Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains

TLDR

Astrocytes are believed to extensively interact with neurons, yet prevailing knowledge—based mainly on GFAP staining and metal‑impregnation—suggests overlapping processes, a view now questioned by the limitations of these techniques. The study aimed to examine the interactions among adjacent protoplasmic astrocytes in CA1 stratum radiatum by injecting them with fluorescent tracers of distinct wavelengths. Fluorescent intracellular tracers were combined with three‑dimensional confocal microscopy and electron microscopy to map astrocyte morphology and contacts. The analyses showed that GFAP labels only about 15 % of astrocyte volume, leading to erroneous conclusions of extensive overlap, while protoplasmic astrocytes actually occupy largely exclusive territories, altering our understanding of their 3D relationships with neurons, oligodendroglia, and vasculature.

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 predominately 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 ∼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.