Glutamate (Glu) stimulation of neuronal cells induces cytosolic Ca²⁺ ([Ca²⁺]_c) increases, which can reach the threshold for activation of the mitochondrial Ca²⁺ uniporter [1]. In this study we used an in vitro model of neuronal ageing [2] which consists of long-term primary cultures of cerebellar granule neurones obtained from Wistar rat pups (P5-8). The aim of the present study was to investigate the effect of such ‘in vitro ageing’ on the relationship between the Glu-evoked [Ca²⁺]_c and mitochondrial Ca²⁺ ([Ca²⁺]_m) load. Neurones loaded with Fura-2-AM (2.5μM) were stimulated with Glu (2-200μM in 0Mg/Gly) followed after a wash period, by the protonophore CCCP (10μM) to unload the mitochondria. The parameters investigated were the amplitude of the Glu-evoked Ca²⁺ signal and the amplitude of the CCCP-induced Ca²⁺ signal. We show that with time in culture there is an increase in the size of the Glu-evoked Ca²⁺ signal. In young neurones (days in vitro (DIV) 7-15), 20μM Glu induced a 2.25-fold increase ±0.32 (S.E.M., n=5 experiments, each with >20 neurones) in the Ca²⁺ signal, whereas in the older neurones (DIV 21-50) the increase was significantly larger (4.28-fold increase ±0.08 S.E.M., n=4; t test, p<0.001). By measuring the amplitude of the CCCP-evoked Ca²⁺ signals post-Glu challenge, we estimated the size of the [Ca²⁺]_m load. We found that the larger increases in [Ca²⁺]_c signals in the older neurones were associated with significantly larger [Ca²⁺]_m loads (in the older neurones: 2.72-fold increase ±0.44 S.E.M., n=4 versus 1.76-fold increase ±0.09 S.E.M., n=5, in the young neurones; t test, p<0.02). The two parameters, Glu-evoked Ca²⁺ signal and CCCP-evoked Ca²⁺ signals, were correlated indicating that the size of the cytosolic Ca²⁺ challenge is one of the most important controllers of the [Ca²⁺]_m load. This is important, since previous work has shown that the mitochondria in aged neurones are chronically depolarised [2,3] and thus potentially less able to accumulate Ca²⁺ due to a decreased electrochemical gradient. Finally, we also investigated whether the changes in Glu-evoked Ca²⁺ responses were associated with changes in vulnerability. Neurones at various DIVs were incubated in Glu for 2h and the amount of neuronal death was dynamically quantified using propidium iodide (50μg/ml) fluorescence. With this method, we show that with increasing age in vitro, there is a clear-cut increase in neuronal vulnerability. In conclusion we report that in vitro ageing increases the amplitude of the Glu-evoked Ca²⁺ signals and increases significantly the [Ca²⁺]_m load. Also, older neurones become more vulnerable to the effects of Glu stimulation and are more susceptible to Glu excitotoxicity. This suggests that the [Ca²⁺]_m load, driven by the [Ca²⁺]_c signal is a key component of the increased vulnerability of aged neurones to Glu.