 Dyma i'r Llyfrant. So, o'r Llyfrant paedgert Llyfrant, Llyfrant paedgert Llyfrant selleidio ar y rai Llyfrant sy'n ddim yn ymddiadau'r llwyddiadau eu llwyddiadau yn ymgyrch yn ffysiologi yn fwylltio'r llwyddiadau'r llwyddiadau. Fynd i'r llwyddiadau'r llwyddiadau'r Llwyddiadau yn ymgyrch yn ddod i'r llwyddiadau llwyddiadau. Mae'n gweithio fel y gallwn i'r 84th paedgert Llyfrant, I add Professor Clare Stanford to our long and imminent list of lecturers. Clare is Professor of Translational Neurofarmacology at UCL. She is an honore fellow and a former trustee of the British Pharmacological Society. She is a past president of both Larson and the British Association for Psychopharmacology and a former member of the Council of the University of London. Although now semi-retired, Clare remains active in pharmacology and has editorial roles on several journals And is currently a member of the Animals in Science Committee, and a Larsa trustee. She's been a long supporter of UAR, most recently being involved in the development of a forced swim test fact sheet and a member of the Openness Award judging committee as you've just seen. So I'm delighted to be able to introduce her to give her lecture a year late. Tort죠os his hairs and other animals on the pathway to anti-depression. Claire? Thank you, Jeremy. It's enormous honour to give this lecture. I can't imagine what I've done deserve to be added to the list, I'm mystified, but thank you. When I've talked about my research in the past and sometimes I've given the lecture, the title, keep calm and carry on. And I think you'll understand why when I tell you about my research in this lecture. But at least it's evidence that in my view there's no such thing as a negative finding. Whether it's negative or positive or not depends on what you do next. And coming from UCL of course I have to mention Jeremy Bentham, who thought that it would be a good idea to have universities that were secular and might even educate women. But what he's mostly known for of course is for considering animal suffering and for suggesting that they should have some sort of legal protection. And that led the path to the Cruelty to Animals Act, which was morphed into ASPA over a hundred years later. And I must admit that Jeremy Bentham still arouses controversy because on the left you see him in his splendid Victorian cabinet. And college recently gave him a makeover, which some staff don't actually think is a great idea. But my story starts with Walter Cannon, beginning of the 20th century, who identified the sympathetic adrenal response, the so-called flight or fight response. And he was collecting plasma samples from cats, which he described as being within earshot of noisy, energetic little dogs. And you can imagine the scenario, a brave man indeed. But he found something in the plasma. In fact it turned out to be two somethings. He was identified much later as adrenaline and noradrenaline. But he found a neuroactive compound which he thought was released in response to the psychological stimulus of hearing these yappy dogs, but also physical stimuli as well. And moreover he thought this neuroactive compound in the plasma was in some way associated with the emotional response of the animals. And moreover that the individual differences in the emotional response was somehow coded by this chemical. And really that summarises this whole lecture, the challenge of finding a causal link between neurobiology and pharmacology and mood and behaviour. Well to start I have to give you a brief tutorial on the life cycle of noradrenaline, which is one of the things that Cannon was measuring. Noradrenaline is synthesised from dietary tyrosine which is taken up into nerve terminals and the rate limiting step in that pathway is the enzyme tyrosine hydroxylates. Tyrosine is intermediate product is dopamine and dopamine is taken into those storage vesicles where the final step in the pathway takes place. Now these storage vesicles fuse with the neuronal membrane when the neurone is excited and extrude their contents into the synaptic cleft. And from there the noradrenaline, some of it diffuses away in the overflow, can reach the plasma or other target cells that are distant. But some of it has taken up on protein transporters and they are one target for antidepressant drugs. And some of the noradrenaline which is taken back into the nerve terminals is metabolised by an enzyme called monoaminoxidase which is also a target for antidepressant drugs. And in this figure I've illustrated two receptors for noradrenaline, there are lots of them, about eleven in all. But the alpha two adrenoseptors are interesting because they operate a feedback inhibition process and when they're excited by extracellular noradrenaline they tend to dampen down release of transmitter. And beta adrenoseptors on target cells are responsible for initiating the response overall. Now this process takes place in both sympathetic nerve terminals but also a huge amount of noradrenaline is released from the adrenal glands. And in fact the adrenal gland was initially the model to study because in Canon's day the assays from noradrenaline were very very insensitive, it was all done by bioassays. And so to get enough noradrenaline to measure you had to use adrenal glands, bovine adrenal glands and try and work out what was going on. And that was justified because adrenal glands really are, the adrenal medala really is like a very big sympathetic nerve terminal and they're derived from the same embryonic tissue. And all the conclusions in those studies have more or less been born out when they've been scaled up for sympathetic nerve terminals. Now I have to slip this in but when it was first realised that the compounds in the plasma were adrenaline and noradrenaline and their molecule was characterised, Michelle Peuge realized that they had a potential to be fluorescent. I don't think it's any relation but it was a brilliant observation because it led to the development of an assay which relied on precisely that process converting noradrenaline into a fluorophore which could be measured by this spectrophorimeter. And I use this every day of my PhD life and I was aware of personally of the ageing process when as a postdoc and I did a brief stint at NIH I actually saw this piece of kit in their museum. But the early studies, when I was working with Marianne Philence in Oxford I did my, did Phil with her and postdoc for a while. And we were trying to follow up on Canon's work on the role of noradrenaline in the stress response using this really super new assay. But laboratories at the time were using stressors like long term immobilisation where animals really were pinned to the bench. They couldn't move at all or quite intense foot shocks, tail shocks and we wanted to use naturalistic stressors. So we started off with cold stress at four degrees C with two rats in a cage which just popped into the cold room for about six hours. So they could cuddle up in the cage but the ambient temperature is cold. And as you can see the noradrenaline that appeared in the plasma, we recorded our intervals, progressed your peak and then dropped off. And that wasn't because the nerve terminals were running out of noradrenaline because when the animals were also treated with disipramine which is a blocker of that transporter, it blocks re-uptake of noradrenaline and so the clearance is delayed. The overall pattern was very similar, it's just plasma concentrations were higher. And we also looked at brief swim stress. These were one minute swim stress about every half hour for six episodes and the general pattern was just the same. But the reason I'm showing you this is because I want you to note the concentrations on the left hand side. Because this shows quite clearly that the plasma concentrations during the swim stress are pretty much the same as plasma concentrations during cold stress. And it's not much warmer outside than four degrees C at the moment. So swim stress is actually not much more stressful than a fferal rat would experience while foraging on a typical December evening. But while this was going on there was a very influential hypothesis. In fact it still is, it's still in all textbooks which was devised by Joseph Shillcrout who is an American psychiatrist. And he noted two things about patients he was seeing. One group of patients were being treated with a drug called Rezepine. And what Rezepine does is it stops those storage vehicles from containing their transmitter straw. So Rezepine essentially strips noradrenergic neurons of their supply of neurotransmitter. And Shillcrout noticed that a proportion of those patients was only about 15%. So it's not simply the loss of transmitter, it's transmission loss past something else. But he noticed that about 15% of those patients experienced suicidal depression. And he surmised that perhaps depression could be caused by a deficit in noradrenergic transmission. And that theory caught on, big time, and it was revised later on to include serotonin. And so it became known as the monoamine theory of depression because they're both monoamines. Now that theory is now deprecated. There's never been any evidence found to support it except perhaps under extreme experimental conditions. But in terms of depressed patients that theory is now dead in the water. But another observation of Shillcrout's was patients were being given Ipriniazid to treat tuberculosis. It still is used to treat tuberculosis. But for a completely different reason it blocks monoamine oxidase, which if you remember is that enzyme which metabolises noradrenin. And a side effect of that, side effect of Ipriniazid was to induce euphoria. In fact it was regarded as an unwanted side effect because if you can imagine you've got these terribly sick tuberculosis patients with some of them bouncing around with their euphoria generally making a nuisance to themselves. But Shillcrout realised that if it made euthymic patients euphoric perhaps it would cheer up depressed patients and they tried that and it worked. And this became the very first anti-depressant drug. Before that all they had was amphetamine which was used for 30 years to treat depression but whereas amphetamine gets you going it will get them out of bed it doesn't cheer you up. So this was a really huge step in the field and Lerd Shillcrout suggests that drugs that boost noradrenin but later changed monoamine transmission could relieve depression. And that theory is alive and kicking because until ketamine came along very recently every single anti-depressant drug in the clinic augment monoamine transmission. Now Shillcrout's problem and of that era was that the assays weren't sensitive enough to measure noradrenin function in the brain. In fact although noradrenin was known to be in the brain the prevailing view was that it was simply there to regulate the cerebral vasculature. Martyr Folk suggested it might have something to do with what the brain does and you regulate movements and mood and cognition and so forth but it was never really an idea that caught on. An evidence of that is Les Iverson who was he did an enormous amount of important work on noradrenin and this book is basically his PhD thesis which was published in 1967 and Les Iverson also went on to do important work on neurokinema receptors which I'm going to be talking about later. But in this book the very last chapter is about noradrenin in the central nervous system and it's very thin compared with the rest of the book and it's got this wonderful quote that in recent years the evidence that noradrenin and dopamine may act as neurotransmitters in the central nervous system has become progressively more convincing. So Les Iverson was really being very cautious there. But three things happened to change all that. One was that Paul Greengard realised that cyclic A&P was a second messenger for lots of receptors but in particular for beta adrenal receptors in the brain and another one was the development of radioligin binding where drugs that bind to receptors are given a radioactive tag and you can use that tag therefore to measure the density, literally count the receptors in tissues. And both these groups reported that after chronic administration antidepressant drugs there is a down regulation of cyclic A&P in the cerebral cortex in the brain and also Eupritjard Nenna found a reduction in the density of beta adrenal receptors in the brain. Now actually that all turned out to be a bit of a red herring but at the time it was hugely important because it made people realise that until then all they'd done was study the acute effects of antidepressant drugs which would tell you a lot about the cellular targets and so forth but in terms of how can they be antidepressants the chronic administration bit was vital because depressants need chronic treatment with antidepressants before the treatment works and a lot of patients it still doesn't work about third of them are treatment resistant but most patients need weeks sometimes months for their therapy to take effect so these groups realise that it's that long term treatment that we really need to be exploring. Well we were doing our share of grinding and binding as it's called because although they'd found down regulation of beta resets in the cortex it wasn't at all clear what was happening to other adrenal receptors if anything and whether the down regulation extended to other brain regions and this graph on left scatchar plot the shift to the left shows that there was actually a down regulation of beta receptors in the cortex that agreed with Pritchard and Enner but nothing happened to alpha 2 adrenal receptors and actually nothing happened to other receptors in other brain regions but what attracted our attention was we thought there was something odd going on with the controls and so we set which were being given a saline injection so we set up an experiment to look at that where a group of animals some of which were simply handled for a minute once a day for 14 days and another batch, not another batch but other animals in that group were given a handling session plus a saline injection which is regarded as a perfectly standard control procedure to any pharmacologist and sure enough what we found was that the activity of tyrosine hydroxylase increased quite dramatically over 60% now that told us that the neurons are synthesising more noradrenaline presumably so that they can maintain the stall to keep up an increased release rate in other words, handling particularly with a saline injection were really quite stressful to these animals and then the density of alpha 2 and beta adrenal receptors that went down and this is consistent with the down regulation that Pritchard and Enner had seen with antidepressants when you soak receptors in with the neurotransmitter they internalise the so-called down regulation so that the target cells can try to maintain some sort of homeostasis so this was telling us even the handling was stressful and was producing changes which actually were well on the way in terms of magnitude to those seen with antidepressant drugs now I have to say we had immense trouble getting this work published I wish I kept some of the referees' comments because some of them were almost actionable actually but we found a home for it eventually with neuroscience letters which I think still only has an impact factor of about one but there we go but now of course we know that handling is stressful to animals and it not only causes biochemical changes in the brain but it affects their behaviour and it affects experimental outcome and it's not just any old handling it's how you handle them can make a difference as well and that is a very important finding and well done Jane Hurst for doing the right experiments that convinced everyone well the third development that made the difference was that of micro dialysis and together with the development of HPLC high pressure liquid chromatography with very sensitive detection techniques like electrochemical detection because noradrenaline is also electroactive and this meant that you can implant a micro dialysis probe in selected brain regions and the noradrenaline which diffuses away from the nerve terminal some of it will find its way to the dialysis membrane at the end of this probe and the probe is perfused with artificial CSF so the solute that penetrates the probe is flushed away and you can measure noradrenaline in aliquots using electrochemical detection now micro dialysis does have a yuck factor attached to it because you do have the probe penetrating above the skull but bear in mind that the diameter of the probe, 250 microns is not much bigger than the diameter of electrodes which are now being implanted routinely in patients for treatment of Parkinson's disease and also in some cases extreme depression also the brain of course has no pain fibres and in fact some surgical procedures are done without anaesthetic to make sure the surgeon is chopping out the right bit and the only bit of the procedure which would cause physical discomfort is creating a small burhole in the skull so that you can insert the dialysis probe into the chosen brain region now that procedure, Trapination has been around for centuries this is her honour spot used to do it routinely they believed it released evil spirits and influences and I suppose this was a medieval way of treating antisocial behaviour but it still happens today here is Amanda Fielding, she did her own she drilled a hole in her own head and she thought it made her just feel absolutely wonderful and in fact she stood as a candidate for a Member of Parliament on the ticket that she thought Trapination ought to be available on the NHS and recently she had another Trapination procedure because she was a bit concerned that the first one had healed over so using micro dialysis we are able to sample changes in extracellular noradrenaline in selected brain regions and with Jeff Daly who is now at Cambridge we went back to our let's use naturalistic non-nogtrious stresses to see what we can measure in the cortex and so the probe is implanted in the cortex and you see that during the day noradrenaline concentration in the accelerated fluid it is an index of release if you like it is fairly stable but when you pick the animals up you get quite a sharp peak and it takes a while to dissipate and then if you pick the animals up and transfer them to a novel cage the peak is even more prominent and takes longer to dissipate if that novel cage is brightly lit then the responses last much longer as does if the novel cage includes a strange rat so this told us that the noradrenaline in the brain is exquisitely sensitive to changes in their environment as well as stress of handling and so forth light intensity is quite important and of course we know this and it underpins the principle now that all animals should have an enriched environment in order to improve their welfare but it just to show how laboratory animal science really has progressed in the last 20 years when the proposal for a enriched environment was first mooted and the popular press got a hold of it this photo caption won the prize in the times that week the press were treating it actually with some derision which was quite distressing ok so an opportunity to look at a brand new antidepressant under development came to light and that was Cybutrami which was being developed by Boots Company and I collaborated with David Heal his photograph is there whom I've known from Oxford days Cybutrami is a noradrenaline and serotonin re-uptaking hibiter and we thought it's a new drug let's see if it's like the others but actually that didn't last very long because Cybutrami stopped being an antidepressant and started being an anti-obesity agent and the reason for that was that in phase 3 clinical trials it worked in the majority of clinical trials but not all of them and because it's a monoamine uptake inhibitor and there are already dozens of those in the clinic some of them have lost their patent protection already in so cheap as chips Cybutrami was never going to be a blockbuster antidepressant but what they noticed was patients in clinical trials all lost weight so they repositioned it as an anti-obesity agent and actually Boots was strongly criticised for fabricating an obesity epidemic simply so that they could flog their drug and this was quite a while ago the media were very cynical but actually to get it licensed as an anti-obesity agent the regulatory authorities were really concerned to ensure that it's not going to be addictive because of course the prototypical anti-obesity agent was amphetamine and if Cybutrami looked anything like amphetamine it was dead as far as clinical approval but actually it didn't look anything like amphetamine with amphetamine you can see it's a releasing agent and dopamine and there's a very fast surge of release which dissipates away progressively whereas Cybutrami because it's an uptake inhibitor you get a progressive accumulation of transmitter in the cleft in response to spontaneous release of transmitter well the FDA were happy with that and a lot of other evidence too I hope to say but they licensed it and if for a long time it was the only essentially anti-obesity agent in the clinic so having failed to mention the good anti-obesity effects of Cybutrami another opportunity came to light and that relied on the substance P preferring receptors so called neurokinin one receptor now substance P was first discovered by Gaddon who was an early head of department of pharmacology at UCL and it was amongst other things it was found in sensory afferent neurons and thought to have a key role in pain and after identifying the receptor a small molecule substance P is quite a well it's a smallish peptide but it's a peptide so it's pharmacologically very difficult but they managed to develop an NK1 receptor antagnist which is a small scaffold which will bind to the receptor and block substance P effects at that receptor and it went into clinical trials as an allogysic antinoseceptive agent but that didn't work out very well and it's quite disappointing in fact what substance P appears to do is regulate stress induced analgesia during intense stress the sense of pain is blunted and substance P seems to be important for that so it wasn't the sort of analgesia that they were looking for but obviously doing broad brush investigation of what these compounds are doing what they noticed was that the NK1 antagnist showed up as a positive result in the force swim test and I'm going to be talking about the force swim test again later that briefly this involves placing a rat or mouse in a container of water from which they can't escape and they swim around for a while but then developing a mobile posture with their noses above the water they don't drown because they float spontaneously but the factor with antidepressants is that all of them increase the latency of animals to develop this immobility and it's called the force salt swim test and here you can see fluoxidine which is prozac that increased immobility and so did this NK1 receptor antagonist which was under development so it looked as there could be a promising antidepressant then Carmen de Felipe working Steve Hunt's group in Cambridge developed the NK1 receptor knockout mouse and they tested that in the force swim test and you can see that that too delays immobility and if you look at the what compare the y axis on these two graphs you can see that the NK1 receptor mouse is really motoring in this test it takes a long while to calm down but the knockout mouse is not actually a knockout because the receptor is still there but they dropped a cassette into the gene for the receptor so it doesn't work anymore so that if you use the amino fluorescence you can't see it in the brain the spinal cord or on western blunts it's just functionally ablated well we used our micro dialysis in those mice to see if they the neuro chemistry fitted with what we know about antidepressants and it looked quite good we started off using anesthetised mice because not having done micro dialysis in mice before we wanted to make sure that our procedures were absolutely up to scratch before we attempted to really moving animals and so both these are in anesthetised animals and you can see that the neurodrenin efflux in the knockout mice is much higher than in the wild types and if you give the animals a dose of antidepressant like desimethalimiprimine the neurodrenin transporter uptaker inhibitor then you get the same response but everything is just raised to a higher concentration in this experiment on the right we were looking at the effects of blocking alpha 2 receptors now blocking those receptors should increase neurodrenin release because those receptors you'll have lost the feedback inhibitory process for neurodrenin release and sure enough in the wild types neurodrenin efflux went up but interestingly nothing happened in the knockouts and we had a story, we had a follow-up of that but it was quite interesting but when we went into freely moving animals it all went pear shaped really because there wasn't any difference in neurodrenin efflux in awake animals and to see whether that was anything to do with the anesthetic we did an experiment starting off in awake animals and then taking them down under this is in the days when halothane was the volatile anesthetic to use and you can see that as the animals undergo anesthesia then the difference in the neurodrenin efflux develops and we think that that difference is actually created by an interaction between the anesthetic and alpha 2 adrenal receptors and this is a cynogism which is exploited in veterinary practice now for short surgical procedures give a dose of an alpha 2 agnus like metatomidine because it greatly reduces the amount of volatile anesthetic you need to use so the animals wake up much more quickly and everything goes very well but that was a bit of a blow that we're expecting increased neurodrenin efflux if it didn't show up in vivo and another disappointment was that when the probe was implanted in the striatum the knockouts had very very much less dopamine release in the striatum than the wild types now dopamine is the reward transmitter dopamine is what makes us think it's worth getting out of bed in the morning so animals that have got impaired dopamine release in the brain are not going to be anti-depressed so we couldn't really understand how this could possibly be a mouse behaving as though it was an antidepress mouse but what we had noticed was that they were really hyperactive strangely that hadn't been noticed before and to record that we placed the animals in the dark zone of a light dark exploration box for an hour and a half because the experiments with Jeff Daly showed us that it takes them at least that time to get the neurodrenin release back down to normal after picking them up and putting them in the enclosure and after that time they're allowed to freely explore both zones and we recorded their activity and you can see that the knockouts are really very busy indeed just to make sure we teamed up with Andy Rammage and Adrian Hobbs Andy is at UCL, Adrian is now at Queen Mary and they did some radio telemetry with these mice and you can see again that these mice really are hyperactive well around this time the development of NK1 receptors antagonists as antidepressants was dropped and the reason for that is pretty much the same as Cybutrymy actually it worked in majority of clinical trials but not all of them and for this company that wasn't good enough because there's just too much competition out there already in the clinic and this article describes some other reasons which may have explained why some of the clinical trials didn't work they got the dose wrong in humans and so forth but anyway it's no longer an antidepressant but we were interested in what happens if you give these mice amphetamine because we knew they got bizarre monoamine regulation in the brain not much dopamine release nor adrenaline is normal under anesthesia it's got the potential to be higher and we know amphetamine increases motor activity so what would happen if we gave the knockout mice amphetamine we wanted to know whether the motor activity is already at a ceiling or whether amphetamine would increase it even more and actually what happened was here as amphetamine increased the activity of the wild types as amphetamine would when we gave it to the knockouts they curled up in the corner of their cage with a good book and we weren't expecting that at all and the only situation at that time where we were aware that amphetamine has a calming effect was with ADHD it's a first line treatment for ADHD attention deficit hyperactivity disorder the only other thing we knew about ADHD was that methylphenidate, acarytinin is also a first line treatment so we tried methylphenidate and we got exactly the same sort of pattern so we're beginning to think that actually we might have a mouse with ADHD rather than an antidepress mouse but we needed to be sure that these abnormalities were really attributed to the lack of functional NK1 receptors and not some extraneous factor so we tried to emulate the lack of NK1 receptors by giving them an NK1 receptor antagonist to the wild types in fact we checked out two antagonists two different ones and we got the same finding so we could turn a wild type into a hyperactive mouse by giving it an NK1 receptor antagonist but if we co-administered anthetamine it blocked that increase in motor activity so anthetamine was preventing the hyperactivity in these mice and that was directly attributable to the lack of functional NK1 receptors so we're quite excited about that and boned up on ADHD and found that it's a terribly interesting disorder in fact it's the most heritable of all psychiatric disorders and there are a range of gastroprope it's not just a childhood disorder about 65% of patients it persists in adulthood and it's the adults really who do have major problems with depression, suicidality, drug abuse sort of about 25% of patients in prisons are actually should have been diagnosed as ADHD possibly because being inattentive and impulsive is not a good combination if you're a driver or whatever but anyway the problems highlighted in red are the ones that I actually want to highlight next there is a very strong association between ADHD and alcoholism but 40% end up as serious alcoholics Now a colleague of ours at UCL Hugh Gurling who sadly died in 2013 was a pioneer in psychiatric genetics and he had already established a strong association between alcoholism and polymorphisms of the TACR1 gene Now a TACR1 gene is the human equivalent of the NK1, the neurokinan a substance P preferring reset to gene in rodents the nomenclature is a mess but anyway they're the same gene and given the association between ADHD and alcoholism alcoholism and human equivalence to the NK1 reset to gene we persuaded Hugh to see whether he would find ADHD patients with polymorphism of that same gene Now he wasn't a researcher on ADHD but fortunately he had lots of colleagues who were and he persuaded them to share their DNA and he said that Karen Cambridge and Philashson Institute of Psychiatry so they pulled all their resources and sure enough they found he described as the strongest snip markers for polymorphisms in the TACR1 NK1R gene that he'd ever seen and that polymorphism association showed up on haplotype analysis as well and this has now been confirmed in subsequent studies so we have hyperactive mice we've got a gene which suggests that we're really onto something but there are three diagnostic criteria for ADHD and we only have hyperactivity the others are inattentiveness and impulsivity and the combination of those problems defines what subgroup of ADHD patients they are in so we set up a study to look at these animals cognitive function and we use the five choice serial reaction time task in which animals are in a chamber and they have to scan the back wall where there are five holes in the wall and at random, at variable intervals a light will shine up above one of the holes signalling that the animal should nose poke in the hole where it intercepts an infrared beam and that can be scored online now it takes rats a long time to learn how to do this and with mice it takes about five months this is only the sort of work an academic would do it's not high throughput screening but anyway, some of these mice sometimes they can't keep their minds on the job and they miss the light or they forget to respond and those are called emissions which is recognised as an index of inattentiveness other mice know they shouldn't nose poke but can't stop themselves and that's a sort of premature response an index of motor impulsivity in rats generally this procedure can be used to measure accuracy but in mice that's more difficult because mice are really careful when they've learnt the task they are nearly 100% accurate it's very difficult to trip them up on accuracy but second part rats take their chances they're quite sloppy so they hover around 60-70% accuracy so you can do decent pharmacology on that and perseveration where the animals instead of going back to collect their water immediately they keep nose poking for a while just to make sure they've got the right hole well using this test we found an increase in emissions premature responses and also increased perseveration which is not a diagnostic feature of ADHD but it's very common in ADHD so we were able to tick the boxes but there was one thing we were a bit bothered about and that's that when these mice had come from Cambridge and after that they were all inbred the wild types of knockouts were treated as two inbred separate strains which is not ideal so we really had to check out whether there had been any genetic drift or anything else of that sort going on so we had the original two inbred strains which we called HOMS and then we crossed them to produce heterozygic parents and then from those we produced homozygic progeny again which we called HEPS and we did a head-to-head comparison of the HOMS and the HEPS this is a collaborating with Stuart Pearson in Oxford and sure enough sure enough both the HOMS and the HEPS were definitely hyperactive during their active period which is our dark phase so that was a great relief so there was also something interesting going on during the end of their light phase is that the wild types of the original colony these wild types were much more eager to get out of bed than all the other groups now these mice are genetically identical they all come from the same angles so the only explanation for that is an interaction between their genome and the environment in other words it's either the mother or the siblings that are responsible for that difference and that could be very important in ADHD as well interactions with others in the environment then we got a portfolio of findings on these mice hyperactivity, impulsivity and so on and we know other things too they've got raised arterial pressure and heart rate and also the males the male knockouts are particularly vulnerable to obesity if you give them what's called lab western diet which is equivalent to feeding them McDonald's and chips all day the knockouts increase their body mass immensely and after a month 35% of their body composition is fat and the females they put on weight but they didn't lay on lard in that way now the association between obesity and ADHD is well recognised they've never ever really been able to track it down to what's behind it is it genes or is it family or is it lifestyle and actually our findings suggest it's a three way interaction between the genome, the sex of the ADHD patient and also the type of diet they're being fed so it's not surprising if it's hard to find but our hypothesis is that if you could if you could find humans with this portfolio of problems and then gene sequence them they should, our prediction is they would have polymorphisms in the tachar 1% genes and then we've got a biomarker because we've looked to the pharmacology of these animals and we know what drugs work so amoxapine relieves their impulsivity methylphenidate relieves their perseveration so we've got a pharmacological profile up to match the animals well given that there are only three diagnostic criteria of ADHD and you can measure them all objectively that really got and yet it's terribly difficult to be confident or convince others that you've got a model of ADHD and that got me thinking about depression and all the research publications that claim to studied animal models of depression and here we have the diagnostic criteria for depression in humans and there are loads of them there are lots of different schemes for diagnosing depression in humans and these are the two prominent ones DSM-5 and ICQ-10 they've got a lot in common there are some differences but they're not major but they group into three main to main psychological, somatic and behavioural but the important point about this is that DSM-5 which is the one people use the most patients have to express at least five of nine symptoms for at least two weeks to qualify for a diagnosis of depression now I don't know any preclinical studies that can match that let's turn that list into something that preclinical scientists like me were used to working with terms like this and starting from the bottom up the purple ones are things that we can measure objectively I don't think there would be any dispute about telling when an animal has got sleep disturbance self-neglect, when it was agitated or not moving around very much appetite disturbances, weight change that sort of thing I've told you that we can already we can measure quite subtle differences in cognitive deficits and I'm going to talk about Anne Haddonia later on but the ones in black I don't think we'll ever be able to measure how can we tell when a rat or mouse is feeling suicidal or when it's feeling guilt or feeling hopeless I just don't think we're ever really going to be able to get there but these are further further difficulties with models of depression and depressive like behaviour because scientists I think who realise that the models really don't stack up to being something analogous to the human condition do describe their model as depression like instead as though that sort of solves the problem but it's never actually really made clear what aspect of the diagnostic criteria for depression is actually being modelled it's plausible that the resident intruder test can model low self-esteem there's a subordinate mouse when it's really on its back possibly and I'm going to talk about Anne Haddonia it's a lot of the models it's not too clear also we have to bear in mind that depression isn't a fixed phenotype the different symptoms wax and wane different patients have different combinations of symptoms the symptoms have different weighting in different patients and some disorders can co-exist and I've already said you get ADHD, co-morbidity with depression and abnormalities aren't confined to specific psychiatric disorders for instance you get cognitive deficits in depression and schizophrenia as well as obviously ADHD so if you pick up a cognitive deficit in your animal model you know what psychiatric disorder is being looked at there that's never really made clear so these are models which are described as either models of depression or cautiously depression-like behaviour and there's a long lecture on just that list but I don't worry the one I really want to draw your attention to is the olfagibol back to me because this is the only one on that list which really has very much validity even the people who developed it John Kelly and Brian Leonard at NUI Galway saying it's good, it's not perfect but it's good-ish and the reason it's good is because it has a range of abnormalities not just behavioural pharmacological endocrine these animals have raised plasma corticosteroid which is equivalent to cortisol in humans which is very very commonly raised in depressed patients there's something wrong with regulation of the adrenal pituitary hypothalamic axis in many depressed patients and the important thing about this model is that the abnormalities are prevented only after treatment with chronic antidepressant drugs which makes it like the situation in depressed patients whereas all the models above this the abnormality is resolved by a single or two or three treatments within 24 hours that said criticism of the bulb back to me is that depressed patients don't have severed olfactory bulbs and absolutely true however there is a group of depressed patients who say that they know when they're going to get a relapse of their depression it's a bit like an aura for a migraine and they know because they lose their sense of smell first so it may well be that these animals are an extremely valuable animal model for that subgroup of patients who show chronic relapsing depressive disorder I'm going to talk a bit about chronic mild stress because it's often not mild at all now chronic mild stress is used to tap into anhedonia rats and mice have very sweet tooth they'll do anything for you for ribena or carnation milk particularly the one with caramel in it they absolutely love that but when the animals have had about a chronic mild stress they lose that sucre's preference and that is interpreted as being analogous to anhedonia the inability to experience pleasure which is a real problem in depressives so depressives it's not just gestatory anhedonia in patients they lose that socialising isn't rewarding they don't find work rewarding nothing is rewarding anymore nevertheless it's plausibly anhedonia in these animals and when developed in the UK by Paul Wilner and as used under the EU directive all the stresses are arguably mild they're not physically causing any discomfort they're more like the sort of daily hassles which can tip a depressive over the edge the washing machine breaking down prying the car on the way to work life just not going very well you don't actually have to have any physical trauma for it absolutely to tip you into a depressive episode and that's how it is but you have to be a really accomplished behavioural scientist to achieve the anhedonia with this I can name the experts in the UK who can get it to work it's not for the faint hearted inexperienced but actually because it's difficult one of the things which is happening is that people outside the EU directive are actually increasing the stress intensity to get their loss of sucres preference and here is an example of just two papers which have been published in leading journals European journals in the last two years and their chronic unpredictable mild stress protocol and I think you'll agree that in many cases individual stresses aren't mild at all I mean look for instance food and water deprivation for 24 hours and then swimming in hot water and swimming in cold water and in this one they have two stresses every day which again I think we would call some of these severe so these stresses really aren't relevant to the sort of human experience which triggers depressive episodes and I would argue is more relevant to a sort of experience in what's called enhanced interrogation techniques which are regarded as ethically unacceptable in humans and next at the forced swim test which I have to mention because there's been a very strong campaign up to get this banned on the basis that it's regarded as a severe stress but on that basis I would remind you of the earlier finding where it's about equivalent to a rat going out to find food on a December evening now there are lots of things about the forced swim test which raise questions about it being a model of depression or even depression-like behaviour points to consider are that depression is a multifactorial chronic relapsing disorder and these rats and mice are absolutely fine when they go back into their cage and they've dried off the process is called behaviour the abnormal behaviour, the inability is called behavioural despair and that sounds like depression doesn't it but there's a lot of debate about whether behavioural despair has got anything to do with depression or whether it's absolutely a passive coping mechanism in the Netherlands believes the immobility is a means of dealing with the stress of the swim not caving into it and that fits with research done by Bill Keating many years ago at Queen Mary, he was a physiologist looking at stream physiology and diving and mountaineering and that sort of thing and he did research on cold water immersion and his advice was if you do fall off a boat into the North Sea don't swim because your survival time goes down, you become hypothermic much more quickly so if you fall into the water float and hope someone comes to rescue now it may be that rats and mice know that innately who knows what really pulls the carpet up under the feet of the ant is that this procedure is sometimes used to model anxiety not depression they're completely separate disorders and as I've mentioned before unicronic administration of antidepressants to treat patients but the immobility in this test is resolved by acute or subacute treatment so this is absolutely nothing about this test is like a model of depression so where do the ideas depression come from well it's in the authors original papers here are some quotes from their early papers we suggested state of despair resembling depression that the immobile behaviour may reflect a state of lowered mood that we hypothesised that the animals had given up hope and was given the name behavioral despair in other words there never was any evidence that this is behavioral despair and that remains the case this is not a model of depression or at least it hasn't been validated as that and it's very unlikely to be however every single antidepressant drug in the clinic prolongs the latency to immobility in the force room test they all do it just one example of a paper from each category of antidepressant drugs for rats and mice and they all have that effect and even a recent addition ketamine even ketamine does that ketamine as you all know I'm sure is an anesthetic you wouldn't normally expect an anesthetic even subanesthetic doses to increase motor activity but it does so the force room test is a very good an indicator of whether or not a drug is likely to have an antidepressant effect on humans so what we have to debate is really all about is a payoff between refinement and reduction it would be lovely to use a procedure which is less stressful but so far there aren't any which have been validated as consistently as the force room test and if they're not as reliable then we will end up using more animals unnecessarily because we'll end up with false positives and false negatives and another reason is that I strongly suspect and got reasons that the stress of the force room test is fundamental for this action of antidepressants to increase the latency to immobility that if it was less stressful that increase whatever it is antidepressants do wouldn't show up so you need a bit of stress to see the effect of the antidepressants so what's been going on and this is a very very common problem in my field and I've set up this absurd example to show you what's happening that if all antidepressants make animals whistle the national anthem then if animals are not whistling the national anthem they must be depressed now that's clearly nonsense even if you're a republican it's a clearly nonsense but that's what's been going on in these sorts of experiments but how can you have a procedure which can find antidepressants that doesn't need an animal model of depression and that's easy to explain if you look on the left this is a bridge at Warrington which if you remember was flushed away in some storms several years ago and the parents on this side of the river had a problem because they couldn't get their children to school which is on the other side of the river and you can call that depression well they solved the problem by getting in their cars driving up the river and going over a different bridge and then back down to the school call that antidepressant so antidepressants may not be curing depression or even targeting the cause of depression they're simply finding a way around the problem and that would fit with how the brain works if we think about sleep and arisal appetite versus satiation pain and antinode deception they all rely on interacting neuronal networks that operate as smoothing circuits allow smooth transitions between one state and another and if one goes wrong then you can get a catastrophic change in function as in depression but it can be resolved by recruiting a separate neuronal network so it's quite likely that the force wind test scores antidepressant and there's no need for it to be a model of depression at all so what would I like to see in the future well obviously one thing I would like people to realise is that if the results of an experiment don't work out it's very important to follow through because you can actually end up in places which are even more interesting and even more important in a way than the place where you were heading originally so always to persevere but for the purpose of the animals it would be really good to see steps incorporated that can help improve animal welfare and successful translation in these sort of models which use routinely in psychopharmacology wouldn't it be good if funders, publishers, journals and referees or encouraged to ensure that the stresses that are used in laboratory are relevant to the human experience especially in terms of their severity and that particularly when cumulative harm is taken into consideration and that severe stress is justified by the experimental objectives so not to use a series of high intensity stresses which certainly cause cumulatively severe stress and then to assign it a label mild stress that's disingenuous it would also be lovely if animal models of multi-factorial psychiatric disorders like depression schizophrenia and so forth the validity of a description of animal's abnormal behaviour as a model of these disorders should be justified bearing in mind the diagnostic criteria that's applying humans now a couple of journals have taken this on board already and I don't think they'd mind me mentioning them one is the German Psychopharmacology and the other is the British Journal of Pharmacology there's no intention to ban anything at all it's just we're trying to nudge authors into doing the right science and to interpreting their data in the right way and it would be absolutely wonderful if other funders and publishers and editorial boards followed suit so thank you for listening and thank you as well as thanking you I must thank all my wonderful collaborators, it's been such fun and so stimulating and interesting working with them and of course I must thank the animals as well who've helped with all this research and the background was the view from my lab at UCL depressing lecture I'm sure you'd answer a few questions if anybody has them I know it's deterring new leaching but this is one there to start with Thanks Claire, that was really interesting a journey through your career and through animal models of depression I'm just curious where are the tortoises you promised us tortoises and I didn't hear anything about them it's the tortoise you showed us a tortoise occasion you showed us some hair I'm quite sure where they were there tortoise and hair is that the tortoise gets there in the end and how I thought it was going to be actually a tortoise model on hanging stress on hanging stress was was hair, very excited Gallupvella 20 years, 30 years new made Janehurst actually did the important work and that's recounted okay, so we haven't got the tortoise tortoises right hi Claire thank you for the talk here if the force wind test is quite predictive in finding antidepressants or antidepressive action on short time scales what can you then know about the antidepressant circuits in the brain of humans that only become effective over a longer period of time absolutely nothing it can tell you about the acute pharmacology but the really important point is that you don't have to have a model of a disorder in order to be able to predict that a drug will end up having an effect in the clinic in humans so I absolutely take your point that the acute effect on the animal's behaviour isn't going to be very helpful in telling us what explains the therapeutic effect of antidepressants because that has to be chronic but it could tell us more about the pharmacology the pharmacological targets what neuronal networks are important in this acute response and then the chronic adaptive changes will follow and maybe that acute response is essential for other things we know go on after long treatment of antidepressants like neurogenesis and that remodelling it may help to find early circuits that trigger those essential responses but no I wouldn't say it's a model of antidepressant either but it's it's balanced towards the antidepressant side that's the point I was trying to get across Hi bit of a lay person here I don't typically know how old mice live to and how old the mice that you're experimenting with but I'm just wondering you've obviously done the knock out and the wild type of baseline measurements I'm just wondering is there a way to monitor cohorts if a group of rodents become gradually more despondent over time and that more human like depression characteristic of it might not be that we're swimming around in hot or cold water but as you say the life gets in the way those sorts of small problems is there a way of monitoring if depression if realistic kind of depression can be captured in rodents have you ever done anything like that I think I've got to relax it first of all first of all because of COVID I haven't actually managed it I can shout it out a bit more so I don't know how old mice live to in a laboratory environment and I'm just wondering how old's the mice are that you're experimenting with the number of the question is is there a way of instigating more depression like characteristics that kind of match with the human condition I don't know I don't know where the mice are at the first or I don't know the anxiety I think it does raise interesting ethical questions actually if you could make an animal perhaps to your thought extent how they work how would they look at that would you really want to create a suicidal improvement that's a real quite severe procedure that's attractive there are sorts of interesting questions there about that where with your factual victimised animal is the only one really that we can say is anything we still don't know what's going on with your head in terms of mood that all the other markers on that animal are very reminiscent of things you find in depressions not every depressions there are things you find incredible impressive the others on that list are simply they'd be described as modern sort of depression but it's not realistic I think the recipe modern for instance when recipe in humans caused depression a small number of patients the lily group went through all those pieces and decided actually that was misinterpretation of the case reports and occasionally patients were overdose and they may have problems but they didn't really mean that's true but recipe induces hypothermia in Romans and also reduces motor activity and that is still used as a modern depression I saw one published last week is a modern depression depressive don't get hypothermia and also the hypothermia is only prevented by norodrone and uptaking uutis and there are dozens of other sorts like serotone, uptaking uutis, ketone, there's a long list of them and the only one that actually works is the tricyclo norodrone and uptaking uutis so we've got a physiological response now which is hypothermia which we don't find in depress patients which are only treated and their motor deficit isn't affected by any depressants at all so there's no way that that particular model could be regarded as anything akin to a depress patient and that sort of thing gets overlooked really but they can still be valid screens but you've got to make sure that the screen is consistently and continually revalidated as new drugs come along and I'm keeping an eye on psilocybin for instance at the moment because a lot of excitement about that being a potential nutrient for depression and it's hard to be tested in the force you decide, I want to know what it does in the force you're testing and hopefully states have found that it does actually have the ability which drugs promising but one paper isn't one swallow doesn't make some ok for a normal question