Three-dimensional approaches to measuring primary cilia in hippocampal neurons: A comparative analysis

Primary cilia are non-motile sensory organelles that detect extracellular signals; disruptions in their functions are linked to neurodevelopmental disorders. Because cilia lengths can rapidly change in response to stressors, they are important for both plasticity and brain homeostasis. Accurate measurement of ciliary length is therefore essential but the absence of standardized methods and the variability introduced by different techniques can compromise measurement reliability and precision. To address this challenge, our study employed two distinct methods to estimate the length of primary cilia in hippocampal subregions in mice. We compared stereology-based 3D quantification, which is considered a methodological gold standard due to its unbiased sampling design and correction for tissue shrinkage, with 3D reconstruction to measure primary cilia length. 3D reconstruction imaging used a 100× oil-immersion objective. With neuronal cilia typically ∼2-10 µm long in hippocampus, a 1-µm z-step provided multiple optical sections per cilium thereby ensuring full structural visualization. Our findings show that both methods allow simple and equally precise measurements of neuronal primary cilia length in hippocampal subregions. Their strong agreement provides researchers with reliable tools for studying primary cilia on immunohistochemically stained sections and supports a consistent methodological framework for investigating cilia dynamics.