Thanks to all who joined our Spring webinar: Controversies of the Elevated Plus Maze
If you missed it, check out the video recording below.
In this webinar, we discuss topics related to testing environment, methodology, and more - especially 'controversial' subjects - meaning areas where there is poor standardization across labs or where researchers have differing opinions on how the test should be run.
Presenters: Chloe Christoforou, M.S., Director of Research & Innovation at BehaviorCloud and Tracy Bedrosian, Ph.D., Co-Founder at BehaviorCloud
TRANSCRIPT (edited slightly for clarity)
CHLOE: Hi Everyone! It's 2:05 pm ET and we're ready to start. We want to welcome you to our webinar, Controversies of the Elevated Plus Maze. Thank you for attending! You might wonder why we wanted to talk about the EPM. Well at BehaviorCloud we provide tools to human and animal researchers who study behavior. We specifically offer a cloud platform that has everything you need to collect, analyze, and share your behavioral data. During the course of the past year, we've seen more and more studies testing anxiety and depression for things like PTSD and traumatic brain injury. So we wanted to host a webinar that would help researchers who are either entering behavioral research or who are wanting to optimize their current set-up.
A little about us: My name is Chloe and I'm your account manager at BehaviorCloud. Feel free to contact me with questions. I have a background in neuroscience studying blast traumatic brain injury at Walter Reed Army Research Institute where we used the elevated plus maze to evaluate our model. I've also helped hundreds of researchers optimize their behavioral labs through consulting, training, and support. And my colleague Tracy is joining us today. She's a co-founder of BehaviorCloud and offers a wealth of animal behavior expertise, particularly in mood disorders. She published multiple peer-reviewed articles and book chapters during her PhD at Ohio State University and her postdoc at the Salk Institute involving behavioral tests including the elevated plus maze.
So let's take a minute to see where everyone is from. Could you all click on the chat box icon and write in there to say where you're from. So we've got someone from University of Toronto… I hope it's not cold there anymore! Oh - University of Alabama. Anyone else? UCLA… I wish I was in California right now. We've got University of Puerto Rico School of Medicine. Sounds like we have a bunch of people from a lot of cool places. I just want to let you know that during the course of this webinar please feel free to utilize the chat box to ask us any questions and we'll do our best to answer all the questions either in the chat window or as we go through the presentation.
We'd like to start this webinar with a poll so I'm going to launch it right now so just take a minute to briefly answer these questions and then we will share the results with you… Well we've got some people studying mouse and rat models. Everyone here is currently doing behavioral tests in their lab and everyone is using the elevated plus maze and animal tracking software. We're going to be here today to teach you more about the elevated plus maze and how you can ultimately optimize your current setup.
So let's get started with the presentation. Tracy, do you want to take the lead?
TRACY: Sure. So we wanted to start off with a brief basic background to make sure everybody is on the same page. The elevated plus maze was first developed by Handley and Mithani in the 1980s and it was developed as a rodent test for anxiety like responses. Since then it's been used very widely to study things like anxiogenic and anxiolytic compounds as well as the effects of different environmental experiences like stressors. The test is based on rodents' natural aversion to open spaces and their tendency to be thigmotaxic, which just means they have a preference to hug the walls or stay in enclosed spaces. The elevated plus maze has two enclosed arms and two open arms, and the open arms are aversive. Typically this test is run on videotape so a camera is mounted above the maze and then the animal's activity is videotaped. Analyzed variables that are looked for are things like time spent in the open versus closed arms, the number of entries that the animal makes into the open arms, and the latency for them to first enter the open arms. This can be analyzed with automatic video tracking software or it can be manually hand scored by a human observer.
So today we're talking about controversies of the elevated plus maze, or in other words, areas where there is poor standardization across labs and researchers tend to have different opinions on how things should be done. We're going to go beyond the opinions today and we're going to see what the data actually say about how testing environment, running the maze, and some other things can affect your overall results.
Alright so we're going to start off with looking at how the testing environment affects your data. Specifically we're going to talk about ambient light and noise, the presence of other subjects, and the presence of the investigator.
So first off we're going to do a quick poll before we discuss these data. Let's see how you do things in your labs. So if you've run the elevated plus maze before which it seems like most everyone has, then let us know in the pop-up poll on your screen whether you run your subjects with the lights on or lights off and we'll let you know the results in a few minutes after we discuss this.
So while we're waiting on the results, let's talk about how light and noise affect your data. Ambient lighting in the testing room can be a point of controversy. Some people run their testing with lights on some with lights off. Other people test under dim light or even in the dark with red light. There's no one right answer here for how to do it because there are considerations about your specific experiment to take into account, but many studies have looked at the effect of lighting on behavior in the elevated plus maze and they show that ambient light has a profound effect on behavior so it's definitely something to consider when you're testing. In low-light rodents are much more likely to explore the open arms, whereas in bright light they're less likely. Bright light really contributes to the aversiveness of the open arms. You can take advantage of this knowledge depending on the goal of your study as long as you apply it consistently to all the subjects, of course. So if you're testing for an anxiogenic effect and you're expecting to measure an increase in open arm time you might want to use low-light to encourage more open arm exploration among your subjects and vice versa.
Now as far as noise in the testing room, rodents are very sensitive to this and their behavior can be easily influenced both by noises coming from outside of the testing room, as well as within. For example, someone might slam a door in the next room and then your subject freezes on the open arm which artificially inflates open arm time for that subject and invalidates your trial. We don't want that, so to avoid this it can be really helpful to use a fan or something similar to create white noise in the test room. Bottom line here: be consistent with your lighting and be mindful that light and noise can both affect your experiment. With that, I think our poll results are probably in.
CHLOE: So everyone said lights on except one person saying lights off. Do you want to suggest that maybe people put signs on their doors to prevent interruptions or things like that.
TRACY: Yeah definitely, it's always good to put up a sign in the hallway outside of your testing room to say to anyone walking around out there, just be mindful that there's testing going on and keep the noise down. Don't open the testing door when you know somebody's running a trial in there.
CHLOE: Yeah, and you mentioned low lighting. How dark is too dark? I know people have sent me videos before where it's basically pitch-black. I don't know if you could shed some light on how dark is too dark or what's the difference between testing in dark and red light.
TRACY: There are some different considerations that go in to that, so if you're using video tracking software there's usually some minimal level of light that's going to be necessary to pick up the subject against the background of the maze. With our software it's actually not too much light - it's pretty minimal. I've seen people that can use an infrared light source and still get pretty good tracking of their subject. You know, the other consideration here is just what experiment you're running. If you're running an experiment where you're doing elevated plus maze during the the dark phase for your rodents, then you want to keep it pretty dark because you don't want to disrupt their circadian rhythm by turning on all the lights in the middle of night. So these are some things where you just have to consider the specifics of your experiment.
CHLOE: Okay.
TRACY: So next up let's talk about how other subjects in the testing room can affect your results. It's very common for a researcher to wheel a big cart full of cages into the test room and run their subjects one by one or even to run multiple mazes side by side. Is this really being efficient or is this being wasteful? What do the data say? Exposure to certain odor cues can be stressful to rodents and therefore alter their behavior. This graph is from an experiment where pain-related behavior was assessed in mice in parallel with exposure to various odor cues in the testing room. The the bar marked CM is exposure to bedding from cage mates in the room, which did not alter behavior. But you can see that bedding from unfamiliar gonadally-intact males of various species all reduced pain behavior because it heightened anxiety in the subjects. This was not the case with odors from castrated males suggesting the effect is related to androgens. This study shows you might want to rethink parking a big cart of cages in your testing room because odor cues from the other animals in the room could cause extra anxiety in your subjects and then affect your downstream data.
Another thing here to consider: what about your own presence in the testing room? Especially if you're male [laughs]. Well that same study looked at corticosterone levels to measure stress in the mice when they were tested by a male versus a female investigator. Male investigators actually elicited more stress among the mice than females. Even exposure to a tee shirt that was worn by a male investigator did the same… unless it was aired out for 30 minutes or more. Exposure to male chemosignals also elicited stress and the effect was equivalent to a restraint stress exposure which is a very strong stressor for mice. So as you might expect this stress translated into a change in behavior in an open field test which is shown here in the graph below. Bottom line here: be aware that you can affect the behavior of your subjects just by your presence in the room. Always randomize subjects really well, especially when different investigators are collecting data for the same experiment. And if possible sit just outside the room during a trial. BehaviorCloud actually makes it really easy to start, stop, and observe your trials remotely just with a mobile device so you can stay out of the room and out of the way.
CHLOE: So Tracy, you brought up some really good points about odor. I know a lot of people have both mouse and rat and you know they showed that in the poll results. So do you recommend that people have their elevated plus maze for mice and rat in separate rooms or the same rooms? Can they use the same maze for both models? What do you think?
TRACY: I think you can use the same room as long as you don't have a lot of other animals in there at the same time when you're running a subject. But generally you have two different elevated plus mazes - a larger size for rats and smaller size for mice. It just fits better with the different body sizes of those animals.
CHLOE: Okay and do you recommend habituating those animals to the room prior to actually starting the experiment or can I just start as soon as I put my cart outside of the room?
TRACY: Oh that's a great point. It's good to always habituate your animals to the room just because there's some novelty there. You're exposing the animals to a new area outside of the housing facility so it's good to bring in your animals about 30 minutes before testing to wherever you're going to have them and let them get used to that space.
CHLOE: Okay so now we're going to talk about important variables to consider when running the maze, specifically starting position, trial length, and what to do with unfortunate events. But first we're going to do another poll so please tell us in the pop-up poll window on your screen how to start your subjects in the elevated plus maze. Is it in the center square facing the open arm or facing a closed arm? Or maybe you start in the middle of one of the arms? We'll share the results in a few minutes after I describe what you should do, so just take a second here to answer the questions.
So the starting position in elevated plus maze is another controversy. Some investigators will put the mice or rats in the closed arm to start, but most agree that starting out in the center square is best because it's a neutral location and the rodent can choose which arm to enter first. What's more controversial is whether you should start them facing a certain direction or switch it up every trial. Well the data are pretty clear in this case that rodents started facing the open arm have a 50% chance of going into the open arm first whereas they only have a 20% chance if they started facing the closed arm. Be careful placing the subject in the maze. When rodents are first placed into the maze they might lurch forward away from the investigator and if they are facing an open arm they just happen to run in the direction of the open arm. The same happens when you place them facing the closed arm they just tend to run into the closed arm but what happens is they don't really venture out into the the open arms. The bottom line here is that you have to make sure that you're consistent about which direction you are starting each subject. Start your subjects facing the same direction and make sure you continue doing that throughout all of your trials and make sure that you're gentle. Let's see what the poll results say: It says that most of you place the animal in the center facing the open arms and then others also said facing the closed arms. Well Wolf and Frey, and many of the other people that actually wrote the method for the elevated plus maze do recommend that you start your animals facing the open arms because if you do face them towards the closed arms they tend to stay in the closed arms and not venture out.
Trial length is another area of confusion. Many researchers do something between a five and ten minute trial but you'll find in the papers that there's no strict standardization. In this case the data show that rodents explore equally each part of the maze at first and then the closed arm tendency emerges after the first two minutes of the exposure to the maze. After that the tendency to prefer the closed arms becomes more pronounced. On second exposure to the maze the effect is already there during the first minute. So what does this mean for you as an investigator? A trial must be at least three minutes and depending on your expected effect size you may want to run a longer trial. Analyzing your data in time bins, which is what is shown here, is easy to do with our software at BehaviorCloud and it will give you a more complete picture of how your effect changes over the course of the trial. So just keep that in mind when you're running your tests in the future.
We have another question here. It's from AFP and it says, what is your opinion about tail pick up versus using the cup method to place the mice in the center of the elevated plus maze? From my experience I've always used the tail pickup. What about you Tracy?
TRACY: I have used the tail method as well, although I was reading a paper recently that said the cup method is actually better because it keeps the animal calmer and they're less likely to lurch away from you and go running into one direction without thinking about it.
CHLOE: So does that mean putting them in a cup or is that like holding them in a cupped hand and putting them down.
TRACY: You can use an actual cup or even like a tube or something plastic and then that way you don't have to handle them too much. Although that is another point that we're going to talk about as we go forward. Handling of the mouse.
CHLOE: Okay and now we're going to talk about unfortunate events. Now there's always the risk of this happening during a trial. One example is when a rodent falls off the open arm. We've all been there and you think that your experiment is ruined. Or maybe your lab mate runs into the room and doesn't see the sign on the door and you think, "What do I do??" Well we always recommend discarding this trial from your data set, but you do want to actually place the animal back onto the elevated plus maze so that it can get the same experience as all the other animals that have gone through the trial. Just make sure that it goes through the allotted time. Say it falls off in minute three, then you want to keep going for another two minutes or however long you're running the test. This is important because if you plan to do a battery of behavioral tests you just want to have each animal go through the same experience as all the other animals. This is the bottom line for most of these points. We just want to have consistency.
TRACY: I actually had a colleague, Chloe, who said that if you sneezed in the middle of a trial you should technically sneeze for all the subsequent trials as well, but that's probably taking it a bit far. [laughs]
Alright, so in our final section we'll talk about other considerations to think about when you're doing elevated plus maze testing. We're going to talk about time-of-day effects, prior handling of your subjects, repeated testing, and how to interpret that elusive center zone. one more poll for you. We'd like to ask, do you test your mice during the light / day phase or the dark / night phase? We'll show these results to you in a few minutes.
As you all know, rodents are mostly nocturnal which means they're most active during the night time and they sleep during the day. Of course this affects other aspects of their behavior as well like memory and cognition. That's to say, would you ace an exam at 3 a.m.? Probably not. So studies like this one we're showing here clearly show that even anxiety behavior is affected by the time of day. Rodents tend to explore the open arms more readily during the dark phase, meaning the night time when they're awake and active, and during their sleep time they exhibit higher anxiety. So this means you've got to be conscious of this fact when you're running an elevated plus maze. You need to know what time is lights on and lights off in your animal housing facility and never let a testing session spill over between the light and dark phase. In fact if you're running a large cohort of subjects you'd really do better to split your testing over a couple of days run during the same general time window rather than do a marathon 8 hour testing session because as you can see here the behavior changes with time. Always randomize your testing order of your subjects. BehaviorCloud actually time stamps all of your data so it's really easy to correlate your results to the testing time after the fact and just make sure that time of day did not affect your result. Now let's see the poll results. It looks like everybody tests during light phase so now just keep in mind when you're doing that to keep your testing within a discrete maybe four hours or so window and don't go too crazy testing all day long because the behavior could change over time.
CHLOE: So Tracy someone else asked a private question about when we mentioned to discard the data if the mouse falls off of the elevated plus maze. What if 80% of the mice do this? Do we discard all that data or what other recommendations can we give to keep the mice from doing this?
TRACY: I experienced this firsthand in my PhD work. We were working with a wild-caught species of mice and those mice are kind of crazy compared to the inbred lab mice. They would just go flying off the elevated plus maze constantly. They were like popcorn jumping, so what we did is we had to adapt to a different anxiety measure in that case. We switched to using the open field test because it kept them contained. If 80% of your mice are falling off you might want to consider open field or maybe light-dark box. There are some other options that could measure a similar variable without having them falling off all the time.
CHLOE: That sounds good.
TRACY: Alright so another thing we're going to talk about is prior handling of your subjects. Some investigators like to handle their rodents for a few days before testing just to habituate them to humans and make the testing less stressful. Other people don't do this at all. This particular study we found showed that prior handling actually eliminated the effect of an anti-anxiety drug in the elevated plus maze, thus reducing the sensitivity of this test. So for elevated plus maze at least, you might want to consider that if you need to run a battery of behavioral tests maybe start with elevated plus maze first so the mice haven't had too much excessive handling, especially if it's your most critical test in your battery. Otherwise just be very consistent with how you handle all of your subjects across groups so that this variable doesn't affect your data.
Another thing to think about is repeated testing. Sometimes you want to measure anxiety behavior in the same set of subjects at different points over the course of an experiment. You'll see this done sometimes but it's really important to consider that the elevated plus maze is subject to something called one trial tolerance. That means open arm exploration decreases markedly upon re-exposure to the elevated plus maze even when mice are treated with anti-anxiety drugs. And this effect lasts for about a month so that makes it difficult to interpret data that's derived from a recent repeat exposure. Keep this in mind for your studies and if you need to get a second anxiety measure within about four weeks of the first elevated plus maze exposure then you can try using a different but analogous test like some of the ones I mentioned. Open field or there's even an elevated zero maze you can use.
CHLOE: Hey Tracy, I'm not sure, what are the differences in the anxiety measures between the elevated plus maze and the open field?
TRACY: So in elevated plus maze you're looking at time spent in the open arms, entries to the open arms, etc. The open field test is based on a very similar principle where the rodents tend to hug the walls of the chamber and the center which is kind of the open exposed area of the field is really the adversive area, so you'll measure time that's spent in the center square of the open field, latency to enter the center square, etc. It's a very similar principle, just a slightly different test.
CHLOE: Great.
TRACY: Alright now for our final controversy: what to do with that pesky Center zone? When you're analyzing elevated plus maze you'll typically compare time spent in the open arms to the closed arms, but there's always some amount of time spent in the center square which is sort of a gray area for analysis because it's really considered neutral territory. Generally we'll recommend excluding time in the center from your analysis and that's totally acceptable, but we wanted to mention that there are versions of this test like the elevated zero maze that actually eliminate this whole confusion completely by having no Center zone at all. It's a circular elevated platform where part of it is enclosed with walls like the closed arms on the elevated plus maze and part of it is open and so there's no center zone to worry about at all. Just something to think about.
CHLOE: We also wanted to mention other uses. If you're thinking that the elevated plus maze could only be used for anxiety and depression in the format that we've already shown you, then you're wrong because there's other ways to use it. So another way to use this maze is the discriminative avoidance test. It's a method for testing learning and memory and as you can see in the picture, one of the closed arms is paired with an adversive stimulus like a bright light or a loud noise and then upon second exposure to the maze the next day you measure how long does the animal spend in the open arms and the other closed arms as well. So it's kind of similar to fear conditioning chambers or anything like that, but it's interesting to see that you can actually use your mazes for something else. You know, sometimes you go through experiments and you might not necessarily need an elevated plus maze for anxiety or maybe you have one gathering dust in your lab and you don't really use it anymore. Maybe this is another application for you.
TRACY: Yeah I wish I had known about this. I would have definitely done that.
CHLOE: Yeah at least you can try it out. We've talked about a lot of things today and it might be a little bit overwhelming, especially if you're new to behavioral research. We talked about the background of the elevated plus maze, why it's used, how it's used, and improvements to optimize your current setup. If you'd like further consulting on what tests are best for you and your lab or to help you with your research, we're happy to help.
We can also provide you with the BehaviorCloud software platform and a complete set up for your behavioral suite including mazes that we mentioned like the elevated plus maze, elevated zero maze, open field and more. If you'd like to see a demo or speak to us in more detail please contact us at the email below: hello@behaviorcloud.com or you can try our free trial at behaviorcloud.com. I think we've gotten everyone's email from this webinar today. We'll be sending this video along with a follow-up survey to our attendees today. This will also include a bibliography of all of the papers we mentioned and we'll also be posting on all of our social media accounts. LinkedIn Facebook Twitter
Please feel free to let us know what topics you want us to cover in the future. This webinar series is our Spring series and it'd be great to get your feedback and your input for our future events. Thank you again for coming please let us know if we can answer any questions in the chat window. We'd be happy to help.
References
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- Pereira, L. O., da Cunha, I. C., Neto, J. M., Paschoalini, M. A., & Faria, M. S. (2005). The gradient of luminosity between open/enclosed arms, and not the absolute level of Lux, predicts the behaviour of rats in the plus maze. Behavioural brain research, 159(1), 55-61.
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- Carobrez, A. P., & Bertoglio, L. J. (2005). Ethological and temporal analyses of anxiety-like behavior: the elevated plus-maze model 20 years on. Neuroscience & Biobehavioral Reviews, 29(8), 1193-1205.
- Andrews, N., & File, S. E. (1993). Handling history of rats modifies behavioural effects of drugs in the elevated plus-maze test of anxiety. European journal of pharmacology, 235(1), 109-112.
- Schneider, P., Ho, Y. J., Spanagel, R., & Pawlak, C. R. (2011). A novel elevated plus-maze procedure to avoid the one-trial tolerance problem. Frontiers in behavioral neuroscience, 5, 43.