Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Begin with a behavior recording chamber containing an anesthetized, transgenic zebrafish larva immobilized in a low-melting agarose.

The larva's midbrain pretectum region and the inferior lobe of the hypothalamus contain modified neurons expressing fluorescent calcium indicators.

Remove the agarose around the larva's head and wash with water to remove any residual agarose.

Place a paramecium as prey near the larva.

Transfer the recording chamber under a fluorescence microscope.

As the prey swims, the larva's eye detects it and sends a signal to the pretectum and hypothalamus regions involved in prey detection.

This opens calcium channels in these neurons, allowing calcium ions to enter and bind to the calcium indicators, leading to fluorescence emission.

Record the time-lapse imaging to detect the fluorescence intensity from neurons along the prey's swimming path and generate a color map.

In this map, arrowheads represent prey trajectories: black arrows show no neuronal excitation, while colored arrows indicate increased neuronal excitation triggered by the prey's movement.

After culturing paramecia and anesthetizing a zebrafish larva according to the text protocol, place the larva into 2% low-melting-point agarose, and immediately remove any excess agarose so that the surface of the agarose looks slightly convex. Using a dissecting needle, quickly orient the larva in an upright position.

Then, using a surgical knife, make a few small cuts in the agarose to remove the agarose around the zebrafish head, which makes space for the paramecium to swim. Carefully remove the residual agarose by gently pouring system water on the chamber. Next, put one paramecium in the recording chamber to serve as a visual stimulus.

Then, place the recording chamber under an epifluorescence microscope equipped with a scientific CMOS camera and select a 2.5x objective lens. Start the image acquisition application for the equipped camera. Click Sequence Pane and select Hard Disk Record on the pane. In the Scan Settings section, set the frame count to 900 or any other total frame number desired.

In the Capture pane, set the Exposure Time at 30 milliseconds and Binning to 2 by 2. Click Live on the capture pane to locate the larva in the camera view and focus, and then click Stop Live and click the Start button. Save the movie in .cxd format and analyze the data according to the text protocol.