 brain and behavior. Good morning everyone, it's a pleasure to be here. What I want to do in the time that's available to me is look at some of the evidence out there to support the view that the microbiome does influence brain development and behavior. Now, assuming that the microbiota is capable of influencing the brain, I suppose one must ask how or what pathways might enable the gut microbexis to actually communicate with the brain. And there are a variety of potential pathways. I mean, the vagus nerve is obviously one potential pathway, the spinal cord. There are numerous potential immune mechanisms by which the microbiota could communicate with the brain. And there are endocrine mechanisms of communication as well. There is an accumulating volume of preclinical evidence looking at some of these mechanisms of communication. The reality is that in humans, we know very little about the potential most important roots of communication between microbes and the brain. If we are to study the influence of the microbiota on the brain, what are the techniques used? Now, this is a summary slide that John Crine and I put together. And it looks at the most common methods that are used in the literature at the moment. Germ-free studies are commonly used. Infection models, where you put an infective agent into the gut and look at the impact on brain function. That's obviously one way of looking at the or exploring the microbiota influence on the brain. We've heard about probiotic studies and indeed prebiotic studies as well. There are a number of antibiotic studies in the literature looking at how antibiotics acting on the gut might influence brain behavior, brain end behavior. And of course, recently, fecal transplantation has become big in relation to C. diff. But of course, it's an approach that could be used to look at the impact of the microbiota on the brain. If we start off with infection studies, back in the 1990s, Mark Leid did a very interesting study where he essentially gave mice a subclinical dose of Campylobacter geogeny that didn't actually cause immune activation. But he showed that acting through the vagus nerve and through the nucleus tractosolitarius that it actually caused anxiety in mice. And since then, there have been a number of studies that have suggested that in effective agents can cause marked alterations in mood or anxiety. The Walkerton study is a study that many of you may be familiar with. It's a naturalistic study. And so far as the town of Walkerton in Canada had its water supply contaminated with E. coli and with Campylobacter. And most of the population of the town suffered a gastrointestinal infection and GI symptoms as a result of infection. Now it was quite interesting to follow the actual patterns of illness that emerged in the years following the actual acute infection. Most of the focus has been on post-infective irritable bowel syndrome, which occurred in a large proportion of the population. But as a psychiatrist, what interests me is the fact that at the end of the first year, a very significant proportion of patients had developed major depressive illness. So it suggests that the effective agent, through whatever mechanism, resulted in the emergence of depression at the end of year one and was sustained in some of these patients for considerable periods of time. Now minocyclin is an antibiotic, a tetracyclic, that is fairly widely used for all sorts of things, including treating acne. And it acts on gram positive and gram negative bacteria. Now clinically, I treat many patients with depression. And some of these patients will have treatment refractory forms of depression. And there is no an emerging, though all of the studies are rather small sampled, but there is emerging evidence that minocyclin, when given in patients who are antidepressant resistant, that you see a response to treatment with the addition of minocyclin. There is also more recent studies suggesting that minocyclin, when given to patients with schizophrenia, has an impact on the negative symptoms of schizophrenia. So a suggestion that modulating the microbiota with an antibiotic might have positive mental health benefits. Fecal transplantation really in psychiatric populations has not taken place. Steve Collins and his group have done some very interesting studies where they've taken animals who have either an anxious phenotype or a depressive phenotype. And they've transplanted the microbiota into germ-free animals. And what they've shown is that transplanting the microbiota from an animal with an anxious phenotype into a germ-free animal gives that animal an anxious phenotype. And likewise, the transplant of a depressive phenotype or the microbiota from an animal with a depressive phenotype into a germ-free animal renders that animal having a depressive phenotype. There has been some speculative evidence put together to indicate that maybe transplantation of the microbiome would be beneficial in things like Parkinson's disease, but it is rather speculative, to say the least. So what about germ-free studies? Well, when we look at germ-free animals, there are a variety of alterations in such animals. Certainly, aspects of cognitive function are altered, aspects of memory are altered in such animals. There are alterations in serotonin centrally. And we've been looking at altered social behavior patterns in such animals as well. This is some of our data here. And it shows that if we look at germ-free animals, if we look at brain-derived neurotrophic factor, which is exceedingly important in areas of the brain involved in cognitive processing, for example, hippocampus, you see that germ-free animals have lower levels of brain-derived neurotrophic factor. You also see that in response to stress, they have altered corticosterone output. They have an exaggerated corticosterone release, which is interesting because behaviorally, actually, these are not anxious animals, but their endocrine response is one of an exaggerated corticosterone release. When we look at the hippocampus also, we see altered levels of 5-HT in the germ-free animals. They have higher levels of 5-HT in the hippocampus, and they've increased 5-HT turnover in the brainstem. Now, recently, we've been looking at social behavior. And some of you may be familiar with the three-chamber associability test. In this particular test, what we do is we place the germ-free animal in a three-chamber, where there is another animal in one of the chambers. And we look at the amount of time the germ-free animal spends with the other animal and the amount of time it spends on its own. And what one sees, essentially, is that in germ-free animals, here you have the germ-free relative to the controls, you see that the germ-free animals will spend more time in the empty chamber than they will with the other animal. However, when we colonize them, when we give them a normal microbiota, you see that they will spend more time with the other mouse. So the behavior can be altered provided the colonization takes place early on. I mean, if it takes place later on in development, you will not see normalization of behavior. Now, we also look at another factor. And it's where you have a germ-free animal and you have two animals in chambers with them. Now, one of the animals is a novel animal that the germ-free animal hasn't come across before. And the other is a familiar animal. Now, most animals, most mice will spend more time with the less, or the non-familiar animal than they will with the familiar animal. But it's interesting when you look at the germ-free animal, you see, in fact, here is the conventionally colonized and they'll spend more time with the novel animal than they will with the one that's familiar. When you look at the germ-free, there's no real discrimination. If you colonize the germ-free, they will spend more time with the novel animal. So, and this is just two aspects. Basically, if you have these animals, we'll spend more time with objects than they will with other animals. So their sociability is significantly altered. They have autistic patterns of behavior. Now, Mark Lytte has put forward a concept that probiotics, in many ways, are delivery vehicles for neurochemicals and I suppose the question is, what sort of neurochemicals do these putative probiotics actually produce or what are they capable of producing? And I was actually quite surprised, I suppose as a psychiatrist, and one who works in neurobiology, I've worked a lot over the years with norepinephrine and with serotonin and so forth. And I was quite surprised that various microbes are capable of actually producing norepinephrine or serotonin or dopamine. Very key monowamy neurotransmitters in the mammalian brain. But not only are they capable of producing neurotransmitters, but they're capable of modulating neurotransmitters as well. There's studies with lactobacillus acidophilus showing the capability to alter cannabinoid receptors. We've worked with bifinfantus, we've done a lot of work with bifinfantus and we've been able to show that bifinfantus increases tryptophan levels in the plasma, which in the psychiatric perspective may be important because tryptophan is obviously the precursor of serotonin. And work with lactobacillus ramnosus which suggests that it alters central GABA receptors. And it's that I want to focus on. Now this is a study that John Krein and I did in collaboration with John Beaninstock in McMaster and John had a strain of lactobacillus ramnosus that he felt was anxiolytic. And we decided that we would look at it in a variety of behavioral models and we would look at its impact on GABA receptor centrally. Now we chose GABA receptors because they're very ubiquitous in the mammalian brain and they are potently, or they have a very significant or potent impact on levels of arousal and anxiety. Now GABA receptors bind things like benzodiazepines and they bind a variety of anesthetic agents. GABA B receptors again are, well they're actually G protein coupled receptors unlike the GABA which are ionotropic receptors. So when we fed lactobacillus ramnosus as opposed to a broth in the earth broth, we essentially found that when the animals were stressed who were given lactobacillus ramnosus that their corticosterone release was decreased. We also found that in behavioral tests like the open field and the elevated plus maze that lactobacillus ramnosus increased exploratory behavior. So it seemed to be having an anxiety effect in much the same way as a benzodiazepine would have. When we looked at GABA receptors, expression in various brain regions if you look here at the infalimbic cortex you find that lactobacillus ramnosus decreases expression of GABA in the pre limbic you get a decrease as well. If you look at the amygdala or sorry, yeah the amygdala which is a very important area in terms of anxiety processing you get very major changes in the expression of GABA A with lactobacillus ramnosus. If you look at the hippocampus here you can see significant increases in GABA A expression there. What about GABA B expression? Again, very significant alterations in GABA B expression in a variety of brain regions. So lactobacillus ramnosus in a placebo controlled study in rodents alters GABA A and GABA B receptor expression. So we wanted to explore how exactly it did this. So we decided that we would do another placebo controlled trial this time in vagotomized animals and in sham operated animals to see if in fact the effects we were witnessing with lactobacillus ramnosus occurred in the presence of the vagotomy. And what we essentially found was when the animals were vagotomized their exploratory behavior was not altered by lactobacillus ramnosus. The increased exploratory behavior that lactobacillus ramnosus brings about was not seen in the presence of a vagotomy. And the changes in GABA A and GABA B expression which you can see here these are hippocampal slices and it's an in situ hybridization study. The changes in GABA A and GABA B expression that we had previously seen with lactobacillus ramnosus treatment did not occur in the presence of a vagotomy. So the effects of lactobacillus ramnosus were mediated through the vagus nerve and the animals needed an intact vagus nerve before one saw anxiolytic effects from the lactobacillus ramnosus and alterations in GABA A and GABA B receptor expression. Now this is a study that Emron Maher has recently published in his group at UCLA and I think it's an extremely important study and it's important because it's the first real attempt to look at in a systematic way the potential impact of probiotics in humans. And what Emron and his colleagues did was they took a group of female subjects and they treated them either with a fermented milk product for four weeks which contained a number of probiotic organisms. So it's a cocktail of probiotic organisms or a non-fermented milk product or no treatment. So you have a group of 15, 12 and 14 and they're treated or not treated for four weeks and they have an FMRI scan pre and post treatment. Now, basically what the study demonstrated was that treatment with the probiotic cocktail altered activity in those areas of the brain that we associate with emotional processing. So the probiotic cocktail was capable of altering activity in those brain regions associated with emotional processing. Now it's the first study of its kind. There are many studies similarly conducted in animals but this is the first study in humans. We've just published a paper in biological psychiatry where we define a psychobiotic as a class of live organism that when ingested in adequate amounts produces a health benefit in patients suffering from psychiatric illness. And I think there is an accumulating volume of evidence that in certain conditions probiotics can have a positive health benefit. So this is a psychobiotic to be distinguished from psychedelic substances or snorting cocaine or whatever. But we do believe that probiotics, they obviously need enormous more research but there is emerging evidence I think to indicate potential from a mental health perspective. And when one looks at the areas where the best evidence is available, undoubtedly irritable bowel syndrome which I suppose gastroenterologists will came as is an entirely gastrointestinal disorder whereas liaison psychiatrists will say it's a psychiatric condition I suppose. That's the product of subspecialization in medicine but whatever the approach one takes to treat irritable bowel syndrome, there is I think an accumulating volume of placebo controlled evidence that probiotics are effective in treating irritable bowel syndrome. If one looks at the data for example with bifidobacterium infantis, there are at least two good placebo controlled trials in the literature indicating efficacy in irritable bowel syndrome and there are other studies with other organisms as well. There is less good data for something like depression. The Masaudi paper is a paper about 18 months ago published which suggests that in a healthy population, a physically healthy population that a combination of L-Helveticus and B-Longum can reduce depression scores and decrease 24 hour urinary free court as all output. Now I think the endocrine aspect of this I find actually a bit more convincing than the measurement of symptoms but it's an interesting preliminary study. There's also a study in chronic fatigue syndrome which is such a difficult syndrome to treat if anyone has been involved in treating it and there's a study looking at L-Casia and finding quite marked reductions in anxiety scores in patients with chronic fatigue syndrome. So there are some of the kind of clinical areas where psychobiotics or probiotics might be of benefit from a mental health perspective. So where are the major gaps? Well one would have to say that when one looks at the impact of the microbiota on the brain and behavior that there's a never increasing and very good quality literature from a pre-clinical perspective but we are seriously lacking good clinical studies and there are not clinical studies that would be necessarily difficult to do. And if one takes the pre-clinical literature we've published and one or two other groups have also shown that if you take an animal model of depression for instance that the microbiota is altered in such models. Steve Collins's group have shown similar changes in an animal model recently. So the microbiota is altered in animal models of depression but there has been no attempt so far to profile the microbiota in patients with major depressive illness. We're about to start such a study but there's nothing in the published literature. Likewise, when it comes to probiotics or psychobiotics as I like to call them there is lots of data pre-clinically in animal models of depression and anxiety that certain strains of bacteria or certain psychobiotics are effective but we have no clinical studies. Now I think one question that is exceedingly important and I haven't really focused on it because for time constraints but it's the issue of are all probiotics the same and do they all have an impact on behavior in the brain and the reality is no they are not. I've shown you one or two studies that we've done where the outcome has been positive but I would say that 99% of the bacteria that we have tried to profile from a behavioral perspective have absolutely no impact whatsoever on behavior. They probably don't even get through the acid in the stomach, they're totally ineffective, they have no impact in animal models but there are some that clearly do have a very dramatic impact in animal models and I think we really do need proper, well controlled clinical trials in patient populations in patients with depression or anxiety and those studies are sadly lacking in the literature at the present time. So I'd like to conclude by acknowledging my colleagues John Kreinger, Clark and my other colleagues in the elementary pharmacist center in Cork and thank you all for your attention. I have one. So very interesting and you briefly mentioned autism and there have been a couple of recent studies that have shown that there is altered microbiome in children with autism. Indeed. So I wonder if you would care to elaborate on that anymore. As you say there have been one or two studies that have suggested an altered microbiota. There were also studies suggesting that one or two antibiotics can significantly improve the behavior in autistic children and there is some suggestion of altered barrier function, gut barrier function in autism. So it may very well be that in some autistic children, I think autism is a heterogeneous entity. I'd be very reluctant to say that it's homogeneous entity but I do think that there are some autistic individuals who have altered barrier function. You get a seepage of LPS and other molecules across the defective barrier. You get a pro-inflammatory phenotype and you do get central, you do get central changes. So would probiotics be beneficial? Perhaps they might. I do think that there is convincing evidence that where there's altered barrier function that some probiotics are capable of improving the barrier function. So it may very well be that autistic subjects might benefit from probiotics as they do from antibiotic therapy but I think we really need very good studies. Autism is an area where there's so much rubbish in the literature. My question is perhaps as much to you as to Dr. Goodman with the notion of psychobiotic beneficial to psychiatric health and yet all the indications that you listed are clinical entities like depression and schizophrenia and irritable bowel syndrome and these clinical trials are really there to treat a disease and that is a different rigor of what is required for such a product. So how do you draw this boundary for yourself? Where is something that is just a beneficial thing which can be taken care of by marketeers and commercial entities transition to become a drug and has a much more hardcore intervention? Oh indeed, you raise a very interesting point and a very important one. It might very well be that there are probiotics or psychobiotics out there that might be of benefit to people's general mental well-being but might be totally ineffective if you put them into a psychiatric population and I think the only way we can really, I'm a psychiatrist so I'm interested in treating depression and anxiety and that's why I focused on that but we really need to employ rigorous methodology. Whichever group, I mean even if we're not going to deal with psychiatric populations I think we do need proper placebo controlled studies if we're going to even demonstrate positive mental health benefit in the general population. I mean that's not, those studies are not too difficult to do and so far most of these organisms of grass status, it's not, they're safe so one isn't going to have to go through the burden of the toxicology that one would with a drug and so forth. I think we do need to do the studies both in the general population and in psychiatric populations and they have to be rigorously controlled studies. Perhaps not toxicology but you still would have to be held to a rigor of what's actually in there. Oh absolutely, I agree entirely. Thank you Ted, that was an elegant talk and probiotic infusions are plausible from the purpose of this meeting within the microbiome framework of actually looking at restocking a habitat for keystone species for microenvironment if you really look at it as restorative or preservation but for all the reasons as you said until we really understand the safety and all of the necessary pathways. I think what's intriguing is you present it for anxiety and depression and that is a particular phenotype but you also generalize it more to let's look at this from the standpoint of a micro habitat for the general population. So I would just like to hear a little more comment from you on the direction in the field for what we might see a probiotic in the future and beyond just the anxiety, depression, disorders but why you really think and do you think it's more likely to be just in the live biologics area or will it encompass diet and foods? Indeed, I think that one of the problems in this area with probiotics is that there are so many people making claims out there which in Europe, EFSA are being exceedingly rigorous in trying to stamp out these claims but until such time as companies cannot make claims spurious claims for which they have no data there is no, there is little really to encourage a legitimate company to actually do the necessary studies to demonstrate the benefits of a probiotic because they'll be up against numerous companies that are marketing very effectively products that often have absolutely no impact. You know, I would stress the fact that the bulk of putative probiotics that we've had anything to do with in our lab and I'm sure that others will attest to this as well who are interested in behavior they do absolutely nothing and yet there are all sorts of crazy and grandiose claims being made by companies out there and I think it's only when that has stopped that there will be I suppose an incentive for companies to do the necessary studies be it in the general healthy population from a mental health perspective or in psychiatric populations. So I think we need to move on as we're running a little bit late so let's thank Ted. Okay the next speaker is Marty Blazer from New York University Medical Center Yeah and...