Name: behavioral assessments of spontaneous locomotion in a murine mptp-induced parkinson's disease model
Uploaded: 2019-01-07T14:00:00
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 &...

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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 ...

In this video, we introduce how to use a cylinder and open field test to narrow motor function in the MPTP model of Parkinson's disease. The primary advantage is that the mice don't need to be trained before the behavioral test. Additionally, this method is efficient and the cost of the equipment is low.Individuals who are new to behavioral tests may underestimate the effect of environmental interference. Keeping quiet and avoiding changes in lining will help. Here, behavioral testing is conducted 24 hours after the final injection of a seven day regimen of MPTP or saline injections and 40 minutes after intragastric administration of L-DOPA to a subset of the animals.If behavioral testing is not performed in the same room where the animals are housed, acclimate the mice to the new room for 30 to 60 minutes before the test. Begin by placing a transparent glass cylinder at the center of a table. Surround the cylinder on three sides with black cardboard, placed four to eight centimeters away from the cylinder to reduce the effect of environmental lighting.Leave one side of the cylinder facing the camera for video recording. Attach a camera capable of at least one million pixel resolution 40 to 60 centimeters away from the cylinder to ensure that the whole cylinder is contained in the field of view. Place one mouse into the cylinder at a time and start filming at once.During this process try to avoid noise or light changes in order to avoid influencing the mouse's behavior. Paw lifts occur when the mouse rears up on it's hind limbs, raises both forelimbs above shoulder level, and lands. Stop filming after three minutes and place the mouse back into the home cage.Clean the cylinder with water and then spray 70%ethnol over the inner wall to sanitize it and remove mouse scents. Wipe the cylinder dry before testing the next mouse. Once testing is completed, play back the video at a rate of 0.5 times the regular speed and count the number of paw lifts against the wall of each mouse.If the mouse raises its forelimbs above shoulder level several times continuously without landing, it should be counted only once. Perform open field testing during the same session as the cylinder test. Prepare a transparent open field reaction box with a wooden plate, covered by a contrasting black or white cloth depending on the color of the mouse.Fix a camera over the field at a height of one meter. Adjust the camera to make sure that the open field reaction box is right in the center of the video. Put one test mouse into the box and let the mouse familiarize itself with the environment for about one minute.Record a five minute video using the camera connected to the computer. Finally, clean up any feces in the open field reaction box, spray 70%ethanol on the box, and wipe it. In the cylinder test, a decrease in the number of rears against the wall was observed in mice treated with MPTP compared with saline treated mice.Increased rears were observed in the MTPT treated mice given L-Dopamine on the day of testing compared to the MPTP treated mice receiving saline. This image show the representative movement trace and the distribution of static time, walking time, and running time in control mice not treated with MPTP. Mice treated with MTPT demonstrated lower movement speed, shorter movement distance, longer static time, and shorter running time then saline treated mice, demonstrating a motor deficit induced by MTPT.MPTP treated mice receiving L-Dopamine showed a higher movement speed, a longer movement distance, a shorter static time, and a longer running time than the mice treated with just MPTP, indicating that L-Dopamine mitigates the MPTP-induced motor deficit. When changing the mice being tested clear the area completely and spray ethanol to eliminate the effect of the scent of the former mice. Following this procedure, other measures such as the beam walking test, pole test can be performed.Data related to balance capacity and motor ability can be gained through these methods. MPTP is neurotoxic for both mice and humans and so strict precautions must be taken to avoid exposure. Extra PPE, such as a mask or respirator should be worn.

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