Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Summary

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

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.

Introduction

Cerebellar granule neurons (CGNs) are well characterized in culture and have served as an effective model to study neuronal death and development ^1,2,3,4,5,6. The early expression of N-methyl-D-aspartate (NMDA) receptors in CGN cultures in vitro makes them an attractive model to study NMDA-induced signalling. Activation of these receptors with NMDA in conjunction with membrane depolarization is used to model physiological neuronal activity stimulation, and has all....

Protocol

This protocol is based on modifications of procedures that have been described previously ^{18,24,25,26,27}. This protocol is approved by the Animal Care Committee at McGill University.

1. Experimental Preparation

NOTE: The following stock solutions can be prepared and maintained until use.

1. Dissection Solution
   1. Dissolve 3.62 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.069 g of sodium phosphate ....

Results

With careful dissection, the intact brain should be removed with minimal damage as seen in Figure 1A-B. Effort should be taken to minimize damage to the brain during removal, particularly damage to the cerebellum. Damaging the cerebellum makes for more difficult identification and complete removal of the meninges, and increases the likelihood of contamination of the neuronal culture. Once the meninges have been removed, the cerebellum can be .......

Discussion

Here we provide a simple method for the culturing of primary mouse cerebellar granule neurons (CGNs), loss and gain of function studies, and modeling different mechanisms of cell death. Several factors affect the reproducibility of the results using this procedure which require close monitoring. These include the purity of the culture including the elimination of glial cells in the culture, the confluence of the culture, and maintaining healthy cells. Introducing variability in these factors can bias the results and chal.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
qPCR lentivitral titration kit	ABM	#LV900
speedy virus purification solution	ABM	#LV999
pCMV-dR8.2	Addgene	#8455
pCMV-VS.VG	Addgene	#8454
Distilled water	Gibco	#15230162
200 mM L-Glutamine	Gibco	#25030081
35 mm Nunc culture dishes	Gibco	#174913
PowerUP SYBR green master mix	life technologies	#A25742
BSA V Solution	Sigma Aldrich	#A-8412
CaCl2 • 2H2O	Sigma Aldrich	#C-7902
Camptothecin	Sigma Aldrich	#C-9911
Chicken Egg White Trypsin Inhibitor	Sigma Aldrich	#10109878001
Cytosine beta-D-Arabino Furanoside	Sigma Aldrich	#C-1768
D-(+)-Glucose	Sigma Aldrich	#G-7528
DNase1	Sigma Aldrich	#11284932001
Eagle-minimal essential medium	Sigma Aldrich	#M-2279
Glycine	Sigma Aldrich	#G-5417
Heat inactivated dialyzed Fetal Bovine Serum	Sigma Aldrich	#F-0392
Hepes Buffer	Sigma Aldrich	#H-0887
Hydrogen peroxide	Sigma Aldrich	#216763
50 mg/mL Gentamycin	Sigma Aldrich	#G-1397
MgSO4	Sigma Aldrich	#M-2643
N-Methyl-D-aspartic acid	Sigma Aldrich	#M-3262
Phenol Red Solution	Sigma Aldrich	#P-0290
Trypsin	Sigma Aldrich	#T-4549
Lipofectamine 3000	Thermo Fisher Scientific	L3000-008
p3000 enhancer reagent	Thermo Fisher Scientific	L3000-008
Opti-MEM I Reduced Serum Medium	Thermo Fisher Scientific	31985070
KCl	VWR	#CABDH9258
NaCl	VWR	#CABDH9286
NaH2PO4H2O	VWR	#CABDH9298
Poly D-lysine	VWR	#89134-858
DMEM	Wisent	#319-005-CL
FBS	Wisent	#080-450