Critiquing LaPlant et al, 2010, in Nature Neuroscience, Part 1. Let's get this going.

So Sci was sitting in a seminar the other day. We were mentioning this paper, some problems we had with it, some of the things we LIKED about it, and various approaches, etc, in our usual sciencey fashion. As the discussion got intense (in a good way), one of the PIs there leaned over and said to Sci "you know, you should TOTALLY blog about this". Sci paused, and said "well...it's probably a little complicated for my readers".

And it is. The title alone. "Dnmt3a regulated emotional behavior and spine plasticity in the nucleus accumbens"?!?! This is complex stuff, much more complex and involved than I usually cover on the blog. It's got DNA methylation, how that affects things like protein transcription, how that affects BEHAVIOR, and what the heck does all that even have to do with SPINE DENSITY!??! It's a lot. It's complicated to understand, because it involves a LOT of background understanding, and it's complicated to explain, for similar reasons.

But then I thought about it. You know, my readers (all two of you) are some smart people. Many ARE in science, if in different fields, and many are interested in science. You guys can get this just fine, if I can EXPLAIN it. And it's an interesting paper, partially because of what it studies, and partially because of what it REVEALS about what scientists think about "emotional behavior" right now.

So. We're going to break it down. We're going to go through the hefty stuff behind this paper, one chunk at a time. We're going to cover the findings, we're going to cover the ideas, and we're going to cover the problems that the other scientists at the table had with this paper. We're going to go into why the problems with this paper exist, and what can be done about them, and we'll talk about what this means for the field of drug abuse and "emotional" behavior in general. We're going to do it over the next few days. Because y'all are PLENTY smart enough to get this, if you only have the background. And gaining expertise in a field is a lot about being able to know your background, where you came from, interpret it correctly, and move it on to the FUTURE.

Let's do this thing.

ResearchBlogging.org LaPlant et al (there's TWENTY SEVEN authors on this thing). "Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens". Nature Neuroscience, 2010.

So let's start with that acronym, Dnmt3a, what it means, what that REALLY means, and why they are interested in it.

Dnmt3a stands for DNA methyltransferase 3a. And to get to what THAT means, we will have to dig deeper.

Welcome to DNA METHYLATION!!!

So. You know you have DNA.

DNA, as you know, makes up genes, which are specific sequences which code for a protein output (someday I will cover how this happens, as it is very cool, but today is not that day). So in order to get a specific protein output, you need to have that gene in your DNA, and that gene also needs to be EXPRESSED. Getting a gene expressed is a long and complicated process, but what we're focused on right now is not the process, but the beginning. Because in order for DNA to be expressed, it has to be AVAILABLE. This means that it can't be tightly wound around histones, for one thing. But even when it's not wrapped around histones, available DNA is not always available.

Let's look at why this is.

Up there at the top of the picture, you can see the base pairs as they make up DNA. What is blown up at the bottom is one of those specific base pairs, cytosine. What DNA methylation does is put a methyl group (highlighted in yellow) onto the cytosine.

It doesn't look like much, but this methyl group is very important. Depending on where it is located, it can either make expression of that gene go DOWN, or it can make it go UP. Too much methylation in some sites can result in cancer, but too much methylation in other sites can silence a gene entirely. Methylation has lots of important functions, including regulation of cell types, cell cycles, and all the way up to things like memory.

So that's basic DNA methylation, what about DNA methyltransferase? DNA methyltranferase (Dnmt) is an enzyme that causes methylation of DNA. There are various types, but the one we are interested in right now is Dnmt3a. And for this paper, what we are interested in is the role that Dnmt3a plays in addiction.

You might say: whoa, we're going from super tiny things changing DNA to addiction, which is whole brain and HUGE! Well, yeah. Little things have big effects. And while histone deacetylation and methylation is today, DNA methylation is forever (or at least for a really long time). Because addiction is such a long-lasting, relapsing disorder, it stands to reason that some semi-permanent to permanent changes are being made. We know that Dnmt3a is expressed in brain areas like the nucleus accumbens, an area that is very closely associated with the rewarding and reinforcing properties of drugs like cocaine.

So to look at whether the DNA methylation could be changed in response to drugs like cocaine, the authors of this study looked for the various DNA methyltransferases in mice that had either received a single injection of cocaine.

And here's what they got. What they got is, at first, not quite what I expected, but when you think about it, it makes sense. Immediately after the cocaine injection, there was a SPIKE in Dnmt3a, but after about 24 hours, the levels of Dnmt3a were decreased. If you've got more Dnmt3a being made (which is what this measured), you presumably have more DNA methylation taking place. So it looks like with one injection of cocaine, you get an brief increase in DNA methylation, that drops off pretty soon after.

What confused me at first was why the levels didn't STAY high, why they dropped off. But this makes perfect sense, really, you don't want whatever changes are happening in response to cocaine to keep happening, and you certainly wouldn't want to go overboard on DNA methylation. So the methylation drops off.

Dnmt3a is not the only Dnmt involved here, but it was the only one that showed significant changes in response to cocaine.

But that's only one injection. What about lots of cocaine over a long period of time?

Here you can see on the left, mice treated with cocaine for 28 days, and THEN give 28 days withdrawal, and on the right, rats self-administering cocaine (taking their own injections their-ownselves), and then given 28 days withdrawal. In both cases, they looked at three different dnmts, and only Dnmt3a was increased, which means that while it spikes once in response to ONE injection, chronic cocaine (given either by injection or self-administered) makes it stay up for a while. That's a lot of DNA methylation going on there.

And now, the critique thus far: So far (the first two figures), we're ok. It's PCR, and you can't really argue too much with PCR. My only critique (and I'll get to this further into the paper as well), is this. Mouse, mouse...rat? why did they do all this PCR in the mouse, and then switch to rat? Or rather, why did they do the RAT self-admin, and then do mouse for everything else? It's much harder to get mice to do coke self-administration, so I don't blame them for using rats. But PCR? You can do PCR on a rat brain just as easily as on a mouse brain. So why the switching back and forth?

So so far, we're through the first two figures, and we can see that DNA methylation may change with just ONE injection of cocaine!! Tune in next time to find out what this does to...reward.

LaPlant Q, Vialou V, Covington HE 3rd, Dumitriu D, Feng J, Warren BL, Maze I, Dietz DM, Watts EL, Iñiguez SD, Koo JW, Mouzon E, Renthal W, Hollis F, Wang H, Noonan MA, Ren Y, Eisch AJ, Bolaños CA, Kabbaj M, Xiao G, Neve RL, Hurd YL, Oosting RS, Fan G, Morrison JH, & Nestler EJ (2010). Dnmt3a regulates emotional behavior and spine plasticity in the nucleus accumbens. Nature neuroscience, 13 (9), 1137-43 PMID: 20729844

17 responses so far

  • Arikia says:

    You are so effing xcore <3

  • aimeemax says:

    *applauds* Sci for a neat neuroscience explanation - I wish you had been my neurochemistry teacher! And yeah, why on earth would they have experimented on rats and meece instead of just rats. Looking forward to the next instalment.

    🙂

  • Kim says:

    Awesome post. As soon as you mentioned DNA methylation, I thought "bring it," scrolled through the intro stuff and got right to the goods. It's awesome to read about DNA without it being genetics, Genetics, GENETICS!!! (In my field, it feels like everything is hinged around it, but that might just be neighboring labs.)

    Didn't know about the rats/mice thing, but that makes sense. One question: how to rats give themselves injections of cocaine? (suddenly images of rats huddled into corners with tourniquets and syringes come to mind...)

    • yud says:

      From what I understand, the rats have something like an IV hooked up to them, and they can hit a button in their cage which administers a dose. They can still move around their cage with the IV line hanging down from above.

    • scicurious says:

      Yup is correct. The way we do self-administration of drugs in rats is this:

      1) Take one rat.
      2) Put a catheter into it's back into a vein (usually the jugular). This catheter is connected to a pump with a syringe, which sits on top of the rat's cage.
      3) The syringe is filled with cocaine, and the pump with the syringe is hooked up to a lever.
      4) Place the rat in the cage (often they live in that cage, in other cases, they only go into it for a few hours a day).
      5) The rat wanders around, and happens to hit the lever. The lever then activates the pump, which delivers a precisely measured amount of cocaine into the rat's back.

      The rat will VERY quickly figure out what's going on, and will soon learn how to press the lever. It doesn't have to be cocaine, though. Rats will press levers for amphetamine, heroin, alcohol, Crisco, sugar pellets, and access to another rat (usually a lady). It's a method that is used in a lot of reward-related testing.

  • Pascale says:

    I just wish that Dnmt interacted with Hmt at some level, so we could discuss Humpty-Dumpty.

  • john says:

    thanks...this is fun. i read these journals some anyway, but wouldn't have done much more than read that abstract as it's hard to know what's influential. looking forward to next posts.

  • RAMcCracken says:

    Didn't I just see (somewhere or other, since I subscribe to several science feeds, but seldom read beyond the headlines or mouseovers) the other day, that some rats are more resistant to cocaine addiction than others, and that they've found some sort of DNA marker associated with this trait? Establish some of the chemistry with cheap little mice, then do more behavioral (self-administration) studies with the rats, maybe?

  • Marcus says:

    Good post. Some of these articles are difficult to get because they never back off the heavy jargon. I know enough to get the basics of this sort of thing. But I always have to go hunting for the "in English" explanation, which doesn't seem to always be there.

  • RAMcCracken says:

    Sorry, hit enter by mistake, or anyway sooner than I planned to! Very little editting on that post. Oopsies! That was supposed to be a thought about why the species switch.

    My first thought was availability of message sequences; since so much more has been done with mice, the parallel rat sequences aren't always available easily, but they have that covered. Personally, I'd go for PCR primers that would work for both species, if possible, and then use mice (cheap! -ish, anyway) except where rats were necessary for whatever reason--like self-administration studies.

    (Nothing like some internal networking to suck down the time. And the sudden appearance of a boss to make me scuttle off unceremoniously. *grin*)

  • Chris says:

    As a non-science person (Biology 101 was as far as I went), I'd like to thank you for turning really interesting but hard-core science-y stuff into really interesting science-y stuff I can (mostly) understand. I have no idea why I find all this neuro-chem stuff so interesting (although I'm guessing it has something to do with my own personal neuro-chemistry!) but I do, and I love your blog.

    Thank you!

  • gerty-z says:

    Great post, Sci! I skimmed through the abstract of this paper and thought that it looked potentially interesting. Can't wait to see how it ends!

    I share Kim's interest in learning how rats self-inject cocaine, and I have one minor point: when talking about DNA methylation, I think you meant cytosine, not cysteine 🙂

  • Quincey says:

    Thanks for the interest in my paper. To answer your critique, I chose to look at Dnmt expression in both species because I wanted more convincing evidence that the subtle regulation that I was observing in mice (less than 20% regulation) happened in another experimental system more relevant to addiction--self administration. Secondly, I also felt that it would be more convincing if cocaine-induced regulation of DNMT3a were evolutionarily conserved across different species (mice and rats) and not just some epiphenomenon unique to mice. It would also be interesting to see if other rewarding stimuli (food, sex, other drugs) regulate Dnm3a in a similar manner--unfortunately, I never got around to doing this experiment.

    I have one minor correction to your commentary: figure 1 demonstrates the Dnmt3a is biphasically regulated following both a single cocaine injection as well as by 1 week of daily cocaine injections (so called "chronic" cocaine injections). Ultimately, I don't know what to make of this odd "see-saw" type regulation that seems to occur with each injection. And to make things more confusing, when animals get a month of coke followed by a month of withdrawal, Dnmt3a is up-regulated (fig2). To my knowledge, there are no other genes that are transcriptionally regulated in such a manner by cocaine.

    In any case, the general idea of this work was to test the role of dna methylation in the nucleus accumbens (NAc). And because we think that the NAc plays an important role in "encoding reward" I took the most rewarding and least rewarding stimuli that I could think of (cocaine and social defeat stress) and tested whether or not DNA methylation appears to be doing anything in this brain structure.

    • scicurious says:

      Hi Quincy, thanks so much for reading!! I usually email the authors of the papers that I blog, but I wanted to wait to contact you until I'd finished the 3-4 part series I was planning to do on your paper. I am going to try to cover the second set of figures tomorrow, as I feel this paper is worth a close look. I am so glad that you found the post! It would be lovely to get your perspective as I go along, and I welcome your addition and corrections.

      As far as your comment, I was actually planning to talk more about the mouse vs rat issue later in the paper, when it becomes clear through your use of floxed mice that you needed to use both, at least in this case. I do think that mouse self-admin might have been more appropriate, but I'm very aware that few laboratories have successfully mastered that procedure. I definitely think that sucrose self-administration, as well as a drug with a different mechanism (morphine, maybe), or even ICSS, would be a great future direction to go in at this point.

      Thanks for clarifying figure 1, I had a very difficult time figuring out how many injections were performed, as they were not listed in the results or legend of the figure, and the methods were in the supplemental data. This is nit-picky, but I think that "sub-chronic" might have been a better description of 7 injections, especially as compared to the 28 day treatment.

      Please do continue to stay in contact as I go through the rest of the paper! I would really appreciate your insights into your rationale behind the work. Would you be willing to get some questions via email?

  • Andrew C. Goforth says:

    this is what I Don't like about bloggers they never fill in the important information, conclusion, what that conclusive material , if any might mean in any other context where there are many. it appears they just like to hear them selves talk about something they want us to think they know something about. there is a scientific method for not only conducting the research but in reporting that research I suggest you read a couple of peer reviewed articles on any topic you care to chose out of any publication that reports on research such as pulmonary medicine, cardiac medicine Renal function and, liver research. Their topics are very defined the criteria they use to define any statistical changes between study groups is carefully looked at to see what other factors could or could not be effecting the out comes of the groups being studied. let me just say that the information is much more realistic even though many of their papers conclude that more research needs to bee done, on either greater number of patients, for longer periods of time or other critical criteria that was not included in the original study must somehow be brought in to the research if for no other reason to rule it out. Bloggers, if its opinion it is not science. If your picking out one part of the research to focus on at the exclusion of how the rest of that research could most profoundly effect the rest . then yep your just a blogger that doesn't know half as much as he or she thinks they do. Right about something you know about not about something that someone else knows about.
    Sincerely

    Andrew C. Goforth RRT

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