Glial Ca²⁺ signalling has been shown to occur in response to physiological and pathological stimuli and Ca²⁺ may in turn act as an intracellular messenger regulating glial function. Previously, we showed that ATP evoked an increase in glial [Ca²⁺]_i in the isolated intact rat optic nerve (James & Butt, 2001). The aim of the present study was to determine whether Ca²⁺ signalling occurred in all glial cell types and which subtypes of purinoreceptors mediated the response to ATP.

Juvenile rats, aged postnatal day (P) 12-28, were humanely killed by CO₂ narcosis, according to Home Office guidelines. Optic nerves were immediately removed to oxygenated artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 133; KCl, 3; CaCl₂, 1.5; NaH₂PO₄, 1.2; MgCl₂, 1.0; D-glucose, 10; Hepes, 10; pH 7.4. Changes in glial [Ca²⁺]_i were measured using fluo-3 imaging and fura-2 ratiometric techniques. For fluo-3, nerves were impaled with a glass micropipette filled with fluo-3 AM (40 µM) and the dye was gently pressure injected beneath the pia into the nerve parenchyma; the nerve was incubated in ACSF for 1 h to allow the dye to be intracellularly sequestered. For fura-2, the pial surface of the optic nerve was carefully split using glass microelectrodes, to facilitate penetration of the dye, and the nerve was incubated for 1 h in fura-2 AM (44 µM). Bath application of ATP and agonists for P2X (α,β-metATP) and P2Y (2MeSATP) purinoreceptors triggered raised glial [Ca²⁺]_i, in nerves loaded with either dye. The responses of individual glial cell somata were distinguished in fluo-3-loaded nerves, and there was no significant difference (P > 0.05, ANOVAR) between cells identified morphologically as astrocytes (discretely distributed single cells with large irregular somata; n = 50) and oligodendrocytes (cells in rows with small rounded somata; n = 50).

Dose-response curves indicated that P2Y receptors were activated at nanomolar concentrations, whereas P2X purinoreceptors were only activated above 10 µM. The rank orders of potency for ATP, 2MeSATP and α,β-metATP were compared with ADP, which acts on the P2Y₁ subtype and UTP, which acts potently on the P2Y₂ and P2Y₄ subtypes. Results were expressed as a percentage of the ATP response in the same nerves (n = 3), and the rank order of potency of the agonists (10 µM; results expressed ± S.E.M.) was 2MeSATP (144 ± 7 %) = ADP (143 ± 3 %) > ATP (100 %) > UTP (86 ± 10 %) ã>> α,β-metATP (17 ± 1 %). The P2Y₁ specific antagonist MRS2179 (1 µM) significantly (P < 0.05, paired t test) reduced the ATP (10 µM)-evoked increase in [Ca²⁺]_i by 70 ± 3 % (n = 3). The results implicate ATP as an important signal in white matter astrocytes and oligodendrocytes in situ, and indicate that the ATP-evoked increase in [Ca²⁺]_i is mediated predominantly via metabotropic P2Y₁ and P2Y_2/4 purinoreceptors, with a significant ionotropic P2X component. It is significant that P2Y receptors were activated at low concentrations of ATP, suggesting a physiological role in signalling, whereas P2X receptors were activated only at high concentrations of ATP, such as might occur following CNS injury.This work was supported by the Special Trustees of Guy’s and St Thomas’s Hospitals.

James, G. & Butt A.M. (2001). J. Physiol. 531.P, 108P.