In hippocampal neurons in culture for > 11 DIV (‘old neurons’) exposure to glutamate (Glu) for 10-15 min causes in a profound increase in [Ca²⁺]_c and mitochondrial depolarisation (MD), which is sustained despite wash-out of Glu (Vergun et al. 1999). The objective of the present work was to test the hypothesis that this post-glutamate [Ca²⁺]_i plateau results from blockade of mitochondrial Ca²⁺ uptake combined with the depletion of intracellular ATP.

Primary cultures of hippocampal neurons were prepared from Wistar rat pups aged 1-2 days post-partum as described earlier (Vergun et al. 1999). Animals were anaesthetized with ether and decapitated. Rat hippocampal neurons in culture were co-loaded with the low affinity [Ca²⁺] indicator, fura-2FF-AM, and rhodamine 123 (Rh123) for simultaneous measurements of [Ca²⁺]_c and mitochondrial potential (ΔC{special}_m), respectively. In old neurons, prolonged Glu challenge resulted in a biphasic [Ca²⁺]_c increase and a profound MD, which persisted after Glu wash-out. In contrast, in neurons at 6-8 DIV (‘young neurons’) a reversible monophasic [Ca²⁺]_c increase was associated with a very small MD.

Application of the protonophore FCCP (0.3 µM) to young neurones 3-5 min after the beginning of the exposure to Glu induced an additional profound MD and [Ca²⁺]_c elevation, which persisted despite Glu washout and only recovered after removal of FCCP. However, when oligomycin (2.5 µg ml^-1) or ouabain (0.5 mM) were applied prior to Glu exposure, then [Ca²⁺]_i decreased upon Glu withdrawal, despite the continuing FCCP-induced MD. Similar effects have been observed previously in cerebellar granule cells and may be explained if ATP required for operation of the plasmalemmal Ca²⁺ pump is depleted by ATP consumption by the reversed F₁F₀-ATP synthase (Khodorov, 2000) or by the Na⁺/K⁺-ATPase (Surin et al. 2000). In contrast, in old hippocampal neurones, treatment with either oligomycin or ouabain failed to prevent either MD or the [Ca²⁺]_c plateau in the post-glutamate period. These data suggest that the sustained [Ca²⁺]_c increase is not simply a consequence of the loss of ΔC{special}_m and ATP depletion and that some intrinsic mechanism that is specific to glutamate exposure inhibits the plasma membrane Ca²⁺-ATPase and may be responsible for the failure of Ca²⁺ extrusion.

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