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

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.

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.

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.