Simultaneous measurements of [Ca²⁺]_i and mitochondrial potential (V_mit) were carried out in cultured rat CGC co-loaded with the low-affinity Ca²⁺ indicator fura-2FF and potentiometric probe rhodamine 123. Primary cultures of cerebellar granule cells (CGC) were prepared from Wistar rat pups aged 7-8 days (Khodorov et al. 1996). Animals were anaesthetized with ether and decapitated. The cells were plated onto poly-D-lysine-coated coverslips in MEM supplemented with 10% fetal bovine serum. In mature cells (15-17 DIV) a prolonged glutamate (100 µM Glu in Mg²⁺-free, 10 µM glycine-containing medium) challenge induced a biphasic [Ca²⁺]_i increase associated with a profound mitochondrial depolarization (MD). Both these changes persisted in the post-glutamate period. In contrast in young cells (6-9 DIV) the same Glu application caused only small monophasic MD and a corresponding reversible [Ca²⁺]_i elevation. Earlier (Vergun et al. 1999) similar relationships were revealed in mature and young hippocampal neurones.

In the present study we found that the above difference between the effects of Glu on young and mature CGC can be eliminated by a prolonged replacement of glucose by its non-metabolised analogue 2-deoxyglucose (DOG, 10 mM) in all the external solutions. A continuous glucose deprivation induced the gradual MD which at the beginning could be abolished by oligomycin (2.5 µg ml^-1). Addition of Glu to the DOG-containing solution 20-90 min after the beginning of cell pretreatment induced a biphasic [Ca²⁺]_i increase associated with a MD which persisted during Glu washout even in the absence of Ca²⁺in the medium.

This effect of glucose deprivation could not be prevented by L-NAME (the inhibitor of NO synthase) or MnTBAP (the scavenger of superoxide and/or hydrogen peroxide) but was greatly attenuated by addition of pyruvate (10 mM) to the DOG-containing medium.

Direct measurements of ATP content ([ATP]) performed in parallel experiments with sister CGC (employing luceferin-luceferase ATP assay) showed that pyruvate effectively diminished the DOG-induced decrease in [ATP] both at rest and during Glu application. This indicates that under conditions when glycolysis is suppressed the pyruvate-dependent respiration can maintain both high V_mit and [ATP] required by young neurons to withstand the toxic Glu challenge. The data suggest that a difference in the resistance of young and mature cultured cerebellar granule neurons to Glu is mainly determined by some peculiarities of their energetic metabolism.

This work was supported by The Physioogical Society UK, INTAS and RFBR.