Using the light spot assay, the behavior for larvae can be tracked before, during, and after entering the light area allowing the detail of the larval movement to be analyzed. This assay is simple and easy to perform. The light response is tested only one time, so that any possible effect of light adaptation can be excluded.
To prepare the agar plates, slowly and evenly cover the bottom of a 15 centimeter Petri dish with an approximately four millimeter thick layer of hot 1%agar. Allow the plate to cool at room temperature until the agar has solidified. To set up the visual stimulation system, clip a prepared LED light source onto an iron frame, taking care that the light projects down toward the desktop.
Tilt the cylinder slightly until the angle between the cylinder plane and the vertical plane is about 10 degrees. Connect the 470 nanometer blue LED light to the LED one plug of the high power LED driver and turn on the driver. Turn the knob in the upper right corner of the driver to select channel 470 nanometers and click LED.
When the screen displays a check, a blue light spot will appear on the desktop. Click OK and turn the knob to adjust the intensity of the light. Move the position of the light source up and down to adjust the diameter of the light spot to two centimeters.
Rotate the knob to select the light intensity according to the experimental requirements. Use a compact power meter console with a standard photo diode power sensor to measure the maximal and minimal light power in the spot. The light power needs to measured in the dark in the actual experiment.
Then record the light power, measure it three times and use the average value. To set up the imagining system, clamp a high resolution web camera with an iron clip about 10 centimeters above the light spot on the desktop and adjust the orientation of the camera lens toward the desktop. Connect the camera to a computer running Windows 7 through a USB interface and place the agar plate on the desktop right below the camera.
Open the AMCap 9.22 software, the light spot will automatically be shown in the AMCap window. Move the camera slightly left or right to ensure that the light spot is near the center of the window. Use a clip to fix an 850 plus or minus three nanometer bandpass filter at five to seven millimeters immediately below the camera.
Place three infrared light generating LEDs with a central wavelength of 850 nanometers evenly around the agar plate about five centimeters away from the edge of the plate and with LED lenses at a 70 degree angle facing toward the plate. Then connect the LEDs to a power source through their AC to DC converters. In the menu of the AMCap software, select options, video device, and capture format and set the pixel size of the captured video to 800 by 600 and the frame rate to 60 frames per second.
Remove the filter and place a ruler under the camera. Adjust the camera focus until the scale line is clear and parallel to the width of the video field of view and click capture, setup, and video capture to select the save path. Then click start recording to record the actual distance corresponding to 600 pixels and calculate the ratio of each pixel to the actual distance.
Before introducing the larva to the imaging setup, acquire a short video of the light position and name the unfiltered control background video light area one. Place the filter back on the camera lens and turn on a light far from the experimental device at as low a setting as possible while still allowing the larvae to be clearly observed. Use a spoon to remove some larvae from the culture medium and gently select a third instar larva.
Wash the larva in distilled water and place the larva in the center of the agar plate. Gently remove any excess water from the larva and turn off the room light. Allow the larva to acclimate for two minutes in the dark environment before turning on the infrared LED light.
The following light avoidance behavior experiment needs to be carried out in the dark. For the convenience of demonstration we filmed in the bright light. Gently brush the larva to the center of the plate.
When the larva begins to crawl straight, rotate the plate to make the larva head towards the light spot. Click capture, setup, and video capture to select the save path and click start recording to record. Allow the larva crawl toward, enter, and leave the light spot until it is nearly out of the field of view, and click stop recording.
Move the filter away from the camera and acquire a short video of the position of the light spot. Then name the post assay unfiltered video light area two and compare this video with the light area one video to ensure that the light spot position is not changed. Here, time curves of the tail speed, body bending angle and angular speed of body bending of a representative larva can be observed.
The size of the larval head cast and, therefore, maximal body bending angle is significantly reduced in third instar larvae with octopaminergic neurons inhibited by tetanus toxic expression compared to parental controls indicating that Tdc2-Gal4 neurons are necessary for a normal larval light response. For successful image processing, make sure that the camera doesn't block the light and that the light spot is round and near the center of the window. Following this procedure, optogenetics can be performed to observe how the larvae react when specific neurons are activated.
Using this assay, the details of larval movement can be analyzed allowing the far impact of environmental and of internal factors on larval light avoidance behavior to be studied.
