Roles of thapsigargin-sensitive Ca²⁺ stores in the survival of developing cultured neurons

The roles of the intracellular calcium pool involved in regulating the Ca²⁺ profile and the neuronal survival rate during development were studied by using thapsigargin (TG), a specific inhibitor of endoplasmic reticulum (ER) Ca²⁺-ATPase in cultured cerebellar granule neurons. Measuring the neuronal [Ca²⁺](i) directly in the culture medium, we found a bell-shaped curve for [Ca²⁺](i) versus cultured days in cerebellar granule neurons maintained in medium containing serum and 25 mM K⁺. The progressive increase in [Ca²⁺](i) of the immature granule neurons (1-4 days in vitro) was abolished by TG, which resulted in massive neuronal apoptosis. When the [K⁺] was lowered from 25 to 5 mM, neither the progressively increasing [Ca²⁺](i) nor the survival of immature granule neurons was significantly changed over 24-h incubation. Similarly, TG caused a dramatic decrease in the [Ca²⁺](i) and survival rate of these immature neurons when switched to 5 mM K⁺ medium. Following maturation, the granule neurons became less sensitive to TG for both [Ca²⁺](i) and neuronal survival. However, TG can protect mature granule neurons from the detrimental effect of switching to a 5 mM K+ serum- free medium by decreasing [Ca²⁺](i) to an even lower level than in the respective TG-free group. Based on these findings, we propose that during the immature stage, TG-sensitive ER Ca²⁺ATPase plays a pivotal role in the progressive increase of [Ca²⁺](i) which is essential for the growth and maturation of cultured granule neurons.