Name: behavioral assessments of spontaneous locomotion in a murine mptp-induced parkinson's disease model
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 5 min 38 s
Description: Watch this Scientific Journal Video about Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model at JoVE.com

Our work is funded by Tianjin Undergraduate Training Programs for Innovation and Entrepreneurship (Grant No. 63183004). This project was initiated in the State Key Laboratory of Medicinal Chemical Biology at Nankai University. The authors declare that there are no conflicts of interests.

This study was performed according to the international, national and institutional rules considering animal experiments. The study protocol was approved by the animal ethics committee of Nankai University.
1. MPTP and L-DOPA administration
NOTE: Ten-week-old female BALB/c mice were provided by the Institute of Zoology, Chinese Academy of Sciences. Mice were housed six per cage under a 12 h light/dark cycle (lights on at 08:00-20:00), a constant temperature of 21-22 &#176;C and a relative humidity of 55% &#177; 5%. Autoclaved standard mice chow of the same formulation and water ad libitum was given to all animals.
<ol>
	<li>After one week of acclimatization, divide the animals into three groups of six mice each.</li>
	<li>To Group 1, administer intraperitoneal injections (see step 1.5) of 250 &#181;L/mouse/day of saline from day 1 to day 7 and then perform intragastric administration (see step 1.6) of 250 &#181;L/mouse of saline on day 8.</li>
	<li>To Group 2, administer intraperitoneal injections of 20 mg/kg/day of MPTP every day from day 1 to day 7 and then perform intragastric administration of 250 &#181;L/mouse of saline on day 8.</li>
	<li>To Group 3, administer intraperitoneal injections of 20 mg/kg/day of MPTP from day 1 to day 7 and then perform intragastric administration of 5 mg/kg of L-DOPA on day 8.</li>
	<li>Perform the intraperitoneal injection as follows.
	<ol>
		<li>Load the drug into a sterile 1 mL syringe with a 26 G needle. Eliminate air from the syringe.</li>
		<li>Scruff the mouse with its belly facing up. Keep the head, neck and body of the mouse in a straight line as well with the head fixed.</li>
		<li>Angle the needle to penetrate the peritoneum. Push the needle for a proper distance until there is little resistance, and then inject the drug. Pull out the needle smoothly.</li>
	</ol>
	</li>
	<li>Perform the intragastric administration as follows.
	<ol>
		<li>Prepare the medication in a sterile 1 mL syringe with an oral-gavage needle. Eliminate air from the syringe.</li>
		<li>Hold the mouse with its belly facing up. Keep the head, neck and body of the mouse in a straight line with the head fixed.</li>
		<li>Keep the needle parallel to the body of the mouse, and insert it from the corner of the mouse&#39;s mouth, pressing the tongue and pushing inward against the upper jaw.</li>
		<li>With little resistance, which shows the needle is entering the esophagus smoothly, carefully push the needle in a proper distance. Before the tip of the needle reaches the bottom part of chest, inject the medication.</li>
		<li>Pull out the needle smoothly.</li>
	</ol>
	</li>
</ol>
2. Cylinder test
NOTE: The behavioral tests were performed on day 8. L-DOPA was injected to the third group of mice 40 min before the behavioral tests. If the behavioral testing is not done in the same room where the animals are housed, animals need to be acclimated to the new room for 30-60 min before test.
<ol>
	<li>Perform the test 24 h after the last dose of MPTP.</li>
	<li>Place a transparent glass cylinder (height = 19.5 cm, diameter = 15 cm, weight &#8805;1 kg) at the center of a table. Surround the cylinder on three sides with black cardboard to reduce the effect of environmental lighting. Leave one side of the cylinder facing the camera for video recording. 
	NOTE: The distance between the cardboard and cylinder should be about 4 to 8 cm.</li>
	<li>Attach a camera (&#62; 1 million-pixel resolution) at ~40-60 cm away from the cylinder to ensure that the full cylinder is visible. 
	NOTE: This is neither too near nor too far so as to not disturb the mouse while recording the video of its movement.</li>
	<li>Place one mouse into the cylinder at a time and start filming at once. Stop filming after 3 min. 
	NOTE: During this process, try to avoid noise or light changes in order avoid influencing the mouse&#8217;s behavior.</li>
	<li>Place the mouse back into the home cage after testing.</li>
	<li>Clean the cylinder with water and then spray 70% v/v ethanol over the inner wall to sanitize it and remove mouse scents. Wipe the cylinder dry before another mouse is placed into.</li>
	<li>Play back the video at a rate of 0.5x the regular speed and count the number of paw lifts against the wall of each mouse. 
	NOTE: Paw lifts occur when the mouse rears up on its hindlimbs, raises both forelimbs above shoulder level and lands. One forelimb lift is NOT counted. Usually, a mouse raises its forelimbs to touch the cylinder walls. If the mouse raises its forelimbs above shoulder level several times continuously without landing, it should be counted only as once.</li>
</ol>
3. Open field test
<ol>
	<li>Perform the open field test 24 h after MPTP dosing. 
	NOTE: It can be carried out at the same time of the cylinder test.</li>
	<li>Prepare a transparent open field reaction box (45 cm L x 45 cm W x 25 cm H) with a wooden plate cover at the bottom by a black cloth. Fix a camera (&#62; 1 million-pixel resolution) over the field at a height of 1 m. 
	NOTE: The color of open field box bottom should be different from the tested mouse color to ensure color contrast in the video.</li>
	<li>Adjust the camera to make sure that the open field reaction box is right in the center of the video.</li>
	<li>Put one test mouse into the box, and let the mouse familiarize itself with the environment for about 1 min.</li>
	<li>Record a 5 min video using the camera connected to the computer.</li>
	<li>Analyze the video using software tools (e.g., MATLAB) to get the movement trace figure, the distribution of static (velocity &#60;1 cm/s), walking (velocity 1-20 cm/s) and running (velocity &#62;20 cm/s) time, the total traveled distance, and average speed of each tested mouse. 
	NOTE: When analyzing the video, we track the whole body of the mouse. The average speed of a body means the total length of path covered, divided by the elapsed time. Therefore, if a mouse is not moving, its instantaneous velocity would be considered as zero.</li>
	<li>Clean up any feces in the open field reaction box. Spray 70% ethanol on the box and wipe it.</li>
</ol>

<ol>
	<li>Dehay, B., Bourdenx, M., Gorry, P., Przedborski, S., Vila, M., Hunot, S., Singleton, A., Olanow, C. W., Merchant, K. M., Bezard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+-synuclein+for+treatment+of+Parkinson's+disease:+mechanistic+and+therapeutic+considerations.">Targeting -synuclein for treatment of Parkinson's disease: mechanistic and therapeutic considerations.</a> Lancet Neurology. 14, 855-866 (2015).</li><li>Weintraub, D., Comella, C., Horn, L., S, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease+-+Part+1:+Pathophysiology,+symptoms,+burden,+diagnosis,+and+assessment.">Parkinson's disease - Part 1: Pathophysiology, symptoms, burden, diagnosis, and assessment.</a> American Journal of Managed Care. 14, 40-48 (2008).</li><li>Ismaiel, A. 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=Metformin,+besides+exhibiting+strong+in+vivo+anti-inflammatory+properties,+increases+mptp-induced+damage+to+the+nigrostriatal+dopaminergic+system.">Metformin, besides exhibiting strong in vivo anti-inflammatory properties, increases mptp-induced damage to the nigrostriatal dopaminergic system.</a> Toxicology and Applied Pharmacology. , 19-30 (2016).</li><li>Bayliss, J. 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=Ghrelin-AMPK+signaling+mediates+the+neuroprotective+effects+of+calorie+restriction+in+Parkinson's+disease.">Ghrelin-AMPK signaling mediates the neuroprotective effects of calorie restriction in Parkinson's disease.</a> Journal of Neuroscience. 36, 3049-3063 (2016).</li><li>Olanow, C. W., Stern, M. B., Sethi, 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+scientific+and+clinical+basis+for+the+treatment+of+Parkinson+disease.">The scientific and clinical basis for the treatment of Parkinson disease.</a> Neurology. 72 (21 Supplement 4), (2009).</li><li>Chiba, K., Trevor, A., Castagnoli, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+the+neurotoxic+tertiary+amine,+MPTP,+by+brain+monoamine+oxidase.">Metabolism of the neurotoxic tertiary amine, MPTP, by brain monoamine oxidase.</a> Biochemical &amp; Biophysical Research Communications. 120 (2), 574-578 (1984).</li><li>Bakay, R. 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=Biochemical+and+behavioral+correction+of+MPTP+parkinson-like+syndrome+by+fetal+cell+transplantation.">Biochemical and behavioral correction of MPTP parkinson-like syndrome by fetal cell transplantation.</a> Annals of the New York Academy of Sciences. 495 (1), 623-638 (2010).</li><li>Kelly, P. H., Seviour, P. W., Iversen, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+apomorphine+responses+in+the+rat+following+6-OHDA+lesions+of+the+nucleus+accumbenssepti+and+corpus+striatum.">Amphetamine and apomorphine responses in the rat following 6-OHDA lesions of the nucleus accumbenssepti and corpus striatum.</a> Brain Research. 94 (3), 507-522 (1975).</li><li>Langston, J. W., Irwin, I., Langston, E. B., Forno, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1-methyl-4-phenylpyridinium+ion+(MPP+):+identification+of+a+metabolite+of+MPTP,+a+toxin+selective+to+the+substantia+nigra.">1-methyl-4-phenylpyridinium ion (MPP+): identification of a metabolite of MPTP, a toxin selective to the substantia nigra.</a> Neuroscience Letters. 48 (1), 87-92 (1984).</li><li>Katila, N., 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=Metformin+lowers+&#945;-synuclein+phosphorylation+and+upregulates+neurotrophic+factor+in+the+MPTP+mouse+model+of+Parkinson's+disease.">Metformin lowers &#945;-synuclein phosphorylation and upregulates neurotrophic factor in the MPTP mouse model of Parkinson's disease.</a> Neuropharmacology. 125, (2017).</li><li>Goldberg, N. R. S., Haack, A. K., Lim, N. S., Janson, O. K., Meshul, 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=Dopaminergic+and+behavioral+correlates+of+progressive+lesioning+of+the+nigrostriatal+pathway+with+MPTP.">Dopaminergic and behavioral correlates of progressive lesioning of the nigrostriatal pathway with MPTP.</a> Neuroscience. 180, 256-271 (2011).</li><li>Schwarting, R. K., Sedelis, M., Hofele, K., Auburger, G. W., Huston, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-dependent+recovery+of+open-field+behavior+and+striatal+dopamine+deficiency+in+the+mouse+MPTP+model+of+Parkinson's+disease.">Strain-dependent recovery of open-field behavior and striatal dopamine deficiency in the mouse MPTP model of Parkinson's disease.</a> Neurotoxicity Research. 1 (1), 41 (1999).</li><li>Shiguang, 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=The+open+field+test+as+a+method+for+ethology+in+Kunming+mice:+test-retest+reliability.">The open field test as a method for ethology in Kunming mice: test-retest reliability.</a> Chinese Journal of Behavioral Medicine and Brain Science. 19 (12), 1093-1095 (2010).</li><li>Roome, R. B., Vanderluit, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paw-Dragging:+a+Novel,+Sensitive+Analysis+of+the+Mouse+Cylinder+Test.">Paw-Dragging: a Novel, Sensitive Analysis of the Mouse Cylinder Test.</a> Journal of Visualized Experiments. (98), e52701 (2015).</li><li>Cotzias, G. C., Van Woert, M. H., Schiffer, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aromatic+amino+acids+and+modification+of+Parkinsonism.">Aromatic amino acids and modification of Parkinsonism.</a> New England Journal of Medicine. 282, 31-33 (1967).</li><li>Cassady, J. C., Johnson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+test+anxiety+and+academic+performance.">Cognitive test anxiety and academic performance.</a> Contemporary Educational Psychology. 27 (2), 270-295 (2002).</li><li>Rogalewski, 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=Semaphorin+6A+improves+functional+recovery+in+conjunction+with+motor+training+after+cerebral+ischemia.">Semaphorin 6A improves functional recovery in conjunction with motor training after cerebral ischemia.</a> Plos One. 5 (5), 0010737 (2010).</li><li>Chen, W., Qiao, D., Liu, X., Shi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treadmill+exercise+improves+motor+dysfunction+and+hyperactivity+of+the+corticostriatal+glutamatergic+pathway+in+rats+with+6-ohda-induced+parkinson's+disease.">Treadmill exercise improves motor dysfunction and hyperactivity of the corticostriatal glutamatergic pathway in rats with 6-ohda-induced parkinson's disease.</a> Neural Plasticity. 2017, 2583910 (2018).</li><li>Colas-Zelin, 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=The+imposition+of,+but+not+the+propensity+for,+social+subordination+impairs+exploratory+behaviors+and+general+cognitive+abilities.">The imposition of, but not the propensity for, social subordination impairs exploratory behaviors and general cognitive abilities.</a> Behavioural Brain Research. 232 (1), 294-305 (2012).</li><li>Spurney, 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=Preclinical+drug+trials+in+the+mdx+mouse:+Assessment+of+reliable+and+sensitive+outcome+measures.">Preclinical drug trials in the mdx mouse: Assessment of reliable and sensitive outcome measures.</a> Muscle Nerve. 39, 591-602 (2009).</li><li>Valle, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+strain,+sex,+and+illumination+on+open-field+behavior+of+rats.">Effects of strain, sex, and illumination on open-field behavior of rats.</a> American Journal of Psychology. 83, 103-111 (1970).</li><li>Tatem, K. 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=Behavioral+and+Locomotor+Measurements+Using+an+Open+Field+Activity+Monitoring+System+for+Skeletal+Muscle+Diseases.">Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases.</a> Journal of Visualized Experiments. (91), e51785 (2014).</li></ol>

The authors have nothing to disclose.

Due to destruction of dopaminergic neurons in the substantia nigra of the brain, MPTP causes Parkinson&#39;s-like symptoms in mice7. L-DOPA is the most preferred drug for PD ever since its clinical use, because it helps in maintaining normal daily activities in patients with PD, with effective suppression of motor abnormalities including akinesia and rigidity15. The mice treated with MPTP showed impairments in the behavioral tests like cylinder test and open field test, whi...

Parkinson&#39;s disease (PD), one of the most common diseases among older individuals, is a long-term neurodegenerative disorder1. Patients always show the phenomenon of shaking, rigidity, slowness of movement and dementia that worsen over time2. Other symptoms including sensory, sleep, and emotional problems are also commonly observed2. The cause of PD is still unclear, but it is generally believed to involve both genetic and environmental factors, which induce loss of dopaminergic neurons in the substantia nigra3, and development of Lewy bodies and Lewy neurites in various regions of the brain4.
Among the studies of PD, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)5 is adapted widely in recreating some PD symptoms in experimental models. In 1984, Langston et al. first found that injections of MPTP in squirrel monkeys resulted in Parkinsonism6. Although the MPTP rodent model doesn&#39;t show the presence of Lewy bodies, which is the biomarker of PD, MPTP causes Parkinson&#39;s-like symptoms by destroying dopaminergic neurons in the substantia nigra of the brain7. Compared to other drug model for PD such as those induced by 6-hydroxydopamine (6-OHDA)8 and 1-methyl-4-phenylpyridinium (MPP+)9, injection of MPTP is easy to execute and the MPTP model takes less time. Mice receive MPTP injections (20-30 mg/kg/day) for seven days, and the behavioral tests are performed on the eighth day10.
The open field test11 was first developed by Calvin S. Hall, an American12. In various studies, different kinds of behaviors are tested. In research which focuses on Parkinson&#39;s diseases, behaviors like locomotion activities and the speed of locomotion are tested to see if the animal&#39;s ability to move around is affected. Compared with other methods used to test the establishment of PD animals, open field test is easy to carry out because the equipment needed is simple, and prototyping and data analysis software (e.g., MATLAB, Excel) can be used to easily collect and graph the data. Also, the coefficient of variation is relatively small13, which means that the result of open field test is reliable. Another advantage over other methods is that the behaviors included in this experiment are easy to distinguish; the mice can be either running, walking or standing still. Usually the open field test can be used on rodents when the researcher needs to evaluate the subject&#39;s mobility.
The Cylinder test is also called the test of asymmetric use of forelimbs. When this test was first designed, it was used to test the asymmetric use of the rat&#39;s forelimbs14. Here, we use this test to analyze the animal&#39;s ability to stretch out and use both of its forelimbs to explore new surroundings. When the substantia nigra and corpus striatum are damaged by MPTP in the brain, the animal tends to arch its back and becomes less likely to stretch out and explore the unknown environment. This test is easy to execute and can give a preliminary result. However, this test has high internal variability, so it is generally used with along with other behavior experiments.
Taking L-DOPA, which is also known as levodopa or L-3,4-dihydroxyphenylalanine, is a common way to treat Parkinson&#39;s disease since one cause of PD is the decrease of dopamine in the . L-DOPA is the precursor to dopamine. But unlike dopamine, it can cross the blood-brain barrier, which means that it will be more efficient in increasing the concentration of dopamine in the brain area. After it crosses the blood-brain barrier, L-DOPA is converted into dopamine by L-amino acid decarboxylase15.
Here we describe the measurement and analysis of motor function in MPTP-induced-PD model mice using a cylinder test14 and a modified open field test. We administer L-DOPA as one example to show how to apply this model in the study of PD drugs. Our results indicate that MPTP induces motor deficit that can be mitigated by L-DOPA.

<table><tbody><tr><td>70% Ethanol</td><td>Ruiboxing Company</td><td>RBX-64175</td><td></td></tr><tr><td>Camera</td><td>BASLER</td><td>acA645-100gm</td><td></td></tr><tr><td>Cylinder test</td><td>Made in-house at Nankai University</td><td>N/A</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td>N/A</td><td></td></tr><tr><td>Levodopa</td><td>Sigma-Aldrich</td><td>72816</td><td></td></tr><tr><td>Matalb 2017a</td><td>Mathworks</td><td>N/A</td><td></td></tr><tr><td>Mice</td><td>Institute of Zoology, Chinese Academy of Sciences</td><td>Balb/c</td><td>Adult female mice(10 weeks)</td></tr><tr><td>MPTP</td><td>Yuanye Biological Technology Company Ltd., Shanghai</td><td>S31504-500mg</td><td></td></tr><tr><td>Open field test</td><td>Made in-house at Nankai University</td><td>N/A</td><td></td></tr><tr><td>Syringe</td><td>Solelybio</td><td>S-xsgwz-w</td><td>Irrigation</td></tr><tr><td>Syringe</td><td>Jiangxi Fenglin Medical Application Co.</td><td>hc3824</td><td>Intraperitoneal injection</td></tr></tbody></table>

behavioral assessments of spontaneous locomotion in a murine mptp-induced parkinson's disease model

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and dementia. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can lead to some Parkinson&#8217;s-like symptoms by destroying dopaminergic neurons in the substantia nigra of the brain. It has been thus used to establish PD models in various animal studies. Here, mice receive MPTP injections (20 mg/kg/day) for seven days and the behavioral tests are performed on the eighth day. This model is adapted efficiently in the study of PD. The behavioral tests here include the cylinder test and the open field test. The cylinder experiment is used to detect the animals&#8217; ability to lift their front paws when put into a different environment. As the PD model mice show arching&#8212;the mouse arches its back&#8212;the number of paw liftings decrease. This test is easy to execute. The open field test is used to detect the amount of time the mice spend on running, walking, and remaining immobile. We analyze animals&#8217; movements in open field using software and obtain data. Lastly, we use L-DOPA, one of the most commonly used PD drugs, as one example to show how to apply this model to the study of PD drugs. Our results indicate that MPTP neurotoxicity induces motor deficit which can be mitigated by L-DOPA.

In the cylinder test, the decrease of rears against the wall was observed in mice (group 2) treated with MPTP from day 1 to day 7 and saline on day 8 as compared with saline-treated mice (group 1), while an increase of rears was observed in the mice (group 3) treated with MPTP from day 1 to day 7 and L-DOPA on day 8 as compared with the mice (group 2) treated with MPTP from day 1 to day 7 and saline on day 8 (Figure 1).
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Watch this Scientific Journal Video about Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model at JoVE.com

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

Parkinson&#8217;s disease (PD) is a common neurodegenerative disorder disease, causing the phenomenon of shaking, rigidity, slowness of movement and ...

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17.7K Views.  Nankai University. We describe establishment of a murine model for Parkinson's disease using MPTP, and behavioral assessments using cylinder and open field tests to measure motor function. We then use L-DOPA as one example to show how to apply this model in the study of PD drugs.

Video: Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Medicine

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Biology

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

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

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

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

Developmental Biology

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

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

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 models.

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

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'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, ...

Neuroscience

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Author Spotlight: A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

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.

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.

Behavioral Assessments of Spontaneous Locomotion in a Murine MPTP-induced Parkinson's Disease Model

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Rozdziały w tym wideo

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

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

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

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

Behavioral Characterization of an Angelman Syndrome Mouse Model

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

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