Selective staining by vital dyes of Muller glial cells in retinal wholemounts

Muller glial cells within the retina may respond to different signaling molecules with an elevation of their intracellular free calcium. To prove the localization of the recorded calcium responses in Muller cells within acutely isolated retinal whole-mounts, retinal pieces from adult animals and humans were exposed to different vital dyes just after the calcium imaging records were finished. The dyes, Mitotracker Orange, Mitotracker Green, Celltracker Orange, Celltracker Green, and monochlorobimane, are all selectively taken up by Muller glial cells, while neuronal cells remain largely devoid of the dyes. By using this method, it can be demonstrated that the free calcium alterations within the wholemounts indeed occur within Muller cells. Moreover, the cross-sectional areas of (dye-filled) Muller glial cell bodies, as well as of (dye-free) neuronal cell bodies, can be measured in retinal wholemounts, and the spatial densities of both types of cells can be determined. The vital dye loading of Muller cells may facilitate investigations of stimulus-induced alterations of retinal glial cell physiology and morphology. (C) 2003 Wiley-Liss, Inc.