Cortical Organotypic Brain Slice Cultures to Examine Sex- and Age-Dependent Astrocyte-Mediated Synaptic Phagocytosis

Astrocytes, the most abundant glial cells in the brain, are an integral part of the synaptic compartment and contribute to synaptic pruning, a key process for refining neural circuits during early postnatal development (PND). Dysregulations in this process are implicated in various neuropsychiatric disorders, including major depressive disorder (MDD). To investigate astrocyte functions in a physiologically relevant context, organotypic brain slice cultures (OBSCs) offer a powerful model, reproducing more closely in vivo conditions than traditional cell cultures and preserving complex brain architecture and interactions. Here, we present OBSCs as an ex vivo culturing method to provide a platform to explore astrocyte-mediated synaptic pruning dynamics in the rat prefrontal cortex (PFC) during PND. Our approach is based on assessing the role of MEGF10, a key protein involved in synaptic pruning, alongside the synaptic markers synaptophysin and PSD95, using Western blotting to analyse the expression levels of these markers in the cortex of developing rat pups. Additionally, we combine immunofluorescence staining with confocal imaging and IMARIS 9.8 software-assisted analysis to investigate the co-localization of the lysosomal marker LAMP1 with synaptic and astrocytic markers to evaluate the precise rate of synaptic engulfment. The methods presented here allow a deeper examination of an astrocyte-mediated synaptic remodelling in healthy and pathophysiological conditions.