Method Article

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

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DOI:

10.3791/70041

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May 15th, 2026

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1Department of Medicinal Chemistry, State Key Laboratory of Discovery and Utilization of Functional Components in Traditional Chinese Medicine, Key Laboratory of Chemical Biology (Ministry of Education), NMPA Key Laboratory for Technology Research and Evaluation of Drug Products, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 2College of Information Engineering, Ningde Normal University, 3Digital Fujian Research Institute of Big Data for Elderly Rehabilitation and Nursing, 4Department of Neurology, Qilu Hospital Cheeloo College of Medicine, Shandong University, 5Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, 6Jinan Microecological Biomedicine Shandong Laboratory

Corresponding Authors: Ning Wang <wn@ndnu.edu.cn>, Xiang Li <lixiang@sdu.edu.cn>

In This Article

Summary

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

Abstract

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.

Introduction

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer'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 µM)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.

Protocol

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

  1. 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.
  2. 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.
  3. 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.

2. Zebrafish embryos preparation and grouping

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.

3. Sleep - wake regulation monitoring

  1. 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).
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
    1. 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.

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.

  1. 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.
  2. Dark-constant light paradigm:
    1. 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.
    2. Open the Tracking-online analysis mode.
    3. Set the light transition program as follows: 0-60 s: Dark, 60-120s: Constant light (1000 lx).
    4. Start the protocol, collect related motion parameters every 6 s throughout the 120 s assay.
    5. 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.
  3. Dark-strobe light paradigm:
    1. 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.
    2. 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.
  4. 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 < 0 indicates freezing behavior; Freeze index > 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.

5. Assessment of thigmotaxis

  1. Select two 6-well plates, fill each well with 4 mL E3 medium.
  2. 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.
  3. Before the thigmotaxis assessment, the well plates should be placed in the appropriate position of Zebrabox 15 min in advance to accommodate.
  4. 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.
    1. Calculate the thigmotaxis with the following formulas:
      Calculate the percent of distance swum in the outer zone:
      Thigmotaxis calculation formula; equation showing behavioral analysis method in research context.
      Calculate the percent of time spent in the outer zone:
      Thigmotaxis equation, ΣFx=0, describing behavioral analysis, includes parameters total dur, an1, an2.
    2. 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.

6. Locomotor assessment

  1. 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.
  2. 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.
  3. 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.
    1. 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.

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.

  1. 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).
  2. Before the startle response test, place the well plate in the test position of the Zebrabox 5 min in advance to adapt.
  3. 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.
  4. 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.
  5. 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.
    1. 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.

8. Light-dark challenge assay

  1. 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).
  2. 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.
  3. On the 10th min, stop behavioral recording and save data. Carefully remove the plate from the Zebrabox.
  4. 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.

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).

  1. 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.
  2. 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.
  3. 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).

Results

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 significantly increased in the MPTP group relative to WTs (p < 0.01, Student's t-test). The number of sleep bouts was decreased in the MPTP group compared to WTs (p < 0.001), while the average sleep bout length was increased (p < 0.0001). Sleep latency did not differ significantly between groups. Wake activity was also impaired in the MPTP group (p < 0.01), and average activity was reduced (p < 0.01).

Photomotor response paradigms
The sensorimotor gating under constant light and strobe light conditions was assessed using a 96-well plate photomotor response assay at 5 dpf. The photomotor response curves are shown in Figure 4A,C. Under constant light, the freezing index of the MPTP-treated group was increased (Figure 4B) compared with the WT group (p < 0.05, Student's t-test), indicating an obvious freezing response. A similar trend was observed under strobe light stimulation (10 Hz), the freezing index of the MPTP-treated group was significantly increased (Figure 4D) compared with the WT group (p < 0.0001, Student's t-test). The reactivity of the MPTP group was reduced, suggesting impaired sensorimotor gating and reflexive movement.

Thigmotaxis assessment
At 5 dpf, the thigmotaxis assessment was conducted using 6 well-plates to record edge-preference behavior, which is a means of evaluating anxiety-like behaviors. Analysis of edge-preference behavior showed that MPTP-treated larvae did not exhibit a strong increase in edge-preference compared to WTs (Figure 5A,B).

Locomotor assessment
At 5 dpf, a locomotor assessment was conducted using 12 well-plates to assess general locomotor and exploratory characteristics of zebrafish larvae. Tracking data from the Zebrabox system indicated reduced locomotor and exploratory behaviors in the MPTP group (Figure 6A). Zebrafish larvae in the MPTP-treated group showed a significant reduction in total swimming distance (p < 0.01, Student's t-test) (Figure 6C). The average swimming speed was also lower in the MPTP group (p < 0.01, Student's t-test) (Figure 6D). In addition, MPTP-treated zebrafish larvae exhibited significant alterations in movement patterns compared with WT larvae. Specifically, the freeze movement time ratio was significantly higher in the MPTP group compared to WT (p < 0.05, Student's t-test) (Figure 6E), suggesting increased immobility or freezing behavior. Conversely, the rapid movement time ratio was significantly lower in the MPTP group than in the WT group (p < 0.01, Student's t-test) (Figure 6F), indicating reduced high-speed locomotor activity potentially reflective of bradykinesia in Parkinson's disease models , which may related to anxiety-like or motor inhibitory deficits observed in dopaminergic dysfunction. These representative results demonstrated that MPTP treatment induces locomotor deficits in zebrafish larvae.

Startle response test
At 5 dpf, a startle response test was conducted using 12-well plates combined with light-flash stimulation to assess the locomotor responsiveness of zebrafish larvae. The startle response curves are shown in Figure 7A. Initiating at 24 s, the introduction of alternating light-dark transitions every 3 s elicited pronounced peaks in velocity in WT larvae, reflecting a robust startle response to photic stimuli. In contrast, MPTP-treated larvae exhibited a consistently diminished velocity with no significant peaks, indicating a profound lack of responsiveness. This discrepancy highlights a dopaminergic-mediated disruption in the neural circuits responsible for stimulus-triggered motor initiation, closely paralleling the hyporesponsiveness and bradykinesia characteristic of Parkinson's disease models. Compared to the WT group, the distance swum post-stimulation was significantly decreased in the MPTP-treated group (p < 0.0001, Student's t-test) (Figure 7B). Meanwhile, the peak velocity post-stimulation was significantly decreased in the MPTP-treated group (p < 0.001, Student's t-test) (Figure 7C), and the MPTP-treated group exhibited a significantly delayed latency to peak speed movement onset (p < 0.05, Student's t-test) (Figure 7D). These results demonstrated the impaired motor reactivity in the MPTP-treated group.

Light-dark challenge assay
At 5 dpf, the light-dark challenge assays were conducted in 24-well plates to evaluate habituation learning, thigmotaxis, and locomotor activity during the light and dark phases. The protocol included a 6 min light acclimation period followed by a 4 min dark test period. The locomotor activity curve (Figure 8A) over time revealed distinct patterns between the MPTP-treated group and the WT group during the light-dark conversion. Neither the WT group nor the MPTP-treated group showed a statistically significant change in habituation learning. However, the WT group exhibited a clear downward trend in locomotor activity over the last 4 min after light-dark conversion, whereas this trend was not obvious in the MPTP-treated group (Figure 8B).

In the light-ON period (100% light intensity), the swimming distance of larvae in the MPTP-treated group did not differ from that of the WT group. In the dark phase, the swimming distance of the MPTP group was significantly lower than that of the WT group (p < 0.0001, Student's t-test) (Figure 8C). Total swimming distance over the 10 min was significantly lower in the MPTP-treated group than in the WT group (p < 0.05, Student's t-test) (Figure 8D). Thigmotaxis was evaluated by measuring the proportion of distance swum in the outer zone. The MPTP-treated larvae did not exhibit a significant change in edge-preference compared to the WT group (Figure 8E).

These results indicate MPTP treatment caused zebrafish larvae to show impaired habituation learning and reduced locomotor activity, which reflects the neurobehavioral defects similar to those of PD patients.

Zebrafish development stages, behavior assays, photomotor response, light/dark challenge diagrams.
Figure 1: Experimental timeline, treatment conditions, and the well plates for behavioral assays. (A) Experimental timeline indicates zebrafish embryo of the MPTP-treated group exposure to 50 µM MPTP from 1 to 5 dpf (the WT group was treated with fresh E3 medium every day instead), sleep/wake behavior monitoring initiated in the evening of 4 dpf, and followed by the other behavioral experiments at 5 dpf: photomotor response paradigms, thigmotaxis assay, locomotor assessment, startle response test, and light-dark challenge assay. (B) 96 well-plate for sleep/wake regulation monitoring and photomotor response assays.(C) 6 well-plate for thigmotaxis assay, with inner and outer zones.(D) 12 well-plate for locomotor assessment and startle response test.(E) 24 well-plate for light/dark challenge assays, white and black bars represent acclimatization and dark challenge phases, respectively. Please click here to view a larger version of this figure.

Activity, waking, sleep patterns in WT vs. MPTP; line graphs; behavioral study comparison data.
Figure 2: Representative sleep-wake activity curves of zebrafish larvae over a 52-h period under a 14 h light/10 h dark cycle. (A) Average activity curve of zebrafish larvae over a 52-h period under a 14 h light/10 h dark cycle. (B) Waking activity curve of zebrafish larvae over a 52-h period under a 14 h light/10 h dark cycle. (C) Sleep curve of zebrafish larvae over a 52-h period under a 14 h light/10 h dark cycle. The shaded bands around each curve represent the standard error of the mean (SEM) for each curve, indicating the variability in the data. On the x-axis, the black and white bars represent dark and light periods, respectively. The numbers marked with "h" (e.g., 2 h, 12h, 26h) denote the hour elapsed since the start of the sleep-wake procedure, with transitions between light and dark periods shown at these time points. Please click here to view a larger version of this figure.

Bar charts comparing WT and MPTP effects on activity, sleep, and latency during day and night.
Figure 3: Parameters of sleep-wake regulation analysis in zebrafish larvae. (A) Average activity (s/min) during day and night. (B) Waking activity (s/min) during day and night. (C) Total sleep duration (min) during day and night. (D) Average sleep bout length (min) during day and night. (E) Number of sleep bouts during the day and night. (F) Sleep latency (min) during the day and night. ** represents p < 0.01, *** represents p <0.001, ****represents p <0.0001 for a Student's t-test. All values represent mean ± SEM (n = 48). Please click here to view a larger version of this figure.

Swim distance and freeze index graphs; WT vs. MPTP; behavioral experiment results; line and bar charts.
Figure 4: Photomotor response paradigms under constant and strobe light conditions. (A) Distance swum (mm) over time (s) under 1 min dark and 1 min constant light conditions for WT (blue) and MPTP-treated (red) zebrafish. (B) Freeze index under 1 min dark and 1 min constant light conditions. (C) Distance swum (mm) over time (s) under 1 min dark and 1 min strobe light conditions with a light transition at 60 s for WT (blue) and MPTP (red) zebrafish. (D) Freeze index under 1 min dark and 1 min strobe light conditions with a light transition at 60 s. * represents p <0.05, **** represents p<0.0001 for a Student's t-test. All values represent mean ± SEM (n = 48). Please click here to view a larger version of this figure.

Bar graphs comparing WT and MPTP groups; distance and time in outer zone (%).
Figure 5: Thigmotaxis behavior analysis in zebrafish larvae. (A) Percentage of distance moved in the outer zone (%). (B) Percentage of time spent in the outer zone (%). All values represent mean ± SEM (n = 24) Please click here to view a larger version of this figure.

Behavioral analysis with WT, MPTP mouse swim paths diagram, velocity graph, statistical results.
Figure 6: Locomotor behavior in WT and MPTP-treated zebrafish larvae. (A) Representative movement traces of WT and MPTP-treated larvae recorded for 5 min. Red traces represent high-speed (>10 mm/s) movement trajectories, while green traces indicate low-speed (<10 mm/s) movement trajectories. (B) Velocity over time (mm/s) across a 300 s recording. (C) Total swimming distance in 5 min (mm). (D) Average velocity (mm/s). (E) Freezing time ratio. Freezing: the larvae at a speed of 0 mm/s. (F) Rapid movement time ratio. Rapid movement: the larvae were moving with a velocity of more than 10 mm/s. * represents p <0.05, ** represents p <0.01 for a Student's t-test and two-way ANOVA. All values represent mean ± SEM (n = 24). Please click here to view a larger version of this figure.

Velocity analysis graph and bar charts compare WT and MPTP mouse response to light stimulation.
Figure 7: Effects of MPTP on startle response and swimming performance in zebrafish. (A) Velocity in mm/s over time in seconds for WT (blue) and MPTP (red) zebrafish. The black arrow represents the first stimulus. (B) Post-stimulus swim distance in mm/s. (C) Post-stimulus peak velocity in mm/s. (D) Startle response latency in seconds for WT and MPTP-treated zebrafish. * represents p <0.05, *** represents p <0.001, **** represents p<0.0001 for a two-way ANOVA and Student's t-test. All values represent mean ± SEM (n = 24). Please click here to view a larger version of this figure.

Swimming distance graphs; WT vs. MPTP in light/dark conditions; behavioral analysis results.
Figure 8: Light-dark challenge assay results in zebrafish larvae. (A) Distance swum (mm) over time (s) during light to dark transition for WT (blue) and MPTP (red) zebrafish. (B) Distance traveled (mm) between 1 min and 4 min after lights-off for WT and MPTP-treated zebrafish, which reflects the habituation learning. (C) Distance swum (mm) during light-on and light-off periods for WT and MPTP-treated zebrafish. (D) Total distance swum (mm) during 10 min. (E) Distance moved in outer zone (%). *represents p < 0.05, **** represents p < 0.0001 for a Student's t-test and two-way ANOVA. All values represent mean ± SEM (n = 24). Please click here to view a larger version of this figure.

Discussion

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 motor and non-motor behavioral changes, including decreased movement, impaired sensorimotor response, and sleep-wake rhythm disorder, which are similar to PD-like symptoms. These findings manifested the robustness of the zebrafish model and the feasibility and availability of this multiple behavioral test method in capturing PD-like symptoms.

The experiment began with zebrafish embryo preparation at 1 dpf. Larvae were exposed to either 50 µM MPTP (MPTP-treated group) or E3 embryo medium (WT group), followed by behavioral assessments conducted from 4 to 6 dpf. The following are the specific results and interpretations of each test:

Sleep-wake regulation monitoring
Beginning at 4 dpf, zebrafish larvae were continuously monitored for 52 h using a 96-well plate to measure the sleep-wake rhythm. The results demonstrated that larvae exposed to MPTP exhibited disorderly sleep patterns, including decreased sleep bout number, increased total sleep time, and average sleep bout duration, indicating consolidated sleep architecture. These findings parallel clinical sleep disturbance in PD patients, reflecting early non-motor symptoms of the disorder.

Photomotor response test (including strobe light stimulation)
Zebrafish larvae treated with MPTP exhibited significantly altered freeze index and delayed response latency, indicating impairments in sensorimotor gating and reflexive movement. These findings were similar to the visual deficits observed in PD patients.

Thigmotaxis test
Although it was initially hypothesized that MPTP-treated zebrafish larvae would exhibit increased thigmotaxis behavior36, manifested as a higher percentage of distance in the outer zone, the currentresults showed no significant difference in edge-preference between the MPTP and WT groups. This finding may indicate that the short duration and open area of the thigmotaxis assay may not have been sufficiently sensitive to detect subtle anxiety-like alterations at the larval stage37,38. Therefore, the absence of a significant difference reflects the complexity of separating motor and emotional behaviors in early larval stages, rather than contradicting the model. Future studies with prolonged observation time, modified arena geometry, or combined pharmacological validation (e.g., using anxiolytic or anxiogenic compounds) may help clarify whether MPTP treatment alters anxiety-like behaviors in zebrafish larvae.

Locomotor assessment
Using 12 well plates at 5dpf, automatic tracking by the Zebrabox system. These results showed that, compared with the WT group, MPTP-treated larvae showed significantly decreased total distance and average speed, which is similar to the typical motor symptom of PD -- bradykinesia. This is consistent with the result in the MPTP model of rodents18, that is, the loss of dopaminergic neurons leads to reduced movement.

Startle response test
Light stimulation in a 12-well plate was used at 5 dpf. Larvae treated with MPTP showed the following characteristics compared with the WT group: weaker reaction, lower peak speed, and increased latency period. These results indicated the impaired motor reactivity of larvae, which is consistent with the characteristics of reflex slowing in PD patients.

Light-dark challenge assay
In the light-dark challenge assay, distance larvae swam, thigmotaxis, and habituation learning were assessed. According to a previous study31, WT larvae are expected to perform increased locomotor activity during the light-to-dark transition. The results showed that WT larvae displayed a significant increase in activity immediately following the light-to-dark transition, whereas MPTP-treated larvae showed no obvious increase (Figure 8A), indicating impaired escape responses. Though MPTP-treated larvae did not exhibit a significant difference with the WT group for the edge-preference, their adaptability to environmental changes was impaired, suggesting cognitive deficits or anxiety commonly associated with PD. Interestingly, it was found that during the light phase, MPTP-treated larvae exhibited even higher locomotor activity than WTs, likely because the assay of the light period was performed under maximal light intensity (100% light power), strong visual stimulation may have driven locomotor activity39,40 and masked dopaminergic deficits in MPTP-treated larvae. In contrast, during the dark phase, when locomotor activity depends primarily on endogenous dopaminergic signaling, MPTP-treated larvae displayed marked reductions in activity, revealing the impact of dopaminergic impairment, indicating motor impairments characteristic of PD pathology. For the habituation learning, no statistically significant difference was observed between the 1st and 4th min of the dark phase in either group. Nevertheless, WT larvae showed a tendency toward decreased locomotor activity over the dark phase, whereas this tendency was less clear in the MPTP-treated group. This result suggests a reduced habituation in MPTP-treated larvae, which may reflect early alterations in non-motor syptoms associated with PD41.

Multiple behavioral tests demonstrated that the MPTP-induced zebrafish larvae model can effectively simulate motor and non-motor symptoms of PD. Compared with the zebrafish studied only with a single behavior assay, our multi-dimensional behavioral test provides a more comprehensive evaluation for the MPTP-induced zebrafish PD model. This overall behavioral phenotype more fully reflects the characteristics of the disease, enhances the transformation potential of the model, and contributes to studying the pathological mechanism of PD and screening candidate drugs.

Advantages and comparison with other studies
Zebrafish larvae are small in size, requiring lower compound or drug consumption than adult zebrafish, which enables large-scale, cost-effective, high-throughput drug screening16. In addition, the rapid growth of larvae allows behavioral experiments to be completed within 1 week, significantly reducing the disease modeling period.

This protocol employs a multi-dimensional, standardized, high-throughput design. MPTP exposure began at 1 dpf, targeting the developing dopaminergic system in zebrafish larvae. Behavioral assessments are conducted from 4 to 6 dpf, offering a relatively short experimental duration. A multi-dimensional behavioral testing protocol allows comprehensive profiling of both motor and non-motor features of PD, thereby providing a deeper understanding of Parkinson's disease phenotypes. Compared with other behavioral tracking systems, Zebrabox offers a variety of configurable parameter settings, enhancing objectivity and reproducibility29. In addition, the behavioral analysis system used in this study outputs data files in standardized formats, which enables custom scripting for efficient and reproducible data analysis.

Limitations
Although this model and program effectively simulated MPTP-induced motor and non-motor deficits, it failed to fully reproduce the chronic and progressive features of human PD. Also, the behavior of adult zebrafish is complex, allowing assessment of behaviors such as novel tank exploration, shoaling, and social preference42, which are different from those measured in larval zebrafish.

Future directions
In the future, we can try to introduce adult zebrafish to the program, so as to establish a chronic PD model that more closely simulates the progression of human diseases. Additionally, gene-editing technologies could be applied to generate PD-related genetic mutation models. When integrated with this behavioral assessment platform, such models may offer new insights into disease mechanisms and facilitate target discovery.

Furthermore, this platform can be employed to screen and validate candidate compounds with neuroprotective properties. By evaluating their effects on various PD-related behaviors, the system may help advance the development of disease-modifying drugs.

Conclusion
This study provided a robust, rapid, and multiple behavioral phenotypic analysis paradigm utilizing the advantages of the zebrafish larvae model. Various behavioral phenotype analysis approaches comprehensively profiled the motor and non-motor deficits induced by MPTP in the zebrafish PD model. This behavioral paradigm may enhance the translational relevance of the model and is expected to facilitate mechanistic research and drug development for Parkinson's disease.

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

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). 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.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
100× E3 stock solutionShanghai Feixi BiotechnologyN/Acontaining 34.8 g NaCl, 1.6 g KCl, 5.8 g CaCl2·2H2O, and 9.78 g MgCl2·6H2O per liter
12-Well Cell Culture PlateNEST Biotechnology712011Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays
24-Well Cell Culture PlateNEST Biotechnology702011Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays
3 mL Pasteur PipetteBiologix30-0238A1
6-Well Cell Culture PlateNEST Biotechnology703011Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays
96-Well Cell Culture PlateNEST Biotechnology701011Non-Treated, Non-Pyrogenic, Sterile, Polystyrene.Used for zebrafish larval behavioral assays
MATLAB R2020aMathWorks, USA N/AMATLAB R2020a, Campus-wide license obtained from Shandong University software portal (softms.sdu.edu.cn).Used for statistical analysis of zebrafish larval behavioral data.
Microsoft ExcelMicrosoftN/AMicrosoft Excel (part of Microsoft Office), version installed via Shandong University licensed software portal (softms.sdu.edu.cn).
MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) Beyotime411252815 mg
Petri DishNEST Biotechnology704202100 mm, sterile, easy grip handle, TC-Treated
Sodium hypochlorite solutionMacklinS817439stock household bleach solution(6-14% active chlorine basis), diluted to ≥10% v/v for surface and waste decontamination; also diluted 1:5 (6% bleach: water) for euthanasia of MPTP-treated zebrafish larvae.
Stereo MicroscopeOlympusSZX2 series
Zebrabox High-Resolution Video Tracking System Viewpoint, France N/A
Zebralab Software Viewpoint, France N/AVersion (3,52,3,87)

References

  1. Elbaz, A., Carcaillon, L., Kab, S., Moisan, F. Epidemiology of Parkinson's disease. Rev. Neurol. (Paris). 172 (1), 14-26 (2016).
  2. Elgueta, D., et al. Analyzing the Parkinson's disease mouse model induced by adeno-associated viral vectors encoding human alpha-synuclein. J Vis Exp. (185), e63313(2022).
  3. Peng, S. Y., Liu, P., Wang, X. W., Li, K. Y. Global, regional and national burden of Parkinson's disease in people over 55 years of age: a systematic analysis of the global burden of disease study, 1991-2021. BMC Neurol. 25, 178(2025).
  4. Zhu, J. Q., et al. Temporal trends in the prevalence of Parkinson's disease from 1980 to 2023: a systematic review and meta-analysis. Lancet Healthy Longev. 5 (7), 464-479 (2024).
  5. Goldman, J. G. Non-motor symptoms and treatments in Parkinson's disease. Neurol Clin. 43 (2), 291-317 (2025).
  6. Kobylecki, C. Update on the diagnosis and management of Parkinson's disease. Clin Med. (Lond). 20 (4), 393-398 (2020).
  7. Zhou, J., Li, J., Papaneri, A. B., Kobzar, N. P., Cui, G. Dopamine neuron challenge test for early detection of Parkinson's disease. NPJ Parkinsons Dis. 7, 116(2021).
  8. Tolosa, E., Garrido, A., Scholz, S. W., Poewe, W. Challenges in the diagnosis of Parkinson's disease. Lancet Neurol. 20 (5), 385-397 (2021).
  9. Salat, D., Tolosa, E. Levodopa in the treatment of Parkinson's disease: Current status and new developments. J. Parkinsons Dis. 3 (3), 255-269 (2013).
  10. Kalia, L. V., et al. Disease-modifying therapies for Parkinson's disease: Lessons from multiple sclerosis. Nat Rev Neurol. 20 (12), 724-737 (2024).
  11. Thirugnanam, T., Santhakumar, K. Chemically induced models of Parkinson's disease. Comp. Biochem. Physiol C Toxicol Pharmacol. 252, 109213(2022).
  12. Crabtree, D. M., Zhang, J. H. Genetically engineered mouse models of Parkinson's disease. Brain Res. Bull. 88 (1), 13-32 (2012).
  13. Hallett, P. J., et al. Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease. Cell Stem Cell. 16 (3), 269-274 (2015).
  14. Dovonou, A., et al. Animal models of Parkinson's disease: bridging the gap between disease hallmarks and research questions. Transl. Neurodegener. 12, 36(2023).
  15. Potts, L. F., et al. Modeling Parkinson's disease in monkeys for translational studies: A critical analysis. Exp. Neurol. 256, 133-143 (2014).
  16. Doyle, J. M., Croll, R. P. A critical review of zebrafish models of Parkinson's disease. Front. Pharmacol. 13, 835827(2022).
  17. Bagwell, E., Larsen, J. A. A review of MPTP-induced Parkinsonism in adult zebrafish to explore pharmacological interventions for human Parkinson's disease. Front. Neurosci. 18, 1451845(2024).
  18. Feng, X. -Z., et al. Behavioral assessments of spontaneous locomotion in a murine MPTP-induced Parkinson's disease model. J Vis Exp. 143, e58653(2019).
  19. Razali, K., Kumar, J., Mohamed, W. M. Y. Characterizing the adult zebrafish model of Parkinson's disease: a systematic review of dynamic changes in behavior and physiology post-MPTP administration. Front. Neurosci. 18, 1432102(2024).
  20. Brinez-Gallego, P., et al. Experimental models of chemically induced Parkinson's disease in zebrafish at the embryonic larval stage: A systematic review. J Toxicol Environ Health B Crit Rev. 26 (4), 201-237 (2023).
  21. Xu, Z. H., et al. Deep clinical phenotyping of Parkinson's disease: Towards a new era of research and clinical care. Phenomics. 2 (5), 349-361 (2022).
  22. Tinaz, S. Functional connectome in Parkinson's disease and Parkinsonism. Curr Neurol Neurosci Rep. 21, 24(2021).
  23. Costa, H. N., et al. Parkinson's disease: A multisystem disorder. Neurosci Bull. 39 (1), 113-124 (2023).
  24. Christensen, C., Thorsteinsson, H., Maier, V. H., Karlsson, K. Æ Multi-parameter behavioral phenotyping of the MPP⁺ model of Parkinson's disease in zebrafish. Front Behav Neurosci. 14, 623924(2020).
  25. Zhang, S., et al. Anti-Parkinson's disease activity of phenolic acids from Eucommia ulmoides Oliver leaf extracts and their autophagy activation mechanism. Food Funct. 11 (2), 1425-1440 (2020).
  26. Maleski, A. L. A., et al. Integrated behavioral and proteomic characterization of MPP⁺-induced early neurodegeneration and Parkinsonism in zebrafish larvae. Int J Mol Sci. 26 (14), 6762(2025).
  27. Vauti, F., et al. All-age whole mount in situ hybridization to reveal larval and juvenile expression patterns in zebrafish. PLoS One. 15 (8), e0237167(2020).
  28. Parichy, D. M., et al. Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish. Dev Dyn. 238 (12), 2975-3015 (2009).
  29. Lee, D. A., Oikonomou, G., Prober, D. A. Large-scale analysis of sleep in zebrafish. Bio Protoc. 12 (3), e4313(2022).
  30. Shen, Q., et al. Rapid well-plate assays for motor and social behaviors in larval zebrafish. Behav Brain Res. 391, 112625(2020).
  31. Rock, S., et al. Detailed analysis of zebrafish larval behaviour in the light-dark challenge assay shows that diel hatching time determines individual variation. Front Physiol. 13, 827282(2022).
  32. Hillman, C., Kearn, J., Parker, M. O. A unified approach to investigating 4 dpf zebrafish larval behaviour through a standardised light/dark assay. Prog Neuropsychopharmacol Biol Psychiatry. 134, 111084(2024).
  33. Best, J. D., et al. Non-associative learning in larval zebrafish. Neuropsychopharmacol. 33 (5), 1206-1215 (2008).
  34. Gomez-Laplaza, L. M., Gerlai, R. Latent learning in zebrafish (Danio rerio). Behav Brain Res. 208 (2), 509-515 (2010).
  35. Winn, A. A., Prestia, K. A., Peneyra, S. M. Testing alternative surface disinfection agents for zebrafish (Danio rerio) embryos. J Am Assoc Lab Anim Sci. 60 (5), 510-518 (2021).
  36. Wagle, M., Nguyen, J., Lee, S., Zaitlen, N., Guo, S. Heritable natural variation of an anxiety-like behavior in larval zebrafish. J Neurogenet. 31 (3), 138-148 (2017).
  37. Maximino, C., et al. Measuring anxiety in zebrafish: a critical review. Behav Brain Res. 214 (2), 157-171 (2010).
  38. Schnorr, S. J., Steenbergen, P. J., Richardson, M. K., Champagne, D. L. Measuring thigmotaxis in larval zebrafish. Behav Brain Res. 228 (2), 367-374 (2012).
  39. Dekens, M. P. S., Fontinha, B. M., Gallach, M., Pflugler, S., Tessmar-Raible, K. Melanopsin elevates locomotor activity during the wake state of the diurnal zebrafish. EMBO Rep. 23 (5), e51528(2022).
  40. Lee, H. B., et al. Key HPI axis receptors facilitate light adaptive behavior in larval zebrafish. Sci Rep. 14 (1), 7759(2024).
  41. Cavanagh, J. F., Kumar, P., Mueller, A. A., Richardson, S. P., Mueen, A. Diminished EEG habituation to novel events effectively classifies Parkinson's patients. Clin Neurophysiol. 129 (2), 409-418 (2018).
  42. Bedrossiantz, J., Prats, E., Raldua, D. Neurotoxicity assessment in adult Danio rerio using a battery of behavioral tests in a single tank. J Vis Exp. 201, e65869(2023).

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Zebrafish Parkinson's ModelMPTP InductionBehavioral PhenotypingLarval ZebrafishLocomotor AssayThigmotaxis AssessmentStartle Response TestLight Dark ChallengeSleep Wake MonitoringAutomated Video Tracking
Phenotypic Profiling of MPTP-Induced Parkinsonian-like Behavioral Phenotypes in Zebrafish Larvae Based on Behavioral Experiment
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