Ageing is a process associated with both neurological and psychological dysfunctions (Rubin et al. 2000). One of the theories that has been proposed to explain such changes is the ‘calcium hypothesis of ageing’, in which changes in any Ca²⁺ homeostatic mechanism would alter the neuronal physiology (Verkhratsky & Toescu, 1998). Mitochondria are intracellular organelles that sustain cellular energetics and are also intimately related to Ca²⁺ homeostasis.

In the present work, we investigated the relationship between mitochondrial function and Ca²⁺ homeostasis in brain slices obtained from mice (CB57Bl/6) that aged normally and were humanely killed in accordance with UK legislation (Schedule I). Parasagittal slices of mouse cerebellum, 250 mm thick, were cut in ice-cold artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 118; KCl, 4.7; CaCl₂, 2.5; NaHCO₃, 25; KH₂PO₄, 1.2; MgSO₄, 1.2; glucose, 11; equilibrated continuously with 95 % O₂ and 5 % CO₂, to maintain the pH at 7.4. During cutting and for the initial period of equilibration (30 min), the ACSF medium was supplemented with 225 mM sucrose to reduce the level of neuronal damage. After 30 min, the slices were transferred to a slice-holding chamber in which they were maintained for different periods of time until use. For Ca²⁺ measurement the slices were loaded with fura-2AM. For monitoring mitochondrial membrane potential, we have used rhodamine 123 (Toescu & Verkhratsky, 2000). Images were captured (rate 0.2Ð3 Hz) and analysed using an intensified GenIV camera (Roper Instruments, UK), fed from a monochromator (Cairn Research Ltd, UK), controlled through the MetaFluor software (Universal Imaging, Inc., USA). The emission was set with a 525 ± 25 nm cut-off filter (Chroma, USA) placed in a Sutter filterwheel installed in front of the camera.

When slices were stimulated (KCl depolarisation) there were significant differences in the type of patterns of Ca²⁺ signal displayed by the young and old cerebellar granule neurones (90 % of the young neurones responded with a single monophasic [Ca²⁺]_i increase, whereas only 55 % of the aged neurones responded in a similar manner. The remainder of the aged neurones showed smaller or no [Ca²⁺]_i increases and rapid [Ca²⁺]_i dysregulation following the cessation of stimulation). More importantly, the aged neurones showed a significant delay in their capacity to recover the resting [Ca²⁺]_i (recovery to 75 % of resting levels after cessation of stimulation: 1.35 ± 0.22 min in young neurones and 2.95 ± 0.45 min for the aged neurones (mean ± S.E.M.; n = 19 and 15 neurones for the two ages, respectively, P < 0.001)). In both young and aged neurones, the cytosolic [Ca²⁺]_i signal was associated with a mitochondrial depolarisation response. In the aged neurones, the mitochondria had a significantly longer repolarization response and quantitative analysis showed a direct correlation between the delays in mitochondrial repolarization and [Ca²⁺]_i recovery, indicating a causal relationship between the two parameters.

The present results thus show that the reported changes in Ca²⁺ homeostasis associated with ageing, which are manifested principally in a decreased capacity of recovering the resting [Ca²⁺]_i values after stimulation, are mainly due to a metabolic dysfunction in which mitochondrial impairment plays an important role.

The financial support of BBSRC (SAGE Initiative) is gratefully acknowledged.

All procedures accord with current UK legislation.