Astrocytes in the neonate rat ventrobasal (VB) thalamus (obtained from humanely killed animals) display spontaneous [Ca²⁺]_i oscillations that are dependent on Ca²⁺ release from intracellular stores and are independent of neuronal activity since they are not blocked by tetrodotoxin. To further clarify the mechanism of these spontaneous oscillations, slices of neonate rat VB were prepared as previously described (Parri et al. 2001), loaded with the calcium indicator fluo-4 and imaged from an area of 206 X 158 µm using a Noran Odyssey confocal microscope. Whole-cell patch-clamp recording was performed using an Axopatch 200B amplifier.

To confirm the intrinsic nature of these oscillations slices were incubated with 4 mM Bafilomycin A1 for 1 h to block vesicular neurotransmitter release. In control conditions synaptic stimulation (5 X 2 ms stimuli of 300 mA at 50 Hz) elicited inward currents of 243 ± 70 pA (n = 4 of 4 cells) in thalamocortical neurones. Currents were abolished in neurons from slices treated with Bafilomycin A1 (n = 4/4), indicating that neurotransmitter release was blocked. In control conditions the number of astrocytes exhibiting spontaneous [Ca²⁺]_i oscillations was 7.25 ± 0.5 (in 600 s) (n = 4 slices), and 7.75 ± 1.9 (n = 4 slices) in Bafilomycin A1-treated slices, showing that oscillations were not dependent on neurotransmitter release. In slices incubated with 20 mM of the phospholipase C inhibitor U73122, the number of cells responding to 50 mM trans-ACPD was reduced from 17.28 ± 2.7 (n = 8) in control conditions to 5.12 ± 0.54 (n = 8), but the number of spontaneously oscillating astrocytes was unaffected (3.37 ± 1.2 in control and 3.87 ± 0.89 (in 300 s) (n = 8) in U73122-treated slices).

The number of cells exhibiting spontaneous oscillations in a 300 s period increased from 2.8 ± 0.8 in 1 mM [Ca²⁺]_o to 4.6 ± 0.55 in 2.5 mM [Ca²⁺]_o and 9.33 ± 0.67 in 5 mM [Ca²⁺]_o (P < 0.005, ANOVA, compared with 1 mM [Ca²⁺]_o) (n = 4 slices). The number of transients exhibited by each spontaneously active astrocyte was 1.64 ± 0.32 in 1 mM, 1.5 ± 0.12 in 2.5 mM and 2.46 ± 0.34 in 5 mM [Ca²⁺]_o (P < 0.05 compared with 1 mM [Ca²⁺]_o). Application of 10 mM of the SERCA blocker cyclopiazonic acid (CPA) resulted in a slow increase in [Ca²⁺]_i levels in 56 ± 23 % (n = 3 slices) of astrocytes, and also induced [Ca²⁺]_i transients in 68 ± 22 % of astrocytes (n = 3 slices), suggesting that an accumulation of cytoplasmic Ca²⁺ can elicit [Ca²⁺]_i release in thalamic astrocytes.

These data show that spontaneous thalamic astrocytic [Ca²⁺]_i oscillations are intrinsic, not dependent on neurotransmitter release, or on receptor activation of PLC pathways. The effects of modifying [Ca²⁺]_o, and the effect of CPA in inducing transients, suggest a role for Ca²⁺ in inducing [Ca²⁺]_i release.

Data are espressed as means ± S.E.M. Statistical significance was measured with Student’s unpaired t test.

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