The development of the first Y-maze was supported by cooperative agreements (15-7412-1155-CA, 16-7412-1269-CA, and 17-7412-1318-CA) between James Madison University (JMU) and the USDA Animal and Plant Health Inspection Service. The development of the Y-maze in Everglades National Park was funded by a cooperative agreement (P18AC00760) between JMU and the National Park Service. We thank T. Dean and B. Falk for their facilitation of this project in Everglades NP and assistance with permitting and funding. We thank W. Kellow for assistance in construction of the USGS Y-maze. C. Romagosa, L. Bonewell, and R. Reed provided administrative and logistical support. We thank the two anonymous reviewers who offered helpful feedback. Funding for the Everglades work and in-kind support was provided by U.S. Geological Survey (USGS) Greater Everglades Priority Ecosystem Science Program, National Park Service (P18PG00352), and USGS Invasive Species Program. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. The findings and conclusions in this publication have not been formally disseminated by the U.S. Department of Agriculture and should not be construed to represent USDA determination or policy.

<ol>
	<li>Fine, J. M., Vrieze, L. A., Sorensen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+petromyzontid+lampreys+employ+a+common+migratory+pheromone+that+is+partially+comprised+of+bile+acids.">Evidence that petromyzontid lampreys employ a common migratory pheromone that is partially comprised of bile acids.</a> Journal of Chemical Ecology. 30 (11), 2091-2110 (2004).</li><li>Hesse, S., Bakker, T. C., Baldauf, S. A., Th&#252;nken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kin+recognition+by+phenotype+matching+is+family-rather+than+self-referential+in+juvenile+cichlid+fish.">Kin recognition by phenotype matching is family-rather than self-referential in juvenile cichlid fish.</a> Animal Behaviour. 84 (2), 451-457 (2012).</li><li>Forester, D. C., Wisnieski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+airborne+olfactory+cues+to+the+recognition+of+home+area+by+the+dart-poison+frog+pumilio.">The significance of airborne olfactory cues to the recognition of home area by the dart-poison frog pumilio.</a> Journal of Herpetology. 25 (4), 502-504 (1991).</li><li>Khannoon, E. R., El-Gendy, A., Hardege, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scent+marking+pheromones+in+lizards:+cholesterol+and+long+chain+alcohols+elicit+avoidance+and+aggression+in+male+Acanthodactylus+boskianus+(Squamata:+Lacertidae).">Scent marking pheromones in lizards: cholesterol and long chain alcohols elicit avoidance and aggression in male Acanthodactylus boskianus (Squamata: Lacertidae).</a> Chemoecology. 21 (3), 143-149 (2011).</li><li>Parker, M. R., Mason, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+sexy+snake:+estrogen+activation+of+female+sex+pheromone+in+male+red-sided+garter+snakes.">How to make a sexy snake: estrogen activation of female sex pheromone in male red-sided garter snakes.</a> Journal of Experimental Biology. 215 (5), 723-730 (2012).</li><li>Wyatt, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pheromones and animal behavior: chemical signals and signatures. , (2014).</li><li>Smith, T. L., Bevelander, G. S., Kardong, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+prey+odor+concentration+on+the+poststrike+trailing+behavior+of+the+Northern+Pacific+Rattlesnake.">Influence of prey odor concentration on the poststrike trailing behavior of the Northern Pacific Rattlesnake.</a> Herpetologica. 61 (2), 111-115 (2005).</li><li>Yosuke, K., Akira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+foraging+for+toxic+prey+during+gestation+in+a+snake+with+maternal+provisioning+of+sequestered+chemical+defences.">Active foraging for toxic prey during gestation in a snake with maternal provisioning of sequestered chemical defences.</a> Proceedings of the Royal Society B: Biological Sciences. 282, 20142137 (2015).</li><li>Parker, M. R., Kardong, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+airborne+and+substrate+cues+from+non-envenomated+mice+during+rattlesnake+(Crotalus+oreganus)+post-strike+trailing.">The role of airborne and substrate cues from non-envenomated mice during rattlesnake (Crotalus oreganus) post-strike trailing.</a> Herpetologica. 61 (4), 349-356 (2006).</li><li>Bezzina, C. N., Amiel, J. J., Shine, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+invasion+success+reflect+superior+cognitive+ability?+A+case+study+of+two+congeneric+lizard+species+(Lampropholis,+Scincidae).">Does invasion success reflect superior cognitive ability? A case study of two congeneric lizard species (Lampropholis, Scincidae).</a> PLoS ONE. 9 (1), 86271 (2014).</li><li>Mason, R. T., Parker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+behavior+and+pheromonal+communication+in+reptiles.">Social behavior and pheromonal communication in reptiles.</a> Journal of Comparative Physiology A. 196 (10), 729-749 (2010).</li><li>Parker, M. R., Mason, R. T., Aldridge, R. D., Sever, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromones+in+snakes:+history,+patterns+and+future+research+directions.">Pheromones in snakes: history, patterns and future research directions.</a> Reproductive Biology and Phylogeny of Snakes. , 563-584 (2011).</li><li>Greene, M. J., Stark, S. L., Mason, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+trailing+behavior+of+the+brown+tree+snake,+irregularis.">Pheromone trailing behavior of the brown tree snake, irregularis.</a> Journal of Chemical Ecology. 27 (11), 2193-2201 (2001).</li><li>Richard, S. A., Tillman, E. A., Humphrey, J. S., Avery, M. L., Parker, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+Burmese+pythons+follow+female+scent+trails+and+show+sex-specific+behaviors.">Male Burmese pythons follow female scent trails and show sex-specific behaviors.</a> Integrative Zoology. 14 (5), 460-469 (2019).</li><li>Richard, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conspecific+chemical+cues+facilitate+mate+trailing+by+invasive+Argentine+black+and+white+tegus.">Conspecific chemical cues facilitate mate trailing by invasive Argentine black and white tegus.</a> PLoS ONE. 15 (8), 0236660 (2020).</li><li>Parker, M. R., Kardong, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+chemical+information+and+context-dependent+post-strike+foraging+behavior+in+Pacific+Rattlesnakes+(Crotalus+oreganus).">Airborne chemical information and context-dependent post-strike foraging behavior in Pacific Rattlesnakes (Crotalus oreganus).</a> Copeia. 105 (4), 649-656 (2017).</li><li>Lutterschmidt, D. I., Maine, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+or+candy?+Neuroendocrine+regulation+of+the+seasonal+transition+from+courtship+to+feeding+behavior+in+male+red-sided+garter+snakes+(Thamnophis+sirtalis+parietalis).">Sex or candy? Neuroendocrine regulation of the seasonal transition from courtship to feeding behavior in male red-sided garter snakes (Thamnophis sirtalis parietalis).</a> Hormones and Behavior. 66 (1), 120-134 (2014).</li><li>Burghardt, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives-minimizing+observer+bias+in+behavioral+studies:+a+review+and+recommendations.">Perspectives-minimizing observer bias in behavioral studies: a review and recommendations.</a> Ethology. 118 (6), 511-517 (2012).</li><li>Holman, L., Head, M. L., Lanfear, R., Jennions, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+experimental+bias+in+the+life+sciences:+why+we+need+blind+data+recording.">Evidence of experimental bias in the life sciences: why we need blind data recording.</a> PLoS Biology. 13 (7), 1002190 (2015).</li><li>Mason, R. T., Gans, C., Crews, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reptilian+pheromones.">Reptilian pheromones.</a> Biology of the Reptilia: Hormones, brain, and behavior. 18, 114 (1992).</li><li>Mason, R. T., Chivers, D. P., Mathis, A., Blaustein, A. R., Haynes, K. F., Millar, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioassay+methods+for+amphibians+and+reptiles.">Bioassay methods for amphibians and reptiles.</a> Methods in Chemical Ecology. 2, 271-325 (1998).</li><li>Mart&#237;n, J., L&#243;pez, P., Rheubert, J. L., Siegel, D. S., Trauth, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromones+and+chemical+communication+in+lizards.">Pheromones and chemical communication in lizards.</a> Reproductive biology and phylogeny of lizards and tuatara. , 43-77 (2014).</li><li>Smith, K. P., Parker, M. R., Bien, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+variation+in+prey+odor+responses+in+northern+pine+snake+neonates+and+adults.">Behavioral variation in prey odor responses in northern pine snake neonates and adults.</a> Chemoecology. 25 (5), 233-242 (2015).</li><li>Parker, M. R., Patel, S. M., Zachry, J. E., Kimball, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feminization+of+male+Brown+Treesnake+methyl+ketone+expression+via+steroid+hormone+manipulation.">Feminization of male Brown Treesnake methyl ketone expression via steroid hormone manipulation.</a> Journal of Chemical Ecology. 44 (2), 189-197 (2018).</li><li>Cooper, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+swab+and+related+tests+as+a+bioassay+for+assessing+responses+by+squamate+reptiles+to+chemical+stimuli.">Evaluation of swab and related tests as a bioassay for assessing responses by squamate reptiles to chemical stimuli.</a> Journal of Chemical Ecology. 24 (5), 841-866 (1998).</li><li>Goetz, S. M., Godwin, J. C., Hoffman, M., Antonio, F., Steen, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eastern+indigo+snakes+exhibit+an+innate+response+to+pit+viper+scent+and+an+ontogenetic+shift+in+their+response+to+mouse+scent.">Eastern indigo snakes exhibit an innate response to pit viper scent and an ontogenetic shift in their response to mouse scent.</a> Herpetologica. 74 (2), 152-158 (2018).</li><li>Clark, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timber+rattlesnakes+(Crotalus+horridus)+use+chemical+cues+to+select+ambush+sites.">Timber rattlesnakes (Crotalus horridus) use chemical cues to select ambush sites.</a> Journal of Chemical Ecology. 30 (3), 607-617 (2004).</li><li>Mart&#237;n, J., L&#243;pez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supplementation+of+male+pheromone+on+rock+substrates+attracts+female+rock+lizards+to+the+territories+of+males:+a+field+experiment.">Supplementation of male pheromone on rock substrates attracts female rock lizards to the territories of males: a field experiment.</a> PLoS ONE. 7 (1), 30108 (2012).</li><li>Downes, S., Shine, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sedentary+snakes+and+gullible+geckos:+predator-prey+coevolution+in+nocturnal+rock-dwelling+reptiles.">Sedentary snakes and gullible geckos: predator-prey coevolution in nocturnal rock-dwelling reptiles.</a> Animal Behaviour. 55 (5), 1373-1385 (1998).</li><li>Parker, M. R., Kardong, K. V., LeMaster, M. P., Mason, R. T., Muller-Schwarze, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rattlesnakes+can+use+airborne+cues+during+post-strike+prey+relocation.">Rattlesnakes can use airborne cues during post-strike prey relocation.</a> Chemical Signals in Vertebrates 10. , 397-402 (2005).</li><li>Parker, M. R., Young, B. A., Kardong, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forked+tongue+and+edge+detection+in+snakes+(Crotalus+oreganus):+an+experimental+test.">The forked tongue and edge detection in snakes (Crotalus oreganus): an experimental test.</a> Journal of Comparative Psychology. 122 (1), 35-40 (2008).</li><li>Greggor, A. L., Thornton, A., Clayton, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neophobia+is+not+only+avoidance:+improving+neophobia+tests+by+combining+cognition+and+ecology.">Neophobia is not only avoidance: improving neophobia tests by combining cognition and ecology.</a> Current Opinion in Behavioral Sciences. 6, 82-89 (2015).</li><li>Candler, S., Bernal, X. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+neophobia+between+cane+toads+from+introduced+and+native+populations.">Differences in neophobia between cane toads from introduced and native populations.</a> Behavioral Ecology. 26 (1), 97-104 (2015).</li><li>Currylow, A. F., Louis, E. E., Crocker, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+response+to+handling+is+short+lived+but+may+reflect+personalities+in+a+wild,+Critically+Endangered+tortoise+species.">Stress response to handling is short lived but may reflect personalities in a wild, Critically Endangered tortoise species.</a> Conservation Physiology. 5 (1), (2017).</li><li>Currylow, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+ecophysiology+of+a+critically+endangered+(CR)+ectotherm:+Implications+for+conservation+management.">Comparative ecophysiology of a critically endangered (CR) ectotherm: Implications for conservation management.</a> PLoS ONE. 12 (8), 0182004 (2017).</li></ol>

While Y-mazes are very powerful tools to investigate chemical ecology in reptiles, their limited design can preclude other avenues of inquiry. However, a diversity of other options is available11,12,20,21,22. For example, tongue-flick assays are simpler to execute and allow simultaneous assessment of behaviors exhibited to an array of chemical stimuli relative to control odors23,24,25,26. Open-field tests are another option where a focal animal freely explores an enclosure until it encounters a source of chemical cues, and its behavioral reactions are subsequently scored27,28. Combinations of these approaches can assess discriminatory capacities of reptiles in varying contexts such as presenting a mix of artificial and natural odors along with refugia29. Y-mazes can also be modified to expose animals to airborne chemical cues alone or in combination with substrate-borne cues16,30, and&#160;post hoc&#160;inference can be used to redesign data collection if archived video data are available31. Bioassays should be designed to simplify data collection and minimize conflicting stimuli, especially when a specific source of cues is being assessed (e.g., chemical cues21).
Researchers in animal behavior often observe and quantify focal animal responses in novel, artificial environments (e.g., an enclosed maze with a featureless landscape), and care should be taken to assess whether a given animal is exhibiting natural, exploratory behavior versus avoidance, agitation, or similar distressed behavior. Distressed animal behavior in experimental apparatuses is primarily attributed to neophobia: fear of novelty32. An example is escape behavior, where the focal animal pushes against the joints or the edges of the apparatus to achieve egress. Another example is shyness, where the focal animal demonstrates reluctance to enter the maze, the degree of which can be quantified by latency of maze entry. Apparatus (re)design can facilitate engagement of the focal animal to avoid these confounding effects of distress. The most common approach is repeated introduction of the focal animal to the apparatus to remove the novelty of the environment before testing begins, and contemporary statistical models (e.g., generalized linear mixed models) allow for test animals to be used in multiple trials. An important aside relevant to ecological considerations in behavioral testing is that reduced neophobia is associated with the success of invasive species33. Thus, depending on&#160;a priori&#160;knowledge of the species in question, neophobia may have variable importance as an experimental design consideration.
Acquisition of behavioral data from videos imposes multiple constraints that become major bottlenecks in experimental timelines. For example, the length of a given trial can exponentially increase data extraction time. One workaround is to analyze behavior only until a threshold is met (e.g., total time active). The threshold can be based on the longest video available for a given trial. Alternatively, machine-based observation (e.g., artificial intelligence) can be developed, although this is time- and resource-consuming with considerable effort required for quality control. Another issue is data management: videos must be of sufficient quality to enable behavioral scoring and assessment, resulting in data storage constraints. While cloud storage is now accessible, upload/download rates are often problematic, especially when data acquisition occurs in remote field locations. Additional challenges manifest in the limitations of recording tools that affect the integrity of behavioral observation. Clear viewing of focal animal behavior is always necessary, but visibility is often impeded by uncontrollable factors (e.g., moisture, insects, wind movement). Further, when recordings come from a single perspective (e.g., bird&#39;s eye view), behaviors occurring in the vertical plane (e.g., head raises14) are difficult to assess. A solution is to provide multiple camera angles per trial. Lastly, the time of day significantly affects behavioral recording. Nighttime behavioral analysis requires a camera with a nighttime mode and minimal light projection to avoid obstructive glare on the Y-maze surface or attraction of insects that can interrupt the camera feed. Considering the above, foreknowledge of the study site or species biology can inform which constraints are likely to occur with what frequency and thus inform desirable sample sizes.
Behavior is tightly coupled with physiology, and the utility of Y-mazes for evaluation of behavioral endocrinology in a variety of species has been demonstrated. However, this paper emphasizes some variation in the execution of these experiments depending on the target species, research question, and resources available. Therefore, the selection of materials and dimensions of each testing setup should be carefully considered for potential subsequent research expansion. Section 2 describes modifications made to materials outlined in section 1, which were incorporated to accommodate future, more complex behavioral trials with tegus. The increased vertical depth of the Everglades mazes will allow new questions about chemical ecology in wild-caught tegus to be answered without unduly protracting project design and setup, further demonstrating the translatability of this experimental apparatus.
When employing the above-described techniques in a relatively remote setting (see section 2), there are several limiting factors that must be considered, and project planning is paramount. Depending on the statistical power needed for the prescribed treatment experiment and biological timing of the target species (e.g., seasonality), the resources and labor required will be affected. Further, if single or repeated use of focal animals are desired, careful attention to reducing potential stressors is necessary. Each of these factors will either extend the project timeline or require increased labor, space, and materials. For example, section 2 presents the use of wild-caught male pythons as focal animals trailing another group of wild-caught and hormonally manipulated males, all of which require approximately 24 h of quiet acclimation time in holding boxes to minimize stress effects. Although these acclimation periods extended trial times to over two days, stress due to captivity and handling affect wild animal behavior and must be minimized to generate clean datasets34,35.
In summary, Y-mazes are powerful, adaptable tools that can be used to investigate the chemical ecology of diverse wildlife under widely variable conditions, provided there is vigilant&#160;a priori&#160;planning. Careful consideration must be taken to choose appropriate questions and to properly design the experimental setup for given taxa and conditions. Researchers and managers can significantly benefit from using Y-mazes to better understand animal chemosensory biology as these tools enable flexible experimental designs that provide large volumes of fine-scale behavioral data, especially when combined with remote monitoring tools.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61858">Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles</a>. An author name was updated.
The name of the tenth author was updated from:
Amy Y. Yackel Adams
to
Amy A. Yackel Adams

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61858">hUsing Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles</a>. An author name was updated.

Erratum: Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Y-mazes are common, simple tools in studies of animal behavior that allow for a variety of questions to be addressed. In addition to being widely used in laboratory studies, Y-mazes are also functionally compatible with various field environments to study wild animals in relatively remote settings. Researchers have examined the behaviors of wild vertebrates using Y-mazes in a wide variety of taxa across similarly diverse field applications (e.g., lampreys1; cichlid fish2; poison frogs3; lacertid lizards4; garter snakes5).
Many researchers are focused on how and to what degree chemical cues drive animal behaviors in reproductive, spatial, and foraging ecology6. A variety of chemical stimuli can be tested in Y-mazes and at fine scales, such as two chemical trails that only differ slightly in concentration7, or detection ability based on the reproductive status of the target species8. Chemical trails&#8212;the principal stimulus used in Y-maze tests&#8212;can be naturally created by conspecifics or specifically placed in the environment by a researcher using a defined chemical source1,5. Stimuli can also be tested in unique combinations to determine multimodal influence of cues such as changing contexts of cue presentation (airborne vs. substrate trails9; visual plus chemical cues10). Although there are many other methods for assessing chemosensory responses in reptiles (see discussion section), Y-mazes allow for searching behavior(s) to be assessed and at multiple temporal and spatial scales, which can lead to greater levels of behavioral inference.
Reptiles have been broadly tested for their reliance on chemical cues in reproductive and foraging ecology, and researchers often employ Y-mazes in these studies11,12. The chemical ecology of reptiles continues to be deciphered by studies employing Y-mazes to address a variety of evolutionary and behavioral questions that are valuable to wildlife managers. For example, recent tests with invasive snake and lizard species have revealed that chemical cues alone can influence choice and time allocation within the novel environment of a Y-maze13,14,15.
The use of large Y-mazes for moderately sized focal animals (e.g., large-bodied reptiles) is generally restricted to laboratory settings where the focal animals can be housed easily over the long term, experimental factors (e.g., climate, light, external stimuli) can be controlled, and access to infrastructure (e.g., power, running water) is unlimited. Studies on wild animals, however, are often restricted to specific locations for various reasons (e.g., logistics, permitting). As a result, challenges arise that must be addressed through creative problem solving and methodological adjustments to maintain consistent and comparable results.
Here, two experimental setups have been described using Y-mazes and remote monitoring tools to assess reproductive chemical ecology of invasive squamate reptiles (i.e., snakes and lizards) in different field scenarios: wild-caught, captive Argentine black and white tegu lizards (Salvator merianae) in Gainesville, FL, and wild-caught Burmese pythons (Python bivittatus) in Everglades National Park, FL. As implied by its name, the Y-maze apparatus creates an experimental environment in which an animal enters a main passageway (the base of the Y; &#34;base&#34;) which then leads to two divergent passageways (the arms of the Y; &#34;arms&#34;). In these experiments, two types of animals are used for a single trial: scent-laying animals (provide the stimulus scent in a restricted area of the maze) and focal animals (data are collected on this animal as it explores the scent trail).
As an experimental apparatus in chemoecological studies, any Y-maze must be constructed in a way that allows easy removal of the animal within and can be dissembled for thorough cleaning and reset. Also discussed are the constraints inherent to these different testing environments (e.g., diurnal vs. nocturnal animals, infrastructure differences) that prompted methodological adjustments. Although the focus was on tegu lizards and Burmese pythons, these designs can be applied to a wide range of reptile species. In this research on invasive reptiles, Y-mazes benefit the rate and scale of inference because they enable rapid collection of data to inform management goals that shift in-step with the invasion threat posed by a given species. In particular, studying chemoecology of invasive species is critical for the development of effective chemical control tools.
Discrimination is the key observation from empirical tests using Y-mazes where a focal animal chooses between two stimuli and that decision-making process is assessed. A swath of behaviors can also be scored in Y-maze trials during the trial itself (live) or after the trial (video) to expand inferential power. The complexity of the&#160;a priori&#160;objectives of a given study dictate whether live observation or archived recordings best suit the design. Here, Y-maze methods have been described in detail for addressing chemoecological questions to inform future studies by researchers interested in similar questions on reptile behavior, especially in chemical ecology.

<table><tbody><tr><td>1&quot; Steel zinc-plated corner brace</td><td>Everbilt, The Home Depot</td><td>13619</td><td>See Supplemental File 1, Step 2.1 &quot;90 degree 2.5 cm steel corner brace&quot;</td></tr><tr><td>121.92cm W x 304.8cm&amp;nbsp; L x 1.27cm H white polypropylene Extended Range High-Heat UHMW Sheet</td><td>TIVAR</td><td>UHMNV SH</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.1. &quot;white polpropylene&quot;)</td></tr><tr><td>182.88 cm L x 81.28 cm W x 0.64 cm Thick Clear Acrylic Sheet</td><td>Plexiglass</td><td>32032550912090</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.1.6. &quot;Acrylic pieces&quot;)</td></tr><tr><td>2.54 cm W x 2.54 cm H x 243.84 cm L Mill-Finished Aluminum Solid Angle</td><td>Steelworks</td><td>11354</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.1.1. &quot;aluminum angle bracket&quot;)</td></tr><tr><td>4.5 kg spool of 5 mm Round Polypropylene Welding Rods</td><td>HotAirTools</td><td>AS-PP5N10</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.1. &quot;heat weld&quot;)</td></tr><tr><td>5 mm Plain Aluminum Rivets</td><td>Arrow</td><td>RLA3/16IP</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.1.1. &quot;rivet&quot;)</td></tr><tr><td>Aluminum angle, 1.9 cm</td><td>Everbilt, The Home Depot</td><td>802527</td><td>See Supplemental File 1, Step 1.2 &quot;aluminum angle (1.9 cm x 1.9 cm x 0.16 cm thick)&quot;</td></tr><tr><td>Aluminum angle, 2.5 cm</td><td>Everbilt, The Home Depot</td><td>800057</td><td>See Supplemental File 1, Steps 1.2 and 2.2.2 &quot;aluminum angle (2.5 cm x 2.5 cm x 0.16 cm thick)&quot;</td></tr><tr><td>Aluminum angle, 3.2 cm</td><td>Everbilt, The Home Depot</td><td>800037</td><td>See Supplemental File 1, Step 1.2 &quot;aluminum angle (3.2 cm x 3.2 cm x 0.16 cm thick)&quot;</td></tr><tr><td>Aluminum flat bar 1&quot; x 1/8&quot; thick</td><td>Everbilt, The Home Depot</td><td>801927</td><td>See Supplemental File 1, Step 3.2.1 &quot;aluminum strap&quot;</td></tr><tr><td>Avigilon 2.0 MP camera</td><td>Avigilon, a Motorola Solutions Company</td><td>2.0C-H4SL-BO1-IR</td><td>See &quot;1.5 Camera set-up and video acquisition&quot; (step 1.5.1 &quot;Avigilon 2.0 MP&quot;)</td></tr><tr><td>Avigilon NVR</td><td>Avigilon, a Motorola Solutions Company</td><td>HD-NVR3-VAL-6TB-NA</td><td>See &quot;1.5 Camera set-up and video acquisition&quot; (step 1.5.3 &quot;NVR&quot;)</td></tr><tr><td>Clear acrylic sheet (5.6 mm thick)</td><td>United States Plastic Corp.</td><td>44363</td><td>See Supplemental File 1, Step 1.3 &quot;clear acrylic sheet&quot; and step 3.2.1 &quot;clear acrylic door&quot;</td></tr><tr><td>Fillet Weld Nozzle 3/16&quot; x 15/32&quot; / 4.5 x 12 mm</td><td>TRIAC</td><td>107.139</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.1. &quot;heat weld&quot;)</td></tr><tr><td>Hanging File Folder Box</td><td>Sterilite</td><td>18689004</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.2.1. &quot;Boxes&quot;)</td></tr><tr><td>HardiePanel HZ10</td><td>James Hardie Building Products</td><td>9000525</td><td>See Supplemental File 1, Step 1.1 &quot;fiber cement siding&quot;</td></tr><tr><td>Heat Welding Gun</td><td>TRIAC</td><td>141.227</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.1. &quot;heat weld&quot;)</td></tr><tr><td>Kraft Butcher Paper Roll, 24&quot;</td><td>Bryco Goods</td><td>24 inch x 175 FT</td><td>See &quot;1.2 Protocol for running scent-laying tegus&quot; (step 1.2.1.2 &quot;butcher paper&quot;)</td></tr><tr><td>Kraft Butcher Paper Roll, 46 cm wide</td><td>Bryco Goods</td><td>BGKW2100</td><td>See &quot;2.3. Protocol for running scent-laying pythons&quot; (step 2.3.4. &quot;scenting paper&quot;)</td></tr><tr><td>Micro-90 Concentrated Cleaning Solution&amp;nbsp;</td><td>International Products Corporation</td><td>M-9050-12</td><td>See &quot;1.4 Breakdown and clean-up&quot; (step 1.4.4 &quot;laboratory-grade soap&quot;)</td></tr><tr><td>MKV ToolNix - Matroska tools for linux/Unix and Windows</td><td>Moritz Bunkus</td><td>v.48.0.0</td><td>See &quot;2.2. Camera setup and video acquisition&quot; (step 2.2.4.2. &quot;movie processing software&quot;)</td></tr><tr><td>Network Camera</td><td>Axis Communications</td><td>M3104-LVE</td><td>See &quot;2.2. Camera setup and video acquisition&quot; (step 2.2.1. &quot;Project camera&quot;)</td></tr><tr><td>Palight ProjectPVC 1/4&quot;</td><td>Palram</td><td>159841</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.2.3. &quot;faceplate&quot;)</td></tr><tr><td>Palight ProjectPVC 1/8&quot;</td><td>Palram</td><td>156249</td><td>See &quot;2.1. Y-maze components and rationale for changes to USDA design &quot; (step 2.1.2.1. &quot;door&quot;)</td></tr><tr><td>Privacy windscreen (green)</td><td>MacGregor</td><td>Size to fit</td><td>See Supplemental File 1, Step 4.2 &quot;green heavy duty shade cloth&quot;</td></tr><tr><td>Protective Glove, Full-Finger</td><td>ArmOR Hand</td><td>HS1010-RGXL</td><td>See &quot;2.3. Protocol for running scent-laying pythons&quot; (step 2.3.11.2. NOTE: &quot;puncture-resistant glove&quot;)</td></tr><tr><td>REScue Disinfectant</td><td>Virox Animal Health</td><td>44176</td><td>See &quot;1.5. Breakdown and clean-up.&quot; (step 1.5.4. NOTE &quot;sanitation solution&quot;)</td></tr><tr><td>Reversable PVC trim, 1/2&quot; x 24&quot;</td><td>UFP Industries, Veranda products</td><td>H120XWS17</td><td>See Supplemental File 1, Step 2.1 &quot;PVC board partition&quot;, and step 3.2.1 &quot;thinner PVC trim boards&quot;</td></tr><tr><td>S4S / Veranda HP TRIM</td><td>UFP Industries, Veranda products</td><td>H190OWS4</td><td>See Supplemental File 1, Steps 1.2, 2.2.2, and 2.2.3 &quot;PVC board&quot;</td></tr><tr><td>S4S / Veranda HP TRIM (1&quot; x 8&quot; Nominal)</td><td>UFP Industries, Veranda products</td><td>827000005</td><td>See Supplemental File 1, Steps 3.2.1 &quot;PVC trim board&quot;</td></tr><tr><td>ScotchBlue 24 in. Pre-taped Painter&amp;rsquo;s Plastic</td><td>3M</td><td>PTD2093EL-24-S</td><td>See &quot;1.2 Protocol for running scent-laying tegus&quot; (step 1.2.1.3 &quot;plastic sheeting&quot;)</td></tr><tr><td>Sterilite 114 L tote box</td><td>Sterilite Company</td><td>1919, Steel</td><td>See Supplemental File 1, Step 3.2 &quot;arm box&quot;</td></tr><tr><td>Sterilite 189 L tote box</td><td>Sterilite Company</td><td>1849, Titanium</td><td>See Supplemental File 1, Step 3.2 &quot;Base box&quot;</td></tr><tr><td>Super Max Canopy</td><td>ShelterLogic</td><td>25773</td><td>See Supplemental File 1, Step 4.3 &quot;white canopy&quot;</td></tr><tr><td>VLC Media Player&amp;nbsp;</td><td>VideoLAN</td><td>v.3.0.11</td><td>See &quot;2.2. Camera setup and video acquisition&quot; (step 2.2.4.3. &quot;media file reviewing program&quot;)</td></tr><tr><td>White Pavilion Tent</td><td>King Canopy</td><td>BJ2PC</td><td>See Supplimental File 2 &quot;3. Enclosure materials and consideratons&quot; (step 3. &quot;pavilion tent&quot;)</td></tr></tbody></table>

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.

A multitude of variables can be recorded and/or scored from Y-maze trials. The design of the study should be primarily driven by the desired outcomes/deliverables. Further, if the study is relying on repeated measures (e.g., repeated use of the same focal animals), proper testing and analysis structures are required. For example, as the USDA trials relied on repeated testing of focal tegus, the planning of experimental trials was fully randomized.
Choice data: The majority of studies using Y-mazes report simple binary choice data and analyze the results with parametric statistics such as a binomial test. The chief limitation here is sample size, which directly affects the power of any statistical analysis. In&#160;Figure 2, a series of statistical thresholds per study sample size are depicted that demonstrate how many &#34;successes&#34; would need to occur for a given binomial test to yield statistically significant results. These are mathematically derived and therefore generalizable to any Y-maze test. Binomial statistics are easy to generate using online freeware. For calculating probabilities, one-tail distributions are used if an&#160;a priori&#160;rationale is given; otherwise, the two-tail distribution should be used.
Choice of an arm is often determined by the distance the focal animal moves in a given arm. The simplest way to set this threshold is by establishing a landmark within the maze. For most Y-maze studies, the landmark is the entrance of the arm box. Because reptiles conduct all chemosensory assessment with the chemical-sensing organs in the anterior region of the head, the head is the focal point during a trial. For example, because Burmese pythons are often longer than the entire maze itself, choice is best and most efficiently determined by the movement of the head past a landmark. Other options for determining choice are time spent in an arm and complete movement of the focal animal into a box. Failure is determined by a focal animal not making a choice within a specific period.
More fine resolution analyses can be derived from choice data in the Y-maze. For example, researchers can generate a choice penalty score16.&#160;Here, researchers must track the degree to which the focal animal explored the non-target arm of the maze. Non-target can be defined as the arm the researchers determine&#160;a priori&#160;that the focal animal will not choose based on the alternative hypothesis tested. The simplest example of a non-target arm would be the unscented arm when only one arm contains a target scent. More complex examples would be the choice between two scents from the same source, but presented at different concentrations7. When the experimental design is multi-level and/or the data go from binary to incremental, as with choice penalty, an appropriate statistical approach should be used such as repeated measures analysis of variance (ANOVA) or other methods used with continuous or proportional datasets.
Behaviors: Throughout the duration of an experiment in which focal animals are observed, a variety of individual behaviors can be quantified. This number of variables can either be determined&#160;a priori&#160;depending on what is known16&#160;or&#160;post hoc&#160;following preliminary observations on a subset of data14,15. The study objectives and their degree of resolution determine what behavioral assessments should be made within the maze, if any (i.e., in many studies, only choice data are quantified17). Behaviors can be assessed throughout the maze, in sections, or during specific time periods; for instance, behaviors seen only in the base or at the junction of the arms may be prioritized8. Video recordings facilitate behavioral scoring, although the resolution of the video and its length&#8212;factors that impose data storage constraints&#8212;should be considered before experimentation begins.
Temporal variables: As with behavioral variables, many temporal aspects of animal performance can be quantified during Y-maze trials. For example, researchers can time latency periods (e.g., latency to emerge from the box8). Most temporal variables are associated with exploration of the maze such as total trailing time or time spent in each arm. These variables are usually analyzed in a multi-factor analysis such as multi-way ANOVA.
Observer bias: With any studies involving animal behavior, observer bias significantly influences data collection18. Therefore, observers should be blind to the treatment being tested. The simplest way to do this is to code the video files numerically and then randomly sort them (e.g., random number generator) prior to assigning them to observers. Controlling for observer bias is difficult-to-impossible when live data collection is the only option. In a field setting, this would require two cooperators: an observer blind to the treatment and a coordinator who sets up the trial. Extensive reviews summarize the effects of experimenter bias on data collection and interpretation in behavioral and ecological studies18,19.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61858/61858fig02.jpg" /> 
Figure 2. Sample sizes and P-values for binomial tests from Y-maze results. Each given sample size represents a set number of trials where a scent is tested in one arm of the Y (target arm) while the other could be a control (non-target). Top number above each bar is the one-tail P-value for that number of target arm choices, bottom is two-tail. Numbers within the top bar represent the maximum number of non-target choices that are still traditionally statistically significant (P &#60; 0.05).&#160;<a href="https://www.jove.com/files/ftp_upload/61858/61858fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1.&#160;<a href="https://www.jove.com/files/ftp_upload/61858/ParkerCurrylowetal-JoVE-R1-supp1.docx" style="font-size: 14px;" target="_blank">Please click here to download this file.</a>
Supplemental File 2.&#160;<a href="https://www.jove.com/files/ftp_upload/61858/ParkerCurrylowetal-JoVE-R1-supp2.docx" style="font-size: 14px;" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles at JoVE.com

Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

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. ...

using-enclosed-y-mazes-to-assess-chemosensory-behavior-in-reptiles

Research

JoVE Journal

Behavior

5.5K Views.  James Madison University. 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.

Video: Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

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 Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

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 ...

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.

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 ...

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, various technologies are being developed as control tools. A subset of these tools focuses on modifying the behavior of arthropods by attraction or repulsion. Therefore, arthropods are often the focus of behavioral investigations. Various tools have been developed to measure arthropod behavior, including wind tunnels, flight mills, servospheres, and various types of olfactometers. The purpose of these tools is to measure insect or arachnid response to visual or more often olfactory cues. The vertical T-maze oflactometer described here measures choices performed by insects in response to attractants or repellents. It is a high throughput assay device that takes advantage of the positive phototaxis (attraction to light) and negative geotaxis (tendency to walk or fly upward) exhibited by many arthropods. The olfactometer consists of a 30 cm glass tube that is divided in half with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the test subjects to make a choice between two potential odor fields in assays involving attractants. In assays involving repellents, lack of normal response to known attractants can also be measured as a third variable.

A vertical, T-maze olfactometer is described for assaying the behavioral response of arthropods. The olfactometer allows the experimenter to measure choices performed by test subjects when subjected to two potential odor fields. Both attraction to and repulsion from odorants can be measured with this device.

Given the economic importance of insects and arachnids as pests of agricultural crops, urban environments or as vectors of plant and human diseases, ...

Neuroscience

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

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 ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

Spatial Memory Testing Using Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this phenomenon in rodents using different types of mazes like Morris water maze, Radial water maze, and Barnes maze. By investigating spatial memory in rodents, neurobehavioral scientists can gain valuable understanding of how these processes are altered following brain damage in humans.
Here, the neurobiology behind spatial memory is briefly reviewed and some general principles behind maze tests are discussed. Then, the video explains generalized protocols on how to conduct trials with specific types of mazes. Lastly, it will explain how behavioral researchers are using these tools to conduct some specific experiments.

Spatial Memory Tests Using Morris Water, Radial Water &amp; Barnes Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this ...

Education

JoVE Science Education

Psychology

Behavioral Science

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Source: Campbell, J. C., et al. <a href="https://www.jove.com/video/55939/a-caenorhabditis-elegans-nutritional-status-based-copper-aversion">A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay.</a> J. Vis. Exp. (2017).
This video introduces chemotaxis in the nematode C. elegans and shows a sample protocol testing chemotactic aversion to copper sulfate.

Source: Campbell, J. C., et al. A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay. J. Vis. Exp. (2017).
This video introduces ...

Using Enclosed Y-Mazes to Assess Chemosensory Behavior in Reptiles

Summary

Explore More Videos

Chapters in this video

Barnes Maze Testing Strategies with Small and Large Rodent Models

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

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

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

Vertical T-maze Choice Assay for Arthropod Response to Odorants

Testing Drosophila Olfaction with a Y-maze Assay

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

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Spatial Memory Testing Using Mazes

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms