Activation of phospholipase C (PLC) by neurotransmitters and hormones leads to the generation of InsP₃, which in turn releases Ca²⁺ from intracellular stores. Depletion of the stores then activates entry of extracellular Ca²⁺ ions, a process termed store-operated or capacitative calcium entry (CCE). This mechanism has been extensively studied in non-excitable cells, although the signal linking depletion of the pools with opening of plasma membrane Ca²⁺ channels is still a matter of speculation. In the case of excitable cells, reports of CCE are scarce, specially for neurones. Though cerebellar granule cells are a model for the study of the PLC/InsP₃ pathway, and in spite of being one of the most common models in neurobiology, there is no evidence for the presence of CCE in this cell type. The aim of our study was to test the presence of CCE in granule cells prepared from 4- to 7-day-old rats killed by decapitation after anaesthesia.

We loaded cells cultured on poly-D-Lys-coated coverslips with 10 µM fura-2 AM for 30 min, followed by other 30 min. To study Ca²⁺ entry we determined Ca²⁺ concentration ([Ca²⁺]_i) as a ratio of fura-2 fluorescence using a microscopic digital imaging system.

After depletion of intracellular Ca²⁺ stores with 1 µM thapsigargin (TPS) in a Ca²⁺-free medium reintroduction of external Ca²⁺ induced a sustained [Ca²⁺]_i increase (0.062 ± 0.007, n = 9, mean ± S.E.M., ratio units). This plateau was due to Ca²⁺ influx from extracellular medium, as shown by the repetitive pattern following removal and readdition of external Ca²⁺, so that it can be used as index of Ca²⁺ entry. Another approach to induce CCE was coapplication of TPS and 100 nM ionomycin in Ca²⁺-free medium, a treatment which induced a CCE response significantly higher than TPS alone (0.09 ± 0.009, mean ± S.E.M., n = 16, P ²le³ 0.05 with respect TPS alone, Student’s unpaired t test). Inclusion of the ionotropic glutamate receptor anatagonists DAP5 and NBQX did not modify the pattern of influx, showing that Ca²⁺ entry is independent of receptor-operated channel activation.

To characterise the pathway for Ca²⁺ influx we used several compounds. 2-APB is accepted as a selective inhibitor for capacitative Ca²⁺ channels (Braun et al. 2001). Application of this drug resulted in inhibition of the CCE-evoked plateau. Consistent with previous reports in other systems, Zn²⁺, Cd²⁺ and Gd³⁺ also reduced the Ca²⁺ entry.

In summary, our data show that depletion of Ca²⁺ stores in cerebellar granule cells evokes a component of capacitative calcium entry with some of the features of the typical capacitative entry system.

This work was supported by SAF-2001-0295.