Quantification of mitochondrial morphology in neurites of dopaminergic neurons using multiple parameters.

New Method

This work describes a method combining immunofluorescence assays with computerized image analysis to measure the mitochondrial morphology within neuritic projections of a specific population of neurons. Six parameters of mitochondrial morphology were examined utilizing ImageJ to analyze colocalized signals.

Comparison with Existing Methods

Our method is a significant improvement of previously described methods. Six morphometric parameters are quantified, providing a gestalt analysis of mitochondrial morphology. Also it can target specific populations of mitochondria using immunofluorescence assay and image analysis.

Methods Fly stocks

All lines were obtained from the Bloomington Drosophila Stock Center (BDSC) unless otherwise noted. Flies were kept at 25°C and raised on standard cornmeal agar diet. A ‘Cantonized’ white eye stock w 118 served as wild-type. Morphometric control experiments used drp1 / CyO -GFP and opa1 / CyO -GFP (gifts from Dr. Leo Pallanck, University of Washington).

Drosophila primary neuronal cultures

Cultures were prepared as previously described in Park and Lee (2006). Briefly, mid-gastrula embryos at developmental stage 7 were harvested in a laminar-flow hood and plated onto round, glass coverslips (Bellco Glass, Inc., Vineland NJ, USA). Cultures were incubated in 4-5% CO 2 at 24-25° C for up to 9 days in vitro (DIV). Culture medium (DDM1) was a mixture of high glucose Hams's F-12/Delbecco's medium (Irvine Scientific, Santa Ana, CA), L-glutamine (2.5mM; Irvine Scientific), HEPES (20 mM), and four supplements: putrescine (100 μM), progesterone (20 ng/mL), transferrin (100 μg/mL), and insulin (50 μg/mL). At 3 DIV, all cultures had 50% of the culture medium replaced with new medium.

Pharmacological treatments

All drugs were added to cultures at 3 DIV after baseline images were acquired, except for experiments with delayed use of rescue therapy. Drug remained in the dish once treated (i.e. was never washed out). Cultures were handled and treated in a laminar flow hood (Forma Scientific, model 1849). Drugs dissolved in ddH2O were sterilized by filtration through a 0.2 μm cellulose acetate filter before use/storage. 1-methyl-4-phenylpyridinium (MPP + ) iodide (Sigma) was prepared as a 40mM stock solution dissolved in ddH2O and stored in darkness at -70° C. MPP + was handled according to guidelines reviewed in Przedborski et al. (2001) .

Immunofluorescence assay

Cultures were fixed with 4% paraformaldehyde for 40 minutes on ice, and then washed 3 times, 10 minutes for each wash. Wash solution was 10mM phosphate buffered saline containing 0.5% bovine serum albumin. All washes were at room temperature (≈25°C). Blocking and permeabilization was performed using 0.1% Triton X-100 and 5% normal goat serum (Sigma) for 30 minutes on ice. After 1 more wash for 10 minutes, permeabilized cultures were incubated overnight (≈16 hours) at 4° C with a 1:1,000 ratio of primary antibody (mouse anti-tyrosine hydroxylase, ImmunoStar) diluted in wash. The next day, primary antibody was removed and cultures were washed 3 times, 10 minutes for each wash. Cultures were next treated with a 1:2,000 ratio of secondary antibody (FITC or TRITC labeled goat anti-mouse, Invitrogen) diluted in wash and placed on ice for 1 hour. Secondary antibody was then removed and cultures were again washed 3 times, 10 minutes for each wash. Coverslips were next mounted onto glass slides upon rows of fluorogel with Tris buffer (Electron Microscopy Sciences), covered with an extra drop of fluorogel, then topped with coverglass (Electron Microscopy Sciences), and edges sealed with clear fingernail polish.

Microscopic detection of mitochondria in dopaminergic neurons

To visualize mitochondria from DA neurons, cultures were stained with both MitoTracker Orange (Invitrogen) and anti-TH antibody (ImmunoStar). At 7 DIV but before staining, cultures were treated with 50 nM MitoTracker Orange (Invitrogen) in the original culture medium for 1 hour at 25° C prior to immunostaining. Culture medium was then removed. Neuronal cultures were fixed and stained as usual (described above) with anti-TH primary antibody and FITC-labeled secondary antibody. The colocalization of signals between MitoTracker and anti-TH staining specifically identified mitochondria in DA neurons for analysis.

Image acquisition

Prepared/stained cultures were viewed under a fluorescence microscope (Olympus IX71 with 100W Mercury lamp). Mitochondria were observed with a LUCPLFLN 40× lens (NA, 0.60). Two fluorescence filter sets were also used as following: Chroma 41017 (FRITC) and 41002 (TRITC). Images were taken using Spot CCD digital camera (2 megapixels, Diagnostic Instruments, Sterline Heights, MI). For a 12-bit image, the intensity ranges from 0 (no signal) to 4095 (maximum saturation). To determine the best threshold for our analysis of mitochondria, the raw MitoTracker signal of several images of mitochondria was compared to its “ideal” threshold level. The ideal threshold level was considered the one where a manual count of the mitochondria from the raw image gave the same results as the Analyze Particles function in ImageJ. The mean ideal threshold level was then set as the threshold to be applied for all future analysis of mitochondria.

Analysis of mitochondrial morphology in dopamine neurons

After images were acquired, they were analyzed using a macro developed for ImageJ ( Rasband 1997-2006 ) software (See Appendix A for code, installation, and use of macro). ImageJ allows for users to write programs (typically macros) in a “Java-like” language. This macro combines a plugin created by Dagda et al. (2009) with the colocalization highlighter plugin from the WCIF version of ImageJ ( http://www.uhnresearch.ca/facilities/wcif/imagej/ ). Analysis of mitochondrial morphology is performed on a merged image between images of the MitoTracker and anti-TH signals. Before the merge, however, both channels are subjected to a threshold to make them binary images. Then an overlap of those two binary images is made. This overlapped image is then used for analysis. The signals are painted by the threshold function in ImageJ based on signal intensity. For mitochondria, this threshold was determined to be 20% of the maximum intensity, which is about 4× the typical background intensity. For the anti-TH signal, we chose a threshold (also around 20% of the maximum intensity) which completely saturated the cell with signal so that all mitochondria within would colocalize. Colocalized signals were measured using a macro adapted from the plugin created by Dagda et al. (2009) . Major differences in the macro from Dagda's original, were changing the threshold to 20% of the maximum intensity and the measured particle range to 5-500 pixels. This macro quantifies mitochondrial morphometrics using four parameters: 1) number of mitochondria, 2) size, 3) interconnectivity, and 4) elongation. These four parameters taken together provide a gestalt “snapshot” of the mitochondrial phenotype. The macro only considers signals within a size range. With our microscope settings and image size, the mean size for mitochondria is around 40 pixels 2 (1.4 μm 2 ), matching what is commonly reported across species (0.75 to 3 μm 2 ; Bereiter-Hahn, 1990 ; Rafelski and Wallace, 2008 ). Thus, the lower and upper limits of the size range had to be decreased from Dagda's original macro. If the lower limit was too low, it would include tiny artifacts as signals, which do not provide meaningful parameter values. The upper limit is less of a concern. In healthy neurons, many of the mitochondria are interconnected, combining to a pixel area much greater than the average mitochondria size, which is the phenomenon that is measured by the interconnectivity score. However, because the soma is typically completely saturated in our images by both the anti-tyrosine hydroxylase antibody and MitoTracker signals, it is not included in analysis. This is easily accomplished by simply drawing around the soma with the selection tool in ImageJ ( Figure 1 ). An upper limit of 500 pixels 2 was used to exclude what are probable artifacts (e.g. the occasional piece of broken glass added during culturing). Thus, a particle size range of 5-500 pixels 2 was chosen for non-somatic mitochondria. Determining the four parameters of mitochondrial morphology requires computation. Counting the number of mitochondria and determining the mean size is a straightforward task; the ImageJ software simply counts each signal and simultaneously determines the pixel area, perimeter, and circularity for each signal. From these measurements, the ImageJ macro calculates a score for interconnectivity and elongation as the following: (1) interconnectivity = mean area mean perimeter (2) elongation = 1 circularity where circularity = 4 ∗ π ∗ ( mean area mean perimeter 2 ) Interconnectivity describes the network of the mitochondria, and is calculated by dividing the mean area by the mean perimeter of all the particles analyzed. Higher scores for interconnectivity signify that mitochondria have more physical connections, while lower scores signify that the mitochondria are more fragmented. Elongation is best thought of as the shape of mitochondria. Higher values are more abstract shapes, while a value of 1 would be considered a perfect circle.

Statistics

All statistics are reported as mean ± 1 SEM. Analysis is performed using either ANOVA with pairwise comparisons using Tukey's Honest Significant Difference correction, or Student's t-test. Significance scores are: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. All distributions are tested for normality and homogeneity of variance before testing. Principal component analysis was performed with R statistical software using the FactoMineR package ( Lê et al., 2008 ). Results were considered significant if the percentage of inertia summing from the first two eigenvalues exceeded values listed in a significance table based on 10,000 analyses with similar numbers of individuals and independent variables ( Lê et al., 2008 ).

ImageJ code for analysis of mitochondrial morphology (immunocytochemistry method) // Displays the colocalization of two images and then measures the morphometrics of the //overlapped signal // Installation notes: // Download and Install WCIF ImageJ package from // http://www.uhnresearch.ca/facilities/wcif/fdownload.html // Save this code as .txt file and move file to >Program Files>ImageJ>macros // Make sure it's the correct ImageJ Folder (the WCIF version) if you have multiple versions // open WCIF ImageJ and install the macro by selecting from the ImageJ toolbar: // Plugins>Macros>Install… and then select the .txt file saved earlier. // Push F8, a window will appear to select a file. // First select the red image // another window will appear, now select the green image // ImageJ will then merge the two images and present a threshold image of the colocalized // points. There will also be the original images as well as merge between the two with // colocalized points as white. The image to be analyzed will be autoselected. // Draw around the area of the image to be analyzed. Only points within this area // will be measured // Push F10. ImageJ will perform the measurements and report the results in a new window. // After copying the results, push F11 to close all windows. // Push F9 and repeat // Code below macro “Close All Windows [F11] ” //F11 used as a shortcut to close all active windows { while (nImages>0) { selectImage(nImages); close(); } } macro “Open Images for colocalization [F8] ” //F8 used as shortcut { open(); //select red image run(“8-bit”); //makes image 8-bit open(); //select green image run(“8-bit”); //makes images 8-bit run(“Colocalization Highligter”, “ratio=50 threshold_channel_1=52 threshold_channel_2=52 display=255 colocalized”); //Colocalization Highlighter is a tool from the WCIF version of //ImageJ, adjust threshold here if necessary //user may wish to use this image for making figures setTool(3); //activates freehand selection tool for drawing around region of interest selectWindow(“Colocalized points (8-bit) ”); //selects image containing only colocalized //signals setThreshold(15, 255); //threshold setting not important here } macro “Measure Morphometrics [F10] ” //F10 used as shortcut { run(“Clear Results”); run(“Set Measurements… ”, “area perimeter circularity redirect=None decimal=2”); run(“Analyze Particles… ”, “minimum=5 maximum=500 bins=100 show=Outlines display summarize”); //set size range of particles here for (i=0; i 1, so absolute value needed here }//loop to assign variables from results { AMP= (MP/i); //calculates mean perimeter AMA= (MA/i); //calculates mean area AMC= (MC/i); //calculates mean circularity Rmorph= (AMA/AMP); //measures the area perimeter ratio, a.k.a. interconnectivity print(getTitle()); print(“Count:” +i); print(“Total Area:” +MA); print(“Avg. Perimeter:” +AMP); print (“Avg. Area:” + AMA); print (“Avg. Circularity:”+ AMC); print (“Area/Perim:”+ Rmorph); selectWindow(“Results”); selectWindow(“Log”); } }