Name: integrating visual psychophysical assays within a y-maze to isolate the role that visual features play in navigational decisions
Uploaded: 2019-05-02T13:00:00
Description: Watch this Scientific Journal Video about Integrating Visual Psychophysical Assays within a Y-Maze to Isolate the Role that Visual Features Play in Navigational Decisions at JoVE.com

We thank Bryton Hixson for setup assistance. This program was supported by the Basic Research Program, Environmental Quality and Installations (EQI; Dr. Elizabeth Ferguson, Technical Director), US Army Engineer Research and Development Center.

All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Environmental Laboratory, US Army Engineer and Research and Development Center, Vicksburg, MS, USA (IACUC# 2013-3284-01).
1. Sensory maze design
<ol>
	<li>Conduct the experiment in a watertight poly methyl methacrylate Y-maze platform (made in-house) set atop of a transparent support platform in a dedicated room. Here the platform is 1.9 cm thick and is supported by 4 7.62 cm beams of extruded ...

<ol>
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Visual cues are known to trigger an optomotor response in fish exposed to black and white gratings13 and there is increasing theoretical and empirical evidence that neighbor speed plays an influential role in governing the dynamical interactions observed in fish schools7,14,15,16,17. Contrasting hypotheses exist to explain how individua...

Animals sense and interpret their habitat continuously to make informed decisions when interacting with others and navigating noisy surroundings. Individuals can enhance their situational awareness and decision making by integrating social information into their actions. Social information, however, largely stems from inference through unintended cues (i.e., sudden maneuvers to avoid a predator), which can be unreliable, rather than through direct signals that have evolved to communicate specific messages (e.g., the waggle dance in honey bees)1. Identifying how individuals rapidly assess the value of social cues, or any sensory information, can be a challenging task for investigators, particularly when individuals are traveling in groups. Vision plays an important role in governing social interactions2,3,4 and studies have inferred the interaction networks that may arise in fish schools based on each individual&#8217;s field of view5,6. Fish schools are dynamic systems, however, making it difficult to isolate individual responses to particular features, or neighbor behaviors, due to the inherent collinearities and confounding factors that arise from the interactions among group members. The purpose of this protocol is to complement current work by isolating how alternative visual features can influence the directional decisions of individuals traveling alone or within groups.
The benefit of the current protocol is to combine a manipulative experiment with computer visualization techniques to isolate the elementary visual features an individual may experience in nature. Specifically, the Y-maze (Figure 1) is used to collapse directional choice to a binary response and introduce computer animated images designed to mimic the swimming behaviors of virtual neighbors. These images are projected up from below the maze to mimic the silhouettes of conspecifics swimming beneath one or more subjects. The visual characteristics of these silhouettes, such as their morphology, speed, coherency, and swimming behavior are easily tailored to test alternative hypotheses7.
This paper demonstrates the utility of this approach by isolating how individuals of a model social fish species, the Golden Shiner (Notemigonus crysoleucas), respond to the relative speed of virtual neighbors. The protocol focus, here, is on whether the directional influence of virtual neighbors change with their speed and, if so, quantifying the form of the observed relationship. In particular, the directional cue is generated by having a fixed proportion of the silhouettes act as leaders and move ballistically towards one arm or another. The remaining silhouettes act as distractors by moving about at random to provide background noise that can be tuned by adjusting the leader/distractor ratio. The ratio of leaders to distractors captures the coherency of the directional cues and can be adjusted accordingly. Distractor silhouettes remain confined to the decision area (&#8220;DA&#8221;, Figure 1A) by having the silhouettes reflect off of the boundary. Leader silhouettes, however, are allowed to leave the DA region and enter their designated arm before slowly fading away once the silhouettes traversed 1/3 the length of the arm. As leaders leave the DA, new leader silhouettes take their place and retrace their exact path to ensure that the leader/distractor ratio remains constant in the DA throughout the experiment.
The use of virtual fish allows for the control of the visual sensory information, while monitoring the directional response of the subject, which may reveal novel features of social navigation, movement, or decision making in groups. The approach used here can be applied to a broad range of questions, such as effects of sublethal stress or predation on social interactions, by manipulating the computer animation to produce behavioral patterns of varying complexity.

<table>
	<tbody>
		<tr>
			<td>Black and white IP camera</td>
			<td>Noldus, Leesburg, VA, USA</td>
			<td></td>
			<td>https://www.noldus.com/</td>
		</tr>
		<tr>
			<td>Extruded aluminum</td>
			<td>80/20 Inc., Columbia City, IN, USA</td>
			<td>3030-S</td>
			<td>https://www.8020.net 3.00&#34; X 3.00&#34; Smooth T-Slotted Profile, Eight Open T-Slots</td>
		</tr>
		<tr>
			<td>Finfish Starter with Vpak, 1.5 mm extruded pellets</td>
			<td>Zeigler Bros. Inc., Gardners, PA, USA</td>
			<td></td>
			<td>http://www.zeiglerfeed.com/</td>
		</tr>
		<tr>
			<td>Golden shiners</td>
			<td>Saul Minnow Farm, AR, USA</td>
			<td></td>
			<td>http://saulminnow.com/</td>
		</tr>
		<tr>
			<td>ImageJ (v 1.52h) freeware</td>
			<td>National Institute for Health (NIH), USA</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>LED track lighting</td>
			<td>Lithonia Lightening, Conyers, GA, USA</td>
			<td>BR20MW-M4</td>
			<td>https://lithonia.acuitybrands.com/residential-track</td>
		</tr>
		<tr>
			<td>Oracle 651 white cut vinyl</td>
			<td>651Vinyl, Louisville, KY, USA</td>
			<td>651-010M-12:5ft</td>
			<td>http://www.651vinyl.com. Can order various sizes.</td>
		</tr>
		<tr>
			<td>PowerLite 570 overhead projector</td>
			<td>Epson, Long Beach CA, USA</td>
			<td>V11H605020</td>
			<td>https://epson.com/For-Work/Projectors/Classroom/PowerLite-570-XGA-3LCD-Projector/p/V11H605020</td>
		</tr>
		<tr>
			<td>Processing (v 3) freeware</td>
			<td>Processing Foundation</td>
			<td></td>
			<td>https://processing.org/</td>
		</tr>
		<tr>
			<td>R (3.5.1) freeware</td>
			<td>The R Project for Statistical Computing</td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>Ultra-white 360 theater screen</td>
			<td>Alternative Screen Solutions, Clinton, MI, USA</td>
			<td>1950</td>
			<td>https://www.gooscreen.com. Must call for special cut size</td>
		</tr>
		<tr>
			<td>Z-Hab system</td>
			<td>Pentair Aquatic Ecosystems, Apopka, FL, USA</td>
			<td></td>
			<td>https://pentairaes.com/. Call for details and sizing.</td>
		</tr>
	</tbody>
</table>

integrating visual psychophysical assays within a y-maze to isolate the role that visual features play in navigational decisions

Collective animal behavior arises from individual motivations and social interactions that are critical for individual fitness. Fish have long inspired investigations into collective motion, specifically, their ability to integrate environmental and social information across ecological contexts. This demonstration illustrates techniques used for quantifying behavioral responses of fish, in this case, Golden Shiner (Notemigonus crysoleucas), to visual stimuli using computer visualization and digital image analysis. Recent advancements in computer visualization allow for empirical testing in the lab where visual features can be controlled and finely manipulated to isolate the mechanisms of social interactions. The purpose of this method is to isolate visual features that can influence the directional decisions of the individual, whether solitary or with groups. This protocol provides specifics on the physical Y-maze domain, recording equipment, settings and calibrations of the projector and animation, experimental steps and data analyses. These techniques demonstrate that computer animation can elicit biologically-meaningful responses. Moreover, the techniques are easily adaptable to test alternative hypotheses, domains, and species for a broad range of experimental applications. The use of virtual stimuli allows for the reduction and replacement of the number of live animals required, and consequently reduces laboratory overhead.
This demonstration tests the hypothesis that small relative differences in the movement speeds (2 body lengths per second) of virtual conspecifics will improve the speed and accuracy with which shiners follow the directional cues provided by the virtual silhouettes. Results show that shiners directional decisions are significantly affected by increases in the speed of the visual cues, even in the presence of background noise (67% image coherency). In the absence of any motion cues, subjects chose their directions at random. The relationship between decision speed and cue speed was variable and increases in cue speed had a modestly disproportionate influence on directional accuracy.

Hypothesis and design 
To demonstrate the utility of this experimental system we tested the hypothesis that the accuracy with which Golden Shiner follow a visual cue will improve with the speed of that cue. Wild type Golden Shiner were used (N = 16, body lengths, BL, and wet weights, WW, were 63.4 &#177; 3.5 mm and 1.8 &#177; 0.3 g, respectfully). The coherency of the visual stimuli (leader/distractor r...

Watch this Scientific Journal Video about Integrating Visual Psychophysical Assays within a Y-Maze to Isolate the Role that Visual Features Play in Navigational Decisions at JoVE.com

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

Here, we present a protocol to demonstrate a behavioral assay that quantifies how alternative visual features, such as motion cues, influence directional decisions in fish. Representative data are presented on the speed and accuracy where Golden Shiner (Notemigonus crysoleucas) follow virtual fish movements.

Collective animal behavior arises from individual motivations and social interactions that are critical for individual fitness. Fish have long inspired ...

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.

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