Light, Sleep & Circadian Rhythms | Russell Foster | Oxford

[00:00:04]many thanks indeed Deborah and also for Mary for inviting me really a great pleasure to be here this morning what I'm going to do is really take up some of the themes that we've heard from Mick and then David and then Julie and talk about light sleep it's regulation and also how the sleep systems are interacting with the light regulation of the clock work and the work I'm gonna present is the product of an interaction between four groups within the sleep and circadian Neuroscience Institute we've got our t AG on earth shri Vasu divan Stewart Pearson myself and what I thought they'd do in the next 30 35 minutes or so is have a brief introduction of just one or two slides here but actually present the whole talk in one summary slide because it's kind of complicated and then when that framework we can then dissect it and talk about the light regulation of / 1 + / 2 these key targets of light regulation of a molecular clock work via two pathways Cree activation and ap1 and the ap1 stuff is unpublished then move on to adenosine regulation of / 1 / 2 and essentially how adenosine sort of hijacks part of the light entrainment pathway and because of the role of adenosine in sleep-wake timing talk about the implications of sleep and circadian interactions and then really ask the question can we use some of this mechanistic understanding to develop new therapeutics to provide in a sense a pharmacological replacement replacement of light for those who are profoundly blind okay so let's kick off with this introduction and overview and as I'm sure everybody here will be aware that of course if you expose a mouse or a human to a light dark cycle and they have no rods and cones then you can still achieve achieve entrainment now it doesn't say that the rods and cones play no role but they are not the primary way in which the retina detects the light dark cycle for the regulation of internal time and the entrainment pathway that's dependent upon a relatively small number of photosensitive retinal ganglion cells which utilize the photopigment melon opsin but the the topic of today really is going to be how do those PRG seas interact with the molecular clock work in the master clock within the suprachiasmatic nuclei and this is essentially that the whole the whole talk so what we'll talk about is light activation the release of glutamate in pay cap from those PRG sees how that increases calcium and cyclic A&P intracellularly in SEN neurons and how that light signal essentially interacts with two regulatory elements on / 1 + / 2 + a p1 and a cree binding domain here's the ybox domain that Nick was talking about that binds clock and b-mail so the first element is is the activation of crab and CRTC 1 which whose proteins bind Kree and you get the first sort of wave of of / production then there's this second limb now force C phosgene has a Kree element so it will also be activated by crab and crtc 1 to produce its protein force but force can't bind to AP 1 alone it requires a binding partner in the form of June but June has to be phosphorylated and that phosphorylation comes about by the activation of erk1 combined jute phosphorylated ink and then by far by nauss and then we get activation of AP 1 and a second wave of production so that's in a sense the light activation pathway and then we'll talk about how that is turned off via sik1 and that raises some interesting issues in terms in terms of internal D synchronization and actually a theme that David kicked off with and then the last part of the talk will be on the fact that there are two types of adenosine receptor on essien neurons and a1 and there's about five times as many a1 receptors as there are a 2a the a ones the inhibitory GI pathway reduce the levels of calcium cyclicamp e upon biting their ligand adenosine whereas the a to a actually excite the production of calcium and cyclic a and P so these two are in opposition collectively this pathway will then change the molecular clockwork and depending upon when you see light or indeed when you see adenosine as we'll discuss you can either advance or delay the clock work and we'll see this this scheme as we go through the talk and I hopefully I'll layer the information okay so the first point is that glutamate and pay cap give rise to an increase in calcium and cyclic KMP within SCN neurons how does that occur well there's three basic pathways that have been elucidated we've known for a long time that light activation gives rise to the release of glutamate and some wonderful work from yen's Hannibal in Denmark on the roll of pay cap glutamate is is detected and elicits a response for our two mechanisms there's the long understood NMDA receptor pathway a voltage gated a ligand gated a calcium channel it opens up upon binding and calcium will flood in from the outside and this is something in fact I worked on four years ago with with Chris Kawa and Mike Monica the second glutamate activation is via a glutamate receptor one pathway and via a Jiki fgq and phospholipase C pathway you get the release of calcium from internal restores so there's two ways in which you can regulate calcium and then as I said pay cap via its receptor and a GS pathway which interacts with adenylate cyclase adenylate cyclase gives rise to increased levels of cyclic a and P so in a sense the light activation and the primary signal is calcium and increase in intracellular calcium and cyclic OMP okay let's now see how that pathway interacts with the per 1 + / 2 regulation and let's kick off with Cree activation and again I'm going to be a sort of illustrating this using this pathway we're going to be talking about calcium Krebs CRTC were CREB crtc 1 and the first wave of production much of this is I'm gonna show you show you has been published in 2013 and led by after yoga nath & Co Pearson and I shall show you some unpublished data that supports these early early findings okay so here's the schematic and then I'll show you the data so as I said you've got the release of glutamate KCAP intracellular calcium and site ek MP now what we knew before is that creb is phosphorylated via PKA as a result of this changed intracellular signal but the key thing is that CREB cannot act alone it cannot bind to the kree domain alone it requires a partner and what we found is that in addition to a phosphorylating creb crtc one is then d phosphorylated by calcineurin and in that that state in the d phosphorylated state it can move into the nucleus and then bind with phosphorylated krebs so these two can interact when CRTC one has lost its phosphate group it can then bind to the kree domain and then you get a wave of per production and you see the rise of per and I've Illustrated / 1 + / 2 here we'll come back to this differential activation later on but the key point about this slide is that both / 1 + / 2 after activation are then turned off so what's the mechanism that essentially makes this system non-responsive to light what's the effects of what's what's the mechanism whereby there's a break on the effects of light on the clock and that break is generated by sick one sick one is a gene that also has a Cree domain so like / 1 / 2 it will bind phosphorylated crab and crtc one in the following way that leads to transcription and translation sick one is a kinase and what it does is reef Oesterle crtc one and of course in the phosphorylated state CRTC one can no longer bind to phosphorylated CREB so it falls off which means you lose the transcriptional drive which means you effectively turn off this whole system and of course CRT a sick one is also being regulated by a Cree domain so it turns off its own transcriptional regulation if you remove light then you've lost your glutamate pay cap signal no more calcium and cyclic MP and the whole system goes back to the resting state okay so let's now look for the evidence for those statements and just to remind you of what I'm talking about is that light hits the molecular clockwork you get the rise of / 1 + / 2 sick one kicks in it phosphorylate crtc 1 and then essentially turns off the transcriptional drive on these 2 genes okay and in a sense what this is is the molecular basis of jetlag and we'll will look at some of the the jetlag protocols fairly shortly ok so this is the in vitro evidence here's our little schematic the first is that what we would expect is that after activation that CRTC one will be able to move into the nucleus where it will then bind with phosphorylated creb these are some lovely pictures from Steve Hughes which I couldn't resist showing you this is pre activation CRTC 1 is distributed evenly throughout this cell after activation it moves beautifully into the nucleus where it has its effects of course so yes the support from that side we'd also predict of course that sic one will be upregulated upon activation of these cells and indeed you see a nice activation of sic one which very broadly parallels the activation of / 1 + / 2 the second thing is that if we knocked down sick one as we've done here what we see is that we see high levels of / 1 in this case induction but boo both / 1 + / 2 and so if you knock a sick one down it's essentially you you've D you've in a way a repression of the system and you get up regulation so again the fact that sick one is is acting to it dis inhibitors it were or rather inhibit per one and perche of production is supported by these data and then finally you would predict that sick one is actually required for the phosphorylation of crtc and these data show that at zero time here there's fairly low levels of sick one and you get this level of phosphorylation of CRTC one hundred and twenty minutes you get high levels of sick one and we see a significant rise in the phosphorylation of CRTC so all the in vitro data fits very nicely with the model but the key thing i think is the in vivo evidence and so what RT did was to use a sort of a knockdown approach where by using double-stranded RNA RNA i to locally inhibit the production of sic one around the suprachiasmatic nuclei and she achieved pretty effective knockdown of sick one so we had a we had a mouse with low levels to vanishingly small small levels of sic one in the sem and this allowed us to do the following experiment and so here we see the jetlag protocol here's the control his the light-dark cycle and in this case the light-dark cycle was advanced by six hours and so you see entrainment the light-dark cycle was shifted here and you see a rien Trainmen t' after around about six days and this of course is the cartoon and i will show you the data in a moment the same protocol for the sick one knockdown the light-dark cycle was advanced by six hours and what was so remarkable and i have to say very pleasing is that what you saw is a very rapid Rhian Trainmen t' took about a day to adjust to the new and shifted light-dark cycle so that's the cartoon here's some actor grams seeing the six-day transition versus the very rapid adjustment okay so that data has been published but some unpublished data using a sick one mutant mouse very generously donated by Philip Cowan and Chris Clarke at the University of Dundee so here we have a mouse that doesn't have sick one at all and so the key thing was can we replicate those knockdown fat findings that we found in the earlier report so again we replicated the jetlag protocol by advancing the light-dark cycle by six hours so we see the six hour advance here and again about six days for this mouse to readjust this is the control the mutant mouse to great pleasure realigned itself just like the RNA I knocked down a mouse and took about one day to adjust so that's a replication of the original findings but because this mouse you know had no sick one we could do longer-term experiments and so the next set of experiments was to use a light dark cycle of different irradiance so here's a light dark cycle where the light portion of the light dark cycle was a hundred Lux we then advanced that light dark cycle but we then lowered the irradiance of the light portion and in the following sort of manner we advanced sequentially but with increasingly low levels of light and basically try to define how our sick one verses control animal would lock on and whether it indeed could lock on to this rapidly changing light dark cycle with diminishing intensities these are the control animals and you see entrained here it takes again this slide to the this this this relatively slow adjustment it gets it adjusts then another shift and you're basically getting into this area here where there's very little evidence that the mass is actually locking on to the shifted dim light dark cycle by contrast the sick one knockout showed an extraordinary and ready realignment to the new light dark cycle in fact not only could it rapidly Rhian train but also it we saw it enhance its sensitivity so it sort of won lux the mouse is perfectly capable of in training to this light dark cycle so sick one mutants do show enhanced retainment to a jet like protocol over a hundred to one lux range so that's sort of where we are with sick one but it raises I think some interesting questions that we may want to address during the question time so why bother to have a break on light at all why not just simply shift the light dark cycle to shift the circadian system to the new light dark cycle immediately and what's the downside of removing this break of sick one and of course where we may be sort of wondering I think is an agenda discussion is internal D synchrony perhaps what's going on in the central versus the peripheral mechanisms are we seeing a similar sort of buffering of entrainment of realignment using a sick one type of a negative feedback loop we don't know yet and indeed it isn't on here but what's happening to the sleep/wake cycle as we advance the the rest activity cycle or that the circadian cycle through those six-hour shifts and we're working with Vlad Vysotsky to address that question and Lois Taylor is is getting answers to all of those those questions but we don't have them yet okay so we've talked about Cree activation and the turning off of the system by sick one let's now go to this second pathway a p1 so this is what we've seen and now what I want to talk about is this activation of Foss it's binding to phosphorylated June and activation of a p1 and this second wave of transcription so the ap1 domain and and its activation by Foss has sort of kind of been around for a long time I mean early studies by Benji roux second and Joe Takahashi led by John kornhauser in the 1990s show that after you hit this year after you hit a mouse or expose a mouse to light you get Foss induction with the Sen and it's been used as a very powerful marker we've known also for a long time that oak activation is required for entrainment at some level and Martin rafts studies show that if you knock down force in a foster smash you would attenuate by half the size of the phase shift that you get in mice but for reasons what I'm not completely sure about a ap1 regulation of / 1 + / 2 fell out of fashion and certainly a lot of the early sort of the foss results were interpreted as well it's simply a marker of light activation it plays their fundamental role we became interested in this whole ap one activation a few years ago as a result of working NRT went to work with early schibler using this star prom technique and I won't bore you with the details but essentially it's a phenomenal technique that can essentially identify transcription factors controlling various pathways and so what we had access to with in previous experiments was essentially the light activated genes within the SCN and I again I won't bore you with the details but in a sense these are our genes that have been activated within the SCN and a significant number of those genes showed activation of the of the ap1 domain so here's the star prom result here's an AP one domain and just for the sake of completeness here / - and you see this AP 1 domain it's very short sequence is highly conserved in humans Mouse dog elephant and an armadillo not quite sure we we had armadillo there but anyway and so so a people ap one was clearly doing something and doing something important and again I'll go through the cartoon I don't have a huge amount of time so I'm not going to provide very much data for this part of the talk but this is all now submitted for publication so you've got to get the details later on so we're sitting in the SCN there's no light exposure and we've got these two genes there there's the C phosgene and c-jun gene the see fast as I said before has credo mains the Jun has an AP one domain so with no light activation not a lots happening of course except for one very important thing is that crab in the unphosphorylated state sits on the AP one domain and actually inhibits transcription so it's sitting there an inhibiting transcription so let's now move to the light activation domain so here's calcium psychic MP our critical signal we've got C foss and so just as we've seen before PKA you phosphorylates a crab crab them binds dephosphorylated crtc one and so we have a first wave of c fast c for transcription and translation so early on you get a big bolus of force induction in response to light via activation of cree there's a slower activation of june so what happens first of all is that PKA phosphorylates CREB and in the phosphorylated state it moves off of the ap1 and essentially stops inhibiting it so we get then low levels of c-jun so okay great we've got the two binding partners here but see Jun can't effectively bind foss until it's phosphorylated and so this calcium pathway here activates Kirk earth then adds a phosphate group to June and in the phosphorylated State we can get binding to the ap1 and increased levels of June production of course with that increased level of June production we then get a reinforcement and we can get even fur so you see that sort of rolling wave of June production which enables it to then bind to phosphorylated June production which enables it to bind to Foss and then the two can work together to activate a p1 on those / 1 + / 2 genes so let's now look at what's going on in total so here's our / 1 + / 2 we've talked about the Cree we're gonna now see how us and Jun interact without a p1 to give that second wave of / 1 + / 2 production so just for the sake of completeness here we have our phosphorylated creb binding D phosphorylated CRTC one binding - Cree and producing a certain level of / production force production is there but it can't actually activate a p1 and tell it combined with phosphorylated Jun and so that takes a while and we again get a second wave of / production so that's how the two regulatory domains of Crean a p1 are generating per production but one thing that struck us has been rather intriguing is that could the differential a a differential activation of Cree and a p1 explained the differences in / 1 + / 2 levels of expression what we've known again for some time and this has happens to be a picture from Mary Harrington's group is that / is induced velocity rapidly and then declines was there's a slower rise and decline of / 1 and that's been sort of turned into a cartoon in this figure here and so what our team then went on to do is look at the Cree and ap1 domains in / 1 + / 2 and what was very intriguing is that if you look at the Cree in ap1 domains what's very clear is that the Cree of regulatory elements in / 1 are much more accessible and in fact whilst there could well be a p1 regulatory domains that consider a p1 regulatory domains in per one they're not absolutely obvious and it's not absolutely clear that / 2 has a Cree domain so what sort of seems to be happening is that / 1 production is being dominated by creer egg elation and / 2 via a p1 and so that will kind of explain that differential activation that rapid turn-on via Cree is producing that sharp rise and then turn off of per one and then via a p1 a slower rise and turn off of per to the biological significance of that differential activation of per one and third to again is something we might want to discuss okay so that's the light activation of the pathway let's now go on to unpublished data on adenosine regulation and and let me stress that this has also been driven forward by RT and shree and again what I want to add to our picture here is the roles of the a to a and the a one adenosine receptors so as many of you will be aware the two process model of sleep regulation is sort of conceptualized in this cartoon here so from the moment we wake in the morning there's an increase in sleep pressure and part of that sleep pressure comes comes about as a result of the role of adenosine so adenosine sleep pressure builds and builds and builds throughout the day and then during sleep sleep pressure and levels of adenosine diminish and evidence for a role for adenosine in the generation of this homeostatic drive for sleep comes from a very nice you I recommend you see this review here published last year but in brief adenosine introduced in if the brain will promote sleep adenosine increases within the brain during wake adenosine dissipates in the brain during sleep and an adenosine receptor antagonist such as caffeine will promote a greater sense of wakefulness now this this this homeostatic drive for sleep is opposed by in a sense the circadian drive for wakefulness so in the morning we see relatively low circadian drive for wakefulness and it doesn't have to sort of work very hard because the circuit of the the homeostatic drive for sleep is fairly low but what happens is that the circadian drive for wakefulness increases throughout the day as the the homeostatic drive for sleep increases and of course in the late afternoon early evening the homeostatic drive for sleep is extremely high but it's opposed by the circadian drive for wakefulness as we fall into sleep the circadian drive for wakefulness diminishes during sleep it's very low but then as we approach wake it increases and of course the sleep window is defined by these interactions between the homeostatic drive and the circadian drive the key point I want to flag up here is the role of a den of adenosine as I said there's an a1 and a2 a receptor within the SCN and the key point is that there's 5 times more and maybe more a 1 receptors compared to the a 2a receptors and the a1 remember are the inhibitory input onto that light regulation pathway so what we see of course is with wake an increased level of adenosine but the net effect is that the adenosine will differentially activate the a1 pathway and diminish the levels of calcium and cyclic OMP so you get lower levels of calcium insightiq MMP as a result of wake and high levels of adenosine which then acts to effectively not turn off but certainly inhibit that whole light induced pathway ok so what I'm saying is wake increases adenosine the a1 receptor you get reduced cyclic AMP calcium or juice / 1 + / 2 smaller responses to light let's test that hypothesis with some data so in this experiment very straightforward we have a six hour sleep deprivation during the second half of the light phase and what we'd predict is that we would increase adenosine and therefore we'd see smaller responses to light we reduce / we'd have see smaller responses to light and certainly you get lower levels of / 1 + / 2 after sleep restriction what happens if so sleep deprivation suppresses / 1 + / 2 entirely consistent with the model I've shown you what happens if we do this experiment so we've sleep-deprived here we see in this case the levels of / - but we're going to use an antagonist which will then block the effects of adenosine on this pathway and the result is a huge increase in levels of / - when you've used an antagonist essentially you've blocked the effects of adenosine on this pathway so adenosine receptor a1 antagonists reverse the effects of sleep deprivation here we see some behavior so in this case we've got a vehicle and we're then going to add an antagonist and again what we said is it will block or reduce the effects of adenosine and particularly of potentially increased light sensitivity so we predict that an antagonist would would produce larger responses to light in terms of behavior and that's what you see you see this beautiful Rhian Trainmen t' - again this shifting jetlag protocol so adenosine receptor antagonists enhance Rhian treatment oh ok and oh yes and so again we would expect that an antagonist sorry an agonist which will mimic the effects of adenosine on this pathway here we see the size of a phase shift in response to light we're then going to add an agonist which will mimic the effects of adenosine and we can actually pretty much abolish the effects of light on the clock work with an adenosine agonist so agonists attenuate the phase shifting responses of light so what we got is lovely evidence for the image I showed you earlier wake drives up levels of adenosine and via this pathway essentially turns off the whole light induced effects are on the molecular clockwork ok let's look at the implications of this for sleep and circadian interaction so the key point is that sleep-wake history encoded by levels of adenosine acting via the a1 and a2 a receptors modulate the responses of the molecular clockwork to light implications for the PRC just for those of you I'm sure you all familiar with these sorts of images but as you know different light pulses at different times either during the middle of the subjective day will produce relatively small effects of no phase shifts the earliest subjective night delays fir further into the subjective a night bigger delays but towards dawn you get phase advances now I guess you'll already be with me on this one what we're saying is that with increased levels of adenosine during wake and diminish levels of adenosine after sleep it will mean that one sleep wake status will have different effects upon the effects of light on the clock work so there'll be at the end of the wake phase with high levels of adenosine a maximal suppression of per whereas after sleep there will be minimal suppression of power so what do we see in a nocturnal species such as a mouse well it's awake at night it then sort of finishes its activity high levels of adenosine it then will have high levels of adenosine at dawn so we'll see small phase shifts at dawn whereas of course after the sleep phase during the day there will be low levels of adenosine and therefore large phase shifts at dusk and indeed what's striking about many nocturnal phase response curves is that there are large delays at dusk and small advances at dawn was of course in a diurnal species such as ourselves you get the reverse so at the end of the wake phase we have high levels of adenosine at dusk and of course after the sleep phase large low levels of adenosine and big phase shifts at dawn and in fact that is a characteristic of many diurnal phase response curves now I'm not saying that a complete phase response curve is being driven by this interaction of sleep-wake but the fine tuning of the phase response curve is clearly being influenced by the sleep history of the organism okay let's just finish up in the last few slides with whether we can use these sorts of information to develop new therapeutics and following on from our previous speaker here's his an act act rest activity profile of a normally cited individual this person has no eyes they're an ophthalmic and we see this beautiful rather tragic for the individual a drifting of the rest activity cycle throw time so is there anything can be done to people with with either radical ocular damage or no eyes at all melatonin of course has been used and melatonin agonists have been used with some level of success can we do something better I guess is the question I'm asking and we use the knowledge of how light interacts with the circadian clock work to help these individuals and I need to have a declaration of interest so I'm part of a spin-out called circadian therapeutics and venture capital has funded some of the experiments that I'm going to talk about now so basically using an adenosine antagonist we get a very effective clock clock modulation so here's the vehicle the vehicle was introduced here at about circadian time six here's the drug the adenosine antagonist and you get a beautiful face advance and so this is the final bit of data I want to show you which is a comparison of this adenosine antagonist with tarsem LT on notice ml t on is a melatonin receptor agonist and so we decided to compare them back-to-back and in fact it's the only FDA approved drug for circadian regulation and here we see an increase in the phase shift on this axis tarsem LT on produces a shift in our mice but it wasn't significant the adenosine antagonist just a nice shift which was highly significant so in a sense I think this this this pathway seems to be a more effective target than the melatonin pathway as far as we can tell but it's still early stages the exciting point I think is that that we're now collaborating with the blind veterans UK and these will be our first clinical population to test this adenosine antagonist and in fact we must talk about using some of your approaches on this population which I think will be really brilliant because there's a large number of individuals with no eyes and lots of different sort of states of retinal damage so we should talk about that okay so what I hope to give you some sense of in this presentation is the light regulation of / 1 + / 2 finally how the molecular clockwork is aligned to the external world via both Cree and ap1 activation and the discovery of this really important element SiC one which acts as this break upon the clock remove sic one in a sense you can shift the circadian system backwards and forwards via light it seems as you like I think the really interesting thing for us has been the role of adenosine adenosine encodes partly once sleep-wake history and what's very clear is that the adenosine pathway is effectively inhibiting the light activation of / 1 + / 2 so sleep wake history and light can interact to fine tune sleep wake timing in ways that we hadn't really appreciated before and then finally I think these pathways and particularly the adenosine pathway and maybe also the sick one pathway provide opportunities for maybe the new therapeutics to try and stabilize sleep wait timing not only in the blind but other clinical populations thank you for your attention Thank You Russell and for sharing some unpublished data bill I have Russell so there's this wood mouse screen where they had the sick 3-kinase yes yes that's also can you know what's interesting is that of course if you knock out sick three or sick - its lethal whereas I think what they had is sort of a slight module modulation but but sick one mutants is not lethal it's very interesting because sick one is the only lighting or the only induced gene whereas the other two are constitutively activated so that I mean there's going to be somewhat some interaction paths I can't integrate at the moment the other six into this system iris oh yes Eunice a very like elegant and elaborate model that is a oneself model for thing but you know a one receptors are highly expressed in astrocyte and there is previous data that support that aesthetic coverage on the right recipient sense of the SCN changes and that cool effect they gate so you can see the well in fact I sort of scan urban masses of data the in vitro stuff well of course it's done in in in cellular assays and a whole range of different cell types you to ourselves for example so so it looks as though the basic biology that we're seeing in SEN cells operates in many other cell types as well many other neuronal types and wouldn't surprise me surprise me if it was within the glia populations as well and i think the the the real power of the sick one mutant mouse is going to be trying to dissect you know all of these various pathways what's happening not just at the level of the SCN but at a range of other cell types and so that's that's what Lewis Taylor is now working on I'm gonna go right to the back as the front people have had a bit of a domination here's I have a question for your just like protocol's yes did you try instead of advancing did you try to delay it yes we have done delays and it works just as well it gets a bit more complicated with delaying because you hit different points of the face response curve but it yes it works both for advancing and delay yeah any other questions I saw some hands here we go hi Russell can you integrate your sik1 kinase result with the vaso repression mutants that also have these yeah these rapid phase responses yeah I mean I I can't at the moment no I mean I think there will be a series of different overlapping mechanisms of course and we unmasked one but but but I offhand I can't integrate that with the vasopressin data but you're quite right I mean there's a very interesting phenotype there as well yeah any other questions one last one yes I did see your hand sorry last question so two very quick questions but basically how do you dissociate the effects of the adenosine agonist or antagonist body temperature so so could we be inducing a phase shift as a result of for example increasing activity which would be one thing and therefore body temperature now we've addressed this I'm just trying to think about how we've dealt with it I can't remember it's a really good question I'm really embarrassed I can't remember what the findings are but but it's but we have addressed it in ways I can't remember and it's a shame that RT isn't here because she would explain it perfectly to you well it's a really good question these are and that's why it's I think quite interesting approach because what we did and what Shree did originally was essentially using in vitro cellular see with purr like reporters and screen essentially all of the fda-approved drugs to see if they had a capacity to shift the clock and what surface there was a whole bunch of fda-approved agonists and antagonists to adenosine so all of them have gone to stage 2 stage 3 clinical trials and they've been developed for things like Parkinson's and they haven't turned out to work very effectively so what we've got are a bunch of either you off patent or slightly on patent drugs which have gone to a clinical trials that that work and so the reason we can move from mice to human so quickly is that these have already been FDA approved in another domain not for circadian phase shifting and I think that's it represents a really quite interesting I should say that five different classes of drugs across the six and a half thousand had an impact upon shifting the clock and when we pursued the adenosine there are four other potential compounds that could be useful but the key point is that they are already drugs in existence and and there are the off patent and and have been clinically validated to cause no harm but to do no good for what they were developed for thanks ask you to save the day for conference next year in Leon this is the European biological rhythm society and on that note I'd like you to all join me in thanking our speakers for this morning and you