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

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