AstroDot: a new method for studying the spatial distribution of mRNA in astrocytes

Astrocytes are morphologically complex and use local translation to regulate distal functions. To study the distribution of mRNA in astrocytes, we combined mRNA detection via in situ hybridization with immunostaining of the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP). mRNAs at the level of GFAP-immunolabelled astrocyte somata, large and fine processes were analyzed using AstroDot, an ImageJ plug-in and the R package AstroStat. Taking the characterization of mRNAs encoding GFAP α and δ isoforms as a proof of concept, we showed that they mainly localized on GFAP processes. In the APPswe/PS1dE9 mouse model of Alzheimer9s disease, Gfap α and δ mRNA density and distribution changed as a function of the region of the hippocampus and the astrocyte9s proximity to amyloid plaques. To validate our method, we confirmed that the ubiquitous Rpl4 (large subunit ribosomal protein 4) mRNA was present in astrocyte processes as well as in microglia processes immunolabelled for ionized calcium binding adaptor molecule 1 (Iba1). In summary, this novel set of tools allows to characterize mRNA distribution in astrocytes and microglia in physiological or pathological settings.