A Streamlined Workflow for Purkinje Cell Labeling and High-Resolution Analyses of Dendrites and Spines in Mice.

Cerebellar Purkinje cells (PCs) exhibit a unique and highly complex dendritic architecture, which plays a crucial role in integrating synaptic inputs and shaping their physiological properties. However, fine morphological investigation of PCs at the single-cell level is challenging due to the densely packed soma and extensive dendritic arborization. Here, we present stepwise procedures for the sparse labeling of PCs via a viral-mediated approach, enabling detailed two-dimensional dendritic Sholl analysis and three-dimensional spine quantification. We demonstrated high-resolution labeling of PCs through the administration of an adeno-associated virus (AAV) carrying a conditional Cre recombinase-dependent cassette (AAV, CAG-FLEX-EGFP). The AAV was delivered through an intracerebroventricular injection in Pcp2-Cre neonatal mice of either sex. Subsequently, we optimized two analytical pipelines to characterize dendritic complexity and spines. As a sample application, we performed analysis on the labeled PCs from anterior (I-V) and posterior (VI-IX) cerebellar lobules in mice of both sexes. In doing so, we detected an age-dependent, lobule-specific dendritic branching pattern difference. PCs from the anterior lobules exhibited a higher spine density but a smaller spine head diameter compared with those from the posterior lobules. Additionally, we examined PC morphological perturbations in a disease model of Dravet syndrome, a disorder caused by SCN1A haploinsufficiency. Here, we identified reduced dendritic complexity and immature spine morphology in the PCs of Scn1a+/- mice of both sexes. Thus, we provide a labeling method that is easily adaptable, efficient, robust, and well suited for examining PC morphology during cerebellar development and related pathogenic conditions.