This work was supported by the Carney Institute for Brain Science at Brown University.

All procedures were in accordance with Brown University Institutional Animal Care and Use Committee guidelines.
1. Gait analysis apparatus
<ol>
	<li>Prepare the gait analysis walkway consisting of a clear plexiglass enclosed walkway (36&#8221; L x 3&#8221; W x 4.5&#8221; H) placed on a clear plexiglass floor (Figure 2A). Make the plexiglass floor semitransparent by covering it with a piece of 16 LB cotton fiber drafting vellum cut to the same width as the walkway.&#160; 
	NOTE: There are other methods for making the floor semitransparent.</li>
	<li>Place a camera with a frame rate of at least 30 frames per second (fps) directly below the walkway to capture the middle of the track (Figure 2B).</li>
	<li>Secure a strip of 12 V LED lights with 18 LEDs/foot approximately 2 inches away from and 1 inch above the floor of the walkway to illuminate the track.</li>
</ol>
2. Animal preparation
<ol>
	<li>Allow the animals to acclimate to the vivarium for at least 1 week prior to handling. Handle the rats for at least 5 days prior to beginning experiment. This study used 8 male Long Evans rats approximately 3 months old.</li>
	<li>Habituate&#160;the animals to the testing room and gait walkway with the room lights turned off
	<ol>
		<li>Place the rat&#8217;s home cage at the surface level at the end of the gait walkway to serve as a goal box. Note that if the home cage is deep, aged or locomotor-impaired rats may benefit from a ramp or step to provide easier access to the home cage.</li>
		<li>Allow the rat to walk from the experimenter&#8217;s hand down the length of the walkway to reach its home cage.</li>
		<li>Rats will often stop at the end of the walkway to look around before jumping down into the home cage. If a rat takes longer than 1 min to exit the walkway, encourage it to enter its home cage with a gentle push.</li>
		<li>If the rat turns around, use a small piece of plexiglass to block the &#8220;start&#8221; end of the walkway. Repeat for a total of 3 runs.</li>
	</ol>
	</li>
	<li>Habituate for at least 2 days or until the rats are comfortable crossing the walkway at a steady pace without freezing.</li>
</ol>
3. Gait testing procedure
<ol>
	<li>Adjust the settings on the webcam software to achieve the clearest picture of the paw prints. Turn off room lights for all gait testing.</li>
	<li>Record each run separately and label appropriately for use with the automated analysis program.</li>
	<li>Ensure there are no spots or debris on the vellum. Begin recording a few seconds before the rat enters the walkway and stop once the rat exits the walkway and enters its home cage.</li>
	<li>Continue until either three acceptable runs are completed or 10 min has elapsed.</li>
	<li>Wipe down the walkway with ethanol in between each rat and replace the vellum as needed. 
	NOTE: An acceptable trial is defined as one in which the animal walks consistently and without pause for the first 4 steps of the run. If this is hard to achieve, adjust the criteria to include trials in which there are 4 consecutive steps at any point in the run that occur without pauses or abrupt acceleration.</li>
</ol>
4. PrAnCER automated analysis
<ol>
	<li>Put all videos to be analyzed in a folder.</li>
	<li>Launch PrAnCER by running the Python script PrAnCER. PrAnCER will analyze the videos based on the steps illustrated in Figure 3 and Figure 4.</li>
	<li>In the pop-up menu, select the specified folder by pressing the Choose a folder button. Select custom options for analysis if desired. Detailed descriptions of each parameter can be found by clicking the question mark next to them. Click Continue when finished.</li>
	<li>Define a region of interest (ROI) on the image of the walkway that appears. To do this, left click to define a top edge and right click to define a bottom edge. If the box that appears is correct, press N to continue. If not, press Z to undo. Once N is pressed, the program will run automatically.</li>
	<li>After PrAnCER is complete, terminate the program by pressing Enter in the terminal.</li>
	<li>To manually review the results&#160;output by PrAnCER, run&#160;the Python script GaitEditorGUI and select&#160;the appropriate .mp4 file for each video. If needed, correct any mis-identified or merged prints.</li>
	<li>To extract spatial and temporal gait parameters, run the Python script ParameterAnalyzer. Choose the number of hind prints to analyze and the folder of videos to analyze, then click Continue. This will output a .csv file for each video containing a number of common gait parameters, which are described in Table 2 and illustrated in Figure 5. 
	NOTE: The full scripts, as well as instructions for reading and analyzing data, are available at the author&#8217;s GitHub (www.github.com/hayleybounds). We implemented this algorithm using the free, open-source Python library OpenCV21. Also included on the GitHub are instructions for building our gait analysis walkway.</li>
</ol>

<ol>
	<li>Lakes, E. H., Allen, K. 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+arthritic+disorders:+reviews+and+recommendations.">Gait analysis methods for rodent models of arthritic disorders: reviews and recommendations.</a> Osteoarthritis and Cartilage. 24 (11), 1837-1849 (2016).</li><li>Lee, H. Y., Hsieh, T. H., Liang, J. I., Yeh, M. L., Chen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+video-based+gait+pattern+analysis+for+hemiparkinsonian+rats.">Quantitative video-based gait pattern analysis for hemiparkinsonian rats.</a> Medical &amp; Biological Engineering &amp; Computing. 50 (9), 937-946 (2012).</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&#8217;s+disease.">Gait analysis in three different 6-hydroxydopamine rat models of Parkinson&#8217;s disease.</a> Neuroscience Letters. 584, 184-189 (2015).</li><li>Batka, R. J., Brown, T. J., Mcmillan, K. P., Meadows, R. M., Jones, K. J., Haulcomb, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+for+speed+in+rodent+locomotion+analyses.">The need for speed in rodent locomotion analyses.</a> Anatomical Record. 297 (10), 1839-1864 (2014).</li><li>Guillot, T. S., Asress, S. A., Richardson, J. R., Glass, J. D., Miller, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treadmill+gait+analysis+does+not+detect+motor+deficits+in+animal+models+of+Parkinson&#8217;s+disease+or+amyotrophic+lateral+sclerosis.">Treadmill gait analysis does not detect motor deficits in animal models of Parkinson&#8217;s disease or amyotrophic lateral sclerosis.</a> Journal of Motor Behavior. 40 (6), 568-577 (2008).</li><li>Williams, M. T., 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=Kaolin-induced+ventriculomegaly+at+weaning+produces+long-term+learning,+memory,+and+motor+deficits+in+rats.">Kaolin-induced ventriculomegaly at weaning produces long-term learning, memory, and motor deficits in rats.</a> International Journal of Developmental Neuroscience. 35, 7-15 (2014).</li><li>Hamers, F. P. T., Koopmans, G. C., Joosten, E. A. J. <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>Kloefkorn, H. E., 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+Gait+Analysis+Through+Hues+and+Areas+(AGATHA):+A+Method+to+Characterize+the+Spatiotemporal+Pattern+of+Rat+Gait.">Automated Gait Analysis Through Hues and Areas (AGATHA): A Method to Characterize the Spatiotemporal Pattern of Rat Gait.</a> Annals of Biomedical Engineering. 45 (3), 711-725 (2017).</li><li>Pereira, J. E., 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+comparison+analysis+of+hindlimb+kinematics+during+overground+and+treadmill+locomotion+in+rats.">A comparison analysis of hindlimb kinematics during overground and treadmill locomotion in rats.</a> Behavioural Brain Research. 172 (2), 212-218 (2006).</li><li>Nakamura, A., 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=Low-cost+three-dimensional+gait+analysis+system+for+mice+with+an+infrared+depth+sensor.">Low-cost three-dimensional gait analysis system for mice with an infrared depth sensor.</a> Neuroscience Research. 100, 55-62 (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>Mendes, C. S., Bartos, I., M&#225;rka, Z., Akay, T., M&#225;rka, S., Mann, R. S. <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>Machado, A. S., Darmohray, D. M., Fayad, J., Marques, H. G., Carey, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+framework+for+whole-body+coordination+reveals+specific+deficits+in+freely+walking+ataxic+mice.">A quantitative framework for whole-body coordination reveals specific deficits in freely walking ataxic mice.</a> eLife. 4, (2015).</li><li>Duty, S., Jenner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Parkinson&#8217;s+disease:+a+source+of+novel+treatments+and+clues+to+the+cause+of+the+disease.">Animal models of Parkinson&#8217;s disease: a source of novel treatments and clues to the cause of the disease.</a> British Journal of Pharmacology. 164 (4), 1357-1391 (2011).</li><li>Kulkarni, S. K., Bishnoi, M., Chopra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+microdialysis+studies+of+striatal+level+of+neurotransmitters+after+haloperidol+and+chlorpromazine+administration.">In vivo microdialysis studies of striatal level of neurotransmitters after haloperidol and chlorpromazine administration.</a> Indian Journal of Experimental Biology. 47 (2), 91-97 (2009).</li><li>Jain, N. S., Tandi, L., Verma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+central+histaminergic+transmission+in+the+cataleptic+and+neuroleptic+effects+of+haloperidol.">Contribution of the central histaminergic transmission in the cataleptic and neuroleptic effects of haloperidol.</a> Pharmacology Biochemistry and Behavior. 139 (Part A), 59-66 (2015).</li><li>Steinpreis, R. E., Anders, K. A., Branda, E. M., Kruschel, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Atypical+Antipsychotics+and+Phencyclidine+(PCP)+on+Rotorod+Performance.">The Effects of Atypical Antipsychotics and Phencyclidine (PCP) on Rotorod Performance.</a> Pharmacology Biochemistry and Behavior. 63 (3), 387-394 (1999).</li><li>Baptista, P. P. A., 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=Physical+exercise+down-regulated+locomotor+side+effects+induced+by+haloperidol+treatment+in+Wistar+rats.">Physical exercise down-regulated locomotor side effects induced by haloperidol treatment in Wistar rats.</a> Pharmacology, Biochemistry, and Behavior. 104, 113-118 (2013).</li><li>Chuang, C. S., Su, H. L., Cheng, F. C., Hsu, S., Chuang, C. F., Liu, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+motor+function+before+and+after+engraftment+of+dopaminergic+neurons+in+a+rat+model+of+Parkinson&#8217;s+disease.">Quantitative evaluation of motor function before and after engraftment of dopaminergic neurons in a rat model of Parkinson&#8217;s disease.</a> Journal of Biomedical Science. 17 (9), (2010).</li><li>Bugalho, P., Alves, L., Miguel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gait+dysfunction+in+Parkinson&#8217;s+disease+and+normal+pressure+hydrocephalus:+a+comparative+study.">Gait dysfunction in Parkinson&#8217;s disease and normal pressure hydrocephalus: a comparative study.</a> Journal of Neural Transmission. 120 (8), 1201-1207 (2013).</li><li>Bradski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The OpenCV Library. , (2000).</li><li>Chen, H., Du, J., Zhang, Y., Barnes, K., Jia, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+a+Reliable+Gait+Evaluation+Method+for+Rodent+Studies.">Establishing a Reliable Gait Evaluation Method for Rodent Studies.</a> Journal of Neuroscience Methods. 283, 92-100 (2017).</li><li>De Ryck, M., Hruska, R. E., 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=Estrogen+and+haloperidol-induced+versus+handling-related+catalepsy+in+male+rats.">Estrogen and haloperidol-induced versus handling-related catalepsy in male rats.</a> Pharmacology, Biochemistry, and Behavior. 17 (5), 1027-1035 (1982).</li><li>Bellardita, C., Kiehn, O. <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+Speed-Associated+Gait+Changes+in+Mice+Reveals+Modular+Organization+of+Locomotor+Networks.">Phenotypic Characterization of Speed-Associated Gait Changes in Mice Reveals Modular Organization of Locomotor Networks.</a> Current Biology. 25 (11), 1426-1436 (2015).</li><li>De Ryck, M., Schallert, T., Teitelbaum, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+versus+haloperidol+catalepsy+in+the+rat:+a+behavioral+analysis+of+postural+support+mechanisms.">Morphine versus haloperidol catalepsy in the rat: a behavioral analysis of postural support mechanisms.</a> Brain Research. 201 (1), 143-172 (1980).</li></ol>

The authors have no conflicts of interest to disclose.

In this study, we tested PrAnCER, a new automated gait analysis system that utilizes contrast-enhanced videos produced using a translucent floor to obscure the animal&#8217;s body and to yield clearly defined paw prints for simple automated detection. PrAnCER accurately identifies paw prints and is sensitive to changes in motor function. We used PrAnCER to assess gait alterations in an acute haloperidol model of PD. Although haloperidol did not induce the expected motor deficits of a robust PD model, we were nonetheless able to demonstrate that PrAnCER can accurately detect changes in gait patterns. Finally, we quantified the accuracy of PrAnCER and demonstrated that its measurement of key gait parameters is comparable to that of manual scoring.
In both haloperidol treated conditions, we observed a high incidence of freezing behavior (akinesia) followed by an escape response of running or bounding forward. While akinesia has been observed at a similar dose (0.25 mg/kg) in several studies16,23, this bounding behavior is not consistent with typical Parkinsonian symptoms3,14,19,24. Interestingly, we found that high dose haloperidol treatment resulted in significantly increased stride length. This finding was initially surprising because other haloperidol models of PD have shown a decrease in stride length3,19. However, they make sense in light of the &#8216;bracing-escape&#8217; behavior pattern described by De Ryck et al. (1980), who reported that rats run to escape after akinetic periods, and that high speed gaits such as running and bounding are associated with increased stride length4,25 (Figure 8C). High dose treatment also resulted in significantly increased maximum contact area of the hind paws. Low dose haloperidol treatment resulted in more characteristic PD gait alterations including a significant increase in stance duration and maximum contact area (Figure 8A-B). These results may be a reflection of the muscle rigidity associated with haloperidol-induced akinesia.
Despite the unusual bracing-escape behavior, we were able to demonstrate that PrAnCER can indeed detect alterations in gait. We showed that in the correct lighting conditions, a translucent floor can produce a highly contrasted and detailed image of the paws. In the present study, we made a transparent floor translucent by covering it with vellum. The same effect could be achieved by placing another translucent covering, such as Mylar, over a transparent floor. Alternatively, the floor itself could be translucent by using, for example, frosted plexiglass. The translucent floor and simple plexiglass walkway are inexpensive and can be constructed in an afternoon. Our edge-detection-based analysis system is resilient to many variations in the apparatus and offers adjustable thresholds to adapt the system to different setups, disease models, or smaller animals such as mice.
Some gait parameter analyses were altered from conventional formulas because of aspects of the walkway. For example, our method of calculating speed differs from other gait studies; the translucent floor combined with LED lighting obscures the view of the body, so it is not possible to track body position to calculate speed as is usually done. For this study, speed was calculated by dividing the distance travelled between two contacts of the same paw by the time from first contact to second contact. Of course, other formulas could be used. For example, if an overall measure of speed is needed, one could divide the distance from the average of forepaw locations at the beginning and end of the run by the duration of the run.
Our analysis confirms that, while not identical to manual scoring, our automated system performs with high accuracy and generates reliable measures of gait. The apparatus described here was optimized for a simple, low-cost analysis of motor function. However, several alterations could be made that would expand the usefulness of PrAnCER. One limitation of our system is that the semitransparent floor, while allowing excellent paw detection, obscures the animals&#8217; body axis. Although we have not found it necessary, this could be addressed by adding an overhead camera to the system. Another improvement would be the use of a video camera with a higher frame rate. While we were able to obtain consistent estimates of temporal parameters, the accuracy of these measures is compromised at frame rates below 100 fps8. Adding a high-speed video camera would require no alteration of the analysis software while increasing the accuracy and precision of temporal measures. Additionally, several other gait systems use a mirror to simultaneously record the lateral and ventral views of the rat2,8,13. Adding this feature to our apparatus would permit more accurate quantification of speed and better observation of behavior during runs.
In this study, we showed that the use of a semitransparent floor effectively isolates paw prints by blocking the visibility of objects not in contact with the walkway floor. We developed an automated scoring system that takes advantage of this high contrast paw print to accurately identify paws. We showed that this system, PrAnCER, quantified gait parameters with an accuracy comparable to commercial systems. We determined that administration of a high dose of haloperidol increased stride length and maximum contact area compared to saline. While this change is the opposite of what we expected, further review of existing literature indicates that it is likely part of the escape behavior observed in response to acute administration of haloperidol. Low dose haloperidol treatment resulted in more typical PD symptoms such as increased stance duration and maximum contact area. We conclude that while acute high dose haloperidol administration is a poor model to study gait impairments associated with PD, our study nonetheless demonstrated the ability of PrAnCER to accurately detect changes in motor function. In the future, we hope to further validate PrAnCER by studying the locomotor changes in other disease models.

Rodents are commonly used as models to study a wide range of diseases and injuries including arthritis1, Parkinson&#8217;s Disease (PD)2,3, neuromuscular disorders4,5, hydrocephalus6, and spinal cord injury7. In these conditions, symptoms such as pain, balance, and motor function can be measured by studying the animals&#8217; gait patterns. These patterns are quantified using a set of spatiotemporal gait parameters that summarize the location and timing of paw prints as well as the area of paw contact on the ground.
Although many options for gait analysis exist, current systems have several drawbacks. In traditional ink and paper testing, an animal&#8217;s paws are coated with ink before it walks across a sheet of white paper (Figure 1A). The resulting paw prints can then be measured for stride length and stance width, but key temporal gait parameters such as speed or step duration cannot be assessed. Modern video-based systems are more reliable, but video analysis requires laborious frame-by-frame scoring unless a suitable automated system is used8. There are many commercial automated scoring systems currently available, but these systems can be prohibitively expensive. Additionally, these systems rely on clear flooring or in some cases, treadmills, both of which alter natural movement. Treadmills have been shown to mask motor deficits in some disease models9, while clear flooring (Figure 1B) causes mice to spend more time on the perimeter of an open field, indicating increased anxiety10. Ideally, a gait analysis apparatus would not rely on either, producing the most natural movement patterns with the least stress to the animal.
Available open-source and commercial options use a variety of methods to overcome the difficulty of isolating a footprint from the animal&#8217;s body despite variable lighting conditions, animal color, and print shapes. Some enhance the contrast of contacting paws using surfaces that release light in response to pressure7,11,12, but these are expensive and technically difficult to construct. Other systems utilize multiple view angles which permit observation of whole-body coordination8,13. While these options offer advantages for measuring additional motor parameters beyond gait, they are unnecessarily complex for simple gait analysis. Further, all of these techniques rely on clear flooring, which alters natural behavior.
PrAnCER is based on what we call Contrast-Enhanced Recordings, which use a combination of lighting and a semitransparent floor to enhance the detection of prints. When viewed from below, this creates a high-contrast image (paw print) while obscuring the view of objects not in contact with the surface (the animal&#8217;s body) (Figure 1D). When viewed from above, the floor appears opaque. The resulting salience of the paws in our method permits accurate identification of a variety of gait and locomotor characteristics by our newly developed automated system. In the present study, we describe the apparatus, our gait analysis protocol, and our automated scoring system, PrAnCER. Our apparatus is easily assembled and PrAnCER can be used to assess motor deficits in a wide range of disease and injury models.
To demonstrate that PrAnCER can be used to detect abnormal gait patterns, we used a haloperidol model of PD, a simple model for transient induction of locomotor changes14. Haloperidol is a dopamine receptor antagonist widely used as an antipsychotic1. It affects motor systems by altering dopamine signaling in the striatum, an important component of the motor pathway in the basal ganglia14. Even a single dose of haloperidol rapidly reduces extracellular dopamine levels in the striatum, causing Parkinsonian-like motor defecits15. The behavioral effects are muscular rigidity, akinesia, and catalepsy, which is defined as an inability to return to a normal posture after being placed in an unusual position11,16. Acute doses of haloperidol cause locomotor deficits identifiable in the rotarod test of motor function17. We reasoned that haloperidol-mediated locomotor impairments would also be evident in a number of characteristics accessible to automated gait analysis.
Although responses to haloperidol vary widely across studies, cataleptic effects of haloperidol emerge at doses of 0.5 mg/kg and higher, while reduced responsiveness and motor impairment is detectable at lower doses (0.1 - 0.3 mg/kg)16,17. In an effort to avoid the cataleptic effects of haloperidol, we decided to test two doses of haloperidol: a high dose (0.30 mg/kg) and a low dose (0.15 mg/kg). As shown in Table 1, Experiment 1 examined the effects of high dose haloperidol, while Experiment 2 tested the effects of low dose haloperidol. We used a within-subject design in which every rat was tested in the high dose, low dose, and control (saline) conditions. The order of condition was counterbalanced across rats. We predicted that acute administration of haloperidol would cause gait impairments similar to those found in other models of PD such as decreased speed, decreased stride length, and longer stance duration3,14,18,19. We observed behavioral changes including akinesia following haloperidol administration at both dosages. In the low dose condition, rats had significantly increased stance duration and hind paw contact area, as expected. These gait changes are comparable to the slow, shuffling steps common among PD patients2,20. In the high dose condition, however, we saw an increase in stride length as well as an increase in paw contact area. Although the increase in stride length was unexpected, further review of the literature indicated that it is likely part of a haloperidol-induced bracing-escape response. We conclude that PrAnCER is indeed capable of detecting Parkinsonian-like changes in rodent gait consistent with the use of neuroleptics.

<table>
	<tbody>
		<tr>
			<td>Gait Walkway Apparatus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34; clear plexiglass</td>
			<td>RISD 3D Store, Providence, RI</td>
			<td></td>
			<td>Approximate Price: $50 
			1 piece cut to 3 1/2&#34; W x 36&#34; L; 2 pieces cut to 4 1/2&#34; W x 36&#34; L 
			Note: We made our walkway 36&#34; long based off of an exisiting plexiglass table we had in house, it could easily be made longer if desired.</td>
		</tr>
		<tr>
			<td>1/4&#34; clear plexiglass</td>
			<td>RISD 3D Store, Providence, RI</td>
			<td></td>
			<td>Approximate Price: $10 
			4 pieces, cut to 1&#34; W by 4&#34; L 
			These will be used to keep the tunnel in place</td>
		</tr>
		<tr>
			<td>10 series 80/20 framing pieces, 1&#34; x 1&#34; T-Slotted Profile</td>
			<td>80/20 Inc.</td>
			<td>1010-S</td>
			<td>Approximate Price: $16 
			2 pieces cut to 36&#34; L</td>
		</tr>
		<tr>
			<td>12V Flexible LED Strip Lights, 16.4ft/5m LED Light Strips, Daylight White</td>
			<td>Amazon</td>
			<td></td>
			<td>Approximate Price: $10</td>
		</tr>
		<tr>
			<td>Bostik Blu-Tack Adhesive</td>
			<td>Amazon</td>
			<td></td>
			<td>Approximate Price: $8</td>
		</tr>
		<tr>
			<td>Clearprint 1000H drafting vellum, 16 LB cotton fiber</td>
			<td>Dick Blick Art Supplies</td>
			<td>11101-1046</td>
			<td>Approximate Price: $50 
			Cut to 4&#34; W x 36&#34; L 
			Note: This particular vellum comes as a roll; we kept it on the roll and cut it to 4&#34; W. 
			Mylar or frosted plexiglass could also be used in place of the vellum, but the camera software detection settings would need to be adjusted.</td>
		</tr>
		<tr>
			<td>Logitech HD Pro Webcam C920, 1080p</td>
			<td>Amazon</td>
			<td></td>
			<td>Approximate Price: $50</td>
		</tr>
		<tr>
			<td>Mobile Laptop Computer Desk Cart Height-Adjustable</td>
			<td>Amazon</td>
			<td></td>
			<td>Approximate Price: $40 
			Small table to place the animals&#39; home cage on at the end of the walkway.</td>
		</tr>
		<tr>
			<td>Plastic ramp</td>
			<td>Pets Warehouse</td>
			<td></td>
			<td>Approximate Price: $6 
			Optional: Ramp to assist the animals descend into home cage</td>
		</tr>
		<tr>
			<td>RetiCAM Tabletop Tripod with 3-Way Pan/Tilt Head</td>
			<td>Amazon</td>
			<td></td>
			<td>Approximate Price: $30</td>
		</tr>
		<tr>
			<td>SCIGRIP #16 solvent cement for acrylic - clear, medium bodied</td>
			<td>Amazon</td>
			<td></td>
			<td>Approximate Price: $8</td>
		</tr>
		<tr>
			<td>Plexiglass table</td>
			<td></td>
			<td></td>
			<td>Approximate Price: $</td>
		</tr>
		<tr>
			<td>15 series 80/20 framing pieces, 1.5&#34; x 1.5&#34; T-Slotted Profile</td>
			<td>80/20 Inc.</td>
			<td>1515</td>
			<td>Approximate Price: $110 
			6 pieces cut to 36&#34; L, 2 pieces cut to 12&#34; With both ends tapped with standard 5/16-18 threads 
			Framing for the plexiglass table top and table legs</td>
		</tr>
		<tr>
			<td>15 series 3 Way - Light Squared Corner Connector</td>
			<td>80/20 Inc.</td>
			<td>14177</td>
			<td>Approximate Price: $24 
			4 connectors 
			To connect the table top and legs</td>
		</tr>
		<tr>
			<td>1/4&#34; clear plexiglass sheet</td>
			<td>RISD 3D Store, Providence, RI</td>
			<td></td>
			<td>Approximate Price: $50 
			Cut at 15&#34; W x 39&#34; L</td>
		</tr>
		<tr>
			<td>5/16-18 x 1&#34; Button Head Socket Cap Screw</td>
			<td>80/20 Inc.</td>
			<td>3118</td>
			<td>Approximate Price: $5 
			Quantity = 12</td>
		</tr>
		<tr>
			<td>Deluxe Leveling Feet, 5/16-18 x 2&#34;</td>
			<td>80/20 Inc.</td>
			<td>2194</td>
			<td>Approximate Price: $50 
			Quantity = 4 
			For table legs</td>
		</tr>
		<tr>
			<td>&#8220;T&#8221; Handle Ball End Hex Wrench, 3/16&#34;</td>
			<td>80/20 Inc.</td>
			<td>6000</td>
			<td>Approximate Price: $5</td>
		</tr>
	</tbody>
</table>

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

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function in many areas of research, the available commercial options have several limitations such as high cost and lack of accessible, open code. To address these issues, we developed PrAnCER, Paw-Print Analysis of Contrast-Enhanced Recordings, for automated quantification of gait. The contrast-enhanced recordings are produced by using a translucent floor that obscures objects not in contact with the surface, effectively isolating the rat&#8217;s paw prints as it walks. Using these videos, our simple software program reliably measures a variety of spatiotemporal gait parameters. To demonstrate that PrAnCER can accurately detect changes in motor function, we employed a haloperidol model of Parkinson&#8217;s disease (PD). We tested rats at two doses of haloperidol: high dose (0.30 mg/kg) and low dose (0.15 mg/kg). Haloperidol significantly increased stance duration and hind paw contact area in the low dose condition, as might be expected in a PD model. In the high dose condition, we found a similar increase in contact area but also an unexpected increase in stride length. With further research, we found that this increased stride length is consistent with the bracing-escape phenomenon commonly observed at higher doses of haloperidol. Thus, PrAnCER was able to detect both expected and unexpected changes in rodent gait patterns. Additionally, we confirmed that PrAnCER is consistent and accurate when compared with manual scoring of gait parameters.

Haloperidol procedure
We developed this gait analysis system to compare gait parameters in control rats to those in experimental rats expected to show a variety of locomotor, gait, and balance impairments. We used a within-subject design in which every rat was tested in the saline, high dose haloperidol and low dose haloperidol conditions. Rats were separated into two groups (A and B) to allow counterbalancing; gait testing was counterbalanced for time of day and order of condition. Each test was separated by 48 h. Rats were lightly anesthetized with isoflurane before receiving intraperitoneal (IP) injections of either saline or haloperidol. Gait was tested 1 h post-injection, at which point the haloperidol should be at peak levels15,16,17.
Behavioral results
We observed prominent behavior changes in animals treated with haloperidol. In the high dose condition, five of the eight rats had periods of immobility at the start of the walkway, during which they were unresponsive to the experimenter touching them and resistant to being moved. In some cases, this state persisted for several minutes until the rat was removed from the walkway. In other cases, the immobile rat would suddenly move rapidly or &#8220;bound&#8221; across the walkway and then return to the immobile state near the end. In the low dose condition, 3 of the 8 rats had similar periods of immobility. At this dosage, there was only one instance of bounding behavior. No bounding was observed when the animals were treated with saline.
We analyzed the effects of haloperidol on the following gait parameters: base of support, stride length, stride speed, stance duration, stance to swing ratio, maximum contact area, and interlimb distance. Because many gait parameters for front and hind limbs are identical and haloperidol generally has uniform impacts on all limbs, we calculated parameters for only the hind limbs and did not separate data for left and right limbs. For each rat, we calculated the mean of each gait parameter from all usable runs from each testing day. All parameters (other than speed variability) were calculated as the mean for the first 4 usable steps of a run. To assess whether each dosage of haloperidol significantly impacted gait, we used a paired sample t-test. In Experiment 1, there was a significant increase in stride length (Figure 6A; t(7) = -2.962, p = 0.021) and maximum contact area (Figure 6A; t(7) = -2.51, p = 0.04) in animals treated with high dose haloperidol. Base of support, speed, stance duration and stance to swing ratio were not significant. In Experiment 2, animals given low dose haloperidol showed a significant increase in stance duration (Figure 6B; t(7) = -2.444, p = 0.044) and maximum contact area (Figure 6B; t(7) = -3.085, p = 0.018) compared to the saline condition. No other gait parameters were significant. Additionally, there was a significant difference between the high dose and low dose haloperidol conditions in base of support (Figure 6C; t(7) =2.651, p = 0.033), maximum contact area (Figure 6C; t(7) = 4.635, p = 0.002) and interlimb distance (Figure 6C; t(7) = 3.098, p = 0.017).
Your location accuracy and errors in the automated system
To assess the accuracy of PrAnCER, we compared its automated analysis to the manual scoring of 21 randomly selected videos from a separate group of 6 control rats. For hand scoring purposes, the videos were converted into a sequence of images, which were then used to manually mark the locations of prints. For efficiency, we focused our analysis on spatial data measured from hind prints only. We extracted the mean stride length and BOS for each video and compared it to the automated values. While mean stride length was not significantly different between manual scoring and PrAnCER analysis (Figure 7B; t(20) = -0.01, p = 0.99), base of support was significant (Figure 7A; t(20) = -2.21, p = 0.038). Though the automated and manual scoring was generally well correlated, the automated system reported a 5% larger BOS on average. This difference may be due to variances in centroid selection rather than detection errors. For manual scoring, the print location was marked drawing an oval around the base of each hind print, as it would be difficult to manually replicate PrAnCER&#8217;s method of center of mass estimation. The clear trend was for PrAnCER to overestimate BOS, perhaps because some animals may splay their toes out in an asymmetric fashion, causing PrAnCER to observe more extreme centroids than manual scoring. Other systems have also noted significant increases in BOS between manual and automated scoring despite consistent stride length measures17. Considering the small differences observed and the consistency with other systems, we conclude that PrAnCER is a reliable measure of gait parameters.
It is important to note that all accuracy analysis occurred after manual correction of the automated output was performed using PrAnCER&#8217;s GUI. As in existing commercial systems, this step is necessary both for correcting errors in scoring and for eliminating runs that do not meet criteria22. We tuned PrAnCER to err on the side of false positives, as these are easier to correct post-hoc. We have never observed PrAnCER fail to detect a real print during manual correction of over 5 hundred videos. Other types of errors, however, were observed. These fell into 3 categories: false detections (detection of a non-print as a print), misclassifications (print mislabeled as front/hind or left/right), and false combinations (two prints incorrectly merged). These errors are easily corrected in the accompanying GUI, and typically occur in only a small percentage of videos filmed under normal conditions. Even with such corrections, PrAnCER markedly decreases the amount of manual labor involved in gait analysis. We estimate that for each video it takes approximately 3 min to run PrAnCER and correct any output errors (if necessary), while it would take nearly 10 min to manually score and analyze the same video.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59596/59596fig1v2.jpg" /> 
Figure 1. Comparison of gait analysis methods. (A) The traditional ink and paper method produces imprecise prints of paw shape and location. (B) Video recording with a transparent floor gives a detailed view of the paw prints but contains many salient features from the rat&#8217;s body that complicates automated scoring. (C) Lightweight paper over a clear floor creates a noisy image and loses details. (D) The use of vellum to create a translucent floor produces highly detailed prints while visually eliminating the body. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig1v2large.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/59596/59596fig2.jpg" /> 
Figure 2. Schematic illustration of the gait walkway apparatus and video recording. (A) The rat walks through a clear walkway with a translucent floor to the home cage goal box while being recorded from below. In this case, the vellum covers a transparent floor to make it translucent. The walkway is illuminated by LED strips placed along its length at a level between the animal&#8217;s feet and body. (B) A screenshot of a video recording demonstrating the effects of the translucent floor. Two paws are clearly visible, but the rat&#8217;s body is essentially undetectable. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig2large.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/59596/59596fig3v2.jpg" /> 
Figure 3. Detection process for one frame of a paw print. (A) The original image is de-noised and then subjected to background subtraction (B). (C) An edge detection algorithm is applied and the results are converted to a series of X, Y coordinates called contours (D). (E) Contours are grouped by proximity and the convex hull (bounding box) of the group is taken to produce a single contour encompassing the print.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig3v2large.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/59596/59596fig4.jpg" /> 
Figure 4. Conversion of individual detections into a classified print. (A) Paw prints are first identified in a set of frames. (B) Individual object detections are given a number that identifies them as a print, representing a single placement of one paw (C). (D) Finally, they are classified as left or right based on their location relative to the midline of the animal&#8217;s path, and front or hind based on their location relative to the previous paw prints. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59596/59596fig5.jpg" /> 
Figure 5. Illustration of gait parameters analyzed. (A) An example output showing the identification and location of paw prints. Original detected edges are shown in black. Final detected paws and approximate area are shown in colors that indicate paw classification. In this figure, yellow: front left, green: hind left, cyan: front right, and magenta: hind right. However, the colors can be changed in the Python script according to user preference. (B) A plot illustrating two major temporal parameters: the amount of time each paw is in contact with the ground (stance phase) and in the air (swing phase). Colored blocks indicate the stance phase and the white spaces indicate the swing phase. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59596/59596fig6.jpg" /> 
Figure 6. Effects of haloperidol on gait. (A) Results of Experiment 1: high dose haloperidol (HalH) significantly increased stride length and maximum contact area compared to the saline condition (Sal). (B) Experiment 2 resulted in more typical parkinsonian symptoms; low dose haloperidol (HalL) significantly increased stance duration and maximum contact area. (C) When comparing the haloperidol treated conditions from both experiments, high dose haloperidol increased base of support, maximum contact area and interlimb distance compared to the low dose condition. Data are means &#177; SEM, n =8. Paired samples t-test differences were as follows: # p &#60; 0.05, ## p &#60; 0.01. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59596/59596fig7v2.jpg" /> 
Figure 7. Accuracy of automated analysis. (A) The automated system significantly differs from manual scoring when measuring BOS, though this may be due to variations in manual centroid selection rather than detection errors. (B) The automated system is not significantly different from manual scoring for stride length. These accuracy results are consistent with those from other available systems. Data are means &#177; SEM, n = 21. Paired samples t-test differences were as follows: # p &#60; 0.05. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59596/59596fig8v2.jpg" /> 
Figure 8. Comparison of temporal parameters. Temporal gait patterns for an animal treated with saline (A) and low dose haloperidol (B). (C) An illustration of the bracing-escape response from a rat given high dose haloperidol. As in Figure 5, colored blocks indicate when the paw was in contact with the ground (stance phase) and the white spaces indicate when the paw was in the air (swing phase). Abbreviations: FL, front left; HL, hind left; FR, front right; HR, hind right. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59596/59596fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Experiment 1</td>
			<td colspan="4">Experiment 2</td>
		</tr>
		<tr>
			<td></td>
			<td>Test 1</td>
			<td>Test 2</td>
			<td>Test 3</td>
			<td>Test 4</td>
			<td>Test 5</td>
			<td>Test 6</td>
			<td>Test 7</td>
			<td>Test 8</td>
		</tr>
		<tr>
			<td>Group A</td>
			<td>HalH</td>
			<td>Sal</td>
			<td>HalH</td>
			<td>Sal</td>
			<td>Sal</td>
			<td>HalL</td>
			<td>Sal</td>
			<td>HalL</td>
		</tr>
		<tr>
			<td>Group B</td>
			<td>Sal</td>
			<td>HalH</td>
			<td>Sal</td>
			<td>HalH</td>
			<td>HalL</td>
			<td>Sal</td>
			<td>HalL</td>
			<td>Sal</td>
		</tr>
	</tbody>
</table>
Table 1. Experimental design. This table illustrates the experimental design used in this study. We used a within-subject design in which every rat was tested in the high dose haloperidol (HalH), low dose haloperidol (HalL) and saline (Sal) conditions. Rats were divided into two groups; testing was counterbalanced for time of day and order of condition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Parameter</td>
			<td>Definition</td>
		</tr>
		<tr>
			<td>Stride Length</td>
			<td>Distance between successive contacts of the same paw</td>
		</tr>
		<tr>
			<td>Step Length</td>
			<td>Distance between successive contacts of contralateral front or hind paws along the axis of the direction of motion</td>
		</tr>
		<tr>
			<td>Base of Support (BOS)</td>
			<td>Distance between successive contralateral front or hind paws perpendicular to axis of the direction of motion</td>
		</tr>
		<tr>
			<td>Maximum Contact Area</td>
			<td>The maximum detected area of a hind print</td>
		</tr>
		<tr>
			<td>Interlimb distance</td>
			<td>Distance between ipsilateral front and hind paws</td>
		</tr>
		<tr>
			<td>Stance Duration</td>
			<td>The length of time a paw was in contact with the ground</td>
		</tr>
		<tr>
			<td>Swing Duration</td>
			<td>The length of time a paw was not on the ground</td>
		</tr>
		<tr>
			<td>Stance to Swing Ratio (SSR)</td>
			<td>Stance duration/swing duration</td>
		</tr>
		<tr>
			<td>Discrete Speed</td>
			<td>Stride length/(stance duration + swing duration) for a paw</td>
		</tr>
		<tr>
			<td>Average Speed</td>
			<td>Average of discrete speeds in the period used in analysis</td>
		</tr>
		<tr>
			<td>Speed Variability</td>
			<td>Percent change in discrete speeds during a run</td>
		</tr>
		<tr>
			<td>Run Speed</td>
			<td>Time to cross the tunnel/length of tunnel</td>
		</tr>
	</tbody>
</table>
Table 2. Description of gait parameters. This table describes the most commonly used gait parameters; those used in this study are indicated in bold.

Watch this Scientific Journal Video about Paw-Print Analysis of Contrast-Enhanced Recordings PrAnCER: A Low-Cost, Open-Access Automated Gait Analysis System for Assessing Motor Deficits at JoVE.com

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 Disease.

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

Gait analysis is used to quantify changes in motor function in many rodent models of disease. Despite the importance of assessing gait and motor function ...

paw-print-analysis-contrast-enhanced-recordings-prancer-low-cost-open

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

8.4K Views.  Brown University. 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’s Disease.

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

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with schizophrenia who are at familial high risk (FHR) may show similar cognitive and emotional changes. However, the neurological changes underlying the emergence of these symptoms remain unclear. This study sought to identify differences in frontal, striatal, and limbic regions in children and adolescents with FHR using functional magnetic resonance imaging. Groups of 21 children and adolescents at FHR and 21 healthy controls completed an emotional oddball task that relied on selective attention and the suppression of task-irrelevant emotional information. The standard oddball task was modified to include aversive and neutral distractors in order to examine potential group differences in both emotional and executive processing. This task was designed specifically to allow for children and adolescents to complete by keeping the difficulty and emotional image content age-appropriate. Furthermore, we demonstrate a technique for suitable fMRI registration for children and adolescent participants. This paradigm may also be applied in future studies to measure changes in neural activity in other populations with hypothesized developmental changes in executive and emotional processing.

This paper describes how to use the emotional oddball task and fMRI to measure brain activation in children and adolescents at familial high risk for schizophrenia (FHR).&#160;FMRI was used to measure differences in fronto-striato-limbic regions during an emotional oddball task. Children with FHR exhibited abnormal functional activation during adolescence.

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with ...

Cortical Source Analysis of High-Density EEG Recordings in Children

EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2, because the composition and spatial configuration of head tissues changes dramatically over development3.&#160;
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how&#160;EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.&#160;

In recent years, there has been increasing interest in estimating the cortical sources of scalp measured electrical activity for cognitive neuroscience experiments. This article describes how high density EEG is acquired and how recordings are processed for cortical source estimation in children from the age of 2 years at the London Baby Lab.

EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis (AFEA) software. Additionally, we demonstrate a method for extracting relevant emotional data output and plotting the emotional response of a population over a specified time frame. By time pairing each participant's treatment response to a control stimulus (baseline), the overall emotional response over time and across multiple participants can be quantified. AFEA is a prospective analytical tool for assessing unbiased response to food and beverages. At present, most research has mainly focused on beverages. Methodologies and analyses have not yet been standardized for the application of AFEA to beverages and foods; however, a consistent standard methodology is needed. Optimizing video capture procedures and resulting video quality aids in a successful collection of emotional response to foods. Furthermore, the methodology of data analysis is novel for extracting the pertinent data relevant to the emotional response. The combinations of video capture optimization and data analysis will aid in standardizing the protocol for automated facial expression analysis and interpretation of emotional response data.

A protocol for capturing and statistically analyzing emotional response of a population to beverages and liquefied foods in a sensory evaluation laboratory using automated facial expression analysis software is described.

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

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

Three-dimensional gait analysis (3DGA) is shown to be a useful clinical tool for the evaluation of gait abnormality due to movement disorders. However, the use of 3DGA in actual clinics remains uncommon. Possible reasons could include the time-consuming measurement process and difficulties in understanding measurement results, which are often presented using a large number of graphs. Here we present a clinician-friendly 3DGA method developed to facilitate the clinical use of 3DGA. This method consists of simplified preparation and measurement processes that can be performed in a short time period in clinical settings and intuitive results presentation to facilitate clinicians&#39; understanding of results. The quick, simplified measurement procedure is achieved by the use of minimum markers and measurement of patients on a treadmill. To facilitate clinician understanding, results are presented in figures based on the clinicians&#39; perspective. A Lissajous overview picture (LOP), which shows the trajectories of all markers from a holistic viewpoint, is used to facilitate intuitive understanding of gait patterns. Abnormal gait pattern indices, which are based on clinicians&#39; perspectives in gait evaluation and standardized using the data of healthy subjects, are used to evaluate the extent of typical abnormal gait patterns in stroke patients. A graph depicting the analysis of the toe clearance strategy, which depicts how patients rely on normal and compensatory strategies to achieve toe clearance, is also presented. These methods could facilitate implementation of 3DGA in clinical settings and further encourage development of measurement strategies from the clinician&#39;s point of view.

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 results.

Three-dimensional gait analysis (3DGA) is shown to be a useful clinical tool for the evaluation of gait abnormality due to movement disorders. However, ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

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

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...