The Replacement Dog: how a veterinarian / dog rescuer / geneticist searched for the right puppy

The death of my fifteen year old golden retriever Jack wasn’t just about my loss. It was about finding someone else to do his job, which for the last five years had been serving as a security blanket for Jenny, my shy collie mix. Jenny depended on him to tell her when people did not intend to eat her, to run interference with well-meaning strangers, and to demonstrate calm at the veterinary clinic. We could not remain a single dog household for long.

Jack and Jenny

I adopted Jenny at age 13 months. She had never been off the farm where she was born until her surrender to a shelter, and the world proved much larger than she expected. Jenny has excellent dog skills but a crippling anxiety upon encountering new people or environments. I adopted her knowing about her idiosyncrasies because I wanted to study anxiety in dogs, and wanted to experience it first hand. And so I have; living with Jenny has informed my understanding of anxiety in a way that reading about it never could have.

In my research, studying the way genetics and environment interact to affect the risk of anxiety has brought me back time and again to the importance of early environment: in utero environment, early maternal care, and puppy socialization. How the brain changes during and after the socialization period turns out to be a huge part of my research interests, and to learn from the source as I had done with Jenny, I would need a puppy, and a very young one at that. I’ve only adopted adult dogs in the past, but now is an excellent time for me to raise a puppy, as I work from home many days.

A puppy who would grow up to fit in well with Jenny had to fit a specific mold: confident around people and other dogs, but not so pushy as to annoy her. Someone she could play with. Someone male, because I didn’t want to deal with girl dog politics for the next ten years.

Jenny

Now, I have counseled others that adopting a very young mixed breed puppy from a shelter or rescue group means you really have no idea at all who you have just brought home, and that there is no shame in purchasing a dog from a responsible breeder. However, in practice, I balked at purchasing a dog. I completed an intense shelter medicine internship at the University of Florida several years ago, and I still feel part of that community. As the distance between the present day and that experience increases, I find myself holding tighter to those connections and looking for new ways to remain a part of sheltering. Purchasing a dog who was not going to be in want of a home felt a bit like eating humanely raised meat: I tell myself it’s okay for others to do it, but when I actually try to do it myself, some part of me rebels.

Yet as I looked at puppies from local rescue groups, in short order I found myself in a panic: could I really adopt a puppy whose genetics were completely unknown, whose parents I most likely couldn’t meet, and who had almost certainly had some early life trauma before ending up in foster care? Genetics and early experience are both critical in shaping the adult personality, and while I hope I could handle dealing with another shy dog, Jenny needed someone dependable, not another neurotic failing to keep it together when the mailman drove past.

When I started to seriously consider purchasing a dog, I had to decide on a breed. I love retriever-collie mixes: ideally the best of both worlds, retriever-social and collie-smart. But finding a responsible breeder of retriever-collie mixes seemed a tall order. Border collies are too intense for me. Australian shepherds have their tails docked so short. And I wanted to find a breed that is not recognized by the AKC, that is absolutely not bred for looks, that possibly even has open stud books to keep the genetics pool large and diverse.

I found the Scotch Collie and the English Shepherd. The Scotch Collie club had an open stud book policy going for it (good for them!). The English Shepherd club had a closed stud book policy (open it up, guys!) but it had been open relatively recently, the breed isn’t recognized by the AKC, and the dogs can’t be shown in conformation classes. The breed is a versatile working breed. Both breeds have lovely breed standards that accept a wide range of phenotypes (for example, 30-80 lbs in adult weight - a wide range!), which in itself tells the story of breeding for temperament and not looks.

In the end, I chose the English Shepherd based on the fact that there are more of them around, so it was easier to find a litter promptly. Waiting a few months would mean potty training a puppy in January in the Midwest, an experience I’ll leave to others.

The English Shepherd club maintained a list of breeders who had available puppies, with lots of information about the parents. It’s a well designed resource, and I link to it not to encourage others to run out and get an ES puppy (they are smart and high energy and not for everyone) but to provide an example of what kinds of information should be provided about available litters.

I screened the descriptions of parents: I discarded those who weighed more than 70 lbs, as managing Jack in his dotage had been hard on my back. I discarded those who were described as “protective” or “taking some time to warm up to people.” I checked that the parents had passed the relevant genetic tests (for this breed, tests for several eye diseases and hip dysplasia). Then I looked at the remaining breeders’ websites.

The breeders I liked talked about how they raised the puppies: giving them lots of positive experiences. They talked about what they did with the puppies’ parents - agility, nosework, herding. They often had long applications for potential owners to fill out, which asked all the questions they should: How will you exercise this dog? Do you have a fenced yard? What will you do if he is destructive?

I found a litter in Virginia with a male who sounded perfect: confident, social, and by the way athletic. My husband and I stuffed Jenny into the car and drove 9.5 hours to pick up our boy. He cost, by the way, probably more than twice what a rescue puppy would have cost, but I have paid for knowing that he is clear of some genetic diseases for which he might have been at risk, and for knowing that he was in the uterus of a calm, happy mother; raised with a litter who had plenty of high quality food and safe places; and had extensive early socialization (including the Early Neurological Stimulation and Early Scent Stimulation programs). He has proven, in his first week and a half with us, to be social and sweet, willing to settle down when asked so long as he is given plenty of exercise and mental stimulation, and terrifyingly smart. He and Jenny are already wrestling for hours daily, laying the foundation for what I trust will be a long friendship.

That is the story of how we found Dashiell.

Dashiell

Rats, dogs, foxes, and the SHRP

Working on my Master’s degree has made me yen for more letters after my name, so I’ve been doing some spare-time reading on subjects that might yield PhD-type projects. My putative interest is in development of the stress system in young dogs. The idea is that if a dog’s stress system develops poorly, whether through bad genetics or a bad early environment, then that dog is more likely to bite people when it grows up. The more we know about how their stress system develops, the more we can know about how to grow healthy dogs with good bite inhibition.

For several months I thrashed around in the literature, reading about development of the stress system in rodents (about whom we know quite a bit, because we are more willing to do experiments on them than on dogs), and reading about socialization periods in dogs. It was hard to find good direction, and I wasn’t quite sure where to start. Recently I have had a breakthrough, however.

First, some orientation. You are walking through the woods. You see a shape on the ground. Your brain interprets the shape: long, thin. Your amygdala (part of the limbic system of your brain) yells SCARY SHAPE SCARY SHAPE and you get a blast of adrenaline in your system. Half a second later your cortex (the thinking, conscious part of your brain) catches up: hey, that looks like a snake. Your hypothalamus (which deals with a lot of hormone regulation) sends a message to your pituitary (which releases a lot of your hormones), and the pituitary releases a hormone which travels down to your adrenals, near your kidneys. Your adrenals release our old friend cortisol, which gets into your blood and tells your body that you are having a stressful experience. Cortisol, you of course remember, is what I like to extract from the saliva of dogs to tell if they are unhappy about being stuck in a noisy hospital run. This whole system is what I’ve been referring to as the “stress system,” more properly called the HPA (hypothalamic-pituitary-adrenal) axis.

If you were a rat or mouse, instead of releasing cortisol, your adrenals would release corticosterone. It is a very similar hormone with similar effects. Dogs actually release equal parts cortisol and corticosterone, but we just study their cortisol levels. I still haven’t figured out why we chose cortisol to focus on in them; there are a lot of tools available for studying cortisol, since humans make it primarily, but also a lot for studying corticosterone, since we study rodents quite a bit.

Now, to get back to my recent reading, very young animals don’t get as frightened by scary things as slightly more mature animals or adults. This phenomenon has been studied intensively in the rat: rats younger than two weeks of age don’t show this corticosterone spike when exposed to something upsetting. This is called the “stress hyporesponsive period,” or SHRP.[1] There has been work on what part of the HPA system is responsible for this blunted response: the amygdala? The hypothalamus? The pituitary? Or are the adrenals themselves not responsive yet?

A good way to stress out an infant rat is to expose it to the odor of an adult male rat. Left to their own devices, adult males will happily eat infants, so the young rats are quite right to fear them. An infant rat, upon smelling a strange adult male, will become immobile. However, a neonatal rat younger than 14 days (in other words, one still in the SHRP) will not become immobile: it hasn’t yet developed the machinery to feel, or possibly just to express, fear. If you remove the infant’s adrenals, so that it is unable to make corticosterone, then even when it matures to older than 14 days it will still not properly become immobile when exposed to the scary smell. Moreover, if you inject corticosterone into one of these pre-14 day rats, it will be able to develop the immobility behavior at age 14 days, just like a normal rat. [2] This suggests that corticosterone is responsible for the immobility behavior. However, if you remove the adrenals of a rat which has already developed the immobility behavior (one which is older than 14 days), it will continue to become immobile in the presence of the scary smell. [3] And if you inject extra corticosterone into a rat too young to have developed the immobility behavior, it will develop it early. [4] This suggests that corticosterone is responsible just for the development of the behavior, not for allowing it to actually happen at specific times once it has initially appeared.

What’s going on up in the brain while all this is happening? When infant rats are too young to express (or possibly feel) fear, are their amygdalas just failing to activate? When neurons in a particular brain region have been recently active, they contain a protein called c-fos. You can check a brain region for the prescence of extra c-fos to see if it has been doing anything in the recent past. This was done with young rats. Rats too young to have developed the fear response did not have amygdala activity (no extra amygdala c-fos) after exposure to the scary smell; if they were injected with corticosterone to cause them to develop the fear response early, then they did have amygdala activity; rats old enough to have developed the fear response did have amygdala activity; and rats whose adrenals were removed prior to developing the fear response did not have amygdala activity. [4] Unfortunately, this study does not appear to have looked at whether rats which were allowed to normally develop the fear response (intact adrenals), but then had their adrenals removed after initial development of the response, still showed amygdala activation. Perhaps that question has been answered elsewhere.

So what does all this mean for dogs? Do dogs have an SHRP? I found one unreferenced assertion that they do, but I have not yet found a study actually examining the canine SHRP. The SHRP does exist in various species, and it seems likely to me that it exists in the dog. Puppies start out fearless, and develop fear later. I suspect that a canine SHRP will prove to be an important part of socialization: the time that puppies don’t yet feel fear may be an important one for introducing them to lots of different kinds of people, so that they can learn that these people are a normal part of puppy life and are not to be feared later on.

The development of the HPA system has been studied in domesticated silver foxes — foxes selectively bred to not fear humans. (These foxes show surprising physical similarities to other domesticated animals in body shape and color, despite not having been bred for these features, leading to speculation that there is some general mechanism of domestication. That general mechanism of domestication is actually what I’d like to get at in a PhD project.) Researchers took two groups of foxes: domesticated foxes, and foxes bred for increased aggressiveness to humans. They tested them for behavioral reactions to humans and cortisol level increases after exposure to humans, at ages 30 days, 45 days, and 60 days. The aggressive foxes did not show aggressive behavior or cortisol spikes at 30 days, but they did show it at 45 and 60 days. The domesticated foxes, on the other hand, did not show aggressive behavior until 60 days, and their behavior at that time was described more as “defensive” than “aggressive.” They never showed the cortisol spike. [5]

Is this the same thing as a silver fox SHRP? I’m not sure that this study exactly gets at that, but it seems suggestive. Questions I’d like to ask about the SHRP in dogs are: Does the SHRP definitely exist in dogs? Is the SHRP length different in dogs and wolves? Does the length of the SHRP affect the socialization of the dog? Is the SHRP length different in different dog breeds? And, most important but most difficult to get at, does length of SHRP have anything to do with a dog’s fearfulness as an adult?


[1] Walker Claire-Dominique, Perrin Marilyn, Vale Wylie, Rivier Catherine. Ontogeny of the Stress Response in the Rat: Role of the Pituitary and the Hypothalamus. Endocrinology. 1986;118:1445-1451.

[2] Takahashi L. K., Rubin W. W. Corticosteroid induction of threat-induced behavioral inhibition in preweanling rats. Behavioral neuroscience. 1993;107:860-866.

[3] Takahashi L. Organizing action of corticosterone on the development of behavioral inhibition in the preweanling rat. Developmental Brain Research. 1994;81:121-127.

[4] Moriceau S. Corticosterone controls the developmental emergence of fear and amygdala function to predator odors in infant rat pups. International Journal of Developmental Neuroscience. 2004;22:415-422. [Free full text.]

[5] Plyusnina I., Oskina I., Trut L. An analysis of fear and aggression during early development of behaviour in silver foxes. Applied Animal Behaviour Science. 1991;32:253-268.

Epigenetics of stress

Last week in journal club I presented "Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses," by Oberlander et al., published in Epigenetics in 2008. This paper tries to get at one small part of the mechanism for how the in utero experience can affect a fetus, possibly even affecting the baby's personality.

Oberlander's study builds on earlier work done in rats. Researchers found that the offspring of particular rat dams were less fearful than average. Specifically, these rat moms were spending extra time licking and grooming their babies, and performing "arched-back nursing." They were dubbed "LG-ABN" dams (licking grooming, arched-back nursing.) Their babies acted less fearful in stressful situations, and had a blunted stress response on the HPA axis.

The HPA axis... This is in large part what I'm studying for my Masters project, so it's hard to limit myself to a short explanation of what it is. In short, one way in which one's brain (or part of it: the hypothalamus, the H in HPA) responds to stress is to send a message to the pituitary (P), which in turns sends a message to the adrenals (A). The adrenals release cortisol, which is known as the stress hormone. Cortisol is what researchers look for in your blood if they want to quantify how stressed you are. Stress out two animals, check their cortisol levels, conclude that the one with higher levels reacted more strongly to the stimulus — that's the formula for any number of experiments, including this one. (It is, as always with physiology, more complicated than that, but the idea that there's a correlation between increased cortisol levels and increased stress is a good start.)

So these babies of LG-ABN moms had a smaller cortisol spike in response to stress. Further work elucidated part of why that happened: the receptors to which cortisol binds in the brain send a message back to the top of the HPA to tell it to stop releasing cortisol ("that's enough, there's plenty out here!") in a negative feedback loop. These receptors (glucocorticoid receptors, abbreviated "GR") were in excessive supply in the brains of these less fearful rats, so the negative feedback loop worked well, and the rats' brains responded to rising cortisol levels by releasing less cortisol — with the result that the spike of cortisol was smaller in response to a stressful stimulus.

Meanwhile, the researchers also found that this trait of decreased fearfulness was not genetic in the normal sense. If they fostered baby rats from non-LG-ABN dams on LG-ABN dams, these babies who were not genetically related to the LG-ABN mom grew up to be less fearful, presumably simply by being raised by her. And they passed the trait on to their offspring! It turned out that the trait was being passed along epigenetically. We all learned in elementary school about genetic traits — getting brown eyes because you got brown eye genes from mom and dad. And we all know that genes are coded on DNA. Epigenetic changes involve not different genes, but changes to the higher-level structure of the DNA. Instead of involving changing the building blocks of the DNA (the genes), epigenetics involve changing the shape of the building, or sometimes tacking something new on to the outside of it.

In this case, it turned out that in order for GR to be produced (remember that receptor for cortisol, necessary for negative feedback?), the machinery for reading genes had to have free access to the GR gene itself. However, an area of that gene had become methylated — in other words, another object was sitting on it, blocking access. The machinery for reading genes couldn't read that gene as well, so fewer GRs were made. Fewer GRs meant less negative feedback and a more easily stressed baby rat.

That's all background. Oberlander, who wrote the paper I presented, wondered whether the same mechanism applied to humans. He knew that human mothers who are depressed during pregnancy often give birth to babies with more reactive HPA axes. Could that be because those babies had fewer GRs, as a result of methylation of the GR gene? He also wondered about the effects of SRI medication, such as Prozac, on this system.

82 pregnant women were enrolled in this study. 33 were taking SRI medication. All were tested using a scale for depression, which resulted in a numeric score; higher scores implied greater depression. Blood was drawn from the moms in their second and third trimesters, and when they gave birth. Blood was taken from the babies' umbilical cords at birth. Then the babies were tested at three months of age for their response to a mild stressor.

The researchers found that the babies of depressed moms did tend to have increased methylation of the GR gene, exactly in the spot that they expected. That increased methylation correlated with an increased cortisol spike when the babies were mildly stressed. SRI exposure in utero didn't have any effect on the size of the spike, although babies whose moms were medicated did tend to have lower cortisol levels in general.

This paper spoke to me on two levels. I enjoy reading about mechanisms; I like imagining how all these little machines in our bodies interact to form our personality and affect how we experience the world. I also liked the study's methods, because I'm interested in finding ways of learning about living individuals. I want to study dogs, so I want to find ways of looking into their brains figuratively, not literally. Examining changes in DNA extracted from a blood draw is cool — it's something I could potentially do to someone's pet, perhaps as part of a study aimed at understanding why some dogs are more easily stressed to the point of biting than are others.

I think that the people who attended journal club found the paper interesting. Two professors who were in attendance work in this area of genetics and behavior, and had useful input for me. One pointed out that the list of variables that the paper's authors checked for in the pregnant women was very small. (It consisted of things like age, whether this was a first pregnancy, whether the pregnancy ended in C-section, whether the woman smoked or drank.) She listed some other things she would have checked for, such as body weight (fat can apparently produce cortisol). She also noted that the baby's blood sample came from umbilical cord blood, which is actually a mix of infant and maternal blood. Also, different parenting strategies weren't taken into account — did depressed mothers treat their babies differently in some way? She concluded that we'd all like to be able to see useful DNA changes just by taking blood samples (which is precisely one of the things that drew me to this paper), but it's actually very hard to do so, so this paper's results should be taken very cautiously.