Measurement of intraneuronal free calcium using the fluorescent probe technique

The distribution of Ca(2+) within the cell is complex and involves binding to cell macromolecules and compartmentalization within the subcellular organelles (1). Normal physiological functions of the cell are regulated by changes in intracellular free Ca(2+) ([Ca(2+)](i)), which ranges from 0.1 to 0.3 µM. This low concentration is regulated by energy-dependent transport systems located in plasma membrane, endoplasmic reticulum, and mitochondria (1). Cytosolic [Ca(2+)](i) within the cell are elevated either through Ca(2+)-influx or by Ca(2+)-release from intracellular stores. Such increases in [Ca(2+)](i) have been reported to activate several intracellular Ca(2+)-dependent reactions including production of second messengers (2,3), spontaneous release of neurotransmitters (4), phosphorylation of proteins (5), and activity of proteases (6). Cells need to maintain a fine balance of these Ca(2+) homeostatic mechanisms in order to function normally, and any major disturbance in these systems will activate destructive events leading to neuronal injury (7-10).