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Postnatal development of the purinergic signaling system in the preBötzinger Complex; implications for the hypoxic ventilatory response
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- Author / Creator
- Reklow, Robert J.
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Low oxygen (hypoxia) evokes the biphasic hypoxic ventilatory response (HVR), in which carotid-body chemoreceptors trigger a rapid increase in ventilation (VE) followed by a centrally-mediated, secondary hypoxic depression of respiration (HRD) and metabolic rate (VO2) that involves adenosine. In adults, VE and VE/VO2 remain above baseline throughout the HRD. However, in neonates VE can fall below baseline, more than the hypoxia-induced decrease in VO2, such that neither VE nor VE/VO2 remain above baseline, which can become life-threatening. Astrocytes in the preBötzinger Complex (preBötC, brain site that generates inspiratory rhythm) sense hypoxia and release ATP, which binds P2Y1 receptors (Rs), increases VE and attenuates the HRD. ATP, however, is rapidly degraded to adenosine, which, via P1Rs, depresses breathing. Thus, the HRD is influenced by an interaction in the preBötC between ATP excitation and adenosine inhibition that changes developmentally. Multiple factors, collectively called the purinome, influence this balance, including P2Rs, P1Rs, ectonucleotidases that terminate ATP signaling and produce adenosine, and transporters and enzymes that terminate the actions of adenosine.
My first aim asked how individual purinome components contribute to postnatal changes in the net signaling actions of ATP in the preBötC by comparing how manipulating specific purinome components affected baseline preBötC behaviour and its responses to P1 and P2R-related agents applied to medullary slices from mice between postnatal day 0 (P0) and P12. My second aim assessed whether developmental changes in the purinome identified in Aim 1 shape the HVR in vivo and contribute to the blunted HVR of neonatal rodents. My final Aim tested the controversial hypothesis that the HVR of neonatal mammals (rodents) is not blunted; i.e., greater reductions in VE during hypoxia in neonates reflects a greater depression of VO2 such that mammals of all ages respond to hypoxia with an adaptive increase in VE/VO2 (hyperventilation).
Data from Aim 1 revealed that ATP excitation of preBötC frequency increased developmentally to match the P2Y1R excitation, which was constant postnatally. Differences between ATP and P2Y1R actions were not due to developmental reductions in ectonucleotidase activity or adenosine inhibition; ATP degradation by preBötC tissue increased with age and the adenosine-evoked decrease in frequency remained constant between P0 and P12. Removing equilibrative nucleoside transporters (ENT1 and 2) that clear adenosine prolonged adenosine inhibition in P0–12 slices, suggesting ENTs are unlikely to mediate developmental changes. In contrast, expression of adenosine kinase (ADK), which metabolizes adenosine and facilitates ENT-mediated adenosine clearance, was barely detectable at P0, and reached adult levels by P21. Functionally, ADK inhibition only affected baseline frequency in older slices.
To test the role of adenosine clearance mechanisms in the development of the HVR (Aim 2), I used plethysmography to measure the development (P0–adult) of ventilatory and metabolic responses evoked by 10% O2 in ENT1/2 knockout mice and ADKtg mice. ENT1/2 knockout had surprisingly modest effects on the HVR. In contrast, neonatal ADKtg mice that express adult levels of ADK responded to hypoxia with an adult-like, sustained hyperventilation, while VE/VO2 did not change in neonatal wild-type mice. These strain differences disappeared by P6, supporting a critical role of ADK in development of the HVR. Surprisingly, the adult-like HVR in neonatal ADKtg mice was not due to an enhanced ventilatory response but a hypoxia-induced decrease in VO2 not seen in wild-types.
To test whether the greater HRD in neonates reflects greater hypoxic depression of VO2 (Aim 3), I measured VE and VO2 responses of C57BL/6J and FVB mice and SD rats to 10% O2 daily starting at P0. Hypoxia never depressed VO2 in P0 rodents; nor did it evoke in P0 mice a sustained increase in VE/VO2. Hypoxia-induced reductions in VO2 emerged after P3 in C57BL/6J mice and at P1 in FVB mice and SD rats, demonstrating that the mechanisms underlying the adaptive depression of VO2 by hypoxia develop after postnatally.
These data demonstrate that puringeric signaling in the preBötC network shifts toward excitation postnatally, in part due to increased ADK activity and enhanced adenosine clearance. ENT and especially ADK activity contribute to a more sustained HVR with development. Most surprising, adult-like ADK expression in neonates bestows an ability for hypoxia to inhibit metabolism. Thus, purinergic modulation of central networks may impact development of adaptive ventilatory and metabolic responses to hypoxia.
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- Graduation date
- Fall 2023
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.