This study investigates the spatial relationships and interactions between protoplasmic astrocytes in the CA1 stratum radiatum of the hippocampus. Using intracellular injection and immunohistochemistry with the astrocytic marker glial fibrillary acidic protein (GFAP), the authors found that GFAP only delineates about 15% of the total volume of the astrocyte, leading to incorrect conclusions about the interaction of neighboring astrocyte processes. To better understand these interactions, the authors used three-dimensional (3D) confocal analysis and electron microscopy to examine groups of adjacent protoplasmic astrocytes injected with fluorescent intracellular tracers. The results show that protoplasmic astrocytes establish primarily exclusive territories, with limited overlap in their processes. This suggests that each astrocyte contributes to the astrocytic component of neuropil in larger regions than previously thought, and that the complex morphology of protoplasmic astrocytes allows them to infiltrate distinct volumes of the neuropil. The findings have important implications for understanding how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other brain structures, including neurons and vasculature.This study investigates the spatial relationships and interactions between protoplasmic astrocytes in the CA1 stratum radiatum of the hippocampus. Using intracellular injection and immunohistochemistry with the astrocytic marker glial fibrillary acidic protein (GFAP), the authors found that GFAP only delineates about 15% of the total volume of the astrocyte, leading to incorrect conclusions about the interaction of neighboring astrocyte processes. To better understand these interactions, the authors used three-dimensional (3D) confocal analysis and electron microscopy to examine groups of adjacent protoplasmic astrocytes injected with fluorescent intracellular tracers. The results show that protoplasmic astrocytes establish primarily exclusive territories, with limited overlap in their processes. This suggests that each astrocyte contributes to the astrocytic component of neuropil in larger regions than previously thought, and that the complex morphology of protoplasmic astrocytes allows them to infiltrate distinct volumes of the neuropil. The findings have important implications for understanding how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other brain structures, including neurons and vasculature.