Name: live imaging of the mitochondrial glutathione redox state in primary neurons using a ratiometric indicator
Uploaded: 2021-10-20T14:30:00
Description: Watch this Scientific Journal Video about Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator at JoVE.com

This work was supported by the Deutsche Forschungsgemeinschaft (BA 3679/5-1; FOR 2289: BA 3679/4-2). A.K. is supported by an ERASMUS+ fellowship. We thank Iris B&#252;nzli-Ehret, Rita Rosner, and Andrea Schlicksupp for the preparation of primary neurons. We thank Dr. Tobias Dick for providing pLPCX-mito-Grx1-roGFP2. Experiments shown in Figure 4 were performed at the Nikon Imaging Center, University of Heidelberg. Figure 2 was prepared with BioRender.com.

All animal experiments conformed to national and institutional guidelines, including the Council Directive 2010/63/EU of the European Parliament, and had full Home Office ethical approval (University of Heidelberg Animal Welfare Office and Regierungspraesidium Karlsruhe, licenses T14/21 and T13/21). Primary hippocampal and cortical neurons were prepared from newborn mouse or rat pups according to standard procedures and were maintained for 12-14 days as previously described13.
<p class="jove_t...

<ol>
	<li>Roede, J. R., Go, Y. M., Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+equivalents+and+mitochondrial+bioenergetics.">Redox equivalents and mitochondrial bioenergetics.</a> Methods in Molecular Biology. 810, 249-280 (2012).</li><li>Turrens, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+formation+of+reactive+oxygen+species.">Mitochondrial formation of reactive oxygen species.</a> Journal of Physiology. 552, 335-344 (2003).</li><li>Lin, M. T., Beal, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+oxidative+stress+in+neurodegenerative+diseases.">Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.</a> Nature. 443 (7113), 787-795 (2006).</li><li>Manfredi, G., Beal, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+the+pathogenesis+of+neurodegenerative+diseases.">The role of mitochondria in the pathogenesis of neurodegenerative diseases.</a> Brain Pathology. 10 (3), 462-472 (2000).</li><li>Mari, M., Morales, A., Colell, A., Garcia-Ruiz, C., Fernandez-Checa, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+glutathione,+a+key+survival+antioxidant.">Mitochondrial glutathione, a key survival antioxidant.</a> Antioxidants &amp; Redox Signaling. 11 (11), 2685-2700 (2009).</li><li>Murphy, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+thiols+in+antioxidant+protection+and+redox+signaling:+distinct+roles+for+glutathionylation+and+other+thiol+modifications.">Mitochondrial thiols in antioxidant protection and redox signaling: distinct roles for glutathionylation and other thiol modifications.</a> Antioxidants &amp; Redox Signaling. 16 (6), 476-495 (2012).</li><li>Dooley, C. T., 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=Imaging+dynamic+redox+changes+in+mammalian+cells+with+green+fluorescent+protein+indicators.">Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.</a> Journal of Biological Chemistry. 279 (21), 22284-22293 (2004).</li><li>Hanson, G. T., 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=Investigating+mitochondrial+redox+potential+with+redox-sensitive+green+fluorescent+protein+indicators.">Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators.</a> Journal of Biological Chemistry. 279 (13), 13044-13053 (2004).</li><li>Gutscher, 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=Real-time+imaging+of+the+intracellular+glutathione+redox+potential.">Real-time imaging of the intracellular glutathione redox potential.</a> Nature Methods. 5 (6), 553-559 (2008).</li><li>Morgan, B., Sobotta, M. C., Dick, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+E(GSH)+and+H2O2+with+roGFP2-based+redox+probes.">Measuring E(GSH) and H2O2 with roGFP2-based redox probes.</a> Free Radical Biology &amp; Medicine. 51 (11), 1943-1951 (2011).</li><li>Marwick, K. F. M., Hardingham, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+in+primary+cultured+neuronal+cells.">Transfection in primary cultured neuronal cells.</a> Methods in Molecular Biology. 1677, 137-144 (2017).</li><li>Kohrmann, 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=convenient,+and+effective+method+to+transiently+transfect+primary+hippocampal+neurons.">convenient, and effective method to transiently transfect primary hippocampal neurons.</a> Journal of Neuroscience Research. 58 (6), 831-835 (1999).</li><li>Depp, C., Bas-Orth, C., Schroeder, L., Hellwig, A., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+protects+neurons+against+calcium-mediated+oxidation+and+contraction+of+mitochondria+during+excitotoxicity.">Synaptic activity protects neurons against calcium-mediated oxidation and contraction of mitochondria during excitotoxicity.</a> Antioxidants &amp; Redox Signaling. 29 (12), 1109-1124 (2018).</li><li>Hauck, B., Chen, L., Xiao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+chimeric+recombinant+AAV+vectors.">Generation and characterization of chimeric recombinant AAV vectors.</a> Molecular Therapy. 7 (3), 419-425 (2003).</li><li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>Zhang, S. J., 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=Nuclear+calcium+signaling+controls+expression+of+a+large+gene+pool:+identification+of+a+gene+program+for+acquired+neuroprotection+induced+by+synaptic+activity.">Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity.</a> PLoS Genetics. 5 (8), 1000604 (2009).</li><li>Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+of+using+fluorescent+probes+to+detect+and+quantify+specific+reactive+oxygen+species+in+living+cells.">The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells.</a> Biochimica et Biophysica Acta. 1840 (2), 730-738 (2014).</li><li>Sies, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress:+a+concept+in+redox+biology+and+medicine.">Oxidative stress: a concept in redox biology and medicine.</a> Redox Biology. 4, 180-183 (2015).</li><li>Lukyanov, K. A., Belousov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+fluorescent+redox+sensors.">Genetically encoded fluorescent redox sensors.</a> Biochimica et Biophysica Acta. 1840 (2), 745-756 (2014).</li><li>Nietzel, T., 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=Redox-mediated+kick-start+of+mitochondrial+energy+metabolism+drives+resource-efficient+seed+germination.">Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (1), 741-751 (2020).</li><li>Albrecht, S. C., 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=Redesign+of+genetically+encoded+biosensors+for+monitoring+mitochondrial+redox+status+in+a+broad+range+of+model+eukaryotes.">Redesign of genetically encoded biosensors for monitoring mitochondrial redox status in a broad range of model eukaryotes.</a> Journal of Biomolecular Screening. 19 (3), 379-386 (2014).</li><li>Albrecht, S. C., Barata, A. G., Grosshans, J., Teleman, A. A., Dick, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+mapping+of+hydrogen+peroxide+and+oxidized+glutathione+reveals+chemical+and+regional+specificity+of+redox+homeostasis.">In vivo mapping of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis.</a> Cell Metabolism. 14 (6), 819-829 (2011).</li><li>Breckwoldt, M. O., 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=Multiparametric+optical+analysis+of+mitochondrial+redox+signals+during+neuronal+physiology+and+pathology+in+vivo.">Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo.</a> Nature Medicine. 20 (5), 555-560 (2014).</li><li>Ricke, K. 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=Mitochondrial+dysfunction+combined+with+high+calcium+load+leads+to+impaired+antioxidant+defense+underlying+the+selective+loss+of+nigral+dopaminergic+neurons.">Mitochondrial dysfunction combined with high calcium load leads to impaired antioxidant defense underlying the selective loss of nigral dopaminergic neurons.</a> Journal of Neuroscience. 40 (9), 1975-1986 (2020).</li><li>Bjornberg, O., Ostergaard, H., Winther, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insight+provided+by+glutaredoxin+within+a+fusion+to+redox-sensitive+yellow+fluorescent+protein.">Mechanistic insight provided by glutaredoxin within a fusion to redox-sensitive yellow fluorescent protein.</a> Biochemistry. 45 (7), 2362-2371 (2006).</li><li>Shokhina, A. G., 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=Red+fluorescent+redox-sensitive+biosensor+Grx1-roCherry.">Red fluorescent redox-sensitive biosensor Grx1-roCherry.</a> Redox Biology. 21, 101071 (2019).</li></ol>

Quantitative and dynamic measurements of the mitochondrial redox state provide important information about mitochondrial and cellular physiology. Several fluorogenic chemical probes are available that detect reactive oxygen species, &#34;redox stress,&#34; or &#34;oxidative stress.&#34; However, the latter terms are not well-defined and often lack specificity9,17,18. Compared to chemical dyes, Grx1-roGFP2 offers several advantag...

Several important mitochondrial enzymes and signaling molecules are subject to thiol redox regulation1. Moreover, mitochondria are a major cellular source of reactive oxygen species and are selectively vulnerable to oxidative damage2. Accordingly, the mitochondrial redox potential directly affects bioenergetics, cell signaling, mitochondrial function, and ultimately cell viability3,4. The mitochondrial matrix contains high amounts (1-15 mM) of the thiol-disulfide redox buffer glutathione (GSH) to maintain redox homeostasis and mount antioxidant defenses5,6. GSH can be covalently attached to target proteins (S-glutathionylation) to control their redox status and activity and is used by a range of detoxifying enzymes that reduce oxidized proteins. Therefore, the mitochondrial glutathione redox potential is a highly informative parameter when studying mitochondrial function and pathophysiology.
roGFP2 is a variant of GFP that has been made redox-sensitive by the addition of two surface-exposed cysteines that form an artificial dithiol-disulfide pair7,8. It has a single emission peak at ~510 nm and two excitation peaks at ~400 nm and 490 nm. Importantly, the relative amplitudes of the two excitation peaks depend on the redox state of roGFP2 (Figure 1), making this protein a ratiometric sensor. In the Grx1-roGFP2 sensor, human glutaredoxin-1 (Grx1) has been fused to the N-terminus of roGFP29,10. Covalent attachment of the Grx1 enzyme to roGFP2 affords two major improvements of the sensor: it makes the sensor response specific for the GSH/GSSG glutathione redox pair (Figure 1), and it speeds up equilibration between GSSG and roGFP2 by a factor of at least 100,0009. Therefore, Grx1-roGFP2 enables specific and dynamic imaging of the cellular glutathione redox potential.
Grx1-roGFP2 imaging can be performed on a wide range of microscopes, including widefield fluorescence microscopes, spinning disc confocal microscopes, and laser scanning confocal microscopes. Expression of the sensor in primary neurons can be achieved by various methods that include lipofection11, DNA/calcium-phosphate coprecipitation12, virus-mediated gene transfer, or use of transgenic animals as the cell source (Figure 2). Pseudotyped recombinant adeno-associated viruses (rAAV) containing a 1:1 ratio of AAV1 and AAV2 capsid proteins 13,14 were used for the experiments in this article. With this vector, maximal sensor expression is typically reached 4-5 days after infection and stays stable for at least two weeks. We have successfully used Grx1-roGFP2 in primary hippocampal and cortical neurons from mice and rats.
In this article, rAAV-mediated expression of mitochondria-targeted Grx1-roGFP2 in primary rat hippocampal and cortical neurons is used to assess basal mitochondrial glutathione redox state and its acute perturbation. A protocol is provided for confocal live imaging with detailed instructions on how to (i) optimize laser scanning confocal microscope settings, (ii) run a live imaging experiment, and (iii) analyze data with FIJI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamide (DA)</td>
			<td>Sigma-Aldrich</td>
			<td>D3648</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Carl Roth GmbH</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose (2.5 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>neoFroxx GmbH</td>
			<td>LC-4522.2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2&#183;6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>442611-M</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methyl-D-aspartate (NMDA)</td>
			<td>Sigma-Aldrich</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>neoFroxx GmbH</td>
			<td>LC-5932.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate (0.1 M stock solution)</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P8574</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Carl Roth GmbH</td>
			<td>4621.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine ethyl ester perchlorate (TMRE)</td>
			<td>Sigma-Aldrich</td>
			<td>87917</td>
			<td></td>
		</tr>
		<tr>
			<td>equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>imaging chamber</td>
			<td>Life Imaging Services (Basel, Switzerland)</td>
			<td>10920</td>
			<td>Ludin Chamber Type 3 for &#216;12mm coverslips</td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope, microscope</td>
			<td>Leica</td>
			<td>DMI6000</td>
			<td></td>
		</tr>
		<tr>
			<td>laser scanning confocal microscope, scanning unit</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic pump</td>
			<td>VWR</td>
			<td>PP1080 181-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, camera</td>
			<td>Hamamatsu</td>
			<td>C9100-02 EMCCD</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, incubationsystem</td>
			<td>TokaiHit</td>
			<td>INU-ZILCF-F1</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, microscope</td>
			<td>Nikon</td>
			<td>Ti microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>spinning disc confocal microscope, scanning unit</td>
			<td>Yokagawa</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td></td>
			<td>https://fiji.sc</td>
			<td></td>
		</tr>
		<tr>
			<td>StackReg plugin</td>
			<td></td>
			<td>https://github.com/fiji-BIG/StackReg/blob/master/src/main/java/StackReg_.java</td>
			<td></td>
		</tr>
		<tr>
			<td>TurboReg plugin</td>
			<td></td>
			<td>https://github.com/fiji-BIG/TurboReg/blob/master/src/main/java/TurboReg_.java</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of the mitochondrial glutathione redox state in primary neurons using a ratiometric indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

Quantification of differences in steady-state mitochondrial redox state after growth factor withdrawal 
To demonstrate the quantification of steady-state differences in mitochondrial redox state, primary neurons grown in standard medium were compared to neurons cultured without growth factors for 48 h before imaging. Growth factor withdrawal results in apoptotic neuronal cell death after 72 h16. Cells were imaged after 48 h to test if this is preceded by changes in mitochondr...

Watch this Scientific Journal Video about Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator at JoVE.com

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

This method allows to investigate how mitochondrial redox state is affected in pathophysiological conditions. It can also be used to assess the efficacy of treatment strategies that aim at protecting mitochondria. The main advantage of this technique is that it allows an organ and specific assessment of dynamic and tracing changes on mitochondria redox state in real time.This method can be combined with additional indicators to simultaneously record, for example, mitochondrial membrane potential or calcium concentrations in addition to redox state. This protocol can be applied to culture cells, tissue explants and slice cultures. Begin by optimizing the scanning confocal microscope settings.To do so, set the detector to 12 bits or 16 bits, activate the sequential scan mode and add a second sequence/track. For both channels, select a pseudo color lookup table that indicates over and underexposed pixels and then select an objective suitable for the object of interest. Mount a coverslip with cells into the imaging chamber.Add one milliliter of imaging buffer and place the chamber on the microscope. Use the eyepiece and transmitted light to focus the cells. Record images with different pixel formats and pinhole sizes.Then record images with different laser intensities and accordingly adjust the detector gain and threshold. Finally, record images with different scan speeds and different numbers of frame averages. For live imaging, set the timelapse interval to 30 seconds and duration to 25 minutes.Then mount the cells, place the chamber on the microscope and focus the cells as demonstrated previously. Switch to scanning mode and use the 488 nanometer channel in the live view to focus and locate the cells for imaging. Optionally, to increase the number of recorded cells per run, use the multi-point function to image two to three fields of view per coverslip.Start the timelapse acquisition and record five images as a two-minute baseline recording. Add 500 microliters of 3X NMDA solution to the chamber to achieve the final concentration of 30 micromolar and record additional 20 images as a 10-minute NMDA response. Next, add 500 microliters of 4X DA solution to the chamber and record six more images.Aspirate the buffer from the imaging chamber and replace it with one milliliter of DTT solution. After recording 10 more images, end the recording and save the image series. For importing the data in the image analysis software, click on plugins, select bio formats, and then bio formats importer.In the dialog box, choose hyperstack in the view stack with, set color mode as default and select auto-scale. Change the image format to 32 bits by clicking on image, selecting type, and then from the options, choose 32 bit. To split the color channels into separate windows, click on image, go to color, and select split channels.Adjust the threshold to select the mitochondria for analysis by clicking on image, selecting adjust and threshold. In the dialog box, select default red dark background and stack histogram. When the selected pixels appear red, click on apply.Then select set background pixels to NAN process all images and perform the same procedure for channel two. For visualizing the 405 to 488 nanometer ratio, create a ratio image by clicking on process and image calculator. In the dialog box, select channel one in image one divide in operation channel two in image two.Then select create new window and process all images. Change the lookup table of the ratio image to pseudo color by clicking on image, selecting lookup tables, and then fire. For analyzing the image, draw ROIs around individual cells or mitochondria on the ratio image.To add the ROIs to ROI manager, go to analyze, click on the tools, select ROI manager, click on add and select show all. To measure the 405 to 488 nanometer ratios of individual cells, click on ROI manager, select all ROIs by pressing Control A, go to more and select multi-measure. In the dialog box, select measure all slices and one row per slice.After exporting the measurements to the spreadsheet software, select the 405 nanometer image, measure the intensities of all ROIs, and again export the measurements to the spreadsheet software. Similarly, measure the ROI intensities of the 488 nanometer image. For saving the ROIs for future reference, select all the ROIs by pressing Control A, go to more and select save.These representative snapshots from a timelapse recording show ratio images of neurons before and after NMDA treatment and after max/min calibration with DA and DTT. Treatment of the neurons with 30 micromolar NMDA induced mitochondria oxidation within a few minutes. Analysis of individual fluorescence channels revealed that NMDA-induced mitochondrial acidosis caused a drop of GFP fluorescence upon both 405 and 488 nanometers excitation.In a control experiment, pretreatment with DA precluded any further mitochondria oxidation by NMDA. And accordingly, the 405 to 488 ratio did not change despite a considerable quenching of redox-sensitive GFP2 fluorescence intensity. This experiment confirmed that the 405 to 488 nanometer ratio is not affected by pH changes.In a separate experiment, mitochondrial membrane potential, redox state, and morphology were assessed in parallel. Treatment of the neurons with 60 micromolar NMDA resulted in the loss of tetramethylrhodamine or TMRE signal and an increase in the 405 to 488 nanometer rho GFP ratio followed by some delayed rounding up of mitochondria. When you begin to use this method, it is very important to take time to carefully optimize microscopic settings.This will help keep the neurons healthy during your experiments. It is also very important to always respect laser safety rules. Make sure not to be exposed to laser radiation when you're adding drugs to tamper during live recordings.

live-imaging-mitochondrial-glutathione-redox-state-primary-neurons

Research

JoVE Journal

Neuroscience

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Measuring Connectivity in the Primary Visual Pathway in Human Albinism Using Diffusion Tensor Imaging and Tractography

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed as candidate sites for misrouting. Since a correlation between the number of lateral geniculate nucleus (LGN) relay neurons and LGN size has been shown, and based on previously reported reductions in LGN volumes in human albinism, we suggest that fiber projections from LGN to the primary visual cortex (V1) are also reduced. Studying structural differences in the visual system of albinism can improve the understanding of the mechanism of misrouting and subsequent clinical applications. Diffusion data and tractography are useful for mapping the OR (optic radiation). This manuscript describes two algorithms for OR reconstruction in order to compare brain connectivity in albinism and controls.An MRI scanner with a 32-channel head coil was used to acquire structural scans. A T1-weighted 3D-MPRAGE sequence with 1 mm3 isotropic voxel size was used to generate high-resolution images for V1 segmentation. Multiple proton density (PD) weighted images were acquired coronally for right and left LGN localization. Diffusion tensor imaging (DTI) scans were acquired with 64 diffusion directions. Both deterministic and probabilistic tracking methods were run and compared, with LGN as the seed mask and V1 as the target mask. Though DTI provides relatively poor spatial resolution, and accurate delineation of OR may be challenging due to its low fiber density, tractography has been shown to be advantageous both in research and clinically. Tract based spatial statistics (TBSS) revealed areas of significantly reduced white matter integrity within the OR in patients with albinism compared to controls. Pairwise comparisons revealed a significant reduction in LGN to V1 connectivity in albinism compared to controls. Comparing both tracking algorithms revealed common findings, strengthening the reliability of the technique.

This manuscript describes deterministic and probabilistic algorithms for white matter (WM) reconstruction, used to examine differences in optic radiation (OR) connectivity between albinism and controls. Although probabilistic tractography follows the true course of nerve fibers more closely, deterministic tractography was run to compare the reliability and reproducibility of both techniques.

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer&#39;s disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...