) prazosin (150 μg/kg) appeared to reduce pLTF (21 ± 9% at 60 min, 26 ± 8% at 90 min), but this effect was not significant. Hypoglossal long-term facilitation was unaffected by intrathecal prazosin, but was blocked by iv prazosin (-4 ± 14% at 60 min, -13 ± 18% at 90 min), suggesting different LTF mechanisms in different motor neuron pools. In conclusion, Gq protein-coupled α1 adrenergic receptors evoke pMF, but they are not necessary for AIH-induced pLTF.