Maternal opioids age-dependently impair neonatal respiratory control networks.

Infants exposed to opioids in utero are an increasing clinical population and these infants are often diagnosed with Neonatal Abstinence Syndrome (NAS). Infants with NAS have diverse negative health consequences, including respiratory distress. However, many factors contribute to NAS, confounding the ability to understand how maternal opioids directly impact the neonatal respiratory system. Breathing is controlled centrally by respiratory networks in the brainstem and spinal cord, but the impact of maternal opioids on developing perinatal respiratory networks has not been studied. Using progressively more isolated respiratory network circuitry, we tested the hypothesis that maternal opioids directly impair neonatal central respiratory control networks. Fictive respiratory-related motor activity from isolated central respiratory networks was age-dependently impaired in neonates after maternal opioids within more complete respiratory networks (brainstem and spinal cords), but unaffected in more isolated networks (medullary slices containing the preBötzinger Complex). These deficits were due, in part, to lingering opioids within neonatal respiratory control networks immediately after birth and involved lasting impairments to respiratory pattern. Since opioids are routinely given to infants with NAS to curb withdrawal symptoms and our previous work demonstrated acute blunting of opioid-induced respiratory depression in neonatal breathing, we further tested the responses of isolated networks to exogenous opioids. Isolated respiratory control networks also demonstrated age-dependent blunted responses to exogenous opioids that correlated with changes in opioid receptor expression within a primary respiratory rhythm generating region, the preBötzinger Complex. Thus, maternal opioids age-dependently impair neonatal central respiratory control and responses to exogenous opioids, suggesting central respiratory impairments contribute to neonatal breathing destabilization after maternal opioids and likely contribute to respiratory distress in infants with NAS. These studies represent a significant advancement of our understanding of the complex effects of maternal opioids, even late in gestation, contributing to neonatal breathing deficits, necessary first steps in developing novel therapeutics to support breathing in infants with NAS.

Maternal Methadone Destabilizes Neonatal Breathing and Desensitizes Neonates to Opioid-Induced Respiratory Frequency Depression.

Pregnant women and developing infants are understudied populations in the opioid crisis, despite the rise in opioid use during pregnancy. Maternal opioid use results in diverse negative outcomes for the fetus/newborn, including death; however, the effects of perinatal (maternal and neonatal) opioids on developing respiratory circuitry are not well understood. Given the profound depressive effects of opioids on central respiratory networks controlling breathing, we tested the hypothesis that perinatal opioid exposure impairs respiratory neural circuitry, creating breathing instability. Our data demonstrate maternal opioids increase apneas and destabilize neonatal breathing. Maternal opioids also blunted opioid-induced respiratory frequency depression acutely in neonates; a unique finding since adult respiratory circuity does not desensitize to opioids. This desensitization normalized rapidly between postnatal days 1 and 2 (P1 and P2), the same age quantal slowing emerged in respiratory rhythm. These data suggest significant reorganization of respiratory rhythm generating circuits at P1-2, the same time as the preBötzinger Complex (key site of respiratory rhythm generation) becomes the dominant respiratory rhythm generator. Thus, these studies provide critical insight relevant to the normal developmental trajectory of respiratory circuits and suggest changes to mutual coupling between respiratory oscillators, while also highlighting how maternal opioids alter these developing circuits. In conclusion, the results presented demonstrate neurorespiratory disruption by maternal opioids and blunted opioid-induced respiratory frequency depression with neonatal opioids, which will be important for understanding and treating the increasing population of neonates exposed to gestational opioids.

Impact of inflammation on developing respiratory control networks: rhythm generation, chemoreception and plasticity.

The respiratory control network in the central nervous system undergoes critical developmental events early in life to ensure adequate breathing at birth. There are at least three "critical windows" in development of respiratory control networks: 1) in utero, 2) newborn (postnatal day 0-4 in rodents), and 3) neonatal (P10-13 in rodents, 2-4 months in humans). During these critical windows, developmental processes required for normal maturation of the respiratory control network occur, thereby increasing vulnerability of the network to insults, such as inflammation. Early life inflammation (induced by LPS, chronic intermittent hypoxia, sustained hypoxia, or neonatal maternal separation) acutely impairs respiratory rhythm generation, chemoreception and increases neonatal risk of mortality. These early life impairments are also greater in young males, suggesting sex-specific impairments in respiratory control. Further, neonatal inflammation has a lasting impact on respiratory control by impairing adult respiratory plasticity. This review focuses on how inflammation alters respiratory rhythm generation, chemoreception and plasticity during each of the three critical windows. We also highlight the need for additional mechanistic studies and increased investigation into how glia (such as microglia and astrocytes) play a role in impaired respiratory control after inflammation. Understanding how inflammation during critical windows of development disrupt respiratory control networks is essential for developing better treatments for vulnerable neonates and preventing adult ventilatory control disorders.

Time and dose-dependent impairment of neonatal respiratory motor activity after systemic inflammation.

Neonatal respiratory impairment during infection is common, yet its effects on respiratory neural circuitry are not fully understood. We hypothesized that the timing and severity of systemic inflammation is positively correlated with impairment in neonatal respiratory activity. To test this, we evaluated time- and dose-dependent impairment of in vitro fictive respiratory activity. Systemic inflammation (induced by lipopolysaccharide, LPS, 5 mg/kg, i.p.) impaired burst amplitude during the early (1 h) inflammatory response. The greatest impairment in respiratory activity (decreased amplitude, frequency, and increased rhythm disturbances) occurred during the peak (3 h) inflammatory response in brainstem-spinal cord preparations. Surprisingly, isolated medullary respiratory circuitry within rhythmic slices showed decreased baseline frequency and delayed onset of rhythm only after higher systemic inflammation (LPS 10 mg/kg) early in the inflammatory response (1 h), with no impairments at the peak inflammatory response (3 h). Thus, different components of neonatal respiratory circuitry have differential temporal and dose sensitivities to systemic inflammation, creating multiple windows of vulnerability for neonates after systemic inflammation.

Viral Mimetic-Induced Inflammation Abolishes Q-Pathway, but Not S-Pathway, Respiratory Motor Plasticity in Adult Rats.

Inflammation arises from diverse stimuli eliciting distinct inflammatory profiles, yet little is known about the effects of different inflammatory stimuli on respiratory motor plasticity. Respiratory motor plasticity is a key feature of the neural control of breathing and commonly studied in the form of phrenic long-term facilitation (pLTF). At least two distinct pathways can evoke pLTF with differential sensitivities to bacterial-induced inflammation. The Q-pathway is abolished by bacterial-induced inflammation, while the S-pathway is inflammation-resistant. Since viral-induced inflammation is common and elicits distinct temporal inflammatory gene profiles compared to bacterial inflammation, we tested the hypothesis that inflammation induced by a viral mimetic (polyinosinic:polycytidylic acid, polyIC) would abolish Q-pathway-evoked pLTF, but not S-pathway-evoked pLTF. Further, we hypothesized Q-pathway impairment would occur later relative to bacterial-induced inflammation. PolyIC (750 µg/kg, i.p.) transiently increased inflammatory genes in the cervical spinal cord (3 h), but did not alter medullary and splenic inflammatory gene expression, suggesting region specific inflammation after polyIC. Dose-response experiments revealed 750 µg/kg polyIC (i.p.) was sufficient to abolish Q-pathway-evoked pLTF at 24 h (17 ± 15% change from baseline, n = 5, p > 0.05). However, polyIC (750 µg/kg, i.p.) at 3 h was not sufficient to abolish Q-pathway-evoked pLTF (67 ± 21%, n = 5, p < 0.0001), suggesting a unique temporal impairment of pLTF after viral-mimetic-induced systemic inflammation. A non-steroidal anti-inflammatory (ketoprofen, 12.5 mg/kg, i.p., 3 h) restored Q-pathway-evoked pLTF (64 ± 24%, n = 5, p < 0.0001), confirming the role of inflammatory signaling in pLTF impairment. On the contrary, S-pathway-evoked pLTF was unaffected by polyIC-induced inflammation (750 µg/kg, i.p., 24 h; 72 ± 25%, n = 5, p < 0.0001) and was not different from saline controls (65 ± 32%, n = 4, p = 0.6291). Thus, the inflammatory-impairment of Q-pathway-evoked pLTF is generalizable between distinct inflammatory stimuli, but differs temporally. On the contrary, S-pathway-evoked pLTF is inflammation-resistant. Therefore, in situations where respiratory motor plasticity may be used as a tool to improve motor function, strategies targeting S-pathway-evoked plasticity may facilitate therapeutic outcomes.

One bout of neonatal inflammation impairs adult respiratory motor plasticity in male and female rats.

Neonatal inflammation is common and has lasting consequences for adult health. We investigated the lasting effects of a single bout of neonatal inflammation on adult respiratory control in the form of respiratory motor plasticity induced by acute intermittent hypoxia, which likely compensates and stabilizes breathing during injury or disease and has significant therapeutic potential. Lipopolysaccharide-induced inflammation at postnatal day four induced lasting impairments in two distinct pathways to adult respiratory plasticity in male and female rats. Despite a lack of adult pro-inflammatory gene expression or alterations in glial morphology, one mechanistic pathway to plasticity was restored by acute, adult anti-inflammatory treatment, suggesting ongoing inflammatory signaling after neonatal inflammation. An alternative pathway to plasticity was not restored by anti-inflammatory treatment, but was evoked by exogenous adenosine receptor agonism, suggesting upstream impairment, likely astrocytic-dependent. Thus, the respiratory control network is vulnerable to early-life inflammation, limiting respiratory compensation to adult disease or injury.

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia.

Plasticity is an important aspect of the neural control of breathing. One well-studied form of respiratory plasticity is phrenic long-term facilitation (pLTF) induced by acute intermittent but not sustained hypoxia. Okadaic acid-sensitive protein phosphatases (PPs) differentially regulate phrenic nerve activity with intermittent vs. sustained hypoxia, at least partially accounting for pLTF pattern sensitivity. However, okadaic acid inhibits multiple serine/threonine phosphatases, and the relevant phosphatase (PP1, PP2A, PP5) for pLTF pattern sensitivity has not been identified. Here, we demonstrate that sustained hypoxia (25 min, 9-10.5% O2) elicits phrenic motor facilitation in rats pretreated with bilateral intrapleural injections of small interfering RNAs (siRNAs; Accell-modified to preferentially transfect neurons, 3.33 µM, 3 days) targeting PP1 mRNA (48 ± 14% change from baseline, n = 6) but not PP2A (14 ± 9% baseline, n = 6) or nontargeting siRNAs (4 ± 10% baseline, n = 7). In time control rats (no hypoxia) treated with siRNAs ( n = 6), no facilitation was evident (-9 ± 9% baseline). siRNAs had no effect on the hypoxic phrenic response. Immunohistochemistry revealed PP1 and PP2A protein in identified phrenic motoneurons. Although PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, we were not able to verify "knockdown" in vivo after siRNA treatment. On the other hand, PP1 and PP2A siRNAs significantly decreased PP1 and PP2A mRNA in PC12 cell cultures, verifying the intended siRNA effects. In conclusion, PP1 (not PP2A) is the relevant okadaic acid-sensitive phosphatase constraining phrenic motor facilitation after sustained hypoxia and likely contributing to pLTF pattern sensitivity. NEW & NOTEWORTHY This study demonstrates that the relevant okadaic acid-sensitive Ser/Thr protein phosphatase (PP) constraining facilitation after sustained hypoxia is PP1 and not PP2A. It suggests that PP1 may be critical in the pattern sensitivity of hypoxia-induced phrenic motor plasticity.

IL-1 receptor activation undermines respiratory motor plasticity after systemic inflammation.

Inflammation undermines respiratory motor plasticity, yet we are just beginning to understand the inflammatory signaling involved. Because interleukin-1 (IL-1) signaling promotes or inhibits plasticity in other central nervous system regions, we tested the following hypotheses: 1) IL-1 receptor (IL-1R) activation after systemic inflammation is necessary to undermine phrenic long-term facilitation (pLTF), a model of respiratory motor plasticity induced by acute intermittent hypoxia (AIH), and 2) spinal IL-1ß is sufficient to undermine pLTF. pLTF is significantly reduced 24 h after lipopolysaccharide (LPS; 100 µg/kg ip, 12 ± 18%, n = 5) compared with control (57 ± 25%, n = 6) and restored by peripheral IL-1R antagonism (63 ± 13%, n = 5, AF-12198, 0.5 mg/kg ip, 24 h). Furthermore, acute, spinal IL-1R antagonism (1 mM AF-12198, 15 µl it) restored pLTF (53 ± 15%, n = 4) compared with LPS-treated rats (11 ± 10%; n = 5), demonstrating IL-1R activation is necessary to undermine pLTF after systemic inflammation. However, in healthy animals, pLTF persisted after spinal, exogenous recombinant rat IL-1ß (rIL-1ß) (1 ng ± AIH; 66 ± 26%, n = 3, 10 ng ± AIH; 102 ± 49%, n = 4, 100 ng + AIH; 93 ± 51%, n = 3, 300 ng ± AIH; 37 ± 40%, n = 3; P < 0.05 from baseline). In the absence of AIH, spinal rIL-1ß induced progressive, dose-dependent phrenic amplitude facilitation (1 ng; -3 ± 5%, n = 3, 10 ng; 8 ± 22%, n = 3, 100 ng; 31 ± 12%, P < 0.05, n = 4, 300 ng; 51 ± 17%, P < 0.01 from baseline, n = 4). In sum, IL-1R activation, both systemically and spinally, undermines pLTF after LPS-induced systemic inflammation, but IL-1R activation is not sufficient to abolish plasticity. Understanding the inflammatory signaling inhibiting respiratory plasticity is crucial to developing treatment strategies utilizing respiratory plasticity to promote breathing during ventilatory control disorders. NEW & NOTEWORTHY This study gives novel insights concerning mechanisms by which systemic inflammation undermines respiratory motor plasticity. We demonstrate that interleukin-1 signaling, both peripherally and centrally, undermines respiratory motor plasticity. However, acute, exogenous interleukin-1 signaling is not sufficient to undermine respiratory motor plasticity.

Cyclooxygenase enzyme activity does not impair respiratory motor plasticity after one night of intermittent hypoxia.

Although inflammation is prevalent in many clinical disorders challenging breathing, we are only beginning to understand the impact of inflammation on neural mechanisms of respiratory control. We recently demonstrated one form of respiratory motor plasticity is extremely sensitive to even mild inflammation induced by a single night (8 h) of intermittent hypoxia (IH-1), mimicking aspects of obstructive sleep apnea. Specifically, phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (AIH) is abolished by IH-1, but restored by high doses of the non-steroidal anti-inflammatory drug, ketoprofen. Since a major target of ketoprofen is cyclooxygenase (COX) enzymes, we tested the involvement of COX in IH-1 suppression of pLTF using the selective COX inhibitor NS-398. Systemic COX inhibition (3 mg/kg, i.p., 3 h before AIH) had no effect on pLTF in normoxia treated rats (76 ±â€¯40% change from baseline, n = 6), and did not restore pLTF in IH-1 treated rats (-9 ±â€¯7% baseline, n = 6). Similarly, spinal COX inhibition (27 mM, 12 µl, i.t.) had no effect on pLTF in normoxic rats (76 ±â€¯34% baseline, n = 7), and did not significantly restore pLTF after IH-1 (37 ±â€¯18% baseline, n = 7). COX-2 protein is expressed in identified phrenic motor neurons of both normoxia and IH-1 exposed rats, but immunolabeling was minimal in surrounding microglia; IH-1 had no discernable effect on COX-2 immunoreactivity. We conclude that the inflammatory impairment of pLTF by IH-1 is independent of COX enzyme activity or upregulated COX-2 expression.

Gestational intermittent hypoxia increases susceptibility to neuroinflammation and alters respiratory motor control in neonatal rats.

Sleep disordered breathing (SDB) and obstructive sleep apnea (OSA) during pregnancy are growing health concerns because these conditions are associated with adverse outcomes for newborn infants. SDB/OSA during pregnancy exposes the mother and the fetus to intermittent hypoxia. Direct exposure of adults and neonates to IH causes neuroinflammation and neuronal apoptosis, and exposure to IH during gestation (GIH) causes long-term deficits in offspring respiratory function. However, the role of neuroinflammation in CNS respiratory control centers of GIH offspring has not been investigated. Thus, the goal of this hybrid review/research article is to comprehensively review the available literature both in humans and experimental rodent models of SDB in order to highlight key gaps in knowledge. To begin to address some of these gaps, we also include data demonstrating the consequences of GIH on respiratory rhythm generation and neuroinflammation in CNS respiratory control regions. Pregnant rats were exposed to daily intermittent hypoxia during gestation (G10-G21). Neuroinflammation in brainstem and cervical spinal cord was evaluated in P0-P3 pups that were injected with saline or lipopolysaccharide (LPS; 0.1mg/kg, 3h). In CNS respiratory control centers, we found that GIH attenuated the normal CNS immune response to LPS challenge in a gene-, sex-, and CNS region-specific manner. GIH also altered normal respiratory motor responses to LPS in newborn offspring brainstem-spinal cord preparations. These data underscore the need for further study of the long-term consequences of maternal SDB on the relationship between inflammation and the respiratory control system, in both neonatal and adult offspring.

The impact of inflammation on respiratory plasticity.

Breathing is a vital homeostatic behavior and must be precisely regulated throughout life. Clinical conditions commonly associated with inflammation, undermine respiratory function may involve plasticity in respiratory control circuits to compensate and maintain adequate ventilation. Alternatively, other clinical conditions may evoke maladaptive plasticity. Yet, we have only recently begun to understand the effects of inflammation on respiratory plasticity. Here, we review some of common models used to investigate the effects of inflammation and discuss the impact of inflammation on nociception, chemosensory plasticity, medullary respiratory centers, motor plasticity in motor neurons and respiratory frequency, and adaptation to high altitude. We provide new data suggesting glial cells contribute to CNS inflammatory gene expression after 24h of sustained hypoxia and inflammation induced by 8h of intermittent hypoxia inhibits long-term facilitation of respiratory frequency. We also discuss how inflammation can have opposite effects on the capacity for plasticity, whereby it is necessary for increases in the hypoxic ventilatory response with sustained hypoxia, but inhibits phrenic long term facilitation after intermittent hypoxia. This review highlights gaps in our knowledge about the effects of inflammation on respiratory control (development, age, and sex differences). In summary, data to date suggest plasticity can be either adaptive or maladaptive and understanding how inflammation alters the respiratory system is crucial for development of better therapeutic interventions to promote breathing and for utilization of plasticity as a clinical treatment.

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-1ß 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.

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.

Isolated in vitro brainstem-spinal cord preparations remain important tools in respiratory neurobiology.

Isolated in vitro brainstem-spinal cord preparations are used extensively in respiratory neurobiology because the respiratory network in the pons and medulla is intact, monosynaptic descending inputs to spinal motoneurons can be activated, brainstem and spinal cord tissue can be bathed with different solutions, and the responses of cervical, thoracic, and lumbar spinal motoneurons to experimental perturbations can be compared. The caveats and limitations of in vitro brainstem-spinal cord preparations are well-documented. However, isolated brainstem-spinal cords are still valuable experimental preparations that can be used to study neuronal connectivity within the brainstem, development of motor networks with lethal genetic mutations, deleterious effects of pathological drugs and conditions, respiratory spinal motor plasticity, and interactions with other motor behaviors. Our goal is to show how isolated brainstem-spinal cord preparations still have a lot to offer scientifically and experimentally to address questions within and outside the field of respiratory neurobiology.

Substance P modulation of hypoglossal motoneuron excitability during development: changing balance between conductances.

Although Substance P (SP) acts primarily through neurokinin 1 (NK1) receptors to increase the excitability of virtually all motoneurons (MNs) tested, the ontogeny of this transmitter system is not known for any MN pool. Hypoglossal (XII) MNs innervate tongue protruder muscles and participate in several behaviors that must be functional from birth including swallowing, suckling and breathing. We used immunohistochemistry, Western immunoblotting, and whole cell recording of XII MNs in brain stem slices from rats ranging in age from postnatal day zero (P0) to P23 to explore developmental changes in: NK1 receptor expression; currents evoked by SP(NK1) (an NK1-selective SP receptor agonist) and; the efficacy of transduction pathways transforming ligand binding into channel modulation. Despite developmental reductions in XII MN NK1 receptor expression, SP(NK1) current density remained constant at 6.1 +/- 1.0 (SE) pA/pF. SP(NK1) activated at least two conductances. Activation of a pH-insensitive Na(+) conductance dominated in neonates (P0-P5), but its contribution fell from approximately 80 to approximately 55% in juveniles (P14-P23). SP(NK1) also inhibited a pH-sensitive, two-pore domain K(+) (TASK)-like K(+) current. Its contribution increased developmentally. First, the density of this pH-sensitive K(+) current doubled between P0 and P23. Second, SP(NK1) did not affect this current in neonates, but reduced it by 20% at P7-P10 and 80% in juveniles. In addition, potentiation of repetitive firing was greatest in juveniles. These data establish that despite apparent reductions in NK1 receptor density, SP remains an important modulator of XII MN excitability throughout postnatal development due, in part, to increased expression of a pH-sensitive, TASK-like conductance.

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.