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 (

BackgroundHypoglossal (XII) motoneurons innervate tongue muscles and are vital for maintaining upper-airway patency during inspiration. Depression of XII nerve activity by opioid analgesics is a significant clinical problem, but underlying mechanisms are poorly understood. Currently there are no suitable pharmacological approaches to counter opiate-induced suppression of XII nerve activity while maintaining analgesia. Ampakines accentuate α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor responses. The AMPA family of glutamate receptors mediate excitatory transmission to XII motoneurons. Therefore the objectives were to determine whether the depressant actions of μ-opioid receptor activation on inspiratory activity includes a direct inhibitory action at the inspiratory premotoneuron to XII motoneuron synapse, and to identify underlying mechanism(s). We then examined whether ampakines counteract opioid-induced depression of XII motoneuron activity.Methodology/Principal FindingsA medullary slice preparation from neonatal rat that produces inspiratory-related output in vitro was used. Measurements of inspiratory burst amplitude and frequency were made from XII nerve roots. Whole-cell patch recordings from XII motoneurons were used to measure membrane currents and synaptic events. Application of the μ-opioid receptor agonist, DAMGO, to the XII nucleus depressed the output of inspiratory XII motoneurons via presynaptic inhibition of excitatory glutamatergic transmission. Ampakines (CX614 and CX717) alleviated DAMGO-induced depression of XII MN activity through postsynaptic actions on XII motoneurons.Conclusions/SignificanceThe inspiratory-depressant actions of opioid analgesics include presynaptic inhibition of XII motoneuron output. Ampakines counteract μ-opioid receptor-mediated depression of XII motoneuron inspiratory activity. These results suggest that ampakines may be beneficial in countering opiate-induced suppression of XII motoneuron activity and resultant impairment of airway patency.

P2 receptor (R) signalling plays an important role in the central ventilatory response to hypoxia. The frequency increase that results from activation of P2Y 1 Rs in the preBötzinger complex (preBötC; putative site of inspiratory rhythm generation) may contribute, but neither the cellular nor ionic mechanism(s) underlying these effects are known. We applied whole-cell recording to rhythmically-active medullary slices from neonatal rat to define, in preBötC neurones, the candidate cellular and ionic mechanisms through which ATP influences rhythm, and tested the hypothesis that putative rhythmogenic preBötC neurones are uniquely sensitive to ATP. ATP (1 mM) evoked inward currents in all non-respiratory neurones and the majority of respiratory neurons, which included inspiratory, expiratory and putative rhythmogenic inspiratory neurones identified by sensitivity to substance P (1 μM) and DAMGO (50 μM) or by voltage-dependent pacemaker-like activity. ATP current densities were similar in all classes of preBötC respiratory neurone. Reversal potentials and input resistance changes for ATP currents in respiratory neurones suggested they resulted from either inhibition of a K + channel or activation of a mixed cationic conductance. The P2YR agonist 2MeSADP (1 mM) evoked only the latter type of current in inspiratory and pacemaker-like neurones. In summary, putative rhythmogenic preBötC neurones were sensitive to ATP. However, this sensitivity was not unique; ATP evoked similar currents in all types of preBötC respiratory neurone. The P2Y 1 R-mediated frequency increase is therefore more likely to reflect activation of a mixed cationic conductance in multiple types of preBötC neurone than excitation of one, highly sensitive group.

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To further our understanding of the role that voltage-activated Ca2+ channels play in the development, physiology and pathophysiology of motoneurones (MNs), we used whole-cell patch-clamp recording to compare voltage-activated Ca2+ currents in oculomotor (III) and hypoglossal (XII) MNs of neonatal [postnatal day (P)1-5] and juvenile (P14-19) rats. In contrast to III MNs that innervate extraocular muscles, XII MNs that innervate tongue muscles mature more rapidly, fire bursts of low frequency action potentials and are vulnerable to degeneration in amyotrophic lateral sclerosis. In neonates, low voltage-activated (LVA) Ca2+ current densities are similar in XII and III MNs but high voltage-activated (HVA) Ca2+ current densities are twofold higher in XII MNs. The HVA Ca2+ channel antagonists (nimodipine and nifedipine for L-type, omega-agatoxin-TK for P/Q-type and omega-conotoxin-GVIA for N-type) revealed that, while N- and P/Q-type HVA Ca2+ channels are present in both MN pools, a 3.5-fold greater P/Q-type Ca2+ current in XII MNs accounts for their greater HVA Ca2+ currents. Developmentally, LVA and HVA Ca2+ current densities decrease in III MNs but remain unchanged in XII MNs. Thus, the differences between these MN pools increase developmentally so that, in juveniles, the LVA Ca2+ current density is twofold greater and the HVA Ca2+ current density is threefold greater in XII compared with III MNs. We propose that this differential expression of LVA and HVA Ca2+ channels in XII and III MNs during development contributes to their distinct physiology and may also be a factor contributing to the greater susceptibility of XII MNs to degeneration as seen in amyotrophic lateral sclerosis.

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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.

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