And so to sleep

Sleep - something that we spend a third of our lives doing, yet, we understand virtually nothing about.  Amita Sehgal from the University of Pennsylvania has been studying the genetic mechanisms involved in sleep behaviour in the model organism, Drosophila melanogaster, and looking into the role of the newly-discovered RYE protein.

Amita -   So, what we were trying to address is how sleep is regulated.  The method we use is called a forward genetic screen.  You mutagenise animals and then you look for ones that are altered in your process or behaviour of interest.  In our case, that behaviour would be sleep.  The system we use for these studies is the fruit fly D. melanogaster because it really lends itself beautifully to this type of approach.

Chris -   And this sounds like a rather daft question, but do flies sleep in the same way that we do?

Amita -   So, I don't know that I would go as far as to say that they sleep in the same way we do, but flies have a rest state that satisfies the criteria for sleep.  We have been using it to figure out how sleep is regulated.  A lot of what we're finding turns out to be also involved in controlling mammalian and human sleep.

Chris -   So, you found some flies that took less sleep.  I presume otherwise this paper wouldn't exist.  So, what did you then do to try to find out why and how that was happening?

Amita -   What we had used to generate these mutations, there's a chemical, it's called ethyl methanesulfonate, EMS for short.  And so, what that does is, it makes changes in the DNA.  So, what you then have to do is go and basically clone the gene that's responsible for the reduced sleep.  And so, in other words, determine which of those many chemical changes in the DNA really accounts for the short sleep, and that is what we did.

Chris -   So, what genes came to fruition?  What did you find when you did this study?

Amita -   So, at the end, we had it narrowed down to a region where there were 7 or 8 genes, and then we went through each of those to determine which was most likely.  For various reasons, we were able to exclude all the others and then the one that we focused on was a change - a mutation - in a nicotinic acetylcholine receptor.

Chris -   Tell us a little bit about that receptor.  Was it linked to sleep before?  What do you think that the mutation is doing to it and how did it affect the flies?

Amita -   So, the nicotinic receptor has been implicated in sleep before.  Acetylcholine is thought to generally be wake-promoting, as is nicotine.  So, what was different about this one is that this turned out to be a receptor that does not promote wakefulness.  It actually increases sleep.  So, when you mutate it, you have less sleep.

Chris -   Does this mean if you increase the expression of that gene where you found this mutation that this makes animals that need more sleep because it's a sleep-promoting gene?

Amita -   Yeah, it's a great question and the answer is, no, with the caveat that you can never be absolutely certain that you have accomplished what you set out to do.  So yes, we put this protein back in the fly.  We tried to increase the expression to get more sleep and we didn't get it, but it could be that our protein never really was increased in expression in let's say, the right cell-type or the right parts of the brain.  And that could be because the animal naturally controls the levels of this.

Chris -   So, is it possible that in a normal fly, the expression of this gene is increased slowly over time as the animal becomes more relatively sleep-deprived until it reaches a threshold point where it makes the animal go to sleep?  Is that what you see, because you would expect to see a sort of linear rise in the expression as an animal got sleepy, if it were the trigger?  Do you see that?

Amita -   That is in fact what we see.  We see that the levels of this protein are high at times of sleep.  The only thing is that, it does not seem to be a gradual rise.  We need to do more experiments to determine whether maybe it is a gradual rise, but our experiments so far haven't seen that yet because we haven't looked at close enough time points.

Chris -   Do you think the alternative is then that something might be tripping it to turn on?  Could it be just not there at very high levels to start with because something else, which is sensitive to how sleep-deprived the animal is, is detecting the sleep deprivation level and when it reaches a critical threshold, it then triggers your protein to turn on and that's what triggers sleep?

Amita -   That is the explanation we're currently favouring, yes.  That it is not what we call the homeostatic signal, but maybe it is controlled by that other signal.

Chris -   So, what have you christened this protein and how do you see it fitting into the genetic domino effect - for want of a better phrase - that we understand, makes the circadian oscillator that's in the brain?

Amita -   So, we call this gene red eye, because that typically is associated with a sleep-deprived state.  With respect to the circadian clock, we think that this is actually independent of the circadian clock.  So, there are two systems that drive sleep.  There's the circadian clock that makes sleep occur with a 24-hour rhythm and then there is the other system that just ensures that you get enough sleep.  We think that this red eye protein is part of that other system.