Behavioral Analysis of Locomotor Dysfunction in Drosophila melanogaster as a Readout for Neurotoxicity

The goal of our research is to develop a high to put cost effective and ethical favorable method for screening of neurotoxic compounds. Traditional methods of toxicology tests often rely on virtual rate models, which are expensive, time consuming, otherwise ethical concerns as we know. We wanted to explore whether we could detect early signs of neurotoxicity using an alternative model as a Drosophila melanogaster that is both sensitive and scalable. This protocol help us to detect behavior phenotypes such as movement patterns alterations and hiperactivity, but also progressive motor decline or security written disruptions, and the wide preference is also tested.

What makes our protocol advantages is that it combines the classic climbing assay with a real time monitoring technology, and this allows us to capture subtle motor impairments that might be missed. By traditional methods providing higher sensitivity and more precise behavioral data.

[Narrator] To begin, gently transfer the flies to an empty bottle. Place the bottle containing the flies inside a box filled with ice, ensuring the entire bottle is covered. After one minute, gently tap the bottle to verify that all flies are anesthetized. Prepare a smaller container filled with ice. Place a Petri dish or a flat clean surface on top of the ice to create a chilled platform. Now invert the tube over the chilled Petri dish and gently tap to release the flies onto the cold surface. Carefully transfer the flies into empty vials. Allow the flies to recover from anesthesia for 30 minutes in a controlled environment at 25 degrees Celsius with a 12 hour light dark cycle, and 50% humidity. Next, for microcapillary feed preparation, use a pipette to manually load 10 microliters of the test solution into each microcapillary tube, ensuring consistent volume across all samples. Add three to five microliters of mineral oil to the open end of each microcapillary tube to prevent evaporation and leakage during the experiment. Insert the prefilled microcapillaries through holes in the cotton vial plugs, ensuring a secure fit. Place the vials containing flies into an incubator set to 25 degrees Celsius with a 12 hour light dark cycle, and leave the vials in the incubator for the duration of the experiment. Every 12 hours, transfer the flies to a clean, empty vial to assess their locomotor activity. Measure and mark line seven centimeters from the bottom of the vial using a permanent marker. This mark will serve as the climbing target. Position a camera or phone in a stable location to record climbing behavior. Ensure proper lighting and a transparent background to enable clear visibility and analysis. Confirm that the experimental setup is compatible with the camera's field of view and start recording. Review the video recordings and measure the time taken by each fly to reach the seven centimeter mark. Prepare the feeding microcapillaries with the sample and mineral oil seal as shown previously. Then prepare individual locomotion chambers using clear plastic straws cut to approximately six centimeters in length. Seal one end of each straw with a transparent film to create an airtight closure. Near the sealed end of the straw, create a small hole to insert the feeding microcapillary, ensuring it fits snugly and does not leak or move during the assay. After anesthetizing and segregating the flies as shown earlier, gently place one anesthetized fly into each tube using a fine paint brush. Seal the open end of each tube with either transparent film or a cotton plug, allowing airflow while preventing escape. Now insert a prefilled microcapillary into each prepared chamber through the designated hole. Place the assembled tubes into the assay arena, making sure the microcapillaries remain accessible for daily replacement. Place the entire setup inside a chamber maintained at 25 degrees Celsius with a 12 hour light dark cycle and 50% humidity. Connect the device to the local tracking system or network. Access the software platform and locate the device assigned to the experiment. Ensure the system is correctly tracking each fly based on visual markers. Enter all experimental metadata and start recording the assay. Climbing ability assessed using the negative geotaxis assay at 24 and 48 hours shows similar performance between the control and treatment groups at 24 hours indicating no immediate motor deficits. By 48 hours treated flies take nearly twice as long to climb compared to controls reflecting significant motor impairment. Continuous activity tracking reveals that treated flies initially display heightened movement, but their activity sharply declines after 12 hours suggesting early hyperexcitability followed by reduced motor function. By 24 to 40 hours, treated flies show reduced movement compared to controls. Overall treated flies have approximately 20% reduced activity across 40 hours. Treated flies initially responded with a delay to the light cues followed by transient circadian alignment, but progressively lost rhythmicity by 48 hours likely as a result of locomotive deficit. Treated flies prefer light zones more than controls.