Science Sunday: The ontogeny of behaviour in the albino rat

Every Sunday, I'd like to post a review of an interesting peer-reviewed science article. To kick things off I'm picking an old favorite, originally posted in 1964! It is certainly well cited, Google Scholar lists the citation count at 452! Indeed this paper was a "Citation Classic" in Current Contents in 1981. At the time the lead author  Robert Bolles, was still living and stated:

"I have always believed in the idea that experimenters should look at their animals...the human eyeball is the instrument of choice if you want to observe a new phenomenon, and particularly if you want to gain a new understanding of it."

Sprague-Dawley Rat

If you have ever wondered why science uses rats the answer is that we know everything there is to know about rats. We know how they age, how they metabolize ... anything, how the respond to stress, how they develop over time, even how they laugh when tickled. Well, our understanding of rat behavior begins in antiquity but is greatly expanded by this 1964 article originally published in the journal Animal Behaviour.

In fact this article describes qualitatively the behaviors of infant rats from birth to about 24 days (rats are weened at day 21). In the first experiment, Bolles and Woods observed 13 litters with an average of 9 pups (117 pups) in their "natural" laboratory environment (cages). The animals were of the Sprague-Dawley line, which is still used today. They did use several different methods of observation and schedules of observation to arrive at a comprehensive guide to the ontogeny of lab rats.

They begin with postural observations, describing three postures that develop over time: lying, sitting and standing. Lying being the default resting position of the rat, often using other bodies for support. Sitting began on day 4 when subjects first began to lift their heads, and was fully developed by day 17 when subjects could sit and perform activities such as grooming. Also beginning on day 4 are the first attempts to support weight on the legs, and by day 10 the animals can support themselves. By day 13 they can run, by day 15 they can stand on three legs and scratch with the fourth. They can rear up on two legs with support for the front legs on day 16 and can rear independent of support (for the purpose of play-fighting with siblings) by day 18.

In similiar fashion reflexes are described. Without relating the specific timeline the reflexes are: twitiching, head waving, stretching and yawning, body flexion, righting reaction, freezing, sniffing, auditory orientation, and visual orientation. When describing startle response int he auditory orientation section there is a great footnote on the word "click:"

*The sound used was relatively well-controlled and constant, but, unfortunately rather poorly defined; it was the sound of a Parker T-Ball Jotter pen being retracted at a distance of approximately 1 foot.

Psychologists are hilarious. Also found it interesting that the animals did not freeze in fear until day 26 and they froze for approximately 15 seconds. I've never seen any rats hold still for that long unless they were sleeping. Following this functional activities are described. Here is the list: sleeping, consumatory behavior, locomotor activity, climbing, grooming, exploration  manipulation, digging, and defecation  Here the theme of development was similar as above, with rudimentary non-functional behaviors appearing first (such as scratching motion without making contact with the skin), that later developed into full-fledged adult-like behavior.

Ultimately we get a description of the social behaviors in the observed rats. Social behavior in young rats is evidenced by chasing and fighting. Bolles, and Woods observed rats begin this social play-fighting on day 14 when their eyes began to open.The activity peaks between day 20 and 30 when the whole litter engages in a high level of activity.

Table 1

In a second experiment Bolles and Woods attempt to quantify the behaviors they observed in the first experiment. Using experimental methods the authors observed 12 rats (2 each from 6 litters) and summarized their behaviors as percentages. To the right is table 1 from the paper. There are many more graphs showing the time course of the development of behaviors and it really is a fascinating reference, but I won't reproduce all of that here.

The first point of discussion and perhaps the most salient is that from these findings we can view rats as a far more social animal than might otherwise be considered. Early social interactions are to wrangle for nursing or comfort, and later become play fighting and chasing. As the authors noted this social behavior likely leads to long lasting changes in the adult organism and "offers interesting possibilities for research in this area." (See the next 50 years of rat studies for more on these possibilities)

Bolles, R., & Woods, P. (1964). The ontogeny of behaviour in the albino rat Animal Behaviour, 12 (4), 427-441 DOI: 10.1016/0003-3472(64)90062-4

A Little Bit of Unpredictable Stress Everyday

That is the recipe for depression. Chronic Mild Stress (CMS). Or at least it is the recipe I used to depress rats. As I mentioned earlier I worked with rats to research the mood-effects of Salvia. One important piece of this research was the idea that a depressed brain is different than a healthy brain and may respond differently to drug exposure. So in order to apply that in rats, I needed a way to create depressed rats. For me, that meant using CMS.

In the CMS model, developed first by Richard Katz, rats exposed to mild stressors everyday showed a decrease in sucrose consumption that was later reversed by administration of an anti-depressant (Katz, 82). Let me explain.

Chronic (happening regularly over time) Mild (not severely traumatic) Stress (a feeling of strain to adapt to changing environmental (or psychological) conditions). Practically this means doing things to rats to make them a little bit uncomfortable everyday. In my research, inspired by work that came out of Paul Willner's lab, this involved the following: overnight strobe-light (2Hz),  cage tilting at 45 degrees, forced paired housing (a strange rat was put in the subject rats cage separated by a wire mesh), continuous illumination (there is no night), overnight food deprivation, overnight water deprivation, and empty water bottle exposure. These were all done briefly enough not to cause excessive stress, remember I was going for uncomfortable not traumatic.

A Long-Evans Lab Rat

There is a hallmark feature of human depression called anhedonia. Anhedonia is the inability to experience pleasure from pleasurable stimuli. Or joylessness. Anhedonia is the reason that depressed people don't laugh at jokes, or smile when a baby giggles, or enjoy doing any of the things they used to enjoy doing. Pleasure doesn't feel good anymore. If you or someone you know has ever been depressed it is likely you witnessed anhedonia.

Happy rats love to drink sugar water. Even when dissolving a very small amount of sugar in water (less than 1% sucrose solution), rats show a strong preference for sweet-water over regular water. We can measure that by giving the rats two water bottles, one with plain drinking water and one with sweet-water. Come back 24-hours later and measure how much of each the rat drank. Happy rats drink around 90% sweet-water. We might say that sweet-water has a greater hedonic value; it is more pleasurable for the rat.

After three weeks of CMS my rats exhibited anhedonia. After three weeks of exposure to the mild stressors listed about the rats no longer show a preference for sweet-water. Again we present the rats with two water bottles, one with plain drinking water and one with sweet-water. 24-hours later we see that they drank about 50% sweet-water. This shows no preference for the sweet-water. Even though the sweet-water might taste better (have a higher hedonic value), the rats don't care. Because they are joyless. They have developed anhedonia. They are depressed (or at least they are now a robust animal model of human depression).

Katz, R. (1982). Animal model of depression: Pharmacological sensitivity of a hedonic deficit Pharmacology Biochemistry and Behavior, 16 (6), 965-968 DOI: 10.1016/0091-3057(82)90053-3

Willner, P. (2005). Chronic Mild Stress (CMS) Revisited: Consistency and Behavioural-Neurobiological Concordance in the Effects of CMS Neuropsychobiology, 52 (2), 90-110 DOI: 10.1159/000087097

The Science Behind Todd Akin's Gaffe

Recently Rep. Todd Akin (R-MO) made a very bad gaffe. It is pretty serious and you might have already heard about it. The quote in question as made during an interview with KTVI on Sunday was:

“If it's a legitimate rape, the female body has ways to try to shut the whole thing down."

Now a lot of people have been very upset about the phrasing of "legitimate rape" and rightfully so (you can already buy "illegitimate rapist" T-shirts). But that's not what I want to write about. I want to talk about the myths regarding pregnancy and rape. There actually is science that has been done specifically on this problem, and as it turns out the opposite is true. Researchers have found that the "per-incident rape-pregnancy rates exceed per-incident consensual pregnancy rates by a sizable margin." (Gottschall & Gottschall, 2003).

The Gottschalls set out to re-evaluate the data gathered by medical and public health professionals regarding the medical and psychological health of rape victims. By carefully screening this data the researchers were able to come up with meaningful comparisons between consensual (albeit unprotected) sex pregnancy rates and rape-pregnancy rates. After adjusting for contraception use the per-incident rate of pregnancy resultant from rape is about 8%. This is compared to the consensual, unprotected intercourse was calculated at 3.1% (Wilcox et al., 2001). But how can we explain that?


One possible explanation is that women, somehow, broadcast their fertility. That is to say that men can tell when women are ovulating, and that this is arousing, and thus triggers the rapist to strike. While there is evidence that men can detect when women are ovulating, I still don't think that fits well here. In general rape is considered a crime of violence not passion. It is about dominance not reproduction. But even still there may be some link between increased arousal and violence in men. A better supported explanation is that of coitus-induced ovulation. This is like what happens in cats; a significant vaginal stimulation triggers ovulation. In fact the very stress of being raped may trigger ovulation. Researchers were able to show that acute stress can trigger ovulation at any point of the menstrual cycle (Tarin et al., 2010).

Certainly more research is to be done on these and other potential mechanisms of this phenomenon. Even still I think it is important for politicians (and all of us) to use the science that is available to us. There is research that we can use to help inform public policy and I feel it is dangerous for us to ignore science.I don't know what this means for Todd Akin (I'm not a political science professor) but hopefully he'll read some science for next time.

Jonathan A. Gottschall, & Tiffani A. Gottschall (2003). Are per-incident rape-pregnancy rates higher than per-incident consensual pregnancy rates? Human Nature, 14 (1), 1-20 DOI: 10.1007/s12110-003-1014-0 
Tarín JJ, Hamatani T, & Cano A (2010). Acute stress may induce ovulation in women. Reproductive biology and endocrinology : RB&E, 8 PMID: 20504303 Wilcox AJ, Dunson DB, Weinberg CR, Trussell 
J, & Baird DD (2001). Likelihood of conception with a single act of intercourse: providing benchmark rates for assessment of post-coital contraceptives. Contraception, 63 (4), 211-5 PMID: 11376648

Researching Diviner's Sage

As I mentioned before I worked in a rat lab, and over the next few weeks I'd like to write a bit about some of the research I did as a graduate student at UMSL. I spent my time depressing rats and treating some of  them with novel plant-derived compounds (some poor rats just got depressed and given placebo). Before I can tell you more about my project I'd like to share with you some of the work that influenced me.

"If I have seen further it is by standing on the shoulders of Giants ." -- Isaac Newton

One of the first sources of inspiration for me was an article I read in Wired about research done by Catherine Willmore (et al.) back in 2007. She was researching a relative of Sage from the Lamiaccae family, Salvia divinorum, AKA Diviner's Sage, AKA Mexican Mint. The plant produces hallucinatory effects in humans and thus has been used by religious and recreational consumers. But since salvinorin A (the main active extract derived from the plant) is selective to kappa-opioid receptors, and is one of the few non-alkaloidal hallucinogens, it has potential to influence the development of a new class of pharmacological drugs and is thus interesting to study. Also because many states lack laws regarding sale and use of Salvia it has grown in popularity amongst the youth. (Don't do drugs)

The researchers used a drug discrimination paradigm in rats to verify that salvinorin A does in fact target kappa-opioid receptors as a primary mode of action. This was my first exposure to this (apparently well-accepted and robust) paradigm. The researchers began by conditioning rats to press a lever when exposed to an established synthetic kappa-opioid agonist (U-69593 obtained from Sigma-Aldrich right here in St. Louis). To do this, they limited the rats' diet, and then put them into a response box with two levers, on days when they got U-69593 they were rewarded with food after pushing the lever on the right, and on days when they got a saline injection they were rewarded when pushing the left lever. Thus once the rats were sufficiently trained on lever pushing then they were assumed to be able to discriminate between a kappa-opioid agonist and control injections. At that point the trained rats were given salvinorin A instead of U-69593 and they still pushed the correct (right) lever, indicating a similar subjective pharmacological experience between U-69593 and salvinorin A. But it is possible the rats had associated "different from saline" with pushing the right lever. So next the researchers injected the rats with nor-BNI (a kappa-opioid antagonist) which will prevent kappa-opioid agonists from having an effect. After treatment with nor-BNI the rats were given a dose of salvinorin A and placed in the response box.  This time the rats pushed the left lever indicating an effect similar to saline.

So I think this falls under the heading of talking to the animals. Not in the Doctor Doolittle sense, but this paradigm asked rats, "Does this drug (salvinorin A), make you feel the same as this other drug (U-69593)?" and the rats kindly answered, "Yes. Yes it does."

Cool paradigm not withstanding, the results of the experiment largely confirm (in an animal model) information that we already suspected. This is the research that introduced me to Salvia and after some digging I found that there hadn't been a whole lot of science done on this mysterious plant. So that gave me an opportunity to ask some new research questions of my own.

Willmore-Fordham CB, Krall DM, McCurdy CR, & Kinder DH (2007). The hallucinogen derived from Salvia divinorum, salvinorin A, has kappa-opioid agonist discriminative stimulus effects in rats. Neuropharmacology, 53 (4), 481-6 PMID: 17681558

Cell Phone Anxiety: Extended Edition

Yesterday I posted some research about cell phone anxiety. You may have noticed that while I referenced a science journal article I didn't talk much about the results. Indeed, the only results I listed were descriptive (basic counting that describes a behavior) and qualitative (as opposed to quantitative). This is because of some fundamental flaws with the data as described in the article. So for those of you interested in such things here is the extended review enumerating three big problems I had with the data:

1. Sampling Error. The author state that they began with 47 participants but 24 had dropped out or otherwise been disqualified before the start of the experiment.  The problem is we can't be sure if there was some other extraneous variable contributing to the high attrition rate. For example, if those 24 dropped out because the idea of being without their cell phone for 3 or 5 days was too threatening then the study missed the opportunity to measure their anxiety. Or maybe not. The thing is that we don't, and can't, know the  possible confound of the sample selection.
2. Median Split. The authors used a median split (M=92) to determine high- vs low- texting participants. The problem here is that someone with 91 texts per day is in one group while someone with 93 is in another. Sure the line has to be drawn somewhere, but when you have a small sample size, it is hard to distinguish the difference between groups with a median split, especially if data is clustered near the median (although we don't know if that was the case here). A better solution might be to use thirds so you have a high-, moderate, and low-texting groups, which might lead to more meaningful comparisons between high- and low-.
3. Low Sample Size. Of course this is the big one. With only 23 participants stretched across four groups there simply is not enough data for meaningful statistical analysis, not with humans anyway. When doing animal research your samples are so homogeneous (there is little individual difference between rats) that you can get away with small sample sizes. In fact to do regression the rule of thumb I'd always heard was that you'd need at least 60 per group. That might be hard to get for some researchers and there are statistical tricks that get used (like bootstrapping) to work around that number. But for this study, the sample is just way to small and, to be fair, the authors acknowledge this in the discussion.

That said the  lit review and theory in the paper were good. Some of the descriptive and qualitative results were interesting. So while statistically almost meaningless, I feel as though the paper is still a valuable contribution to the relatively barren landscape of research into cell phone restriction anxiety.

• Dorothy Skierkowski & Rebecca M. Wood (2012). To text or not to text? The importance of text messaging among college-aged youth Computers in Human Behavior, 28 (2), 744-756 DOI: 10.1016/j.chb.2011.11.023

Cell Phone Anxiety

Editor's Selection Icon Earlier I wrote about cell phone rudeness in the classroom. In that post I mentioned that asking students to go without cell phones is like asking them to go without friends. That statement generated a lot of interest from faculty and students int he comments, on facebook, and in person so I thought I would share a little bit of research that backs up my statement.

In a recently published article (see below for reference) Dorothy Skierkowski and Rebecca Wood tracked college-aged youth's anxiety over a 3- or 5-day texting restriction. That is to say, for three or five days participants in the study were not allowed to use text messaging. In spite of the studies extremely small sample size (n=23), they had some interesting results. They found that participants that were identified has high-volume texters (greater than 92 texts per day on average) thought about texting an average of 47 times per day during the restricted period. Even the low-volume texters thought about it 23 times on average. In psychology we might consider those repetitive worrisome thoughts as rumination, a hallmark of anxiety.

Another important finding from this study involved the open-ended survey responses from students collected during the restriction. The authors sum those responses with the following sentence:

 "Clearly, asking students to restrict their texting behavior made a powerful impact on most study participants, to the extent that some were not able to refrain from doing so, a large number believed their relationships had worsened over the duration of the study, and most endured the effects of texting restriction with a moderate to high degree of annoyance, anxiety, and/or stress. "

So it is because of this and similar studies that I think we need to review the idea of the cell-phone ban in the classroom setting. While this study involved restricted use over a matter of days, it was still observed that students frequently thought about texting and failed to comply with text-messaging restrictions. So my fear is that banning cell phones might be just as detrimental to learning as allowing them in class. I decided to do my own experiment to gather data on how cell phones impact learning in MY classroom. I let you know how that turned out in a future post.

UPDATE: If  you are interested in statistics you might like my follow-up to this post which more closely examines the statistics in this article.

Dorothy Skierkowski & Rebecca M. Wood (2012). To text or not to text? The importance of text messaging among college-aged youth Computers in Human Behavior, 28 (2), 744-756 DOI: 10.1016/j.chb.2011.11.023