This work is supported by Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research grants to A.J.-A.

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.
<ol>
	<li>Dissection Solution
	<ol>
		<li>Dissolve 3.62 g of sodium chloride (NaCl), 0.2 g of potassium chloride (KCl), 0.069 g of sodium phosphate (NaH2PO4 &#8729; H2O), and 1.306 g of D-(+)-Glucose in 500 mL of distilled H2O. Then add 12.5 mL of HEPES Buffer, 450 &#181;L of Phenol red solution, and 20 mL of BSA fraction V to the solution. Adjust to pH 7.4 using 1 N sodium hydroxide (NaOH).</li>
		<li>Sterilize with a 0.22 &#181;m filter and store at 4 &#176;C. This solution will remain stable for up to one week to 10 days.</li>
	</ol>
	</li>
	<li>Trypsin
	<ol>
		<li>Prepare a stock solution at a concentration of 25 g/L of trypsin in 0.9% sodium chloride. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor
	<ol>
		<li>Prepare a stock with a concentration of 10 mg/mL by dissolving 1 g of chicken egg white trypsin inhibitor in 100 mL of 1 mM HCl. Store this solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>DNase
	<ol>
		<li>Dissolve 100 mg of DNase 1 in 10 mL of sterile water to generate a 10 mg/mL stock. Store the solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>MgSO4
	<ol>
		<li>Add 6.02 g of magnesium sulfate (MgSO4) to 50 mL of distilled water (H2O) to generate a 1 M stock. Store the solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>CaCl 2
	<ol>
		<li>Dissolve 0.735 g of calcium chloride (CaCl2&#8729; 2H2O) in 50 mL of distilled H2O to a stock concentration of 100 mM. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>KCl
	<ol>
		<li>Dissolve 3.7275 g of KCl in 50 mL of distilled H2O to a stock concentration of 1 M. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>D-(+)-Glucose
	<ol>
		<li>Dissolve 1.802 g of D-(+)-glucose in 50 mL of distilled H2O to a stock concentration of 100 mM. Store solution at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cell Culture Media
	<ol>
		<li>Prepare cell culture media as follows: 5 mL of heat inactivated dialyzed Fetal Bovine Serum, 500 &#181;L of 200 mM L-Glutamine, 100 &#181;L of 50 mg/mL Gentamycin and 1 mL of 1.0 M KCL should be added to 45 mL of filter sterilized Eagle&#39;s minimal essential media (E-MEM) that is supplemented with 1.125 g/L D-Glucose to generate 50 mL of cerebellar granule neuron culture media. Store media at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cytosine Beta-D- Arabino Furanoside
	<ol>
		<li>Dissolve 48 mg of cytosine beta-D-arabino furanoside in 10 mL of growth media to a stock concentration of 20 mM. Store solution at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Poly-D-Lysine
	<ol>
		<li>To generate a 2 mg/mL poly D-lysine stock, add 10 mL of sterile H2O to 20 mg of poly D-lysine. Stock poly D-lysine should be stored at -80 &#176;C in 100 &#181;L aliquots.</li>
	</ol>
	</li>
	<li>NOTE: The following solutions should be prepared prior to dissection and maintained at 4 &#176;C until use.</li>
	<li>MgSO4 Supplemented Dissection Solution
	<ol>
		<li>Add 300 &#181;L of stock MgSO4 to 250 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Dissection Solution
	<ol>
		<li>Add 100 &#181;L of stock trypsin and 12 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor Solution 1
	<ol>
		<li>Add 164 &#181;L of stock trypsin inhibitor, 250 &#181;L of stock DNase 1 and 12 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Trypsin Inhibitor Solution 2
	<ol>
		<li>Add 1040 &#181;L of stock trypsin inhibitor, 750 &#181;L of stock DNase 1 and 12 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>CaCl 2 supplemented dissection solution
	<ol>
		<li>Add 10 &#181;L of stock CaCl2 and 25 &#181;L of stock MgSO4 to 10 mL of dissection solution.</li>
	</ol>
	</li>
	<li>Poly D-Lysine Coated Culture Dishes
	<ol>
		<li>To coat culture dishes, dilute 100 &#181;L of stock poly D-lysine in 40 mL of sterile H2O. For 35 mm Nunc culture dishes, add 2 mL of diluted poly D-Lysine solution. For 4 well plates, add 300 &#181;L of diluted poly D-Lysine to each well.</li>
		<li>Let the poly D-lysine sit for 1 h before aspirating and washing each well with 4 mL or 600 &#181;L (for 35 mm culture dishes or 4 well plates, respectively) of sterile H2O. Then aspirate the H2O and let the dishes air dry for 1 h.</li>
	</ol>
	</li>
</ol>
2. Brain Extraction and Isolation of Cerebellum
<ol>
	<li>Decapitate a 6-7 day old mouse pup using decapitation scissors. Perform the dissection of the brain in a sterile tissue culture hood.</li>
	<li>To remove the brain, grasp the head using a pair of forceps and cut through the skin toward the anterior of the head using microdissection scissors as demonstrated in Figure 1. Then cut through the skull bone using a new pair of scissors to minimize risk of contamination. Remove the skull covering the brain using forceps and tease out the brain using forceps or a spatula.
	<ol>
		<li>As needed, cut through the optic nerve to facilitate getting the brain out as a whole.</li>
	</ol>
	</li>
	<li>Place the brain in the dissection solution which is supplemented with MgSO4. Keep the solution and the brain on ice.</li>
	<li>Following brain removal, under a dissection microscope, dissect out the cerebellum from the brain while still in MgSO4 supplemented dissection solution, and remove the meninges using fine forceps. Turn the cerebellum to its ventral side and insure removal of the choroid plexus.</li>
	<li>Pool the cerebella into a 35-mm dish containing ~1 mL of MgSO4 supplemented dissection solution.</li>
	<li>Chop the tissue into small pieces and transfer into a 50 mL tube containing 30 mL of MgSO4 buffered dissection solution.</li>
</ol>
3. Mouse Cerebellar Granule Neuron Isolation and Culturing
<ol>
	<li>Centrifuge the 50 mL tube containing the chopped cerebral tissue for 5 min at 644 x g and 4 &#176;C.</li>
	<li>Remove the supernatant and add 10 mL of trypsin dissection solution. Then shake the tube at high speed for 15 min at 37 &#176;C.</li>
	<li>Add 10 mL of trypsin inhibitor solution 1 to the tube and rock gently for 2 min.</li>
	<li>Centrifuge the tube for 5 min at 644 x g and 4 &#176;C.</li>
	<li>Remove the supernatant and add 2 mL of trypsin inhibitor solution 2, and then transfer to a 15 mL tube.</li>
	<li>Triturate the tissue in the 15 mL tube until the solution becomes murky. Then let settle for 5 min.</li>
	<li>Remove the clear supernatant and transfer the supernatant to a new tube containing 1 mL of CaCl2 supplemented dissection solution.</li>
	<li>Add another mL of trypsin inhibitor solution 2 to the bottom of the tube containing the pellet from step 3.6. Triturate again and let settle for 5 min. Remove the supernatant and add it to the tube containing the supernatant from step 3.7 (That is a repeat of steps 3.6-3.7). Repeat this process until most of the tissue is mechanically dissociated.</li>
	<li>Add 0.3 mL of CaCl2 supplemented dissection solution to the supernatant collection for every mL of supernatant.</li>
	<li>Mix the contents of the tube and then centrifuge for 5 min at 644 x g.</li>
	<li>Remove the supernatant and add 10 mL of fresh media to the pellet and mix.</li>
	<li>Cells may then be counted and diluted to a concentration of 1.5 x 106 cells/mL. Please note that in general 10 million cells are expected from each dissected brain, dependent on trypsinization time and efficiency of dissection.Plate cells on previously made poly D-lysine plates. For 4-well plates, plate 0.5 mL, giving 7.5 x 105 cells per well. For 35-mm dishes, plate 4 mL, giving 6 x 106 cells per plate.</li>
	<li>After 24 h, add cytosine-&#946;-arabino furanoside (AraC) to the plates to reduce glial contamination. For each mL of media 0.5 &#181;L of 20 mM AraC is required. Addition of AraC does not require a media change. If cells are to be maintained for 7-8 days, repeat this treatment on day 3.</li>
	<li>Maintain cultures in a 5% CO2 incubator at 37 &#176;C and feed with 100 &#181;L of 100 mM glucose added to the culture for every 2 mL of media every 2 days past 5 days. A complete media change is not required.</li>
</ol>
4. Lentivirus Packaging, Purification and Titration
NOTE: The protocol for the packaging, concentration, purification and titration of lentivirus has previously been described in detail without the use of a kit 28, including alternative methods for titration, such as flow cytometry 29. Here we briefly present the protocol used in our lab for the production of lentivirus to study neuronal injury using speedy virus purification solution and a qPCR lentiviral titration kit.
<ol>
	<li>Plate Hek293T cells in 10 cm culture dishes with Dulbecco&#39;s modified Eagle&#39;s minimal essential medium (DMEM) supplemented with 10% FBS. For obtaining a high viral titer, grow at least 5 plates of Hek293T cells for each transfection. Make sure on the day of transfection cells are 70% confluent. Change media three hours prior to transfection.</li>
	<li>For each transfection, prepare two tubes. In tube 1 add 1.5 mL MEM-reduced serum media and 41 &#181;L of transfection reagent, mix well. In tube 2, add 1.5 mL of MEM-reduced serum media and 6 &#181;g of transfer plasmid, 6 &#181;g of pCMV-dR8.2 vector (packaging plasmid), 3 &#181;g pCMV-VSV-G vector (envelope plasmid), and 35 &#181;L of p3000 enhancer reagent (supplied with lipofectamine). Mix well, and combine tubes 1 and 2, vortex and incubate for 15 min.</li>
	<li>Remove 50% of the media from Hek293T plates and add DNA-lipid complexes to cells. Incubate for 6 h at 37 &#176;C, 5% CO2. Remove media and replace with 5 mL of fresh DMEM, incubate overnight.</li>
	<li>At 24 h post-transfection, collect the first batch of viral supernatant from each plate. Keep the viral media at 4 &#176;C and add 5 mL of fresh media into each plate of Hek293T cells. Incubate overnight.</li>
	<li>48 h post-transfection, collect the second batch of viral supernatant, combine it with the first batch and centrifuge combined viral supernatant for 10 min at 600 x g.</li>
	<li>Filter the supernatant through a filter with 45 &#181;m pore size.</li>
	<li>Purify the virus using a speedy virus purification solution. For every 45 mL of viral supernatant, add 5 mL of lentiviral binding solution, mix, and centrifuge for 10 min at &#62;5,000 x g and 4 &#176;C.</li>
	<li>Remove supernatant carefully so as not to disturb the pellet.</li>
	<li>Add 20-40 &#181;L of medium and re-suspend the pellet. Aliquot and store at -80 &#176;C.</li>
	<li>To obtain the lentiviral titer, a qPCR lentiviral titration kit is used. Dilute 2 &#181;L of purified virus in 500 &#181;L of PBS. Add 2 &#181;L of the diluted virus to 18 &#181;L of virus lysis buffer (provided in the kit).</li>
	<li>Set up three different RT-q-PCR reactions in triplicates and label them as, viral lysate, positive control 1 (STD1), and positive control 2 (STD2). For each reaction add, 12.5 &#181;L of 2x qPCR MasterMix, 2.5 &#181;L of either viral lysate or STD1 (provided in the kit) or STD2 (provided in the kit), and 10 &#181;L of reagent-mix (provided in the kit) in 0.2 mL PCR tubes.</li>
	<li>Set the PCR program as follows: 20 min at 42 &#176;C for reverse transcription, 10 min at 95 &#176;C for enzyme activation, 40 cycles of 15 s at 95 &#176;C for denaturation and 1 min at 60 &#176;C for annealing/extension.</li>
	<li>Calculate the viral titer using this formula: 
	Titer of viral lysate = 5 x 107/23(Ctx-Ct1)/(Ct2-Ct1). 
	Ctx = average Ct value of unknown sample, Ct1 = average value of STD1, Ct2 = average value of STD2. 
	NOTE: CGNs can be best transduced with lentiviruses or infected with adenoviruses depending on the mode of injury being studied. Addition of lentiviruses at an MOI of 2-3 to cell culture solution at the time of plating is used to study NMDA induced signalling at day 7 of the culture. This results in more than 80% transduction and is appropriate to pursue with biochemical analyses of these neurons (Figure 2). Addition of adenoviruses at day 5 of culture (at MOI 50) and studying NMDA induced signalling at day 7-8 is used for other techniques such as imaging. This treatment results in less than 10% infection of neurons, making it ideal for imaging and co-localization of proteins. Adenoviruses can be used at the time of plating to study DNA damage induced cell death by addition of camptothecin at day 2-3 or oxidative stress by addition of hydrogen peroxide. The presence of fluorescence proteins (GFP, RFP, CFP or YFP) tagged to the gene of interest can be used to estimate percent transduction and potential toxicity. With the recommended MOI, we see minimal toxicity and maximum transduction (Figure 3).</li>
</ol>
5. Modeling Neuronal Injury
<ol>
	<li>To induce NMDA induced neuronal excitotoxicity, following 7 days in vitro, treat CGNs with 100 &#181;M NMDA and 10 &#181;M glycine for 1 h. Replace all the medium with conditioned medium from parallel cultures with no treatment. This concentration results in 50% cell death at 24 h post treatment (Figure 3C). Mitochondrial fragmentation is evident at 6-8 h post treatment (See Jahani-Asl et al.26,27).</li>
	<li>To induce ROS-induced cell death (oxidative stress), treat neurons with 75-100 &#181;M hydrogen peroxide (H2O2) and switch to conditioned media from parallel cultures following 5 min exposure to H2O2. Note, due to the instability of H2O2, optimize the concentration to a level that induces 50-70% cell death in culture following 24 h of treatment.</li>
	<li>To induce DNA damage induced cell death, treat CGNs with 10 &#181;M camptothecin. This concentration induces more than 50% cell death in 24 h. Mitochondrial fragmentation and early apoptotic signalling occurs 2-3 h after addition of camptothecin at this concentration (See Jahani-Asl et al.18)</li>
	<li>To induce low K+ induced neuronal apoptosis in CGNs, change media containing 25 mM K+ to low potassium media with 5 mM K+ following 7 days in vitro.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. (32), (2009).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> J Vis Exp. (23), (2009).</li><li>Holubowska, A., Mukherjee, C., Vadhvani, M., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+cerebellar+granule+neurons+in+vitro+and+in+vivo+to+study+neuronal+morphology+and+migration.">Genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration.</a> J Vis Exp. (85), (2014).</li></ol>

The authors have nothing to disclose.

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...

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 allowed for research into the mechanisms of synaptic plasticity 7,8. On the contrary, over-stimulation of these receptors by NMDA ligand can be used to model excitotoxicity, a major mechanism of neuronal loss in acute brain damage and neurodegenerative diseases 9. One mechanism for the induction of excitotoxicity is through ATP starvation with reduced oxygen, as seen with acute neuronal injury. This results in membrane depolarization and elevated levels of glutamate release at the synapse. The subsequent overstimulation of the NMDA receptor by elevated glutamate results in excessive Ca2+ influx via these receptors, which in turn activates several pathways including Ca2+-activated proteases, phospholipases, and endonucleases, resulting in the uncontrolled degradation of critical cellular components and cell death. Additionally, high intracellular Ca2+ leads to the generation of oxygen free radicals and mitochondrial damage 10,11.
While the majority of neuronal loss following NMDA-induced neuronal excitotoxicity is due to calcium influx and is Bax/Bak independent, other mechanisms of cell death cannot be excluded from this model. The appearance of both necrotic and apoptotic like cell death due to excitotoxicity is partially due to the generation of reactive oxygen species (ROS) and DNA damage caused by high intracellular Ca2+ levels 12. DNA damage results in neuronal death through apoptotic mechanisms, being correlated with hallmarks of apoptotic cell death, such as the appearance of chromatin masses and apoptotic bodies. Induction of apoptosis is mediated through the release of cytochrome c from the mitochondria, and has been shown to be dependent on Bax/Bak oligomerization 13. Bax/Bak oligomerization promotes pore formation in the outer mitochondrial membrane, resulting in cytochrome c release and the activation of pro-apoptotic regulators as seen with mild ischemic injury 14.
Generation of ROS is a significant issue in the brain due to the low endogenous levels of antioxidants, coupled with the large oxygen requirement for neuronal functioning 15. When exposed to an ischemic event, nitric oxide synthase is upregulated , producing nitric oxide and increasing reactive oxygen species 14. The increased concentration of oxygen radicals can result in DNA damage and indirectly cause energy starvation. High levels of DNA double-stranded breaks are remedied by the activation of poly ADP-ribose polymerase-1 (PARP-1), an eukaryotic chromatin-bound protein responsible for catalyzing the transfer of ADP-ribose units from NAD+, a process integral to DNA repair 16. However, with excessive damage due to oxidative stress, PARP-1 activation can cause energy starvation due to the increased drain on NAD+, a necessary substrate for ATP production through oxidative phosphorylation. Ultimately, oxidative stress will trigger apoptosis in a Bax/Bak dependant manner leading to mitochondrial cytochrome c release, and has been shown to induce mitochondrial remodelling in CGNs 17.
Finally, changes in concentration of potassium chloride (KCl) in CGN cultures can be used to model low potassium/depolarization mediated apoptosis 18,19,20. When exposed to low levels of K+, CGNs undergo distinct physiological changes, resulting in reductions of both mitochondrial respiration and glycolysis, attributed to decreased cellular demand 21, as well as reduction in levels of nuclear factor-&#954;B (NF&#954;B) which regulates activities including inflammation and synaptic transmission 22. This model is of particular interest for the study of cell death during neuronal development. The low K+ environment more closely resembles physiological conditions, and causes hallmarks of cell death seen during neuronal development 23.
In summary, CGNs provide a longstanding model to investigate the underlying molecular mechanisms of neuronal death and degeneration. The following protocol will allow isolation and culturing of CGNs, expression or repression of a particular genetic pathway using viruses and the induction of neuronal death via different mechanisms representing neuronal injury and degeneration.

<table><tbody><tr><td>qPCR lentivitral titration kit&amp;nbsp;</td><td>ABM</td><td>#LV900</td><td></td></tr><tr><td>speedy virus purification solution&amp;nbsp;</td><td>ABM</td><td>#LV999</td><td></td></tr><tr><td>pCMV-dR8.2</td><td>Addgene</td><td>#8455</td><td></td></tr><tr><td>pCMV-VS.VG</td><td>Addgene</td><td>#8454</td><td></td></tr><tr><td>Distilled water&amp;nbsp;</td><td>Gibco</td><td>#15230162</td><td></td></tr><tr><td>200 mM L-Glutamine&amp;nbsp;</td><td>Gibco</td><td>#25030081</td><td></td></tr><tr><td>35 mm Nunc culture dishes</td><td>Gibco</td><td>#174913</td><td></td></tr><tr><td>PowerUP SYBR green master mix</td><td>life technologies</td><td>#A25742</td><td></td></tr><tr><td>BSA V Solution</td><td>Sigma Aldrich</td><td>#A-8412</td><td></td></tr><tr><td>CaCl2 &amp;bull; 2H2O</td><td>Sigma Aldrich</td><td>#C-7902&amp;nbsp;</td><td></td></tr><tr><td>Camptothecin</td><td>Sigma Aldrich</td><td>#C-9911</td><td></td></tr><tr><td>Chicken Egg White Trypsin Inhibitor&amp;nbsp;</td><td>Sigma Aldrich</td><td>#10109878001</td><td></td></tr><tr><td>Cytosine beta-D-Arabino Furanoside</td><td>Sigma Aldrich</td><td>#C-1768</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma Aldrich</td><td>#G-7528</td><td></td></tr><tr><td>DNase1&amp;nbsp;</td><td>Sigma Aldrich</td><td>#11284932001</td><td></td></tr><tr><td>Eagle-minimal essential medium</td><td>Sigma Aldrich</td><td>#M-2279</td><td></td></tr><tr><td>Glycine</td><td>Sigma Aldrich</td><td>#G-5417</td><td></td></tr><tr><td>Heat inactivated dialyzed Fetal Bovine Serum&amp;nbsp;</td><td>Sigma Aldrich</td><td>#F-0392</td><td></td></tr><tr><td>Hepes Buffer&amp;nbsp;</td><td>Sigma Aldrich</td><td>#H-0887</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma Aldrich</td><td>#216763</td><td></td></tr><tr><td>50 mg/mL Gentamycin&amp;nbsp;</td><td>Sigma Aldrich</td><td>#G-1397</td><td></td></tr><tr><td>MgSO4&amp;nbsp;</td><td>Sigma Aldrich</td><td>#M-2643</td><td></td></tr><tr><td>N-Methyl-D-aspartic acid</td><td>Sigma Aldrich</td><td>#M-3262</td><td></td></tr><tr><td>Phenol Red Solution&amp;nbsp;</td><td>Sigma Aldrich</td><td>#P-0290</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Sigma Aldrich</td><td>#T-4549</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Thermo Fisher Scientific</td><td>L3000-008</td><td></td></tr><tr><td>p3000 enhancer reagent</td><td>Thermo Fisher Scientific</td><td>L3000-008</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>KCl&amp;nbsp;</td><td>VWR</td><td>#CABDH9258</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>VWR</td><td>#CABDH9286</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>VWR</td><td>#CABDH9298</td><td></td></tr><tr><td>Poly D-lysine&amp;nbsp;</td><td>VWR</td><td>#89134-858</td><td></td></tr><tr><td>DMEM</td><td>Wisent</td><td>#319-005-CL</td><td></td></tr><tr><td>FBS</td><td>Wisent</td><td>#080-450</td><td></td></tr></tbody></table>

modeling neuronal death and degeneration in mouse primary cerebellar granule neurons

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.

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 ...

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Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

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.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

modeling-neuronal-death-degeneration-mouse-primary-cerebellar-granule

Research

JoVE Journal

Neuroscience

8.0K Views.  Jewish General Hospital. Dit protocol beschrijft een eenvoudige methode om te isoleren en primaire muisknop cerebrale submodule neuronen (CGNs) van 6-7 dag oud pups, efficiënte transductie van CGNs voor verlies en winst van functie studies, kweken en modellering NMDA-geïnduceerde neuronale excitotoxicity, lage-kalium-veroorzaakte celdood, DNA-schade en oxidatieve stress met behulp van de zelfde cultuur-model.

Video: Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Developmental Biology

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Biology

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...

Modeling Neuronal Injury in Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates the generation of a neuronal injury model in mouse primary cerebellar granule neurons. It outlines the steps involved in inducing neuronal injury using NMDA, an excitatory neurotransmitter receptor agonist, and glycine, a co-agonist. NMDA and glycine bind to specific neuronal receptors, causing their overstimulation and resulting in a massive intracellular calcium influx, which leads to neuronal cell damage and death.

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

	Dissection Solution
	
		Dissolve 3.62 g of ...

JoVE Encyclopedia of Experiments

Neuropathology

Trauma

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase activation, resulting in neuronal apoptosis.

Neurodegenerative Diseases

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

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