Integrating Visual Psychophysical Assays within a Y-Maze to Isolate the Role that Visual Features Play in Navigational Decisions

Our protocol enables us to mimic visual features that a freely moving fish may experience when traveling in a group and quantify how those features influence their navigational decisions. This technique can be used to link individual level directional decisions in response to a sensory cue to see how those decisions change based on the actions of others. Our methods provide insight into how animals assess and navigate their environment and the emergent properties associated with collective decision making, artificial intelligence, and complex engineered systems.

Conducting psychophysical assays on freely moving individuals is challenging. We continuously strive to maximize control over the animal's sensory experience without biasing their behavior through unwanted cues from the equipment. Video documentation is an excellent format for clarifying complex experimental methods as it increases transparency and hopefully reproducibility.

Begin by placing a custom constructed water-tight poly methyl methacrylate Y maze platform on top of the transparent support platform in a dedicated room. The Y arm holding and left and right domain areas should be identical in construction. For example, here the Y maze arms are 46 centimeters in length, 23 centimeters in width and 20 centimeters in depth, with a central decision area of approximately 46 centimeters in diameter.

Install an overhead black and white gigabyte ethernet camera to record the maze from above and to record the behaviors of the fish and the visual projections with nine meter internet protocol cables attached to a computer with a one gigabyte ethernet card in a control room. Then, use the software of the connected computer to adjust the camera settings so that the image clarity is optimized for visualizing the relevant behaviors. Install four overhead track lighting systems along the walls of the experimental room, positioning the lights to avoid reflections on the maze.

Secure a short throw projector to the bottom edge of the maze's support structure and set the projection resolution to 1440 by 900 pixels. Then use an image processing program to measure and correct any light distortion created by the projector. Before beginning the experimental trials, set the environmental conditions in the maze to that of the holding system and transfer the animals to the domain for up to 40 minutes per day for five days without the computer generated visual stimuli.

During this time, select, assign, weigh, measure, and transfer the subject fish to the experimental tanks. To initiate a psychophysical assay, turn on the room projector and the light emitting diode light track systems to the predetermined level of brightness to allow the bulbs to warm. Open the camera viewer program and load the settings for aperture, color, and recording saved during the setup to ensure the best quality of video can be obtained and activate the camera to be used for recording.

Select Load Features from the Camera dropdown menu and navigate to the saved Camera Settings folder. Open the saved settings to confirm the video quality and click Continuous Shot before closing the viewer program. Using the predetermined experimental schedule with randomized subject treatment exposures over the course of the experiment, enter the values selected for the current trial and record the treatment combination data into the lab notebook and hit the return key in the Vfish.

pde window to initiate the visual projections. When the virtual fish appear in the domain, log the clock time and lift the holding gate. End the trial when 50%of the subject fish's body moves into a choice arm or when the designated experimental assay period elapses.

Log the clock time, the start and stop times from the stopwatch, and the fish's choice. Stop the video recording and press pause in the visual stimuli program to prompt the user for the trial outcome data. Upon confirmation of the selection, the program will return to the first screen and await the values for the next experimental trial.

Then, return the fish to its holding tank and place the next fish into the holding area Y arm for the next trial. At the conclusion of a session, press stop in the program once the last fish of the session has made a decision to write the session data out to file. Repeat the water exchange at the conclusion of the morning session to ensure water quality stability between trials and review the lab notebook to make any needed notes.

After the last afternoon trial, press stop in the visual stimuli program to output the collected data to the data out folder. In this representative experiment, the majority of the fish showed a significant improvement in the accuracy with which they followed the directional stimuli as the speed of those stimuli increased. The trend in decision speed was inconsistent and highly variable across stimulus speed levels however, suggesting that it took subjects on average anywhere from five to 20 times longer to make their decision when the stimuli were moving than when they were not.

Our methods can be expanded to include tests of the effects of sublethal stress on response timing and consistency on how social information is exchanged and transferred between individuals. Our technique has successfully integrated Y mazes and a random dot motion assay, aiding in uncovering the sensory rules that individuals follow when navigating their surroundings. When working with water and electricity, use safety precautions, such as GFCI switches, drip loops, and shock resistant non-slip footwear to reduce the chance of electrical shock and slipping.