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 dendritic spine quantification. We demonstrated high-resolution labelling of PCs at the single-cell level 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 (ICV) 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 quantitative analysis on the labelled 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 to those from the posterior lobules. Additionally, we applied our workflow to examine PC morphological perturbations in a disease model of Dravet syndrome (DS), 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.Significance Statement Obtaining specific labeling of cerebellar PCs in vivo that is compatible with high-resolution imaging and straightforward analysis of dendritic structure and fine spine features remains challenging. The present study demonstrates that combining the Pcp2-Cre transgenic strain and Cre-dependent AAV-FLEX-GFP enables sparse and well-defined labeling of PCs. Using our optimized analytical pipeline, we detected slightly different branching patterns and spine morphology between the anterior and posterior vermis. We also identified dendritic atrophy and immature spine morphology in the PCs from Scn1a+/- mice. We demonstrate that our labeling method and the accompanying analytical pipeline are reliable, straightforward, and efficient. This work establishes an optimized sparse labeling strategy and streamlined analytical toolkit that will enable deeper investigation of Purkinje cell biology in vivo during cerebellar development and in disease-relevant conditions.