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

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.

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.

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.