The structure and function of the endoplasmic reticulum (ER) in neurons and glia are being investigated using newly developed techniques which make it possible to investigate the dynamic properties of the ER in living cells by video and laser scanning confocal microscopy. Several new techniques have been developed using the sea urchin egg as a model system. The sea urchin egg has the advantages of being readily available, having prominent ER, and being easy to microinject. A novel technique for specifically staining ER in living cells shows that the ER is probably a single compartment, that it undergoes movements in the absence of microtubules, that a striking change its organization occurs at the time of calcium release, and that the appearance of microtubules has a profound effect on its organization. We are currently adapting these techniques so they can be applied to understand the organization and dynamics of the ER in neuronal cell bodies and axons. A second initiative uses calcium-sensitive fluorescent indicators to investigate calcium regulation by the ER. In a cell-free preparation derived from sea urchin eggs, it was demonstrated that the ER is the source of Inositol l,4,5, triphosphate (InsP3)-induced calcium release. Cytosolic calcium is being investigated with the laser scanning confocal microscope using a novel improvement in fluorescent calcium indicators. Application of these sensitive calcium indicators to the question of the distribution of calcium entering axons is now being investigated.
