INTEGRATING ASTROCYTE-SPECIFIC GENETIC MANIPULATION WITH IMAGE QUANTIFICATION TOOLS

Astrocytes arborize with thousands of fine branches that are critical for driving synapse formation, neuronal migration, and blood-brain barrier formation during brain development. Genetic manipulation and quantification of astrocyte structures are fundamental tools in identifying key regulators of astrocyte morphogenesis and function. However, the astrocyte specificity and efficiency of many tools remain limited, with significant off-target effects. To address this limitation, we developed a novel combination of genetic and quantitative approaches to study astrocyte development in vivo and in vitro. GEARBOCS is a modified CRISPR/Cas9 system packaged within a single AAV vector that allows for astrocyte-specific genetic knockouts. We combined this approach with postnatal labeling by electroporation (PALE), which sparsely labels cortical astrocytes early in postnatal development, enabling visualization of complex morphological changes in fine astrocytic processes. PALE effectively delivers plasmids exceeding AAV packaging limits, enabling the incorporation of biosensors and fluorescent reporters into astrocytes. Using this integrated system, we knocked out candidate genes involved in astrocyte development and mitochondrial degradation. Astrocyte morphology was quantified using IMARIS-based 3D Sholl analysis to assess complexity. In conjunction, we used a specialized MATLAB program to quantify mitochondrial degradation in astrocytes by analyzing changes in a fluorescent mitochondrial reporter, reducing subjective bias. By integrating our astrocyte-specific tools, we identified molecular drivers of astrocyte morphology and mitochondrial degradation. Together, these tools provide precise, cell-type-specific methods for manipulating and examining astrocyte structure and function, offering insights into how astrocyte morphology and mitochondrial changes contribute to synapse regulation during brain development.