Mitochondrial polarisation status and [Ca2+]i signalling in rat cerebellar granule neurones aged in vitro

Mitochondrial membrane potential is a major factor that controls, ultimately, the cellular energy supply. By use of a mitochondrial membrane potential dye (rhodamine 123, R123) and image analysis we show that during long-term (>3 weeks) culture of primary neurones (cerebellar granule neurones) there is a gradual and time-dependent depolarisation of neuronal mitochondria. This process was demonstrated by analysing the changes in the heterogeneity of the cytosolic rhodamine 123 fluorescent signal as a function of the age in culture and by measuring the amplitude of the rhodamine 123 fluorescence evoked by the addition of a mitochondrial protonophore (FCCP). The relationship between cytosolic [Ca(2+)](i) and mitochondrial membrane potential was assessed by recording both parameters simultaneously, in neurones loaded with fura-2 and rhodamine 123. Neuronal stimulation (KCl-evoked depolarisation) induced a mitochondrial depolarisation response resulting from the entry of cytosolic Ca(2+) into mitochondria. In young cultures (10 DIV), the mitochondrial membrane potential recovered fully within 30s from the start of the stimulation, despite the continuous presence of the depolarisation stimulus and the maintained cytosolic [Ca(2+)](i) signal. In contrast, in older neurones (DIV 22), the mitochondrial response was of smaller amplitude and displayed a much longer repolarization period. Also, in these older neurones, the threshold [Ca(2+)](i) level required for the initiation of the mitochondrial depolarisation response was increased by 50%. Thus, the present results indicate that neuronal maturation and ageing in conditions of long-term in vitro culture determine significant changes in the mitochondrial polarisation status that are manifest both in resting conditions and during stimulation and could explain some of the reported changes in neuronal homeostasis in long-term neuronal cultures.