Correlated measurements of free and total intracellular calcium concentration in central nervous system neurons

Transient changes in the intracellular concentration of free calcium ([Ca-i (2+])) act as a trigger or modulator for a large number of important neuron al processes. Such transients can originate from voltage- or ligand-gated f luxes of Ca2+ into the cytoplasm from the extracellular space, or by ligand - or Ca2+-gated release from intracellular stores. Characterizing the sourc es and spatio-temporal patterns of [Ca2+](i) transients is critical for und erstanding the role of different neuronal compartments in dendritic integra tion and synaptic plasticity Optical imaging of fluorescent indicators sens itive to free Ca2+ is especially suited to studying such phenomena because this approach offers simultaneous monitoring of large regions of the dendri tic tree in individual living central nervous system neurons. In contrast, energy-dispersive X-ray (EDX) microanalysis provides quantitative informati on on the amount and location of intracellular total, i.e., free plus bound , calcium (Ca) within specific subcellular dendritic compartments as a func tion of the activity state of the neuron. When optical measurements of [Ca2 +](i) transients and parallel EDX measurements of Ca content are used in ta ndem, and correlated simultaneously with electrophysiological measurements of neuronal activity, the combined information provides a relatively genera l picture of spatio-temporal neuronal total Ca fluctuations. To illustrate the kinds of information available with this approach, we review here resul ts from our ongoing work aimed at evaluating the role of various Ca uptake, release, sequestration, and extrusion mechanisms in the generation and ter mination of [Ca2+](i) transients in dendrites of pyramidal neurons in hippo campal slices during and after synaptic activity. Our observations support the long-standing speculation that the dendritic endoplasmic reticulum acts not only as an intracellular Ca2+ source that can be mobilized by a signal cascade originating at activated synapses, but also as a major intracellul ar Ca sink involved in active clearance mechanisms after voltage- and ligan d-gated Ca2+ influx. (C) 1999 Wiley-Liss, Inc.