HIGH-RESOLUTION FLUORESCENT LABELING OF LIVING CEREBELLAR SLICES

In the present study, we extend previous research on staining of livin g brain slices with fluorescent phospholipids. This new procedure allo ws high-resolution staining of specific cell types, in particular, Pur kinje cells, in the cerebellar slice while not affecting the intrinsic electrical activity of the tissue. Four different nitrobenzoxadiole ( NBD)-phospholipids were incorporated into living cerebellar slices via loading from small unilamellar vesicles (SUVs), composed of a carrier and the fluorescent lipid. The labeled acidic phospholipid, NBD-phosp hatidic acid (NBD-PA), produced the highest resolution images with exq uisite labeling of the dendritic fields. The label was incorporated pr edominantly into the Purkinje cell body (excluding the nucleus), with more diffuse staining in other cell types, including stellate, basket and granule cells. The labeled lipid concentration and composition of the carrier Lipid were significant in determining the specificity of l abeling. Labeling, which was optimal after a 1 h incubation, was prese nt throughout the depth of the slice. This procedure provides a promis ing approach to fluorescent labeling that will allow simultaneous moni toring of changes in cellular morphology and electrophysiology of livi ng brain slices.