Name: evaluation of motor impairment in c. elegans models of amyotrophic lateral sclerosis
Uploaded: 2021-09-02T13:30:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis at JoVE.com

We thank the reviewers for helpful comments and suggestions. We thank Aleen Saxton, Brandon Henderson, and Jade Stair for outstanding technical assistance. We thank Brian Kraemer and Rebecca Kow for assistance developing these assays. This material is the result of work supported with resources and the use of facilities at the VA Puget Sound Health Care System. This work was supported by a grant from the United States (U.S.) Department of Veterans Affairs (VA) Biomedical Laboratory Research and Development Service [Merit Review Grant #I01BX004044 to N.F.L.]

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The authors declare that they have no disclosures.

Radial locomotion: 
The resolution of this assay is easily controlled by changing the time variable. Increasing the length of time makes it easier to observe differences between animals with severe phenotypes, thereby identifying subtle differences. However, because this assay measures displacement, if the assay time is extended too long, animals with normal motility, such as N2, will travel to the edges of the plate, and foraging behavior will lead to backtracking. This will artificially decrease the measurement of distance traveled. Time periods that are too long may result in the disappearance of differences between strains, particularly between animals with less severe motor phenotypes, as animals become evenly dispersed across the plate. Shortening the time variable will prevent more active worms from finding the edges of the plate. This method does not track the total distance traveled for each worm but compresses the distance traveled for each worm into a linear distance from the center of the plate. As such, it is inherently less robust than a method that records the total track length of individual worms. However, the radial locomotion assay requires very little researcher training, uses relatively affordable reagents that are commonly available in most worm labs, and is sensitive enough to produce significant and reproducible results. For labs that prefer automated video tracking, several methods have been previously established to track and analyze crawling movements12, or the software parameters used for the swimming assay in this paper could be altered to permit crawling detection and analysis.
This experiment is usually done in triplicate independent replicates, with a set of 30-40 worms per replicate. Each replicate is split onto two different 100 mm or 150 mm plates, with 15-20 worms per plate. Using more worms than recommended per plate can make it hard to score efficiently. A total number scored of 90+ is sufficiently powered to establish significance for mild, moderate, or severe motility impairment. Being consistent with timing between strains scored is essential for accuracy and reproducibility. 30 minutes is generally long enough to establish differences between moderate to severe phenotypes such as transgenic strains expressing human mutant TDP-43 compared to wild-type worms (Figure 4). If the time variable will be extended, it is advisable to also increase the size of the plate from 100 mm to 150 mm. Environmental factors such as temperature and humidity can affect this assay, which is typically conducted at ambient room temperature, so it is important to always use a wild-type (N2) control when comparing across replicates. In addition, this assay can measure the motility of some strains that do not exhibit normal swimming behavior in liquid (thrashing), making it a useful complement to the swimming assay.
Swimming assay: 
The use of the imaging and acquisition system to automate tracking and analysis of worm swimming allows for rigorous and unbiased data. However, there are several factors during initial setup of the experiment that needs to be controlled between samples. These include the time to acclimate to liquid before beginning recording, ambient conditions (i.e., temperature, humidity), and consistent light and recording settings. On the recording stage, there are several features that help reduce variability between plates. These include an integrated track-mounted camera and bright-field stage that makes recording video consistent between plates, shielding around the stage that prevents reflections, glare, and air movements while recording, and a robust software package that reliably detects worms and allows for manual correction of tracks in video post-processing. In this protocol, videos of a 35 mm plate with worms are recorded for 1 minute and then processed using the software package. After processing, manual correction of tracks ensures that worm behaviors are recorded accurately without confounding tracking errors. Turn count and track duration data are used to determine turns per minute as a final readout. To ensure reproducibility, data is collected over a minimum of 3 independent replicate experiments, each with 40-50 animals scored, to achieve a combined final number of animals 120-150. This number is sufficient to discriminate small differences in swimming behavior from control worms. Some worms have motor deficits too severe to be captured by swimming assays. For example, if the animals placed in a liquid medium curl instead of performing the expected thrashing response, this assay will not record those movements accurately and another movement assay, such as radial locomotion, may better capture those motility defects. The provided protocol uses a commercially available imaging system (see Table of Materials for more details), but other worm tracking systems may provide a similar utility, with some being open source12. Previously published methods describe manual scoring of worm thrashing13. While the automated analysis produces a number of metrics for each individual worm, detection of body bends which is measured in thrashes per minute, provides consistent results between experiments and tracks well with conventional scoring of worm thrashes by eye.

Amyotrophic Lateral Sclerosis (ALS) is a debilitating, aging-related neurodegenerative disease with a particular impact on motor neurons. The disease features loss of motor neurons in the brain and spinal cord and progressive motor impairment. This results in major functional disability and premature death, typically within 3-5 years after diagnosis1. Mutations in at least 38 genes can cause ALS; however, most patients with ALS accumulate ubiquitinated inclusions of the protein TDP-43 as their primary pathology in neurons and glial cells2,3,4. A number of animal models have been developed to study the underlying mechanisms causing or contributing to ALS in vivo (reviewed in5). In C. elegans, these models include genetic loss-of-function mutations in homologs of ALS-causing genes or transgenic expression of human ALS genes. There are numerous advantages to modeling ALS in C. elegans. C. elegans are a tractable simple animal with a differentiated nervous system, well-characterized behavioral paradigms, and considerable genetic homology to humans6,7. Many tools exist for working with C. elegans, including robust genome editing capabilities, in vivo fluorescent reporters of neurodegeneration, RNAi screening paradigms, tractable genetics, and established behavioral and phenotypic assays. C. elegans models of ALS recapitulate aspects of human disease, including accumulation of insoluble protein, neurodegeneration, and early death8,9. Further, motor dysfunction featuring disturbance of both crawling and swimming behaviors is present in many C. elegans ALS models.
This protocol describes two methods to characterize C. elegans motor phenotypes: the radial locomotion assay for evaluating crawling on a solid surface and the assessment of swimming in liquid (thrashing) using the WormLab automated tracking and analysis. These sensitive methods for characterizing motor deficits allow comparisons of severity and offer tools for measuring suppression and enhancements of motor phenotypes. The radial locomotion assay quantifies differences in crawling motility (sinusoidal movement on a solid surface) among populations of worms. This assay takes advantage of C. elegans natural unstimulated exploration behavior by placing worms in a single location on a plate and marking their final location after a given period of time10. Alternatively, swimming in liquid (thrashing) assays count body bends of individual worms over a set period of time. The manual counting of body bends by the human eye is time-intensive and typically exhibits considerable variability between experimenters. The use of computer-assisted automated tracking and analysis can eliminate much of that variability. In addition to the characterization of baseline motor impairment of ALS models, both radial locomotion and swimming assays can detect modulation of distinct locomotor phenotypes from genetic or small molecule interventions. These methods have utility for studying ALS&#160;models and any C. elegans strain that exhibits altered motility.

<table><tbody><tr><td>C. elegans</td><td>Caenorhabditis Genetics Center (CGC)</td><td>-</td><td>Aquire your strains as desired, N2 is a useful control strain</td></tr><tr><td>Disposable pasteur pipets, borosilicate glass</td><td>VWR</td><td>14673-010</td><td>Glass pipet used to create worm pick - hold glass pipette in one hand and ~1&quot; of platinum wire (held by pliers) in the other over a flame to join.&amp;nbsp;</td></tr><tr><td>Disposable petri dishes, 35x10mm</td><td>VWR</td><td>10799-192</td><td>Assay plates for WormLab Imaging System</td></tr><tr><td>Disposable petri dishes, 60x15mm</td><td>VWR</td><td>25384-090</td><td>Stock plates for worms</td></tr><tr><td>Disposable petri dishes, 100x15mm</td><td>VWR</td><td>25384-302</td><td>Standard radial locomotion assay plate</td></tr><tr><td>Disposable petri dishes, 150x15mm</td><td>VWR</td><td>25384-326</td><td>Longer time frame radial locomotion assay plate</td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>M80</td><td>Scope for maintaining worms and setting up radial locomotion assays</td></tr><tr><td>Fine-tipped markers</td><td>VWR</td><td>52877-810</td><td>Need at least 2 colors for radial locomotion assays. Fine tips required for accuracy.</td></tr><tr><td>Flatbed Scanner</td><td>Amazon</td><td>Epson Perfection V850</td><td>Optional for radial locomotion assay. Protocol assumes a resolution of 300dpi, most scanners would work fine</td></tr><tr><td>ImageJ</td><td>NIH</td><td>-</td><td>Optional free software provided by the NIH - https://imagej.nih.gov/ij/</td></tr><tr><td>M9 buffer</td><td>VWR</td><td>IC113037012</td><td>Medium used for swimming assay. Can be made from scratch, see WormBook: Maintenance of C. elegans</td></tr><tr><td>NGM (Nematode Growth Medium)</td><td>VWR</td><td>76347-412</td><td>Medium used to cultivate C. elegans. Can be made from scratch, see WormBook: Maintenance of C. elegans</td></tr><tr><td>OP50&amp;nbsp; bacteria</td><td>Caenorhabditis Genetics Center (CGC)</td><td>OP50</td><td>Primary food source for C. elegans</td></tr><tr><td>p1000 pipettor</td><td>VWR</td><td>76207-552</td><td>Pipettor, used in swimming assay</td></tr><tr><td>p1000 tips</td><td>VWR</td><td>83007-384</td><td>Tips for pipettor, used in swimming assay</td></tr><tr><td>Platinum wire, 0.2032mm diameter</td><td>VWR</td><td>BT136585-5M</td><td>Fine gauge platinum wire used to create worm pick - hold glass pipette in one hand and ~1&quot; of platinum wire (held by pliers) in the other over a flame to join.&amp;nbsp;</td></tr><tr><td>Ruler</td><td>VWR</td><td>56510-001</td><td>Need to score radial locomotion assays</td></tr><tr><td>WormLab Imaging System</td><td>MBF Bioscience</td><td>WormLab</td><td>The Imaging System includes WormLab hardware (bright field stage, camera, and housing) and WormLab software. https://www.mbfbioscience.com/wormlab-imaging-system</td></tr></tbody></table>

evaluation of motor impairment in c. elegans models of amyotrophic lateral sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor impairment that worsens with time. While considerable advances have been made in determining genetic drivers of ALS for a subset of patients, the majority of cases have an unknown etiology. Further, the mechanisms underlying motor neuron dysfunction and degeneration are not well understood; therefore, there is an ongoing need to develop and characterize representative models to study these processes. Caenorhabditis elegans can adapt their movement to the physical constraints of their surroundings, with two primary movement paradigms studied in a laboratory environment- crawling on a solid surface and swimming in liquid. These represent a complex interplay between sensation, motor neurons, and muscles. C. elegans models of ALS can exhibit impairment in one or both of these movement paradigms. This protocol describes two sensitive assays for evaluating motility in C. elegans: an optimized radial locomotion assay measuring crawling on a solid surface and an automated method for tracking and analyzing swimming in liquid (thrashing). In addition to the characterization of baseline motor impairment of ALS models, these assays can detect suppression or enhancement of the phenotypes from genetic or small molecule interventions. Thus, these methods have utility for studying ALS&#160;models and any C. elegans strain that exhibits altered motility.

Both radial locomotion and swimming assays offer sensitive detection of motility impairment (Figure 4 and Figure 5). To investigate the mechanisms underlying pathological TDP-43 in ALS, C. elegans models have been developed that express wild-type or ALS-mutant human TDP-43 pan-neuronally. These animals display molecular and cellular characteristics reminiscent of ALS, including motor dysfunction9. Importantly, they exhibit moderate motility impairment with the expression of wild-type human TDP-43 and more severe motility impairment in animals expressing ALS-mutant TDP-43 using both radial locomotion and swimming assays. Some mutant or transgenic animals will have greater impairment in crawling than swimming, or vice versa. By using two different motility assays, a clearer picture of phenotypic differences between strains is obtained.
Radial locomotion 
When placed on a seeded agar plate, C. elegans explore their surroundings, including seeking out the boundaries of their food source. Radial locomotion assays are one way to take advantage of that behavior as a metric for physical fitness. By analyzing the locomotor behavior (crawling on a solid surface) in a controlled and quantifiable way, the radial locomotor assay offers a simple and effective tool for assessing the severity of motor deficits and other motor-related phenotypes. Radial locomotion assays capture differences in motility in moderately or severely impaired ALS model worms and offer a baseline to compare modulation of motility phenotypes or changes in motility with age (Figure 6). This strategy can be applied to quantify the crawling behavior of any strain that has altered movement from wild type (N2) or control worms. However, this method may not be a good choice to assess animals unable to crawl normally, such as roller mutants or animals that are paralyzed. Typically, wild-type worms will present an average displacement between 200-300 &#181;m/min when raised and tested at 20 &#176;C. The example data presented in Figure 4 show expected results comparing N2, two different transgenic strains expressing wild-type human TDP-43 with a mild phenotype [TDP-43(WT-mild), CK402 (bkIs402[Psnb-1::TDP-43; Pmyo-3::GFP])] or stronger phenotype [TDP-43(WT-moderate), CK410 (bkIs402[Psnb-1::TDP-43; Pmyo-2::GFP])], a transgenic strain expressing mutant human TDP-43 with a severe phenotype [TDP-43(M337V), CK423 (bkIs423[Psnb-1::TDP-43; Pmyo-2::GFP]), and a transgenic strain expressing another neurodegenerative-disease associated protein, wild-type human tau [tau(WT), CK144 (bkIs144[Paex-3::tau(4R1N); Pmyo-2::GFP])]. The two strains expressing wild-type TDP-43 have different degrees of impairment as detected by radial locomotion. TDP-43(WT-low) is not significantly different from N2, while TDP-43(WT-high) does exhibit clear differences in motility. TDP-43(M337V) and tau (WT) strains have more severe impairments in motility.
Swimming assay 
C. elegans engage in stereotypical swimming (thrashing) movement when immersed in liquid. Immediately upon submersion, worms begin to characteristically bend head and tail towards each other with a bend angle of approximately 45&#176;, with the angle vertex being the midpoint of the worm. Worms alternate bending in the ventral and dorsal directions. One thrash, as measured by the software, represents the movement of going from straight to a body bend angle of 20&#176; or greater, irrespective of directionality (angle thresholding can be adjusted post-processing within the Analysis and Plotting window [Workflow | Analyze Data | Body Shape | Bending Angle | Mid-Point | Amplitude Threshold: # degrees]. The swimming assay described here uses automated computer-based tracking and analysis to provide unbiased scoring of swimming activity. It is expected that wild-type (N2) worms will average between 150-200 thrashes per min when raised and recorded at 20 &#176;C. The example data presented in Figure 5, shows expected results comparing N2, two different transgenic strains expressing wild-type human TDP-43 with a mild phenotype [TDP-43(WT-mild), CK402 (bkIs402[Psnb-1::TDP-43; Pmyo-3::GFP])] or stronger phenotype [TDP-43(WT-moderate), CK410 (bkIs402[Psnb-1::TDP-43; Pmyo-2::GFP])], and a transgenic strain expressing another neurodegenerative-disease associated protein, wild-type human tau [tau(WT), CK144 (bkIs144[Paex-3::tau(4R1N); Pmyo-2::GFP])]. The transgenic strain expressing ALS-mutant TDP-43 [TDP-43(M337V)] does not thrash in liquid, and so it is denoted on the graph as ND (no data). This assay can discriminate different phenotypes than the radial locomotion assay shown in Figure 4. For example, in the radial locomotion assay (Figure 4), TDP-43(WT-low) was not significantly different from N2. However, in the swimming assay (Figure 5), both TDP-43(WT-low) and TDP-43(WT-high) are significantly different from N2, as well as significantly different from one another. Also, despite both TDP-43(M337V) and tau(WT) having severe crawling impairments by radial locomotion (Figure 4), only tau(WT) is able to thrash enough to be tracked by the software (Figure 5). TDP-43(M337V) animals are unable to thrash, and software-based analysis does not detect or track these worms accurately. Thus, data on these worms were not collected (ND, no data).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62699/62699fig01.jpg" /> 
Figure 1: Radial locomotion assay workflow. Panels A-E show the generalized steps of the radial locomotion assay. The steps are as follows: (A) NGM Plates, with OP50 seeded to the edges, are prepared by labeling and marking central dot, (B) worms are placed in the center of the agar as marked by the central dot, (C) worms are allowed to move freely for a set amount of time, (D) plate is flipped bottom up and the final location of each worm is marked in a different color than the central dot, (E) The distance from the center dot to each final worm location in measured either by hand or digitally. <a href="https://www.jove.com/files/ftp_upload/62699/62699fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62699/62699fig02.jpg" /> 
Figure 2: Digital measurement of final location using ImageJ.&#160;Distance from center measurements can be measured by hand or digitally, to measure digitally using ImageJ (A)&#160;scan backside of the plate with a ruler in the frame. (B) Draw a known length using the line tool. (C) Use the known length drawn in (B) to set scale [Analyze | Set Scale&#8230;]. (D)&#160;Use the line tool to draw a line from the center point to a final location mark, repeat for each mark. (E)&#160;A paintbrush line marking the first mark measured aids in scoring (squiggly black line near the 1 mark). Final location marks are numbered in yellow to illustrate the directionality of scoring. (F)&#160;Measurements are recorded in the Results window. Save results elsewhere for statistical analysis. <a href="https://www.jove.com/files/ftp_upload/62699/62699fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62699/62699fig03.jpg" /> 
Figure 3: Materials and hardware adjustments for using the software. (A)&#160;Materials used for the software-assisted swimming assay. (B)&#160;General set up of the software and needed materials, note that the housing is in the raised position. (C) The lens mounting bracket height can be adjusted. This image shows the adjustable track and a tape indicator marking the preferred height for performing swimming assays. It is necessary to adjust the brightfield light and camera focus before recording. (D) A 35 mm assay plate is placed on the stage, animals in M9 are added to the plate, and a 1 min timer is started. (E)&#160;It is sometimes advantageous to gently swirl the plate to move worms closer to the center - observe location on the live capture screen; alternatively, a few drops of M9 from a pipettor can be used to separate worms. (F) Adjust the focus ring on the camera lens, observe worms on the screen to determine optimal focus. <a href="https://www.jove.com/files/ftp_upload/62699/62699fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62699/62699fig04.jpg" /> 
Figure 4: Radial locomotion assays detect differences in crawling speed. Unstimulated dispersion of developmentally staged L4 larvae was measured using the radial locomotion assay described above and graphed as &#181;m/min traveled (A-B).&#160;The same data plotted in (A)&#160;and&#160;(B) demonstrate two different possible graphical presentations.&#160;In (A),&#160;data are displayed as a bar graph, making relative differences between strains clearer. In (B),&#160;the final displacement of each worm isplotted within the graph, allowing the variation within the population to be better visualized. To evaluate significance among strains tested, one-way analysis of variance (ANOVA) with Tukey&#39;s multiple comparison test was used. **p=0.0022, ****p&#60;0.0001, ns=not significant. TDP-43(M337V) and tau(WT) are also significantly different from N2, p&#60;0.0001. Error bars in (A)&#160;are standard error of the mean (SEM) and in (B)&#160;are standard deviation. <a href="https://www.jove.com/files/ftp_upload/62699/62699fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62699/62699fig05.jpg" /> 
Figure 5: Swimming assays detect differences of thrashing in liquid. Rates of swimming (thrashing or undulation frequency in liquid) were measured using unbiased computer-assisted scoring and analysis described above and graphed as thrashes/min (A-B). Same data plotted in (A)&#160;and (B). In (A),&#160;data are graphed as a bar graph, which makes relative differences between strains easier to see. In (B),&#160;data from each individual worm scored are plotted within the graph, allowing the variation within the population to be better visualized. To evaluate significance among strains tested, a one-way analysis of variance (ANOVA) with Tukey&#39;s multiple comparison test was used. ****p&#60;0.0001, ns=not significant. TDP-43(WT-moderate) and tau(WT) are also significantly different from N2, p&#60;0.0001.Error bars in (A)&#160;are standard error of the mean (SEM) and in (B)&#160;are standard deviation. <a href="https://www.jove.com/files/ftp_upload/62699/62699fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62699/62699fig06.jpg" /> 
Figure 6:&#160;Mutant TDP-43 worms travel less than wild-type worms. Plates showing a representative difference in the final location of staged L4 N2 (wild-type) and TDP-43(M337V) (CK423 (bkIs423[Psnb-1::TDP-43; Pmyo-2::GFP]), a strain expressing mutant human TDP-43 with severely impaired motor function after 1 hour of unstimulated crawling at room temperature. Plates are marked with a red dot for the center point and blue dots for the final location of the animals. <a href="https://www.jove.com/files/ftp_upload/62699/62699fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Recording swimming behavior using the imaging and tracking software. (A)&#160;The tracking workflow and the location of the video capture icon. (B) The Video Capture window is shown in Live View, and the most important aspects are highlighted. The green &#34;Live view&#34; words will change to a red &#34;Recording&#34; when the record button is activated. <a href="https://www.jove.com/files/ftp_upload/62699/Currey2021_JoVEManuscript_SuppFigure1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Preparing and tracking swimming behavior. This figure shows a series of screenshots to aid in setting up a video image sequence for tracking. The general protocol for preparing a video sequence is to open each workflow menu in this order: Import Image Sequence | Set Sequence Info | Adjust Image | Detect and Track | Save Project. The Analyze Data window can only be used after a project file is tracked. (A) shows an opened .avi (video) file after importing the sequence into the software from within the Track workflow. (B)&#160;The Set Sequence Info window provides a place for notes, set/change the scale, and to check the metadata for a video sequence. Instructions for changing the scale are found in this window and should be followed when the camera is lowered or raised (B). Image (C) shows the Adjust Image window settings. (D)&#160;Screenshot showing the detection tab of the Detect and Track window. Detecting worms is used to train the software and must be set for each video sequence. Additional detection parameters can be set here if desired. (E) Screenshot of Tracking tab of the Detect and Track window with the recommended settings for tracking swimming behavior. This is the last step in setting up a video sequence. Save the sequence as a project using the Save Project window. Repeat these steps for each video sequence. Settings can be saved as a configuration to reduce workload and ensure that all sequences are treated the same (not shown). To track project files for analysis, navigate to the Batch Workflow. (F)&#160;shows the icon used to navigate to the Batch workflow, and (G)&#160;shows the batch processing window, highlights the add and start buttons, and shows the expected appearance of a project file that has been tracked. <a href="https://www.jove.com/files/ftp_upload/62699/Currey2021_JoVEManuscript_SupFigure2.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Swimming behavior analysis. After a project file has been tracked, it can be opened using [File | Open Project]. Worms will appear in green as shown in (A). Scrubbing through the video offers a quick check that the video processed as expect, green highlighting will disappear while scrubbing. The Data Analysis and Plotting window is opened from the Analyze Data workflow item. (B) shows the Data Analysis and Plotting window with Position view opened (default), all tracks are highlighted. Below the datapoints is a plot showing the recorded track for each highlighted worm. The track summary analysis is used to calculated turns per minute. (C-D) show the track summary report and export details. (E-F) show the track summary in a spreadsheet and how to calculate turns per minute, the output measured in this assay. <a href="https://www.jove.com/files/ftp_upload/62699/Currey2021_JoVEManuscript_SuppFigure3.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis at JoVE.com

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility.

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

The neurodegenerative disease amyotrophic lateral sclerosis (ALS) features progressive loss of motor neurons accompanied by muscle weakness and motor ...

evaluation-motor-impairment-c-elegans-models-amyotrophic-lateral

Research

JoVE Journal

Neuroscience

3.9K Views.  Veterans Affairs Puget Sound Health Care System. This protocol describes two sensitive assays for discriminating among mild, moderate, and severe motor impairment in C. elegans models of amyotrophic lateral sclerosis, with general utility for C. elegans strains, with altered motility.

Video: Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

Behavior

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Medicine

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening protocol, demonstrated in video herein, which reliably assesses the neuromuscular function of SOD1-G93A mice in a quick manner. This protocol encompasses a simple neurological scoring system (NeuroScore) designed to assess hindlimb function. NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in SOD1-G93A mice. The protocol developed by ALS TDI provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression. In this report, the combination of a detailed manuscript with video minimizes scoring ambiguities and inter-experimenter variability thus allowing for the protocol to be adopted by other laboratories and enabling comparisons between studies taking place at different settings. We believe that this video protocol can serve as an excellent training tool for present and future ALS researchers.

This video protocol describes a sensitive, reliable, and quick method for evaluating the neuromuscular deficits in a transgenic mouse model of amyotrophic lateral sclerosis.

The SOD1-G93A transgenic mouse is the most widely used animal model of amyotrophic lateral sclerosis (ALS). At ALS TDI we developed a phenotypic screening ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Evaluation of Motor Impairment in C. elegans Models of Amyotrophic Lateral Sclerosis

Summary

Explore More Videos

Chapters in this video

Assaying β-amyloid Toxicity using a Transgenic C. elegans Model

Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

A Caenorhabditis elegans Model System for Amylopathy Study

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

A Quick Phenotypic Neurological Scoring System for Evaluating Disease Progression in the SOD1-G93A Mouse Model of ALS

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Methods to Assess Subcellular Compartments of Muscle in C. elegans