Glutamate‐induced mitochondrial depolarisation and perturbation of calcium homeostasis in cultured rat hippocampal neurones

Abstract

The objective of this study was to clarify the relationships between loss of mitochondrial potential and the perturbation of neuronal Ca2+ homeostasis induced by a toxic glutamate challenge. Digital fluorescence imaging techniques were employed to monitor simultaneously changes in cytoplasmic Ca2+ concentration ([Ca2+]i) and mitochondrial potential (ΔΨm) in individual hippocampal neurones in culture coloaded with fura‐2 AM or fura‐2FF AM and rhodamine 123 (Rh 123). In most cells (96 %) at 6‐7 days in vitro (DIV) and in a small proportion of cells (29 %) at 11‐17 DIV the [Ca2+]i increase induced by exposure to 100 μm glutamate for 10 min was associated with a small mitochondrial depolarisation, followed by mitochondrial repolarisation, and a degree of recovery of [Ca2+]i following glutamate washout. In the majority of neurones at 11‐17 DIV (71 %), exposure to glutamate for 10 min induced a profound mono‐ or biphasic mitochondrial depolarisation, which was clearly correlated with a sustained [Ca2+]i plateau despite the removal of glutamate. Addition of glutamate receptor antagonists (15 μm MK‐801 plus 75 μm 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX)) to the washout solution did not affect the post‐glutamate [Ca2+]i plateau in neurones exhibiting a profound mitochondrial depolarisation but greatly improved [Ca2+]i recovery in those neurones undergoing only a small mitochondrial depolarisation, suggesting that the release of endogenous glutamate delays [Ca2+]i recovery in the postglutamate period. Cyclosporin A (500 nM) or N‐methyl Val‐4‐cyclosporin A (200 nM) delayed or even prevented the development of the second phase of mitochondrial depolarisation in cells at 11‐17 DIV and increased the proportion of neurones exhibiting a small monophasic mitochondrial depolarisation and [Ca2+]i recovery upon glutamate removal. We have thus described a striking correlation between mitochondrial depolarisation and the failure of cells to restore [Ca2+]i following a toxic glutamate challenge. These data suggest that mitochondrial dysfunction plays a major role in the deregulation of [Ca2+]i associated with glutamate toxicity.

Relationships between superoxide levels and delayed calcium deregulation in cultured cerebellar granule cells exposed continuously to glutamate

“…Cyclosporin A is problematic as a permeability transition diagnostic with intact cells due to its ability to inhibit calcineurin and hyperphosphorylate proteins (Hallak et al 2001). The more selective methyl-4-valine cyclosporin did not afford convincing protection in our hands (Castilho et al 1999) although others report partial protection against deregulation (Khaspekov et al 1999;Vergun et al 1999;Alano et al 2002).…”

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons

“…It is characterized by a dramatic increase in [Ca 2 þ ] i following the activation of Ca 2 þ -permeable receptors, particularly glutamate receptors, even after the stimulus has ended, thought to be due to loss of the mitochondrial ability to buffer intracellular Ca 2 þ . [28][29][30][31] More recently, it was demonstrated that, in cerebellar granule neurons, NCX also participates in this process of secondary Ca 2 þ overload, 14 due to its loss of the ability to pump Ca 2 þ out of the cell, following its proteolytic inactivation by calpains upon exposure to glutamate, providing a new hypothesis for the origin of delayed Ca 2 þ deregulation. Hoyt et al 32 have shown in cultured rat forebrain neurons that, although reversal of NCX contributes to Ca 2 þ transients and to the immediate rise in intracellular Ca 2 þ levels upon exposure to glutamate, it has no effect in the mean peak increase caused by a prolonged exposure to glutamate nor does it contribute to neuronal injury.…”

Normal brain ageing: models and mechanisms

“…Another study, using a similar experimental model and looking at the NMDA-evoked electrophysiological and [Ca 2C ] i responses, showed that with ageing (26 months old rodents) there was a significant increase in responsiveness when compared with the 'middle-aged' (nine months old) neurons (Cady et al 2001). Interestingly, during attempts to develop an 'ageing in the dish' experimental model, it was reported that several of the functional features of the aged neurons as recorded in acute brain slice preparations from the aged animals (either hippocampal or cerebellar) developed during longer term primary neuronal cultures (Blalock et al 1999;Vergun et al 1999;Xiong et al 2004).…”