Fig. 6.

Stimulation of axons in the optic nerve evokes calcium signalling in astrocytes and NG2-glia. Optic nerves from GFAP-EGFP mice and NG2-DsRed mice aged P15 were isolated intact and loaded with the calcium-sensitive dye fura-2, as described previously (James & Butt, 2002). Cells were imaged on an Olympus upright microscope (BX50W1), and visualized using an Achroplan ×20 water immersion lens and excited alternately at 340 and 380 nm, using a Cairn monochromator (Cairn Research Ltd, UK). Emissions were detected at 510 nm using a Photometrics S-Coolsnap CCD camera (supplied by Cairn Research Ltd, UK). The monochromator and the CCD camera were controlled and synchronized by Axon Imaging Workbench 5.1 (Axon Instruments), and quantitative measurements were made using the same program. (A) Measurements were made from EGFP+ astrocytes and DsRed+ NG2-glia, and calcium variations expressed as the change in ratio after background subtraction (F340:F380). Spontaneous [Ca²⁺]_i oscillations were observed in both EGFP+ astrocytes and DsRed+ NG2-glia. (B) Electrical stimulation of the optic nerve to evoke axonal action potentials resulted in a rapid increase in [Ca²⁺]_i in EGFP+ astrocytes and DsRed+ NG2-glia, which was sustained for the duration of the stimulus. (C) Bath administration of 100 µm ATP or glutamate evoked raised [Ca²⁺]_i in EGFP+ astrocytes and DsRed+ NG2-glia. (D) Mechanical stimulation of the optic nerve with a sharp microelectrode triggered raised glial [Ca²⁺]_i that was inhibited by the purinoreceptor antagonist suramin, indicating that ATP released by astrocytes propagates a calcium wave that passes to NG2-glia.