Gut microbiota composition inpacts brain chemistry
The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action.
Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion.
We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and adjunctive treatment of neuropsychiatric disorders.
There is now compelling evidence for a link between the enteric microbiota and brain function. The proliferation of the Bifidobacteria and Lactobacilli strains in the large intestine, have anxiolytic and mnemonic effects in both rodents ( Li et al., 2009 and Bravo et al., 2011) and humans ( Messaoudi et al., 2011a,Messaoudi et al., 2011b, Rao et al., 2009 and Cryan and Dinan, 2012). The intake of these bacteria as live cultures (probiotics) alters the expression of genes integral to neurodevelopment and complex behaviours in rodents. For instance, the oral administration of Bifidobacteria to rats elevated hippocampal brain-derived neurotrophic factor (BDNF) ( Bercik et al., 2011a and O’Sullivan et al., 2011), which may underlie some antidepressant actions ( Kerman, 2012). At present, only several probiotics have been examined, but it seems likely that of the 40,000 species in the gut ( Forsythe and Kunze, 2012), there will be others with psychotropic properties. Thus, intuitively, augmenting the growth of intrinsic gut microbiota with prebiotics (nutrients for intestinal bacteria) may afford greater benefits to the brain ( Burnet, 2012).
The prebiotics, fructo-oligosaccharide, (FOS) and galacto-oligosaccharides, (GOS) are soluble fibres which are digested by, and result in the proliferation of, the Lactobacilli and Bifidobacteria in the gut. Increasing the proportion of these bacteria with prebiotics has many beneficial effects on the gut and the immune system (Drakoularakou et al., 2010, van Vlies et al., 2012, Vulevic et al., 2008 and Vulevic et al., 2013), and increase circulating gut peptides such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), which benefit metabolism ( Delmee et al., 2006 and Overduin et al., 2013). However, the central effects of prebiotic administration have not been explored. Interestingly, selective antimicrobials which elevate the levels of intrinsic gut Lactobacilli, also increase brain BDNF concentrations in mice ( Bercik et al., 2011a). It is possible therefore, that prebiotic-mediated microbiota proliferation has similar effects, and the measurement of brain BDNF in rodents administered with these compounds would provide the necessary proof-of-principle. Additional evidence suggests that gut bacteria may also influence glutamate neurotransmission in the brain.
Mice devoid of gut microbiota from birth have reduced levels of N-methyl-d-aspartate receptors (NMDARs), specifically the NR1 and NR2A subunits, in the hippocampus (Sudo et al., 2004), or NR2B subunits in the amygdala (Neufeld et al., 2011 and Kiss et al., 2012). To our knowledge, the effect of increasing gut microbiota on brain NMDARs has not been explored, and such information may have therapeutic relevance (Collingridge et al., 2013). Interestingly, germ-free mice also lack circulating d-alanine, a bona fide NMDAR co-agonist which is rich in bacterial cell walls ( Konno et al., 1993), and their inoculation with bacteria restoredd-alanine concentrations, which were then increased further by an additional administration of aBifidobacteria. It is reasonable to propose, therefore, that an elevation of central d-alanine, and perhaps other amino acids associated with glutamate neurotransmission, would follow prebiotic administration, and thereby present as a strategy to increase brain NMDAR signalling.
The three major aims of this study were to: (1) test if prebiotic administration to rats altered brain levels of BDNF; (2) examine whether central NMDARs and associated amino acids were altered by prebiotics; and (3) provide initial evidence for neuroactive blood-borne molecules that may affect central BDNF levels after prebiotic feeding. We orally administered water, FOS or GOS to rats for five weeks and measured BDNF NR1, NR2A and NR2B subunits in the frontal cortex and hippocampus, and encoding mRNAs in the hippocampus. The concentrations of glutamate, glutamine, and serine and alanine enantiomers in the plasma, cortex and hippocampus were also quantified. Finally, we measured the levels of PYY and GLP-1 in plasma from prebiotic-fed rats, and tested their effect on BDNF release from SH-SY5Y neuroblastoma cells.
Studies have shown that probiotics have psychotropic effects, but the neurobiological consequences of prebiotic intake have not been explored. The aim of the current study was to provide unequivocal evidence for neurochemical and molecular changes in the rat brain following prebiotic consumption, to prelude future functional analyses. The specific hypotheses tested were, first, that Bifidobacteria proliferation by prebiotics is associated with an increase in brain BDNF, as evinced with probiotics; and second, that prebiotic augmentation of commensal microbiota elevates central NMDAR subunits, given that these receptors are reduced in germ-free rodents. Our data support both suppositions and present substantial grounds for exploring the utility of prebiotics in the modulation of brain function. We have also offered an initial indication for the involvement of PYY, and potentially other gut hormones, in the augmentation of BDNF signalling following prebiotic intake. The interpretations and relevance of our findings are discussed in turn.
The elevated expression of BDNF and encoded protein in rats fed with FOS, is consistent with the effect of aBifidobacterium probiotic ( Bercik et al., 2011a and O’Sullivan et al., 2011) and the selective proliferation of these species with antimicrobials ( Bercik et al., 2011a). Thus, FOS administration may have augmented the colonization of similar psychotropic strains, within the moderate overall increase in Bifidobacteria numbers relative to GOS fed rats. In view of these observations therefore, it was surprising that GOS did not alter the levels of hippocampal BDNF protein and, moreover, by a greater magnitude than FOS.
Unaltered BDNF protein after GOS feeding may have reflected the reciprocal change in BDNF mRNA in the dentate gyrus and CA3 region of the hippocampus. If these alterations were translated to protein, then it is unlikely that an overall change in BDNF would be detected in whole hippocampal homogenates. The measure of BDNF in regionally dissected hippocampus would test this possibility. Arguing against a causal link between gut bacterial densities and BDNF gene expression, is a similar increase of BDNF mRNA in the dentate gyrus of both FOS and GOS rats, in spite of the fewer numbers of Bifidobacteria in the FOS group. The possibility that prebiotics alter brain signalling independently of the gut microbiota cannot be ruled out, and a direct interaction between oligosaccharides and the gut mucosa has been shown to influence the response of the immune system ( Bode et al., 2004 and Eiwegger et al., 2010), which may then impact on brain chemistry. The elevation of gut hormones after prebiotic intake might also reflect a direct effect of oligosaccharides on the gut (see below).
The physiological relevance of a reciprocal change in BDNF mRNA in two regions of the hippocampus after GOS intake is difficult to interpret without functional measures. However, an elevation of BDNF gene expression in the dentate gyrus has been associated with antidepressant action (Kerman, 2012). A similar elevation of BDNF mRNA after FOS and GOS administration is, therefore, in keeping with a potential antidepressant/anxiolytic property of gut bacteria (Bercik et al., 2011a). The concomitant decrease of BDNF mRNA in the CA3 is more difficult to interpret, though one possibility is that we were observing the differential, activity-dependent expression of BDNF mRNA splice variants in each hippocampal subfield (Chiaruttini et al., 2008). Regional molecular and electrophysiological analyses of the rat hippocampus after GOS administration are, therefore, required.
The potential mechanisms underlying the elevation of cortical NR1 subunits after GOS feeding remains elusive, and although they correlate with Bifidobacteria numbers (see Section 3), elevated NR1 may have also been mediated by the direct physiological response of the gut to GOS such as the release of PYY (see discussion below). Nevertheless, the significant elevation of hippocampal NR1 mRNA after FOS and GOS administration is intuitive in view of data showing reduced NR1 in mice devoid of gut bacteria ( Neufeld et al., 2011), though it is not clear why a parallel change in hippocampal NR1 protein occurred only after FOS feeding. Given the potential complexities of prebiotic actions already encountered (cf: reciprocal changes of BDNF mRNA abundance in GOS-fed rat hippocampus), unaltered hippocampal NR1 protein in GOS rats may be authentic, and mechanistically associated with the increase of NR2A subunits, which was not apparent after FOS intake. Altered NR1:NR2 subunit ratios in the rodent hippocampus following pharmacological and genetic manipulations are not unusual ( Owczarek et al., 2011 and Kato et al., 2012), and in the latter study, an increase of NR2A, but not NR1, subunits is associated with significant functional outcomes. Similarly, unaltered NR2B subunits after prebiotic administration may suggest that Bifidobacteria and/or the metabolic response to GOS ingestion may not affect the neurophysiological processes, such as long-term depression, which are associated with dynamic changes in NR2B subunits ( Liu et al., 2004). Of course, the lack of functional information to support our interpretations of these data is the major caveat of this study.
The demonstration of elevated faecal and plasma d-alanine concentrations after GOS feeding is consistent with studies showing that gut bacteria, including Bifidobacteria, are a source of this d-amino acid ( Konno et al., 1993). The significant reduction of blood l/d-alanine ratios confirms that this d-alanine was not directly derived from circulating l-alanine. In spite of this, central d-alanine concentrations remained unaltered after prebiotic feeding, and this may be because, higher plasma concentrations are required to initiate the appropriate kinetics for d-alanine uptake into the brain ( Morikawa et al., 2007).
The current investigation also revealed that GOS-fed rats contained greater amounts of cortical d-serine than controls. Since the same animals also showed greater levels of cortical NR1 subunits which are the d-serine binding moiety, it is reasonable to propose that NMDAR mediated signalling was elevated in the frontal cortex. Of course, only direct electrophysiological analysis of NMDAR signalling after GOS feeding can prove this. Of note, the increase of cortical d-serine did not appear to arise from the plasma but may have been synthesized locally, as suggested by a slight, though not significant, rise of cortical l-serine after GOS feeding. That is, the synthesis of d-serine from l-serine by serine racemase (Sikka et al., 2010), may have been a homeostatic response (preservation of l/d-serine ratios) to either local elevations of the l-enantiomer, and/or a greater demand for d-serine. The possibility that d-alanine is linked to an elevation of central d-serine signalling, is suggested by studies demonstrating augmented d-serine release from neurons in the presence of d-alanine (Rosenberg et al., 2010).
We have confirmed that the administration of GOS to rats increases circulating concentrations of PYY (Overduin et al., 2013). It is reasonable to suggest, therefore, that the effects of GOS on brain signalling may be mediated by gut hormones. Earlier work has shown that PYY has the potential to affect the brain either directly (Connor et al., 1997 and Nonaka et al., 2003) or via vagal nerve activity (Hernandez et al., 1994). Ourin vitro data suggest that elevated brain BDNF expression after GOS intake, may have been mediated by plasma PYY. Another study has shown that plasma from mice administered with Bifidobacterium longum, did not change the expression of BDNF mRNA in cultured SH-SY5Y cells ( Bercik et al., 2011b), and this was reasoned to be further evidence of a vagal, rather than a ‘blood-borne’ mediation of probiotic central effects. It is reasonable to assume that our discrepant data reflect the different parameters measured in both studies (i.e. BDNF release versus gene expression) and/or different actions of B. longum and prebiotics on brain BDNF. However, the presence of neuroactive substances in blood does not preclude the involvement of gut-brain vagal circuitry after prebiotic ingestion. The enteric secretion of PYY after GOS intake may directly affect local BDNF signalling in myenteric neurons of the gut ( Boesmans et al., 2008), which innately influence vagal nerve activity ( Murphy and Fox, 2010). Of course, since circulating PYY can also directly access brain PYY receptors ( Hernandez et al., 1994 and Nonaka et al., 2003), in all likelihood, the central effects of PYY would involve direct and indirect mechanisms. However, the mechanisms underlying the increase of hippocampal BDNF after FOS intake remain elusive. Whether PYY or, indeed, other factors such as short-chain fatty acids ( Jakobsdottir et al., 2013), and/or the immune system ( Vulevic et al., 2008 and Vulevic et al., 2013), underlie the changes in BDNF and NMDAR signalling after prebiotics, requires further examination.
Our results have provided the necessary ‘proof-of-principle’ for the central actions of prebiotic consumption. The increase of hippocampal BDNF after prebiotic intake is consistent with a probiotic effect, and may have been a direct consequence of elevated gut Bifidobacteria numbers. However, an additional effect of gut hormones (e.g. PYY) or other mediators, such as the immune system resulting from direct oligosaccharide-gut interactions, cannot be ruled out. The elevation of NMDAR subunits after prebiotics is intuitive given their reduction in the brains of germ-free animals. Furthermore, the strong correlation between Bifidobacteria numbers and cortical NR1 levels presented in this report, further supports a link between the microbiota and central glutamate neurotransmission. Mechanistic investigations beyond the scope of the present study, are now required to ascertain the systems underlying the observed changes, and will also reveal if vagal nerve modulation is involved. Moreover, behavioural analysis in rats will ascertain if the changes in BDNF after prebiotics impart an anxiolytic action, or that increased NMDAR subunits translate to improved cognitive performance. Importantly, our study has provided sufficient cause to warrant further exploration into the utility of prebiotics in therapies of neuropsychiatric illness and which, by virtue of their ability to proliferate gut bacteria and stimulate neuroendocrine (and other) responses, may even prove to be more potent than probiotics.