Friday, September 30, 2011

Ecological Field Techniques

As you can tell from my previous posts, my primary interest in Chile is to collect a lot of blood samples so I can determine cortisol concentrations. But my time down here isn’t just spent bleeding degus; in order to get some of my desired blood samples, I have to employ several ecological field techniques. And in addition to collecting these blood samples, I am also gathering other types of data to complement my primary research. Let me describe some of the techniques that I have been using this field season:

Widespread trapping:

For my primary project investigating the effects of poor maternal care on the pup stress response, I have to find and determine several social groups. To do this, my collaborators and I set out 300 live Tomahawk traps over a large area during early August. We tried to place the traps in areas where we found active burrow systems; a good way to determine whether a burrow system is active is to look for fresh feces and dust-bathing sites. If a fecal pellet is fresh, then it should be soft and green when you break it open. Dust-bathing sites are near burrow openings and are little sandy areas that are free of rocks and vegetation. Watching degus dust-bathe is rather cute; they quickly flip onto their side, roll in the dust, and jump back into an upright position all within a second.
An open Tomahawk trap
Anyway, after setting out our traps, we trapped for several hours a day for about 3 weeks. We would typically open and bait the traps in the morning (we use rolled oats for bait, the degus absolutely love it) and then check and re-bait the traps every hour or so. If we caught a degu, we would take it back to the truck, put an ear tag on each ear, weigh the animal, and then check their reproductive condition if they were a female. We would then return the degus to their respective burrows at the end of our trapping period. We weren’t getting many degus at first, so we spent a few days perched on the hillside, watching degus through our binoculars so we could figure out how to better place our traps.
Ear-tagged degu

Male degu

Female degu
Getting weighed

Nighttime Radio-telemetry:

To determine which degus belong to which social groups, it is necessary to supplement trapping numbers with nighttime radio-telemetry data. Figuring out social groups is tricky because degus use multiple burrows, and some degus move around more than others. The accepted rule for determining social group membership is to establish whether a group of animals spend 80% or more of their nights in the same burrow together. So, in order to figure out my social groups, I had to radio collar and track my females for two weeks. Earlier this month I re-trapped my degus and removed the collars so it wouldn’t be an extra burden during their last few weeks of pregnancy. After the degus give birth (about two-thirds of my degus have already given birth) we then re-collar them and do more tracking because social group membership can frequently change composition.

In order to radio-collar an animal, the first thing I had to do was to make sure that the collar was still functioning properly. After checking that the collar had a strong, clear signal, I then took a piece of special wire and flamed some heat-shrink tubing onto it. I then threaded the wire through the transmitter, threaded one end of the wire through a piece of hard, plastic tubing that protects the transmitter’s antenna, and then pulled the two wire ends through a crimp. With one person holding the degu, I then flipped the collar onto the degu’s neck and used two pairs of pliers to tighten the collar. The degus necks are always smaller than they appear because of their thick fur, so I had to rotate the collar to get the fur out of the way and continually test whether I could push the collar over the degu’s head. It’s important to make sure the collar’s tight enough so the degu can’t get it’s front paws stuck in it, but it’s also important to make sure that the collar isn’t too tight because it can irritate the degu’s neck and cause an infection. Once the collar was the right tightness, I then crimped it, cut the wire ends, and returned the degu to its burrow. Every subsequent time that I caught the degu I checked to make sure that the collar wasn’t irritating the skin.

And a radiocollared degu

Actually tracking the radio-collared degus wasn’t too difficult. By using a receiver attached to a tracking antenna, I would type in the frequency of the degu’s collar (each radio collar has its own, unique frequency) and then wander around listening to the loudness of the beeping. The receiver and collars are usually sensitive enough to pinpoint the degu’s location within a meter or so, but large obstacles like rocks and trees can sometimes bounce the signal.
Modeling the radiotelemetry equipment

Vegetation Sampling:

Some of the degus that I’m working with are part of my Chilean collaborator’s long-term study, and one of the measurements that he has always taken is relative food abundance around the different burrow systems. To do this, we take vegetation samples from two areas per burrow system. To determine which areas to measure, we choose a direction at random (N, S, E, or W) and then mark a spot 3 and 9 meters from the center of the burrow system. We then lay down a 20cmx20cm grid and harvest all of the plants within it. This sounds pretty easy, but we also have to take off the roots from the plants and then separate them into monocots and dicots. The samples are then weighed after being dried in a drying oven for 72 hours.

Hematocrit, Soil Hardness, and Fecal Water Content:

One of the other students (a recent Tufts graduate) is doing a side-project to examine the correlation between hematocrit and cortisol levels. Hematocrit is the proportion of red blood cells in the blood and has been linked to hydration, stress, altitude, etc. Measuring hematocrit is fairly easy; when I bring a blood sample back to the lab I have to spin it in the centrifuge to separate the plasma from the red blood cells (I collect the plasma because that’s where the hormones are). The blood samples are in little, glass tubes and after spinning them each tube separates into a bottom layer of red blood cells and a top layer of clear, yellow-ish plasma. I then use some calipers to measure the lengths of the two layers and, presto! There’s the hematocrit measurement.

Because hematocrit is linked to hydration, we’re also measuring rainfall (as determined from local weather stations), soil hardness, and fecal water content. Soil hardness is quick and simple; we just take a penetrometer (it looks like a long, metal tube with a spike sticking out the bottom), place it on our soil site, and push it all the way to the ground. If the spike digs in far, then the soil is soft and we’ll have a low reading. If the spike doesn’t sink into the soil much, then the soil is hard and we’ll have a high reading. To measure fecal water content, we first collect the feces from the degus (you just put a paper towel under the trap and let the degus work their magic, it doesn’t take very long) and then weigh the feces, dry them in a drying oven for 8 hours, and then weigh them again.
Collecting feces

Degus, like many rodents, are often infested with fleas and other ectoparasites. Ectoparasite level is a good indicator of general health and may even be correlated to immune function. To measure ectoparasite levels, we have one person hold a degu over a yellow bucket while another person brushes the degu with a flea comb. We first comb the degu’s back in the direction of the fur 15 times and against the fur 5 times. Then we comb each side with the fur 5 times and against the fur 5 times, and finally we comb the head against the fur 5 times. Before we comb the degu we spray some alcohol into the bottom of the bucket to instantly kill the parasites, which we then collect and store in alcohol.

Friday, September 23, 2011

Seasonality of the stress response

The natural world is dynamic, ever-changing environment. As the seasons progress, organisms experience fairly predictable changes in temperature, precipitation, and photoperiod (length of daylight). Just as the environment is not a static world, neither is an animal’s ability to cope with environmental challenges.

Research has shown that many animals display seasonal patterns in both baseline and stress-induced glucocorticoid concentrations. For the white-crowned sparrow (WCS), the highest CORT (in this case, corticosterone) levels occur during the breeding season while the lowest CORT levels are seen during molt. Previous research on degus has shown that the highest concentrations of CORT (cortisol) occur during lactation.

There are many different hypotheses for why we see these seasonal differences in the stress response, but here are the main three:

  •  Energy mobilization hypothesis: Because CORT’s metabolic effects help increase the amount of available energy, this hypothesis predicts that high levels of CORT will occur during energetically costly periods. This is the case for both WCS and degus, as breeding (for WCS) and lactation (for degus) are very energetically costly life history stages.
  • The behavior hypothesis: CORT also has many functions on behavior- this hypothesis states that animals modulate their CORT levels depending on the type of behavior that the season requires. This is probably best explained by an example: If a bad storm comes through when a WCS has just laid eggs (this means it’s the beginning of the breeding season), then it makes sense for the WCS to abandon its nest and flee to safety since their reproductive effort is, at the time, rather low, and the WCS can start another clutch later. But, if a bad storm occurs when a WCS has several-day old chicks (this would be during the late breeding season), then it is probably in the best interest of the WCS to try to ride out the storm since their reproductive investment is rather high. According to this hypothesis, we would expect to see high levels of CORT during the early breeding season (since high levels of CORT are correlated with movement) and low levels of CORT during the late breeding season. As it turns out, this is exactly what we see in wild WCS. But, what is unexplained is why levels during the late breeding season are still higher than any of the non-breeding life history stages.
  • The preparative hypothesis: In my previous blog post I mentioned that CORT helps prime the fight-or-flight response, another essential component of the stress response. In addition to priming the fight-or-flight response, elevated baseline levels of CORT also have permissive functions on the immune, metabolic, and reproductive systems in order to help an animal get ready for a stressful period.  The preparative hypothesis predicts that animals increase baseline levels of CORT during life history stages when there’s a predictably greater risk of encountering severe stressors. This hypothesis hasn’t been as well studied as the other two because determining the risk of experiencing adverse conditions is difficult to define and measure.
While the seasonality of the stress response has been relatively well studied in birds, there are fewer field studies examining the seasonal stress hormone profiles of mammals. Of these mammalian studies, few actually measure baseline glucocorticoid levels. Measuring baseline CORT is difficult because CORT starts to increase after the onset of a stressor, so most animals must be bled within 3 minutes of capture (the 3-minute rule is a pretty universal rule in field endocrinology). Trapping mammals is difficult because you often need many, many traps spread across a large area, so most researchers collect blood samples after trapping for a period of a few hours.

One of the aims of my research is to determine the seasonal stress profile of the degu. I am hoping to improve upon a previous study that measured CORT after a trapping period of 2 hours. Here are the four blood samples that I take from each animal:

Baseline: This sample is taken within 3 minutes of capture. This sample is the most important of the four because it allows me to determine what kind of CORT levels an animal is typically experiencing on a day-to-day basis.

Stress-induced: I take this blood sample 30 minutes after an animal is trapped. Capture is a major stressor (it’s basically a simulated predation event from the animal’s point-of-view), so this sample will help me determine how high an animal’s CORT levels can get after encountering a significant stressor.

DEX: After I take the stress-induced sample, I then give the animal an injection of dexamethasone (DEX). DEX is a synthetic glucocorticoid and binds to CORT receptors, thus initiating negative feedback. After waiting for 90 minutes, I then take another blood sample, which will tell me how well the animal can turn of its stress response. The ability to turn off the stress response is really important because high, continuous levels of circulating glucocorticoids can start to cause problems (this is termed chronic stress, and some of the pathologies include reproductive suppression, immunosuppression, and muscle-wasting). In fact, one of the hallmarks of a chronically stressed animal is poor negative feedback.

ACTH: After taking the DEX blood sample, I then inject that animal with adrenocorticotropic hormone (ACTH). ACTH is released from the anterior pituitary, travels through the bloodstream, and binds to receptors in the adrenal glands to cause an increase in CORT production and release. 15 minutes after injection, I take another blood sample. This sample will tell me the maximum amount of CORT an animal can produce. It will be interesting to compare this sample to the stress-induced sample to see whether animals are reaching their full adrenal output 30 minutes after capture.

The blood samples that I’m collecting are not the full story of the stress profile, though. Changes in levels of the carrier protein, corticosterone binding globulin (CBG), could affect the availability of biologically active CORT. The role of CBG is unclear, though; many biologists claim that because CBG-bound CORT is unable to bind to receptors, then high levels of CBG decrease the amount of “free CORT.” On the other hand, CORT is a steroid hormone (it’s lipid soluble, not water soluble), so in order to circulate through the bloodstream, it may be necessary to have a protein carrier in order to reach all of the target tissues.

Density and distribution of CORT receptors may also play a large role in the seasonal regulation of the vertebrate stress response. CORT can only exert its physiological effects by binding to receptors, so if changes in CORT receptors mirror that of changes in CORT concentrations, then an animal’s stress response may not be changing over the seasons. Measuring CORT receptors takes a lot of time because the receptor protocol needs to be optimized for each tissue type. I won’t be attempting this, but my lab-mate is currently doing some really cool, cutting-edge research on the seasonal regulation of CORT receptors in the house sparrow.

So far, I have collected blood samples from male and female degus during the breeding/early pregnancy and late pregnancy time points. I probably will not have time to take full stress series on the lactating females this year, but I hope to return to Chile next September to get these samples. 

Field site in winter (June)
Field site in spring (September)

I think you can guess which degu is the pregnant female and which degu is the male.

Friday, September 16, 2011

What is stress and how does it help us?

Let’s talk about stress.

When we think of stress, we typically think of events that challenge our comfort or happiness- things like big exams, arguments with a significant other, or financial problems. When stuff like this happens to us, we often tell people that we’re “stressed out.” But where did the word “stress” come from? Prior to the early 1900s, “stress” was a term that belonged to the lingo of engineers and physicists (think of bridges and buildings). Our current connotation of stress can be attributed to an Austro-Hungarian endocrinologist named Hans Selye.  Selye was injecting rats with various organ extracts when he noticed that regardless of what type of organ extract he used, all of his rats developed ulcers, atrophied thymus glands, and enlarged adrenal glands. It turns out that Selye was a very clumsy scientist and often dropped his rats and had to chase them around the room while he was trying to inject them. Selye realized that the constant handling, chasing, and actual injections made the rats…well… stressed. And so that’s how “stress” came to be.

But what is stress? A good, basic definition is that stress is the physiological and behavioral response to unpredictable or noxious stimuli (meaning, stress is what helps you run away from a charging llama). There are two kinds of stress: acute stress and chronic stress. Acute stress may be running away from a territorial llama, but then getting to safety and revising your walking route so you never have to pass by it again. Chronic stress may be when you have to pass by an aggressive llama two times a day, everyday for a month, in order to get to your field site and there’s no alternative route because of an electric fence. This kind of stress is more long term, and the stress of dealing with the llama bypasses the actual encounter because you spend a good part of your day just thinking and worrying about the llama (note: this is just an example, there are no aggressive large animals at my field site). I’ll talk more about chronic stress in future posts, but for now, let me discuss how the stress response works:

There are two waves to the stress response. The first wave, termed the fight-or-flight response, happens within seconds of perceiving a stressor and helps the animal take immediate action to avoid or deal with the stressor. The second wave of the stress response is slower and is mediated by glucocorticoids. In my research, I mostly focus on the physiological responses caused by the glucocorticoids, but the fight-or-flight response is important and is frequently glossed over, so let me tell you a little more about it.

OK, so say a llama charges you, do you flee or do you fight? This instantaneous response is mediated by a group of hormones called catecholamines. The two main catecholamines responsible for the fight-or-flight response are norepinephrine and epinephrine (also called noradrenaline and adrenaline). When your brain perceives something as dangerous, it activates your sympathetic nervous system (SNS). The SNS activates preganglionic sympathetic nerves that innervate the adrenal medulla (the adrenal medulla is the inner part of the adrenal gland, you have two adrenal glands that sit on top of each of your kidneys). These nerves form synapses with cells that produce norepinephrine and epinephrine (these are called chromaffin cells, each individual cell can produce only norepinephrine or epinephrine, never both).  Activated preganglionic sympathetic nerves release acetylcholine into the synapse, which causes chromaffin cells to increase their membrane conductance for Ca2+, which then causes intracellular Ca2+ to rise, which then results in exocytosis of norepinephrine and epinephrine into the bloodstream. Norepinephrine and epinephrine then circulate through the bloodstream and bind to adrenergic receptors in many different tissues (question to think about: if catecholamines are protein hormones, where do you think the adrenergic receptors are located in the target cells?). Catecholamines cause a variety of physiological effects including vasoconstriction, increase in heart rate, bronchodilation, inhibition of insulin secretion, stimulation of glycolysis in muscle cells, and increase of lipolysis in fat cells (question to think about: If a person with bee allergies gets stung, how does their EpiPen help them survive?). The net result of the fight-or-flight response is to help an animal activate their muscles (and other tissues) and to mobilize as much energy as possible so they can confront or flee their stressor.

So while your fight-or-flight response is kicking into high gear to help you flee the charging llama (let’s be real here, most of us would probably run away from a charging llama rather than try to fight it), the second wave of the stress response is getting ready to help you deal with some of the more long-term effects of the stressor. The second-wave of the stress response starts when your brain perceives a stressor and then causes increased secretion of corticotropin-releasing hormone (CRH) from the hypothalamus. CRH travels down to the anterior pituitary where it causes increased release of adrenocorticotropic hormone (ACTH). ACTH then travels through the bloodstream to the adrenal glands where it stimulates increased secretion of glucocorticoids (in humans and degus, the main glucocorticoid is cortisol, in most other animals the main glucocorticoid is corticosterone. From now on I’ll refer to cortisol and corticosterone as CORT). CORT then circulates through the bloodstream and binds to receptors in a variety of different tissues. Because CORT is a steroid hormone, most of its receptors are intracellular (although there is evidence that CORT also has some extracellular receptors). Since the majority of CORT’s actions are mediated through intracellular binding, most of the physiological actions of CORT take a long time to take effect (we’re talking at least 30 minutes, here, but many physiological changes are only seen after several hours). Increased concentration of CORT causes an amazing variety of physiological effects to take place:

  •  CORT has a permissive function (this means that they help other physiological mediators take full effect) on the action of catecholamines in the cardiovascular system. This means that CORT helps prime your flight-or-fight response for the next stressor that comes around.
  • During hemorrhage, CORT has a suppressive function on secretion of arginine vasopressin (AVP) and renin. Without CORT, your body would produce way too much AVP and renin in response to hemorrhage and you would probably die from too much vasoconstriction in the liver and heart.
  • The effects of CORT on the immune systems are very complicated. But, to generalize, CORT has mostly suppressive effects on the immune system and helps prevent over-activation of immune function (question to think about: what do you use cortisone cream for?).
  • CORT helps mobilize energy stores for quick consumption by inducing lipolysis, increasing gluconeogenesis (synthesizing glucose from amino acids and fats), making cells more resistant to insulin, and stimulating appetite.
  • High CORT concentrations can suppress the reproductive system by inhibiting release of gonadotropin-releasing hormone (GnRH) and decreasing the effects of luteinizing hormone (LH) on the gonads.

To put it all together, the second wave of the stress response helps animals recover from stressors and get prepared for new stressors. In terms of our llama escape, we can think about it this way:

After we get away from the llama, our high levels of CORT help us recover from the stressor by helping us not overdo our hemorrhaging (because there’s a chance that we may have been wounded in the stressful encounter) and stimulating our appetite so we can replenish our energy stores. Our increased CORT levels are also helping us prepare for the next llama interaction (since the llama may be searching for us as we recover) by priming our fight-or-flight response, suppressing our inflammatory response so our limbs remain movable, increasing the amount of available glucose, and getting our mind off of reproduction. Basically, the body is diverting energy from unnecessary functions to preparatory functions- after the stressor is gone your body can start to worry about storing glucose, fixing a wound, and having sex.

This was my attempt to give you a basic overview of the purpose of the stress response. Having a basic understanding of the stress response is important for understanding the aims of my research, so continue to bear with me and I’ll eventually tell you more about the degus. If you’re interested in learning more about stress, here are some suggested readings:

-For an unintimidating, highly entertaining introduction to stress and its effects on human health, you must read: “Why Zebras Don’t Get Ulcers” by Robert M. Sapolsky.
-If you’re still wondering “What is stress and how do we define it?” check out this review paper: Romero, L.M., Dickens, M.J., Cyr, N.E. (2009) The reactive scope model- a new model integrating homeostatsis, allostasis, and stress. Hormones and Behavior 55, 375-389.
-For a more in-depth overview of the fight-or-flight response, try reading pgs. 333-339 in “Animal Physiology: Mechanisms and Adaptations” by Randall, Burggren, and French.
-Want to know more about glucocorticoids? This comprehensive review has it all: Sapolsky, R.M., Romero, L.M., Munck, A.U. (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocrine Reviews 21, 55-89.

And here is a picture of a one-day old degu:

Baby degu!


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