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...

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The authors declare that they have no conflict of interest.

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

2.6K ビュー.  Heidelberg University. この方法は、ミトコンドリア酸化還元状態が病態生理学的状態でどのように影響を受けるかを調べることができます。また、ミトコンドリアの保護を目的とした治療戦略の有効性を評価するために使用することができます。この技術の主な利点は、リアルタイムでミトコンドリア酸化還元状態の動的およびトレースの変化の器官および特異的な評価を可能にすることで?...