Thursday, December 24, 2009

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.

Wednesday, December 23, 2009

Jack’s contribution to veterinary education

Today I got an email from a tech at my school, asking if the radiology department could use my dog Jack for a teaching exercise. My roommate had suggested Jack as a good dog for this particular class. He would be sedated, and students would radiograph (x-ray) him. In exchange for allowing them to practice on him, I would be able to request that any body part be radiographed, and have a free analysis of the image by a radiologist.

Jack does have marginally bad hips, but he hasn’t shown any discomfort lately, so I don’t think that getting rads of his hips is worth the annoyance to him of having to spend a day in the hospital and be sedated. Recovering from sedation isn’t awful, but it isn’t as fun as, say, not having been sedated in the first place, either. So the lure of free rads isn’t compelling to me.

I replied that I would be willing to let them use him, however, and this is why. I believe in use of student dogs, particularly mellow dogs like Jack who aren’t traumatized by being in the hospital for a day and being handled by strangers. What’s the alternative, after all? Students might learn these procedures on client dogs, who are actually sick or are less comfortable with strangers than Jack is. As my advisor once said, “When a veterinarian performs a procedure on your pet, how many times do you want him to have practiced that procedure on other animals?” The answer, of course, is “One thousand times.” We don’t want our animal to be the first.

Who else could vet students practice on besides their own dogs? One option would be to purchase animals for this use, or to use ex-research animals which are maintained on university campuses for this reason. Such animals are well cared for, of course, but it is not as good a life as being a pet. I believe that every dog should get to be loved by a human.

So Jack will volunteer yet again to help out. (He has already given blood to a study on hemangiosarcoma in golden retrievers, and gotten a cardiac ultrasound to help veterinary cardiologists better define values in normal dogs.) He will be annoyed. But if the visit goes like his previous volunteer appointments, he’ll get ice cream afterwards. That’s his payment for helping to advance veterinary medicine.

Monday, December 21, 2009

Letting go of dominance theory

Recently I took my dog Jack along on a visit to a friend who was dog sitting. My friend’s resident dog has always gotten along with Jack pretty well, but this guest dog, Ally, had the habit of rushing at Jack when he entered the room, barking at him and generally behaving in a manner that alarmed all the humans involved. Ally was also hesitant around strange humans, in this case myself and my boyfriend. She would come up to us only gingerly, and was easy to scare away with sudden movements or loud noises.

My friend theorized that Ally’s issue with Jack was that she wanted to be the alpha dog, because dogs see the world in terms of a pack structure, like wolves. He felt the way to fix the problem was to let Ally know who was really boss, so whenever Ally rushed at Jack he would yell at her. The idea that a problem in dog training can be solved by asserting dominance is known as dominance theory; use of dominance theory has been publicized recently by Cesar Millan, the Dog Whisperer. A recent article in the Boston Globe addresses the controversy about Millan’s approach, and has this quote from Karen Pryor, who popularized clicker training, which is a very different approach:

...while his critics dispute Millan’s claim that domestic dogs are pack animals and should be treated as such, Pryor proposes that Millan’s hard-line message speaks to the real pack animal in the room. “We’re the ones who care very deeply about who’s boss and we don’t want to stop believing that humans are superior,’’ Pryor says. “We’re primates that have gone strongly in the direction of hierarchies. Dogs? They don’t care about that at all.’’

This quote really tickled me, even though in my opinion it goes a little too far. No offense to Pryor; I think her book Don’t Shoot the Dog should be required reading for any pet owner. But I think saying dogs don’t care about hierarchies at all may be overstating the case. In my house, it’s clear which dog is in charge, though the other two don’t seem to much care who comes second and who comes third. When I sat in on sessions with a veterinary behaviorist last year, I saw dogs diagnosed with dominance aggression, and I agreed with the diagnosis.

However, I do object to the blanket application of dominance theory to all problems in dog training. Wild dogs don’t run in packs the way wolves do; even if they did, to say that dominance issues are the answer to every canine behavioral problem is silly. Dogs are more complicated than that. In Ally’s case, I don’t think she was trying to show Jack that she was in charge; I think she was scared of him, and trying to deal with the situation in the best way she could come up with.

And who developed the idea that yelling is what makes a good leader? Are managers who regularly yell at their subordinates considered good bosses? My favorite bosses were always people who helped me solve my problems, not people who got angry at me when I couldn’t find a solution on my own.

The American Veterinary Society of Animal Behavior has a position statement on dominance theory, in which they provide a good framework for approaching its use in training. I do believe that overuse, or misuse, of dominance theory will gradually fade from the way we as a society manage our dogs, but for now, it seems to be hard for us as humans to let go of it.

Monday, December 7, 2009

Links post

Friday, December 4, 2009

But what do you do with the spit after you get it?

Last night, before bed, I collected some saliva from my dog Jack and my roommate’s dog Casey. Jack has, by this point, become inured to the idea that occasionally I am going to grab his muzzle and stick a sponge on a stick inside his lip and swab around for two minutes. He was ready for bed when I did it last night and decided he might as well just doze through it.

Casey is another matter. When I cornered him and grabbed his muzzle, he freaked out.


Me: Casey, I have done this to you five times. Why do you always act like it is going to hurt?


Me: I have done it to myself. I know it doesn’t hurt.


Me: I have done it to about 30 dogs and it didn’t seem to hurt any of them much.

Casey: Oh.

Most dogs are annoyed when I try to insert the swab, but then discover it doesn’t hurt and just deal with it. Casey inevitably squeezes his eyes shut, hyperventilates, and sometimes even collapses to the ground. (You might at this point ask “how do you know there isn’t something special about Casey?”, to which I would reply that, while there are certainly many special things about Casey, he also reacts this way to having his nails clipped, so I am pretty sure he is not experiencing any unusual physical sensations when I collect his saliva.)

I imagined Casey asking me “Why are you doing this to me?” and what my answer might be: “To find out how stressed you are.” As bizarre as that might be from a dog’s perspective, in fact my plan was to analyze his saliva to find out how much cortisol was in it. Cortisol has been called the “stress hormone,” and is an indicator of how much stress an animal is under.

Because I’ve never worked in a lab before, I wanted to do a few sample assays with unimportant data (i.e., saliva from my own dog that is easy to collect) before using the irreplaceable data collected from hospital dogs. It has taken me four months to successfully enroll 24 dogs; if I waste those samples, I am SOL. (Six dogs a month? Well, I’m a lot better at it than I used to be, and I had to take some time off in the middle. I am actually averaging 3-4 dogs a week at this point.) I performed my test assay this morning.

The cortisol assay is a kit costing a couple of hundred dollars which I purchased from the ever-helpful Salimetrics. It is an ELISA, or enzyme-linked immunoabsorbent, assay. The main equipment in the kit is a little plate (I was surprised by how tiny it was) made up of 96 wells, into each of which one might pour a very small amount of liquid. Each of the wells has some cortisol antibodies in the bottom (or so I’m told, since obviously antibodies are too small to see). These will grab on to any cortisol they see and hold on to it.

When I got into the lab this morning, I took the kit out of the fridge, and some older samples out of the freezer, so that they could warm up to room temperature. Then, while last night’s samples from Casey and Jack centrifuged, I sat down with the lab tech’s computer and figured out where everything was going to go on the plate. Aside from all my samples, I had to have room for the standards, which are samples given to you with the kit containing a known amount of cortisol. If your assay doesn’t tell you that the well with the 3.000 standard has about 3.000 µg/dL of cortisol in it, you know you’ve done something wrong. There are also the controls, which contain an arbitrary “high” and “low” amount of cortisol, and are also useful for telling you when you’ve messed up.

Then there are the “zero” and “blank” wells. The zero wells contain nothing but the diluent, or the liquid used to dilute some of the chemical agents in the kit. When the calculations are done at the end, you need to have this well to be able to tell the difference between a real effect and an effect caused by the diluent. And, finally, there are the blank wells, which don’t have any cortisol binding antibodies in them, and therefore will behave differently than the zero wells when the plate is read.

For me, the hardest part of the process is making sure that everything goes where it’s supposed to. This is more complicated than it sounds, involving lots of finding the right well in a small plate that is packed with small wells; remembering to leave space for a sample that I’m going to add later, after it has been diluted; getting the standards and controls in the right places; etc. Doing something that doesn’t require a lot of brainpower but does require a lot of precision, over and over and over, is hard for me.

When all the standards, controls, and samples were in place, and diluent was in the zero wells and the blank wells, I added conjugate to everything. The “conjugate” is cortisol which is attached to an enzyme. Of course, the whole point of the exercise is that you are putting in your saliva samples which contain cortisol, and now you’re adding this new source of cortisol as well. The two sources of cortisol are going to compete for the chance to bind to the cortisol-binding antibodies in the bottom of each well. When there is more cortisol in the sample, less of the conjugated cortisol (with its attached enzyme) can bind, and vice versa.

I put the plate with its samples and conjugate on to a rotator. This is a small device with a flat top which basically waves the plate around so that its contents mix. Every time I use it I am terrified that the plate is going to go flying off and spread my samples across the lab floor. It has not happened yet.

After 55 minutes for the whole competition thing to happen, I washed the plate off (four times), which includes blotting: picking the plate up, turning it upside down, and slamming it on to an absorbent pad. This is also a scary procedure, especially as sometimes strips of wells break off and you have to figure out in which direction to reattach them. Attach them upside-down, and you won’t know which sample is which.

At this point, in theory, the wells were mostly full of bound cortisol. Some of the cortisol, which came from the conjugate I added, had enzyme bound to it. Some of the cortisol, which came from my samples, did not. The wells which contained samples high in cortisol had less of the bound enzyme, and the wells which contained samples low in cortisol had more of the bound enzyme. Next I added TMB (tetramethylbenzidine), which reacts with the bound enzyme to change its color. Obviously, the wells with more bound enzyme will change to a different color than the wells with less.

More plate rotating is next, and then a brief time (sequestered in the dark) for the plate to let the reaction run its course. When I took the plate out of its light-proof bag at the end of this waiting period, the wells were all full of a lovely blue liquid, and different wells were visibly different shades. I added stop solution to stop the reaction, which immediately changed the color to yellow. And then I put the plate in the plate reader, which is a small machine attached to a Windows computer on the lab tech’s desk. It made some thumping noises as it determined the optical density of each well — its color, expressed as a number. It relayed these numbers to the waiting computer, which did the calculations to convert the numbers into concentrations, and I exported the results into Excel.

So how did I do? This was actually my second attempt at doing this assay. Both times, the results were fairly close to what I expected, based on the standards. However, part of what I wanted to figure out today was whether I could dilute small samples and still calculate values which were close to undiluted values. (In other words, if I dilute one sample by 50%, and multiple the calculated concentration by 2, will the result be similar to what I get when I put an undiluted version of that sample in a different well?) My dilutions were way off, so that’s something I need to figure out. I’m hoping to find a lab on campus which does these assays frequently, and ask if I can work with them for a little while, to get some experience.

By the way, the above implies that I competently performed this entire operation on my own, which is completely untrue: I worked under the close guidance of the extremely talented hospital lab technician. I am very lucky that she enjoys teaching; she told me today that she once considered being a school teacher. Her patience and good nature made the day much more enjoyable than it might have been.

Thursday, December 3, 2009

Opening up the local meat bottleneck

There have been some interesting developments recently in local meat processing in New England.

First, some background. I am a proponent of raising food animals on pasture on small farms. Specifics about why I believe in this approach are beyond the scope of this post; I was convinced by The Omnivore’s Dilemma by Michael Pollan and the Pew Report, also known as “Putting Meat on the Table: industrial farm animal production in America.” One of the main obstacles to success as a small scale food-animal farmer in New England, where I live, is the lack of processing operations (slaughterhouses) set up to handle small-scale farmers. There are plenty of people who want to raise animals on pasture and there are plenty of people who want to buy the meat or eggs, but there is a real bottleneck between the two, and building new slaughterhouses is expensive.

Mobile poultry processing units (MPPUs) are an inexpensive solution to the problem. MPPUs will actually come to your farm or to one near by to process your chickens. There are currently two MPPUs operating in Massachusetts, and more in surrounding New England states. Regulators are still somewhat wary of these units; they understand the need for them, but are more used to applying regulations to large scale plants than to slaughterhouses run out of flatbed trailers. Jen Hashley is the driving force behind one of the Massachusetts units. She has been working with regulators to make sure they’re happy with the unit, and looking for funding for getting more units built.

Recently, Grist reported that Whole Foods is considering getting into the mobile processing business by building their own fleet of MPPUs. Since Jen Hashley’s recent application for funding for new units was denied, this seems at first like a great solution to the problem. However, Whole Foods would allow use of the units only by farmers who have contracts with Whole Foods to grow chickens according to Whole Foods guidelines. Which is just contrary to the whole point of local food: that the farmer has the power to choose how he raises his animals, given his unique personal circumstances and beliefs. The consumer than has the power to choose which farmer to support, based on whether he agrees with that farmer’s choices. Not enough farmers around to allow the consumer to pick and choose? Well, that’s why it would be nice to have more mobile units, available to anyone who wants to pay to use them, to support the existence of more farmers making more varied choices.

I say:

Dear Whole Foods: Building MPPUs is a wise enterprise for you! However, you have already been criticized by Michael Pollan, the Foodie King, for your lack of support of local production, in his book The Omnivore's Dilemma and then in your open discussion with him. Allowing only limited access to your MPPUs will not be taken well by the locavore community, who will rightly see the move as an attempt to establish control overchicken production in this region. Why not allow open access to them? You’ll have fewer chickens grown exactly the way you want, of course, but aren’t consumers ready to pick and choose the kind of farmer they want to buy their chicken from? Some of these farmers will raise chickens your way, and you can sell those. Some of them won’t, and you don’t have to sell those — but why not give it a shot? Develop a labelling scheme which presents the relevant specifics to the consumer. On pasture? In a barn? Antibiotics? Organic feed? Different people care about different things. Some of us care a lot about choice.

Meanwhile, in Vermont, Walter Jeffries is responding to the lack of processing options by building an on-farm processing unit for his pastured pigs. He writes in great detail about the reasons he's choosing this solution. It’s a ballsy move. Building a slaughterhouse can cost literally millions of dollars. He has to try to balance doing so affordably, and complying with all the regulations necessary to convince the USDA to come inspect his meat. If the USDA refuses to inspect, he can’t sell his meat by the pound or across state lines. While MPPUs are the answer in some places, they aren’t the answer everywhere, and they are definitely not right for farmers who are raising animals of the four-legged variety.

I have no idea how either of these stories will end. Will Whole Foods’ choices, whatever they may be, support or limit local chicken production in New England? Will Jeffries be able to lure a USDA inspector to his plant when it’s done, or will it languish unused? It’s an interesting time to be watching news about local meat.