Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains

Abstract

Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominantly on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only approximately 15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.

Fig. 1.

Protoplasmic astrocyte of CA1 stratum radiatum iontophoretically filled with the fluorescent dye Alexa 488.A, Optical slice reveals the dense spongiform processes of these astrocytes. A clear but complex boundary is evident in the extent of these processes. B, Via manual segmentation (red), it was possible to delineate the extent of and calculate the neuropilar volume occupied by each astrocyte. Scale bar, 10 μm.

Fig. 2.

GFAP immunolabeling of LY-filled astrocytes in CA1 stratum radiatum. Dye-filled astrocytes (red) consistently display a GFAP cytoskeleton (green).A, An optical slice through double-labeled astrocytes reveals the limited degree of interdigitation or contacts between GFAP skeletons. B, A maximum projection through this volume of tissue (∼25-μm-thick) demonstrates the limited extent of GFAP throughout individual astrocytes. Relationships between GFAP skeletons are more difficult to resolve. C, In a 3D perspective projection through the same volume, the ability of astrocytes to fill space in the neuropil with their processes is apparent, as is the lack of lacunas in the felt work of GFAP processes, in which GFAP-negative variants of protoplasmic astrocytes could possibly reside. Volume thickness is ∼30 μm. Scale bar, 20 μm.

Fig. 3.

The overall morphology of astrocytes often appeared to be strongly influenced by their neighbors.A, Optical slice through an astrocyte filled with the Alexa 568. B, Alexa 488-filled astrocytes in the same optical slice. C, Overlay of A andB. The astrocyte in the middle avoids overlap of its processes with its two neighbors. Scale bar, 15 μm.

Fig. 4.

Optical slices through neighboring protoplasmic astrocytes filled with distinct fluorescent dyes. A, The major processes (arrows) of astrocytes were commonly seen to extend tangentially to the approaching processes of neighboring astrocytes. B, The processes emerging from two astrocytes with adjacent somata radiate parallel to or away from each other. C, Blood vessels (arrowhead) appeared to be capable of influencing the arrangement of astrocyte processes as they attempted to form end feet. Astrocyte on thefar right is highly elongated as it reaches for passing vessel. The center astrocyte (green) shows little overlap with its neighbors.D, One astrocyte (green) is seen to have its process “invade” the territory of its neighbor as both astrocytes form end feet on passing blood vessels. Scale bars, 15 μm.

Fig. 5.

High-magnification view of interface region between the fine processes of neighboring astrocytes. A, Optical slice through the interface region between two adjacent protoplasmic astrocyte. Fine processes intermingle in a limited region at the interface zone. A′, x–z view of astrocytes in A. B, The processes of an oligodendrocyte-like cell (red) are seen to interdigitate extensively with the processes of an adjacent protoplasmic astrocyte. B′, x–z view ofB. Scale bars, 10 μm.

Fig. 6.

The discreet region of interaction (yellow) between the fine processes of protoplasmic astrocytes. Pixels containing both greenand red were determined using the colocalization routine (see Materials and Methods) and then pseudocolored in bright yellow to mark their presence. x–y(large panel), x–z (bottom panel), and y–z (right panel) slices through the area in which two adjacent astrocytes interface. Scale bar, 20 μm.

Fig. 7.

Another example of colocalization between adjacent astrocytes. A, Slice through adjacent astrocytes, as seen in Figure 6. B, Stereo pair of the same group of astrocytes. 3D views of colocalization reveal sheets in which neighboring astrocytes interact with each other. Scale bar, 20 μm.

Fig. 8.

Electron microscopic examination of the boundary regions of a photooxidized astrocyte. A,B, Tomographic reconstructions of astrocyte boundary regions were generated using a 0.5-μm-thick section through a photoconverted astrocyte. Computational slices through the resulting volumes demonstrate an abrupt decrease in the density of fine astrocytic processes at the boundary to extent of the astrocyte. Scale bars, 1 μm.