Purinergic and Glial Signalling in the Hypoxic Ventilatory Response

• Author / Creator

  Rajani, Vishaal

• The hypoxic ventilatory response comprises of an initial increase in ventilation, followed by a secondary depression that can be life threatening in premature infants. During hypoxia, ATP is released in the ventrolateral medulla, where it is suggested to attenuate the secondary hypoxic respiratory depression. The objective of my thesis is to explore the contribution of ATP to the hypoxic ventilatory response, specifically exploring the (i) cell-types, (ii) site, (iii) receptor subtype and (iv) signalling pathways that underlie the actions of ATP. I first tested the contribution of glia. I disrupted vesicular release proteins that are necessary for gliotransmission via bilateral injection of an adenovirus controlling glial-specific expression of tetanus light chain protein (TeLC) into the ventral respiratory column (VRC). TeLC caused a decrease in ventilation during the initial phase I component of hypoxic ventilatory response when ventilation increases quickly, and also during the phase II component after ventilation has plateaued after the secondary depression. I next tested which receptor subtype is responsible for attenuating the hypoxic ventilatory depression and where in the VRC this effect was mediated. In adult rats, in vivo unilateral injection of P2Y1 receptor agonist into the preBötzinger Complex (preBötC, central site of inspiratory rhythm generation) caused an increase in respiratory frequency, while in vivo injection of P2Y1 receptor antagonist into the same site increased in the secondary hypoxic respiratory depression. In vivo injection of adenosine, the end product of ATP breakdown, into the preBötC had no effect on ventilation. To explore signaling cascades underlying the excitatory effects of P2Y1 receptor activation in the preBötC, I tested the effect of selective pharmacological blockers on the P2Y1 receptor mediated frequency increase in the neonatal rhythmic slice preparation. Experiments revealed that the P2Y1 receptor mediated excitation is sensitive to calcium chelation, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition, antagonism of IP3 receptors, and protein kinase C (PKC) inhibition. Similar to the P2Y1 network response, I show that the ATP activation of cultured preBötC glia via intracellular Ca2+ increases are also sensitive to SERCA inhibition. These findings suggest that ATP, through a process that involves gliotransmission, acts via P2Y1 receptors in the preBötC to attenuate the secondary hypoxic respiratory depression in vivo, via signaling cascade that involves release of calcium from intracellular stores and PKC activation. This work provides in vivo verification of multiple hypotheses previously developed from in vitro preparations, and lays a foundation for future research in understanding the underlying mechanism of purinergic signalling and the contribution of glia to respiratory network modulation.

• Subjects / Keywords

  • ATP
  • PreBötzinger Complex
  • Astrocytes
  • Hypoxia
  • Glia
  • Purinergic receptor
  • Hypoxic Ventilatory Response
  • P2Y1

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3P844189

• 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.