The effect of D-alanine and a prebiotic on rat brain N-methyl-D-aspartate (NMDA) receptors and executive function

Lead Research Organisation: University of Oxford
Department Name: Psychiatry

Abstract

The NMDA glutamate receptor is important for brain functions such as memory and problem solving. A naturally occurring chemical called D-alanine can activate this receptor and may control the way we think, but its levels in the brain are quite low. We have often wondered whether increasing the amounts of D-alanine in the brain would activate the NMDA receptor more, and therefore make the thinking processes work better. Animals and people can drink a lot of D-alanine in water without making them feel ill. We have found that when rats drink D-alanine for a couple of weeks, it causes chemical changes to the NMDA receptor in the brain. Scientists have shown that the sorts of changes we see, make the NMDA receptor more active. We therefore want to prove that D-alanine increases NMDA receptor function, and if this also improves thinking. This information will be useful to people who are interested in stopping the decrease in brain function which happens as the brain gets older or when it is affected by an illness. We will feed rats with D-alanine for two and eight weeks and see if they are better at problem solving than animals not given the compound (controls). This will be done with the attentional set-shifting task which basically tests how many go's it takes the rats to work out where food is buried, by using the information they were taught before the actual test. After the tests we will use standard methods to measure chemical changes to NMDA receptors and the amounts of other brain molecules which sometimes change when the receptor is active. All this information should tell us if D-alanine helps the thinking process, and if it is related to certain chemical alterations in the brain. In separate experiments, rats will be fed with D-alanine as before, and while they are asleep we will measure their brain electrical activity produced by adding the chemical NMDA. We believe that the brains of D-alanine fed rats compared to controls will have more electrical activity after adding NMDA because, the NMDA receptor is more active in these animals. Scientists have known for a while that D-alanine in our bodies actually comes from the good bacteria in our guts. Some drinks and yoghurts, called prebiotics, make these bacteria grow more, and this makes us healthy because the bugs help breakdown the food we eat. When we fed rats with a prebiotic called fructo-oligosaccharide (FOS), we saw the same changes in the NMDA receptor in the brain as we previously saw with D-alanine feeding. We now want to see if FOS feeding does actually increase levels of D-alanine in the brain, and if it changes rat behaviour and brain electrical activity. Finally, we will add D-alanine to isolated brain cells (cell culture) and measure chemical changes to the NMDA receptor. If nothing happens, we will know that D-alanine can only change NMDA receptor chemistry when the brain is in the body. This means that other molecules, pathways and brain areas, which are not present in cell culture, are needed for D-alanine to affect the receptor. We will also test if D-alanine added to cells affect other chemicals without causing changes in NMDA receptor chemistry. Overall, the study will tell us if the thinking process and NMDA receptor activity can be increased after drinking D-alanine or a prebiotics. This is important to know because a lot of people take prebiotics with their food, and it is possible that these, or just D-alanine alone, can help brain function as we get older or develop a brain disease.

Technical Summary

Brain N-methyl-D-aspartate receptors (NMDARs) are integral to the neural circuitry underpinning memory and cognition. D-alanine, an NMDAR co-agonist, is thought to arise from the natural breakdown of bacteria in the mammalian gut. Brain levels of D-alanine are low compared to the major NMDAR co-agonist D-serine. A repeated administration of D-alanine results in its stable accumulation in the cerebral cortex. This build up of brain D-alanine may lead to long-term augmentation of NMDAR activity, and possibly, improvement in executive brain functions mediated by the frontal cortex. Thus, D-alanine intake may be beneficial in some situations, such as aging, where NMDAR function progressively declines. In preliminary experiments we observed elevated levels of phosphorylated NR1, NR2B, but not NR2A, NMDAR subunits in the rat frontal cortex after a 2 week administration of D-alanine, or the prebiotic fructo-oligisaccharide (FOS), which promotes lactobacilli growth in the gut. We know plan to examine the functional consequences of elevated NMDAR subunit phosphorylation by D-alanine and FOS at the level of electrophysiology and behaviour. Our proposed research will: 1) Test if rats fed for 2 and 8 weeks with D-alanine and FOS show improvements in the attentional set-shift task which is a measure of medial prefrontal cortex function; 2) measure rat brain NMDAR phosphorylation after behavioural tasks; 3) confirm if FOS administration leads to increased levels of gut bacteria and brain D-alanine; 4) evaluate NMDAR activity in the frontal cortex of experimental and control rats with in vivo electrophysiology; 5) explore in cell cultures the direct effects of D-alanine on the expression of NMDARs and the signalling pathways (ERK-MAP kinases, CREB) which are modulated by NMDAR activation. If successful, the study will provide novel insights into NMDAR modulation, and may reveal the utility of D-alanine and prebiotics in the maintenance of healthy cognitive function.

Planned Impact

Who will benefit and how? In addition to the immediate beneficiaries from academia, our proposed project, if successful, is likely to benefit the commercial private sector with a particular interest in prebiotics. We believe that manufacturers of such products would be attracted to such research as it offers the potential to develop a new market strategy for prebiotics as agents capable of promoting brain health, in addition to their more widely accepted role on digestive and immune well-being. If we also show in our study that D-alanine itself promotes cognitive improvement then it to may be commercialised as an active ingredient which may be incorporated into other foods. Understanding more about how the gut bacteria and D-alanine may modulate normal brain function and age-related cognitive decline will help agencies (Food Standards Agency, Department of Health, Department for Environment, Food and Rural Affairs) inform the public and encourage them to make more sensible healthy food choices. For example, if our research yields encouraging data with D-alanine, the health benefits of cow's milk, vegetables and fruit which are all high sources of this compound could be further pursued. It is conceivable that the whole population within a 10 to 15 year period may experience the benefits of healthy diet with children, and succeeding generations, gaining most from an improved quality of health and life expectancy. The individual consumer may also directly benefit from our work shows that prebiotics improve working memory. In this regard, the active compound we will test (the prebiotic FOS) is a common ingredient in many commercially available products. Therefore once disseminated through public engagement channels listed below - one could encourage the uptake of such products and their related benefits. Thus, the potential of our research to impact on the health of the UK could be realized in a short time. Since the proposal is highly translational, and the compounds tested are greatly tolerated, human studies could proceed very soon after our data in animals are analysed. This would immediately preclude the use of rodents for this type of research, and result in their direct 'replacement' (cf the 3Rs) with human subjects. Staff engaged in the project will be trained and expected to acquire proficiency in several research skills including molecular biology, neural cell culture, microbiology, electrophysiology, behavioural neuroscience and animal welfare. Moreover, the post-doctoral researcher will develop an aptitude in management and organization so that the several components of the study between three institutions can be efficiently co-ordinated. Importantly, the appointed staff will experience research within both an academic and Industrial environment, and thus allow them to make a founded decision about the scientific career that suits them best. All these attributes will provide the researcher with a multiple of scientific as well as interpersonal skills, which would be appreciated in several employment sectors. Exploitation plans: To ensure that the identified groups will benefit, the results of the proposed investigation would be disseminated through publication in peer-reviewed journals and presented at national and international scientific meetings. In addition, the results would be communicated more widely to the general public via the channels of public meetings (e.g. at schools and charitable organizations), and the general print and broadcast media in which the applicants are active. Our Industrial partner has an additional duty to distribute potentially beneficial information via their established channels and policies, thereby extending dissemination coverage. We will actively pursue companies interested in prebiotics in order to fund additional work in this area, in particular human intervention studies which are costly to implement but which will directly benefit all parties involved.

Publications

10 25 50
 
Description 1) We hypothesized that the ingestion of prebiotics (dietary fibres that promote the growth of beneficial intestinal microbiota) will augment brain chemistry and function by increasing central concentrations of the D-amino acid, D-alanine. This D-amino acid is only found in bacteria, and so when these breakdown in the gut, D-alanine is released and enters the blood stream. D-alanine is also an activator of the N-methyl-D-Aspartate receptor (NMDAR), which is found at high levels in the CNS and is crucial for healthy brain function. In pilot studies we observed that the direct oral administration of D-alanine to rats increased the levels of brain NMDARs. The project therefore, tested the effects of D-alanine and prebiotics on NMDAR neurobiology, function and related behaviours. We found specifically that: i) Feeding rats with fructo-oligosaccharides (FOS) or galacto-oligosaccharides (Bimuno, BGOS), increased the levels of NMDARs in brain areas crucial for memory and problem solving. The numbers of the beneficial microbe, Bifidobacteria, in the rat gut were four-fold greater than those in control animals, and 2.5x greater than in FOS-fed rats. ii) Although BGOS feeding to rats increased D-alanine levels in the blood, brain concentrations of the D-amino acid did not change. We therefore concluded that prebiotic-mediated changes in central NMDARs were not related to D-alanine. ii) Rats administered with D-alanine or BGOS demonstrated better problem solving abilities in the attentional set-shifting task, compared to controls. This is in-keeping with the previously observed elevation of rat brain NMDARs following dietary supplementation with D-alanine or BGOS. iv) In electrophysiological experiments, neurons in the brains of rats fed with BGOS were more responsive to NMDAR agonists compared to controls. This showed that the elevation of brain NMDARs observed after prebiotic feeding were functional and increased the activity of cells. 2) Since the project found robust actions of BGOS on the brain, we extended our studies to further explore the utility of prebiotics to maintain brain health, and test the role of the immune system. Studies have demonstrated that the growth of good gut bacteria suppresses harmful inflammatory responses. Such responses have been shown to be detrimental to brain chemistry and behaviour. We therefore investigated whether BGOS supplementation could 'protect' a mouse from the biochemical and behavioural effects of an acute infection. We administered a single injection of lipopolysaccharides (LPS), which are contained in pathogenic bacteria. We found that: i) Mice receiving a single LPS injection displayed a decrease in locomotor activity which was followed 24hrs later by anxious behaviour. However, mice fed on BGOS prior to an LPS injection showed relatively normal LMA, and no anxious behaviour. ii) The brains of mice injected with LPS and on a normal diet, contained higher levels of the inflammatory molecules, TNFa, IL-1b, IL-6 compared to animals injected with saline. However, mice fed with BGOS contained the same levels of inflammatory markers compared to BGOS supplemented mice receiving a saline injection, and those on a normal diet injected with saline. iii) Together these data suggest that BGOS supplementation attenuates the molecular and behavioural responses to an acute infection. This is in-keeping with our supposition that the proliferation of gut bacteria modulates the host's immune system, and benefits brain function. 3) In a supplementary investigation using a small amount of grant funds set aside for contingency studies (in the event of failed objectives), and with an additional contribution from Clasado Ltd, we investigated the effect of FOS and BGOS on emotional processing and the modulation of the stress hormone, cortisol, in healthy volunteers. In a double-blind placebo controlled study, we found that: i) Participants ingesting BGOS once every day for 3 weeks, had lower levels of salivary cortisol compared those who had ingested a placebo or FOS for the same amount of time. ii) Subjects who had been provided with BGOS demonstrated less anxiety in a computer-based psychometric test compared with the other groups. iii) Together these data show that BGOS supplementation has a mild anxiolytic action in healthy subjects, and this is corroborated by the observed reduction of salivary cortisol.
Exploitation Route The prebiotic BGOS is commercially available, and so the general public can benefit from its mild anxiolytic properties, and potentially, pro-cognitive effects. Throughout aging there is a natural decline in cognitive abilities, and control of the stress response; indeed, both processes maybe interlinked. The population most likely to benefit from dietary supplementation with BGOS therefore, are the elderly. Our finding that D-alanine has a significant effect on cognitive function in rats, also suggests its potential application as a supplement for cognitive deficits in the aged. The amino acid is naturally occurring, and earlier work in schizophrenia patients shows that it is well tolerated in humans. Further studies are needed to test the utility of D-alanine supplementation in the elderly.

Since the last submission of this report, the findings from this reward have been tested for their 'translatability'. This award was crucial for establishing the effect and potential underlying mechanisms of prebiotics on cognition in rodents, but these findings are only of societal and economic benefit if they can be applied to the human population. We therefore ran a proof-of-concept study to test if the pro-cognitive effect of the commercially available BGOS prebiotic could be seen in volunteers. Based on the assumption that we would be more likely to see an improvement in cognitive function in the presence of a pre-existing deficit, we chose to recruit people with psychosis because they display cognitive impairment, even when stable and on medication. We confirmed that the enhancement in cognitive performance revealed in rats, was reproduced in humans. We think our findings should now be taken forward by 1) examining the effect of prebiotics on cognitive deficits in normal aging, and 2) exploring other prebiotic formulations which might be more effective in some subjects than others.
Sectors Agriculture, Food and Drink,Communities and Social Services/Policy,Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The basic scientific findings of our research has led us to test the effect prebiotics on emotional processing in healthy human subjects. We have found that a commercially available compound has mild effects on psychological mechanisms that underlie anxiolysis in people. These observations have been accepted for publication, and have been covered by the International media, as well as causing a spike in sales of Bimuno at Boots Ltd in January 2015. We are now testing the compounds on cognitive health in children who may have mild learning difficulties. We hope this will lead eventually to a significant societal impact
First Year Of Impact 2015
Sector Agriculture, Food and Drink,Retail
Impact Types Economic

 
Description Prebiotic effects on the brain 
Organisation Clasado BioSciences
Country Jersey 
Sector Private 
PI Contribution We have demonstrated that dietary supplementation with prebiotics: 1) alters the chemistry and electrical responses of neurons in the rodent brain; 2) improves problem solving abilities of rats, which corresponds to the chemical and electrophysiological changes in the brain; and 3) reduces stressful behaviour in rodents and healthy human subjects. This collaboration has now extended to include investigations into the effects of early-life prebiotics and manipulation of gut bacteria, on adult brain function.
Collaborator Contribution Clasado Ltd, measured fecal gut bacteria numbers in test groups and also provided prebiotic and placebo compounds for all studies. The company also made intellectual contributions to the study (microbiology, immunity, etc)
Impact See specific research outcomes
Start Year 2010
 
Title Prebiotic intervention in schizophrenia to manage weight gain and cognitive impairment 
Description Based on the findings of the BBSRC funded animal work, we are testing if the pro-cognitive effects of the Bimuno prebiotic will improve functioning of schizophrenia patients. In additional studies we have found that the prebiotic also attenuates weight gain in rats that have been administered antipsychotic drugs. We are therefore also assessing weight gain and metabolism in these patients. The study is a proof-of-concept investigation that is funded by a PhD studentship and university funds. 
Type Preventative Intervention - Nutrition and Chemoprevention
Current Stage Of Development Initial development
Year Development Stage Completed 2016
Development Status Under active development/distribution
Impact The first stage showing that nurturing beneficial gut bacteria improves cognitive function has been published. A study of the attenuation of drug-induced weight gain by the same prebiotic is being published. Impact on the development has therefore been on the scientific community so far. 
URL https://clinicaltrials.gov/ct2/show/NCT03153046
 
Description Food on the Brain 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Many questions after talk, followed by a forum with in depth discussions

Requests for me to send original articles and information on the companies that supplied prebiotics.
Year(s) Of Engagement Activity 2014
 
Description Invited speaker at two neuropharmacology conferences (Amsterdam, Ireland), two nutrition conferences (Amsterdam, Rome), and the 1st microbiome and brain conference (Amsterdam) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact My talks led to discussions with Industrial representatives (food biotech) and media, as well as academics
Year(s) Of Engagement Activity 2015,2016
 
Description The pros and cons of cognitive enhancers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact This was a public engagement excercise with 'Science Oxford'. I was invited as a brain expert to visit secondary schools and share scientific knowledge about compounds that increase problem solving abilities. My prebiotic research was also discussed, and the general issue of drug safety was emphasized.

Schools asked for further information
Year(s) Of Engagement Activity 2012
 
Description media engagement (eg newspapers, BBC radio 4) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Led to other requests for interviews and participation in possible future food science TV programme- to be aired in may 2017
Year(s) Of Engagement Activity 2015,2016