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 2ϩ 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.
Key pointsr The role of metabotropic purinergic receptors (P2YRs) in modulating motor output from the CNS is virtually unknown, despite the fact that many motoneurons, including respiratory motoneurons, express P2YRs.r Using rhythmically active brainstem-spinal cord and medullary slice preparations, we demonstrate that compared to the 4th cervical spinal nerve (C4) inspiratory output controlling the diaphragm, P2YR activation is >10 times more efficacious at potentiating the hypoglossal nerve (XII) inspiratory output controlling airway muscles.r P2YR potentiation of inspiratory output appears largely mediated by P2Y 1 R. r Whole-cell recordings from XII motoneurons (MNs) suggest that the P2Y 1 R-mediated potentiation of inspiratory synaptic inputs, glutamate currents, and persistent inward currents, results in part from potentiation of a transient receptor potential cation channel, subfamily M, member 4 (TRPM4)-mediated, calcium-activated, non-specific cation current, I CAN .r The low sensitivity of phrenic output to P2YR activation questions its physiological significance in modulating diaphragm activity. However, the greater sensitivity of XII MNs, combined with observations that ATP is often co-released with noradrenaline and that noradrenergic neuron activity decreases in sleep, makes it tempting to speculate that loss of purinergic modulation contributes to state-dependent reductions in XII MN excitability.Abstract PreBötzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y 1 R) activation. Despite this, and the fact that inspiratory MNs express P2Y 1 Rs, the role of P2Y 1 Rs in modulating motor output is not known for any MN pool. We used rhythmically active brainstem-spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y 1 R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y 1 R agonist, 0.1 mM) potentiated XII inspiratory burst amplitude by 60 ± 9%; 10-fold higher concentrations potentiated C4 burst amplitude by 25 ± 7%. In whole-cell voltage-clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca 2+ -dependent non-selective cation current I CAN ; high intracellular concentrations of BAPTA, which buffers Ca 2+ increases necessary for activation of I CAN ; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for I CAN ). Real-time PCR analysis of mRNA extracted from XII punches and laser-microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y 1 R modulation than p...
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-β-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.
Despite the enormous diversity of glutamate (Glu) receptors and advances in understanding recombinant receptors, native Glu receptors underlying functionally identified inputs in active systems are poorly defined in comparison. In the present study we use UBP-302, which antagonizes GluR5 subunit-containing kainate (KA) receptors at ≤ 10 μM, but other KA and AMPA receptors at ≥ 100 μM, and rhythmically active in vitro preparations of neonatal rat to explore the contribution of non-NMDA receptor signalling in rhythm-generating and motor output compartments of the inspiratory network. At 10 μM, UBP-302 had no effect on inspiratory burst frequency or amplitude. At 100 μM, burst amplitude recorded from XII, C1 and C4 nerve roots was significantly reduced, but frequency was unaffected. The lack of a frequency effect was confirmed when local application of UBP-302 (100 μM) into the pre-Bötzinger complex (preBötC) did not affect frequency but substance P evoked a 2-fold increase. A UBP-302-sensitive (10 μM), ATPA-evoked frequency increase, however, established that preBötC networks are sensitive to GluR5 activation. Whole-cell recordings demonstrated that XII motoneurons also express functional GluR5-containing KA receptors that do not contribute to inspiratory drive, and confirmed the dose dependence of UBP-302 actions on KA and AMPA receptors. Our data provide the first evidence that the non-NMDA (most probably AMPA) receptors mediating glutamatergic transmission within preBötC inspiratory rhythm-generating networks are pharmacologically distinct from those transmitting drive to inspiratory motoneurons. This differential expression may ultimately be exploited pharmacologically to separately counteract depression of central respiratory rhythmogenesis or manipulate the drive to motoneurons controlling airway and pump musculature.
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.
Breathing in premature infants often stops briefly (apnea) because the brainstem network that controls breathing is immature (apnea of prematurity, AOP). Apneas cause hypoxia, triggering the hypoxic ventilatory response (HVR), which comprises an initial increase in ventilation followed by a centrally mediated 2° decrease. In adults, breathing remains above baseline during this 2° phase, but in the very young it falls below baseline leading to a life‐threatening feedback loop where apnea causes hypoxia, respiratory depression, further hypoxia and so on. Understanding the cause of this powerful hypoxic inhibition of breathing in the very young, a major question in perinatal physiology, could inform new therapies for AOP. The transmitter ATP is released in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation) during hypoxia where it increases breathing and attenuates the 2° depression. However, extracellular ATP (ATPe) is degraded to adenosine (ADO), which inhibits breathing and is implicated in AOP; e.g., caffeine blocks ADOe actions and is the first line treatment for AOP. We hypothesize that the greater 2° inhibition of breathing in prematurity is due to an immature system for clearing ADOe. ADOe‐mediated inhibition stops with its removal from the extracellular space, which depends on i) equilibrative nucleoside transporters (ENTs) that move ADOe across cell membranes down its concentration gradient, and ii) ADO kinase (ADK), an enzyme that keeps the level of ADO inside cells lower than outside so ENTs clear ADOe. How ENT activity develops in the preBötC is unknown. However, the form of ADK that influences ADOe in the brain is minimally functional until 2 weeks of age in rodents. To assess the importance of ADOe clearance in the development of the HVR, we used plethysmography to measure the HVR (10% O2) in 0‐56 day old (P0‐14) wild‐type (WT), ENT knockout (KO), and ADKtg mice engineered to have functional ADK throughout life. In WT and ENT KO neonates (P0‐2), ventilation fell below baseline after ~7 min in hypoxia, but the hypoxia‐evoked frequency increase was markedly reduced in the ENT KO. Strikingly, P0‐2 ADKtg mice showed the mature breathing response; ventilation remained above baseline levels throughout hypoxia. At P12 in WT and ENT KO, the HVR had still not matured; ventilation fell below baseline during the 2° phase, yet it remained above baseline in P12 ADKtg mice. To assess the importance of ENT and ADK specifically in the preBötC, we isolated the preBötC network in rhythmic brain slices from P0‐12 mice. The ADO inhibition of preBötC frequency lasted twice as long in P0‐12 ENT KO mice compared to WT. Conversely, the decrease in basal frequency caused by ADK inhibition (ABT‐702) increased with age (P0‐2, 15%; P9‐11, 23%). These data suggest that i) ENTs are important for ADO clearance in the preBötC after birth and ii) low ADK activity at birth is a major contributor to the greater hypoxic respiratory depression in early development.
Exposure to low oxygen (hypoxia) evokes a biphasic hypoxic ventilatory response (HVR) characterized by a carotid‐body‐mediated increase in ventilation followed by a centrally‐mediated secondary hypoxic respiratory depression (HRD). The HRD is strongest and life‐threatening in premature infants (e.g., Apnea of Prematurity). Purinergic signaling plays a prominent role in shaping the HVR; hypoxia evokes release of ATP from astrocytes in the preBötzinger Complex (preBötC, key site of inspiratory rhythm generation), which increases breathing and attenuates the HRD. However, ATP is metabolized into adenosine (ADO), which inhibits the preBötC and is implicated in the HRD. Thus, the net effect of ATP in the preBötC during hypoxia is determined by the balance between the actions of ATP and ADO. The objective of this study is to determine during development how mechanisms important in clearing extracellular ADO (ADOe) influence preBötC activity and the HVR. First, we focused on equilibrative nucleoside transporters (ENTs) that passively move ADO across cell membranes down its concentration gradient. We compared the HVR (10 min 10% O2) of WT and ENT1/2 double KO mice (P0–3, P6–8, and P12–14) using whole‐body plethysmography. The HRD of ENT KO mice was significantly greater compared to WT mice at all ages, reflecting greater reductions in breathing frequency and a likely role for ENTs in removing ADOe during hypoxia. We then applied the ENT inhibitor NBMPR (100 μM) to the solution bathing rhythmic, preBötC‐containing medullary slices from neonatal mice to directly assess the influence of ENTs on basal preBötC rhythm. A significant, NBMPR‐mediated, 16% ± 7% (n=5) increase in frequency suggests that ENTs elevate basal ADOe tone, causing a modest reduction in frequency. In contrast, the reduction in inspiratory frequency evoked by injecting ADOe into the preBötC (to simulate a high ADOe load as may occur in hypoxia) was almost 2‐fold greater in slices from ENT1/2 KO compared to WT mice; i.e., under high load, ENTs appear to clear ADOe. We next examined ADO kinase (ADK), an enzyme that converts ADO into AMP, lowering intracellular ADO levels and facilitating clearance of ADOe by ENTs. Inhibition of ADK with ABT‐702 (10 μM) produced an adenosine‐mediated (i.e., the effect was reversed with the A1R antagonist, DPCPX, 100 nM) decrease in the baseline frequency of rhythmic medullary slices that was 16% ± 2% (n=7) at P0–2 and 24% ± 5% (n=8) by P9–11.In summary, these data suggest that under basal conditions ENTs and ADK are important in controlling ADOe tone, and that under conditions of high ADOe load, as produced here via local injection of ADO in vitro or hypoxia in vivo, ENTs and ADK become important in clearing ADOe, which increases ventilation. Finally, the observation in vitro that ADK activity in the preBötC appears to increase during development raises the intriguing possibility that the greater HRD of premature infants is due, at least in part, to an immature system for clearing ADOe.Support or Funding InformationCIHR, NSERC, WCHRI, AIHS, CFI, Alberta Lung AssociationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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