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. 2022 Oct 26;3(4):101787.
doi: 10.1016/j.xpro.2022.101787. eCollection 2022 Dec 16.

A two-choice assay for noxious light avoidance with temporal distribution analysis in Drosophila melanogaster larvae

Bibi Nusreen Imambocus  1 , Andrey Formozov  2 , Fangmin Zhou  3 , Peter Soba  4
Affiliations

A two-choice assay for noxious light avoidance with temporal distribution analysis in Drosophila melanogaster larvae

Bibi Nusreen Imambocus et al. STAR Protoc. .

Abstract

Two-choice assays allow assessing of different behaviors including light avoidance in Drosophila larvae. Typically, the readout is limited to a preference index at a specific end point. We provide a detailed protocol to set up light avoidance assays and map the temporal distribution of larvae based on analysis of larval intensities. We describe the assay setup and implementation of scripts for analysis, which can be easily adapted to other two-choice assays and different model organisms. For complete details on the use and execution of this protocol, please refer to Imambocus et al. (2022).

Keywords: Behavior; Model organisms; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Light avoidance setup (A) A digital camera (A) is mounted in a darkbox. (B and D) An infrared LED frame (B) is placed aroud the arena containing two agar plates (D). (C) The outer half of each plate is illuminated with a white light source (C) from below, which is fitted underneath the acrylic glass plate. (E) The camera is connected to a computer (E) via a frame grabber for video recording. A simplified cartoon of the setup is shown on the right side.
Figure 2
Figure 2
Workflow of the video analysis In the first step, using the ffmpeg software with the reprocess_video.bat script or another equivalent approach, ∗.mp4 format video files are (1) reduced for frame number (e.g., every 200th frame), (2) cropped to remove unnecessary parts of the video, and (3) and converted into the ∗.avi format. In the second step, using Fiji and the reprocess_avi_and_get_means.ijm script, the processed ∗.avi videos are (4) masked to generate binary, black-and-white images, and then (5) analyzed by generating ROIs measuring the intensity of the respective dark and light side of each arena. Generated data tables (∗.csv files) are then used to draw the graphical output of the data.
Figure 3
Figure 3
Intensity-based analysis of time-dependent larval distribution in control and upon Dp7 neuron silencing in light avoidance assays (A) Distribution of larvae in controls (Dp7-LexA x w1118) at indicated time points. (B) Intensity plots for dark and light sides and the sum of both. (C) Intensity-based PI of control showing preferred distribution to the dark side in light avoidance assays. (D) Distribution of larvae with Dp7 neuron silencing (Dp7-LexA x LexAop-Kir2.1) at indicated time points. (E) Intensity plots for dark and light sides and the sum of both. (F) Intensity-based PI of larvae with Dp7 neuron silencing showing loss of dark preference in light avoidance assays.
See this image and copyright information in PMC

References

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