Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Abstract

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to ionizing radiation

“…Primary CGNs culture. Primary CGNs were isolated from 6-7 days old mouse pups by mechanical and enzymatic dissociation 67 . Pups were euthanized according to the McGill animal use and care committee guidelines.…”

“…Neurons were maintained at 37°C in a 5% CO2 incubator for up to 7 days. 24 h following plating, 10 μM cytosine-β-arabino furanoside (Sigma-Aldrich, #C-1768) was added to reduce glial contamination 67 . To overexpress OSMR, CGNs were infected with adenovirus bearing pAdenoG vector that contains the coding sequence of mouse OSMR (abm, #218317 A) at the time of plating.…”