KS and HF thank the Michael J Fox Foundation for Parkinson&#39;s Research for support of their work on Parkinson&#39;s disease.

All animal studies were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (IACUC).
1. Gait apparatus
NOTE: Modeled from the MouseWalker14, the RatWalker was designed with dimensions in proportion to the difference in step length between rats and mice. It consists of a side illumination backlight, walkway enclosure, optical waveguide walkway, mirror, and camera (Figure S1). LED strips, oriented in a staggered position, were used on each side of the walkway and backlight waveguides to accommodate the extra material. The materials needed to build the modified gait apparatus can be found in Table S1.
<ol>
	<li>Use a backlight (Figure S2) to create a silhouette of the animal which is utilized by the software to assign position, direction of movement, and morphometric qualities. Construction is comprised of a layered panel of an acrylic diffuser, optical waveguide, reflector, and LED light strips assembled within a stock aluminum frame (Table S1).</li>
	<li>Use a walkway enclosure (Figure S3) to guide the animal along the platform and direct the animal to the home-cage. Construction consists of clear acrylic sheets solvent welded with dichloromethane (Table S2).</li>
	<li>Use the walkway (Figure S4) to provide the medium to generate lit footprints. The walkway is constructed from clear acrylic, which is side-lit with strip LEDs and housed in aluminum angle (Table S3).</li>
	<li>Place a mirror (Figure S5) directly under the walkway at a 45-degree angle to reflect the underside of the walkway for videography. It is constructed from a 1/4&#34; thick glass mirror supported by acrylic, and angled brackets arranged in a row (Table S4).</li>
	<li>Perform videography using a tripod-mounted, domestic-quality, high-speed action camera.</li>
</ol>
2. Equipment Setup
<ol>
	<li>Align the backlight, walkway, and mirror according to Figure S1, on top of a countertop, workbench, or stable cart. Ensure each component is centered with respect to the walkway.</li>
	<li>Using a level, make certain that the components are horizontally plumb.</li>
	<li>Place the walkway enclosure on top of the walkway.</li>
	<li>Clean all contact surface areas with 70% ethanol. Make sure to use a nonabrasive towel to prevent scratching of the walkway.</li>
	<li>Mount the high-speed camera onto a 57-inch tripod and place it mid-line to the mirror, spaced far enough to capture the entire walkway inside of the field of view. From the video settings menu, ensure that the high-speed camera is set to linear acquisition in 1080p mode at 120 frames per second (fps) with any type of auto-adjust or optimizations turned off.</li>
	<li>Plug in and turn on the LED strip lights for the backlight and walkway. It may be necessary to dim the backlight to reduce background capture.</li>
</ol>
3. Animal Acclimation
NOTE: One week prior to the first experiment, run the animals through the modified gait apparatus.
<ol>
	<li>Position a home-cage at the terminus of the walkway.</li>
	<li>With the enclosure installed and the lights off, place the rat at the end of walkway opposite the home-cage and allow it to walk across the walkway in an unforced manner.</li>
	<li>Run each rat through the modified gait apparatus several times, until they can smoothly cross the entire walkway.</li>
	<li>Repeat the process two days before the experiment.</li>
</ol>
4. Gait Procedure
<ol>
	<li>Place a home-cage at the end of the walkway before the start of each run to serve as a positive cue for the rat to traverse the walkway.</li>
	<li>Turn off the room lights, power on the camera, and start recording several seconds before the rat is placed on the platform. 
	NOTE: Be certain to use a memory card that is officially recommended by the camera manufacturer. An unlisted memory card may still work but is not guaranteed to capture at the purported frame rate.</li>
	<li>With the enclosure installed, place the rat at the end of the walkway opposite the home-cage and allow it to walk across the walkway in an unforced manner.</li>
	<li>Stop recording once the animal reaches the terminus of the walkway.</li>
	<li>Clean the walkway using 70% ethanol and a nonabrasive towel in between runs and after an animal urinates or defecates, then allow ethanol to evaporate before introducing another animal.</li>
	<li>Run the rats through the walkway a total of 7 times during each observation period, taking the first three runs that score as passing for analysis.</li>
	<li>Score a run as passing if the animal makes four or more consecutive steps in the direction of the home-cage without interruption due to grooming, pausing, or errant movements. 
	&#8203;NOTE: It is good practice to record the mass of the animals before each round of measures. For our study, WT (n = 7) and DKO (n = 8) weighed 200.3 &#177; 21.67 g and 296.6 &#177; 3.85 g, respectively (p = 0.004, Unpaired t test with Welch&#39;s correction). We do not see an issue with rats of any age or size.</li>
</ol>
5. Video preprocessing
NOTE: The videos captured by the high-speed camera are rendered in mp4 format at 120 fps and a resolution of 1080p. To ease the burden on the analytical software downstream, first trim unnecessary footage and strip the audio from each video using LosslessCut software (version 3.23.7, https://github.com/mifi/lossless-cut), then convert the mp4 video stream into a png image sequence using the open-source software FFmpeg (version 4.2, http://ffmpeg.org/). Note: other Lossless formats such as tiff can be utilized in place of png.
<ol>
	<li>Create a directory for the videos on a PC running Windows 7 or higher, then transfer the videos from the high-speed camera&#39;s storage device to the newly created directory. In addition, copy ffmpeg.exe to the same location.</li>
	<li>In LosslessCut, drag the videos to the interface to open. Discard the audio, set the start and end cut points to include only the analytically relevant portion of the video, set the capture frame format to png, and export. Once the video is exported, rename the video file using any naming convention followed by &#34;_trimmed&#34;.</li>
	<li>To batch convert the videos to image sequences, open a command prompt, set the working directory to the location of the videos with &#34;cd [path to directory]&#34;, and run the following commands: 
	for %i in (*) do mkdir &#34;%~ni_cropped&#34; 
	for %i in (*) do mkdir &#34;%~ni_trimmed&#34; 
	for /f &#34;tokens=1 delims=.&#34; %a in (&#39;dir /B *_trimmed.MP4&#39;) do ffmpeg -i &#34;%a.MP4&#34; &#34;%a/%a_%04d.png&#34;</li>
	<li>&#160;After the batch process completes, open each image sequence in ImageJ Fiji23 and crop the sequence to th&#160;e region of interest (ROI) encompassing the area of the floor within which the rat is observed.</li>
	<li>To reduce background from the walkway illumination, increase the color balance minimum of the cyan channel to 76.</li>
	<li>Save as image sequence and change the &#34;_trimmed&#34; suffix to &#34;_cropped&#34;, saving the files in their respective &#34;_cropped&#34; folder.</li>
</ol>
6. Gait Processing
NOTE: Gait data is processed and quantified using the freely available software, MouseWalker (http://biooptics.markalab.org/MouseWalker/)14.
<ol>
	<li>Unpack and install the MouseWalker software onto a PC running a 64-bit windows environment with Microsoft Excel installed.</li>
	<li>After launching MouseWalker.exe, perform an initial scale calibration for each set of runs. Load an image sequence and using landmarks or a ruler captured in the video, measure two points of known distance. Calculate the number of pixels per centimeter in the video frame and enter this value into the parameters section of the settings form along with the video acquisition frame rate.</li>
	<li>Similarly, measure the head, tail, and feet of the rat to determine head length, maximum tail width and area, minimum and maximum foot area, and other features necessary to complete the tracking parameters section of the MouseWalker settings form. See http://biooptics.markalab.org/MouseWalker/&#160;for the user manual and other documentation.</li>
	<li>To obtain the body area values, open the same image sequence in ImageJ, draw a selection outlining the rat, and perform a region of interest (ROI) pixel count.</li>
	<li>Parameters and settings used for this publication (Figure S6). 
	&#8203;NOTE: Parameters are provided for illustration and are dependent on the scale of the video, acquisition hardware, and conditions. Software calibration and adjustment is required each time the camera or equipment is re-positioned. Capturing a measuring device within the acquisition improves accuracy and facilitates calibration.</li>
	<li>Following calibration, load each image sequence. Selecting auto will start the autonomous assignment of footprints.</li>
	<li>Scroll through each frame of the sequence, manually correcting miss-assigned footprints. Save once this step is complete.</li>
	<li>Lastly, select evaluate to process the footprint position and pressure data. A series of graphs, images, and a spreadsheet with quantitative gait metrics will export into a results folder.</li>
</ol>
7. Data Analysis
<ol>
	<li>Use the spreadsheet exported at the end of each evaluation that contains quantitative gait data for each run. Concatenate data from each run and average per rat. Plot the mean data and test for significance using GraphPad Prism version 7.0a.</li>
</ol>

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The authors declare no competing financial interests.

Gait disturbances, including decreased arm swing, slower walking speed, and shorter steps, are a defining feature of PD, and occur early during disease course1,5. Several methods have been developed over the years to observe and record footfalls for gait analysis in rodent models of PD, with manual techniques for quantifying footfall position leading to automated approaches that are more sensitive and capable of capturing dynamic parameters. Some static approaches involve &#34;inking&#34; rodent&#39;s paws with an indicator to be tracked onto media placed on a walkway24,25,26. The residual footfalls are later quantified by hand. These methods are often used in conjunction with videotaping for manual kinematic scoring. Autonomous methods of capturing and quantifying gait patterns were introduced more recently15,16, and are available commercially. Autonomous gait quantification adds temporal attributes to otherwise static footprints, allowing investigators to look for abnormalities attributed to velocity and time in addition to distance and angle27. Pressure is also quantifiable when coupled with the FTIR method.
Gait pattern abnormalities have also been reported in toxin-induced rat models of PD. In particular, gait changes have been reported in the 6-OHDA lesion model using a commercial gait analysis platform28,29,30. The most prominent alteration in this model has been declines in walking speed and cadence. Further, the commercial gait analysis platform has been used to assess a number of dynamic and static gait parameters of PD models, such as the impact of unilateral 6-OHDA-induced lesions and how transplantation of dopaminergic neurons rescues the gait alterations31. Additionally, a study has analyzed the ratio of parameters from 6-OHDA lesions in injured versus uninjured sides of the brain, while correcting for speed in related parameters such as hindlimb step cycle, hindlimb print area, and step sequence, all of which are significantly altered when comparing different types of lesions to saline controls32. However, it is important to note that the pathogenetic background of toxin-induced models is of a toxic nature rather than a neurodegenerative process as in human PD6, and as such gait assessment following lesion induction might mimic advanced PD when neurons are lost but make studies into early motor changes more difficult.
Gait geometry has been measured in Pink1 KO, Parkin KO, and DJ-1 KO rats22, models of hereditary PD, that exhibit a progressive nigral dopaminergic neuron loss with aging. Gait was measured using the commercial NeuroCube apparatus, where the rats are allowed to walk in a circle, looking at geometry and dynamic features. Pink1 and DJ-1 KO rats showed shorter duration in stride, swing, and stance when compared to WT at 4 and 8 months.
Because commercial gait analysis systems are costly and come with proprietary analysis pipelines, we sought an open alternative for our study of hereditary rat models of PD. The MouseWalker system14, which comes with build instructions and open-access software, captures all of the parameters of commercial equipment designed for small rodents. Since the platform was too small to consistently achieve passable results with adult rats, i.e. four uninterrupted steps during locomotion at walking speed, we re-scaled the hardware to accommodate rats. In addition, we utilized a domestic action camera in place of a commercial high-speed video solution.
Lower frame density was a potential pitfall of using an action camera in place of a high-speed camera. However, the quality threshold in domestic videography is rapidly rising and is capable of recording at 120 fps in high resolution. Furthermore, lens distortion can be corrected during recording by camera software producing a consistently linear field of view (FOV).
We were initially concerned about the pressure dynamics of using a similar thickness of acrylic for the walkway with a wider base and heavier animals, and the ability of the software to process video of larger animals in a wider FOV. We speculate that the range of mass between mice and rats falls within the range of sensitivity of the acrylic FTIR platform allowing us to measure rats during their entire life cycle. Furthermore, it is possible that potential pixel dilution may be offset by the higher surface area of the rats&#39; pawprints relative to the area captured in the FOV, if there is any significant difference at all. With proper calibration, as documented here, the freely available software14 was able to process rat gait video as described.
With this protocol, we were able to demonstrate that the FTIR mouse gait system14 modified here for rats can detect alterations in gait prior to the visual observation of hindlimb dragging in male DKO rats. Of note, the overt visually apparent motor impairment (hindlimb dragging) observed in DKO rats has been previously reported in male Pink1 single KO rats22. While male DKO rats exhibit visually observable hindlimb dragging starting at 4-6 months of age, the gait analysis uncovered locomotion changes at 2 months of age. In particular, changes in hindlimb gait parameters were found. DKO rats exhibit increased hindlimb (both left and right) swing speed and an associated decrease in hindlimb (both left and right) swing duration. Further, the DKO rats place their left hindpaw closer to their body during paw touchdown, while the right hindpaw is further away from their body during paw takeoff. While we did not uncover changes in stride or stance duration in DKO rats at 2 months, the swing duration was shorter in DKO rats as is similarly reported in Pink1 and DJ-1 KO rats22. Overall, these changes suggest the hindlimb gait parameters are altered prior to the development of hindlimb dragging in male DKO rats. Future longitudinal gait studies tracking the development of locomotion alterations will help pinpoint the age at which gait changes become significant.
In this study, we showed that a FTIR mouse gait system14 modified here for the study of rats can be used to distinguish changes in gait parameters in 2-month-old male DKO rats, a model for hereditary PD, as compared to age-matched WT rats. Previous studies in PD patients revealed reduced stride length and lower average swing time, as well as increased stride time variability and swing time variability4. Thus, our findings of changes in swing time in DKO rats, and previous reports of swing time alterations in Pink1 KO and DJ-1 KO rats22, appear relevant to PD progression.

PD, the most common age-related neurodegenerative movement disorder, is caused by the loss of DA neurons in the substantia nigra pars compacta. This loss of nigral DA neurons and the DA inputs into the striatum lead to the observed motor function impairments seen in patients with PD1,2. The defining motor characteristics of patients with PD, known collectively as Parkinsonism, include rigidity, resting tremor, bradykinesia, postural instability, and micrographia3. Furthermore, gait disturbances, which are common in PD patients, appear early in the course of disease1,4,5. While certain lifestyles are suggested to help slow the progression of PD, such as healthy eating and regular exercise, there is currently no cure for PD, only medications to manage the symptoms. This leaves room for the need of further investigation in hopes of improved therapeutics. Thus, characterization of the gait pattern in PD animal models is a crucial tool to characterize the relevance of the model as well as how therapeutic treatments aimed at controlling PD are preventing or improving motor impairments.
There are various PD animal models that have been used to test therapeutic treatments, however each one has their limitations. For example, animal models treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) have yielded a great wealth of information about processes important for nigral DA neuron loss and subsequent striatal adaptations, and pointed to the role of mitochondria in PD pathogenesis; however, the pathogenetic background of the MPTP model is of a toxic nature rather than a neurodegenerative process as in human PD6. Additional chemically inducible models include 6-hydroxydopamine (6-OHDA) and rotenone. 6-OHDA was the first agent used to induce PD by selective accumulation of the drug in the DA neurons, which eventually kills the neurons and leads to PD like symptoms. This model was first used for the tracking of DA depletion by examining the behavior in response to amphetamine and apomorphine7. This method of PD induction has proved to be useful for the screening of pharmacological agents that impact DA and its receptors8. While the 6-OHDA model is a great model for tracking quantifiable motor deficits, this model does not show how the gradual loss of neurons and formation of Lewy bodies impact the animal. The other method of induction, rotenone, has been shown to have progressive degeneration of nigrostriatal neurons with the loss of tyrosine hydroxylase and DA transporter, allowing for a better model to track loss of neurons over time9. The rotenone treated rats showed bradykinesia, postural instability, and unsteady gait10. However, this method has been found to be widely variable between different strains of rats, which has provoked questioning whether or not rotenone is a reliable PD model11,12,13. While gait analysis has been shown to be impacted by the induction of PD in rats, to date, genetically induced PD rat models have not been readily used for gait analysis by freely walking down a runway.
One way to analyze motor impairment in freely walking rodents is kinematic gait analysis, which can be performed by utilizing FTIR imaging. This established method uses an optical touch sensor based on FTIR, which records and tracks the footprints of the rodents as they move down the runway14,15,16. As compared to other methods, FTIR does not depend on any markers on the animal&#39;s body that could interfere with the paw prints. Generation of the video data produces digital paw prints of all four limbs that can be combined to create a dynamic and reproducible walking pattern for various rodent models. The principle of imaging-based gait analysis is to take each individual paw and measure the contact area over time as the rodent walks down the runway. Each 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 proceeded by the swing phase, which is when there is no paw signal detected. After evaluation of the video, several parameters are generated that can be used to compare wild-type (WT) versus PD model. Some examples of the parameters are step length (distance the paw covers in one step), swing duration (duration of time the paw is not in contact with the runway), swing speed (step length as a function of swing duration), and step pattern (diagonal steps, lateral steps, or girdle steps).
To demonstrate the utility of FTIR to uncover early gait pattern changes in rats, we used a genetic rat model of PD. While most cases of PD are idiopathic; the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control17, that could be harnessed to create animal models18. Unfortunately, mice are resistant to neurodegeneration upon loss of these proteins (single and combined)19,20,21. In rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairments22, but without complete penetrance. Therefore, we generated a combined Pink1/Parkin double knockout (DKO) rat model, which displays the overt visually apparent hindlimb dragging phenotype reported in male Pink1 KO rats22 but now at a higher rate: 100% versus 30-50% of males between 4-6 months.
While this method works well for analyzing motor deficits in mice14, FTIR imaging gait system specifications to accommodate the size and weight of rats was previously unavailable noncommercially. Here we explain how to build the RatWalker, a modified FTIR gait imaging system modeled after the MouseWalker14, except adapted for the size and weight of rats. This system utilizes an optical effect, FTIR, to provide a method to visualize and subsequently record animal footprints for analysis. Contact of an animal&#39;s foot with the optical waveguide (platform) causes disruption in the light path resulting in a visible scattering effect, which is captured using domestic-grade, high-speed videography and processing using open-source software. This study demonstrates the power of FTIR imaging in studying gait changes in genetic rat models of PD. For example, while overt visually apparent motor changes (i.e. hindlimb dragging) are observed in male DKO rats at 4 months at the earliest, using FTIR we are able to uncover gate abnormalities in male DKO rats at 2 months of age.

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applying the ratwalker system for gait analysis in a genetic rat model of parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Rat Colony Maintenance 
The generation and characterization of Pink1 and Parkin single KO rats has been described previously22. The Pink1 and Parkin single KO rats were obtained from SAGE Labs (and now available from Envigo). DKO rats were generated by crossing Pink1-/- rats with Parkin-/- rats to obtain Pink1+/-/Parkin+/- rats, which were interbred to obtain Pink1-/-/Parkin-/- rats (will be available from Envigo). To confirm the deletion of 26 bp in Park6 (gene encoding Pink1), genotyping was performed using 5&#39;-CCCTGGCTGACTATCCTGAC-3&#39; forward and 5&#39;-CCACCACCCACTACCACTTACT-3&#39; reverse primers. Deletion of 5 bp in Park2 (gene encoding Parkin) was tested after DNA was amplified using a forward 5&#39;-GGTGTCTTGGCTCAGTGTGA-3&#39; and reverse 5&#39;-GCCACCCAGAATAGCATCTC-3&#39;. Amplified Polymerase Chain Reaction (PCR) samples were sent to ACGT Inc (Wheeling, IL) for sequencing (Figure S7). All rats were kept on the Long Evans Hooded (LEH) background. DKO rats were viable and fertile; however, there was a high rate of mortality among DKO dams at the time of parturition (approximately 30%). Only male rats were utilized in these experiments. Rats were kept in a temperature-controlled environment with a 12-hour light/dark cycle and free access to rat chow and water.
Outcomes 
To serve as an example of the utility of the FTIR gait analysis system for rats, adapted from14, we performed gait analysis on male WT and Pink1/Parkin DKO rats at 2 months of age to determine if the use of kinematic gait analysis could uncover subtle motor impairments not observed with human visual perception prior to the appearance of gross motor problems starting at 4 months of age.
Similar to previous gait studies in mice14, the adapted FTIR system was able to display the footprint pattern created by the walking rat as well as the path created by the body center (Figure 1A). Despite the increased weight of the DKO rats as compared to WT (Figure 1B), the foot pressure applied to the walking surface (visualized as heat maps) as determined by the intensities of the FTIR signal, was unaltered (Figure 1C). Upon assessment of several gait parameters as a function of walking speed (Figure 1D-H), we observed that the walking speed and step lengths were similar between WT and DKO rats (Figure 1D). However, the variation between WT and DKO rats became apparent in stance phase and swing duration at slower walking speeds (Figure 1E, F). The fraction of the step cycle where the leg is in the stance phase (stance duration / period) is the duty factor, and this parameter highlights more time spent in the swing phase than in the stance phase as the duty factor decreases, typical of running (Figure 1G). Again, differences are highlighted at lower speeds. Further, while swing speeds increase with increased speed in WT animals, the correlation is blunted in DKO rats (Figure 1H).
The FTIR gait analysis also allowed for plots of stance phase traces of each leg relative to the body in freely walking rats (Figure 2A, B). The stance traces are normalized to body length and are defined as the position of the foot relative to the body center from paw touchdown (anterior extreme position, AEP) to the end of the stance phase (posterior extreme position, PEP). Upon comparing the paw positioning, we observed significant changes in AEP (left hindlimb) and PEP (right hindlimb) suggesting the left hindlimb is closer to the body during paw touchdown (AEP), while the right hindlimb is further away from the body during paw takeoff (PEP) in DKO as compared to WT rats (Figure 2C).
Several additional parameters were significantly changed in the DKO rats as compared to WT. In particular, changes in hindlimb swing patterns were uncovered. The swing speed of both the left and right hindlimbs was increased in DKO rats as compared to WT rats (Figure 3A), while the swing duration of both left and right hindlimbs was decreased (Figure 3B). Of note, step length was unaltered (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62002/62002fig01.jpg" /> 
Figure 1. Footprint pattern and step parameter analysis. Representative footprint patterns for (A) WT and (B) DKO rats showing (top panel) footprint heat map representing pixel intensity and horizontal line representing the body path as well as (bottom panel) individual feet labeled with different colors: left fore (LF, yellow), left hind (LH, blue), right fore (RF, orange), and right hind (RH, green). (C) Average walking speed for each WT (n = 7) and DKO (n = 8) rat. Mean with SEM. Not significant. (D-H) Step parameters as a function of speed in WT (n = 7) and DKO (n = 8) rats. Linear regression lines and R square values included. (D) Step length increases with speed in WT and DKO rats. (E) Swing duration is inversely proportional to speed in WT rats, but not in DKO rats (R not significant). (F) Stance duration decreases with speed in WT and DKO rats. (G) Duty factor is inversely proportional to speed in DKO rats, but not WT rats (R not significant). (H) Swing speed increases linearly with speed in WT rats, but not DKO rats (R not significant). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62002/62002fig01large.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/62002/62002fig02.jpg" /> 
Figure 2. Stance traces and paw positioning analysis. (A) Representative walking track analysis for a WT rat (before and after background correction) visualized with nose (red solid), head contour (blue dashed), tail contour (green dashed), body center (white dashed), and footprints (circles: green, RF and light blue, LH). (B) Representative plots of stance traces for freely walking WT and DKO rats. (C) Paw positioning in WT (n = 7) and DKO (n = 8) rats are shown. AEP, anterior extreme position; PEP, posterior extreme position; L, left; R, right; F, forepaw; H, hindpaw. Mean with SEM. Significant compared to WT (p &#60; 0.05*, 0.001***) using two-way ANOVA and Sidak&#39;s multiple comparison test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62002/62002fig02large.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/62002/62002fig03.jpg" /> 
Figure 3. Hindpaw swing parameters altered in DKO rats. Forepaw and hindpaw measurements of the (A) speed at which paws are moving and the (B) time paws are airborne in WT (n = 7) and DKO (n = 8) rats. L, left; R, right; F, forepaw; H, hindpaw. Mean with SEM. Significant compared to WT (p &#60; 0.01**) using Student&#39;s unpaired two-tailed t-test with Welch&#39;s correction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62002/62002fig03large.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/62002/62002fig04.jpg" /> 
Figure 4. Step length unaltered in DKO rats. Forepaw and hindpaw measurements of the step length in WT (n = 7) and DKO (n = 8) rats. L, left; R, right; F, forepaw; H, hindpaw. Mean with SEM. Significant compared to WT (not significant) using Student&#39;s unpaired two-tailed t-test with Welch&#39;s correction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62002/62002fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62002/Supplementary Figures S1-7.docx" target="_blank">Please click here to download this file.</a>
Supplemental Table. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62002/Supplementary Tables S1-4.docx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease at JoVE.com

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

applying-ratwalker-system-for-gait-analysis-genetic-rat-model

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.7K Views.  University of Nebraska Medical Center. Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters. 

Video: Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.