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.
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.
