Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, C85

Oral Communications

Evidence of a role for astrocyte purinergic signalling in regulation of extracellular potassium and action potential propagation in central nervous system white matter

V. Bay1, P. Hurst1, A. Butt1

1. University of Portsmouth, Portsmouth, United Kingdom.

It is now recognized that astrocytes in the central nervous system (CNS) communicate with each other by an ATP-mediated rise in intracellular calcium ([Ca2+]i), called Ca2+ signaling. Astroglial Ca2+ signaling has been studied extensively in grey matter synaptic regions, but its functional significance remains unclear. A key function of astrocytes is considered to be uptake of K+ released by axons during action potential propagation, which is essential for sustained neuronal activity. Therefore, we examined whether glial ATP-mediated communication has a role in K+ regulation and maintenance of axonal excitability in the rat optic nerve, a CNS white matter tract. Wistar rats aged postnatal day (P) 15-30 were killed humanely in accordance with the UK Animals (Scientific Procedures) Act (1986), and optic nerves were isolated and maintained in a brain slice chamber continuously perfused with artificial cerebrospinal fluid (aCSF). Changes in glial [Ca2+]i, nerve compound action potential (CAP) and [K+]o were measured in the presence of a range of purinergic receptor (P2R) modulators, for both ionotropic P2XR and metabotropic P2YR: (100μM unless otherwise stated) general P2XR/P2YR antagonist suramin; P2XR antagonist PPADS; P2X7R antagonist oATP; P2X7R agonist BzATP (10μM); P2Y1 antagonist MRS2179; thapsigargin (10μM) to block Ca2+ release from intracellular stores. Calcium imaging in fura-2 loaded nerves indicated that ATP evoked raised glial [Ca2+]i predominantly via P2Y1R and P2X7R, and was significantly decreased by suramin, MRS2179, PPADS, oATP and thapsigargin. In addition, stimulation of axonal action potentials at 35Hz evoked a rise in glial [Ca2+]i that was significantly reduced by suramin (p<0.05, t-tests, n≥5). Electrophysiological measurement of [K+]o (using K+-selective microelectrodes) and the CAP showed that increasing the stimulation frequency to 35Hz for 120s results in an activity-dependent increase in [K+]o and decay in nerve conduction, followed by a post-stimulus clearance of K+ and nerve hypoexcitability, and finally K+ redistribution and recovery of nerve conduction. Treatment with the P2 purinergic receptor antagonists suramin, PPADS, MRS2179, oATP, or with thapsigargin or zero [Ca2+]o, resulted in a significantly greater activity-dependent decay in the CAP compared to aCSF controls (p<0.01, t-tests, n≥5). We tested the effects of suramin, oATP and thapsigargin on [K+]o during 35Hz stimulation and all resulted in a significantly greater rise in [K+]o compared to controls (p<0.05, paired t-tests, n≥5). The results show K+ regulation and nerve conduction are reduced when P2R are blocked. The study provides evidence that ATP-mediated astrocyte Ca2+ signaling is important for K+ regulation and maintaining axonal activity in CNS white matter.

Where applicable, experiments conform with Society ethical requirements