The effect of early-life prebiotic feeding on adult rat hippocampal function, central and peripheral metabonomics and microbial metagenomics

Lead Research Organisation: University of Oxford
Department Name: Psychiatry

Abstract

Scientists have shown that growing good bacteria in our guts can improve some brain functions, such as memory. We have found that when rats eat a compound called BGOS, good gut bacteria grow and there is an increase in the levels of two molecules, called the NMDA receptor and BDNF, in a brain area called the hippocampus. These molecules and the hippocampus are very important for memory function. We have also found that rat pups that eat BGOS shortly after they are born, contain higher levels of NMDA receptors and BDNF in their hippocampus when they are adults compared to animals that did not eat BGOS when they were young. We therefore, wonder whether the changes in NMDA receptors and BDNF we see in the older brain after BGOS at a younger age, actually change the function of the brain. We are also interested in whether BGOS feeding in rat pups changes the chemical processes, called metabolism, in the brain and body when they get older. Finally, we would like to see if rats being fed BGOS at a young age, contain different types of gut bacteria when they get older, compared to rats that were not given BGOS.
Rat pups will be fed with BGOS when they are 3 days old, and then once every day until they 21 days old. While they are given BGOS, the pups will be allowed to stay with their mothers and breast-feed. Other rats will be given a sugar compound (placebo) which is like BGOS but does not grow gut bacteria. When all the rats are 22 days old, they will be put into different cages, given normal adult food and water, and allowed to grow. We will then do four different experiments:
1) When some rats are 22, 56, 112 or 196 days old, they will be humanely killed and their brains removed. The hippocampus of rats at each age that were fed BGOS or placebo, will be taken out for electrophysiology. This is a technique that measures the electrical activity in brain tissue generated when neurotransmitters travel between brain cells. Scientists have shown that changes in the levels of NMDA receptors and BDNF change electrical activity in the hippocampus and affects memory. We therefore, predict that BGOS feeding increases hippocampus electrical activity compared to the hippocampus from placebo fed animals.
2) Rats that had been fed BGOS or placebo at a young age, will be subjected to memory tests when they are 22, 56, 112 or 196 days old. In these tests, the animals will be put into mazes where they have to learn and remember how to get a food reward. The hippocampus is important for this test, and scientists have shown that changes in the levels of NMDA receptors and BDNF in the hippocampus can change the activity of this brain area and how well a rat can remember. We predict that rats that had been fed BGOS will do the memory tasks better than placebo fed animals at all ages.
3) We will look at the concentration of molecules that are made from metabolism, in the brain, liver, blood and urine of all rats at all ages. The approach we will use is called metabonomics, which is a fast way to look at thousands of molecules in one sample, and hundreds of samples at the same time, in order to detect changes that tells us about metabolism.
4) We will use a method called metagenomics to measure the different type of bacteria, and their amounts in the droppings of all rats at all ages. This approach is a bit like metabonomics, except that thousands of bacteria, instead of metabolic molecules, can be identified and counted in one sample, and hundreds of samples can be processed together. This experiment will show us if taking BGOS at an early age will make good gut bacteria continue grow as the rat gets older.
Overall, the study will tell us if BGOS feeding from an early age improves memory, metabolism and the growth of good gut bacteria as the body and brain get older. If it does, then growing good bacteria with BGOS in children might make them more healthy in life and resistant to some diseases.

Technical Summary

The influence of the intestinal microbiota on adult brain function has been convincingly demonstrated in rodents and humans. Nurturing the growth of the beneficial gut bacteria with probiotics and prebiotics alters brain chemistry, and improves cognitive performance in the healthy host. There are also some suggestions that the early-life establishment of gut microbial communities influences brain development. Therefore, supporting the proliferation of the commensal microbiota in early-life, may ensure healthy brain development and reduced susceptibility to the age-related, progressive decline in executive brain functions.
In pilot studies, we have observed that the administration of the prebiotic BGOS (Bimuno, galacto-oligosaccharides) to neonatal rats from post-natal day (PD) 3-21, increases the levels of hippocampal glutamate NMDAR receptor, NR2A subunits, Brain Derived Neurotrophic Factor (BDNF), and the synaptic protein, synaptophysin, at PD22, 56 and 112, relative to controls. These proteins are crucial for normal brain development and function, and our data suggest that early-life BGOS feeding may improve adult brain performance. Early-life dietary supplementation with prebiotics may also influence the microbiota-dependent metabolic processes in later-life.
Our proposed research will: 1) test if feeding neonatal rats with BGOS alters glutamate receptor dependent excitatory post-synaptic potentials in the hippocampus at PD22, 56, 112 and 196; 2) evaluate hippocampus-dependent spatial memory in adult rats at the aforementioned ages following neonatal BGOS supplementation; 3) examine the metabolic profile of rats at all ages after early-life BGOS feeding, using metabonomic technology; and 4) apply metagenomic technology to monitor gut microbial communities in the aging rat following neonatal BGOS intake. Overall, the current proposal will provide new insights into how early-life gut microbiota influences adult microbial colonization and host physiology.

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 maintaining brain and metabolic health from an early age, in addition to their more widely accepted role on digestive and immune well-being. Thus, if we show in our study that neonatal prebiotic administration prevents an age-related decline in cognitive behaviour, then it may be commercialised as an active ingredient which may be incorporated into other foods, including infant milk formulations.
Understanding more about how the gut bacteria may modulate normal brain function in early-life and through the life-course, 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, many natural foods including, asparagus, oatmeal and legumes, contain high amounts of prebiotics. 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 BGOS) is a commercially available product. 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 microbiology, metagenomic and metabonomic technologies 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 two 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 their 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 We have fed sucking rat pups with a prebiotic or a 'placebo', daily for 3 weeks until they were weaned (post-natal day [PD] 22). We then allowed rats to grow without any further manipulation. At PD23, 56 128 and 196, we evaluated their emotional and cognitive (learning, memory, problem solving) behaviours using standard test mazes. We found that at PD23, rats that had received the prebiotic in early-life were less anxious than those that had been fed on the placebo. However, these animals did not show any improvements in cognition. When we tested the other ages, we did not see the same effect of the prebiotic as we did see at PD23. This showed that the anxiolytic action of the prebiotic only occurred when it was being ingested by the rat, and so did not change the actual development of the brain. This finding is important for 2 reasons: 1) it demonstrates that, in spite of the suckling rat pups receiving natural prebiotics from the mothers milk, nurturing the good bacteria with an additional prebiotic still improves brain function, suggesting that the effects of the young gut microbiome on the brain is not saturating. 2) the effect is reversible and only occurs during prebiotic intake. This means that long term changes in brain architecture after prebiotics do not seem possible, and therefore lowers the risk of adverse effects occurring from sustained prebiotic supplementation in early-life. the impact these findings have on human health and well being are discussed below. There have also been unexpected results from this study. The first is the lack of changes in cognitive behaviours. This is surprising because we have found that early-life prebiotic feeding increases molecules (glutamate receptors) in the brain that are crucial for cognitive function. However, we have detected changes in emotional behaviours measured on the elevated plus maze at PD23, 128 and 196, with a trend at PD56. These data suggest that improved emotional behaviour after early-life prebiotic supplementation is sustained in adulthood. The increase in glutamate receptors after neonatal prebiotic feeding were confirmed at the later ages (PD 56, 128, 196), we do not see a change in the function of their function. Surprisingly, metabolomics and metagenomics have not yielded any data that could generate hypotheses about the potential mechanisms that underlie the observed molecular and specific behavioural changes. All data are currently being consolidated and finalised.
Exploitation Route We have investigated the effect of the prebiotic on reading and learning in school children. Our previous BBSRC funded work has shown procognitive effects of this prebiotic, but that effect was mediated by the prefrontal cortex of the brain, not the hippocampus which we have been studying in our current rat pup supplementation project. Furthermore, our early-life study in rats indicates an anxiolytic action of the prebiotic which we have not seen in adult animals. Some children have difficulties in learning because they may be anxious at school, so anxiety was also assessed in our study. The study is now completed, but no effect of the prebiotic on cognition and emotional behaviour in school children was observed. However, we can not rule out the involvement of gut bacteria on neurodevelopment since the age group of the children was not equivalent to the neonatal stage of development when our data in rodents was accrued. Future studies in humans, therefore, should focus on infants which has societal relevance given that prebiotics are included in infant formula milk.
Sectors Agriculture, Food and Drink,Communities and Social Services/Policy,Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Metabolomics (Dr Jonathan Swann) 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution In the current grant we will prepare tissues to transport to London and investigate the concentration of metabolites in central and peripheral areas of adult rats following early-life prebiotic administration.
Collaborator Contribution Will be analysing samples in Year 3 of grant.
Impact No outcomes yet
Start Year 2015
 
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
 
Description metagenomics (Prof Philip Poole, Plant Sciences) 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We will extract faecal DNA from adults rats which have been neonatally fed with a prebiotic. This will then be used by our Plant Sciences collaborators to run metagenomics.
Collaborator Contribution Microbial DNA will be analysed by this collaborator in Year2 of the grant.
Impact no outputs yet
Start Year 2015
 
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
 
Title Prebiotic supplementation in school children 
Description Based on previous findings from BBSRC funded animal work, and the preliminary findings from the current award, we are testing if the pro-cognitive effects of the Bimuno prebiotic will improve reading and learning in healthy school children. This is, in essence, a translational extension of the present BBSRC funded project in that it is testing if early-life prebiotic supplementation has beneficial effects on neurodevelopment. In addition, given the huge positive response from the general public to the "The Truth about Sleep" programme where the presenter's sleep improved following prebiotic intake (see Engagement section), we are measuring sleep in these children using actigraphy. We hope to publish the results of the study by the end of the year. 
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 demonstration of the pro-cognitive actions of the prebiotic, and its long-term effects on the expression of brain genes when administered in early life, are the basis of this experimental medicine study in children. The findings in rats have been published and at the moment target the scientific community. 
URL https://clinicaltrials.gov/ct2/show/NCT02926508
 
Description European Researchers Night (curiosity carnival, Oxford Natural History Museum) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact We had a stall called 'Bugs on the Brain' in the Oxford Natural History Museum where visitors participated in a computer-based quiz to learn facts about gut bacteria, and how we can feed the 'good' ones through high fibre diets. We also presented and discussed our work that shows that modulating beneficial microbes with prebiotics improves psychological process of the brain. We also discussed how the current BBSRC grant is looking into how gut bacteria might influence neurodevelopment. At the end of their visit, participants could sample a small amount of prebiotic powder. This was especially popular with the children. The main comments were that some participants wanted to learn more about diet and the brain, whilst other thought they would review their dietary habits.
Year(s) Of Engagement Activity 2017
 
Description How food and gut bacteria can affect our brain (Oxford University Museum of Natural History) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Invited to speak to the general public about research on the gut bacteria and brain, including evidence that diet can affect mood and cognition through intestinal microbes.
Year(s) Of Engagement Activity 2019
URL https://oumnh.web.ox.ac.uk/event/the-gut-brain-axis-and-how-what-we-eat-affects-how-we-feel
 
Description Interview for BBC Radio 4 series on bacteria in human evolution and development 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This was a radio interview for a four-part science program on how bacteria may have/and continue to influence human development. I was approached in recognition of our publications on prebiotics and brain health and neurodevelopment The discussion were focussed on how gut bacteria might affect brain development, and how different we would be in a germ-free environment, based on animal work.
Year(s) Of Engagement Activity 2018
URL https://www.bbc.co.uk/programmes/b09zxl63
 
Description Interview for BBC1 television science program. 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact The program was an investigation into sleep and how it can be enriched. The presenter, Dr Michael Mosley, read that prebiotics improved sleep in rats but wanted to test if the same was true for humans. My study of prebiotics in healthy volunteers, which was performed based on data from BBSRC funded animal work, was familiar to the presenter through earlier media coverage (guardian newspaper etc) and publication searches. An experiment was therefore designed to test if a short course of a prebiotic improved Dr Mosley's sleep, which was measured by actigraphy. At the beginning of the experiment, when in repose, Dr Mosley spent about 25% of his time awake (1.6hrs awake, 4.9hrs asleep), but after a daily ingestion of prebiotic for one week, this improved to only 10% awake (40mins awake, 6hrs asleep). Since this program I have been receiving a lot of emails about the study and more anecdotal evidence for prebiotics improving sleep. I have been invited to conferences and institutions to give presentations on our work on gut bacteria and the brain, in some cases the TV appearance has been instrumental.
Year(s) Of Engagement Activity 2017
URL https://www.bimuno.com/prebiotics-and-sleep
 
Description Interview for a health magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact An article was written about 'psychobiotics' and brain health, and I was featured as one of the leading experts in the field together with Prof John Cryan, University of Cork, Ireland. The interviewer stated that the article was commissioned in response to the general public expressing a lot of interest in the field of microbiome-gut-brain axis research, and the potential to maintain and/or improve brain health with prebiotic and probiotic supplements. However, the interviewer also expressed an interest on the value of prebiotic supplementation in early-life and how it could affect brain development. She was aware of my current BBSRC funding, which I confirmed and explained the research plan, but I did not divulge current data. I mentioned that we hope to publish something in this area sometime this year. It was clear from the proceeding questioning that early-life prebiotic supplementation was also a topic of general interest, and I was made aware of the reasons eg anecdotes of colleagues' behaviours, anxieties and poor learning performance at school.
Year(s) Of Engagement Activity 2017,2018
 
Description Interview for national news (Guardian newspaper) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact This was an interview about our work on gut bacteria modulation of brain function, and how this might be relevant to the treatment of mood disorders. There is more and more interest in this area and the general public are interested if supplements such as prebiotics alone can treat mental illness. My view was that this is not the case, but diet can improve the efficacy of psychotropic against for those who do not fully respond to them.
Year(s) Of Engagement Activity 2017
URL https://www.theguardian.com/lifeandstyle/2017/nov/06/microbiome-gut-health-digestive-system-genes-ha...