Monday, October 27, 2014

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?

Saturday, October 18, 2014

Domestication and human evolution, streaming!

Domesticated humans, domesticated canids, domesticated finches! The Center for Academic Training & Research in Anthropogeny held a conference on domestication and human evolution, and live streamed it. I watched live, refusing to speak to my husband or dogs during it, which didn’t go over well. You, however, can watch the archive on YouTube.

The conference was a series of short talks from researchers. These included:

Robert Wayne, “The transformation of wolf to dog: history, traits, and genetics”

Wayne’s lab published a recent genetics paper on exactly where the dog was domesticated, and in this talk he stepped us through their findings. Previous work (and there has been plenty of it) on the location of dog domestication has begun with the assumption that dogs evolved from a population of wolves which still exists basically unchanged, inhabiting the same range now as it did then. Previous work has suggested that this occurred in Asia or the Middle East. Wayne’s group argues that the population of wolves which gave rise to dogs no longer exists, but lived in Europe about 20,000 years ago. It’s a different perspective on a question which is very difficult to answer, because dogs continue to interbreed with wolves so freely that these genetic studies are awfully hard to interpret.

Anna Kukekova, “Fox domestication and the genetics of complex behaviors”
This talk came out of the lab where I work and I got to see my own name on the list of contributors at the end of the talk. Kukekova gave an overview of the history of the fox domestication project, in which lines of foxes were selectively bred for tame temperament or aggressive temperament, and recent research. Our lab digs in to the question of what it is in the genetics of the tame foxes that makes their personalities so different from those of conventional farm foxes. Since this conference was about domestication, and the tame foxes are the best known and longest running domestication study, speakers returned to the foxes throughout their talks. They are a tough nut to crack. Behavior is exceedingly complex mechanistically and we (by which I mean all behavioral geneticists, not just our lab) are still trying to figure out how to get a handle on the genetics that affect it.

Robert Franciscus, “Craniofacial feminization in canine and human evolution”
Craniofacial feminization means that your face is flat, basically. Look at the reconstruction of a Neanderthal face: the chin juts out. Look at a modern human face: flat. Look at a chimpanzee face: jutting chin. Look at a baby chimpanzee: flat. Do humans look like baby chimps? We kind of do. Is there a significance to this? Franciscus argued convincingly that there is, and discussed differences in dog versus wolf muzzle length (and got quite technical about how his group investigated them). We don’t know why this feminization or neotenization (childlike changes) happens in domestication, but it seems to be a recurrent theme. This was the first talk that really grappled with the idea that humans are domesticated, with changes compared to our recent ancestors that parallel changes between dogs and wolves, or between tame and conventional foxes.

Terrence W. Deacon, “The domesticated brain”
Do domesticated animals have smaller brains than their wild counterparts? This is certainly the case in dogs and wolves. Is it the case in humans and our ancestors? Deacon’s group has studied Neanderthal brain size based on their skulls, and they conclude that modern humans do not have clearly smaller brains. He noted that the tame foxes also do not appear to have smaller brains than their conventional counterparts. Why does the difference in brain size show up in only some, but not all, examples of domestication? Is it perhaps not a necessary part of the domestication process?

Phillipp Kaltovich, “Neotenous gene expression in the developing human brain”
It is pretty difficult to study gene expression in human brains, because you have to cut up brains to get your data. Kaltovich did get his data from somewhere, though, and it was really interesting to see his comparisons of gene expression in young versus older brains. The question was whether gene expression changes with age, which helps get at the bigger question, are there gene expression differences in domesticated species compared to their wild counterparts, and are these expression differences similar to the differences in young versus mature animals? In other words, are domesticated species basically enternally young (neotenized)? He did find differences, but his group will have a long way to go to put them together into findings that really illuminate the domestication question. I have a lot of sympathy for how hard this particular approach is, as my research is currently also focused on brain gene expression.

Tecumseh Fitch, “The domestication syndrome and neural crest cells: a unifying hypothesis”
In a recent paper, Fitch’s group put forth the concept of a domestication syndrome, a set of changes associated with domestication: flatter face, behavioral changes, white markings, etc. Subsets of these changes are seen in all domesticated species. Fitch’s group hypothesizes that a particular kind of cell involved in early development is involved in all of these changes. This cell, the neural crest cell, migrates through the growing embryo and develops into many different structures and cell types, including coloration cells (explaining white markings), teeth (explaining dentition changes), and the adrenal medulla (source of adrenaline, explaining behavioral changes). It’s an interesting hypothesis and I’ll be curious to see where this group goes with validating it.

Kazuo Okanoya, “Domestication and vocal behavior in finches”
Okanoya’s group studies a species of domesticated finch and its very closely related wild ancestor. The wild finch has a simple song, while the domesticated species has a quite complicated one. Okanoya’s group investigates the difference in these songs, as a model for the development of complex language in domesticated humans. He played both songs, wild and domesticated, and the difference was dramatic. He linked the changes in the song between species to sexual selection.

Richard Wrangham, “Did Homo sapiens self domesticate?”
The question of self domestication was one of the recurring themes of the conference, and for me this was the most transfixing session. Wrangham studies chimpanzees and bonobos, two closely related species with very different aggression levels, as models of the difference between humans and our more aggressive, non-domesticated ancestors. He defines domestication as the reduction of reactive aggression. Reactive aggression is different from proactive aggression: humans are quite good at controlling our reactive aggression, as we are able to tolerate strangers and live in large groups very well. But we do still show significant proactive aggression, which he described as aggression performed in cold blood, such as armed robbery. Wrangham suggests that a reduction in aggression is the trait evolutionarily selected for in self-domestication, and the other parts of the package (flatter faces, white markings) come along for the ride as associated traits. Self-domestication is often seen in island species, and he gave the example of the red colobus with a neotonous (childlike) island version compared to the mainland population.

The whole conference was really fascinating. It’s available on YouTube now, so go, check it out!

Thursday, October 16, 2014

State of the Zombieverse

I haven’t been blogging much lately, and it’s mostly because I’ve been writing so much for more mainstream media outlets:
  • “Neutering without a Scalpel” in the summer issue of the Whole Dog Journal. This story was about Zeuterin, a new product for performing chemical castration on a dog — in other words, non-surgical neutering. I tried to cover all the possible pros and cons of using Zeuterin versus the traditional surgical approach. I don’t think either solution is going to be right for every dog or every situation, and it’s nice to see new options coming out.
  • “Testing the Tests“ in the fall issue of The Bark. I read so many journal articles about canine behavioral assessments (also known as temperament tests) for this piece. You know, the tests that are often used in shelters to try to identify behavior problems in dogs before they’re put up for adoption. It was fascinating reading and ended up telling an interesting story about researchers’ attempts to figure out whether behavioral assessments are good at predicting canine behavior. The answer: not really, but some new research approaches have appeared recently which have shed a lot of light on how to interpret these tests and how to approach building better tests.
  • I’ve also been writing for DVM360 (I have two pieces going through the pipeline there right now, one about shelters and one about stress), I have another piece pending for the Whole Dog Journal, a guest post for a new dog site, and you already know about my upcoming ultra short, ultra fun online class for APDT, right?
So with all of that, I haven’t been blogging much, but I keep thinking about it and missing it. I figured I should at least report here on where to find me.

Wednesday, October 15, 2014

Journal articles, stress, and anti-depressants with me and APDT

I’m teaching another online course for APDT: Analyzing Journal Articles: Pre-Natal Stress and Anti-Depressants. I’m trying something new with this one. It's just one week long. Basically, I’m going to be walking through a recent journal article that I thought had interesting implications for dogs. There will be several short lectures at different experience levels. Some will provide background in the area to students who don’t have an extensive science background. Some will be aimed at students who do know a lot of science and want to dig deeper into stuff that isn’t often covered in online courses. And some will be straightforward “what is this article about?”

It's an interesting article, about whether stress before birth can affect an animal’s personality and whether anti-depressant medication change reverse those effects. Hopefully it isn’t hard to see how this could be relevant to dogs with behavior issues.

If we don't get at least ten students signed up, we won’t do it, so go sign up now! You will get CE! If people do sign up and do enjoy it, I’m hoping to do more like this -- providing a way for dog trainers to keep up on recent research and get a better feel for what it’s like to read a scientific paper. So this is my test case. Will people be interested? I really hope so!

[ETA: Although the last time I checked, the APDT web page for this class lists it as "no CEUs," I checked with their education coordinator, and students completing this class will earn 4 CEUs. So yes, you will get CEU credit for it!]

Tuesday, August 12, 2014

Veterinary super heroes!

V-FORCE from the AVMA


I was tickled to discover that the AVMA published a comic book. V-Force! Veterinarians to the Rescue! Did you know that veterinary superheroes have x-ray vision and use it to diagnose leptospirosis? Did you know that some of them (us? can I say some of us?) even have microscopic vision so that they can see microbes and diagnose infection with West Nile Virus? Why didn't I get these super powers when I graduated?

My favorite scene bar none is the one in which the doctor (that is doctor-of-humans not doctor-of-animals) says "I'm just glad the veterinarian was around to help link the cause of the disease." Words spoken by no doctor ever. More fantastical than teleporting veterinarians?

The point of the comic is to leave it lying around in veterinary waiting rooms for small children to pick up, so that they learn a) some of the alternate careers vets can have besides working as small animal clinicians and b) that veterinary medicine isn't just about helping animals, it's about helping people, too. (OneHealth to the rescue! I want a super hero named OneHealth Man!)

I like this effort by the AVMA to communicate some important information about veterinary careers, but I do wonder if a somewhat heavy-handed comic is the best way to do it. You're not going to get a lot of bang for your buck with it (although on the other hand it probably didn't cost all that many bucks to produce, relatively). Hey, AVMA, are you listening to me? (AVMA reps have commented on my blog posts in the past when I've made enough noise, so consider my voice raised now.) I have an idea.

Did you see the recent SyFy show Helix? Which actually starred a veterinary pathologist, and yet didn't do a great job of portraying what veterinary pathology is really like or why it's an exciting career. I think the AVMA, if they are serious about this goal of getting the public to better understand what veterinarians are capable of, should be trying to get more veterinarian characters on TV shows and in movies, and make sure they are well characterized. I suggest one way to do it is to offer a liaison service to Hollywood: the AVMA would provide a veterinarian consultant appropriate for the particular role, and would pay their salary and expenses for the job. The consultant would help the writers make the role realistic.

I'm completely serious about this: I think Hollywood (or part of it) is starting to realize that a realistic depiction of scientists is something its audience is actually interested in, but it's got to be hard to find someone who can speak to the daily life of a veterinary pathologist. Make it easy for them. Because a favorite character on a TV show is something that makes kids, and even adults, consider their future career options in a different light, not a comic book with a clunky story.

Wednesday, August 6, 2014

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.

Monday, July 28, 2014

What percent nature? What percent nurture?

The Nature versus Nurture debate is over: we no longer ask if genetics governs personality or if environment does. They work together, and it’s hard to pick their effects apart. But surely we can pick their effects apart a little? For example, if a dog trainer is trying to impress upon their students the importance of getting a puppy from a good breeder who takes behavior into account — or conversely, the importance of bringing a new puppy to a puppy class: what should she tell them? 50/50? 60/40? Surely there are some numbers we can cite?

It’s a tough question, but one that researchers have tackled. The concept is called heritability: the measurement of how much of a trait is due to genetic influences, and how much is due to environment.

Human researchers have it easier than dog researchers, because humans sometimes produce identical twins, and twin and adoption studies form the basis of human heritability studies. Some twins are identical (100% identical genetics), some are fraternal (around 50% similar genetics); some are raised in the same home, and some are adopted out and raised separately. You can do some complex math to all of these situations and come out with conclusions about particular traits. Identical twins more similar than fraternal twins for a particular trait? Strong genetic component. Raised together twins more similar than raised apart twins? Strong environmental component.

These studies have given us some numbers: IQ (how someone scores on a particular standardized test) is about 40-50% heritable. Environment does the rest.

Dog studies are harder. Dogs don’t have identical twins. Theoretically, the best way to study the heritability of personality traits in dogs would be to breed parents who do or do not show the trait in question and assess the puppies, then rinse, wash, and repeat for several generations. But this is expensive and somewhat ethically fraught to do in a laboratory, so we fall back on finding populations of dogs whose personality traits have been well measured and whose pedigrees are well known.

How often does that happen? Not very. But there is a test, the Swedish Dog Mentality Assessment (DMA), which is given to a large percentage of dogs in Sweden and some other European countries. Those crazy, overly-responsible Europeans measure their dogs’ personalities before breeding them, to make sure they're breeding stable dogs. Researchers have mined this resource repeatedly to learn more about the heritability of a variety of personality traits.

As lucky as we are to have this resource, it’s not an ideal one. The DMA is a suite of behavioral assessments which are given to a dog on a particular day in a strange environment by a judge who doesn’t know the dog well. Ideally, personality is best measured over time, by someone who knows the animal very well — its owner. And, in fact, every study I read that evaluated heritability of personality using the DMA noted that one of the most important factors was not genetics but the identity of the judge who gave the test. Did some judges tend to judge more severely than others? Did dogs respond differently (more or less fearfully, perhaps) to different judges? Hard to say, but we know that the reliability of the test suffered as a result.

Perhaps more alarmingly, we’re not really sure about the validity of the test, either. What are these assessments actually measuring? They’re measuring the response of a dog to a particular stimulus in a particular situation. Can this response be generalized to a personality trait? If the dog reacts fearfully to a person wearing a sheet over his head so he looks like a ghost, does that mean the dog is fearful or just that this was a particularly surprising experience? The DMA asserts that it measures playfulness, chase-proneness, curiosity/fearlessness, and most interestingly, aggressiveness. But does it? Studies of the validity of behavioral assessments in shelter dogs — a similar situation in which a series of small tests are given to a dog by a stranger in a strange situation — have repeatedly shown that the subtleties of personality are really hard to measure in this way.

Ideally, a personality heritability study would be designed using the canine behavioral assessment and research questionnaire (C-BARQ), a questionnaire which relies on the dog's owner to assess the dog’s personality through 101 questions. This test has been found to be valid and reliable. And the University of Pennsylvania has a database of the results of this test when given to thousands of different dogs. Except... they don’t have the pedigree information for many (or perhaps not for any) of these dogs. So this isn’t a practical solution, either.

So it’s hard, and I don’t really trust the studies that are out there as a result. What do these studies find? Most studies out there use the DMA or tests like it, and find roughly 20%-50% heritability for most personality traits studied. These numbers might be artificially low, though, because the tests may not be testing real traits — behavior that is stable over time.

I was able to find one study using the C-BARQ, which had much higher heritabilities, around 70%-100%. It's a dramatic difference, but I would hesitate to assign the responsibility for that difference entirely to the C-BARQ. This study used a non-random set of samples, selecting aggressive golden retrievers and dogs related to them. With no control set of non-aggressive goldens and unrelated animals, it’s hard to know how to interpret the study’s results.

So what are the real numbers? I still want to wriggle away from an answer. I don’t think we really know. I’d love to see a C-BARQ study using a random sample — maybe by finding pedigrees for dogs already in their database, if that’s possible. Until then, I’ll guess that the real answer falls in the 30%-60% range for most traits. But, in the end, does it really matter? Genetics are important and environment is important. The best genetics can fail in the face of a poor environment, and the best environment can fail in the face of poor genetics. We can’t predict everything about our next dog; we can just do our best to make a good decision, and then provide the best possible environment for whoever comes home with us.

I owe the inspiration for this post to my students in APDT's Canine Behavioral Genetics course, who asked about the balance of nature versus nurture and would not be satisfied with vague answers.

References
  • Strandberg E. & Peter Saetre (2005). Direct genetic, maternal and litter effects on behaviour in German shepherd dogs in Sweden, Livestock Production Science, 93 (1) 33-42. DOI: http://dx.doi.org/10.1016/j.livprodsci.2004.11.004
  • Liinamo A.E., Peter A.J. Leegwater, Matthijs B.H. Schilder, Johan A.M. van Arendonk & Bernard A. van Oost (2007). Genetic variation in aggression-related traits in Golden Retriever dogs, Applied Animal Behaviour Science, 104 (1-2) 95-106. DOI: http://dx.doi.org/10.1016/j.applanim.2006.04.025