Stress and Anxiety, aka CRF and 5-HT2

Today's post comes to you from several tweets that Sci received way back in the mists of time (that is...two weeks ago. Three? Something like that). Sci got wind of this paper and has been meaning to blog it for a while, but other things get in the way, like other things will. And when those other things finally get out of the way, Sci sometimes finds that she's so SLEEPY she doesn't know if she can make it through any more dry, sciency prose (sciency prose, even at the best of times, is pathetically dry. It's why Sci blogs for you. See how she cares).
Like right now, when Sci is SO SLEEPY she just wants to lie down next to the cat and sack out. Scicat is currently reclining in a truly relaxed manner on the floor and isn't making this any easier. But for the sake of stress, anxiety, depression, and a large glad of iced Moroccan mint green tea, SCI BLOGS ON.

(Sci's determination very much resembles that of the bottom biting bug pictured here. A friend of mine showed this to me about a year ago, and it may still remain the oddest thing I have ever seen on the internet. Sci also finds it hilarious that every time anyone in Japan apparently trains for ANYTHING, they must at some point sit under a waterfall, and always end by looking determined on the top of Mt. Fuji. It's like the Rocky Steps of Japan.) Magalhaes, et al. "CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling" Nature Neuroscience, 2010.

Anxiety and depression have become increasing visible, and increasingly diagnosed, problems in our society. Whether the increased diagnostic is a good or a bad thing, Sci can't say, but in her mind, the increased visibility can only be a good thing, helping to alleviate some of the stigma that surrounds these and other psychiatric disorders, disorders which often have no visible symptoms. And the increased visibility of these disorders has also been important in promoting research to find the causes behind psychiatric disease, and of course to hopefully, some day, find cures.
One of the things that has really interested the scientific community with regard to disorders like anxiety and depression is the role of stress. Believe it or not, it's only recently that the role of stress, and particularly the role of stress HORMONES, has become established as something that can promote, bring on, or otherwise exacerbate a whole host of psychiatric disorders. So first things first, Sci's gonna give you a little background on what exactly it is that's got you so stressed.

Stress, in biological terms, is what happens when you can't respond effectively to the environment around you. Stress can be a lot of things. In the animal world, stress can be as simple as it being too cold or too hot, too light or too dark, too hungry or too thirsty. In human terms, stress can refer to all of those, but it can also refer to times with little sleep, time with lots of exams or grants, and times in which other humans have high expectations you feel unable to match.
But regardless of the cause of the stress, your body is going to respond the same way, with the release of a hormone from the hypothalamus of the brain. This hormone is corticotropin-releasing factor (CRF). Vasopressin is released as well. By their powers combined (and put very, very simply), these hormones cause the release of other hormones (many of those involved themselves in stress) which can increase your heartrate, temporarily decrease inflammation, and generally place your body in a state of alarm.
Now this state of alarm is a very good thing in the short term. But scientists have realized that the effects of these hormones, particularly CRF on the brain may be...a cause for alarm.
CRF is one of the current targets (one of many, really) under investigation by scientists for a cause of anxiety. It is possible that people with anxiety disorders may release too much CRF in response to stress, or be more sensitive to the effects of the CRF they release, causing an anxiety response that is a lot higher than you would normally expect.
But the question is, HOW does CRF cause this anxiety response? And now we get in to the hypothesis of the paper, the 5-HT2 receptor.
Serotonin (5-HT) 2

Sci has blogged a good bit on and its role in things like depression, but it plays a role in disorders like anxiety as well. To sum up, serotonin is a chemical messenger that is released from neuron to neuron, and helps to convey information. BUT, chemicals messengers (we call them neurotransmitters) can only have effects if they hit a receptor specific for that neurotransmitter (for more on this, you can see this post I wrote on neurotransmission, and this one I wrote on serotonin). In the case of the serotonin system, there are between 14 and 17 (this paper says 15 but I know that's still up for debate) currently known serotonin receptors, and there are probably going to end up being more than that. Each serotonin receptor results in a different effect in the body, due to both the specific placement of that receptor (obviously a receptor in the stomach is going to have a different effect than one in the brain, and different parts of the brain are going to have different results as well), and the type of the receptor involved (you can have a stimulatory and an inhibitory receptor in the same part of the brain, and depending on which one gets stimulated, you can end up with different effects).
The one receptor that we are concerned with today is called the serotonin-2 receptor (5-HT2). There are actually 3 types of 5-HT2 receptors, 5-HT2A, 5-HT2B, and 5-HT2C. In particular, the 5-HT2A and 5-HT2C receptors have been thought to be involved with anxiety.
But yet again, the question is HOW?
So for this paper, the scientists took mice, and injected CRF (the stress hormone we mentioned before) into the brains of mice. They did this by injection, rather than stressing the mice behaviorally, to try and keep the results specific to the brain. What they found (the first half of the paper is all binding curves, which unfortunately aren't so great to look at if you're not a scientist) is that CRF injection, which in turn activates CRF receptors (remember that chemicals in the brain are only as good as their receptors), ALSO increases the number of 5-HT2 receptors that are present on the cell membranes of neurons. They found this out via binding studies with the 5-HT2 receptors. The authors showed that CRF treatment (which can be described as a 'stress' because it's the addition of a stress hormone) caused 5-HT2 receptors which had just been hanging out in the cell before to be recruited to the cell membrane and thus to be active.
The actual mechanism behind this is a little complicated. Basically, CRF receptors are stimulatory to certain actions in a cell. Those stimulatory actions stimulate the movement of 5-HT2 receptors to the membrane. The 5-HT2 receptors then have increase INHIBITORY actions at the surface of the neuron. So stimulation by CRF increases inhibition via 5-HT2 receptors. Sounds complicated, but it's not so bad when you break it down.
And of course, that's nice, there's a cool mechanism (which we neuroscientists love) but what kind of effect does it have on BEHAVIOR?? After all, we can't prove that CRF did anything important unless we see an increase in 5-HT2 receptors having an effect on behavior.
Enter the elevated plus maze.
The elevated plus maze is one of the common tests for looking at anxiety in rodents. How do you tell if a mouse is anxious? Of course it's anxious! It's a MOUSE! It's a little pile of squeaky panic! But you can test it with this maze. It's in the shape of a large plus sign which is placed flat, several feet off the floor. Two of the arms (let's say the up and down arms) are just open and flat, while the other two (the side to side arms) have walls on them. Put your mouse in the middle and see where he goes.
Mice, as you may know, like the dark. They also don't really like to walk around something narrow which they might fall off. So the open arms (with no walls) are not very nice, and an anxious mouse will spend more of his time in the arms with the walls, where he feels safer. If you have a REALLY anxious mouse, he will spend all his time in the walled arms, while if you have a less anxious mouse, he will spend more time exploring the open arms of the maze, because he's feeling brave.
So what the scientists did here was to use the maze to test the anxiety of mice that had been treated with CRF (the hormone for stress), and then treated with a 5-HT2 receptor agonist, which would stimulate the receptors that were sensitized following CRF treatment. And they got this:

This is what data looks like from an elevated plus maze. What you're looking at is how long it took the mouse to enter the open arms of the maze (looks like a really simple measure, and it is, but trust Sci, behavioral data isn't easy, there are a lot of little things you have to keep your eye one). The first three bars up there show vehicle treatments, just the 5-HT2 agonist (they used a really low dose to check for sensitivity) and then just the CRF. But the FINAL bar, you can see they used CRF treatment and then a 5-HT2 agonist. The animals were much more sensitive to the 5-HT2 agonist AFTER they had been treated with CRF. Since we know from the first studies they did that CRF increases 5-HT2 receptors, this means that the increased numbers of 5-HT2 receptors on the neuron membranes were having a behavioral effect in how the mouse reacted. Basically, the 5-HT2 agonist at a higher dose makes mice more anxious, and it takes them longer to enter the open arms of the maze. Now, these mice have higher levels of 5-HT2 receptors, and are more sensitive to the 5-HT2 agonist, and are thus more anxious at a lower dose.
What's really nice about this study is that the authors were then able to reverse the effect, using a 5-HT2 antagonist. Since a 5-HT agonist makes mice MORE anxious, a 5-HT2 antagonist should make them LESS anxious, right? And so it did.

This graph looks more complicated but the take home message is the same. This time, in the far left two sets of bars, they started out with a dose of 5-HT2 agonist that made normal mice super anxious, so anxious that they never ever went in the open arms of the maze. They then tried to BLOCK that effect in control mice, and in mice treated with CRF, using a 5-HT2 antagonist. In the control mice, the antagonist reduced their anxiety a little, but in the CRF treated mice it reduced their anxiety a LOT, showing that the 5-HT2 receptors are more sensitive to agonists and to antagonists, and that both of these have behavioral effects.
They showed a couple more things in this article (it's thick, like Nature Neuroscience papers often are), but the take home message is this:
1) CRF is a stress hormone involved in anxiety.
2) 5-HT2 is a receptor involved in anxiety.
3) CRF (which is technically a kind of stress) INCREASES the numbers of active 5-HT2 receptors
4) When CRF increases the numbers of 5-HT2 receptors, mice are more sensitive to them.
What does all this mean? It means that these mice may be more anxious (though the behavioral data didn't really bear this out, the mice really only responded to the agonists and antagonists, this could just be the sensitivity of the test). But what's really important about this is that these authors have found one mechanism by which stress may contribute to feelings of anxiety. If you've got problems with high 5-HT2 receptors already, AND your more sensitive to stress, this may bring on some major high anxiety when you're in a stressful situation.
But what it ALSO means is that, using this mechanism, scientists may be able to find new therapies to treat anxiety. For example, they may be able to use 5-HT2 antagonists in people who have higher levels of 5-HT2 receptors in their neurons. They may be able to use antagonists to block the effects of CRF so that anxious people are not as sensitive to stress. We are a long way from these treatments still (though some 5-HT2 antagonists are on the market and work pretty well), but knowing how these things work can go a long way to helping us help people with anxiety.
Magalhaes, A., Holmes, K., Dale, L., Comps-Agrar, L., Lee, D., Yadav, P., Drysdale, L., Poulter, M., Roth, B., Pin, J., Anisman, H., & Ferguson, S. (2010). CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling Nature Neuroscience, 13 (5), 622-629 DOI: 10.1038/nn.2529

7 responses so far

  • pinus says:

    Great write up. One minor point. I think there is an important distinction being made in the field with respect to the peripheral hormonal actions of CRF, compared to the central neuromodulatory actions of CRF. One might argue that CRF signaling in regions that have 5HT2 receptors that regulate anxiety is not hormonal, but rather due to CRF release from local and projecting neurons.

  • nikipedia says:

    Great post. It kind of irritates me to see this kind of thing published without any discussion of sex differences, though -- they only looked at male mice and I would be shocked to find identical results in females. See this recent article in Molecular Psychiatry finding sex differences in CRF receptor trafficking and signaling: (doi: 10.1038/mp.2010.66 if the link doesn't work).

  • sjs says:

    you are one of the best writers on the internet. seriously.

  • Nerissa Belcher says:

    The results only make sense if the so-called 5HT2 antagonist M100907 actually works as a partial agonist. Leading me to wonder if a person under high stress might benefit from thinking about something which is still stressful but less so. I.e. cognitively inducing their own partial agonist. Reviewing the link provided in reply #2 by SJS is suggestive this would be most effective with women. I.E. men naturally lower CRF receptor activity over time when under stress and women maintain CRF receptor activity. So men are on autopilot in stress control and women have to do something. Specifically think/compain about relatively trivial things like their husband often being late, their weight and more.
    Obsessively thinking and complaining about stressful things is called rumination. This is much more common in women than men.
    As an example of the benefit of rumination consider a woman whose boss is hinting that woman is likely to be fired before the day is over. My idea suggests if the woman complained to everyone that would listen, as she often did, about being overweight or what a jerk her boyfriend was, she'd get through the workday with overall less stress. Consider the implications. That woman might stay heavy or stay with the jerk in order to have a mechanism to handle higher stress elsewhere.

  • Dr Becca says:

    I'm a little confused by figure 2. You said "they started out with a dose of 5-HT2 agonist that made normal mice super anxious," but there's no difference between veh/DOI and veh/veh--it looks like it's the 5-HT2 antagonist that's making the normal mice anxious in those two left sets of bars (regardless of whether they received the 5-HT2 agonist). Also, shouldn't we expect the CRF/veh controls in fig 2 (white bars) to be exactly the same as CRF/veh in fig 1? It looks like there's about a 3-fold difference between the two groups. What's up?

  • Scicurious says:

    Nerissa: it's an interesting idea, but I think to work, you'd have to prove that stressful thoughts really ARE partial 5-HT2 agonists, and I think the reality is a lot more complicated than that. 5-HT2 receptors may contribute to anxiety, but I don't think they are the only thing.
    Dr. Becca: you know, that figure confused the crap out of me when I first saw it, and then I thought I got it, and now I'm worried again. 🙂
    Ok. Here goes.
    The open bars are the vehicle vehicle treated, remember, the BLACK bars are the dose of M100907 (the antagonist). Hmmm...AH HA. I think I was backward. It looks like the M100907 antagonist causes total latency, which is then partially ameliorated by CRF and then further by DOI...hmmmm...ok it looks like DOI is the anxiety reducing agent here, while M100907 is inducing, and I'll bet that's because DOI had 5-HT2C activity and that's why we're getting the effect in this particular case, though it makes me wonder why they used this particular drug at all, and how they are interpreting the data as they do. The cRF treated mice are still more sensitive...The 5-HT2A receptor and CRF appear to interact with each other in all cases, making the mouse more anxious. I think the important thing is this figure is that M100907 in the fourth set of bars ATTENUATED the affects of DOI and CRF together (as compared to the open barred vehicle group). I think. This is where the whole thing got very confusing for me, and I think the way the authors expressed the conclusion is also confusing. Anyone else got other ideas on this? M100907 is really pretty selective, but DOI is not...

  • Dr Becca says:

    Thanks, Sci! You're right--the interaction is clearly there, which I guess is the take-home message. Still, though, I'm troubled by the difference between CRF/veh mice and veh/veh mice in the control condition for panel 2, a difference that isn't there in panel 1. Did they give the mice a higher dose of CRF in the 2nd experiment?
    Sigh...I guess I should just read the damn paper!
    Unfortunately, I know very little about serotonin receptor subtypes, so I can't really comment on what non-selectivity-related effects might be going on. Hopefully someone else can chime in!