From the genetics of dog breeds to stress and reproduction

The other morning I was talking to my husband in bed in an attempt to help him wake up.

Me: So I ran into our friend who walks those three goldens separately yesterday and we had a nice conversation. She said she’d read my blog and had a dog genetics question for me.

Him: mmmppphh

Me: She said she’d heard that 1% of dog genes account for all the differences between breeds and asked me if it was true. I pointed out that 1% of 20,000 is still a lot of genes, and also explained that it's really hard to use statistics like that to describe genomic differences, because you can measure those differences in so many different ways.

Him: Did you tell her that humans and chips are 98% similar genetically?

Me: Yes I did.

Him: But I’ve been seeing that for at least 10, maybe 20 years. Is it still true?

I consulted the internet on my phone.

Me: Let's see... The Smithsonian Institute says we're 1.2% different from them. I think I'll skip this link to the Institute for Creation Research -- is that really the second hit on “human chimp genetic similarity”?! Ah, Wikipedia gives more information: “The alignable sequences within genomes of humans and chimpanzees differ by about 35 million single-nucleotide substitutions. Additionally about 3% of the complete genomes differ by deletions, insertions and duplications. Since mutation rate is relatively constant, roughly one half of these changes occurred in the human lineage.” Well, that’s not true.

Him: What?

Me: Mutation rate isn’t constant.

Him: It’s not?

Me: Well it is closer to constant in specific areas, like parts of the mitochondrial DNA, which we like to use as clocks. But over the whole genome, which is what they're talking about here, no. Different areas evolve at different rates. There are hotspots that go faster. And then the whole species might change faster when its environment suddenly changes. Like if you're in a lovely sunny valley and you're well adapted to it and then suddenly an Ice Age starts and your valley fills with ice and you suddenly have intense selection pressure to change your coat length and thickness and your diet and things like that. The stress itself can change your mutation rate.

Him: Stress can’t change your mutation rate! How would that even work? If a female is stressed, it’s too late, her eggs are already made.

Me: Her grandkids then? Or only sperm have more mutations? Hmm, that’s good point.

I consult the internet again. I find and discard an article about yeast evolving more quickly under stressful conditions. Yeast don't make eggs or sperm as part of their reproductive process.

Me: Here you go. Flies. Close enough to mammals for you? Stress does cause flies to have offspring with more mutations. It makes sense because if you’re stressed, it means you're probably not well adapted to your environment, so you should do the random shuffle with your kids’ genetics in the hopes that something, anything, different will give them a better shot. Mostly they’ll be worse off, but at that point it’s worth if it a few are better off and can pass on those genes.

Him: But how does it work with female flies having already made their eggs before they’re stressed?

Me: I dunno... Hang on... Here we are. OK, so the researchers mutated the males, their sperm.

The reason the researchers mutated the males has to do with how DNA is fixed in male and female fruit flies. There is almost no DNA repair in sperm. But the egg can repair DNA in any sperm that fertilizes it.

So the researchers were basically asking how much of the mutated DNA from the male could slip through the repair processes in the egg. The answer was that eggs from stressed females let a lot more mutations through.

Why would stressed female eggs not fix DNA as well? Probably because fixing DNA perfectly costs lots of energy. And these stressed females may not have had enough energy to spare.

There are two different kinds of DNA repair out there. The one that fixes the DNA perfectly costs a lot of energy. The other kind gets rid of any gross problems but leaves errors behind. This costs less energy but leads to more mutations.

The idea is that stressed females can't afford to use the perfect DNA repair system. So they use the other one. Their kids survive but they have more mutations.

—Stanford at the Tech, Understanding Genetics

 Me: Oh crap now I’m late to take Jack to physical therapy.

...Kind of makes you wonder about puppies conceived in puppy mills or animals conceived in hoarding situations, doesn’t it? Might they have more mutations than animals conceived in less stressful environments?

How antidepressants work: the good parts version

[Author’s note: Please consider my last post, How do antidepressants work (in dogs and the rest of us)?, to be the director’s cut of this topic: fairly long and juicy, with some bits in which I indulge my inner geek and perhaps go into more detail than is truly necessary. This, then, is the good parts version: the same material, but presented as an overview from a higher altitude, with fewer details and assuming less scientific knowledge. These posts are both intended as material for my upcoming online class with APDT, and I want to make sure students of all levels of science background are covered. Also, it’s good for me to take a step back from time to time and remember that not everyone wants to know every gory detail about this brain stuff.]

We don’t fully understand what causes depression in humans, and we don’t fully understand how the medications we use to treat depression work. We do know that those medications work well in dogs just as they do in us. In dogs, however, they are more often used to treat fearfulness or aggression. We know that antidepressants generally take effect only after several weeks of constant use, and that they work much better if they are paired with behavior modification training in dogs or therapy in humans. And we actually do know enough about how they work to take a guess at why that's true.

You can buy your very own Prozac bone sticker!

Depression in humans and fearfulness and aggression in dogs are related to stress: something in our lives that we can't control and can’t quite adjust to. In humans, that might be extended unemployment or long term caretaking for a sick family member. In dogs, it can be the inability to control who comes to visit your house (that terrifying mailman) or perhaps a lack of understanding of the big scary world (for undersocialized dogs).

Sometimes training or therapy aren’t enough to help us deal with these problems; some problems are too hard for our brains to cope with on their own. Antidepressants seem to help our brains adapt, however. A part of the brain deeply involved in learning and memory, the hippocampus, tends to be smaller in people who are depressed and tends to get larger again when they take antidepressants. This change may be associated with an improved ability to make new mental connections.

So that’s why antidepressants take weeks to take effect: that part of the brain is growing and changing, which doesn’t happen after just one pill. And that may also be why antidepressants work so much better in the context of training or therapy. It’s nice for your brain to be more able to learn new ways of coping with a difficult world, but the ability to learn is not the same as actual learning. To learn, you have to get out there and do: talk through your problems and find the way to feel differently about them and take new approaches to solutions if you’re a human, or get to practice new ways of interacting with the mailman if you’re a dog.

The take home message for dog owners? Don’t expect your dog to respond to antidepressants immediately; it will take a few weeks. And don’t expect your dog to respond without behavior modification. Antidepressants aren’t magic bullets and they won’t fix the problem on their own. But they will make it easier for all the training you do to take effect.

How do antidepressants work (in dogs and the rest of us)?

There are plenty of humans and dogs on antidepressants, and we believe that the mechanisms of these medications are much the same in both human and dog brains. But despite the fact that these are widely used medications, we aren’t completely clear how they work. Yes, this is going to be another post in which I ask a question and then don’t really tell you the answer. But I’ll tell you what we do know.

You can buy your very own Prozac bone sticker!

What is depression?

There are probably many different kinds of depression, so that the disease is slightly different in many (or all) people and dogs. As a result, getting a handle on the mechanisms of depression and its treatments is difficult. So studies about depression and antidepressants have to speak in generalities, such as “this is true for 50% of people with depression.”

In general, then, depression is triggered by chronic stress, which results in increased levels of stress hormones. The major stress hormone in humans and dogs is cortisol, so that’s the term I’ll use in this post. The major stress hormone in mice and rats is the closely-related corticosterone, so if you delve into more of the research in this area, you may find that hormone mentioned as well. It’s basically the same as cortisol. Note that while I’ll talk about depression in this post, in dogs we more commonly perceive stress-related problems as behavior problems, such as shyness or aggression. These problems, in certain cases, can be very successfully treated with antidepressants in combination with training.

Depression and the hippocampus

The area of the brain which is the most sensitive to increased levels of cortisol is the hippocampus, a part of the limbic system which is involved in learning, memory, and emotion. The cells of the hippocampus are armed with little widgets called glucocorticoid receptors, or GR, which grab cortisol molecules as they float by. Once a GR has attached itself to some cortisol, it becomes active, and takes itself over to the cell’s DNA. Here it tells the cell to activate some genes and deactivate other genes. This is how cortisol effects stress-related changes in our body: by telling the massive recipe-book inside our cells which genes to cook up and which ones to leave idle.

Image courtesy of Wikipedia

So the first generality about depression is that it results in more cortisol than normal. The second generality is that it also results in a smaller hippocampus than normal. We can guess, though we’re not sure, that this is somehow related to all that GR activation. We know that in depression, fewer new neurons are born in the hippocampus, so that is probably part of the answer. However, it’s not the whole answer, because even in healthy people, new neurons are created at a very low rate, not fast enough to explain this decrease in size of the hippocampus. Another possibility is that the shape of individual neurons changes. Neurons branch out like trees to touch lots of other neurons, and the neurons of depressed people have fewer branches. So the problem could be caused by fewer neurons, or by neurons that have fewer branches and therefore less communication with other neurons. We’re not sure which, but either way, the changes are significant.

Antidepressants and the GR

Antidepressants affect many substances in the brain (most famously, the class of antidepressants of which Prozac is a member affect serotonin levels). We have trouble picking out cause and effect here. We assume that antidepressants aren’t affecting all of these substances directly; we assume that some of these affects are indirect, in other words, side effects. We’d like to know which substances antidepressants directly affect in the brain, in other words, what their mechanisms are, but we’re still not sure.

We do know that they affect the GR, though we don’t know if they do so directly or indirectly. So far, we haven’t found any direct effects on the GR. One theory is that antidepressants affect another widget, one which pumps cortisol out of cells so that the GR can’t grab it and become active. The decrease in number of active GR, then, causes the changes in the brain which result in mood improvement.

We do know that depressed people on antidepressants start to have increased birth of new neurons in their hippocampus, which itself increases in size. Why does this affect mood? We don’t know, but we can hypothesize that improved ability to make new mental connections and learn is at the heart of the change. This helps us understand why antidepressants typically take several weeks to work: our brains are changing, growing, and that takes time.

Antidepressants and dogs
 
As usual, all the research on how this stuff works was done in rats, mice, and humans. But we do think that the mechanisms are similar in dogs, and indeed in most or all mammals. Many dogs are on antidepressants with positive effects, including my shy dog Jenny, who receives both buspirone and lots of counter-conditioning. As research continues to get us more answers about how these medications actually work in the brain, we will do better and better at understanding which kinds of antidepressants are better for which individuals, when to start them, and when to stop them.



Anacker C., Livia A. Carvalho & Carmine M. Pariante (2011). The glucocorticoid receptor: Pivot of depression and of antidepressant treatment?, Psychoneuroendocrinology, 36 (3) 415-425. DOI: http://dx.doi.org/10.1016/j.psyneuen.2010.03.007

For further reading, check out related posts by Scicurious:

• If low serotonin levels aren't responsible for depression, what is?
• Stress and antidepressants: by their powers combined

Can prenatal stress be reversed?

I was scanning the titles of new journal articles a while back, and came across one that made me think, hey, that may be about rats, but it is totally relevant to dogs. And then I thought, why don’t I teach a class on it? Read and interpret this really interesting journal article with a group of dog trainers and dog lovers?

I will be teaching the class Prenatal Stress and Anti-Depressants for APDT the week of November 18 (and you are invited to take it). This post will be used as reading material for it. In the class, we will talk about this article and what conclusions we can draw from it and apply to dogs. So I may not draw as many the conclusions for you in this post as I usually do; the plan is for the students to do that together in class. But it was a fascinating paper and there’s lots of good material in it, so read on if you want a conclusion-free summary of it!

Pereira-Figueiredo I., Juan Carro, Orlando Castellano & Dolores E. Lopez (2014). The effects of sertraline administration from adolescence to adulthood on physiological and emotional development in prenatally stressed rats of both sexes, Frontiers in Behavioral Neuroscience, 8 DOI: http://dx.doi.org/10.3389/fnbeh.2014.00260


Why prenatal stress?
So what’s prenatal stress and why is it important to dogs? The authors of the article don’t provide much background on this phenomenon, but it’s an interesting one: when a mammal undergoes unusually high stress during her pregnancy, the personality of her offspring can be affected. We believe that the stress hormones rising in her bloodstream can pass through her placenta to the fetus or fetuses, and can change how their brains develop at this very early stage of life. That’s prenatal stress: stress experienced before birth.

Part of what this paper investigates is exactly how prenatal stress affects the developing personality, because we don’t yet fully understand how this stuff works. In general, though, we expect prenatally stressed animals to be more anxious and less confident than animals who were not prenatally stressed.

Does prenatal stress affect dogs? We don’t know for sure, but we think it is something that can affect most or all mammals. Would it happen commonly? Hard to say, but I imagine a pregnant dog who is stray or in a shelter and highly stressed, and I wonder what effects this might have on her puppies.

What can you do about it?
If you have a puppy that you believe was prenatally stressed and whose personality you thought was adversely affected, what are your options? Careful socialization and good enrichment are always an excellent choice, but in some cases you might consider medication. This study looks at whether a particular anti-depressant, sertraline, might help change the individual’s personality long-term if given in adolescence. Sertraline is an SSRI, in the same class of medications as Prozac. These are widely used medications believed to be fairly safe, but one of the questions these researchers ask is whether it is safe when given throughout an animal’s entire adolescence.

SSRIs such as sertraline affect the levels of serotonin in your brain. Serotonin is a chemical which affects mood; depressed people tend to have less of it, as do aggressive people. As a result, it is the target of a fair number of anti-depressants, which work to increase its levels. Prenatal stress is known to disrupt the serotonin system in the brain, so a medication which affects serotonin is a reasonable choice for prenatally stressed individuals.

So the idea is: give these prenatally stressed animals a medication which increases their serotonin levels while they are adolescents and their brains are still developing. The hope is that they will develop into more normal adults than they would have without the medication. So, exactly how do you investigate such a question?

Methods: how the study worked
First, the researchers stressed some pregnant rats by putting them in clear tubes to restrain them, and shining bright light on them. This was repeated for forty-five minutes at a time, three times a day. They also kept control rats, who were not stressed during their pregnancy. The pups born to these two sets of rats were then in two categories: prenatally stressed pups and non-stressed pups.

The rat pups began their anti-depressant treatment with sertraline when they were one month old. Now there were four groups of rat pups:

prenatal stress anti-depressants	prenatal stress no anti-depressants
no prenatal stress anti-depressants	no prenatal stress no anti-depressants

Having these four groups allowed the researchers to pick apart the two different effects, the effect of prenatal stress and the effect of anti-depressants during adolescence.

The pups were tested at two months of age, the beginning of rat adolescence, to see if the prenatal stress had affected their personalities. They were assessed for how they dealt with startling noises and being exposed to open space (scary for a rat!). Their blood was also tested to see how their immune systems were developing, because immune systems develop differently in animals who have been subjected to high stress. All these tests were run again one month later, at the end of rat adolescence, to see how the anti-depressant given throughout adolescence had affected treated rats compared to the control groups.

Results: what they found

• Although we may think of prenatal stress as mostly affecting an animal’s behavior, it’s been shown to also affect metabolism, so this study looked at birth weight. Interestingly, prenatal stress only reduced the birth weight of the female rat pups, not the males. The weights of these females had equalized compared to non-stressed pups by weaning age. After weaning, though, the prenatally stressed females continued to gain weight and ended up heavier as adults than the non-stressed females. When prenatally stressed females were given the anti-depressant sertraline, however, this weight difference was reduced.
• The pups were also tested for their startle response when they heard a sudden sound. Prenatally stressed rat pups did seem to have a larger startle amplitude (size) compared to controls, but this wasn’t statistically significant, and was not reversed by sertraline treatment.
• Prenatally stressed females did not habituate to the startling sound after several exposures as well as rats from other groups did; treatment with sertraline reversed this effect.
• The pups’ behavior in an open space was tested. No difference was seen between prenatally stressed and non-stressed pups, except in males on their first time being tested (not on later tests).
• In the open space test, only non-stressed females explored more (became more confident) on repeated testing; males and prenatally stressed females did not become more confident with repeated exposure to the open space.
• The pre-natally stressed rats showed a significant decrease in their number of white blood cells. This change was reversed when they were treated with sertraline.

Discussion: what does it all mean?
The study’s main conclusions are that effects of prenatal stress can be seen in rats, and that giving sertraline during adolescence did not harm them.

The rapid weight gain in the pre-natally stressed females is an effect that’s been seen before, and seen in humans. Children born with low birth weights often grow to have issues with their weight and can suffer from diseases related to a poorly regulated metabolism. This loss of control of energy balance has been associated with dysregulation of serotonin in humans, adding additional support to the choice of sertraline, an anti-depressant which interacts with the serotonin system.

It is interesting that no anxiety-like behavioral changes were seen in the prenatally stressed rats. Prenatal stress is known to cause anxious personalities in many cases. However, these rats were as confident (or as anxious!) in the open space test as rats who had not been prenatally stressed. The researchers comment that this particular test has been done on prenatally stressed rats in other studies, and that those rats didn’t show anxiety in the open space test either, so this does seem to be a real result, rather than a statistical error.

They did see some differences, though. Male rats who had been prenatally stressed did show some additional reluctance to explore (i.e. anxiety) on their first day only in the open space test. After that they explored equal amounts compared to other groups.

The researchers also note that while most of the rats that they tested were equally anxious on all days that they were tested in the open space, females who had not been prenatally stressed appeared to begin to explore more on repeated tests, as if they were learning to be less anxious as their surroundings became more familiar. This was not the case in male rats or in rats who had been pre-natally stressed.

Remember also that female rats who were prenatally stressed did not habituate to startling sounds as well as rats from other groups. Is it possible that with this particular model of prenatal stress, the personality effects of prenatal stress appear not as classical anxiety, but as difficulty habituating to new situations or stressors such as loud noises?

Finally, the researchers looked at effects of pre-natal stress on the immune system, and found significant effects (decreased numbers of white blood cells) which were reversed by treatment with the anti-depressant sertraline. Why did they care about the immune system? Because the immune system and the stress system are closely intertwined. Stressed animals show changes in the numbers of their white blood cells just as the prenatally stressed rats did. The researchers were using the changes in the immune system as markers for changes in the stress system.

There are two possibilities for why these prenatally rats showed stress-associated changes in their immune systems: either because they themselves had high stress levels, or because their immune systems were developing prenatally (in utero) in a high stress environment due to their mother’s stress levels. Either way, it is interesting that treatment with sertraline reversed these effects, suggesting that it may have either changed current stress levels in these adolescent rats (even though the only serious stressors they had undergone were before their birth!), or had counteracted other effects from that prenatal stressor.


Conclusions
It can be hard to know exactly what conclusions to draw from a scientific paper. What do you think? What are the most important findings in this paper (maybe just two or three of them)? Do you think those findings are real phenomena, or maybe just statistical mistakes? If they’re real, do you think they can be extrapolated from rats to humans or dogs?

Can a father's stressful experiences affect his offspring's stress system?

I am again indebted to my APDT students, who asked a very interesting question which turned into a blog post. This time it was: “Can a father’s stress be passed on epigenetically to his offspring through sperm?” Warning: epigenetic geekery ahead. If you are not in the mood for some technical terminology, this may not be the post for you.

Source: Wikipedia

I’ve blogged about epigenetics before (on the epigenetics of fear and of stress) and there are summaries of what epigenetics is in those two posts, but basically it’s changes in the DNA that don’t involve the sequence of bases. We’ve been so focused on the importance of DNA sequence as we’ve learned more and more about genetics, but in recent years the importance of other factors has started to become obvious. It’s like saying that the content of a book isn’t the only thing controlling whether the book gets read or not — it also matters whether the cover is appealing, how much it costs, and whether it’s shelved where people can find it.

One of my students tried to answer her own question and found this fascinating article:

Rodgers A.B., S. L. Bronson, S. Revello & T. L. Bale (2013). Paternal Stress Exposure Alters Sperm MicroRNA Content and Reprograms Offspring HPA Stress Axis Regulation, Journal of Neuroscience, 33 (21) 9003-9012. DOI: http://dx.doi.org/10.1523/jneurosci.0914-13.2013

Essentially, Rodgers et al stressed out some male mice and then tested their offspring six ways from Sunday to see if the effects had been passed on. And they had, but in some surprising ways.

The mice

Male mice were chosen because we have evidence that environmental effects can be passed on epigenetically through the sperm. Because sperm are made throughout the male’s lifetime, they can easily serve as messengers to pass information about the environment on from fathers to their offspring. Mothers can’t pass this information on through their eggs (so far as we know), because eggs are made before a female is born, and can’t easily be changed later. Of course, a mother has plenty of other chances to pass on information about the environment to her offspring: while they’re in utero and while they’re dependent on her. But for dad, sometimes he just has that one chance; he may never interact with his offspring in any other way than through the information in his sperm.

(Why do parents want to give their offspring information about the  environment? To let them know, at formative times in their lives, how to develop. If the world is a safe one, you don’t need a highly reactive stress system. But if it’s a dangerous place, you need your store of cortisol ready to go. It’s easiest for these sorts of developmental decisions about how to tune the stress system to be made early in development — in utero or post-natally — so that’s why parents have systems to pass the information on early, early, early.)

Male mice, then, were chosen for this study. In addition to the control group of unstressed mice, there were two groups of stressed mice: mice who were stressed in adolescence, and mice who were stressed as adults. Epidemiological research in humans suggests that adolescence is an important time for epigenetic changes in sperm.

The stressors

The males were subjected to a variety of stressors. In reading the list, I was torn between sympathy for the mice, and bemusement at the entries:

Stressors, selected because they are nonhabituating, do not induce pain, and do not affect food or water intake, included the following: 36 h constant light, 15 min exposure to fox odor, novel object (marbles) overnight, 15 min restraint in a 50 ml conical tube, multiple cage changes, novel 100 dB white noise (Sleep Machine; Brookstone) overnight, and saturated bedding overnight.

Wet beds! Scary white noise! Scary marbles! And yet yes, probably very stressful to a mouse, and I should not make fun.

Breeding
The males were given time to recover from the stress and then bred. They were removed from the cage as soon as they had mated with the female, which took 1-3 days, to minimize their interactions with her, so that their stress levels could not affect her. (However, the smart reviewers at F1000 [warning: not open content] note that a stressed male might have been more aggressive in mating, which could cause the female to alter her care of her offspring.)

Offspring stress response
The offspring of stressed males, it turned out, had a less responsive stress response than the offspring of unstressed males. In other words, when these mice were stressed by being restrained in a conical tube for 15 minutes, the ones whose fathers had undergone the variety of stressors had a smaller cortisol response compared to the ones whose fathers had not been stressed. The result was almost exactly the same whether the fathers had been stressed as adolescents or as adults, which surprised the researchers.

Now, if a mouse receives information from his father (or his father’s sperm, but you know what I mean) that the world he’s going to live in is a stressful place, I would have expected that that mouse would develop a more reactive stress system, not less. Worried that terrifying marbles or a wet bed are going to attack you at any moment? Then you had better have your stress response at the ready, right?

The stress system is, of course, much more complicated than that. We don’t understand yet why some models of stress system dysregulation show less reactive responses and some show more reactive responses. For example, humans with depression or PTSD can both show either more or less reactive stress responses than mentally healthy humans. So what exactly does this mean for these particular mice? The next thing I would do is to look at their behavior. Do they act more stressed?

Offspring behavior
The offspring were subjected to quite a few and quite varied tests to see if their stress behaviors were different. The researchers tested things like how much the offspring startled in response to a loud sound; how fearful they were of being in a brightly lit box versus a dark box (mice feel safer in darkness); how long they struggled when suspended by their tails; and more. Really surprisingly (to me, at least), there were no behavioral differences between the offspring of the stressed fathers and the offspring of unstressed fathers, despite this significant difference in stress system responsiveness. So what does that mean?

The researchers tested a bunch of other stuff that left them empty handed as well, like gene expression differences in the brains and adrenals of the different sets of mice. All nothing. But what did they find that was different? They found an epigenetic difference in the fathers’ sperm.

microRNA changes in sperm
Epigenetics is all about gene expression: determining which genes are used frequently to make their associated proteins, and which are left to lie dormant. The two best understood epigenetic mechanisms, acetylation and methylation, affect how much messenger RNA (mRNA) is transcribed from a particular gene. If there is more messenger RNA for a particular gene, then it’s easier to go the next step and make more of the protein that that gene codes for, and that gene’s expression increases. The mechanism that these researchers looked at is different. Instead of methylation and acetylation, they looked at microRNAs (miRNA) in the sperm of these mice. Where methylation and acetylation affect how much messenger RNA is generated, microRNAs attach to the messenger RNA itself after it has been created, and silence it.

The way it works is this: since RNA is basically half of a double strand of DNA, it’s really sticky. It wants to find something that looks like its complement and stick to it. So microRNAs are little bits of RNA that stick to particular messenger RNAs. Then when the cell takes those messenger RNAs and tries to use them to make a protein — it can’t. Because there’s this microRNA stuck to it, blocking the sequence of the message. So microRNAs reduce the expression of a gene, but they do it one step later on in the gene to protein pathway than methylation or acetylation does.

Back to our book example, it’s like if you have a cookbook (the DNA). You copy out a recipe on a piece of paper for later use (the RNA). Then you use the recipe to make cookies (the protein). Methylation puts big rocks in front of the bookshelf so you can't get to it and get at the cookbook. Acetylation glues the pages of the book together so you can’t read it. But microRNAs are your obnoxious husband who draws in marker all over your copied recipe, so you have to go back and copy it out again. (Disclaimer: while my husband is quite capable of being obnoxious, he has never defaced any of my recipes. He has scribbled notes on the medication list for my dogs in the face of my express requests to the contrary, however. Rosie hasn’t been on ciprofloxacin for six months but it still says “cipro” on her meds list. It’s like he’s incapable of thinking ahead.)

There is a lot we don’t know about microRNAs. The whole epigenetics field is like this: we are getting to the point where we can detect these changes, but we still don’t really know what they mean. So in this study, they found that 9 microRNAs were expressed at different levels in sperm of the stressed mice versus the unstressed mice. We can make some predictions, using computer algorithms, about which messenger RNAs these microRNAs were going to stick to and silence, but we don’t know for sure that that’s what they were actually going to do.

Still, the predicted list is pretty interesting, because it contains the messenger RNA for the enzyme which controls methylation. Methylation! Another epigenetic mechanism! So is there some epigenetic chain going on here? The dad passes on microRNAs which will result in the DNA of the offspring being more or less methylated. It’s so hard to know what that means, because methylation has very different effects depending on which gene is affected, and this change is a more global change. But it’s a really intriguing finding, isn’t it?

Conclusions
This study is exciting, but I still felt a bit of disappointment as I read it. No behavior changes? Really? Is it really significant without the behavior changes? I mean, do we really care about stress system changes if there are no behavior changes? Of course we do, and I wonder if future studies will investigate different behaviors, or behaviors at different points in the mouse’s life, and then we’ll understand this system a little better.

What does it mean for dogs? Of course it is immediately applicable to the question: if a male dog is stressed, will this stress affect his offspring? The answer is a nice solid maybe. In some way that we can’t really predict or define.

But at another level, this is another step in our progress towards understanding how genes and the environment interact. Stressful situations change gene expression in the stressed individual and possibly their offspring. How, why? How can we measure it? How can we use our knowledge to help an animal who has been traumatized, or undersocialized? Watching the field of epigenetics unfold is so much fun: everything is new, we understand so little, but the new technologies are coming so fast that we’re learning more and more.

Shelter dogs, movement, and stress

The always-awesome group of researchers at the Center for Shelter Dogs (associated with the Animal Rescue League of Boston, MA) has just published a new paper. They took a bucketful of different kinds of stress measurements of dogs in their shelter and looked to see if there were correlations between the different kinds. They are working on the same problem that I tackled in my Master’s work: it is awfully hard to tell which dogs in a population are stressed; can we use some kind of easy marker (like observing behavior) to do it? They got similar results to mine: yes, it’s super hard! There is no silver bullet answer. But they provide some interesting insight into how to move forward in the quest to improve stress detection methods.

Jones S., Dowling-Guyer S., Patronek G.J., Marder A.R., Segurson D’Arpino S. & McCobb E. (2014). Use of Accelerometers to Measure Stress Levels in Shelter Dogs, Journal of Applied Animal Welfare Science, 17 (1) 18-28. DOI: 10.1080/10888705.2014.856241

The biggest contribution this paper makes, I think, is the use of an accelerometer to test activity levels in shelter dogs. They attached this device to the collars of dogs when they first came in to the shelter, and got a report of how much the dogs moved around over the course of 24 hours. Because cortisol is such a difficult measurement of stress to interpret, it’s important to supplement cortisol measurements with other measurements, to sort of triangulate your answer. Movement in the kennel is one that I haven’t seen measured before in shelter dogs, and is something that might provide some interesting answers: do dogs move around a lot when they are stressed (pacing) or very little (depression)? I’m really happy to see this new measurement entering the shelter dog literature.

The researchers also attempted to provide a stress score of the dogs by watching them and subjectively assessing stress levels based on a minute’s worth of behavior. This is the holy grail of stress studies: can we look at a dog and tell how stressed it is? If so, we wouldn't need to do all this correlation with cortisol levels. I think we all want to say that someone who really understands dog body language can tell if a dog is stressed by looking at it. I know that I believe that I can estimate the average dog's stress level by looking at it. If you see a dog with low body posture, refusing to meet your eyes, maybe shaking, it’s stressed, right? How hard is that?

Well, if you try to compare your observations (particularly over a very short period of time — in my work, I had more luck with 20 minutes than with 2), the answer is, it’s extremely hard. In this study, they tried to assess an average level of stress in the dogs by taking multiple cortisol measurements, both salivary and urinary. Salivary cortisol changes so very fast that I wouldn’t expect it to correlate well to a day’s worth of exercise, or even to behavioral observations unless they were taken within just a few minutes of the saliva sample, but as this study involved multiple saliva samples over time, I was interested to see if a better average measure was obtained. The researchers also compared salivary cortisol samples with urinary cortisol samples, and since urine builds up in the bladder over time, I’d expect urinary samples to produce a better average as well.

So what did they find? To the question of movement (measured by the accelerometer) vs cortisol (salivary and urinary):

• Maximum activity level correlated with salivary cortisol (p = 0.025)
• Maximum activity level did not correlate with urinary cortisol
• Mean (average) activity level correlated with mean urinary cortisol (p = 0.028)
• Mean activity level did not correlate with salivary cortisol

To the question of the behavioral scoring: no correlation with cortisol of either kind.

So how we interpret all this?

First off, this study ended up enrolling only 13 dogs, taking quite a few measurements of each dog (I haven’t actually covered all of their findings here), and trying to draw conclusions. On the one hand, I want to emphasize that this is how stress studies in dogs are done. I did exactly the same thing in my stress study of hospitalized dogs. It is just extremely hard to enroll enough dogs. If you read the paper itself you get a feel for what the primary researcher went through, as she lists the reasons she had to exclude dogs from the study. It reminded me of my intense frustration during my Master’s work as I had to give up on dog after dog for a variety of reasons. This is par for the course in all these studies: it is almost impossible to get the time and funding to enroll enough subjects to have solid statistical findings. So I am not in any way criticizing these researchers, who did a great job introducing some interesting findings.

But on the other hand, it’s important to recognize that with so many questions and such a small sample set, it is almost impossible to trust the statistical significance of the results. If you do a study in which you ask 100 questions, and you set your p value at 0.05 (which is usual), then you are saying that you expect 5 of your answers to look significant even though they are not. That's what a p value is: setting the bar at which you accept a few false positives. One way around this is to have more subjects, which will lower your p values. Then you can say you'll only accept p < 0.01 or something even more stringent. This makes your findings a bit more trustworthy.

For the number of questions this study asked and the size of their sample set, I would take their findings with a grain of salt. Does activity level correlate with cortisol level? I think it's likely that it does. But I also think that what this study tells us is “this is an interesting area which is worth more study,” not “you should trust that these findings are absolutely true.”

Moreover, what are high cortisol levels telling us about dogs who move around a lot? Exercise itself can increase cortisol levels. This can be a good stressor. So are these dogs distressed or not? This is a problem which is going to be very hard to pick apart. I think a lot of the dogs in a shelter who pace incessantly are indeed very distressed, but some, as this paper points out, are coping with their distress by means of that exercise and are doing better than the dogs who don’t move. The paper also asks the question about how to interpret movement in small versus large dogs. Small dogs simply have relatively more room to move in a little shelter kennel. So how does that change the equation?

As for the lack of correlation between the behavioral stress score and cortisol levels: I feel pretty confident interpreting that one despite my comments about statistics above, because this is a question that has been asked before, and is always answered the same way. We can’t tell what a dog’s cortisol level will be by looking at its behavior. Why not? Is it because we don’t know what the dog's inner experience is (we don’t know if the dog is feeling stress)? Or is it the cortisol level that is lying to us, doing a terrible job of telling us about stress levels, and we’re interpreting the behavior just fine? We don't know, but we do keep trying to find out. Hopefully one day we will.

In the end, I enjoyed this paper. Kudos to the researchers for exploring stress levels in a variety of ways, instead of just one or two. This study was well designed, in my opinion; it just needed a lot more dogs. Hopefully this group or another one will be able to pull together a bigger study going forward.

Designing stress studies, part 3: how do you get the pee?

Having discussed how to choose what substance to test for cortisol (blood, saliva, urine, feces, hair), and how to get the blood or saliva, I now move on to how to collect the —

Urine

I don’t have any personal experience with collecting urine for stress studies. How hard can it be, though, right? I certainly was sent to collect urine from patients fairly frequently as a vet student, and have fond memories of chasing male dogs around a yard with a cup while they would spray just two or three drops at a time. The best vet clinics have long-handled soup ladles which you can use to collect the pee. I have certainly never used my own soup ladle to collect pee from my own dogs to take in for analysis when they were doing poorly.

One of my professors this past semester analyzed estrogen in baboon urine. Apparently one waits on the ground while the baboon is in a tree and watches. Eventually the baboon pees out of the tree. It falls on the ground and voila. Confused, I asked, “But doesn’t it soak into the ground? How do you collect it?” She explained that usually it fell onto a leaf and you could use a syringe to get it from there. I thought to myself: your world is not my world.

Getting pee from cats is a whole separate story. You provide them with a litter box with nonabsorbable pellets, and collect the pee from that. It sounds simple in practice, but in my experience many cats will refuse to pee on a non-absorbable surface.

Of course, if all else fails, you can extract urine directly from the bladder of a dog or cat using a needle. This procedure, called a cystocentesis, obviously requires trained personnel, who may not be available to all studies.

No post on pee would be complete without input from the queen of pee, Julie Hecht. When asked, Julie had quite a bit of advice about urine collection in dogs. She pointed out that when the study in question is being performed using laboratory animals rather than pets, you can teach the dogs to pee on command. This is super convenient, but you’re less likely to have that option with pet dogs. She listed some pitfalls that she found with colleting pee from pet dogs:

• Timing! If you need to collect pee before and after the particular event that you’re studying, it is problematic if the animal doesn’t feel the need to go at the right time.
• If you are out walking with the owner and the dog, try not to act weird. Dogs notice when you act weird. Then they don’t feel like peeing. So make casual conversation, even though all you are thinking about is collecting that lovely, lovely pee.
• Wind sucks. Wear plastic gloves.
• If a dog has a lot of fur, finding the urine stream can be hard. She says succinctly: “That stinks.”

So that is the lowdown on pee collection, and the conclusion of my series on designing stress studies!

Designing stress studies, part 2: how do you get your sample?

I recently posted about how to choose what bodily substance to use to test for cortisol in a stress study: blood, saliva, urine, feces, or hair. Once you have your substance of choice, though, you have to actually extract it from the dog. This can present more or fewer challenges, you know, depending.

Blood

When people first started measuring cortisol, they used blood to do it. Blood is where cortisol shows up first. All the other substances that we measure cortisol in have had their cortisol levels compared to blood cortisol levels, to make sure that they correlate strongly. Researchers had to do studies to prove that these other substances worked for this measurement, which cost a lot of effort and money. They did this because blood is pretty hard to get hold of, in most cases. Sticking a needle in a dog will usually stress it out, and it's hard to get the blood extracted before the stress of the restraint starts changing the blood cortisol levels.

But even aside from that, sometimes a blood draw is simply out of the question. For my Master’s work, I had to cold-call hospital clients and convince them to let me enroll their dog (already in the hospital for some procedure or other, in other words, already having a bad day) in my study. If I had told them that the dog would need a blood draw too, I guarantee that most of them would have said no.

In a comment on the previous post in this series, Tegan pointed out that animals can be trained to submit calmly to blood draws. For some studies, this approach would be invaluable. For my study, again, it wouldn’t have worked. Training an animal to accept a needle is an arduous process, and I had access to those dogs once, on one night. For most shelter dog studies, this would also be an impossible hurdle. But it’s a pretty cool thing to do, if you can do it.

I wish I had a video of another approach to stress-free blood draws. I have seen other vets slide a needle into the lateral saphenous vein, the vein that bulges out of the side of a dog’s hind leg just above the hock. If the dog is distracted (say by someone feeding it), a competent venipuncturist can get it done using this vein with little to no stress. I have seen this technique used in shelter dogs who would not allow restraint for a more traditional draw. But it takes a dog with short, smooth fur and a particularly lovely bulgey vein. It does not work in little dogs. And it definitely requires a competent person to do the draw. After a few years of practice in blood draws, I was just getting to the point during my internship where I could do this one. There can’t be too much poking around to find the vein, or the game is up.

(I did find a video of a technician drawing from the lateral saphenous of a dog who is lying on his side, with an assistant holding off. This is the same vein as the one I am talking about, but in the procedure I’ve seen, the dog can be standing and you actually don’t need someone else to hold off the vein. You come at the vein from above, not below, in a standing dog. Just in case any of you blood-drawers out there want to try this yourself.)

Since blood was such a pain to get, people started trying other substances, figuring anything had to be easier than a blood draw.

Saliva

Saliva is now used much more often than blood in human cortisol studies. You hand a person a cup and they drool into it. No needles, no added stress. Dogs are not so easy. You can’t ask a dog to drool into a cup; you have to get the drool out yourself.

For my study, I used Sorbettes, also known as eye sponges. The instructions say to put one Sorbette into the dog’s mouth for 30-60 seconds, and voila, it has enough saliva on it for an assay. You then put the Sorbette into a tube and spin the tube in a centrifuge to get the saliva out. You only need 25µg, which is hardly anything! What could go wrong.

Sorbettes

First of all, when you are analyzing the saliva later on, you use 25µg per well in the plate of saliva samples, and you get one cortisol value per well. But it turns out that the assay is fairly imprecise, and gets it wrong a decent percent of the time, sometimes close to 10% of the time. So it makes sense to use two wells per sample (now we are at 50 µg per dog). This way, if you get two very different answers for your two wells, you know that the assay went wrong and not to use one of the samples. Wait, which sample is good and which sample is bad? To avoid that problem, just use three wells per sample (now 75µg per dog). Then you can throw out the bad one and keep the two good ones. I had to do this maybe 4-5 times total out of my 90-odd samples. Every time, I was really glad that I had three wells. With two wells I would have had to discard that sample (and that dog) from the study. With one well I would have included bad data in my results.

So 75µg is still not all that much saliva, but it turns out that it is enough to be pretty difficult to get, especially from dogs who are stressed out in a hospital. I used three Sorbettes and rolled them around in the dogs’ mouths for up to four minutes, at which point I had to stop in case the stress of restraint was affecting the cortisol levels. Even then, I had a lot of dry sponges. It was incredibly disheartening. In the end, we saved most of my samples by a) diluting them and changing our calculations, and b) showing the dogs cans of cat food to make them salivate.

I am currently engaged in an email exchange with other researchers who are having similar problems, particularly in small breed dogs and puppies. These days, the new tech to use to get saliva out of dogs is a small rope which the dog can chew on. I like that better than the little sponge-on-a-stick, which dogs could possibly break off and swallow (I had one come perilously close to doing just that). But even so, the problem of getting enough spit remains.

Could you give the dogs food? There is a study suggesting that cheese will not interfere with the cortisol assay, and would be safe to give. [1] It makes me nervous, though.

Could you condition the dogs to salivate when you present the little rope? This is currently under discussion, but some of us are concerned that messing around with the dog’s experience of sampling would invalidate the sample. It’s worth a small study to test it out, though, for sure. I hope someone does it.

By the way: I heard a story, which may be apocryphal, but I will repeat it anyways (and maybe someone out there can corroborate): supposedly a rhino salivary cortisol study used the procedure of collecting saliva with a very long-handled spoon. If true, it is awesome.

To come: urine, feces, and hair, oh my.

References

[1] Ligout S., Wright H., van Driel K., Gladwell F., Mills D.S. & Cooper J.J. (2010). Reliability of salivary cortisol measures in dogs in training context, Journal of Veterinary Behavior: Clinical Applications and Research, 5 (1) 49. DOI: 10.1016/j.jveb.2009.09.027

Designing stress studies, part 1: what do you sample?

Apparently I am an expert in designing stress studies in dogs using cortisol, because I have published one paper about it. Here are some of the words of wisdom I have to share from my extensive experience. You may also be interested in my previous post from several years ago, Why cortisol sucks as a measurement of stress. As I have so many words of wisdom to share, I am going to start with a post just on what you should sample in order to get some cortisol levels. (I intend more posts to follow. But you know how these things go.)

You can measure cortisol in blood, saliva, urine, feces, or hair. We consider the blood (plasma) measurement to be the gold standard: when the adrenals release cortisol, they release it into the blood. This is the hardest to get (you have to stick a needle into the dog) and the fastest to change. Blood cortisol starts increasing only 3 minutes after the onset of a stressor. Practically, this means that since sticking a needle into a dog is likely to stress the dog, you have to complete the blood draw (probably including catching and restraining the dog, unless it is a very mellow dog) in under 3 minutes! [1] This can be possible to do with some dogs and impossible with others. Either way, it requires someone who is very competent at blood draws.

After cortisol is released into the blood, it diffuses into the saliva. This process takes about a minute, so you should collect the saliva less than 4 minutes after you stress the dog by restraining it. [1] If the dog really doesn’t mind the restraint, you can take longer, but I found that sticking things in a dog’s mouth to collect saliva tended to get them excited. In a hospital, just walking into the dog’s run got most dogs excited!

Blood and saliva are the best ways to measure the immediate response to a stressor: take a baseline measurement (in under 3-4 minutes), stress the dog, wait some period of time, then take the post-stress measurement (in under 3-4 minutes, in order to be sure you’re measuring the correct stressor). Taking a single measurement of blood or saliva is not going to tell you as much: there is no known baseline of cortisol for any species, including dogs. It varies too much hour to hour, not to mention that some individuals just start at a different level when they are unstressed. [2]

So take one sample before the stressor starts. After the stressor starts, how long do you wait to sample again? Definitely the same amount of time for each dog. Studies have mapped the time course of cortisol’s rise and fall after a stressor: it seems to go up for an hour or so and then come back down [3]. This is almost certainly dependent on the stressor, of course. My personal rule of thumb is that 20 minutes is a good amount of time to wait to make sure that the cortisol levels have come up enough to be a good reflection of the dog’s reaction to the stressor you’re measuring. (So, just to be super clear: the 3-4 minute rule is just about the beginning of the rise in cortisol levels. The rise will continue for a while.)

If you are interested in how an animal is responding to a chronic stressor, like a few days or weeks in a shelter environment, you’ll be more interested in some measurement of cortisol which covers a longer time period than 20 minutes. Saliva and blood are awful for this kind of study, because their cortisol levels change so fast that you aren’t getting a good overall picture of daily cortisol level; you’re getting more of a snapshot. You could take hourly samples, but that would be difficult in terms of collection and expensive in terms of analysis.

For this kind of study, most people use urinary cortisol. Technically this is the cortisol to creatinine ratio: what is the ratio of cortisol to a standard urine molecule, creatinine? Measuring cortisol this way standardizes your measurement so that it isn’t affected by how dilute the urine is. Urinary cortisol levels will provide something like an average cortisol measurement over however long the dog has been filling up its bladder, probably about 4-6 hours. Urinary cortisol has  been used as a measurement for chronic stress in shelter dogs [4], where you are interested in average stress levels, not an immediate stress response. (For more on measuring stress in shelter dogs using cortisol, see the excellent recent review by Hennessy. [5])

One interesting study looked at elevations in urinary cortisol after dogs had had a trip to a veterinary clinic [6]. In this case, I worry that measuring a specific stressor that has a beginning and an end prior to urine collection is difficult with this method. When did the dogs start making that urine? Before they got stressed, while they were stressed, after they stopped being stressed? When you are comparing different dogs’ urinary cortisol, are you comparing the same thing?

I rarely see studies using fecal cortisol to assess stress in dogs, beyond the proof of concept study [2]; these studies are mostly done in wild animals, because poop is the only thing you can easily collect from them. I have always thought that fecal cortisol might actually be a really good approach to stress measurement in shelter dogs, though: easier to collect than urine, and measuring a longer period of time than urine (since dogs urinate more often than they defecate), so therefore presumably getting a better average. Today as I was looking on Mendeley for some references for this post, I encountered a new study using fecal cortisol to assess stress in cats. [7] Cool.

You can actually measure cortisol in hair as well! I have not seen this done in dogs. It would be a good measurement of even longer term stress levels, over months. One fascinating study measured cortisol levels in archaeological hair, to determine cortisol levels in prehistoric humans. [8]

So, in summary: saliva or blood are good samples to take for a response to an acute stressor, usually one you have control over. Take a sample before the stressor begins and then about 20 minutes after the stressor has begun. Be careful to take your samples very promptly to make sure you are not measuring the stress of the sampling. Urine and feces are better measurements for chronic stressors, and provide a several hour summary of what the cortisol has been doing in the dog’s blood. You can take just one sample of these to compare to your control group.

References

[1] Kobelt A.J., Hemsworth P.H., Barnett J.L. & Butler K.L. (2003). Sources of sampling variation in saliva cortisol in dogs, Research in Veterinary Science, 75 (2) 157-161. DOI: 10.1016/S0034-5288(03)00080-8

[2] Schatz S. & Palme R. Measurement of faecal cortisol metabolites in cats and dogs: a non-invasive method for evaluating adrenocortical function., Veterinary research communications, PMID: 11432429

[3] Vincent I.C. & Michell A.R. (1992). Comparison of cortisol concentrations in saliva and plasma of dogs, Research in Veterinary Science, 53 (3) 342-345. DOI: 10.1016/0034-5288(92)90137-Q

[4] Stephen J.M. & Ledger R.A. (2006). A longitudinal evaluation of urinary cortisol in kennelled dogs, Canis familiaris, Physiology & Behavior, 87 (5) 911-916. DOI: 10.1016/j.physbeh.2006.02.015

[5] Hennessy M.B. (2013). Using hypothalamic–pituitary–adrenal measures for assessing and reducing the stress of dogs in shelters: A review, Applied Animal Behaviour Science, 149 (1-4) 1-12. DOI: 10.1016/j.applanim.2013.09.004

[6] Vonderen I.K., Kooistra H.S. & Rijnberk A. (1998). Influence of Veterinary Care on the Urinary Corticoid: Creatinine Ratio in Dogs, Journal of Veterinary Internal Medicine, 12 (6) 431-435. DOI: 10.1111/j.1939-1676.1998.tb02146.x

[7] Gourkow N., LaVoy A., Dean G.A. & Phillips C.J.C. (2014). Associations of behaviour with secretory immunoglobulin A and cortisol in domestic cats during their first week in an animal shelter, Applied Animal Behaviour Science, 150 55-64. DOI: 10.1016/j.applanim.2013.11.006

[8] Webb E., Thomson S., Nelson A., White C., Koren G., Rieder M. & Van Uum S. (2010). Assessing individual systemic stress through cortisol analysis of archaeological hair, Journal of Archaeological Science, 37 (4) 807-812. DOI: 10.1016/j.jas.2009.11.010

Links post

I’m catching up on science blogs reading after a few days off. I was awfully busy recovering from defending my thesis! I am now almost done with the MS part of my dual-degree program, though there will be some thesis edits to do. Then I settle in for the final two years of the DVM program.

Anyways, links!

• The PepsiGate linkfest (A Blog Around the Clock): so comprehensive, he even linked to me.
• Mesozoic Blogosphere (Chasmosaurus): David Orr considers the usefulness of topic-based networks. The comments suggest aggregators to achieve this goal.
• “Dominance” mythologies, Suzanne Hetts (The Other End of the Leash): More on dominance theory in dog training from Patricia McConnell
• Tick news? It ain’t good, Dr. Flea tells AVMA audience (Pet Connection)
• Environmental enrichment is key to happy, healthy animals (Pet Connection): This seemed like a relevant link after The Thoughtful Animal’s recent post about behavioral differences in pigs in enriched environments.
• On detecting stress endocrines in hamster poop (C6-H12-O6): need I say more?
• Learning to speak dog (Dog Star Daily): the usefulness of understanding canine body language, and some good pointers
• You are what you eat – how your diet defines you in trillions of ways (Not Exactly Rocket Science): Nice post about how populations of gut bacteria are influenced by diet in different life stages and in different cultures. “As we learn more about our bacterial partners, we might eventually find ways of influencing them to improve our health, just as breast milk appears to selectively nourish helpful species.” He suggests inoculating people with appropriate gut bacteria, which makes me a little sad. I’d rather see people change their eating habits. Anyone for some research on the effects of fresh whole foods on populations of gut bacteria?
• Under Pressure: The Search for a Stress Vaccine (Wired): What is it today with links to articles about fixing problems with injections? Actually, this is a really good article about Robert Sapolsky, who did ground-breaking work on the effects of chronic stress on health. Apparently Sapolsky is now working on a vaccine to counter the neural effects of chronic stress. I have to admit that I find that a little scary. It sounds like a great answer to the problem of a society full of highly-stressed people, but the stress response is so complex and affects so many parts of our metabolism that it just can’t work without horrible side effects, can it? (The article addresses some of the issues.)
• Virginia Heffernan Is Our Target Audience (Uncertain Principles): For those who don’t know the background, Heffernan wrote a piece in the New York Times in which she criticized Scienceblogs.com for having some snarky people on it, and said as a result of its tone, she didn’t find it to be a good place to go to learn about science. Various science bloggers have opined that she’s dumb and no one should change what they are doing. Here, Uncertain Principles suggests perhaps science bloggers should be trying harder to speak to this particular audience. I’m not going to write a whole blog post about it, but I vote with UP and the others who’ve voiced this particular opinion. Who cares who’s right? The important thing is getting your message across, and it’s pretty clear that some members of the audience find a less snarky message to be easier to absorb.

Why do other measurements of stress suck worse than cortisol?

After an overwhelming number of requests (2) for a sequel to my post Why cortisol sucks as a measurement of stress, I am obliging. The fact that I am in the middle of writing this particular section of my thesis and need some high-level perspective on it might also have something to do with it. So: why do other measurements of stress suck worse than cortisol?

When I left you, you were trying to design a study of stress in hospitalized dogs using cortisol as your marker of psychological distress. You were confounded by the fact that cortisol measures both psychological and physiological distress, and that it varies a lot between individuals. I haven’t been around to keep an eye on you lately, so you have started investigating other approaches to measuring stress other than cortisol.

Cortisol is a messenger used by the HPA (hypothalamic-pituitary-adrenal) axis, for the brain to send a message about stress levels out to the body, for the body to pass that message along to the organs that need to change their operations as a result, and for the body to then report back to the brain that the message has been received, so the brain can stop yelling about it. There are multiple levels in this axis; cortisol comes from the bottom-most level, the adrenals. Why not go up one level, to the pituitary? It is actually in the brain, so it is closer to the source of the message and might be less distorted by the game of telephone.

The hormone that the pituitary gland releases as part of the HPA axis is ACTH (adrenocorticotropic hormone, or “the hormone that makes the adrenal cortex change”). ACTH causes cortisol release. Why don’t you measure ACTH release directly? Unfortunately, ACTH can only be measured in the blood; it doesn’t get into the saliva. (Or urine, hair, or feces, three other places you can go to get an estimate of cortisol levels.) The owners of your hospitalized dogs aren’t going to be happy if you tell them you need to draw blood from their dogs for your study. And remember, you’d have to draw the blood pretty quickly in order to get it before the brain mounted a stress response as a result of having a needle stuck into the body. Cortisol levels change in under three minutes. I don’t actually know how long it takes ACTH levels to change, but I will hazard a guess that since they are farther up the telephone chain, they change faster.

What about farther down the chain? CBG (corticosteroid binding globulin, a.k.a. transcortin) is a protein that carries cortisol around in the blood. The body uses CBG as a way of regulating the stress response. When there is less CBG, cortisol is more able to jump inside cells and do its work. OK, no one actually uses CBG to measure stress levels, because we have no real idea how it works. But it is a very cool system that I’m really curious about. And stress researchers would do well to remember that it is there. If the dogs you are studying are very sick, they might not be able to make as much CBG as a healthy dog would, and that would affect their cortisol levels.

That pretty much exhausts using the HPA. Luckily there is an entire second axis for you to mine: the SAM (sympatho-adrenomedullary) axis. This is the series of chemicals that regulate the well-known “fight or flight” response. This particular game of telephone includes adrenaline (epinephrine), the effects of which which many people enjoy abusing when they go on roller coasters. This axis works much more quickly than the HPA. If you hear a sudden loud noise, you will get an adrenaline rush within a second. So you can try to measure adrenaline levels in the blood, but there is just no way you will be able to get the blood out fast enough to not have the stress of the needle (damn needle) affecting them. If you had a very controlled population of animals, with catheters already placed that they were used to, so that you could draw out blood without stressing them, that might work, assuming you could catch the animals without stress. (Catch a mouse without stressing it: difficult. Catch a dog without stressing it: actually, when I went into the runs with the hospitalized dogs I was studying, they definitely experienced eustress, or happy stress.)

You can also measure adrenaline levels in pee! This turns out not to be useful, though. Adrenaline levels go up and down, as we’ve said, very quickly, in response to individual stressors. Pee collects all those changes and averages them out over however many hours (say six). So this approach is definitely not good for measuring responses to specific stressors, like a sudden loud noise. It might be better at measuring something longer term (hey, like the response to being in a hospital!) but initial studies haven’t shown it to work very well at that, either. Adrenaline is just the most interesting when you can map it as it goes up and down, not when you have to look at an average and guess about what was smoothed out.

What about the other end of the SAM? When you get an adrenaline rush, you have some physical changes. Among many other things, your heart rate gets faster. Can you measure that? Well, again, good luck measuring that in a dog without having the excitement of interacting with a human confound your measurement! And heart rate is very sensitive to physical changes; you might be measuring whether the dog is standing up versus lying down, rather than its level of distress.

It turns out that what is a better way to measure physiologic changes from SAM activation is heart rate variability. Your heart rate normally speeds up a little when you breathe in, and slows down a little when you breathe out. (I actually did notice this in a dog once, in a lab where I was supposed to be learning how to find abnormal heart rhythms, and I had to call a vet over to ask if it was actually normal, because it sounded so weird once I noticed it.) When you are stressed (physically or psychologically), this variability goes away. This is not a bad way to measure stress, but you can’t measure it with a stethoscope; you have to hook up equipment to the dog in the form of a little vest with a monitor attached. This is expensive (too expensive for you to use, because your project is on a shoestring budget!). You would also have to get the dog used to the vest, so that you were sure you wouldn’t be measuring stress from having clothing on when the dog is used to being naked. It is therefore not a good measurement for hospitalized dogs on their first day in the hospital, but it is a good measurement for some studies. It’s best when used in conjunction with cortisol, so that the two measurements can catch each other’s mistakes.

That uses up the SAM, but there is a system that is the opposite of the SAM. When your body is not in “fight or flight” mode, it is in “rest and digest” mode. This mode is regulated by the parasympathetic branch of the ANS (autonomic nervous system). (The SAM is the sympathetic branch of the ANS.) Can you measure parasympathetic activity? It should increase when stress decreases, and vice versa. It turns out that when your body is thinking it’s time to rest and digest, it releases a digestive protein into your saliva, known as α-amylase. This protein is useful for pre-digesting carbohydrates. More α-amylase suggests less stress. And it’s even in the saliva, so it can be measured non-invasively! You are very excited until you find a paper from the 1950s (I am not kidding) which is the last time anyone bothered to look for α-amylase in dog saliva. Dogs don’t make it. Because they are not meant to eat lots of carbs? Oh wait, this isn’t a post about nutrition.

(For those of you who say “OK, but what about measuring stress via α-amylase in humans?” — I didn’t delve any deeper into this one after I learned it wasn’t useful in dogs. My guess is that it suffers from similar problems to measuring cortisol: it measures more than just [lack of] distress. It also has been less widely used than cortisol, so we understand its pitfalls less. This would be another good measurement to use as a complement to measuring cortisol. If you want to use it in humans, read lots studies that have used it before you commit.)

So much for the ANS. But you know that increases in stress cause decreases in parts of the immune system. In fact, that’s partly why we care about stress in hospitalized dogs — stressed dogs may not heal as quickly or as well. Can we measure the immune system?

We can. Your saliva normally contains a kind of antibody called IgA. This presumably provides a first line of defense against the bugs on your food. When you are stressed, you make less of it. (At a guess, this is because when you’re running from a lion, you’re not likely to be eating. You’re more likely to be getting bitten, so your immune system needs to focus on defenses against open wounds instead of microbes in food.) Salivary IgA is known as “sIgA.” Can you measure that in dogs? You can, and it is being fairly widely used in humans, in fact. Only some initial work has been done on it in dogs, though. It seems to be prey to some of the same issues cortisol is — varying regularly throughout the day, varying irregularly between individuals — so it’s not yet clear if it’s really a better option. It might be a good way to go for a long term project. For something short, though, it might be better to stick with what is well-understood.

Are there any other ways to measure immune system function as it relates to stress? As I said, your immune system reorients when it thinks you’re running from a lion, to protect against open wounds. It does this in part by packing the blood full of a kind of white blood cell called a neutrophil. Neuts are the first line of defense against microbes coming in through open wounds. You can measure their ratio to another kind of white blood cell, a lymphocyte, to measure stress. A greater N : L (neutrophil : lymphocyte) ratio implies greater stress levels. In some ways, this is a really great measure of stress, because it takes a little while — an hour or so — for the N : L ratio to change after a stressor. So when dogs first come in to the hospital, if you can get blood right away, you could actually measure their unstressed baseline. A later blood sample could provide a comparison. Then you could ignore all that annoying individual variability, because you would be measuring the difference pre- and post-stressor in the same individual. I would have loved to have use this measurement.

But, as always, good luck getting an owner to consent to not one but two unnecessary blood draws. I am not sure I would have felt good about adding that much stress to an already stressed dog’s hospital visit, either. For a different kind of study, this might be a really good option, though as always, it measures the effects of multiple systems, so there is going to be some extra variability to account for.

And that is why, though cortisol is a really appalling way to try to measure stress (looking at my salivary cortisol data right now, I keep saying “why does anyone use this hormone?!”), it is still the most widely used approach. As we learn more about how all these systems interact, it is possible that some day we will develop a method of taking multiple kinds of measurements and basically triangulating distress. Or maybe we’ll develop hand-held fMRI scanners and be able to directly measure activation of specific parts of the brain. For now, we are stuck with spit.