Confocal imaging of glial cells in the intact rat optic nerve

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Abstract
Astrocytes and oligodendrocytes in the isolated intact mature rat optic nerve have been computer imaged in three dimensions by laser scanning confocal microscopy of single cells, dye-filled with lysinated rhodamine dextran (LRD). Our results illustrate the first application of these techniques to an intact CNS white matter tract and provide comparative data for previous studies on neonatal rat optic nerve (Butt and Ransom: Glia 2:470–475,1989; Butt and Ransom: J Comp Neurol 338:141–158, 1993). The combined use of intracellular injection of LRD and confocal imaging significantly improves the resolution of glial cell structure, particularly that of mature astrocytes, for a number of reasons. (1) Single mature dye-filled glia can be imaged, because LRD does not pass through gap junctions. (2) The entire process field of astrocytes can be visualized in a single two-dimensional image. (3) Cell images can be rotated through 360° in all planes to provide a new perspective of glial cell structure in the intact tissue. (4) Reconstruction of optical sections, within a narrow focal plane, provides a high definition and resolution of the finer details of glial morphology. Using these techniques, three astrocyte subclasses were distinguished on morphological criteria. It is the conclusion of this study that the majority of these forms represent a single population of fibrous astrocytes which are well-suited to perform the multiple functions attributed to astrocytes in the CNS. The morphology of mature myelin-forming oligodendrocytes was also described. The results confirm that the structural characteristics of both astrocyte subtypes and oligodendrocytes described in younger rat optic nerves by Butt and Ransom (Glia 2:470–475,1989; J Comp Neurol 338:141–158, 1993) accurately reflect those of the mature glial population.