Many recent studies on bi-directional communication between astrocytes and neurones suggest astrocytes have a form of excitability based on intracellular Ca²⁺ fluctuations. This study investigates the spontaneous Ca²⁺ fluctuations in astrocytes from the stratum radiatum. Wistar rats (P8-16) were killed by a Home Office approved humane procedure and brain slices prepared. Astrocytes were loaded with calcium green-1 AM (Kang & Nedergaard, 1999). Patch-clamp recording confirmed their identity as astrocytes (15/15 cells patched).

Astrocytes display spontaneous Ca²⁺ transients that persist in the absence of neuronal action potentials: control, 3.1 ± 0.7 mHz; 0.5 µM TTX, 4.4 ± 0.8 mHz (n = 230, 12 slices). Inhibition of the sarco(endo)plasmic reticulum Ca²⁺-ATPases by cyclopiazonic acid (CPA, 50 µM) abolished the spontaneous calcium transients: control, 2.8 ± 0.4 mHz; CPA, 0.12 ± 0.05 mHz, P < 0.001; wash, 1.3 ± 0.3 mHz (n = 101, 5 slices), suggesting that the spontaneous transients are due to Ca²⁺ release from intracellular stores.

Inhibition of either Ca²⁺-induced Ca²⁺ release (CICR) or IP₃-induced Ca²⁺ release with ryanodine (20 µM) or 2-APB (100 + 200 µM), respectively, did not inhibit the spontaneous transients. The involvement of nicotinic acid adenine dinucleotide phosphate has still to be investigated, thus the intracellular receptor mediating these events is still unknown. Many metabotrophic receptors signal via PLC, although the PLC inhibitor U73122 (10 µM) did not inhibit the spontaneous Ca²⁺ transients: control, 7.9 ± 1.0 mHz; U73122, 10.1 ± 1.9 mHz (n = 161, 6 slices).

Application of ATP or glutamate resulted in an increase in intracellular Ca²⁺. In addition, ATP (100 µM) caused an increase in frequency of the spontaneous transients: TTX, 3.6 ± 0.7 mHz; TTX + ATP, 9.9 ± 2.2 mHz (n = 171, 8 slices, P ▓le│ 0.05). In contrast, glutamate (100 µM) resulted in an initial increase in frequency followed by a significant reduction in the frequency of the transients during the glutamate-induced plateau. This persists for several minutes after glutamate washout and the resting Ca²⁺ had returned to control values: TTX, 2.1 ± 0.6 mHz; initial glutamate, 3.7 ± 0.7 mHz (P ▓le│ 0.05); glutamate plateau, 1.0 ± 4.7 mHz (P ▓le│ 0.05); wash, 1.9 ± 0.5 mHz (n = 219, 9 slices). Therefore it is likely that the spontaneous transients share a similar intracellular mechanism as the glutamate-induced rise in Ca²⁺ with ATP modifying the frequency of these transients. Values are means ± S.E.M.; significance levels were determined by Student’s paired t tests or ANOVA.

This work was supported by TR Golden Charitable Trust, King’s Medical Research Trust, HeadFirst, Patrick Berthoud Charitable Trust and Community Fund.