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.

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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.

Inspiratory rhythm generating networks in the preBötzinger Complex (preBötC) are ~100‐fold more sensitive to ATP than XII MNs that control airway patency. This is believed to reflect in part that the effects of ATP in the preBötC are primarily mediated by P2Y1Rs while P2XRs receptors dominate the actions of ATP in XII MNs. However, the effects of P2Y1R signaling on XII motor output and MN properties have not been examined. To address this deficit, we applied nerve and whole‐cell recording techniques to rhythmic medullary slices of neonatal rat.Local application of MRS2365 (P2Y1R agonist) to the XII nucleus of rhythmic slices increased tonic discharge and evoked 22±2 (0.1 mM) and 33±4% (1 mM, n=16) increases in inspiratory burst amplitude. MRS2365 also increased the frequency and amplitude of spontaneous PSCs in non‐inspiratory and inspiratory MNs alike. In inspiratory MNs, it also increased the charge transferred per inspiratory cycle (12±6%, n=5). The increase in synaptic activity was superimposed on a small, slow, inward current that was similar in all XII MNs, averaging 27±7 pA (1 mM, n=10). Following block action potentials with TTX, MRS2365 had no effect on membrane current or resistance.These data suggest that P2Y1Rs do not directly affect XII MN membrane properties, but potentiate inspiratory XII nerve output by specifically modulating synaptic function.Supported by CIHR, NSERC, CIHR‐MFN, AHFMR, CFI, ASRA.

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