This work was supported by S&#227;o Paulo Research Foundation (FAPESP, grant 2017/00003-0). We are grateful for the Coordination for the Improvement of Higher Education Personnel (CAPES). We thank Dr. Anthony R. West, Dr. Heinz Steiner, and Dr. Kuei Y. Tseng for support and mentoring.

All experiments were performed in accordance with The Ethics Committee of the Faculty of Philosophy, Sciences, and Letters of Ribeir&#227;o Preto (CEUA/FFCLRP 18.5.35.59.5).
1.&#160;6-OHDA lesion
<ol>
	<li>Use Sprague-Dawley male rats weighing 200-250 g at the beginning of the experiments (6 weeks). House the animals (2-3 per cage) under standard laboratory conditions (12:12 h light/dark cycle, lights on at 06:00 h, temperature-controlled facilities (22-24 &#176;C), with food and water available ad libitum. 
	NOTE: A complete description of how to generate severe 6-OHDA-lesioned rats is described elsewhere42.</li>
	<li>Appropriately acclimate animals before the beginning of any experimental procedure.</li>
	<li>Administer (intraperitoneally, i.p.) the norepinephrine transporter inhibitor imipramine (20 mg/kg, dissolved in 0.9% saline solution) 30 min before surgery. 
	NOTE: Imipramine administration will increase 6-OHDA selectivity for dopaminergic neurons.</li>
	<li>Administer ketamine/xylazine (70/10 mg/kg) intraperitoneally.</li>
	<li>Make sure the animal is deeply anesthetized by the lack of response to toe pinch and positioned in a prone position in the stereotaxic apparatus on the top of a heating pad.</li>
	<li>Remove the fur on the head and disinfect the surgical area 3 times, alternating between an iodine-based scrub and alcohol.</li>
	<li>Use a scalpel to make an incision (approximately 1 cm) at the region where the microinjection will occur.</li>
	<li>Clean the skull region with cotton swabs and ensure the Bregma and Lambda are exposed.</li>
	<li>Ensure MFB stereotaxic coordinates are taken from bregma43,44,45: -4.3 mm anterior, 1.6 mm lateral (right side), and 8.3 mm ventral (from the dura mater).</li>
	<li>Administer the 6-OHDA at a rate of 0.4 &#181;L/min (10 &#181;g in 4 &#181;L of saline solution containing 0.1% ascorbic acid) unilaterally in the right medial forebrain bundle (MFB) using a 50 &#181;L Hamilton glass syringe.</li>
	<li>At the end of the surgery, suture the scalp incision and rehydrate the animal with warm (~37 &#176;C) sterile 0.9% saline solution (~10 mL/kg, s.c.).</li>
	<li>Remove the animal from the stereotaxic frame and place it in a warmed recovery cage. Monitor until consciousness is regained.</li>
	<li>Assess the effectiveness of the dopaminergic lesion 4 weeks post-lesion using a stepping test46,47. 
	&#8203;NOTE: In this test, the akinesia of the forelimb contralateral to the lesion is assessed through the number of adjusting steps with the forepaw contralateral to the lesion. Rats presenting three or fewer adjusting steps with the contralateral forelimb are included in the study as putative severely 6-OHDA-lesioned rats48.</li>
</ol>
2. L-DOPA chronic treatment
<ol>
	<li>Start the chronic treatment on Monday, 4 weeks post-6-OHDA lesion. 
	NOTE: Since AIMs will be videotaped for 3 consecutive days per week (Wednesday, Thursday, and Friday) for 3 weeks, it is recommended to start the treatment on Monday.</li>
	<li>Treat the rats with freshly prepared L-DOPA (via subcutaneous (s.c.), 1 mL/kg) plus benserazide hydrochloride (5 mg/kg and 12.5 mg/kg, respectively) for 3 weeks, once daily from Monday to Friday45. 
	NOTE: Once established, AIMs will manifest with each administration of L-DOPA. Therefore, L-DOPA administration 2-4 times a week after the 3-week induction period is sufficient to maintain a stable expression of AIMs48.</li>
</ol>
3. AIMs recording and scoring
<ol>
	<li>Perform experiments between 09:00 h and 17:00 h. Ensure that the investigator is entirely unaware of the rats&#39; identity and pharmacological treatment.</li>
	<li>Carefully put the rat inside a transparent cylinder (20 cm diameter x 40 cm tall) and allow it to acclimatize for at least 15 min. Ensure that the floor is covered with the bedding material. Mirrors must be positioned behind the cylinder so the experimenter can observe the animal from all possible angles. 
	NOTE: If there is more than one rat per experiment, use a cylinder for each rat. Manipulation of the animals between sessions can interfere with the behavioral analysis.</li>
	<li>Position a high-resolution video camera in a way that favors the viewing of axial, limb, and orolingual AIMs. Since rotational behavior is present under these experimental conditions, the mirrors behind the cylinder will allow tracking AIMs at a 360&#176; angle. Use a tripod or fix the camera directly to the bench. 
	NOTE: Orolingual AIMs can be challenging to score, especially if there is a high incidence of rotational behavior. Capturing videos with the camera positioned slightly below the plane where the animals are at a 15&#176; angle will help observe this movement subtype.</li>
	<li>Gently remove the animal from the cylinder and administer L-DOPA (5 mg/kg combined with 12.5 mg/kg of benserazide, s.c.).</li>
	<li>Put the animal back in the cylinder and start a timer to track AIMs after L-DOPA injection. Use a video camera to record AIMs for 180 min after the injection of L-DOPA. Although the analysis can be carried out by direct observation of the animal, offline scoring is preferred. 
	NOTE: Ensure the timer is also videotaped so that AIMs can be scored at precise times after L-DOPA administration.</li>
	<li>Score AIMs at 30 min intervals up to 180 min post-L-DOPA injection (see Table 1 for definitions) as initially described20,23. Scores must be given over 1-2 min epochs and classified as axial, limb, or orolingual (Figure 1).</li>
	<li>Carefully observe each AIM and attribute scores for severity and amplitude. Use Table 1 for instructions. Do not include normal behaviors, such as rearing, sniffing, grooming, and gnawing in the rating. There will be a total of six observation periods (30, 60, 90, 120, 150, and 180 min).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SUBTYPES</td>
		</tr>
		<tr>
			<td>Axial: torsion of head, neck, and trunk</td>
		</tr>
		<tr>
			<td>Limb: involuntary movements of both distal and proximal forelimb</td>
		</tr>
		<tr>
			<td>Orolingual: involuntary movements of the orofacial muscles</td>
		</tr>
		<tr>
			<td>SEVERITY</td>
		</tr>
		<tr>
			<td>0: Absent</td>
		</tr>
		<tr>
			<td>1: Occasional (with interruptions, present in less than half of the observation time)</td>
		</tr>
		<tr>
			<td>2: Frequent (with interruptions but present in more than half of the observation time)</td>
		</tr>
		<tr>
			<td>3: Continuous but interrupted by external sensory stimuli</td>
		</tr>
		<tr>
			<td>4: Continuous and not interrupted by external sensory stimuli</td>
		</tr>
		<tr>
			<td>AMPLITUDE</td>
		</tr>
		<tr>
			<td>Axial</td>
		</tr>
		<tr>
			<td>1: Torsion of head and neck at approximately 30&#176; angle</td>
		</tr>
		<tr>
			<td>2: Torsion of head and neck at approximately 30&#176; &#60; angle &#8804; 60&#176;</td>
		</tr>
		<tr>
			<td>3: Torsion of the head, neck, and upper trunk at approximately 60&#176; &#60; angle &#8804; 90&#176;</td>
		</tr>
		<tr>
			<td>4: Torsion of head, neck, and trunk at &#62; 90&#176; angle, often causing the rat to lose balance</td>
		</tr>
		<tr>
			<td>Limb</td>
		</tr>
		<tr>
			<td>1: Small involuntary movements of the distal forelimb</td>
		</tr>
		<tr>
			<td>2: Movements of low amplitude causing translocation of both distal and proximal forelimb</td>
		</tr>
		<tr>
			<td>3: Involuntary movements of the whole limb, including shoulder muscles</td>
		</tr>
		<tr>
			<td>4: Strong limb and shoulder movements, often similar to ballism</td>
		</tr>
		<tr>
			<td>Orolingual</td>
		</tr>
		<tr>
			<td>1: Small involuntary movements of the orofacial muscles</td>
		</tr>
		<tr>
			<td>2: Orofacial movements of high amplitude with tongue protrusion</td>
		</tr>
	</tbody>
</table>
Table 1: AIMS definition and rating criteria.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62924/62924fig01.jpg" /> 
Figure 1: Sequence of photos showing each AIM subtype. (A to A&#39;&#34;) Axial AIM showing dystonic-like movements in different angles (A: 30&#176; angle, A&#39;: 30&#176; &#60; angle &#8804; 60&#176;, A&#34;: 60&#176; &#60; angle &#8804; 90&#176;, and A&#39;&#34;: &#62; 90&#176; angle). (B to B&#34;) Limb AIM (black arrows) showing involuntary movements of the distal (B and B&#39;) and whole forelimb (including shoulder, B&#34;). (C) Orolingual AIM (red arrow) of high amplitude with tongue protrusion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62924/62924fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Data analysis
<ol>
	<li>Compute axial, limb, or orolingual AIMs subtypes by multiplying the severity and amplitude scores for each observation period after L-DOPA administration (i.e., six observation periods) as previously described20. The theoretical maximum score that one animal could accumulate in one testing session is 240 (maximum score per observation period is 40, see Table 1 for details).</li>
	<li>Plot AIMs scores for all the six observation periods recorded on Wednesday, Thursday, and Friday over the 3 weeks of chronic L-DOPA administration (Figure 2). AIMs can be summed up to indicate each animal&#39;s maximum score on each scoring day (Figure 3) or each week (Figure 4) over the 3 weeks of chronic L-DOPA administration.</li>
	<li>Verify if the data has a close-to-normal distribution before selecting the statistical analysis approach. 
	NOTE: AIMs rating scales consist of ordinal data and can be analyzed with nonparametric statistics. Parametric tests are widely used when comparing AIMs scores across experimental groups following repeated testing sessions40.</li>
</ol>

The authors have no conflicts of interest.

This protocol demonstrates how to induce and analyze AIMs in the rat model of PD induced by unilateral microinjection of 6-OHDA in the MFB. Chronic daily administration of low doses of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) produced the development of AIMs over the 3 weeks of treatment. Temporal analysis revealed a significant increase of AIMs, and the peak-dose dyskinesia is observed between 30 and 90 min after L-DOPA administration. AIMs are repetitive and purposeless movements affecting axial, limb, and orolingual muscles. The protocol and the data presented here agree with the previous literature22,23,49,50,51.
The axial, limb, and orolingual AIMs rating scale is very sensitive because it rates the amount of time (i.e., severity) that the rodents display dyskinetic-like movements and indicates the amplitude of these movements20. AIMs scores can be expressed in different temporal resolutions (see Figure 2, Figure 3, Figure 4)17,18,19,52. Several studies have addressed the role of L-DOPA dosage on the development of AIMs. Chronic administration of lower doses of L-DOPA (5-20 mg/kg) favors a gradual development of AIMs over the treatment, while high doses of L-DOPA (25 mg/kg or more) result in the rapid and robust development of AIMs23,25,27,53. Interestingly, a proportion of severe 6-OHDA-lesioned animals may not develop AIMs (or have very low AIMs scores) following chronic administration of low doses of L-DOPA40,49,53. It is also essential to consider the L-DOPA&#39;s route of administration. Although AIMs share similar onset, time profile, and severity grade40, it has been demonstrated that dose-failure episodes frequently occurred in rats treated intraperitoneally27. Also, i.p. administration of L-DOPA produced inter-individual variations in plasma levels, an effect that was not observed after s.c. injections27.
In this study, AIMs were evident in animals with severe nigrostriatal damage (over 90% of dopaminergic depletion). Massive loss of striatal dopaminergic innervation is characterized by a reduction in the number of adjusting steps performed with the forelimb contralateral to the lesion48. Therefore, we recommend the stepping test to select animals with putative severe 6-OHDA lesions at the nigrostriatal pathway. The amphetamine-induced rotation test is commonly used to select putative 6-OHDA-lesioned rats. Amphetamine increases dopamine release in the intact striatum, and thus it increases the existing imbalance between the innervated and denervated striatum. However, an interesting study conducted in 312 6-OHDA-lesioned rats11 revealed no correlation between amphetamine-induced rotation and tyrosine hydroxylase positive cells in the ipsilateral substantia nigra pars compacta. This same study showed a poor correlation between amphetamine-induced rotation and the severity of AIMs.
There is a clear relationship between the phenomenology of AIMs and the pattern of nigrostriatal dopaminergic depletion20. The 6-OHDA rat model of PD we used in this protocol develops severe unilateral depletion of nigrostriatal dopamine terminals following 6-OHDA infusion in the MFB. Fully dopamine-depleted 6-OHDA-lesioned rats have a higher incidence of AIMs compared to partially dopamine-depleted animals. A detailed analysis of striatal expression of the neuropeptides dynorphin and enkephalin following 6-OHDA lesion and chronic L-DOPA administration is described elsewhere45 and is consistent with previous literature23,25.&#160;At the molecular level, dyskinetic rodents share molecular markers associated with the dyskinesia evident in nonhuman primate models of PD and PD patients, such as striatal upregulation of &#916;FosB transcription factor26,32,33,54. Also, the rodent model of PD shows increased microvessel density in the striatum in response to L-DOPA treatment55 in a similar way observed in the putamen of PD patients56. Rodent models of LIDs respond to antidyskinetic pharmacological approaches used in the clinic, such as the NMDA receptor antagonist amantadine22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37, 38,39. Altogether, these pieces of evidence confirm the face validity and predictive validity of the 6-OHDA rat model to characterize the pathophysiology of LIDs and for the screening of compounds with antidyskinetic properties. The mice model of LIDs is also a valuable tool to study several aspects of LIDs in transgenic mice. Protocols for the mice model of LIDs can be found in papers from different research groups30,57,58.
It is important to note that the 6-OHDA-lesioned rodent model of LIDs has limitations. 6-OHDA-induced dopaminergic depletion is acute compared to the progressive nature of nigrostriatal depletion in the PD patient5. Also, it can be challenging to find an excellent therapeutic window for using an antidyskinetic drug in patients because increasing the dosage of a drug may interfere with the beneficial effect produced by L-DOPA or induce side effects that may not have been observed in the animal model59,60. Another limitation of the rat model of LIDs is the lack of physical similarity between AIMs in the rodents and the manifestation of the choreiform movements observed in patients and nonhuman primates10,61,62. Another potential limitation of the protocol presented here is that AIMs are scored every 30 min after L-DOPA administration, but this issue can be managed if the experimenter videotapes all animals for 180 min after L-DOPA administration. The experimenter can then adjust the scoring protocol for more frequent scoring (e.g., every 10 or 20 min). This approach is advantageous in experiments that require a more dynamic picture of AIMs time course (e.g., the combination of AIMs scoring and in vivo electrophysiological or neurochemical recordings).
In conclusion, the 6-OHDA rat model of LID reproduces the pathophysiology of disease and develops AIMs that can be modulated by drugs clinically used. Although it has limitations, the 6-OHDA rat model of LID still represents an invaluable preclinical tool to identify effective antidyskinetic interventions with translational potential.

The dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) represents the most effective treatment for the motor symptoms of Parkinson&#39;s disease (PD)1. L-DOPA therapy may ameliorate motor symptoms associated with PD but loses effectiveness with time. Motor fluctuations such as &#34;wearing-off fluctuation&#34; or &#34;end-of-dose deterioration&#34; manifest clinically as a shortened duration of the effect of single L-DOPA doses2. In other cases, clinical manifestations consist of slow twisting movements and abnormal postures (dystonia)3 and occur when dopamine levels are low (off-period dystonia)4. On the other hand, L-DOPA-induced dyskinesias (LIDs) appear when dopamine levels in the plasma and the brain are high5.
LIDs produce debilitating side effects that include motor complications such as choreiform, dystonic, and ballistic6 movements. Once established, LIDs occur after every L-DOPA administration. Motor complications occur in 40%-50% of PD patients undergoing L-DOPA therapy for 5 years, and the incidence increases over the years7. Although the pathophysiological mechanisms involved in the development of LIDs in PD patients are not yet fully elucidated, the extent of dopaminergic denervation, pulsatile L-DOPA administration, downstream changes in striatal proteins and genes, and abnormalities in non-dopamine transmitter systems are factors that contribute to the development of these unwanted side effects6,8,9,10.
The neurotoxin 6-hydroxydopamine (6-OHDA) is a well-characterized tool to study PD in rodents11,12,13,14. Since 6-OHDA does not cross the blood-brain barrier, it must be injected directly into the nigrostriatal pathway. 6-OHDA-induced dopaminergic depletion is concentration- and site-dependent15. Unilateral administration of 6-OHDA at the medial forebrain bundle (MFB) can produce severe (&#62;90%) nigrostriatal damage in rodents16,17,18,19. Chronic administration of L-DOPA to severe unilaterally 6-OHDA-lesioned rodents causes the appearance of dyskinetic-like movements named abnormal involuntary movements (AIMs). Dyskinetic-like movements in rodents share similar molecular, functional, and pharmacological mechanisms related to LIDs in PD patients5. Therefore, 6-OHDA-lesioned rats20 and mice21 are valuable preclinical models to study LIDs. When treated chronically (7-21 days) with therapeutic doses of L-DOPA (5-20 mg/kg), unilaterally 6-OHDA-lesioned rats and mice show a gradual development of AIMs that affect the forelimb, trunk, and orofacial muscles contralateral to the lesion17,18,19,20,22,23,24. These movements are presented at a time course similar to L-DOPA-induced peak-dose dyskinesias in PD patients25 and are characterized by hyperkinetic movements and dystonia5. AIMs are usually scored based on their severity (e.g., when a specific AIM is present) and amplitude (e.g., characterized by the amplitude of each movement)5,23,25.
6-OHDA-lesioned rodent models of LIDs present face validity (i.e., the model has several characteristics that look like the human condition)5,11,26,27,28. Rodent AIMs, similar to what occurs in PD patients, are seen as hyperkinetic (forelimb and orolingual) and dystonic (axial) movements29 and mimics peak-dose dyskinesia. At the molecular and functional level, rodent models share many pathological characteristics with PD patients5, such as upregulation of FosB/&#916;FosB19,26,30,31,32,33 and serotonin transporter (SERT)34,35. Concerning predictive validity, drugs that reduce LIDs in PD patients (e.g., the N-methyl-D-aspartate (NMDA) receptor antagonist amantadine) present antidyskinetic efficacy in the rodent model22,36,37,38,39.
The rodent AIMs rating scale was created based on four AIMs subtypes that include AIMs affecting the head, neck, and trunk (axial AIMs), hyperkinetic forelimb movements (limb AIMs), and dyskinetic-like orolingual movements (orolingual AIMs). Although contralateral rotation (locomotive AIMs) is also present in unilaterally lesioned rodents20,22,23,25,40, it has not been scored as a dyskinetic-like movement since it may not represent a specific measure of LIDs22,37,41.
Here, we will describe how to induce and analyze dyskinetic-like movements (axial, limb, and orolingual AIMs) in the severe (&#62;90%) unilaterally 6-OHDA-lesioned rat model of PD. We organized our protocol based on the previous literature and our laboratory expertise.

<table><tbody><tr><td>6-hydroxydopamine hydrobromide</td><td>Sigma-Aldrich, USA</td><td>H6507</td><td>Neurotoxin that produces degeneration of catecholaminergic terminals</td></tr><tr><td>Benzerazide hydrochloride</td><td>Sigma</td><td>B7283</td><td>Peripheral dopa-decarboxylase inhibitor</td></tr><tr><td>Camera Bullet IR Turbo HD (HD-TVI)&amp;nbsp; 2.8mm B</td><td>HIKVISION</td><td>DS-2CE16C0T-IRP</td><td>Camera used to record all behavior</td></tr><tr><td>Imipramine hidrochloride</td><td>Alfa Aesar</td><td>J63723</td><td>Norepinephrine transporter inhibitor (NET) used to protect noradrenergic neurons from 6-OHDA</td></tr><tr><td>Ketamine hydrochloride</td><td>Ceva Animal Health</td><td></td><td>Anesthesia for surgical intervention</td></tr><tr><td>L-3,4-dihydroxyphenylalanine (L-DOPA) methyl ester (hydrochloride)</td><td>Cayman Chemical Company</td><td>16149</td><td>Dopamine precursor</td></tr><tr><td>Mirrors</td><td></td><td></td><td>Used to observe the behavior of animals during experiments in all directions</td></tr><tr><td>Needles 0.30 x 13 mm</td><td>PrecisionGlide</td><td></td><td>Needles used to inject drugs</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Samtec</td><td></td><td>Salt</td></tr><tr><td>Syringes 1 ml Sterile</td><td>BD Plastipak</td><td></td><td>Syringes used to inject drugs</td></tr><tr><td>Transparent cylinders</td><td></td><td></td><td>Used to record animal behavior during experiments</td></tr><tr><td>Xylazine hydrochloride</td><td>Ceva Animal Health</td><td></td><td>Sedative, analgesic and muscle relaxant for surgical intervention</td></tr></tbody></table>

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

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). The most common pattern observed in the clinic is the peak-dose dyskinesia which consists of clinical manifestations of choreiform, dystonic, and ballistic movements. The 6-hydroxydopamine (6-OHDA) rat model of PD mimics several characteristics of LIDs. After repeated L-DOPA administration, 6-OHDA-lesioned rats exhibit dyskinetic-like movements (e.g., abnormal involuntary movements, AIMs). This protocol demonstrates how to induce and analyze AIMs in 6-OHDA-lesioned rats with 90%-95% dopaminergic depletion in the nigrostriatal pathway. Repeated administration (3 weeks) of L-DOPA (5 mg/kg, combined with 12.5 mg/kg of benserazide) can induce the development of AIMs. The time course analysis reveals a significant increase in AIMs at 30-90 min (peak-dose dyskinesia). Rodent models of LIDs are an important preclinical tool to identify effective antidyskinetic interventions.

Although the AIMs patterns observed in rats are simpler and limited compared to those observed in humans and nonhuman primates, this model reproduces both hyperkinetic and dystonic-like movements induced by chronic L-DOPA administration. Here we present data collected from a group (n = 10) of unilaterally 6-OHDA-lesioned rats chronically treated with L-DOPA (5 mg/kg combined with 12.5 mg/kg of benserazide) for 3 weeks (Monday to Friday). Note that the data presented in Figure 2, Figure 3, and Figure 4 are from the same animals.
Figure 2 shows the time course analysis for scores applied to axial, limb, and orolingual AIMs over 3 weeks of chronic L-DOPA administration. Each AIM score subtype at a specific observation period is reported as the multiplication of severity and amplitude scores in this analysis. This type of analysis can be convenient to explore the effect of drugs that interfere with the temporal profile of AIMs.Note that L-DOPA-induced peak-dose dyskinesias occur between 30-90 min with a gradual decrease after 120 min post-injection (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62924/62924fig02.jpg" /> 
Figure 2: Time course analysis showing axial, limb, and orolingual AIMs over 3 weeks of chronic L-DOPA administration (5 mg/kg combined with 12.5 mg/kg of benserazide) to 6-OHDA-lesioned rats. (A) Axial, (B) limb, and (C) orolingual AIMs were scored on Wednesday, Thursday, and Friday over 3 weeks of chronic L-DOPA administration. Each AIM subtype is reported as the multiplication of severity times amplitude on each of the six observation periods. Each AIM subtype can be analyzed individually or summed up as indicated in (D). Note that the peak severity of the AIMs will gradually increase during the first week of chronic L-DOPA administration. Data are expressed as mean &#177; SEM (n = 10). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62924/62924fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3 shows the sum of AIMs scores presented for all the six observation periods depicted in Figure 2 (datasets are color-coded for easy comparisons). This analysis indicates each animal&#39;s maximum score on each scoring day (Wednesday, Thursday, and Friday) over the 3 weeks of chronic L-DOPA administration. This type of analysis can be advantageous to explore the effect of compounds with potential antidyskinetic profiles, especially because AIMs scores are stable over weeks 2 and 3 of chronic L-DOPA administration. Furthermore, weekly AIMs scores (Figure 4) can be generated from the sum of scores attributed to 6-OHDA-lesioned rats on Wednesday, Thursday, and Friday.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62924/62924fig03.jpg" /> 
Figure 3: Sum of axial, limb, and orolingual AIMs on individual scoring days over 3 weeks of chronic L-DOPA administration (5 mg/kg combined with 12.5 mg/kg of benserazide) to 6-OHDA-lesioned rats. In this figure, AIMs scores presented in each of the six observation periods (see details in Figure 2 legend) were summed. Individual datasets were color-coded to compare with the data displayed in Figure 2. This analysis indicates each animal&#39;s maximum score on each scoring day (Wednesday - W, Thursday - T, and Friday - F) following 3 weeks of chronic L-DOPA administration. (A) Axial, (B) Limb, and (C) orolingual AIMs. (D) Sum of axial, limb, and orolingual AIMs. One-way repeated measures ANOVA and Holm-Sidak post-hoc test revealed that AIMs increase gradually over the chronic administration of L-DOPA. Note that AIMs scores are stable over weeks 2 and 3 of chronic L-DOPA administration (*p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 vs. Wednesday (W) on Week 1). Data are expressed as mean &#177; SEM (n = 10). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62924/62924fig03large.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/62924/62924fig04.jpg" /> 
Figure 4: Sum of weekly axial, limb, and orolingual AIMs throughout chronic L-DOPA administration (5 mg/kg combined with 12.5 mg/kg of benserazide) to 6-OHDA-lesioned rats. In this figure, AIMs scored each week (Wednesday, Thursday, and Friday - see Figure 3) were summed. This analysis indicates each animal&#39;s maximum score on each week of chronic L-DOPA administration. (A) Axial, (B) Limb, and (C) orolingual AIMs. (D) Sum of axial, limb, and orolingual AIMs. One-way repeated measures ANOVA and Holm-Sidak post-hoc test revealed that AIMs increase over the chronic administration of L-DOPA (*p &#60; 0.05 vs. Week 1). Data are expressed as mean &#177; SEM (n = 10). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62924/62924fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Rating L-DOPA-Induced Dyskinesias in the Unilaterally 6-OHDA-Lesioned Rat Model of Parkinson's Disease at JoVE.com

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

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

L-DOPA-induced dyskinesias (LIDs) refer to motor complications that arise from prolonged L-DOPA administration to patients with Parkinson's disease (PD). ...

rating-l-dopa-induced-dyskinesias-unilaterally-6-ohda-lesioned-rat

Nanyang Technological University

Research

JoVE Journal

Biology

2.6K Views.  University of São Paulo. Rodent models of L-DOPA-induced dyskinesias are invaluable tools to identify therapeutic interventions to attenuate the development or alleviate the manifestations that emerge due to the repeated administration of L-DOPA. This protocol demonstrates how to induce and analyze dyskinetic-like movements in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease.

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

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

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

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

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 ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

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

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

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

Neuroscience

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

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

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

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

Induction of Levodopa-Induced Dyskinesias in a Rat Model of Parkinson's Disease

Source: Caulfield, M. E., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62970">Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#39;s Disease.</a> J. Vis. Exp. (2021).
This video demonstrates the process of inducing levodopa-induced dyskinesias in a Parkinson&#39;s rat model by administering levodopa alongside a dopamine conversion inhibitor. Repeated injections overstimulate the brain neurons, resulting in motor dysfunction.

Indukcja dyskinez wywołanych lewodopą w szczurzym modelu choroby Parkinsona

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;
1. ...

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

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