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.

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All authors contributed to the experimental design, analyses and writing the paper. A.C.U. and C.M.W. setup and collected the data. The authors have nothing to disclose.

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 individuals in groups integrate neighbor movements, such as reacting proportionally to all discernible cues14, adopting a motion-threshold response17, or monitoring collision times18. A first step in testing these alternative hypotheses is validating their underlying assumptions. Here we demonstrated the utility of our protocol in identifying the role that a particular sensory feature can have on guiding directional decisions.
We isolated how individuals of a social fish species, the Golden Shiner, responded to changes in the relative speed of visual stimuli designed to mimic conspecifics in a school. Golden Shiner directional accuracy did improve with increases in the relative speed of the visual stimuli, but the functional relationship between these variables was only marginally disproportionate. The relationship between decision speed and stimulus speed, while significant, was highly variable and inconsistent. The results do demonstrate, however, that a speed difference found in images scattered across the field of view of these fish does play an important role in triggering a response and guiding their overt attention. Teasing apart how individuals select among the actions of specific neighbors could be probed with the current design by introducing conflicting directions in the stimuli.
In a recent experiment with Zebrafish, Danio rerio, we found no evidence of indecisiveness in solitary trials7, yet Golden Shiner in this demonstration displayed a greater reluctance to leave the holding area. The differences between these two species may be explained by their life history strategies and the relative strength of their social tendencies (or reliance). Zebrafish appear to display more variable social coherency than Golden Shiners (e.g., facultative vs. obligate schoolers3). It is likely that the stronger social coherency in Golden Shiner may have contributed to subjects showing higher levels of shyness, or hesitancy within the domain than their zebrafish counterparts.
The order of the steps is subtle yet critical in the protocol. The process of balancing the lights, the projector, and program filter can take more time than often anticipated for new domains. In this protocol, lessons learned have been included to reduce setup and light balance time, such as use of track lights that reflect off the wall (not on the domain), adjustable light controllers, and program-generated filters for the projector. Consider also that what may appear to be visually acceptable to the human eye will not be viewed by the camera and software the same way, thus your lighting conditions may require additional adjustments. Even slight changes in monitor angles will result in background gradient changes. Thus, detailed note taking and saving file settings will greatly reduce the likelihood of changes occurring during the experiment. Moving through the process from physical to filtering, as presented here, yields the fastest steps to success.
The use of a ST projector enables greater spatial flexibility over a monitor, but this approach creates an unwanted visual anomaly called a &#8220;hotspot&#8221;. A hotspot is a bright spot on the projection surface created by the proximity of the projector&#8217;s bulb. In the protocol, Section 4 was dedicated to the creation of background filters and checking for homogeneous lightning across the domain. The steps provided here will help users avoid, or minimize, the unwanted effects of the hotspot by modeling any unwanted gradient and using the model to reproduce an inverse gradient to counter the effects. Lastly, the ST projector model may vary, however, image adjustments (rotate, flip, front or rear screen projection) and keystone correction (&#177; 3-5 degrees) are useful features to ensure the desire image fits the domain and can be adjusted for distortion.
Over time, the experimental rooms were updated for ease by changes in the hardware (i.e., cameras, cabling, video cards, monitors). It is noteworthy to mention that hardware changes will likely result in additional start-up time to balance lighting and work through any potential program issues. Therefore, it is recommended that any hardware be dedicated to a system until completion of the desired experiments. Most challenges have been tied to performance differences between monitors, video cards and the cameras resulting sometimes in alteration of the programming code. Since the time of this work, new domains have been developed in which the inner test domain can be removed and switched for other test domains. We recommend this flexibility be considered when designing the experimental domains and support structures.
The current protocol allows investigators to isolate and manipulate visual features in a manner that both reflects the visual environment expected within a school, while also controlling for confounding factors that accompany exposure to real conspecifics (e.g., hunger, familiarity, aggression)7. In general, computer animation (CA) of virtual fish (i.e., silhouettes) is a practice that is becoming more common place due to its distinct advantages in stimulating behavioral responses19,20,21. CA allows one to customize visual cues (direction, speed, coherency, or morphology), while introducing a level of standardization and repeatability in the desired stimulus that exceeds what can be achieved when using live animals as the stimulant. The use of virtual reality in behavioral studies, on both animals22 and humans23, is also steadily increasing and promises to become a powerful empirical tool as the technology becomes more available and tractable. Taken together, these virtual approaches also replace and reduce the live animal requirements of animal ethics in science (e.g., IACUC, AAALAC, and ACURO)24, while concomitantly lowering laboratory costs and burdens.

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&quot; X 3.00&quot; 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 ratio) were fixed at 0.67, while we manipulated the speed at which our motion cues (i.e., the leaders) moved with respect to their distractors. Speed levels of the leader silhouettes that provide the directional cues ranged from 0-10 BL/s (in increments of 2), which spans the range of speeds typically considered to reflect sustained, prolonged, or burst swimming modes of activity in fish12. At the control level, 0, the leader silhouettes were oriented towards a destination arm among the randomly oriented distractors, but none of the silhouettes moved. The destination arm was chosen at random for each trial by the program. Distance units are in body length, which was defined by the mean standard length of our subjects, and time is in seconds. The current representative analysis focuses on measuring primary response variables (decision speed and accuracy), yet the design of the experiment also enables investigators to extract added information by tracking subject movements and analyzing their kinematics.
Our fish subjects were housed following section 6 of the protocol. Each subject was exposed to one level of the treatment per day. We randomized both within subject treatment level (cue speed) across days and the order in which subjects were tested on each day. Linear and generalized linear mixed effects models (LMM and GLMM, respectively) were used to test the effects of leader silhouette speed on the speed and accuracy with which subjects followed the visual stimuli. Subject id was included as the random effect in both models.
Data and findings 
In the absence of any motion cues Golden Shiner acted as expected and chose their direction at random (stimulus speed = 0, binomial test, nLeft = 33, nRight = 40, = 0.45, P = 0.483). While most subjects showed no signs of stressful behavior within the domain and made a decisive decision within the allotted time (5 min), 22% of the subjects showed a reluctance to leave the holding area or enter the decision area. Data from these indecisive fish were not included in the analysis. The remaining 78% of our subjects showed a significant improvement in the accuracy with which they followed the directional stimuli as the speed of those stimuli increased (GLMM, z = 1.937, P = 0.053). Figure 5A shows the nature of this relationship, where we find a 1.2-fold increase in directional accuracy for each increase in stimulus speed level. This relationship is only modestly disproportionate and is not, by itself, suggestive of a threshold response to changes in cue speed. Increases in stimulus speed also led to a significant increase in decision speed (LMM, F1,56 = 4.774, P = 0.033). However, as evident in Figure 5B the trend in decision speed was inconsistent and highly variable across stimulus speed levels. What is apparent in these decision speed data is that it took subjects, on average, anywhere from 5-20x longer to make their decision when the stimuli were moving than when they were not (decision speeds of 4.6 &#177; 2.3 s and 81.4 &#177; 74.7 s for stimulus speeds of 0 and 8, respectively, &#177; standard deviation, SD). Indeed, without the control level we found no significant change in decision speed as a function of stimulus speed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59281/59281fig1.jpg" /> 
Figure 1. Y-Maze domain. A. Image of the Y-maze apparatus for decision-making test. Annotations represent the following: Holding Area (HA, green), Decision Area (DA, blue), Left Decision Arm (LDA), and Right Decision Arm (RDA). B. Image of the Y-maze and room with overhead adjustable track lighting and GigE camera placement (only one of the four overheads lights strips are visible). C. Image of the Y-maze (side-view) including the projector placement which is locked by the sliding carriage to eliminate movements during, or between, trials. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59281/59281fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59281/59281fig2.jpg" /> 
Figure 2. Background and stimulus calibration. A. Image of the illuminated Y-maze with a uniform background color and a pixel intensity transect (green line) between the holding area and the Decision Area, DA (mean pixel intensity 112 &#177; 1278). The light gradient generated by the projector&#8217;s bulb (hotspot) is clearly visible. B. Image showing the alignment of the projections with the DA. C. Image of the maze with the filtered background and a solitary silhouette projected in the center of the DA for calibration (size, speed). The addition of the counter gradient background in (C) results in a darker background (mean pixel intensity 143.1 &#177; 5.5) and far less spatial variability (coefficient of variation drops from 11.4 (A.) to 0.03 (C.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59281/59281fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59281/59281fig3.jpg" /> 
Figure 3. Schematic of the general flow of operations in the visualization program used in the experiments. For additional procedural details see7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59281/59281fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59281/59281fig4.jpg" /> 
Figure 4. Experimental trial with both real and virtual fish silhouettes. A. Image a (live) Golden Shiner leaving the holding area (green circle). B. Image of a (live) Golden Shiner in the decision area (green circle) among the virtual fish silhouettes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59281/59281fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59281/59281fig5.jpg" /> 
Figure 5. Accuracy and speed of directional responses to changes in the relative speed of motion cues. A. Graph of the fish decision accuracy with which Golden Shiner followed the &#8216;leader&#8217; silhouettes plotted against the stimulus speed (BL/s). B. Graph of the fish decision speed plotted against the stimulus speed (BL/s). Data are means &#177; standard errors, SE. Groups of 15 virtual silhouettes were randomly distributed throughout the decision zone with a 67% coherency level (10 of the 15 silhouettes acted as Leaders, the remaining 5 silhouettes acted as distractors) and we varied the speed of the leaders from 0-10 BL/s. Distractor speeds remained fixed at 1 BL/s at all speed levels, except the control in which none of the silhouettes moved. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59281/59281fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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6.1K Views.  US Army Engineer Research and Development Center. Ons protocol stelt ons in staat om visuele kenmerken na te bootsen die een vrij bewegende vis kan ervaren tijdens het reizen in een groep en te kwantificeren hoe deze functies hun navigatiebeslissingen beïnvloeden. Deze techniek kan worden gebruikt om individuele niveau directionele beslissingen te koppelen in reactie op een zintuiglijke cue om te zien hoe deze beslissingen veranderen op basis van de acties van anderen. Onze methoden geven inzicht in hoe dieren hun omgeving beoordelen en nav...

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

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.&#160;bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.&#160;distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.&#160;random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.&#160;shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

The dry-land Barnes maze is widely used to measure spatial navigation ability in response to mildly aversive stimuli. Over consecutive days, performance (e.g.&#160;latency to locate escape cage) of control subjects improves, indicative of normal learning and memory. Differences between rats and mice necessitate apparatus and methodology changes that are detailed here.

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land ...

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze1,2&#160;that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.
In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal's behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.
We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Neuroscience

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a ...

Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. Decrypting the scent-trailing behavior of vertebrates, particularly invasive species, enables the discovery of cues that induce exploratory behavior and can aid in the development of valuable basic and applied biological tools. However, pinpointing behaviors dominantly driven by chemical cues versus other competing environmental cues can be challenging. Y-mazes are common tools used in animal behavior research that allow quantification of vertebrate chemosensory behavior across a range of taxa. By reducing external stimuli, Y-mazes remove confounding factors and present focal animals with a binary choice. In our Y-maze studies, a scenting animal is restricted to one arm of the maze to leave a scent trail and is removed once scent-laying parameters have been met. Then, depending on the trial type, either the focal animal is allowed into the maze, or a competing scent trail is created. The result is a record of the focal animal&#39;s choice and behavior while discriminating between the chemical cues presented. Here, two Y-maze apparatuses tailored to different invasive reptile species: Argentine black and white tegu lizards (Salvator merianae) and Burmese pythons (Python bivittatus) are described, outlining the operation and cleaning of these Y-mazes. Further, the variety of data produced, experimental drawbacks and solutions, and suggested data analysis frameworks have been summarized.

Y-mazes enable researchers to determine the relevance of specific stimuli that drive animal behavior, especially isolated chemical cues from a variety of sources. Careful design and planning can yield robust data (e.g., discrimination, degree of exploration, numerous behaviors). This experimental apparatus can provide powerful insight into behavioral and ecological questions.

Reptiles utilize a variety of environmental cues to inform and drive animal behavior such as chemical scent trails produced by food or conspecifics. ...

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze shape, thus requiring several separate mazes in different configurations. The maze geometry cannot be reconfigured in a single environment to accommodate scalability and reproducibility. The reconfigurable maze is a unique approach to address the limitations, allowing quick and flexible configurations of maze pathways in a repeatable manner. It consists of interlocking pathways and includes feeders, treadmills, movable walls, and shut-off sensors. The current protocol describes how the reconfigurable maze can replicate existing mazes, including the T-shaped, plus-shaped, W-shaped, and figure-eight mazes. Initially, the T-shaped maze was constructed inside a single experimental room, followed by modifications. The rapid and scalable protocol outlined herein demonstrates the flexibility of the reconfigurable maze, achieved through the addition of components and behavioral training phases in a stepwise manner. The reconfigurable maze systematically and precisely assesses the performance of multiple aspects of spatial navigation behavior.

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze ...

Game-Based Exploration: Unlocking Human Spatial Navigation Abilities

Assessing Human Spatial Navigation in a Virtual Space and its Sensitivity to Exercise

Spatial navigation (SN) is the ability to locomote through the environment, which requires an understanding of where one is located in time and space. This capacity is known to rely on the sequential firing of place cells within the hippocampus. SN is an important behavior to investigate as this process deteriorates with age, especially in neurodegenerative disorders. However, the investigation of SN is limited by the lack of sophisticated behavioral techniques to assess this hippocampal-dependent task. Therefore, the goal of this protocol was to develop a novel, real-world approach to studying SN in humans. Specifically, an active virtual SN task was developed using a cross-platform game engine. During the encoding phase, participants navigated their way through a virtual city to locate landmarks. During the remembering phase, participants remembered where these reward locations were and delivered items to these locations. Time to find each location was captured and episodic memory was assessed by a free recall phase, including aspects of place, order, item, and association. Movement behavior (x, y, and z coordinates) was assessed through an asset available in the game engine. Importantly, results from this task demonstrate that it accurately captures both spatial learning and memory abilities as well as episodic memory. Further, findings indicate that this task is sensitive to exercise, which improves hippocampal functioning. Overall, the findings suggest a novel way to track human hippocampal functioning over the course of time, with this behavior being sensitive to physical activity training paradigms.

Author Spotlight: Investigating the Effects of Mind-Body-Movement Practices on Brain Function

Here, we present a novel, brief, and active spatial navigation task that assesses both spatial navigation and episodic memory ability. Importantly, spatial navigation and episodic memory were associated with one another, and this task demonstrated sensitivity to exercise.

Spatial navigation (SN) is the ability to locomote through the environment, which requires an understanding of where one is located in time and space. ...

Behavioral Training Procedures for Head-fixed Virtual Reality in Mice

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays to be performed in head-fixed mice. This enables precise behavioral recordings while incorporating various neurophysiological techniques that require head-fixation to minimize movement-related signal noise during neural recordings. However, despite the growing use of VR, there is little published data on the detailed methodology of how to implement it. In this study, a training protocol is developed whereby male and female C57B16/J mice are trained to run down a virtual linear corridor, the length of which is increased from 1-3 m over multiple training sessions. Building upon this foundation, this study investigated the feasibility of mice performing complex behaviors within VR using a Y-maze paradigm. The task required navigating to the arm with black walls from the choice point in the Y-maze. After reaching a criterion of two consecutive days equal to or greater than 70% correct, the mice progressed to increasingly difficult sensory discrimination. The findings provide important details on the methodologies useful for the successful training of mice in VR and demonstrate that mice exhibit learning capabilities in navigating the Y-maze. The methodology presented not only offers insights into training duration in VR-based assays but also underscores the potential for probing intricate behaviors in mice, opening avenues for more comprehensive neuroscience investigations.

The article describes the experimental procedures for the commonly used linear track virtual reality (VR) paradigm in mice as well as determining the feasibility of running complex VR tasks by testing a Y-shaped signal discrimination task.

Virtual reality (VR) combined with head-fixation is increasingly being utilized in behavioral neuroscience studies as it allows complex behavioral assays ...

A Visuospatial Planning Task Coupled with Eye-Tracker and Electroencephalogram Systems

The planning process, characterized by the capability to formulate an organized plan to reach a goal, is essential for human goal-directed behavior. Since planning is compromised in several neuropsychiatric disorders, the implementation of proper clinical and experimental tests to examine planning is critical. Due to the nature of the deployment of planning, in which several cognitive domains participate, the assessment of planning and the design of behavioral paradigms coupled with neuroimaging methods are current challenges in cognitive neuroscience. A planning task was evaluated in combination with an electroencephalogram (EEG) system and eye movement recordings in 27 healthy adult participants. Planning can be separated into two stages: a mental planning stage in which a sequence of steps is internally represented and an execution stage in which motor action is used to achieve a previously planned goal. Our protocol included a planning task and a control task. The planning task involved solving 36 maze trials, each representing a zoo map. The task had four periods: i) planning, where the subjects were instructed to plan a path to visit the locations of four animals according to a set of rules; ii) maintenance, where the subjects had to retain the planned path in their working memory; iii) execution, where the subjects used eye movements to trace the previously planned path as indicated by the eye-tracker system; and iv) response, where the subjects reported the order of the visited animals. The control task had a similar structure, but the cognitive planning component was removed by modifying the task goal. The spatial and temporal patterns of the EEG revealed that planning induces a gradual and lasting rise in frontal-midline theta activity (FM&#952;) over time. The source of this activity was identified within the prefrontal cortex by source analyses. Our results suggested that the experimental paradigm combining EEG and eye-tracker systems was optimal for evaluating cognitive planning.

The study of cognitive planning combining EEG and eye-tracking systems provides a multimodal approach to investigate the neural mechanisms that mediate cognitive control and goal-directed behavior in humans. Here, we describe a protocol for investigating the role of brain oscillations and eye movements in planning performance.

The planning process, characterized by the capability to formulate an organized plan to reach a goal, is essential for human goal-directed behavior. Since ...

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

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