We thank Dr. Gonzalo Quasollo for his invaluable help with imaging and Dr. Andrea Pellegrini for cell culture technical support.
This research was supported by grants from: the National Institute of Health, USA (#RO1AG058778-01A1, Subaward Agreement No 165148/165147 between UTHSCSA-Instituto Investigaci&#243;n M&#233;dica M y M Ferreyra) and from the National Agency of Agencia Nacional de Scientific and Technological Promotion, Argentina (ANPCyT, PICT 2017 #0618).

The animal procedures are performed following approved protocols of the National Institute of Health Guide for the Care and Use of Laboratory Animals. Approval to conduct the study is granted by the Animal Care and Ethics Committee (CICUAL) of INIMEC-CONICET-UNC (Resolution numbers 014/2017 B and 006/2017 A).
1. Primary rat cortical neuron cultures
<ol>
	<li>Anesthetize E18 Wistar pregnant rats in a CO2 chamber with a mixture of 80% CO2/20% O2 for 60 s of exposure and then sacrifice then by dislocate the cervical vertebrae.</li>
	<li>Perform the following steps in a laminar flow hood. Extract encephalon from the embryos with a forceps style # 3 and straight sharp small spring scissors (Table of Materials) to Hanks solution in cell culture dishes.</li>
	<li>Dissect the tissue under 20x magnifying glass, using two fine-tipped forceps style #5, separating the frontal cortex from the meninges. Transfer the frontal cortex to a 15 mL conical tube and incubate the tissue with 3-4 mL of Trypsin-EDTA (0.25%) for 15 min, at 37 &#176;C for chemical digestion.</li>
	<li>After digestion, dilute it with 3-4mL of Dulbecco&#39;s modified eagle medium (DMEM) plus 10% fetal calf serum (FCS). Then wash it 3 times with Ca2+/Mg2+ free Hanks Balanced Salt solution and 0.1% glucose by centrifugation (3000 g for 5 min).</li>
	<li>Mechanically dissociate the tissue 10 times by pipetting up and down using a 1000 &#181;L tip, in&#160;DMEM&#160;plus 10% FCS&#160;(Table of Materials). Then centrifuge the homogenate at 100 x g for 1 min, collect the supernatant and complete the remaining volume to 1000 &#181;L.</li>
	<li>Plate the cell suspension on cell culture dishes at a density of 520 cell/mm2 with 2 mL of DMEM containing 10% FCS, 1% penicillin/streptomycin and 1% of an essential aminoacid additive (see Table of Material). Then incubate at 37 &#176;C in a humidified environment with 5% CO2 for 2 h.
	<ol>
		<li>Organic matter removal and poly-L-lysine coating of cell culture dishes
		<ol>
			<li>Remove the organic matter by placing the glass material in a reaction chamber and add 96% ethyl alcohol to just about cover the material. Then add 68% (w/v) nitric acid drop by drop until brownish-greenish bubbles start to appear. Once this happens, partially cover the container and allow the reaction to continue until explosion ceases. 
			NOTE: Remove the organic matter under a gas extraction cabin to avoid exposure to noxious gases.</li>
			<li>Before poly-L-lysine coating, rinse the glasses with sterile Mili-Q water about ten times. Then incubate the culture glasses and dishes with 0.1 &#956;g/&#956;L poly-L-lysine for 4 h before using them. After incubation, rinse the glasses with sterile Mili-Q water about ten times.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>To allow neural cell differentiation, change the medium for a serum-free neurobasal medium, with the serum-free supplement (Table of Materials). Keep cultures at 37 &#176;C with 5% CO2 until treatment.</li>
</ol>
2. Lentivirus production
NOTE: The oligonucleotides containing the sequences targeting the 3&#39;UTRs of either the CN-A&#945; isoform or CHOP and with the non-targeting sequences (scrambles) are listed in Table 1.
<ol>
	<li>For annealing, mix two single-stranded oligonucleotides with complementary sequences in equal molar amounts, using Annealing Buffer (10 mM Tris-HCl, pH 7.5-8.0, 50 mM NaCl, 1 mM EDTA).</li>
	<li>Heat at 95 &#176;C for 2 min and then cool slowly by transferring samples from the heat block or water bath to a bench-top at room temperature. Dilute the resulting product [short hairpin RNA, (shRNA) with cohesive ends] to a final concentration of 0.5 &#181;M, aliquot them and store at -20 &#176;C.</li>
	<li>Clone the shRNA insert into lentiviral vector pLKO.3G, with GFP as a green fluorescent marker. For this, digest 1 &#181;g of vector with EcoRI and PacI restriction enzymes by mixing the following in a sterile tube: 2 &#181;L of 10x restriction enzyme buffer (100 mM bis-Tris propane-HCl, pH 6.5, 100 mM MgCl2, 1 mg/mL bovine serum albumin), 1 &#181;L of 1 &#181;g/&#181;L pLKO.3G, 0.5 &#181;L of 10 U/&#181;L ECoRI, 0.5 &#181;L of PacI (10 U/&#181;L), and 16 &#181;L of sterile ionized water.
	<ol>
		<li>Mix gently by pipetting. Incubate the reaction for 3-4 h at 37 &#176;C. 
		NOTE: Overnight digestions are generally unnecessary and may result in DNA degradation.</li>
	</ol>
	</li>
	<li>For ligation, mix the digested vector and insert in a molar ratio of 3:1 and incubate it with the T4 ligase and ligase buffer (provided by manufacturer) overnight at 16 &#176;C. 
	NOTE: At this point ligation reaction can be stored at 4 &#176;C until further use.</li>
	<li>Transform DH5&#945; competent cells (Table of Materials) or another suitable E. coli strain with the ligation reaction as follows.
	<ol>
		<li>Mix competent E. coli with the total volume of ligation reaction and chill it on ice for 15 min, place it in a 42 &#176;C bath for 2 min and then chill it on ice again for 15 min.</li>
		<li>Transfer the total volume subjected to heat shock to a 15 mL tube and complete the volume to 1000 &#181;L with Luria-Bertani (LB) liquid medium that has been previously maintained at 37 &#176;C. Shake the bacteria for 90 min at 37 &#176;C in an orbital shaker at approximately 330 rpm.</li>
		<li>Centrifuge them at 5,000 x g for 5 min at 4 &#176;C and discard 900 &#181;L of supernatant. Use the remaining supernatant to resuspend the pellet. Spread bacteria suspension evenly over a solid ampicillin (100 &#181;g/mL) LB plate.</li>
		<li>Incubate at 37 &#176;C overnight, then check bacterial growth. At this point, the bacteria may be stored at 4 &#176;C for 4-5 days.</li>
		<li>Pick a single colony to transfer into a 50 mL tube with 10 mL of LB liquid medium and ampicillin (100 &#181;g/mL). Shake the bacteria ON at 37 &#176;C at approximately 330 rpm.</li>
		<li>Centrifuge the culture to pellet the bacteria at 5,000 x g for 5 min at 4 &#176;C, discard the supernatant and conserve the bacterial pellet. This can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Purify the plasmid with a DNA purification kit from bacteria pellet following manufacturer&#39;s instructions (Table of Materials). Calculate the DNA concentration by measuring the absorbance at 260 nm and multiplying by the dilution factor, using the following relationship: A260 of 1.0 = 50 &#181;g/mL pure double strand (ds) DNA.</li>
</ol>
3. Lentivirus infection
NOTE: Perform the lentivirus generation procedure in a Class II laminar flow hood.
<ol>
	<li>24 h before transfection, seed 1-5 &#215; 106 HEK 293 cells into 100 mm of diameter culture dishes in 8 mL of growth medium and incubate them at 37 &#176;C, 5% CO2 overnight.</li>
	<li>Mix 14 &#181;g of pKLO.3G vector with specific insert, 10.5 &#181;g of packing plasmid psPAX and 3.5 &#181;g of envelope plasmid pMD2.G (Table of Materials). Dilute 45 &#181;L of plasmid transfection reagent (Table of Materials) in 700 &#181;L of reduced serum media (Table of Materials) and incubate for 5 min at room temperature. Then combine the DNA mixture with the diluted plasmid transfection reagent, mix gently, and incubate for 20 min at room temperature.</li>
	<li>Meanwhile, change for fresh DMEM the medium of the culture dish of HEK 293 cell cultures (70% confluent at the time of transfection). Then add the entire volume of the DNA solution drop by drop. Rock the dish gently.It is not necessary to add/change medium after transfection.</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 for 48 h- 72 h to allow shRNAs to reach their optimum transduction checked by GFP fluorescence using a fluorescence microscope. Afterwards, collect the medium with lentivirus and store at 4 &#176;C.</li>
	<li>Filtrate viral supernatant with a 0.45 &#181;m nylon membrane. Aliquot the flow-through in 1 mL. Next, centrifuge at 17,000 x g for 4 h. Discard the supernatant and conserve the pellet. Allow invisible pellet to dry, and, once dried, store at - 80 &#176;C.
	<ol>
		<li>Perform viral titration by flux cytometry. Seed 4 x 105 HEK 293 cells in 24-well plates and incubate at 37 &#176;C for 24 h. Then resuspend the viral particles in 200 &#181;L of DMEM culture medium (Table of Materials) and add 0.05, 0.02, and 0.005 &#181;L to each well.</li>
		<li>Incubate viral infection for 72 h, remove medium and trypsinize (0.05% trypsin, 1 mL) for 3 min. Add 1 mL of DMEM in 10% FCS solution and centrifuge at 500 x g for 5 min. Discard the supernatant and wash the pellet twice with PBS.</li>
		<li>Resuspend the pellet with 100 &#181;L of PBS and fix the cells with 100 &#181;L of 2% paraformaldehyde (PFA) in PBS. Analyze the cells with a flux cytometer and determine the percentage of GFP fluorescent cells. Collect this data to calculate viral titration using the following formula: 
		Viral Titration 
		<img alt="Equation1" src="/files/ftp_upload/61974/61974eq01.jpg" /></li>
	</ol>
	</li>
</ol>
4. Primary neuron cultures stressed with ganglioside GM2 accumulation and immunocytochemistry using anti-MAP2 antibody
<ol>
	<li>Infect the primary cortical neuron cultures (15 days in vitro) with specific shRNA oligonucleotides targeting the 3&#39; UTRs of either CN-A&#945; or CHOP and incubate them at 37 &#176;C with 5% CO2 for 1 day. 
	NOTE: Primary cortical neuron cultures of 7-8 days in vitro could also be used if the cells show high neurite growth.
	<ol>
		<li>Prepare a 500 &#181;M stock solution of ganglioside GM2 in ethanol and sonicate it for 1 h.</li>
		<li>Add GM2 stock solution to the culture dishes medium at a final concentration of 2 &#181;M. Allow to incubate at 37 &#176;C with 5% CO2 for 16, 24, and 48 h.</li>
	</ol>
	</li>
	<li>Use some cultures to prepare cellular homogenate for western blot analysis (for antibody specification, see the Table of Materials).</li>
	<li>Follow steps 1.4. and 1.5. and then fix the cells with 4% PFA and 120 mM sucrose in PBS for 20 min at 37 &#176;C, and then wash it with PBS. Perform this procedure on an anti-slipping membrane in a wet chamber. Afterwards, add permeabilization solution (0.2% Triton X-100 in PBS) for 5 min and wash with PBS.
	<ol>
		<li>Use 5% BSA diluted in PBS for 45 min to allow blocking, then incubate with anti-MAP2 antibody, diluted 1:800 in blocking solution, at 4 &#176;C overnight.</li>
		<li>At the end of the incubation time, wash twice with PBS and incubate with secondary antibody (Table of Materials) for 1 h. Then wash the cells with PBS and mount the glasses onto slides using an aqueous mounting medium (Table of Materials).</li>
	</ol>
	</li>
</ol>
5. Neurite atrophy analysis
<ol>
	<li>Obtain images with a 20x air lens (NA 0.8) using an epifluorescence-inverted microscope equipped with a CCD-camera. Analyze images with ImageJ plug-ins. For this, load the picture to ImageJ and subtract the background by clicking on Process | Subtract Background.</li>
	<li>Click on Image | Adjust | Threshold or type the following keys: Ctrl (or Cmd) + Shift + T. A window pops open to adjust minimum values, allowing path and soma tracings to be distinguishable. Click on the binary image (neurites and somas are white colored). On this image, use freehand selection to delete somas; the selected soma area turns black again.</li>
	<li>Select traces and click Edit | Selection | Create Selection. Get the ROI by clicking Ctrl (or Cmd) and T keys of the selection and conserve it. Open the original image, then click on the ROI manager panel and select the belonging ROI, previously obtained. Then click on the original image.</li>
	<li>Once the ROI traces on the image, type Ctrl (or Cmd) and M keys (Analyze | Measure). A window pops open; copy the mean value (arbitrary units a.u.) to process statistical analysis. 
	NOTE: To obtain values as &#181;m, click on Analyze | Set Scale. A window pops up open to set values. Be sure to add the correct scale (Analyze | Set Scale) depending on the characteristics of the microscope used.</li>
</ol>

<ol>
	<li>Schroder, M., Kaufman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+unfolded+protein+response.">The mammalian unfolded protein response.</a> Annual Review of Biochemistry. 74, 739-789 (2005).</li><li>Cui, W., Li, J., Ron, D., Sha, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+PERK+kinase+domain+suggests+the+mechanism+for+its+activation.">The structure of the PERK kinase domain suggests the mechanism for its activation.</a> Acta Crystallographica. Section D, Biological Crystallography. 67, 423-428 (2011).</li><li>Bollo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+interacts+with+PERK+and+dephosphorylates+calnexin+to+relieve+ER+stress+in+mammals+and+frogs.">Calcineurin interacts with PERK and dephosphorylates calnexin to relieve ER stress in mammals and frogs.</a> PLoS One. 5, 11925 (2010).</li><li>Chen, Y., Holstein, D. M., Aime, S., Bollo, M., Lechleiter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin+beta+protects+brain+after+injury+by+activating+the+unfolded+protein+response.">Calcineurin beta protects brain after injury by activating the unfolded protein response.</a> Neurobiology of Disease. 94, 139-156 (2016).</li><li>Harding, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+stress+response+regulates+amino+acid+metabolism+and+resistance+to+oxidative+stress.">An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.</a> Molecular Cell. 11, 619-633 (2003).</li><li>Hetz, C., Saxena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+stress+and+the+unfolded+protein+response+in+neurodegeneration.">ER stress and the unfolded protein response in neurodegeneration.</a> Nature Reviews Neurology. 13, 477-491 (2017).</li><li>Lin, J. H., Li, H., Zhang, Y., Ron, D., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+effects+of+PERK+and+IRE1+signaling+on+cell+viability.">Divergent effects of PERK and IRE1 signaling on cell viability.</a> PLoS One. 4, 4170 (2009).</li><li>Virgolini, M. J., Feliziani, C., Cambiasso, M. J., Lopez, P. H., Bollo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+atrophy+and+apoptosis+mediated+by+PERK+signaling+after+accumulation+of+GM2-ganglioside.">Neurite atrophy and apoptosis mediated by PERK signaling after accumulation of GM2-ganglioside.</a> Biochimica et Biophysica Acta - Molecular Cell Research. 1866, 225-239 (2019).</li><li>Ferreira, A., Busciglio, J., Landa, C., Caceres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglioside-enhanced+neurite+growth:+evidence+for+a+selective+induction+of+high-molecular-weight+MAP-2.">Ganglioside-enhanced neurite growth: evidence for a selective induction of high-molecular-weight MAP-2.</a> The Journal of Neuroscience. 10, 293-302 (1990).</li><li>Moore, C. B., Guthrie, E. H., Huang, M. T., Taxman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA+(shRNA):+design,+delivery,+and+assessment+of+gene+knockdown.">Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown.</a> Methods in Molecular Biology. 629, 141-158 (2010).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocols. 1, 2406-2415 (2006).</li><li>Rusnak, F., Mertz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcineurin:+form+and+function.">Calcineurin: form and function.</a> Physiological Reviews. 80, 1483-1521 (2000).</li><li>Hogan, P. G., Chen, L., Nardone, J., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+by+calcium,+calcineurin,+and+NFAT.">Transcriptional regulation by calcium, calcineurin, and NFAT.</a> Genes &amp; Development. 17, 2205-2232 (2003).</li><li>Endo, M., Mori, M., Akira, S., Gotoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C/EBP+homologous+protein+(CHOP)+is+crucial+for+the+induction+of+caspase-11+and+the+pathogenesis+of+lipopolysaccharide-induced+inflammation.">C/EBP homologous protein (CHOP) is crucial for the induction of caspase-11 and the pathogenesis of lipopolysaccharide-induced inflammation.</a> Journal of Immunology. 176, 6245-6253 (2006).</li></ol>

The authors have no competing financial interests.

We describe an experimental system that enables molecular modulation of the transition from survival to apoptotic UPR phases in a neuronal cell model.
For a proper analysis of neurite atrophy, it is essential to obtain primary neuron cultures with numerous, long, highly branched processes9,11. This facilitates the examination of neuron process extension, allowing the clear differences between treatments to be detected. It is important ...

Endoplasmic reticulum (ER) stress is defined as any perturbation that compromises protein-folding capacity in the organelle. The accumulation of unfolded proteins within the ER lumen activates a transduction cascade signal called the unfolded protein response (UPR). This complex signaling pathway is orchestrated by three stress sensors: PERK (protein kinase RNA [PKR]-like ER kinase), IRE1 (inositol-requiring enzyme 1) and ATF6 (activated transcription factor 6). All together attempt to restore homeostasis. But if stress persists, UPR eventually induces cell death by apoptosis1.
PERK, an ER transmembrane protein, upon ER stress, leads the phosphorylation of eukaryotic initiation factor-2 alpha (eIF2&#945;), reducing global protein synthesis and thus protein load in the ER2. We demonstrated that calcineurin A/B (CNA/B), a heterodimer Ca2+ phosphatase, directly binds the cytosolic domain of PERK, increasing its auto-phosphorylation and significantly enhancing inhibition of protein translation and cell viability3,4. Interestingly, CNA/B is abundant in the mammalian brain, distinguishing two isoforms of the subunit A of CN: &#945; and &#946;.
Under sustained ER stress, the PERK signaling pathway is the only UPR branch that remains activated, thus mediating both the pro-survival and the apoptotic response. In the chronic phase, one major downstream event is the induction of the transcription factor, CHOP (CCAAT/enhancer binding protein homologous protein)5. Chronic ER stress is also increasingly recognized as a common contributor to an extensive range of pathological disorders, including neurodegenerative diseases6. It is important to understand how UPR can facilitate cytoprotective signaling instead of cell death 7. However, at present little is known about the exact mechanism controlling the transition between these two UPR phases.
Recently, we found that, in cultured neurons, ganglioside GM2 accumulates in ER membranes and induces luminal calcium depletion. This in turn activates PERK signaling, which mediates neurite atrophy and apoptosis 8. In this study, the GM2 build-up in cultured neurons is used as a cell system model of ER stress-induced neurite atrophy. Specifically, two PERK factor expressions are manipulated, CN-A&#945; and CHOP, which switches the transition between early/protective events and a chronic/apoptotic phase. To accomplish this, the respective genes are silenced; thus, primary cortical neuron cultures are infected with lentivirus-delivered specific shRNA. Western blot analysis reveals a significant reduction of CN-A&#945; and CHOP expression levels in comparison with the control cells, which are infected with lentivirus that carry a scrambled shRNA. After this treatment, neurons are subjected to different incubation times of exogenous GM2, fixed, and immunostained with anti-microtubule associated protein 2 (MAP2) antibody9. Images are obtained with an epifluorescence microscope. The total neurite outgrowth is evaluated relative to total cell number.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 anti-Mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>#R37120</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CHOP</td>
			<td>Thermo Fisher</td>
			<td># MA1 - 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CN-A&#945;</td>
			<td>Millipore</td>
			<td># 07-067</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GM2</td>
			<td>Matreya</td>
			<td>#1961</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-MAP2</td>
			<td>Sigma Aldrich</td>
			<td># M2320</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-&#946;-actin</td>
			<td>Thermo Fisher</td>
			<td># PA1 - 183</td>
			<td></td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>#3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 200 epifluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>#17504944</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>#11966025</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>Promega</td>
			<td>#R6011</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Life Technologies</td>
			<td>#16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-tippeds forceps&#160; style #5</td>
			<td>Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep style #3</td>
			<td>Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293</td>
			<td>ATCC</td>
			<td>#CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680 CW secondary antibody</td>
			<td>LI-COR Biosciences</td>
			<td>#92632221</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680 secondary antibody</td>
			<td>LI-COR Biosciences</td>
			<td>#92632220</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800 CW secondary antibody</td>
			<td>LI-COR Biosciences</td>
			<td>#92632210</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800 CW secondary antibody</td>
			<td>LI-COR Biosciences</td>
			<td>#92632211</td>
			<td></td>
		</tr>
		<tr>
			<td>lentiviral envelope plasmid pMD2.G</td>
			<td>Addgene</td>
			<td>#12259</td>
			<td></td>
		</tr>
		<tr>
			<td>lentiviral packing plasmid psPAX2</td>
			<td>Addgene</td>
			<td>#12260</td>
			<td></td>
		</tr>
		<tr>
			<td>lentiviral vector pLKO.3G</td>
			<td>Addgene</td>
			<td>#14748</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin hemisulfate</td>
			<td>Santa Cruz Biotechnology</td>
			<td>#295358</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine LTX &#38; Plus Reagent (plasmid transfection reagent)</td>
			<td>Life Technologies</td>
			<td>#A12621</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION shRNA</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monosialoganglioside GM2</td>
			<td>Matreya</td>
			<td>#1502</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Life Technologies</td>
			<td>#21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane 0.45 &#181;m</td>
			<td>BIO-RAD</td>
			<td>#1620115</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey infrared imaging system</td>
			<td>LI-COR Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OneShot Top 10</td>
			<td>Life Technology</td>
			<td>#C404010</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (Reduced serum media)</td>
			<td>Life Technologies</td>
			<td>#105802</td>
			<td></td>
		</tr>
		<tr>
			<td>PacI</td>
			<td>BioLabs</td>
			<td>#R0547S</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Life Technologies</td>
			<td>#15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Santa Cruz Biotechnology</td>
			<td>#45036</td>
			<td></td>
		</tr>
		<tr>
			<td>phenylmethylsulfonyl fluoride</td>
			<td>Santa Cruz Biotechnology</td>
			<td>#329-98-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>sigma aldrich</td>
			<td>P#2636</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight sharp small spring scissors</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega</td>
			<td>#M1801</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %</td>
			<td>Life Technologies</td>
			<td>#25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibra-Cell Ultrasonic Liquid Processor (VCX 130)</td>
			<td>Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard plus SV Minipreps DNA purification system</td>
			<td>Promega</td>
			<td>#A1330</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular modulation by lentivirus-delivered specific shrnas in endoplasmic reticulum stressed neurons

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis.
There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation.
Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy.
We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress.
This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

Here, we address the question of whether silencing two PERK downstream components affects the transition phase of UPR in an ER stress cell model.To achieve this, we silence the CN-A&#945; gene as well as the CHOP gene by two specific shRNA sequences for each (Table 1) in primary neuron cell culture for 1 day10. The expression is analyzed by Western blotting (Figure 1 and Figure 2). A clear inhibition is o...

Watch this Scientific Journal Video about Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons at JoVE.com

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

In the present study, the expression is knocked down of two downstream signaling components of the PERK pathway, the cytoprotective calcineurin and the pro-apoptotic CHOP, by using specific shRNAs. In opposite ways, these modulate the susceptibility of primary cortical neurons to neurite atrophy after induction of endoplasmic reticulum stress.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) ...

molecular-modulation-lentivirus-delivered-specific-shrnas-endoplasmic

Research

JoVE Journal

Neuroscience

903 Views.  Universidad Nacional de Córdoba. In the present study, the expression is knocked down of two downstream signaling components of the PERK pathway, the cytoprotective calcineurin and the pro-apoptotic CHOP, by using specific shRNAs. In opposite ways, these modulate the susceptibility of primary cortical neurons to neurite atrophy after induction of endoplasmic reticulum stress. 

Video: Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

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.

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 ...