This work was supported by the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme [600-IRMI/FRGS 5/3 (033/2019)].

The present study has been approved by the Committee on Animal Research and Ethics (CARE), Universiti Technologi MARA (UiTM) [Reference No: UiTM CARE 346/2021, dated 7 May 2021].
NOTE: The published protocols22,25,26 for standard husbandry and maintenance of the 6-OHDA-lesioned adult zebrafish PD model were utilized. Experiments were conducted with adult male zebrafish (Danio rerio) aged more than five months old with a standardized length of 3.2-3.7 cm.
1. Zebrafish maintenance and pre-ICV microinjection preparations
<ol>
	<li>Maintain the fish in an aerated water tank under a controlled temperature of 28 &#177; 1.0 &#176;C. For zebrafish husbandry and maintenance, use distilled water mineralized with commercial sea salt (1 g/L) throughout the experiment27.</li>
	<li>House a maximum of 25 fish per 45 L tank or one fish per 1.8 L water and expose them to a schedule of 14 h light and 10 h dark photoperiod. Feed the fish at least twice per day with food pellets supplemented with freeze-dried worms.</li>
	<li>Prepare a concentrated stock solution of tricaine methanesulfonate (MS-222) by dissolving 2.5 g of MS-222 and 5 g of sodium bicarbonate in 250 mL of distilled water. Dilute 2 mL of stock solution to produce 200 mL of working anesthesia solution.</li>
	<li>Prepare 99.96&#160;mM of 6-OHDA by first dissolving 0.2 mg of ascorbic acid in 1 mL of 0.9% w/v sterile-filtered NaCl. Filter the solution with 0.2-micron filter before adding 25 mg of 6-OHDA in powder form into the solution. Prepare the solution fresh before each injection and store it in dark at 4 &#176;C. 
	CAUTION: Wear appropriate personal protective equipment (i.e., gloves, laboratory coat, and face mask) and practice good laboratory practices when handling the chemicals. All handlings of the chemicals should be done within a biosafety cabinet.</li>
</ol>
2. Anaesthetisation and ICV injection of zebrafish
<ol>
	<li>Fast the fish for 24 h to avoid regurgitation during anesthesia. Anaesthetise the fish by immersing it into a container containing 0.01% w/v of MS-222 solution for approximately 1 min or until all visible muscular movement ceases.</li>
	<li>Position the anesthetized fish on a water-soaked sponge placed under a stereomicroscope and wet the fish regularly.</li>
	<li>Identify the position for injection based on the intersection between the metopic suture (MS), coronal suture (CS), and sagittal suture (SS) that connects the frontal and parietal skull of the zebrafish brain.</li>
	<li>Make a small hole of 1.0 mm2 area using a sharp 27 G needle in the skull guided by the specific anatomical position on the zebrafish skull (Figure 1A,B).</li>
	<li>Lower the microcapillary injector at a 60&#176; angle until it reaches a depth of 1,200 &#181;m from the cranial roof of the zebrafish skull (Figure 1C). Press the Z limit to fix the position.</li>
	<li>Set the initial injection pressure to 4000 hPa and compensation pressure to 10 hPa. Set the duration of injection to 0.3 s. Lower the intensity of injection with each subsequent injection.</li>
	<li>Inject 0.5 &#181;L of 99.96&#160;mM neurotoxin 6-OHDA (or 0.9% w/v saline for sham control group) and let the microcapillary rest for 20 s. Continue to wet the fish with distilled water throughout the injection process to prevent drying out.</li>
	<li>Slowly remove the microcapillary and resuscitate the fish under running distilled water. Place the fish in an isolated recovery tank and remove any distractions that can potentially disturb the recovery process.</li>
	<li>Flush the microcapillary before the next injection to clear the blockage and ensure that the intensity of injection is sufficient to yield the desired volume of 0.5 &#181;L of 6-OHDA.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63355/63355fig01v2.jpg" /> 
Figure 1: Injection site of neurotoxin, 6-OHDA. (A) The point of microcapillary entry is guided by the intersection between the metopic suture (MS), coronal suture (CS), and sagittal suture (SS) that connects the frontal and parietal skull of the zebrafish brain (plan view). (B) A schematic drawing (plan view) of the zebrafish skull and brain shows the microcapillary, which is lowered directly above the habenula (Hab), and its point of entry at the intersection between hemispheres. (C) A schematic drawing (sagittal section) of the zebrafish brain shows the angle of injection and depth of penetration. The black dot represents the lesioned site that is situated above the targeted area, the ventral diencephalon. Abbreviations: 6-OHDA: 6-hydroxydopamine, CS: coronal suture, Dn: diencephalon, Hab: habenula, Hyp: hypothalamus, MS: metopic suture, OB: olfactory bulb, POA: preoptic area, PT: posterior tuberculum, SS: sagittal suture, Tec: tectum, and Tel: telencephalon. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63355/63355fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Locomotor assessment
NOTE: Locomotor assessment of zebrafish (n = six / group; sham vs lesioned) was assessed individually via the open tank test using established protocols28,29 at day three and day 30 post-6-OHDA lesion.
<ol>
	<li>Video recording
	<ol>
		<li>Place the experimental tank (length 20&#160;cm, width 11.5&#160;cm, height 13&#160;cm) with its walls covered with white paper on a raised platform (Figure 2A).</li>
		<li>Illuminate the tank from the bottom using a light source. Fill the tank with distilled water (80%-90% full) and maintain the temperature at 28 &#177; 1.0 &#176;C. Measure the temperature using a thermometer and regulate it using a commercial aquarium heater.</li>
		<li>After a minimum of 2 min of acclimatization, record the fish swimming behavior from a plan view on the 2-dimensional (2D) plane of the experimental arena using a video camera for 5 min (Figure 2B). To avoid inconsistency in the swimming behavior of the earlier and the last batch of recordings, do not exceed the acclimatization by 10 min30.</li>
		<li>Analyze the videos using video tracking software with the open-tank protocol for the acquisition of distance traveled (cm) and mean speed (cm/s) of each subject.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63355/63355fig02.jpg" /> 
Figure 2: Experimental setup of an open tank test for assessment of zebrafish locomotor behavior. (A) The experimental tank (front view) is placed on a raised platform that is illuminated from below. The four walls of the tank are covered with white paper and the recordings are captured axially. The temperature is measured using a thermometer and regulated at 28 &#177; 1.0 &#176;C using a commercial aquarium heater. (B) Screenshot (plan view) of video recording that is captured using the setup. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63355/63355fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Data analysis
	<ol>
		<li>Double click on the icon to open the video tracking software. Click on the File tab and select Create New Empty Experiment. This will allow the user to customize the experiment parameters according to the aims of the investigation.</li>
		<li>Click on the Protocol tab, select Video Sources, and click on Add New Video Source. Click on the available drop-down list and select the Video File option. This will prompt the file browsing pop-up from which the video recordings of interest can be selected.</li>
		<li>Click on the Apparatus subtab and select the Rectangular icon to set up the apparatus. Drag the rectangular icon to cover the whole experimental arena. Set the scale bar accordingly and input the numerical value of the scale measurement used in the length of the ruler section. The present experiment used a 10 mm scale for the open tank test.</li>
		<li>Set the animal color by selecting The Animals are Darker than The Apparatus Background. Leave the other available options in Tracking to the preset default settings.</li>
		<li>In the Zones subtab, click on the previously drawn apparatus. This zone is set as the standard zone of which the position is the same for all tests.</li>
		<li>Select the following options under test scheduling and test data report: Test Duration, Total Distance Traveled, and Average Speed. Other available tests on the list are optional and dependent on the researcher&#39;s investigative interest.</li>
		<li>In the Experiment tab, assign the animals according to their test group by typing in the group name under the Name section and the number of animals per group in the Number of Animals section.</li>
		<li>Switch to Tests tab to run the experiment. Click on the Start Test icon and wait until all the videos are analyzed.</li>
		<li>In the Results tab, click on the View the Report icon to view the locomotor data in text report form.</li>
	</ol>
	</li>
</ol>

The authors declare no conflicts of interest.

The present work successfully demonstrated the locomotor assessment of the established 6-OHDA-induced, adult zebrafish-based PD model. The entire experiment involved three major steps: pre-ICV microinjection preparations, ICV microinjection of zebrafish, and locomotor assessment. To ensure the healthy recovery of adult zebrafish following the ICV microinjection procedure and good experimental outcome, some good practices for each step have been recommended in the present study.
Pre-ICV microinjection preparation: Animal selection was best performed the day before the experiment. The gender was identified and the length of the fish was measured. Male adult zebrafish with a standardized length of 3.2-3.7 cm were placed in a separate experimental tank. Additionally, the fish should undergo 24 h fasting period to avoid regurgitation during anaesthesia33. A fish tank (with its four walls covered with white paper) with a standing water tank set up should be prepared before the experiment to lessen the exterior stress and help the recovery process of the zebrafish. All chemicals were prepared fresh before the start of each experiment as they could deteriorate rapidly over time and become unstable at room temperature34,35.
Microinjection of 6-OHDA:Clean and gentle handling of zebrafish should be performed throughout the process to prevent the introduction of unnecessary injury and infection to the fish. The fish should be placed on top of a wet sponge and kept in a moist condition to avoid drying out36. A small incision was made using a sterile needle with firm and appropriate force to avoid extra pressure that may crack the zebrafish skull. This incision should allow entry of the microcapillary into the brain cavity. The microcapillary was then lowered until a depth of 1,200 &#181;m from the entry point, which is in between two hemispheres-the telencephalon and tectum (Figure 1B,C). The entry point was chosen between these hemispheres to prevent any additional laceration of neurons37. This technique involved the use of a microinjector, the pressure and timing of the delivery should be calibrated to ensure delivery of 0.5 &#181;L of neurotoxin. This calibration can be performed by measuring the size of the droplet formed on the filter paper38. Our practice usually involved the following set up of programmable parameters whereby the intensity of injection was lowered with each subsequent injection (injection pressure: 4000 hPa, duration of injection: 0.3 s, and compensation pressure: 10 hPa). In order to avoid the neurotoxin from leaking out of the brain cavity, a 20 s interval was applied between injection and withdrawal of the microcapillary35. Due to the small size of the capillary, the microcapillary may be blocked after each injection. As such, the microcapillary should be essentially flushed prior to the next injection so as to clear the blockage and ensure that the intensity of injection is sufficient to yield the desired volume of 0.5 &#181;L of 6-OHDA. The fish would then be transferred into a recovery tank maintained at 28 &#177; 1.0 &#176;C. If the fish fails to recover within 30 s, flush out its gill and mouth with distilled water until full recovery of muscle movements occurs.
Locomotor Assessment: To ensure good locomotor assessment on 6-OHDA-induced adult zebrafish, the behavioral study should be conducted within the same time frame for each time point. Each behavioral study should allow for a minimum of 2 min acclimatization period and should be performed within 4 h39. The present experiment was carried out in the early morning between 8 am to 12 pm as zebrafish were more active during this period40. A longer acclimatization period is necessary if zebrafish show any abnormal behavior with clear signs of stress and anxiety (freezing and erratic behavior)41. However, to avoid inconsistency in the swimming behavior of the earlier and the last batch of recordings, the acclimatization should not exceed 10 min30. For the open field test, an experimental tank of any size, color, shape, and texture could be used for recordings that range from a minimum of 5 to 30 min42,43. Zebrafish behavior is greatly influenced by the temperature of its surroundings. Small fluctuations of more than 4 &#176;C could greatly impact the swimming speed44. Hence, the temperature of water in the experimental tank should be strictly maintained under a controlled temperature of 28 &#177; 1.0 &#176;C using a commercial heater, and the water level was kept around 12 cm deep throughout the experiment. The walls of the tank were covered with white paper to create contrast between the test subject and experimental arena as well as to reduce external stimuli that may cause an unprompted reaction from test subjects45. The fish from each group were tested individually in accordance with the current standard practice for zebrafish neurobehavioural research23,37,46. Given the zebrafish propensity to social interactions, there is concern that isolation during the testing period might impact their behavior47. However, the current experimental setup was limited to a maximum of 10 min per trial and this short period of isolation was found to not have any effect on the locomotor activity of adult zebrafish48. In order to provide accurate data collection for the behavioral study, the assessment was performed by randomly selecting the zebrafish from different experimental groups (i.e., alternating two zebrafish from either the sham and 6-OHDA lesioned group until n = 6) during the open tank test49. The recorded videos were analyzed using a video tracking system that is commonly used for rodents&#39; behavioral tracking. As zebrafish is an emerging animal model, the behavioral tests conducted using zebrafish are usually adapted from the established scientific literature on rodents50. Here, we demonstrated the ability of the video tracking software to automatically track zebrafish in the experimental arena and effectively compute the desired parameters. The video tracking software stood out from the other available software due to the variety of video files supported by the software, regular update package releases, and support provided for different operating systems51.
One of the limitations of existing animal-based PD models is the lack of mechanistic similarities that mimic the motor impairment as observed after dopaminergic neuronal loss in the substantia nigra pars compacta of the human brain52. The emergence of the adult zebrafish-based PD model may, however, address this particular limitation. As observed in the present study, the reduced swimming speed corresponded to our previous cellular findings whereby more than 85% dopaminergic neuronal loss in the ventral diencephalon of 6-OHDA-induced adult zebrafish model three days postlesion22. It appears that specific ablation of dopaminergic neurons in this area of interest is required to disrupt the descending motor signaling from the brain, causing slowness in movement53. L. J. Caldwell, et al.23&#160;who performed ICV injection of 6-OHDA at the optic tectum (point of entry), for instance,&#160;observed only changes in zebrafish shoaling and mating behavior. Ablation of DpN in ventral Dn is crucial as the population of DpN in the Dn of adult zebrafish acts as the only dopamine source for zebrafish motor neurons. This is analogous to the human substantia nigra54. The present study also observed faster-swimming speed and longer distance traveled by lesioned zebrafish in later postlesion time points, indicating continued and ultimately full restoration of dopamine signaling that governs zebrafish swimming behavior. These findings thus validated the capability of newly regenerated dopaminergic neurons in regaining their functional activities in lesioned adult zebrafish.
The current application route of 6-OHDA involved a slightly invasive injection paradigm that required insertion of microcapillary deep into the brain, toward the lesion area of ventral Dn. This method is slightly laborious in comparison to peripheral injection and needs to be performed within 3 min per fish to reduce the risk of mortality following injection. As such, prior practice of ICV injection is required to ensure that the method can be performed within the critical duration at the targeted area (Dn). In order to achieve a valid locomotor assessment of adult zebrafish, the open tank test is limited to only 4 h of assessment period per day. Hence, prior planning is required in an experimental framework that involves a large number of animals whereby extra time should be allocated to ensure that the setup met the minimum requirements for each recording (e.g., temperature and water depth). This planning is especially crucial in time-based experiments such as that of the current study, as each recording needs to be performed on the intended timepoint. The present experimental setup was limited to the study of two swimming parameters that specifically assessed the zebrafish motor function. Other behavioral parameters such as shoaling and anxiety-like behavior, however, required other experimental setups and different analytical methods. In summary, this is a reproducible and useful method to study the DpN neuroregeneration process in 6-OHDA-induced adult zebrafish which may yield important insights into cell replacement treatment strategies against PD.

Parkinson&#39;s disease (PD), a disease distinctively characterized by muscle rigidity, resting tremor, and bradykinesia, is the fastest growing neurological disease in the world1,2. The risk and prevalence of PD increase rapidly with age especially in individuals aged 50 years and above3. The etiology and pathogenesis of PD hitherto remain poorly understood. This has often left the early-onset of PD undiagnosed. At present, the lack of dopamine and the loss of dopaminergic neurons (DpN) in PD patients are strongly linked to the manifestation of motor symptoms4. Capitalizing on this relationship, several treatments have been designed either to act directly as dopamine replacement (i.e., levodopa) or to compensate for the loss of DpN (i.e., deep brain stimulation). Although these treatments bring about symptomatic benefits, they do not modify the deteriorating course of the disease5. In view of this significant weakness, cell replacement therapy has been proposed. The efficacy of this approach is, however, inconsistent given the challenges of graft preparation, cell growth control, and phenotype instability. Cell replacement therapy, which had raised ethical concerns, also poses the risk of inducing brain tumors and unwanted immune reactions6,7.
The limitations of current therapeutic strategies have led to a greater emphasis on the regeneration of DpN as a potential approach in treating PD. Regeneration of DpN or neuroregeneration has emerged as one of the promising breakthroughs in the management of PD, not only due to its potential as a new therapeutic method but also as means to understand the mechanism of the disease8,9. This approach focuses on the restoration of neuronal function through differentiation, migration, and integration of existing progenitor cells into the lesioned circuitry10. In order to further explore neuroregeneration, various in vivo studies have been undertaken. It was found that vertebrates such as mammals, amphibians, and reptiles generate new brain cells following injury11,12. Among the vertebrates, mammalian animals are more sought after given their genetic resemblance to human beings. Mammals, however, exhibit limited and poor reparative capacity in the central nervous system (CNS) that can last through adulthood following a brain lesion13. In general, mammals are unsuited as animal models for understanding neuroregeneration given that the low number of neurons produced will not be sufficient to restore damaged neural circuits observed in PD. As such, the teleost-based model, specifically in zebrafish, is greatly favored for its high proliferative rate, capability to continuously self-renew, and close brain homology with humans14,15.
Zebrafish is most commonly used to study disordered movement in PD16. The zebrafish-based PD model is usually induced by neurotoxins, which include 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA)17. Although effective in inducing specific loss of DpN and decrease of dopamine levels, MPTP-based models do not closely mimic the conditions of PD as the DpN loss is not restricted solely to the CNS18. The inability of 6-OHDA to cross the blood-brain barrier restricted its effects on cellular and functional changes within the brain when it is administered intracranially as opposed to intramuscularly19. Peripheral administration of 6-OHDA caused a global reduction of dopamine levels throughout the nervous system20. While administration of 6-OHDA into the cerebrospinal fluid caused ablation of DpN throughout the CNS21, which does not mimic the condition as seen in PD whereby the loss of DpN occurs specifically at the substantia nigra of the human brain. ICV administration of 6-OHDA, on the contrary, specifically induced significant ablation of DpN in the area of ventral Dn in the zebrafish brain, which closely resembled substantia nigra22. Interestingly, recovery of DpN was reported 30 days post 6-OHDA-induced lesion and these neurons survived over the course of life23,24. The functional recovery of DpN was demonstrated through a locomotor assessment of distance traveled (cm) and mean speed (cm/s) using the 6-OHDA-induced adult zebrafish-based PD model22.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6-Hydroxydopamine (6-OHDA)</td><td>Sigma-Aldrich, Missouri, USA</td><td>162957</td><td></td></tr><tr><td>Ascorbic acid</td><td>Thermo Fisher Scientific, California, USA</td><td>FKC#A/8882/53</td><td></td></tr><tr><td>Disposable pasteur pipette, 3 mL</td><td>Thermo Fisher Scientific, California, USA</td><td>FB55348</td><td></td></tr><tr><td>Microcentrifuge tube, 0.2 mL</td><td>Eppendorf, Hamburg, Germany</td><td>30124332</td><td></td></tr><tr><td>Nice conical flask, 100 mL</td><td>Evergreen Engineering &amp;amp; Resources, Semenyih, Malaysia</td><td>SumYau0200</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, Missouri, USA</td><td>P4417</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich, Missouri, USA</td><td>S5761</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck, Darmstadt, Germany</td><td>106404</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon, Tokyo, Japan</td><td>SMZ745</td><td></td></tr><tr><td>Tricaine methanesulfonate (MS-222)</td><td>Sigma-Aldrich, Missouri, USA</td><td>E10521</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ANY-maze software</td><td>Stoelting Co., Illinois, USA</td><td>-</td><td>version 7.0; video tracking software</td></tr><tr><td>Cubis II Micro Lab Balance</td><td>Sartorius, G&amp;ouml;ttingen, Germany</td><td>SE 2</td><td></td></tr><tr><td>FemtoJet IV microinjector</td><td>Eppendorf, Hamburg, Germany</td><td>5192000035</td><td></td></tr><tr><td>Femtotip II, sterile injection capillary</td><td>Eppendorf, Hamburg, Germany</td><td>5242957000</td><td></td></tr><tr><td>InjectMan 4 micromanipulator</td><td>Eppendorf, Hamburg, Germany</td><td>5192000027</td><td></td></tr><tr><td>LED Portable Lamp</td><td>MR. DIY, Selangor, Malaysia</td><td>9023251</td><td>20 mAh</td></tr><tr><td>PELCO Pro Superalloy, offset, fine tips</td><td>Ted Pella, California, USA</td><td>5367-12NM</td><td></td></tr><tr><td>Shanda aquarium heater</td><td>Yek Fong Aquarium, Selangor, Malaysia</td><td>SDH-228</td><td></td></tr><tr><td>Thermometer</td><td>Sera Precision, Heinsberg, Germany</td><td>52525</td><td></td></tr><tr><td>Video camera</td><td>Nikon, Tokyo, Japan</td><td>D3100</td><td></td></tr></tbody></table>

locomotor assessment of 6-hydroxydopamine-induced adult zebrafish-based parkinson's disease model

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that can restore these neurons. Much effort is currently directed toward a better understanding of neuroregeneration using preclinical in vivo models. This regenerative capability for self-repair is, however, inefficient in mammals. Non-mammalian animals like zebrafish have thus emerged as an excellent neuroregenerative model due to its capability to continuously self-renew and have a close brain homology to humans. As part of the effort in elucidating cellular events involved in neuroregeneration in vivo, we have established the 6-hydroxydopamine (6-OHDA)-induced adult zebrafish-based PD model. This was achieved through the optimized intracerebroventricular (ICV) microinjection of 99.96&#160;mM 6-OHDA to specifically ablate dopaminergic neurons (DpN) in the ventral diencephalon (Dn) of zebrafish brain. Immunofluorescence indicated more than 85% of DpN ablation at day three postlesion and full restoration of DpN at lesioned site 30 days postlesion. The present study determined the impairment and subsequent recovery of zebrafish swimming behavior following lesion by using the open field test through which two parameters, distance traveled (cm) and mean speed (cm/s), were quantified. The locomotion was assessed by analyzing the recordings of individual fish of each group (n = 6) using video tracking software. The findings showed a significant (p &#60; 0.0001) reduction in speed (cm/s) and distance traveled (cm) of lesioned zebrafish 3 days postlesion when compared to sham. The lesioned zebrafish exhibited full recovery of swimming behavior 30 days postlesion. The present findings suggest that 6-OHDA lesioned adult zebrafish is an excellent model with reproducible quality to facilitate the study of neuroregeneration in PD. Future studies on the mechanisms underlying neuroregeneration as well as intrinsic and extrinsic factors that modulate the process may provide important insight into new cell replacement treatment strategies against PD.

The present experiment assessed the changes in adult zebrafish swimming behavior following ICV microinjection with 6-OHDA. The reason for using 6-OHDA as the neurotoxin of choice was due to its inability to cross the blood-brain barrier, which produced specific and targeted ablation of DpN in the area of interest-ventral diencephalon (Dn)16. The DpN subpopulation here holds anatomical resemblance to the DpN subpopulation in the human&#39;s substantia nigra pars compacta31.
As per our previous work22, the cellular effect of 6-OHDA ICV microinjection against DpN of adult zebrafish was confirmed through immunohistostaining of DpN marker-tyrosine hydroxylase (TH). The main brain region of interest was the Dn, made up of the preoptic area (POA), posterior tuberculum (PT), and hypothalamus (Hyp). It was found that 99.96&#160;mM 6-OHDA resulted in a 100% survival rate of the adult zebrafish with the lowest number of TH-immunoreactive (TH-ir) in Dn. It was also found that more than 85% (p &#60; 0.01) of TH-ir DpN in the Dn was ablated on day three postlesion. The number of TH-ir DpN then increased by more than 50% at day 14 postlesion before achieving full regeneration 30 days postlesion (Figure 3). This data supports the regenerative capabilities of DpN subpopulation in Dn of adult zebrafish following ablation32.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63355/63355fig03.jpg" /> 
Figure 3: Regeneration of DpN in the Dn region of zebrafish lesioned by 99.96&#160;mM 6-OHDA. (A) The number of TH-ir DpN in three main areas of the Dn region, POA, PT, and Hyp, over four data points: sham, 3, 14, and 30 days post-lesioning by 99.96&#160;mM 6-OHDA neurotoxin. Each bar represents mean &#177; SD of n = 6 independent experiments; *p &#60; 0.05. (B) Representative confocal microscope images of sagittally sectioned zebrafish brain of sham (I, I&#39;, and I&#39;&#39;), 3 days post-lesioning (II, II&#39;, and II&#39;&#39;), 14 days post-lesioning (III, III&#39;, and III&#39;&#39;), and 30 days post-lesioning (IV, IV&#39;, and IV&#39;&#39;) stained with TH (DpN; green) and DAPI (nuclei; blue). Scale bar = 50 &#181;m. Abbreviations- DAPI: 4&#8242;, 6-diamidino-2-phenylindole, 6-OHDA: 6-hydroxydopamine, Dn: diencephalon, DpN: dopaminergic neurons, Hyp: hypothalamus, POA: preoptic area, PT: posterior tuberculum, SD: standard deviation, and TH-ir: tyrosine hydroxylase immunoreactive. Adapted from Vijayanathan et al.22. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63355/63355fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
We then performed locomotor assessment using the open tank test to investigate changes in distance traveled (cm) and mean speed (cm/s) of adult zebrafish following ICV microinjection of 6-OHDA and sham. Experimental fish were then assessed on day three postlesion (least number of TH-ir DpN observed) and day 30 postlesion (fully restored DpN reported at lesion site). Analysis of zebrafish swimming behavior using a video tracking software indicated that both the mean speed (cm/s) and distance traveled (cm) of the lesioned group on day three postlesion were significantly reduced (p &#60; 0.001) to &#60;45% when compared to sham (Figure 4). The lesioned group exhibited recovery of motor function 30 days postlesion with no significant difference of both the mean speed (cm/s) and distance traveled (cm) when compared to sham.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63355/63355fig04.jpg" /> 
Figure 4: Changes in swimming behavior following intracerebroventricular injection by 6-OHDA. Swimming behavior of adult zebrafish was assessed before lesioning, on day three and day 30 postlesion by 99.96&#160;mM 6-OHDA. Parameters that were assessed included: (A) mean speed (cm/s) and (B) distance traveled (cm). Each bar represents mean &#177; SD of six fish; ****p &#60; 0.0001 (Student t-test). Abbreviations: 6-OHDA: 6-hydroxydopamine, SD: standard deviation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63355/63355fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model at JoVE.com

Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

The limitations of current treatments in delaying dopaminergic neuronal loss in Parkinson&#39;s disease (PD) raise the need for alternative therapies that ...

locomotor-assessment-6-hydroxydopamine-induced-adult-zebrafish-based

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.0K Views.  Universiti Teknologi MARA (UiTM), Cawangan Selangor, Kampus Puncak Alam. The present protocol describes the intracerebroventricular (ICV) injection of adult zebrafish with neurotoxic 6-hydroxydopamine (6-OHDA) at the ventral diencephalon (Dn) and the assessment of the impairment and subsequent recovery of swimming behavior postlesion by using the open tank test, which is accompanied by analysis using a video tracking software.

Video: Locomotor Assessment of 6-Hydroxydopamine-induced Adult Zebrafish-based Parkinson's Disease Model

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Developmental Biology

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Environment

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.

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 ...

Behavior

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

We present a behavioral chamber designed to assess cognitive performance. We provide data showing that once acquired, zebrafish remember the task 8 weeks later. We also show that hyperglycemic zebrafish have altered cognitive performance, indicating that this paradigm is applicable to studies assessing cognition and memory.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Biology

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 ...

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 ...

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Behavioral Test Battery to Assess Neurotoxic Effects in Adult Zebrafish

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

Author Spotlight: Assessment of Environmental and Drug-Induced Behavioral Changes in Adult Zebrafish 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.