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Background  Besides their neuronal support functions, astrocytes are active partners in neuronal information processing. The typical territorial structure of astrocytes (the volume of neuropil occupied by a single astrocyte) is pivotal for many aspects of glia–neuron interactions.    Methods  Individual astrocyte territorial volumes are measured by Golgi impregnation, and astrocyte densities are determined by S100β immunolabeling. These data are compared with results from conventionally applied methods such as dye filling and determination of the density of astrocyte networks by biocytin loading. Finally, we implemented our new approach to investigate age-related changes in astrocyte territories in the cortex and hippocampus of 5- and 21-month-old mice.    Results  The data obtained by our simplified approach based on Golgi impregnation were compared to previously published dye filling experiments, and yielded remarkably comparable results regarding astrocyte territorial volumes. Moreover, we found that almost all coupled astrocytes (as indicated by biocytin loading) were immunopositive for S100β. A first application of this new experimental approach gives insight in age-dependent changes in astrocyte territorial volumes. They increased with age, while cell densities remained stable. In 5-month-old mice, the overlap factor was close to 1, revealing little or no interdigitation of astrocyte territories. However, in 21-month-old mice, the overlap factor was more than 2, suggesting that processes of adjacent astrocytes interdigitate.    Conclusion  Here we verified the usability of a simple, versatile method for assessing astrocyte territories and the overlap factor between adjacent territories. Second, we found that there is an age-related increase in territorial volumes of astrocytes that leads to loss of the strict organization in non-overlapping territories. Future studies should elucidate the physiological relevance of this adaptive reaction of astrocytes in the aging brain and the methods presented in this study might be a powerful tool to do so.

Versatile and Simple Approach to Determine Astrocyte Territories in Mouse Neocortex and Hippocampus