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

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.