A Cell-Based Assay Optimized for High-Content Cilia Imaging with Primary Rat Hippocampal Neurons

Abstract

Summary Genetic manipulations of dissociated rodent neurons provide translatable in vitro models for disease-driven phenotypes. Cilia are cellular antenna with a role in neuronal maturation and function often perturbed in neurodevelopmental disorders. Efforts for automated imaging of these microscopic protrusions are crucial given the role of cilia in the brain. We developed a cell-based assay to monitor cilia in rat hippocampal neurons using lentiviral-mediated shRNA-based gene silencing. This optimized platform can be used for high-throughput cilia imaging, disease modeling, and drug screening. For complete details on the use and execution of this protocol, please refer to Di Nardo et al. (2020) .

DEPDC5-dependent mTORC1 signaling mechanisms are critical for the anti-seizure effects of acute fasting

“…Cultures were prepared as we previously described (Di Nardo et al, 2020b). Briefly, hippocampi and cortices from 18-day-old rat embryos (Charles River CD1) were isolated under the microscope and collected in Hank's Balanced Salt Solution containing 10mM MgCl2, 1mM kynurenic acid, 10mM HEPES and penicillin/streptomycin.…”

“…Rat cortical neurons were plated at a density of 750,000 cells/well onto six-well plates and 1,000,000 cells/well coated with 20 mg/mL poly-D-lysine (PDL). Transduction of the neurons was performed with lentivirus as previously described (Di Nardo et al, 2020b). For immunoblotting, neurons were washed with cold PBS on ice and lysates collected in RIPA buffer with protease and phosphatase inhibitors.…”

Phenotypic characterization of Cdkl5-knockdown neurons establishes elongated cilia as a functional assay for CDKL5 Deficiency Disorder

“…Controls were sex- and age-matched littermates. Rat hippocampal neurons were cultured as previously described ( Di Nardo et al, 2020a , Di Nardo et al, 2020b ). Lentiviral stocks were prepared as previously described ( Di Nardo et al, 2020a ).…”

“…To assess functional consequences of Cdkl5 gene silencing, we imaged cilia in cultured neurons with an assay that we previously developed for unbiased cilia quantification (cilia HCA ) ( Di Nardo et al, 2020a , b ). We optimized cilia HCA to measure cilia length (hereafter ciliaL HCA ) using the ImageXpress Micro Confocal (XM-C) platform ( Molecular Devices ) at 13 days in culture (DIV13).…”