Scientists mimic natural conditions in the lab to more accurately test stress
By Seth Palmer
August 23, 2013
Animal models play a crucial role in our understanding of human biology, but the validity of conclusions drawn from studying these models is – as in all research – highly dependent on scientists’ developing appropriate, accurate paradigms through conscientious methodology; this is especially true for studies of animals’ cognition and behavior, where such due diligence is critical in avoiding anthropomorphic bias.
Recently published in the journal Frontiers in Human Neuroscience, a particularly methodologically driven study led by Huck Institutes researcher Victoria Braithwaite and graduate student Lauren Chaby which uses rats as a model for investigating long-term effects of stress lends credence to the notion that the events of adolescence can change individuals in dramatic ways that persist well into adulthood.
“The results of our study suggest that exposure to chronic, unpredictable stress during adolescence leads individuals to respond more negatively and impulsively to letdown and ambiguity as adults. Even though we’ve only done this work with rats, it’s still interesting as it shows that stress during the really vulnerable stage of adolescence can have a huge long-term impact,” says Braithwaite, a professor of fisheries and biology at Penn State and co-director of the Center for Brain, Behavior and Cognition, part of the Huck Institutes of the Life Sciences. “Lots of people have looked at the short-term consequences, but very few have followed them through to adulthood and seen these effects. What we seem to be seeing is that there are pretty permanent changes occurring as a result of stress during adolescence, and I think that’s significant.”
To avoid distorting their results when working with animal models, researchers must be mindful of the ways in which the animals will view the experimental tasks at hand (or, rather, “at paw”). Given the obvious inability of lab animals and scientists to speak to one another, this is no small task, even for an experienced scientist like Braithwaite, and both she and Chaby stress the importance of designing studies naturalistically.
“We’ve learned – and we found out the hard way,” Braithwaite says, “the importance of coming up with tests that are really obvious to the animal. Some of the stuff we’re trying to get at here is quite complicated – we’re basically trying to peer into the mind of the animal – and so we’ve got to ask the animal questions in ways that it can understand what the question is, and it can be really difficult getting at that. Interestingly, some of our more successful tests have ended up using really simple things that the animals are naturally doing and that we can easily measure. But when we’re looking at choice behavior – how the animals are choosing to do one thing or the other – that gets more complicated, and we have to be quite careful how we set that up and how we interpret the results.”
“A lot of what drives what we do,” Braithwaite continues, “is thinking about how the natural history of a system influences it, for example, variation in predation pressure or variability of the environment – how those kinds of ecological factors have an impact – and then trying to come up with something in a lab model that can mimic that. It's how we got the idea for this study of using multiple stressors – both social and physical – which makes this study different from a lot of others, since most labs tend to concentrate on one or the other.”
“We try to think about things in terms of the rodent’s natural life cycle,” adds Chaby, the lead author of the paper and a dual-degree candidate under Braithwaite’s mentorship in the Huck Institutes’ Ecology and Neuroscience graduate programs. “A lot of the stressors we use, try to mimic relatively realistic circumstances that you could imagine a rat encountering – which is difficult; this isn’t always done in other studies, and it’s another aspect that makes this study unique.”
While other studies have included stressors such as restraint, confinement, or electric shock – natural correlates of which, such as entrapment or lightning strike, may not have been routinely encountered in the animals’ wild ancestral environment – Braithwaite and Chaby opted instead to use more-naturalistic physical and social stressors including damp or foreign bedding, crowding, and isolation.
“Rather than employing more-commonly used stressors like restraint and shock,” Chaby says, “we tried to use stressful stimuli that were more ecologically relevant.”
“Since stressors such as restraint and shock aren’t the kind of stimuli that rats are likely to come across naturally,” Braithwaite adds, “we avoided them. There are some really quite harsh treatments used to induce stress, and I feel quite strongly that if you don’t need to use those kinds of extreme stressors, then I’m not sure of the value – it’s not very naturalistic.”
In that same vein, Braithwaite and Chaby have also observed a relationship between naturalistic laboratory ecology and quality data – where things like environmental complexity and variability have a significant effect, as does social interplay, both among the animals, themselves, and between the animals and the researchers.
“The ecology of the lab is interesting in its own right,” says Braithwaite, “because while the natural world varies, these lab animals are often put in constant, homogeneous conditions – the most mundane, boring environment you can imagine – and then tested as though they will truly reflect the way that people might behave given lots of change and variation; there’s a mismatch there, and even though we’re using domesticated strains of lab rodents, I think that in many of those cases you’ve got animals that aren’t behaving in the way they’ve been selected to do in a natural environment.”
As a direct result of this line of thinking, the animals – upon delivery to the lab – are paired in object-enriched environments and given time to explore and adjust to their new surroundings before any tests are initiated.
“A social partner gives these animals a certain amount of stimulation,” Braithwaite says, “but even when they are given a social partner, it’s really important to have enrichment items in the cages – something for them to do, something to chew on or play with, a tube to hide in and run in and out of – just to help keep them occupied. We know that these sorts of things promote neural growth in key areas of the brain that are important for decision-making and even cognitive bias, so providing these enrichment items makes a big difference that can affect your results: the behaviors you see from these animals are going to be more robust and more reliable.”
“In addition to behavioral effects,” Chaby adds, “enrichment also affects physiological measures. It’s been shown that even basic enrichment can change the structural representation of the forepaw in the brain of a rodent – so when you’re talking about sensory capacity and acuity, these things can differ depending on the amount of enrichment and stimulation an animal was exposed to during its development.”
Leading up to and through the course of a given study, the researchers make a point of interacting frequently with the animals for further socialization and to help them acclimate to the goings-on of the lab; Braithwaite, in particular, feels that these practices are beneficial to the animals and improve results.
“We handle the animals regularly,” says Braithwaite, “because we’ve found that the more they’re handled, the more they become used to us and to the experimental paradigms and setups; if they’re not handled very much, they find anything new to be stressful and they respond badly, they don’t learn things very well, and a whole suite of other knock-on consequences. There’s actually quite a knack to knowing how to interact with the animals once you’ve got them in the lab, in order to get the best and the most information out of them. Unfortunately, there’s still some reluctance in the scientific community to do these things, because people think that it’s going to mess up the results – but I think the evidence so far to date suggests that actually it improves the robustness of the data you collect.”
Results and follow-up
To minimize the influence of any unintended stressors on their results, the researchers allowed a week for the animals’ socialization and acclimation after their delivery to the lab and prior to the stress phase, which lasted 40 days – until the onset of adulthood – and was followed by nearly two months of normal, stressor-less conditions before the final tests.
“All the tests of cognitive bias and impulsivity were done after about two months of the test animals’ being back on standard conditions,” Braithwaite says, “so that they were just like the control animals during that phase, doing what rats normally do in a lab. We wanted to be absolutely sure that any effects we observed during the testing phase were associated solely with the stressors from the adolescent phase, which really is important because this shows that what’s happening during adolescence – during that sensitive developmental phase – has long lasting effects.. In the end, we were surprised that it had as big of an impact as it did over so many different tests – the differences we were able to detect in terms of things like the animals’ emotional state, how sensitive it was to not getting an expected reward, and whether it had a more negative interpretation of ambiguous situations.”
Moving forward, Braithwaite and Chaby plan to continue their current investigations, but with an additional focus on neural connectivity, which may help them to explain the physiology underlying the behaviors they’ve observed.
“The speed with which the animals corrected the wrong choice is much faster in those animals that have had this chronic, unpredictable stress,” says Braithwaite. “We think that this could be an indication of impulsivity, and there are a number of mechanisms that might underpin that. I think our work is pointing at a number of mechanisms that we can explore, looking within the brain at the connectivity that is going on between the areas that control emotion and decision-making, and trying to determine where these differences are coming about. We suspect that what is going on is that the connections which are strengthened and solidified during adolescence in a normal animal are probably not wired as well or are weaker somehow in these stressed animals, which – using tools such as MRI – is something we should be able to measure.”
“We know that adolescence is a period when people exhibit a high level of impulsivity,” Chaby adds, “and if we’re seeing a scenario where impulsivity is retained into adulthood, then this could have serious consequences for risk-seeking and other behaviors that can potentially be detrimental to the welfare of an animal or a human being. We’re interested in looking in more detail at what’s happening with impulsivity – what types of impulsivity might be affected and what kind of an impact increased impulsivity may have on other behaviors.”
“What we’re potentially doing here,” Braithwaite continues, “is providing an animal model that would allow us to investigate these questions in a system where we can do more than we could with a human population – more-controlled experiments, specifically looking at sensitive time windows when exposure to these things might have their biggest effects, and then trying to extrapolate and figure out how to control or how to rescue, potentially, some of those effects. We really need animal model systems to have that capacity to play with those sorts of questions, and this work underlines how fundamentally important stress is during development, on so many different levels – the stress state is a very nebulous state where lots of things are going on that are having a mammoth effect in terms of development. There are lots of things that can be spun off from this research, and in a way, it’s amazing to me that we’re only still scratching the surface in terms of understanding the impact that stress can have.”
Other researchers involved in this study include Sonia Cavigelli, a Huck Institutes co-funded faculty member of the Neuroscience graduate program and the Center for Brain, Behavior and Cognition and an associate professor of biobehavioral health at Penn State; and undergraduate students Amanda White and Kayllie Wang of the Department of Biology at Penn State.
This research was supported by the Huck Institutes of the Life Sciences and the Eberly College of Science at Penn State University and by the Pennsylvania Department of Health using Tobacco CURE funds.