Name: Phenotypic Profiling of MPTP-Induced Parkinsonian-like Behavioral Phenotypes in Zebrafish Larvae Based on Behavioral Experiment (Video) | JoVE Journal
Uploaded: 2026-05-15T12:20:00.000+00:00
Duration: 10 min 33 s
Description: Learn a multiparametric behavioral protocol for profiling motor and non-motor Parkinsonian phenotypes in larval zebrafish using MPTP-induced models. This method enables comprehensive drug screening and neurotoxicity assessment.

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5 minutes

This work was supported by grants from the National Natural Science Foundation of China (No. 21906095), Shandong University Young Scholar Future Program Sponsorship, the Shandong Natural Science Foundation (No. ZR2019BB018), the Fundamental Research Funds of Shandong University (No. 2017GN0030), the China Postdoctoral Science Foundation (No. 2018M640640). It is also supported by both the 2025 Open Research Fund from the Digital Fujian Institute of Big Data for Elderly Rehabilitation and Nursing (Grant No. BDRI202502) and the 2025 Open Research Fund from the Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University (Grant No. SWHX-202503). Research Project of Jinan Microecological Biomedicine Shandong Laboratory (Grant No.JNL-2023002KF).&#160;The authors would like to thank the Laboratory Animal Center, Qilu Hospital of Shandong University, and Dr. Sun Chengxi from the Department of Clinical Laboratory, Qilu Hospital of Shandong University, for their assistance during the experiment.

This study involving zebrafish was conducted in compliance with institutional guidelines for animal care and use, and was approved by the Ethical Committee of the School of Pharmaceutical Sciences, Shandong University (No.YXDW2025-0056). The reagents and the equipment used are listed in the Table of Materials.
1. MPTP Preparation
<ol>
	<li>Prepare 1× E3 medium by diluting 100× E3 stock solution 1:100 with ultrapure water (e.g., mix 5 mL of 100× E3 with 495 mL of ultrapure water to obtain 500 mL of 1× E3 medium). 
	NOTE: Prepare E3 medium freshly on the day of use.</li>
	<li>Prepare a 1 mM MPTP stock solution by adding 5.0 mg of MPTP powder to a 50 mL centrifuge tube and adding E3 medium to a final volume of 25 mL (use the centrifuge tube as a mixing container, but no centrifugation is required). Label the solution as 1 mM MPTP stock solution. Store the stock solution in the dark.</li>
	<li>Preparation of 50 µM MPTP solution: transfer 2.5 mL of the 1 mM MPTP stock solution into a clean container and dilute with 47.5 mL of E3 medium to obtain a final volume of 50 mL at a final concentration of 50 µM MPTP. The remaining part of the stock solution continued to be stored in the dark. 
	CAUTION: MPTP is a potent neurotoxin and must be handled with extreme caution. Always wear appropriate personal protective equipment (PPE), including gloves, a laboratory coat, and protective eyewear. Disposal of MPTP waste according to institutional biosafety guidelines.</li>
</ol>
2. Zebrafish embryos preparation and grouping
<ol>
	<li>Select sexually mature male and female adult zebrafish and perform controlled breeding using breeding tanks with removable dividers. At 8:00 pm, place male and female zebrafish separately in the breeding tanks at a ratio of 2:3, and separate them with dividers.</li>
	<li>On the following day, remove dividers at 8:00 am to allow mating, thereby initiating spawning. Collect embryos after 1 h, and wash them three times with clean system water.</li>
	<li>House embryos in 100 mm Petri dishes with fresh E3 medium, and incubate them at 28 °C under a 14 h light / 10 h dark cycle (lights on from 08:00 am to 10:00 pm). 
	NOTE: Do not exceed 50 embryos per dish with 20 mL medium to ensure normal development.</li>
	<li>At 1 dpf, carefully remove the unfertilized, malformed, and abnormal embryos using a Pasteur Pipette, and replace the solution containing zebrafish embryos. For the WT group, replace the existing medium with fresh E3 embryo medium. For the MPTP-treated group, replace the medium with 50 µM MPTP solution (Figure 1A), then return both groups to the incubator.</li>
	<li>For both groups, replace the solution and remove any dead or unhealthy embryos every 24 h. For the MPTP-treated group, replace the MPTP solution daily from the morning of 1 dpf until the morning of 4 dpf (Figure 1A). Then, transition the MPTP-treated group to E3 medium beginning on the morning of 5 dpf until the end of the experiment. 
	NOTE: Replace the solution gently using a Pasteur Pipette. Aspirate the medium slowly from the edge of the Petri dish and add fresh solution along the wall of the Petri dish to minimize disturbance to the embryos.</li>
</ol>
3. Sleep - wake regulation monitoring
<ol>
	<li>At 4 dpf, select zebrafish larvae from each group at 6:00 pm, and transfer them to a 96-well plate using a modified 3 mL Pasteur Pipette with the tip cut off, one larva per well (Figure 1B). The first four rows represent the WT groups, and the last four rows represent the MPTP groups (48 zebrafish in each group). 
	NOTE: To avoid stimulating or damaging zebrafish, modify Pasteur Pipettes by cutting the tip (10 mm) to provide a wide bore larger than the size of 4 dpf zebrafish larvae (body length exceeds 3.7 mm27,28).</li>
	<li>Observe each well with a stereo microscope to ensure the appearance of each zebrafish larva is healthy, the fins are open and regularly swung, the body is upright, and the swim bladder is inflated29. After ensuring that the zebrafish in each well are in a healthy state, fill each well with E3 medium. 
	NOTE: Fill each well with E3 medium to near bulging, ensuring the surface tension is maintained. Carefully add the liquid to prevent bubbles and cross-well overflow.</li>
	<li>After transferring the fish and liquid into a 96-well plate, place it in the Zebrabox chamber to acclimate for 30 min prior to the sleep-wake behavioral test. 
	NOTE: Before placing the 96-well plate in the Zebrabox chamber, clean up the bottom of the plate carefully to avoid dirt affecting the video tracking results.</li>
	<li>Open the Zebralab video tracking software, select the Quantization mode, and ensure that the 96-well plate is positioned centrally on the bottom of the Zebrabox chamber.</li>
	<li>Set the light adjustment to turn off the light at 10: 00 pm and turn on the light at 8: 00 am, which is consistent with the light conditions when the zebrafish is reared. During the daytime, white light is generally around 1000 lux. Set the software parameters as follows29: detection threshold, 40; burst, 25 (threshold for on movement); freeze (threshold for on movement), 4; bin size, 60 s; total monitoring time, 52 h (8: 00 pm on the first day to 00: 00 pm on the third day). Start the sleep-wake procedure at 8: 00 pm. 
	NOTE: Once the procedure is started, prohibit any noise and vibration from the outside throughout the experiment, so as not to affect the normal sleep-wake rhythm of zebrafish. To prevent water evaporation during long-term monitoring, cover the 96-well plate with transparent plastic wrap.</li>
	<li>After 52 h, stop the sleep-wake experiment, and save the data file. Remove the 96-well plate from the ZebraBox. 
	NOTE: Using MATLAB and Excel, necessary parameters such as number of sleep bouts (sleep bout is defined as periods of inactivity lasting at least 1 min)29, average sleep bout length (average duration of sleep bouts), total sleep duration (all duration of sleep bouts over the monitoring period), sleep latency (time from a light transition to the onset of the first sleep bout), average activity (defined as locomotor activity during all the monitoring period), and waking activity (defined as locomotor activity during only the awake period, excluding the sleep bouts) were extracted and analyzed from the data file.
	<ol>
		<li>Use a Student's t-test to compare the differences between the WT group and the MPTP-treated group. Additionally, plot the sleep curve, waking activity curve, and average activity curve of zebrafish over the 52 h using MATLAB software.</li>
	</ol>
	</li>
</ol>
4. Photomotor response paradigms
NOTE: Photomotor response paradigms (including strobe light) are high-throughput behavioral tests in 5 dpf larvae to quantify sensorimotor and reflexive locomotor changes in response to a sudden transition of light. This test was conducted using a 96-well plate, including 2 paradigms: (1) a light transition from 1 min of darkness to 1 min of constant light (1000 lx), and (2) a strobe light response involving a transition from 1 min of darkness to 1 min of strobing light at 10 Hz.
<ol>
	<li>Place the 5 dpf larvae in a 96-well plate (1 larva per well) (Figure 1B), and fill each well with E3 medium. Divide a total of 96 larvae into the WT group and the MPTP group on average.</li>
	<li>Dark-constant light paradigm:
	<ol>
		<li>Place the 96-well plate with 5 dpf larvae into the Zebrabox and allow larvae to acclimate for 5 min in the dark before the assay.</li>
		<li>Open the Tracking-online analysis mode.</li>
		<li>Set the light transition program as follows: 0-60 s: Dark, 60-120s: Constant light (1000 lx).</li>
		<li>Start the protocol, collect related motion parameters every 6 s throughout the 120 s assay.</li>
		<li>Stop the protocol at 120th s, export the data file. Place the larvae in full darkness for 5 min to acclimate the environment, preparing them for the following dark-strobe light assay.</li>
	</ol>
	</li>
	<li>Dark-strobe light paradigm:
	<ol>
		<li>Set the light transition program as follows: 0-60 s: Dark, 60-120s: strobe light (1000 lx) at 10 Hz. 
		NOTE: A 10 Hz strobe corresponds to 10 light pulses per second. In ZebraLab, implement this by alternating the light intensity from 0 to 1000 lx every 100 ms.</li>
		<li>Upon completion of the 5 min dark adaptation, start the dark-to-strobe light assay. Collect motion parameters every 6 s throughout the 120 s assay.</li>
	</ol>
	</li>
	<li>Data analysis: Once the assay ends, export the data file. Use MATLAB software to extract the swimming distance every 6 s. Plot the total distance (mean value per 6 s) swum in the 2 min period for both assays. Calculate freeze index by subtracting the total distance traveled in 1 min in the dark from the total distance traveled in the light30. 
	NOTE: Freeze Index &lt; 0 indicates freezing behavior; Freeze index &gt; 0 indicates escape; Freeze index = 0 means no reaction. Use a Student's t-test and two-way ANOVA to compare the differences between the WT group and the MPTP-treated group. A total of 48 zebrafish larvae (n = 48) were used for each group.</li>
</ol>
5. Assessment of thigmotaxis
<ol>
	<li>Select two 6-well plates, fill each well with 4 mL E3 medium.</li>
	<li>At 5 dpf, zebrafish larvae from the WT group and MPTP-treated group were collected and put in two 6-well plates (one is the WT group, and the other is the MPTP-treated group, 1 larva per well) (Figure 1C). 
	NOTE: Ensure the initial position of the larvae is in the center of the wells.</li>
	<li>Before the thigmotaxis assessment, the well plates should be placed in the appropriate position of Zebrabox 15 min in advance to accommodate.</li>
	<li>Data collection and analysis: Using the Draw Area function, draw two concentric circles in the observation area of each well (the radius of the center circle accounts for 1 / 2 of the radius of the hole) (Figure 1C), and the remaining parameters are set to be the same as locomotor assessment. Start the protocol for 5 min thigmotaxis assessment, and do a parallel experiment. 
	NOTE: After the end of the program, Zebralab software will generate the trajectory of zebrafish larvae within 5 min and various related motion parameters. In the data file, an1 represents the whole area of each well, and an2 represents the inner zone of each well. Thus, total dis(an1) and total dur(an1) denote the total swimming distance and total time spent in the entire well, while total dis(an2) and total dur(an2) represent the distance and time spent in the inner zone, respectively.
	<ol>
		<li>Calculate the thigmotaxis with the following formulas: 
		Calculate the percent of distance swum in the outer zone: 
		<img alt="Thigmotaxis calculation formula; equation showing behavioral analysis method in research context." src="/files/ftp_upload/70041/70041eq1.jpg" style="margin: auto;" data-altupdatedat="1780977605000" data-alt="Equation 1"> 
		Calculate the percent of time spent in the outer zone: 
		<img alt="Thigmotaxis equation, ΣFx=0, describing behavioral analysis, includes parameters total dur, an1, an2." src="/files/ftp_upload/70041/70041eq2.jpg" style="margin: auto;" data-altupdatedat="1780977605000" data-alt="Equation 2"></li>
		<li>Employ a Student's t-test to compare the differences of thigmotaxis between the WT group and the MPTP-treated group. A total of 24 zebrafish larvae (n = 24) were used for each group.</li>
	</ol>
	</li>
</ol>
6. Locomotor assessment
<ol>
	<li>At 5 dpf, select zebrafish larvae from the WT group and MPTP-treated group, and respectively transferred to two 12-well plates (one is the WT group, and the other is the MPTP-treated group, 1 larva per well, with 2 mL E3 medium) (Figure 1D). 
	NOTE: Ensure the sizes of zebrafish larvae are similar, and larvae selected should have open fins, upright posture, good expansion of the swim bladder, and regular movement.</li>
	<li>Before the locomotor assessment, place the well plate in the test position of the Zebrabox 15 min in advance to adapt. 
	NOTE: Ensure the light conditions during adaptation are consistent with the light conditions during the test.</li>
	<li>After a 15 min acclimation period, select the tracking mode-online analysis, record the locomotor activity of each larva for 5 min using an automated computerized video-tracking system. Using Zebralab software to collect movement tracks and a variety of related locomotor parameters every 60 s, export the complete data file, and use MATLAB to analyze the total distance and average speed of each larva in 5 min.
	<ol>
		<li>Use a Student's t-test and two-way ANOVA to compare the differences between the WT group and the MPTP-treated group. A total of 24 zebrafish larvae (n = 24) were used for each group.</li>
	</ol>
	</li>
</ol>
7. Startle response test
NOTE: This test measures the visual startle response (VSR), also known as dark-flash induced startle, elicited by sudden transitions to darkness. This startle response test assesses a photic-evoked visual startle induced by abrupt, millisecond-scale dark flashes. Unlike the dark-strobe light paradigm, which involves repeated light pulses to evaluate sustained behavioral responses, this assay focuses on rapid, reflexive motor reactions to an acute visual stimulus.
<ol>
	<li>Preparations: at 5 dpf, select zebrafish larvae from the WT group and the MPTP-treated group, and transfer them respectively to two 12-well plates (one is the WT group, and the other is the MPTP-treated group, 1 larva per well, with 2 mL E3 medium) (Figure 1D).</li>
	<li>Before the startle response test, place the well plate in the test position of the Zebrabox 5 min in advance to adapt.</li>
	<li>Set the stimulation parameters: open Zebralab software, run the Tracking-online analysis mode, open the Light-triggering interface, select Use one of the 3 triggering methods below, enable Enhanced stimuli, set to When session is started; in the transition schedule, the dark flash rhythm is defined as follows ( the program starts to trigger after 21 s ): Transition 1: 5 ms, 100% light intensity; transition 2: 2890 ms, 100% light intensity; transition 3: 5 ms, 0% light intensity; transition 4: 100 ms, 0% light intensity; tick Repeat list of transitions to ensure periodic flashing.</li>
	<li>Start protocol: ensure that the Numeriscope video recording function is turned on, high-throughput automated tracking will generate a multi-dimensional dataset encompassing 30 behavioral endpoints in this 90 s test.</li>
	<li>Data analysis: After completion of the startle response assay, export the raw data files and import them into MATLAB software for analysis. Calculate the swimming distance of each larva in 3 s intervals, along with the average swimming speed.
	<ol>
		<li>Identify the peak movement distance, peak velocity, or response latency following the flash stimulus for each group. Use a Student's t-test and two-way ANOVA to compare the differences between the WT group and the MPTP-treated group. A total of 24 zebrafish larvae (n = 24) were used for each group.</li>
	</ol>
	</li>
</ol>
8. Light-dark challenge assay
<ol>
	<li>At 5 dpf, transfer zebrafish larvae individually into two 24-well plates (Figure 1E), with one larva per well and 1 mL of E3 medium per well. Each plate contained 24 larvae, and the two 24-well plates were evenly divided into the WT group and the MPTP-treated group (n = 24 per group).</li>
	<li>Before testing, place the 24-well plate into the designated testing area inside the Zebrabox for a 5 min light acclimatization period to adapt the testing environment. Activate the Tracking-Online Analysis mode. Configure the light program as follows: 6 min of constant light followed by 4 min of complete darkness (Figure 1E). The total testing time is 10 min, and the interval time for collecting data points is 60 s. 
	NOTE: The global 6 min of light is for acclimatization and habituation, ensuring stable baseline locomotor and special light-dark responses31,32.</li>
	<li>On the 10th min, stop behavioral recording and save data. Carefully remove the plate from the Zebrabox.</li>
	<li>For each larva, extract the swimming distance at each 60 s interval. Calculate the distance traveled during the light phase (0th-6th min), the dark phase (7th-10th min), and the total 10-min period. Quantify the thigmotaxis behavior and habituation learning performance in the dark phase. 
	NOTE: Habituation learning is defined as the reduction of a behavioral response when an animal is exposed to a continuous stimulus/ environment33,34, and it was assessed by statistically comparing the mean total distance moved (mm) during the 1 st min and the 4th min following the light-to-dark transition. Use a Student's t-test and two-way ANOVA to compare the differences between the WT group and the MPTP-treated group. A total of 24 zebrafish larvae (n = 24) were used for each group.</li>
</ol>
9. Disposal of MPTP waste
NOTE: After each behavioral experiment, spray and wipe MPTP-contacted surfaces (e.g., lab benches and the ZebraBox) with freshly prepared sodium hypochlorite (bleach; ≥10% v/v).
<ol>
	<li>Collect the liquid waste (including remaining exposure solutions and rinsing media) and solid waste (e.g., Pasteur pipettes, gloves, Petri dishes, and multi-well plates) of MPTP. Clearly label them as hazardous neurotoxic waste.</li>
	<li>Treat contaminated liquid waste and solid materials with freshly prepared sodium hypochlorite (bleach; ≥10% v/v) for at least 24 h to ensure decontamination, followed by disposal in accordance with institutional regulations.</li>
	<li>Euthanize MPTP-treated zebrafish larvae by immersing in a diluted sodium hypochlorite solution (e.g., 1:5 ratio of 6% bleach: water) for 5 min35. Confirmed death by lack of movement and heartbeat. Treat the decontaminated larvae and media as hazardous biological waste for institutional disposal (following institutionally approved protocols).</li>
</ol>

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<li>Xu, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+clinical+phenotyping+of+Parkinson%27s+disease%3A+Towards+a+new+era+of+research+and+clinical+care%5BTitle%5D)+AND+%22Phenomics%22%5BJournal%5D)">Deep clinical phenotyping of Parkinson's disease: Towards a new era of research and clinical care.</a> Phenomics. 2 (5), 349-361 (2022).</li>
<li>Tinaz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+connectome+in+Parkinson%27s+disease+and+Parkinsonism%5BTitle%5D)+AND+%22Curr+Neurol+Neurosci+Rep%22%5BJournal%5D)">Functional connectome in Parkinson's disease and Parkinsonism.</a> Curr Neurol Neurosci Rep. 21, 24(2021).</li>
<li>Costa, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parkinson%27s+disease%3A+A+multisystem+disorder%5BTitle%5D)+AND+%22Neurosci+Bull%22%5BJournal%5D)">Parkinson's disease: A multisystem disorder.</a> Neurosci Bull. 39 (1), 113-124 (2023).</li>
<li>Christensen, C., Thorsteinsson, H., Maier, V. H., Karlsson, K. Æ <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-parameter+behavioral+phenotyping+of+the+MPP%E2%81%BA+model+of+Parkinson%27s+disease+in+zebrafish%5BTitle%5D)+AND+%22Front+Behav+Neurosci%22%5BJournal%5D)">Multi-parameter behavioral phenotyping of the MPP⁺ model of Parkinson's disease in zebrafish.</a> Front Behav Neurosci. 14, 623924(2020).</li>
<li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-Parkinson%27s+disease+activity+of+phenolic+acids+from+Eucommia+ulmoides+Oliver+leaf+extracts+and+their+autophagy+activation+mechanism%5BTitle%5D)+AND+%22Food+Funct%22%5BJournal%5D)">Anti-Parkinson's disease activity of phenolic acids from Eucommia ulmoides Oliver leaf extracts and their autophagy activation mechanism.</a> Food Funct. 11 (2), 1425-1440 (2020).</li>
<li>Maleski, A. L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+behavioral+and+proteomic+characterization+of+MPP%E2%81%BA-induced+early+neurodegeneration+and+Parkinsonism+in+zebrafish+larvae%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Integrated behavioral and proteomic characterization of MPP⁺-induced early neurodegeneration and Parkinsonism in zebrafish larvae.</a> Int J Mol Sci. 26 (14), 6762(2025).</li>
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<li>Parichy, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+table+of+postembryonic+zebrafish+development%3A+staging+by+externally+visible+anatomy+of+the+living+fish%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Dev Dyn. 238 (12), 2975-3015 (2009).</li>
<li>Lee, D. A., Oikonomou, G., Prober, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+analysis+of+sleep+in+zebrafish%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">Large-scale analysis of sleep in zebrafish.</a> Bio Protoc. 12 (3), e4313(2022).</li>
<li>Shen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+well-plate+assays+for+motor+and+social+behaviors+in+larval+zebrafish%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Rapid well-plate assays for motor and social behaviors in larval zebrafish.</a> Behav Brain Res. 391, 112625(2020).</li>
<li>Rock, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detailed+analysis+of+zebrafish+larval+behaviour+in+the+light-dark+challenge+assay+shows+that+diel+hatching+time+determines+individual+variation%5BTitle%5D)+AND+%22Front+Physiol%22%5BJournal%5D)">Detailed analysis of zebrafish larval behaviour in the light-dark challenge assay shows that diel hatching time determines individual variation.</a> Front Physiol. 13, 827282(2022).</li>
<li>Hillman, C., Kearn, J., Parker, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+unified+approach+to+investigating+4+dpf+zebrafish+larval+behaviour+through+a+standardised+light%2Fdark+assay%5BTitle%5D)+AND+%22Prog+Neuropsychopharmacol+Biol+Psychiatry%22%5BJournal%5D)">A unified approach to investigating 4 dpf zebrafish larval behaviour through a standardised light/dark assay.</a> Prog Neuropsychopharmacol Biol Psychiatry. 134, 111084(2024).</li>
<li>Best, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-associative+learning+in+larval+zebrafish%5BTitle%5D)+AND+%22Neuropsychopharmacol%22%5BJournal%5D)">Non-associative learning in larval zebrafish.</a> Neuropsychopharmacol. 33 (5), 1206-1215 (2008).</li>
<li>Gomez-Laplaza, L. M., Gerlai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Latent+learning+in+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Latent learning in zebrafish (Danio rerio).</a> Behav Brain Res. 208 (2), 509-515 (2010).</li>
<li>Winn, A. A., Prestia, K. A., Peneyra, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testing+alternative+surface+disinfection+agents+for+zebrafish+%28Danio+rerio%29+embryos%5BTitle%5D)+AND+%22J+Am+Assoc+Lab+Anim+Sci%22%5BJournal%5D)">Testing alternative surface disinfection agents for zebrafish (Danio rerio) embryos.</a> J Am Assoc Lab Anim Sci. 60 (5), 510-518 (2021).</li>
<li>Wagle, M., Nguyen, J., Lee, S., Zaitlen, N., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heritable+natural+variation+of+an+anxiety-like+behavior+in+larval+zebrafish%5BTitle%5D)+AND+%22J+Neurogenet%22%5BJournal%5D)">Heritable natural variation of an anxiety-like behavior in larval zebrafish.</a> J Neurogenet. 31 (3), 138-148 (2017).</li>
<li>Maximino, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+anxiety+in+zebrafish%3A+a+critical+review%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Measuring anxiety in zebrafish: a critical review.</a> Behav Brain Res. 214 (2), 157-171 (2010).</li>
<li>Schnorr, S. J., Steenbergen, P. J., Richardson, M. K., Champagne, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+thigmotaxis+in+larval+zebrafish%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Measuring thigmotaxis in larval zebrafish.</a> Behav Brain Res. 228 (2), 367-374 (2012).</li>
<li>Dekens, M. P. S., Fontinha, B. M., Gallach, M., Pflugler, S., Tessmar-Raible, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Melanopsin+elevates+locomotor+activity+during+the+wake+state+of+the+diurnal+zebrafish%5BTitle%5D)+AND+%22EMBO+Rep%22%5BJournal%5D)">Melanopsin elevates locomotor activity during the wake state of the diurnal zebrafish.</a> EMBO Rep. 23 (5), e51528(2022).</li>
<li>Lee, H. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Key+HPI+axis+receptors+facilitate+light+adaptive+behavior+in+larval+zebrafish%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Key HPI axis receptors facilitate light adaptive behavior in larval zebrafish.</a> Sci Rep. 14 (1), 7759(2024).</li>
<li>Cavanagh, J. F., Kumar, P., Mueller, A. A., Richardson, S. P., Mueen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diminished+EEG+habituation+to+novel+events+effectively+classifies+Parkinson%27s+patients%5BTitle%5D)+AND+%22Clin+Neurophysiol%22%5BJournal%5D)">Diminished EEG habituation to novel events effectively classifies Parkinson's patients.</a> Clin Neurophysiol. 129 (2), 409-418 (2018).</li>
<li>Bedrossiantz, J., Prats, E., Raldua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotoxicity+assessment+in+adult+Danio+rerio+using+a+battery+of+behavioral+tests+in+a+single+tank%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Neurotoxicity assessment in adult Danio rerio using a battery of behavioral tests in a single tank.</a> J Vis Exp. 201, e65869(2023).</li>
</ol>

The authors declare no conflicts of interest.

This study developed and verified a comprehensive protocol to evaluate and analyze MPTP-induced PD-like behavioral phenotype. This protocol integrated 6 different tests, locomotor assessment, thigmotaxis assay, startle response test, photomotor response paradigms (including strobe light stimulation), light-dark challenge assay, and sleep-wake regulation monitoring, to evaluate and depict the motor and non-motor defects of PD. The results indicated that compared with the WT group, MPTP-treated zebrafish larvae showed moto...

Parkinson&#39;s disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer&#39;s disease1. Its prevalence and the number of individuals affected have been steadily increasing in recent years, presenting a significant public health challenge worldwide2. The incidence rate of PD notably escalates with age3,4. Clinically, PD manifests through both motor symptoms -- such as dyskinesia, bradykinesia, resting tremor, and postural instability -- and a variety of non-motor symptoms, including cognitive impairment, anxiety, and sleep disturbances5. Currently, the diagnosis of PD primarily depends on the presence of motor symptoms6; however, by the time these symptoms become evident, over 60% of the dopamine neurons in the substantia nigra have already been lost7. Although non-motor symptoms frequently appear earlier, the absence of specific and objective assessment criteria complicates early diagnosis and intervention8. Presently, the primary treatment for PD involves the administration of levodopa9, which is converted into dopamine in the brain, thereby replenishing dopamine levels in the striatum. While this treatment can temporarily alleviate symptoms, it does not halt the progression of the disease10. Consequently, further research into the pathogenesis of PD and drug screening is essential.
Building appropriate animal models is crucial for the research on the pathogenesis of PD and anti-PD drug screening. Now, there are three main models for PD studies: chemical induction model11, gene editing model12, and human xenotransplantation model13. Commonly used model animals include rodents, such as mice14, and non-human primates like monkeys15. Recently, zebrafish (Danio rerio) have gained increasing attention in PD studies for their advantages such as small size, rapid growth, high genetic homology with humans, and transparent embryos16. Using neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to induce dopamine neuron damage17,18, is a classical method to construct a PD model. Zebrafish exposed to sublethal MPTP concentrations exhibit various PD-like behavioral phenotypes19.
However, most zebrafish PD studies nowadays mainly concentrate on basic locomotor behaviors or simple light-dark transitions16,20, and single-dimensional behavioral assays are difficult to fully reflect the complex behavioral characteristics of PD, especially the behavioral changes in non-motor function. Considering PD is a disease that involves multiple neurobehavioral systems beyond motor control21,22,23, a multiparametric behavioral phenotype evaluation method is urgently needed to capture its multisystem phenotypes24.
Accordingly, this study builds a multiple, parametric, and rapid behavioral phenotyping assay protocol to more comprehensively describe the PD-like behavioral phenotypes in MPTP-treated zebrafish larvae. A sublethal dose of MPTP (50 &#181;M)25 was applied to larvae from 1 to 5 days post-fertilization (dpf)16,20,26. From 4 to 6 dpf16,20,24, we conducted 6 behavioral tests: sleep-wake regulation monitoring, photomotor response paradigms, thigmotaxis assessment, locomotor assessment, startle response test, and light-dark challenge assays. These behavioral assessments cover both motor and non-motor aspects, such as movement deficits, sensorimotor, habituation learning, risk-taking, stress responsiveness, and sleep-wake rhythm.
Utilizing the Zebrabox high-throughput video tracking system and quantitative analysis method, we extracted and quantified various behavioral parameters and compared the behavioral differences between the MPTP-treated group and the wild-type (WT) group by statistics. Compared with existing zebrafish PD assays, this multiparametric behavioral battery captures motor and non-motor phenotypes within a standardized framework. The automated tracking system ensures objective behavioral data acquisition and high reproducibility. In addition, the small body size of larvae allows individual animals to be placed in multi-well plates, enabling high-throughput assessment. The above advantages are beneficial for deeply analyzing the pathological mechanism of PD, screening the candidate drugs or compounds, and exploring the influence of environmental factors on the occurrence or development of PD.

<table><tbody><tr><td>100&amp;times; E3 stock solution</td><td>Shanghai Feixi Biotechnology</td><td>N/A</td><td>containing 34.8 g NaCl, 1.6 g KCl, 5.8 g CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O, and 9.78 g MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O per liter</td></tr><tr><td>12-Well Cell Culture Plate</td><td>NEST Biotechnology</td><td>712011</td><td>Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays</td></tr><tr><td>24-Well Cell Culture Plate</td><td>NEST Biotechnology</td><td>702011</td><td>Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays</td></tr><tr><td>3 mL Pasteur Pipette</td><td>Biologix</td><td>30-0238A1</td><td></td></tr><tr><td>6-Well Cell Culture Plate</td><td>NEST Biotechnology</td><td>703011</td><td>Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays</td></tr><tr><td>96-Well Cell Culture Plate</td><td>NEST Biotechnology</td><td>701011</td><td>Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays</td></tr><tr><td>MATLAB R2020a</td><td>MathWorks, USA&amp;nbsp;</td><td>N/A</td><td>MATLAB R2020a, Campus-wide license obtained from Shandong University software portal (softms.sdu.edu.cn).Used for statistical analysis of zebrafish larval behavioral data.</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td>N/A</td><td>Microsoft Excel (part of Microsoft Office), version installed via Shandong University licensed software portal (softms.sdu.edu.cn).</td></tr><tr><td>MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)&amp;nbsp;</td><td>Beyotime</td><td>41125281</td><td>5 mg</td></tr><tr><td>Petri Dish</td><td>NEST Biotechnology</td><td>704202</td><td>100 mm, sterile, easy grip handle, TC-Treated</td></tr><tr><td>Sodium hypochlorite solution</td><td>Macklin</td><td>S817439</td><td>stock household bleach solution(6-14% active chlorine basis), diluted to &amp;ge;10% v/v for surface and waste decontamination; also diluted 1:5 (6% bleach: water) for euthanasia of MPTP-treated zebrafish larvae.</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZX2 series</td><td></td></tr><tr><td>Zebrabox High-Resolution Video Tracking System&amp;nbsp;</td><td>Viewpoint, France&amp;nbsp;</td><td>N/A</td><td></td></tr><tr><td>Zebralab Software&amp;nbsp;</td><td>Viewpoint, France&amp;nbsp;</td><td>N/A</td><td>Version (3,52,3,87)</td></tr></tbody></table>

phenotypic profiling of mptp-induced parkinsonian-like behavioral phenotypes in zebrafish larvae based on behavioral experiment

Phenotypic Profiling of MPTP-Induced Parkinsonian-like Behavioral Phenotypes in Zebrafish Larvae Based on Behavioral Experiment

This article introduces a unique, multiparametric behavioral phenotyping protocol for MPTP-induced Parkinson's disease (PD) in larval zebrafish. Chemical induction of PD-like pathology in zebrafish provides a high-throughput, genetically tractable system, yet most studies have focused narrowly on basic locomotor assays or simple light-dark transitions. Such limited approaches fail to capture the broader spectrum of PD-related dysfunctions, ranging from bradykinesia and sensorimotor deficits to anxiety-like behaviors and sleep disturbances. To address this gap, this study developed an integrated battery of 6 complementary assays: locomotor test to assess general locomotor and exploratory drive; thigmotaxis assessment to detect anxiety-related edge-preference behaviors; startle response test to record bradykinesia and impaired motor reactivity; light-dark challenge assays to evaluate risk-taking and stress responsiveness; photomotor response paradigms (including strobe light stimulation) to quantify sensorimotor and reflexive movement; and sleep-wake regulation monitoring to measure sleep-wake behavior. Larval zebrafish are exposed to sublethal concentrations of MPTP from 1 day post-fertilization (dpf) to 5 dpf, with behavioral assessments initiated from 4 to 6 dpf. High-resolution automated tracking generates several behavioral endpoints per assay. This approach uncovers previously undetected PD-like alterations, such as disorderly sleep architecture, impaired habituation learning, sensorimotor and reflexive movement, reflecting early nonmotor symptoms. By combining these assays, researchers obtain a comprehensive behavioral symptom that more closely mirrors the complexity of human PD. This protocol expands the paradigm for studying PD pathogenesis, screening candidate therapeutics, and exploring environmental modulators. It exemplifies the collection's goal of presenting innovative animal models that enhance translational relevance through robust, multifaceted phenotyping.

Sleep-wake regulation monitoring 
The 52-h sleep-wake regulation monitoring began at 4 dpf and was conducted using a 96-well plate. Zebrafish in MPTP-treated showed significant disruptions in sleep structure compared to the WT group. Representative sleep curves, wake activity plots, and averaged activity traces are shown in Figure 2. Parameters of sleep-wake regulation analysis in zebrafish larvae are shown in Figure 3. Total sleep time was s...

This protocol establishes a behavioral phenotyping paradigm in MPTP-treated zebrafish larvae to evaluate Parkinson's disease-like motor and non-motor deficits.

This article introduces a unique, multiparametric behavioral phenotyping protocol for MPTP-induced Parkinson's disease (PD) in larval zebrafish. Chemical ...

We present a Multiparametric behavior protocol to comprehensively profile motor and non-motor Parkinsonian phenotypes in larval Zebrafish. This protocol applies to Parkinson's disease modeling, drug screening, environmental neurotoxicity, studies using the fish larvae. To begin, wear appropriate personal protective equipment, including gloves, a laboratory coat, and protective eyewear.Dilute 100x E3 stock solution at a one to 100 ratio with ultrapure water to prepare a single strength medium. Now select sexually mature male and female adult zebrafish. Using breeding tanks with removable dividers, perform controlled breeding.At 8:00 PM, place male and female zebrafish into separate sections of the breeding tanks at a ratio of two males to three females using dividers to keep them apart. The next morning at 8:00 AM, remove the dividers to allow mating. Collect the embryos one hour later and wash them three times with clean system water.House the embryos in 100 millimeter Petri dishes containing 20 milliliters of fresh E3 medium. Then incubate the embryos at 28 degrees Celsius under a 14-hour light and 10-hour dark cycle. At one day post fertilization, carefully remove unfertilized, malformed, and abnormal embryos using a pasture pipette.Aspirate the medium slowly from the edge of the Petri dish and add fresh E3 embryo medium along the wall of the Petri dish to minimize disturbance to the wild type embryos. For the MPTP treated group, use 50 micromolar MPTP solution. Four days post fertilization, modify a three-milliliter pasture pipette by cutting 10 millimeters from the tip to create a wide bore.At 6:00 PM, use the modified pipette to select zebrafish larvae from each group and transfer one larvae per well into a 96-well plate. Observe each well under a stereo microscope to confirm that each zebrafish larva appears healthy. Verify that the fins are open and regularly swinging, the body is upright, and the swim ladder is inflated.Next, clean the bottom of the plate carefully to prevent dirt from affecting video tracking results. Then transfer the 96-well plate centrally to the bottom of the zebra box chamber. Select the quantization mode in the video tracking software.For sleep wake regulation monitoring, set the light adjustment to turn off the light at 10:00 PM and turn on at 8:00 AM.Configure the software parameters so that the detection threshold is set to 40, burst to 25, freeze to four, total monitoring time to 52 hours, and then sized to 60 seconds. Start the sleep wake procedure at 8:00 PM.To monitor photo motor responses, place five day post fertilization larvae into a 96-well plate with one larva per well. Then set the light transition program to zero to 60 seconds of darkness, followed by 60 to 120 seconds of constant light.Start the protocol and collect motion parameters every six seconds throughout the 120-second assay. Stop the protocol at 120 seconds and export the data file. Then place the larvae in complete darkness for five minutes to acclimate them for the subsequent dark strobe light assay.Set the light transition program to deliver strobe light at 10 hertz for 60 seconds. To assess thigmotaxis, select two six-well plates. Fill each well with four milliliters of E3 medium.At five days post fertilization, collect zebrafish larvae from the wild type group and the MPTP treated group and place them into separate six well plates. Before the thigmotaxis assessment, place the well plates in the zebra box for 15 minutes to allow acclimation. Then use the draw area function to draw two concentric circles in each well with the inner circle radius equal to half the radius of the well, and start assessment.For a locomotor assessment, transfer five-day-old zebrafish larvae from the wild type group and the MPTP treated group into two separate 12-well plates. After a 15 minute acclimation, select the tracking online analysis mode and record locomotor activity for five minutes using the automated video tracking system. For the startle response test, transfer five day old larvae individually into two 12-well plates with one larva per well and two milliliters of E3 medium.Place the plates into the zebra box for a five minute light acclimation period. Launch the ZebraLab software and activate the tracking online analysis mode. Open the light triggering interface, then select, use one of the three triggering methods below, and enable enhanced stimuli.Then, set the starting method to when session is starting. In the transition schedule, define the dark flash rhythm. Set the program to trigger after 21 seconds, then set transition one to five milliseconds with 100%light intensity.Transition two to 2, 890 milliseconds with 100%light intensity. Transition three to five milliseconds with 0%light intensity, and transition four to 100 milliseconds with 0%light intensity. Tick the box for repeat list of transitions to ensure periodic flashing, and then start the protocol.For the light dark challenge assay, transfer zebrafish larvae into two 24-well plates with one larvae per well and one milliliter of E3 medium. After a five minute acclimation in the zebra box, turn on the tracking analysis online mode. Then configure the light program for six minutes of constant light, followed by four minutes of complete darkness.At the 10th minute, stop behavioral recording and save the data. Carefully remove the plate from the zebra box. After each behavioral experiment, spray and wipe all MPTP contacted surfaces with freshly prepared sodium hypochlorite solution.Collect liquid and solid waste containing MPTP and clearly label them as hazardous neurotoxic waste. Treat all contaminated materials with freshly prepared sodium hypochlorite solution. Treated zebrafish larvae showed disrupted sleep and wake activity traces compared to the wild type group.Total sleep time was also significantly increased in the treated group. The number of sleep outs was significantly decreased in the treated group while the average sleepout length increased. Wake activity and average activity were significantly impaired in the treated group.Under constant light, the freezing index of the treated group was significantly increased. It also significantly increased under strobe light stimulation at 10 hertz. Photo motor response curves showed reduced reactivity in the treated group.Tracking results indicated reduced locomotor and exploratory behavior in the treated group. Total swimming and average swimming speed were also significantly reduced in the treated group. The freeze movement time ratio was significantly higher in the treated group, but the rapid movement time ratio was significantly lower.Alternating light and dark transitions produced pronounced velocity peaks in wild type larvae. The distance swum after stimulation and peak velocity after stimulation were significantly decreased in the treated group. Latency to peak speed movement onset was also significantly delayed in the treated group.The key challenge is minimizing internal determinants while accounting for individual variability to ensure accurate and reproducible behavior analyze in the zebrafish level. Behavioral clustering and phenomic profiling can be applied to uncover subtle neurotoxic effects and gain deeper magnetic insights. Future studies can apply this platform to hazard approved drug screening and the pharmacological evaluation in zebrafish Parkinson's Disease models.

phenotypic-profiling-mptp-induced-parkinsonian-like-behavioral

Research

JoVE Journal

Behavior

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Our laboratory developed a novel high-throughput automated imaging system that is useful for the detection of several different behaviors in 7-day-old zebrafish larvae. The system can be used for detecting subtle changes in behavior after the larvae have been exposed to environmental toxicants or pharmaceuticals.

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

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.

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

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.

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

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.

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

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.

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

This protocol presents a reliable method to assess blood-brain barrier (BBB) integrity in adult zebrafish (Danio rerio) model using Evan's blue dye-based assessment, enabling both qualitative and quantitative analysis of neurovascular disruption.

Visualizing the Gatekeeper: Evan's Blue Dye-Based Assessment of Blood-Brain Barrier Permeability in Adult Zebrafish

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

A procedure to study the capacity of an alcohol associated environmental context to trigger the renewal of alcohol-seeking behavior in rats is described.

A Procedure to Observe Context-induced Renewal of Pavlovian-conditioned Alcohol-seeking Behavior in Rats

Learn a multiparametric behavioral protocol for profiling motor and non-motor Parkinsonian phenotypes in larval zebrafish using MPTP-induced models. This method enables comprehensive drug screening and neurotoxicity assessment.

Phenotypic Profiling of MPTP-Induced Parkinsonian-like Behavioral Phenotypes in Zebrafish Larvae Based on Behavioral Experiment

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