We thank animal caretakers at the ENI&#39;s Animal facility for help with breeding, and Dr. Nuno Raimundo for useful comments on the manuscript. I.M. is supported by the grants from the German Research Foundation (DFG) through the collaborative research center SFB-889 (project A8) and SFB-1190 (project P02), and the Emmy Noether Young Investigator Award (1702/1). C.M.R. is supported by the fellowship from the G&#246;ttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB).

All animal experiments reported here are conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval by the Nieders&#228;chsisches Landesamt f&#252;r Verbraucherschutz und Lebensmittelsicherheit (LAVES), registration number 14/1701.
1. Study Design
<ol>
	<li>As animal behavior work requires careful planning, consider the following parameters while designing the experiment.
	<ol>
		<li>Number of animals needed per group.
		<ol>
			<li>Use a statistical software (e.g., PASS, EDA, or GPower) to calculate the required group size. 
			NOTE: The group size depends on the variation between animals and the severity of the phenotype. For the kinematic gait analysis, the number of mice is usually 10 -&#160;20 per group.</li>
		</ol>
		</li>
		<li>Sex of the experimental animals.
		<ol>
			<li>Consider the effect of estrogen levels on the experiment, depending on the animal strain. 
			NOTE: Many behavior studies focus on males in order to avoid the influence of estrogen levels on the experiment. These influences are more or less strong depending on the animal background strain.</li>
			<li>If both sexes will be used, test for sex influence and evaluate the two sexes independently when necessary.</li>
		</ol>
		</li>
		<li>Age of the experimental animals.
		<ol>
			<li>Use adult animals (2 months of age, or older) if only one-time point is needed.</li>
			<li>Select several time points when change in motor behavior with advancing age is to be studied. The earliest possible time point is 1 month, after mice are weaned from their mothers. Test the animals in regular intervals, e.g., every 1, 2, or 3 months.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Apply for authorization from the local authorities to perform animal behavior testing.</li>
	<li>Make plans for procuring the test animals.
	<ol>
		<li>Make a breeding plan or contact an animal distributor in a timely manner so that enough experimental animals are available on the day when the experiments start.</li>
		<li>Allow the animals to habituate for one week if they are kept in a new room/setting during the experiments.</li>
	</ol>
	</li>
</ol>
2. Video Recording
NOTE: To illustrate the use of kinematic gait analysis, here a commercially available imaging system with its accompanying imaging and analysis software (see the Table of Materials) are used.
<ol>
	<li>Start the computer and the imager software.</li>
	<li>Determine the health status and well-being of each animal by observing it in its home cage, and weighing it on a balance.</li>
	<li>When needed, gently apply red finger paint to the animal&#39;s paws with a brush. Allow the paint to dry for ~5 min in a spare clean cage. 
	NOTE: Avoid painting the animal&#39;s abdomen as the paint is used to enhance the contrast between paws and body. It is useful to have black finger paint handy for corrections. This step is needed for animals with brown fur, or in case the paws have been tattooed for identification. If chosen to paint the paws of one animal, all animals in the same group and control group need to be painted as well.</li>
	<li>Set the speed of the treadmill on the top right panel of the apparatus; If more than one running speed will be applied, start with the slowest speed first.</li>
	<li>Place the animal in the test chamber (avoid clamping the tail or paws when closing the chamber). Cover the chamber with a dark cloth and allow each animal to adjust for 1 - 2 min.</li>
	<li>Turn on the light in the test chamber by turning the treadmill light rotary switch to the &#34;on&#34; position. Turn the treadmill rotary switch to &#34;forward&#34; to start the treadmill, then click the &#34;record&#34; button in the imager software. 
	NOTE: While the treadmill is running, it is important to observe animal performance carefully and constantly: stop the treadmill immediately if the animal cannot keep up with the treadmill speed, or shows secondary symptoms non-related to locomotion (e.g., epileptic seizures). Testing conditions may need to be readjusted.</li>
	<li>When the animal runs stably (no quick escapes to the sides, front, or back), record for at least 5 s before stopping the treadmill. Stop recording by clicking &#34;stop&#34; on the imager software, and turn the treadmill rotary switch back to the &#34;off&#34; position. 
	NOTE: To avoid unstable running of animals it can be helpful to let them run for several seconds, or to allow them to run in the other direction (by turning the treadmill rotary switch to &#34;reverse&#34; instead of &#34;forward&#34;).</li>
	<li>Click the &#34;processing&#34; button in the imager software to open a menu in which the start and the end point of the video section (to be used for analysis) can be set. To do this, use the slider on the bottom of the screen to navigate through the video.</li>
	<li>To select the current time point as the start or end point, click &#34;from frame #&#34; and &#34;to,&#34;&#160;respectively. Make sure the section contains at least 7 steps/paw (14 steps in total) of the animal running stably at a constant speed.</li>
	<li>Enter the animal identification, birth date, weight, and sex. Save the data on a desired location on the computer or server. Click &#34;camera&#34; to return to the recording interface.</li>
	<li>If multiple running speeds need to be recorded, repeat steps 2.6 - 2.10 with the desired running speeds. Before recording the next video, ensure that the red paint is still present on the paw, otherwise repeat step 2.3.</li>
	<li>After recording, release the animal to its home cage. After removing an animal, clean the treadmill belt thoroughly with soapy water followed by disinfectant to prepare it for the next experimental animal.</li>
</ol>
3. Video Processing
<ol>
	<li>Start the analysis software and click &#34;select study folder&#34; to select the folder with the recorded videos.</li>
	<li>Select one video, or several videos that can be processed consecutively, and click &#34;go.&#34;</li>
	<li>Use the &#34;redraw&#34; function to select the area where the mouse is running; this section should only contain the mouse and white background.</li>
	<li>If the &#34;reverse&#34; treadmill function was previously used, choose &#34;Check if subject&#39;s nose is to your right &#62;&#62;&#62;&#34; to mirror the video since the software is designed to only analyze animals running to the left. Click &#34;accept&#34; to proceed.</li>
	<li>Use the &#34;refresh&#34; function to see the default mask and paw print that the software detects. 
	NOTE: The original video is displayed on the left, and a black and white image of the proposed paw prints is on the right.</li>
	<li>Enter values in the &#34;length&#34; and &#34;width&#34; boxes to change the mask that excludes the red area around the snout of the animal for analysis; as the color is similar to the paws, not masking that area could result in the software accidentally classifying the snout area as a paw.</li>
	<li>Adjust the sliders &#34;filter noise&#34; and &#34;filter fur and dark patches&#34; to optimize the black and white paw print. Set the &#34;filter noise&#34; slider to ~800 - 950 for black animals and to ~700 - 800 for brown or white animals, depending on the exact fur color of the animal. Select &#34;ok&#34; when the settings are satisfactory. 
	NOTE: The &#34;filter fur and dark patches&#34; slider depends on how &#34;red&#34; the paw is. For painted paws, the value is usually around 100 - 120, and for non-painted paws the best value is around 50 - 100. These settings depend on the color shadings of the fur and paws, and need to be optimized for every animal. The black and white paw print should have clear representations of the paws with as little background noise as possible.</li>
	<li>Select one or several videos that passed the first adjustment (labeled with &#34;@@&#34; before the video name) and select the &#34;go&#34; function to start the analysis of these videos. 
	NOTE: The analysis takes 2 - 5 min per video. It is possible to run the analysis of several videos overnight since this step requires no input from the experimenter.</li>
	<li>Select an analyzed video (labeled with &#34;@@@&#34;) and click &#34;go.&#34;&#160;Note that the paw area (in cm2) in contact with the belt over time (gait dynamics ) for each separate paw can now be seen. To compare the original video and the calculated paw print for a selected area, use the &#34;play video&#34; function.</li>
	<li>Use the following (three) tools to correct small mistakes made by the software.
	<ol>
		<li>Use the &#34;correct&#34; option to delete a wrong signal, e.g., when the software records a signal even though the corresponding paw is not in contact with the belt. Click once to zoom into the relevant area, and mark the left border of the object to remove with the second click and the right border with the third click.</li>
		<li>Use the &#34;connect&#34; option to combine two signals, e.g., when no signal is recorded for a few frames even though the paw is in contact with the belt. Click once to zoom into the relevant area and double-click in the middle of the two objects to combine.</li>
		<li>Use the &#34;delete&#34; option to remove time points from the analysis completely. Use this option only if the mistake cannot be fixed with the &#34;correct&#34; or &#34;connect&#34; function, e.g., when a signal from the left forelimb paw is accidentally recorded for the left hind limb paw. Click once to zoom into the relevant area, and mark the left border of the area to remove with the second click and the right border with the third click. 
		NOTE: The tools may only be used to correct small mistakes; systematic failures (e.g., if the signal from one paw was extremely weak) cannot be corrected: the video should be excluded from analysis and the recording of the respective animal repeated, when possible. Note that the &#34;play video&#34; option is no longer available after the &#34;correct,&#34;&#160;&#34;connect,&#34;&#160;or &#34;delete&#34; option has been used, and clicking the &#34;undo&#34; button will reset all 3 editing tools.</li>
	</ol>
	</li>
	<li>Select &#34;next limb&#34; to proceed through the 4 limbs; when &#34;next limb&#34; is clicked after the last paw, the software completes the analysis and shows the results for this animal on 4 screens.</li>
</ol>
4. Gait Analysis
<ol>
	<li>When all videos from one experiment are analyzed, select all videos and click &#34;re-organize results&#34; to export the results (a list of parameters in spreadsheet files).</li>
	<li>Open the file with the ending &#34;reorganized_stride_info&#34; and add information that is not included in this spreadsheet: group information (e.g., genotype, treatment), age, and the measurements of animal length and width which are saved in another spreadsheet file with the ending &#34;SFI_TFI_PFI_reorganized_stride_info.&#34;</li>
	<li>Normalize the gait parameters to animal width or length where necessary, e.g., SL to animal length and SW to animal width.</li>
	<li>Sort the results by group, age, and running speed: analyze all these conditions independently. 
	NOTE: Different ages or running speeds cannot be combined within a same group.</li>
	<li>Calculate the average (mean) values, standard deviation, and standard error of the mean for each parameter for all experimental conditions.</li>
	<li>Perform statistical analysis according to the experimental design, e.g., use a 2-tailed t-test to compare mutant/treated animal to a wild-type (WT)/control, or ANOVA to compare several independent groups.</li>
	<li>Look at all measured parameters: it is helpful to plot each parameter to better visualize the results. If there are statistical differences in a given parameter, check if other dependent parameters change correspondingly. 
	NOTE: For example, if the SL is significantly decreased in a certain test group, this will also cause a higher stride frequency (since the running speed is the same) and may result in an increased SW (in order to maintain posture stability).</li>
	<li>Select parameters that are most relevant for a model, and/or are comparable to observations in the human disease. For a presentation, create representative videos for each group and complement them by graphs showing the readout for the relevant parameters, since subtle gait changes are often not obvious from the videos.</li>
</ol>
5. Troubleshooting
NOTE: Some animals, especially mouse models with an anxiety phenotype, may have difficulties to perform even a simple task like running on a treadmill. The following are steps that can be taken to lower anxiety levels and encourage running.
<ol>
	<li>Habituation and positive enforcement.
	<ol>
		<li>At 2 - 3 days before the first test, place the mouse in the test chamber, cover it with a dark cloth, and leave the light turned off. Let the mouse adjust to the new environment for ~5 min. Add chow or chocolate/nut butter (e.g., Nutella) to the test chamber so a positive association may be formed.</li>
	</ol>
	</li>
	<li>Negative enforcement by air puffs/rear boundary.
	<ol>
		<li>Mice do not like air puffs or a movement behind them, and will run away from the disturbance. To motivate running, use mild air puffs, or rhythmic movement of the flexible bar that forms the rear boundary of the test chamber, to encourage the mouse to run towards the front part of the test chamber.</li>
	</ol>
	</li>
	<li>Slow start.
	<ol>
		<li>When testing fast running speeds, start the treadmill at a lower speed and then slowly increase the treadmill speed towards the desired testing condition.</li>
	</ol>
	</li>
	<li>Minimize free movement.
	<ol>
		<li>The test chamber length is limited by two adjustable bars in the front and back. If a test animal keeps up with the running speed but does not run steadily, limit the chamber&#39;s length to result in more steady running.</li>
	</ol>
	</li>
	<li>If the above-mentioned measurements are not successful, record running on the next day. If the animal still refuses to run after testing on three days, record this as the finding, and exclude the animal from further testing. 
	NOTE: The results of the gait analysis depend on good-quality video recording. There is no reason to exclude videos during the analysis if the videos have been recorded carefully. If the video quality is insufficient, it will become obvious during step 3.6 when the parameters for the creation of the digital paw print are being set. If any other body part except the paws and snout appears red (e.g., due to the missing fur around the genitals or finger paint sprinklings on the abdomen), the quality drops significantly. The adjustments in step 3.6 allow correcting only small issues, and if this cannot bring the video to an acceptable signal/noise ratio, the video needs to be excluded from the analysis, and recording needs to be repeated. Thus, it is recommended to analyze videos soon after recordings are performed.</li>
</ol>

The authors declare no competing financial interests.

Studying the motor coordination is a useful approach in the characterization of models of neurodegenerative diseases, especially for diseases like PD in which motor coordination is severely affected. With the help of a kinematic gait analysis functional assay, we can identify subtle changes in the gait of animals at the onset of locomotion problems, or in models with weak neurodegeneration and hence relatively modest phenotype. Given the wide range of phenotypes in various models of neurodegenerative diseases that encomp...

Neurodegenerative diseases impose a significant burden on patients, families, and society, and will become of even greater concern as life expectancy increases, and the world population continues to age. One of the most common symptoms of neurodegenerative diseases are balance and mobility problems. Thus, characterization of motor behavior in aging mammalian (e.g., rodent) models, and/or models showing neurodegenerative phenotypes, is a valuable tool to demonstrate the in vivo relevance of the specific animal model(s), or therapeutic treatments that aim to improve the disease symptoms. Almost every approach to treat neurodegenerative diseases ultimately requires testing in an animal model before initiation of a clinical trial in humans. Therefore, it is crucial to have reliable, reproducible behavior tests that can be used to consistently quantify disease-relevant phenotypes along age progression, in order to ensure that a candidate drug, which showed potential in an in vitro model, can effectively ameliorate the phenotype in a living animal.
One aspect of motor behavior assessment in rodents is kinematic gait analysis, which can be performed by VPI (also called ventral plane videography)1,2. This established method capitalizes on continuous recording of the underside of the rodents walking atop a transparent and motorized treadmill belt1,2,3,4. Analysis of the video feed data creates &#34;digital paw prints&#34; of all four limbs that dynamically and reliably recapitulate the rodent&#39;s walking pattern, as originally described by&#160;Kale et al.2 and Amende et al.3.
The principle of imaging-based gait analysis is to measure the paw area in contact with the treadmill belt over time, for each individual paw. Every stance is represented by an increase in paw area (in the braking phase) and a decrease in paw area (in the propulsion phase). This is followed by the swing phase in which no signal is detected. Swing and stance together form a stride. In addition to gait dynamics parameters, posture parameters can also be extracted from the recorded videos. Exemplary parameters and their definition are listed in Table 1 and include stance width (SW; the combined distance from the fore or hind paws to the snout-tail axis), stride length (SL; average distance between two strides of the same paw), or paw placement angle (the angle of the paw to the snout-tail axis). The posture and gait dynamics data allow drawing conclusions on animal balance (by posture parameters and their variability over several steps) and coordination (by gait dynamics parameters). Other parameters, such as ataxia coefficient (the SL variability calculated by [(max. SL&#8722;min. SL)/mean SL]), hind limb shared stance time (time that both hind limbs are in contact with the belt), or paw drag (total area of the paw on the belt from full stance to paw lift-off) can also be extracted, and have been reported to be changed in various neurodegenerative disease models5,6,7,8 (see Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Unit</td>
			<td>Definition</td>
		</tr>
		<tr>
			<td>swing time</td>
			<td>ms</td>
			<td>duration of time the paw is not in contact with the belt</td>
		</tr>
		<tr>
			<td>stance time</td>
			<td>ms</td>
			<td>duration of time the paw is in contact with the belt</td>
		</tr>
		<tr>
			<td>% brake</td>
			<td>% of stance time</td>
			<td>percentage of stance time the paws are in the brake phase</td>
		</tr>
		<tr>
			<td>% propel</td>
			<td>% of stance time</td>
			<td>percentage of stance time the paws are in the propulsion phase</td>
		</tr>
		<tr>
			<td>stance width</td>
			<td>cm</td>
			<td>combined distance from the fore or hind paws to the snout-tail axis</td>
		</tr>
		<tr>
			<td>stride length</td>
			<td>cm</td>
			<td>average distance between two strides of the same paw</td>
		</tr>
		<tr>
			<td>stride frequency</td>
			<td>strides/s</td>
			<td>number of complete strides per second</td>
		</tr>
		<tr>
			<td>paw placement angle</td>
			<td>deg</td>
			<td>angle of the paw in relation to the snout-tail axis of the animal</td>
		</tr>
		<tr>
			<td>ataxia coefficient</td>
			<td>a.u.</td>
			<td>SL variability calculated by [(max SL-min SL)/mean SL]</td>
		</tr>
		<tr>
			<td>% shared stance</td>
			<td>% of stance</td>
			<td>hind limb shared stance time; time that both hind limbs are in contact with the belt at the same time</td>
		</tr>
		<tr>
			<td>paw drag</td>
			<td>mm2</td>
			<td>total area of the paw on the belt from full stance to paw lift-off</td>
		</tr>
		<tr>
			<td>limb loading</td>
			<td>cm2</td>
			<td>MAX dA/dT; maximal rate of change of paw area in the breaking phase</td>
		</tr>
		<tr>
			<td>step angle variability</td>
			<td>deg</td>
			<td>standard deviation of the angle between the hind paws as a function of SL and SW</td>
		</tr>
	</tbody>
</table>
Table 1. Definition of key gait parameters that can be tested by ventral plane imaging.
Assessing the motor behavior of rodent models for neurodegenerative diseases can be challenging depending on the severity of the phenotype of a specific model at a given age. Several diseases, most prominently PD, show strong motor behavior (locomotion) deficits, both in patients and in animal models. One of the four key symptoms in PD is bradykinesia, which progresses with aging and manifests in severe gait impairments already in early stages of PD9. Studies of the acute PD model, rodents treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP), have already used VPI gait analysis10,11,12. However, given the acute nature of this model, these studies do not address the age-related progression of motor deficits. Several recent studies have conducted gait analysis in aged mice with neurodegenerative changes, for example13,14,15, emphasizing the relevance of understanding the disease progression with advancing age.
In addition to motor deficits, animal models of neurodegenerative diseases often have difficulties focusing on the examination tasks and show prominent cognitive impairments, in particular with advancing age. Such a phenotype can influence the result of motor behavior tests. Namely, one of the most widely used tests to examine motor deficits, the rotarod test16, relies on cognition, attention, and stress17,18. While the willingness to walk on a motorized treadmill also depends on these factors, the recorded read-out is running, which is a more standardized feature and far less influenced by altered cognition. Effects of stress and attention may be visible in specific parameters, like swing/stance time for stress, and SL for attention19,20, but not in overall running ability.
The kinematic gait analysis approach further offers the advantage of having options to adjust the challenge for rodent models. The treadmill with adjustable angle and speed allows walking speeds from 0.1 - 99.9 cm/s, so that rodents with severe walking impairments may still be able to run at a slow speed (~10 cm/s). Non-impaired animals can be measured at faster running speeds (30 - 40 cm/s). The observation of whether or not the tested animals are able to run at a certain speed provides a result by itself. Further, the rodent can be additionally challenged to run up an incline, or down a decline, by tilting the treadmill to a desired angle with the help of a goniometer, or by attaching a weighted sled to mouse or rat hind limbs.
In addition to numerous studies of single proteins that are mutated in patients, there is a recent increasing awareness of the links between defective endocytosis process and neurodegeneration13,21,22,23,24,25,26,27,28. Mouse models with reduced levels of endophilin-A (henceforth endophilin), a key player in both clathrin-mediated endocytosis13,21,29,30,31,32,33,45 and clathrin-independent endocytosis34, were found to show neurodegeneration and age-dependent impairments in locomotor activity13,21. Three genes encode the family of endophilin proteins: endophilin 1, endophilin 2, and endophilin 3. Notably, the phenotype resulting from depletion of endophilin proteins varies greatly depending on the number of missing endophilin genes13,21. While triple knock-out (KO) of all endophilin genes is lethal just a few hours after birth, and mice without both endophilin 1 and 2 fail to thrive and die within 3 weeks after birth, single KO for any of the three endophilins shows no obvious phenotype for tested conditions21. Other endophilin mutant genotypes show reduced lifespan and develop motor impairments with increasing age13. For example, endophilin 1KO-2HT-3KO mice display walking alterations and motor coordination problems (as tested by kinematic gait analysis and rotarod) already at 3 months of age, while their littermates, endophilin 1KO-2WT-3KO animals, display a significant reduction in motor coordination only at 15 months of age13. Due to the vast diversity of phenotypes in these models, it is necessary to identify and apply a test that can integrate a variety of challenges corresponding to the animal&#39;s motor and cognition abilities, as well as the age. Here, we detail the experimental procedures that capitalize on the kinematic gait analysis to assess the onset and progression of motor impairments in a mouse model that shows neurodegenerative changes (i.e., endophilin mutants). This includes measuring gait parameters at various ages and different severities of locomotion impairments.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DigiGait</td>
			<td style="width:123px;">Mouse Specifics, Inc., Framingham, Massachusetts, USA</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">DigiGait Imager and Analysis Software are included with the hardware</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">non-transparent blanket or dark cloth</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">cover the test chamber to reduce the animal&#39;s feeling of exposure/stress</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">balance</td>
			<td style="width:123px;">e.g. Satorius</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">balance with 0.1 g accuracy and a maximum load of at least 100 g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">red finger paint</td>
			<td style="width:123px;">e.g. Kreul or Staedtler</td>
			<td style="width:123px;"></td>
			<td>for increasing the contrast between paws and animal&#8217;s body</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">small paint brush</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">soft brush to apply finger paint to the animal paws</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">diluted detergent</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">for cleaning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">disinfectant, e.g. Meliseptol or 70% ethanol</td>
			<td style="width:123px;">e.g. B.Braun</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">for desinfection</td>
		</tr>
	</tbody>
</table>

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.

To illustrate the use of kinematic gait analysis, we have performed gait analysis on WT C57BL/6J mice with advancing age, as well as several endophilin mutant lines, using commercially available instrumentation and software (please refer to the Table of Materials). In this setup, a high-speed camera under a transparent treadmill records the running of a mouse (Figure 1A). The software then recognizes the contrast between the red colored paws ...

Watch this Scientific Journal Video about Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration at JoVE.com

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

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. ...

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11.8K Views.  European Neuroscience Institute (ENI). 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).

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...