In this video, we show how to use a seven point scoring system to quantify changes to dopaminergic neuron dendrite morphology in C.elegans. There's currently a large variation in analytical methods of quantifying neurodegeneration in worms. Additionally, some existing skills focus only on cell bodies and are not sensitive to certain types and levels of damage.
The purpose of introducing this scoring system is to provide the ability to capture a comprehensive picture of neurodegeneration at all levels of damage, and to provide a universal system to support consistency across C.elegans dopaminergic neurodegeneration research. Here, we will demonstrate how to image neurons under fluorescence, assign scores to those neuron's dendrites, and preview how to display and interpret the results. For each experimental group, pipette or pick 20 to 30 worms to an imaging platform compatible with the imaging microscope.
Most common platforms include 2%agarose pads mounted on glass slides with a cover slip, and 96-well plates containing well volumes at or less than 100 microliters of liquid medium. Allow worms to paralyze completely. This may take several minutes.
Load the prepared imaging platform into an imaging microscope capable of z-stack captures. Locate the worm's head region first in bright field, then under a single color GFP fluorescence using the imaging microscope. The worm is shown here in a 400x magnification.
Scroll through the focus to find upper and lower bounds where the dendrites are clear. Set these as upper and lower bounds for z-stack image capture. Select desired pitch.
Here we use one micrometer. Click to capture z-stack images in the GFP fluorescence channel. For each z-stack, open the image file using either the microscope software or an external image analysis software.
Load the stack into the software and compress the stack into a single flattened image. Align images between and within treatment groups manually or using automatic winding software. Work with one neuron image at a time.
Choose one of the four CEP dendrites to assess for blebs, breaks, and irregularities, including bends, kinks, and curves. Scoring from left to right when the nose is at the top of the image is recommended to ensure repeatability in scoring. Using these guidelines, assign one score value to each dendrite.
Assign a score of zero for dendrites with no visible damage, a score of one for dendrites with irregularities but no blobbing or breakage, a score of two for dendrites with less than five blebs, a score of three for between five and 10 blebs, a score of four for over 10 blebs and/or breakage removing up to 25%of the dendrite, a score of five for breakage removing 25 to 75%of the dendrite, and a score of six if more than 75%of the dendrite is absent. If multiple criteria are met within a single dendrite, consider the criteria corresponding to the highest score. These representative scoring images found in Figure 1 of the article associated with this video may be used as a reference.
To demonstrate scoring, we will walk through assigning scores to a few example neuron images. Start with the top most dendrite, there are no visible blebs, breaks, or irregularities. Assign a score of zero.
The second dendrite has around 14 blebs and no visible breaks. Assign a score of four. The third dendrite cannot be seen in its entirety due to overlap with dendrite two and cannot be scored.
The bottom most dendrite has about 11 blebs and a few breaks. Assign a score of five. In this image, only three dendrites are scorable, since one dendrite was not fully captured in the z-stack capture.
There is no blebbing on the three visible dendrites. So this would be scored from top to bottom as a zero, a zero, and due to the kink here, a one. Finally, in this image, all four dendrites are more than 75%lost.
All dendrites here receive a score of six. Record all scores. Scores may be unblinded at this time.
The score template used here can be found in the Supplementary Files. Divide neurodegeneration score tallies by the total number of dendrites scored in the treatment group. Present data as proportion of dendrites within the treatment group at each neurodegeneration score.
Our scoring system was used to assess neurodegeneration in L4 larval stage BY200 C.elegans after rotenone exposure. The data is displayed here as with the proportions of the total number of dendrites at each score value for each experimental group. We consider each dendrite scored as n 1.
In this figure, a dose-dependent neurodegeneration response to rotenone exposure can be appreciated, and the specific breakdown of the score distribution is displayed clearly. These experimental groups were statistically different according to a Chi-squared test for independence supplemented by a Bonferroni correction for multiple comparisons. In this video, we have demonstrated how to use the seven point scale developed in our laboratory to quantify levels of dopaminergic neuron morphologic alteration and degeneration in C.elegans.
Using the scoring method promotes consistency in analysis and allows for comparison between neurodegeneration research studies in worms. Our scoring system may be used to analyze data derived from C.elegans experiments that use cell-specific fluorescent reporters, such as dat-1 dopamine transporter gene that allow for visualization of dopaminergic neurons. Now, please find a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method in this article's supplementary material.
