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 str...

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+reproducible+rotenone+model+of+Parkinson's+disease.">A highly reproducible rotenone model of Parkinson's disease.</a> Neurobiology of Disease. 34 (2), 279-290 (2009).</li><li>Quary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+strain+differences+in+response+to+chronic+systemic+administration+of+the+mitochondrial+toxin+3-nitropropionic+acid+in+rats:+implications+for+neuroprotection+studies.">Major strain differences in response to chronic systemic administration of the mitochondrial toxin 3-nitropropionic acid in rats: implications for neuroprotection studies.</a> Neuroscience. 97 (3), 521-530 (2000).</li><li>Cicchetti, F., Drouin-Ouellet, J., Gross, R. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gait+parameters+in+freely+walking+rodents.">Quantification of gait parameters in freely walking rodents.</a> BMC Biology. 13, 50 (2015).</li><li>Hamers, F. P., Lankhorst, A. J., van Laar, T. J., Veldhuis, W. B., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+quantitative+gait+analysis+during+overground+locomotion+in+the+rat:+its+application+to+spinal+cord+contusion+and+transection+injuries.">Automated quantitative gait analysis during overground locomotion in the rat: its application to spinal cord contusion and transection injuries.</a> Journal of Neurotrauma. 18 (2), 187-201 (2001).</li><li>Hamers, F. P., Koopmans, G. C., Joosten, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CatWalk-assisted+gait+analysis+in+the+assessment+of+spinal+cord+injury.">CatWalk-assisted gait analysis in the assessment of spinal cord injury.</a> Journal of Neurotrauma. 23 (3-4), 537-548 (2006).</li><li>Pickrell, A. M., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+PINK1,+parkin,+and+mitochondrial+fidelity+in+Parkinson's+disease.">The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease.</a> Neuron. 85 (2), 257-273 (2015).</li><li>Dawson, T. M., Ko, H. S., Dawson, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+animal+models+of+Parkinson's+disease.">Genetic animal models of Parkinson's disease.</a> Neuron. 66 (5), 646-661 (2010).</li><li>Gispert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson+phenotype+in+aged+PINK1-deficient+mice+is+accompanied+by+progressive+mitochondrial+dysfunction+in+absence+of+neurodegeneration.">Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration.</a> PLoS One. 4 (6), 5777 (2009).</li><li>Goldberg, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkin-deficient+mice+exhibit+nigrostriatal+deficits+but+not+loss+of+dopaminergic+neurons.">Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons.</a> Journal of Biological Chemistry. 278 (44), 43628-43635 (2003).</li><li>Kitada, T., Tong, Y., Gautier, C. A., Shen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+nigral+degeneration+in+aged+parkin/DJ-1/PINK1+triple+knockout+mice.">Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice.</a> Journal of Neurochemistry. 111 (3), 696-702 (2009).</li><li>Dave, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+recessive+gene+knockout+rat+models+of+Parkinson's+disease.">Phenotypic characterization of recessive gene knockout rat models of Parkinson's disease.</a> Neurobiology of Disease. 70, 190-203 (2014).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Hruska, R. E., Kennedy, S., Silbergeld, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+aspects+of+normal+locomotion+in+rats.">Quantitative aspects of normal locomotion in rats.</a> Life Sciences. 25 (2), 171-179 (1979).</li><li>de Medinaceli, L., Freed, W. J., Wyatt, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+index+of+the+functional+condition+of+rat+sciatic+nerve+based+on+measurements+made+from+walking+tracks.">An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks.</a> Experimental Neurology. 77 (3), 634-643 (1982).</li><li>Kunkel-Bagden, E., Dai, H. N., Bregman, B. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+Analysis+Methods+for+Rodent+Models+of+Osteoarthritis.">Gait Analysis Methods for Rodent Models of Osteoarthritis.</a> Current Pain and Headache Reports. 18 (10), 456 (2014).</li><li>Boix, J., von Hieber, D., Connor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+Analysis+for+Early+Detection+of+Motor+Symptoms+in+the+6-OHDA+Rat+Model+of+Parkinson's+Disease.">Gait Analysis for Early Detection of Motor Symptoms in the 6-OHDA Rat Model of Parkinson's Disease.</a> Frontiers in Behavioral Neuroscience. 12, 39 (2018).</li><li>Zhou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+analysis+in+three+different+6-hydroxydopamine+rat+models+of+Parkinson's+disease.">Gait analysis in three different 6-hydroxydopamine rat models of Parkinson's disease.</a> Neuroscience Letters. 584, 184-189 (2015).</li><li>Vandeputte, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+quantitative+gait+analysis+in+animal+models+of+movement+disorders.">Automated quantitative gait analysis in animal models of movement disorders.</a> BMC Neuroscience. 11, 92 (2010).</li><li>Chuang, C. 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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 approache...

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-/- ...

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&#039;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.

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 ...

This method makes it possible to reproducibly quantify differences in gait parameters. The gait parameters can be assessed in freely-walking rodents, and the technique does not rely on invasive steps such as dipping the feet with ink. Demonstrating the procedure will be Ben Lamberty.Begin by placing 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. Turn off the room lights. Power on the camera and start recording several seconds before the rat is placed on the platform.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. Stop recording once the animal reaches the terminus of the walkway. Clean the walkway using 70%ethanol and a non-abrasive towel in between runs and after an animal urinates or defecates.Then, allow ethanol to evaporate before introducing another animal. Run the rats through the walkway a total of seven times during each observation period, taking the first three runs that score as passing for analysis. 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.Gait analysis was performed on male wild-type and Parkin DKO rats at two months of age to determine if the use of kinematic gait analysis could uncover subtle motor impairments prior to the appearance of gross motor problems. Despite the increased weight of the DKO rats, the foot pressure applied to the walking surface was unaltered. Upon assessment of several gait parameters as a function of walking speed, the walking speed and step lengths were found to be similar between wild-type and DKO rats.The variation between wild-type and DKO rats became apparent in stance phase and swing duration at slower walking speeds. The fraction of the step cycle where the leg is in the stance phase was the duty factor. More time was spent in the swing phase than in the stance phase as the duty factor decreased.Furthermore, while swing speeds increased with increased speed in wild-type animals, the correlation was blunted in DKO rats. The FTIR gait analysis was also used to create plots of stance phase traces of each leg relative to the body in freely-walking rats. Upon comparing the paw positioning, significant changes in AEP and PEP were observed.Several additional parameters were significantly changed in the DKO rats as compared to wild-type. The swing speed of both the left and right hind limbs was increased in DKO rats, while the swing duration of both left and right hind limbs was decreased. Consistent setup of equipment, including camera position with relation to RatWalker, is important for the successful implementation of this protocol.After performing this protocol, animals are available for any additional behavior or experimental endpoints necessary. For example, gait changes can be correlated with histological alterations. This technique can be used to assess gait in other non-PD rodent models, such as models of anxiety and depression.

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Research

JoVE Journal

Neuroscience

2.6K Views.  University of Nebraska Medical Center. This method makes it possible to reproducibly quantify differences in gait parameters. The gait parameters can be assessed in freely-walking rodents, and the technique does not rely on invasive steps such as dipping the feet with ink. Demonstrating the procedure will be Ben Lamberty.Begin by placing 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. Turn off the room lights. Power on the camera and start r...

Video: 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.

low-cost gait analysis for behavioral phenotyping of mouse models of neuromuscular disease

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse...

the 6-hydroxydopamine rat model of parkinson's disease

The 6-hydroxydopamine Rat Model of Parkinson&#039;s Disease

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...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

paw-print analysis of contrast-enhanced recordings (prancer): a low-cost, open-access automated gait analysis system for assessing motor deficits

Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

We describe a novel gait analysis system, Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER), an open-access automated system for the quantification of gait characteristics in rats that utilizes a novel semitransparent floor to automatically quantify gait. This system was validated using the haloperidol model of Parkinson&#8217;s...

Paw-Print Analysis of Contrast-Enhanced Recordings (PrAnCER): A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits

induction and assessment of levodopa-induced dyskinesias in a rat model of parkinson's disease

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#039;s Disease

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...

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

gait analysis in an angelman syndrome mouse model

Gait Analysis in an Angelman Syndrome Mouse Model  

a quick phenotypic neurological scoring system for evaluating disease progression in the sod1-g93a mouse model of als

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

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

3d kinematic gait analysis for preclinical studies in rodents

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

generalized psychophysiological interaction (ppi) analysis of memory related connectivity in individuals at genetic risk for alzheimer's disease

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;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...

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

clinical-oriented three-dimensional gait analysis method for evaluating gait disorder

Clinical-oriented Three-dimensional Gait Analysis Method for Evaluating Gait Disorder

In this study, a clinician-friendly three-dimensional gait analysis method, which was designed to be performed in the rehabilitation clinic, is presented. The method consists of a simplified measurement method and intuitive figures to facilitate clinicians&#39; understanding of the...

home-based monitor for gait and activity analysis

Home-Based Monitor for Gait and Activity Analysis

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular...

gait analysis of age-dependent motor impairments in mice with neurodegeneration

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...

3d kinematic analysis for the functional evaluation in the rat model of sciatic nerve crush injury

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

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...

use of rotorod as a method for the qualitative analysis of walking in rat

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue...

rating l-dopa-induced dyskinesias in the unilaterally 6-ohda-lesioned rat model of parkinson's disease

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson&#039;s Disease

Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's...

Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease

histological examination of mitochondrial morphology in a parkinson's disease model

Author Spotlight: Establishing a New Fluorescence-Based Protocol for In Vivo Mitochondrial Morphology Analysis in Parkinson&#039;s Disease

This study presents a method to analyze the morphology of mitochondria based on immunostaining and image analysis in mouse brain tissue in situ. It also describes how this allows one to detect changes in mitochondrial morphology induced by protein aggregation in Parkinson's disease...

Histological Examination of Mitochondrial Morphology in a Parkinson's Disease Model

measuring constipation in a drosophila model of parkinson's disease

Author Spotlight: Exploring Non-Motor Symptoms in Parkinson's Disease 

This protocol presents an assay for modeling constipation in an alpha-synuclein based Drosophila model of Parkinson's disease.

Measuring Constipation in a Drosophila Model of Parkinson's Disease

development of an alpha-synuclein based rat model for parkinson's disease via stereotactic injection of a recombinant adeno-associated viral vector

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system.  The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

genetic studies of human dna repair proteins using yeast as a model system 

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

a model of chronic nutrient infusion in the rat

A Model of Chronic Nutrient Infusion in the Rat

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological...

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 ...

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, primarily in the substantia nigra. The disease causes motor deficiencies, which present as rigidity, tremors and dementia in humans. Rotenone is an insecticide that causes oxidative damage by inhibiting the function of the electron transport chain in mitochondria. It is also used to model Parkinson&#8217;s disease in the Drosophila. Flies have an inherent negative geotactic response, which compels them to climb upwards upon being startled. It has been established that rotenone causes early mortality and locomotion defects that disrupt the flies&#8217; ability to climb after they have been tapped downwards. However, the effect of rotenone on spontaneous movement is not well documented. This study outlines two sensitive, reproducible, and high throughput assays to characterize rotenone-induced deficiencies in short-term startle-induced locomotion and long-term spontaneous locomotion in Drosophila. These assays can be conveniently adapted to characterize other Drosophila models of locomotion defects and efficacy of therapeutic agents.

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson&#039;s Disease Model of Drosophila

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, causing locomotion defects. Rotenone models Parkinson&#8217;s disease in Drosophila. This paper outlines two assays that characterize both spontaneous and startle-induced locomotion deficiencies caused by rotenone.

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

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 ...

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 ...

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.

Analyzing the Parkinson&#039;s Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &amp;#945;-Synuclein

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.