Fetal, neonatal, and infant microbiome: Perturbations

Seminars in Fetal & Neonatal Medicine xxx (2016) 1e8

Contents lists available at ScienceDirect

Seminars in Fetal & Neonatal Medicine journal homepage: www.elsevier.com/locate/siny

Review

Fetal, neonatal, and infant microbiome: Perturbations and subsequent effects on brain development and behavior Rochellys Diaz Heijtz* Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden

s u m m a r y Keywords: Commensal gut microbiota Neurodevelopment Germ-free mice Autism spectrum disorder Cognition Antibiotics Breastfeeding

The human gastrointestinal tract harbors a diverse and complex community of microbes, termed gut microbiota, that normally assemble during the first postnatal years of life. This evolution-driven process has been shown to contribute to the developmental programming of epithelial barrier function, gut homeostasis, and angiogenesis, as well as the development and function of the immune system. Research over the last few years has revealed that the actions of the gut microbiota have much wider effects on host physiology and development than originally believed, including the modulation of brain development and behavior. This article briefly reviews recent findings on the impact of the gut microbiota on brain development, and how disturbances in the assembly and maturation of the gut microbiota may impact development of motor, social, and cognitive functions. The potential link between microbiota and metabolic requirements of the developing brain is also considered. © 2016 Published by Elsevier Ltd.

1. Introduction Human brain development is a protracted process that begins in utero and continues at least through late adolescence [1]. Critically, this process involves more than just a simple unfolding of a genetic blueprint, but rather a complex interaction between genetic and environmental factors. It is now recognized that the epigenome, through which environmental factors regulate gene expression, is responsible for long-lasting programming effects of environmental experiences (e.g. early life stress) on brain and behavior [2]. Over the past several decades, it has become clear that environmental influences during early life may profoundly affect brain development and later life structure and function. One such external environmental factor is the commensal gut microbiota that over evolutionary time has adapted to coexist not merely in a commensal, but rather a mutualistic relationship with mammals [3]. During birth and rapidly thereafter, the newborn is massively colonized with trillions of bacteria. This postnatal microbial colonization process has been shown to contribute to developmental programming of epithelial barrier function, gut homeostasis, and angiogenesis, as well as the development and function of the gut immune system [4]. A growing

number of animal studies have revealed that the gut microbiota has effects on host physiology and development outside the gastrointestinal (GI) system, including the early-life programming of brain circuits involved in the control of stress response, motor activity, anxiety-like behavior and cognitive functions [5e10]. These findings have raised the possibility that perturbations of the developing infant gut microbiota may directly, or indirectly, modify developmental trajectories of the human brain and subsequent function in later life. The discovery of the size and complexity of the human microbiome (see other contributions in this issue of Seminars), in combination with the aforementioned preclinical studies have given way to a paradigm shift in our conceptualization of the origin of human neurodevelopmental and psychiatric disorders [11]. The precise mechanisms mediating the interactions between the gut microbiota and the developing brain remain largely unknown, but are likely to involve multiple direct and indirect pathways [12]. This article begins with a general overview of early postnatal human brain development and the impact of the gut microbiota on key neurodevelopmental processes, followed by a consideration of how some factors that perturb the developing gut microbiota might affect brain development and subsequent behavior. In addition, the potential link between the gut microbiota and metabolic demands of the developing brain is also considered.

* Address: Department of Neuroscience, Karolinska Institutet, Retzius v€ ag 8, SE171 77, Stockholm, Sweden. Tel.: þ46 (08) 524 87886. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.siny.2016.04.012 1744-165X/© 2016 Published by Elsevier Ltd.

Please cite this article in press as: Diaz Heijtz R, Fetal, neonatal, and infant microbiome: Perturbations and subsequent effects on brain development and behavior, Seminars in Fetal & Neonatal Medicine (2016), http://dx.doi.org/10.1016/j.siny.2016.04.012

2

R. Diaz Heijtz / Seminars in Fetal & Neonatal Medicine xxx (2016) 1e8

2. Gut microbiota acts as “environmental agent” shaping brain development The first years of postnatal life represent a time of rapid changes in brain structure and function. The neonate brain grows from about 36% to about 80e90% of its adult volume by the age of 2 years [1,13]. During this period, there is massive outgrowth of dendrites and axons, followed by the formation of new synapses (synaptogenesis), expansion of glia cells, and myelination. Rapid overproduction of synaptic connections continues during the first two postnatal years, peaking between 3 and 24 months depending on the cortical region [14], before synaptic refinement and elimination occur in late childhood continuing beyond adolescence [15]. By the end of the second year, the overall pattern of adult myelination is established [16]. However, myelination continues, at a slower rate, into the second decade of life, with prefrontal regions being among the last brain areas to attain mature levels [17]. Interestingly, the maturation of the gut microbiota also occurs during the first two to three years of postnatal life, coinciding with this critical window of early brain development [18]. The importance of a normal gut microbiota for synaptogenesis is exemplified by our studies using the germ-free (GF) mouse model (devoid of any microbiota throughout development). These mice show higher expression of synaptic-related proteins, i.e. synaptophysin and PSD-95 in the striatum compared to conventionally raised mice (specific-pathogen-free; SPF) [5]. Conventionalization of GF mice early in life normalized the expression levels of these two synaptic-related proteins, suggesting that host microbes modulate synaptic development (either the production of new synapses or the pruning of existing ones). Recent evidence indicates that myelination in the prefrontal cortex can be affected by the commensal gut microbiota. In the absence of microbiota, mice displayed increased levels of several key myelin-associated genes (e.g. myelin basic protein, myelin oligodendrocyte protein, and myelin-associated glycoprotein), and hypermyelination as indicated by a decreased g-ratio (quantification of myelin thickness relative to axonal diameter) [19]. Although further experiments would be required to elucidate how the gut microbiota regulates synaptogenesis and myelination, these results indicate that brain regions important for motor control and cognitive functions are shaped during development by the host microbiota. Interestingly, accelerated brain growth in infancy has been associated with a broad range of developmental delays in motor, language, and cognitive functions [20,21]. For example, neurodevelopmental disorders, including autism spectrum disorder (ASD), have been associated with “atypical brain connectivity patterns” involving higher-order association neocortical regions. Moreover, many children with ASD also suffer from gastrointestinal (GI) problems such as abdominal pain, gases, diarrhea, and constipation. In fact, some studies have found a strong positive association of autism severity with GI dysfunction [22,23]. The causes of ASD-associated GI problems remain unclear, but may be linked to an imbalance of commensal bacteria in the gut, as several studies have reported that children with ASD exhibit altered composition of the gut microbiota [24]. It will therefore be of great interest to investigate the potential link between the “relative degree of microbiota maturity” (both in terms of its composition and metabolic capacity) and the connectome of infants with low and high risk for autism in a multicenter prospective longitudinal study. In parallel, the use of gnotobiotic mouse models could provide a powerful tool to examine the effects of gut microbiota from these infants on underlying cellular and molecular determinants of atypical brain connectivity. Previous studies have demonstrated that microglia, the resident immune cells of the central nervous system (CNS), play an important

role not only during inflammation, but also in shaping neural circuits in the developing brain [25]. In a recent study, Erny et al. discovered that indigenous microbes control microglia maturation and function [26]. In the absence of microbiota, microglia display altered cell proportions and an immature phenotype, and they have a diminished response to bacterial (i.e. lipopolysaccharide) or virus (i.e. lymphocytic choriomeningitis virus) challenges, indicating that a microbiota is required to prime the innate immune system in both the periphery and central CNS. A breakthrough study by Clarke et al. [27] showed that commensal microbiota is a source of peptidoglycan (PGN; a major component of the bacteria cell wall) that systemically primes the innate immune system, enhancing killing by bone marrow-derived neutrophils of pathogens (e.g. Streptococcus pneumoniae and Staphylococcus aureus). The pattern recognition receptor (PRR) nucleotide-binding oligomerization domain-containing protein-1 (Nod1), which recognizes meso-diaminopimelic acid (mesoDAP)-containing PGN found predominantly in Gram-negative bacteria, was identified as the homeostatic regulator mediating the systemic effects of PGN. Interestingly, some Gram-negative bacteria from the Proteobacteria phylum, such as Escherichia and Shigella spp., are among the most abundant microbes during the first year of life [28]. It is therefore tempting to speculate that, during early postnatal development, the meso-DAP-containing PGN found in Escherichia and Shigella spp. may play a role in priming the host immune system, and the long-term programming of brain and behavior. In accordance with this hypothesis, we recently reported that during critical windows of development, PGN-derived from the commensal gut microbiota can be translocated into the developing brain, and sensed by specific PRRs of the innate immune system [29]. These findings underscore the need to further study and characterize the influence that endogenous microbial-derived products such as PGN may have on the developing brain. 3. Potential prenatalematernal exchange of microbiota in utero Until recently, the traditional view has been that the intrauterine environment of a healthy pregnancy is free of any bacteria. Recent studies, however, have challenged the idea of a sterile intrauterine environment by demonstrating the presence of bacterial DNA in the amniotic fluid, umbilical cord blood, meconium, placenta and fetal membranes from healthy pregnancies without any indication of infections or inflammation (for review, see [30]). For example, Satokari et al. found the presence of DNA derived from the commensal intestinal bacteria in full-term placenta from healthy pregnant women collected after elective cesarean section (C-section) without rupture of membranes [31]. Moreover, Aagaard et al. have demonstrated that the human placenta harbors a unique, low-abundance, but metabolically rich microbiome that is composed of commensal bacteria from the Firmicutes, Tenericutes, Proteobacteria, Bacteriodetes, and Fusobacteria phyla. The placenta microbiome, which resembles the oral microbiome, appears to be highly sensitive to the maternal health status, as its composition varies in association with maternal history of antenatal infection, antibiotic exposure, and preterm birth [30]. Consistent with these findings, previous studies have shown that oral inoculation of pregnant BALB/c mice with a genetically modified Enterococcus faecium strain resulted in a transfer of this strain to the amniotic fluid [32]. Although there is still some skepticism regarding some aspects of the human placenta microbiome concept, and about the origin and viability of the bacteria found in the intrauterine environment [33], the aforementioned findings do raise the intriguing possibility that transfer of maternal microbiota to the fetus may occur in utero, thereby initiating the colonization of the fetal gut. The potential role of the placenta microbiome on the developing fetal brain needs further investigation.



To date, the best experimental evidence for an influence of gut microbiota on fetal brain development comes from a recent study in GF mice by Braniste et al. investigating the development of the bloodebrain barrier (BBB) [34]. The BBB, which ensures an optimal microenvironment for neural development, begins to develop early in fetal life and continues to mature during the early postnatal stages of life. These investigators found that GF mice, beginning in fetal life and continuing into adulthood, showed increased BBB permeability compared to SPF mice with a normal gut microbiota. The increased BBB permeability was associated with reduced expression of tight junction proteins (occludin and claudin-5) known to regulate barrier function in endothelial tissue. During prenatal development, the developing fetus receives all nutrients from the maternal circulation, and therefore it is tempting to speculate that the metabolic output of the maternal gut microbiota might include compounds that can cross the placenta barrier and influence fetal brain development (see Section 5). 4. Factors influencing the assembly of the neonate gut microbiome: implications from brain development and behavior 4.1. Mode of delivery One critical factor that affects the natural assembly of the neonate gut microbiota is the mode of delivery [35]. In a seminal study, Dominguez-Bello et al. clearly demonstrated that the delivery mode has a marked influence on the acquisition and structure of the bacterial community in the infant gut [36]. Whereas vaginally delivered infants harbor bacterial communities resembling their own mother's vaginal microbiota (e.g. Lactobacillus, Prevotella, Senathia spp.), infants delivered by C-section (without membrane rupture) miss this normal source of bacterial colonization. Instead, their intestines are colonized by microbiota derived from their mother's skin (e.g. Staphylococcus, Corynebacterium, and Propionibacterium spp.) and other environmental sources including health-care workers and hospital environment. Infants delivered by C-section show decreased gut microbiota diversity and delayed colonization of Bacteroides and Bifidobacterium spp. These infants are also more often colonized with Clostridium difficile compared with vaginally delivered infants. Importantly, alterations in the microbial composition introduced by the delivery mode have been shown to persist well beyond infancy, and some studies have detected minor differences in specific bacteria species even after 7 years of age (for review, see [37]). However, the implications of this delay or aberrant colonization of the infant gut with regard to human brain development and function are not yet fully known. A recent systemic review and meta-analysis of the impact of mode of delivery on ASD and attention deficit hyperactivity disorder (ADHD) reported that birth by C-section is associated with a 23% increased risk of ASD, and perhaps a small increased risk of ADHD (albeit there were limited numbers of studies available for this meta-analysis) [38]. Recently, a large Swedish population-based cohort and sibling design study (with 2.6 million births) found birth by elective C-section to be associated with a 20% increased risk of ASD, which is comparable with the aforementioned meta-analysis [39]. However, the association disappeared when using sibling controls, indicating that the association between C-section and ASD is due to unmeasured familial influences (genetic and/ or environmental). Consistent with these findings, a recent cohort study from the UK (including ~13,000 children) found no association between planned C-section and ASD or ADHD [40]. Other research has shown an association between elective C-section and developmental delay in social and gross motor domains at 9 months of age, but not at 3 years [41], suggesting a possible link between C-section

3

and transient delays in motor and cognitive development. Interestingly, a recent study reported differences in spatial attention between C-section and vaginally delivered infants. Infants delivered by elective C-section (without rupture of membranes) exhibited slower initiation of saccades driven by visual stimulus spatial properties compared to vaginally delivered infants [42]. Although the sample size was limited, the results suggest that the mode of delivery may have an impact on early stages of brain functioning. A larger sample size will be needed to confirm these findings and to determine whether restoration of the infant microbiota via vaginal microbial transfer immediately after birth, as recently shown [43], could normalize deficits in attention processes of infants born by C-section. In animal models, the mesolimbic and mesostriatal dopaminergic pathways appear to be particularly vulnerable to a wide range of perinatal perturbations, including delivery by C-section (for review, see [44]). For example, rats born by C-section show persistent blunting of stress-induced dopamine release in the right prefrontal cortex and increased D1 receptor expression in the ventral striatum. Similarly, GF mice also show alterations in the dopaminergic system (e.g. increased dopamine turnover in the striatum and alterations in the expression of D1 receptors both in striatum and hippocampus regions) [5], thus suggesting that the developing dopamine system is especially sensitive to early perturbations of the gut microbiota. Recently, it was reported that in comparison to vaginally delivered mouse pups, animals delivered via C-section displayed a number of behavioral abnormalities, including increased anxiety-like behavior, social deficits, and repetitive behaviors, which have been previously associated with ASD [45]. Whereas these rodent models highlight the influence that mode of delivery has on brain development and behavior, we also need to keep in mind that the clinical situation is more complex, and that animal models do not completely mimic all aspects of Csection birth (e.g. pups delivered by C-section are also crossfostered to another dam). 4.2. Exposure to antibiotics in early life Mounting evidence indicates that antibiotic exposure early in life disrupts the development of neonatal intestinal microbiota (for review, see [33,37]). For instance, intrapartum antibiotic prophylaxis (e.g. penicillin or amoxicillin) for the prevention of perinatal group B streptococcal disease has been associated with reduced diversity and lower abundance of lactobacilli and bifidobacteria in the neonatal gut. The laboratory of Martin Blaser has elegantly demonstrated that early life-disruption of the gut microbiota by antibiotics may lead to long-lasting alterations in the composition and metabolic activity of the gut microbiota, resulting in adult-onset obesity (for review, see [46]). These findings illustrate that even transient perturbations of the gut microbiota, during critical time windows of development, can have long-lasting effects on the host. Surprisingly, there has been a relative paucity of information regarding the consequences of antibiotic treatment during prenatal or early postnatal life on brain development and subsequent function and behavior; this is probably due to the fact that these drugs have long been considered safe. In a recent study, we demonstrated that oral administration of a low-dose antibiotic (ampicillin) to pregnant C57BL/6 mice during the 3-week pregnancy leads to an increase in motor activity in both male and female offspring, and altered social behavior, but only in the male offspring [47]. It is worthwhile to stress that this low-antibiotic regimen produced no changes in the body weight of the dams or of their offspring. On the other hand, a recent study reported that treatment with a cocktail of oral antibiotics (neomycin, bacitracin, and pirimacin) to C57BL/6 pregnant mice during E9eE16 produced a transient reduction in locomotor activity of their offspring during


4


the prepubertal period, which was associated with a decrease in the relative abundance of the bacteria order Lactobacillales [48]. In contrast to our findings, this cocktail of antibiotics produced a reduction in body weight in both the dams and their prepubertal offspring. Interestingly, other investigators have found that neonatal antibiotic treatment (vancomycin from postnatal days 4e13) selectively affects visceral pain in adulthood without impacting cognitive or anxiety-related behaviors in male rats [49]. Taken together, these findings clearly illustrate that the impact of antibiotics on brain development and behavior are highly dependent upon factors such as the dose regimen used and the developmental time window when such exposure(s) occurred. In light of evidence that the vaginal [30] and maternal gut microbiota [50] undergo extensive remodeling during pregnancy, antibiotic exposure could perturb the maternal gut microbiota and may thereby influence the developing neonatal microbiome (via vertical mothereneonate transfer) and subsequent “early-life” microbiomeebrain interactions.

4.3. Breastfeeding vs formula After birth, breastfeeding is another important source of bacterial exchange between the mother and infant that promotes the colonization and maturation of the infant microbiome (for review, see [51]). Breast milk contains bacteria (e.g. Staphylococcus and Streptococcus spp.) and a number of bioactive compounds, such as human milk oligosaccharides, that stimulate the growth of beneficial microbial communities, including Bifidobacterium spp. However, feeding practices (e.g. introduction of infant formula) may perturb the developing gut microbiota, and may thereby attenuate the beneficial health effects of exclusive breastfeeding (for review, see [37]). For example, the gut microbiota of formula-fed infants is more diverse, and contains higher proportions of Clostridium difficile and Escherichia coli than that of breastfed infants. Results from a recent population-based cohort study indicate that breastfeeding, especially when it is prolonged (at least 4e6 months), is associated with better cognitive development of the newborn ([52] and references therein). Importantly, the effect is more striking for preterm-born infants, who have an increased risk for behavioral problems and cognitive impairments later in life [52]. However, a large nationally representative survey of US children (including 37,900 children) concluded that, although breastfeeding may support cognitive development more generally, there does not appear to be a specific contribution of breastfeeding to the prevention of ASD [53]. A new and exciting study examined how breastfeeding and genetic variation contribute to emerging social behaviors in 7month-old infants [54]. These authors investigated individual differences in infant attention to social eye cues (i.e. fearful, angry, or happy eyes) in the context of genetic variation in the ectoenzyme Cluster of Differentiation 38 (CD38 rs3796863 polymorphism; which has been previously implicated in social behavior and oxytocin release), and the duration of exclusive breastfeeding. The attention of infants to angry vs happy eyes was found to vary as a function of exclusive breastfeeding experience and genetic variation in CD38. Infants who had had extended durations of exclusive breastfeeding showed increased attention to happy eyes and decreased attention to angry eyes, but this was more pronounced in infants with a genotype previously associated with decreased availability of oxytocin and an increased rate of autism (i.e. homozygous for the C allele of rs3796863). These findings indicate that extended breastfeeding may enhance socio-emotional development, particularly in infants at high risk for social dysfunction. Although this study did not consider the influence of infant gut microbiota per se, this research points to early emerging,

genetically predisposed social skills in humans that may be shaped by mothereinfant bacteria exchange in early postnatal life. 4.4. Prenatal stress One form of early life adversity that has received increasing recognition with regard to the assembly of the neonate microbiome is “maternal psychological stress” during pregnancy or prenatal stress. A significant number of pregnant women suffer from various forms of psychological disturbances during pregnancy, including maternal depression and anxiety [55]. Epidemiological studies indicate that children of prenatally stressed mothers show more impulsivity, anxiety problems, and worse cognitive and psychological development [56], and have increased risk for neurodevelopmental disorders such as ADHD, and ASD [57]. These findings are supported by experimental animal studies demonstrating that exposure to prenatal stress can alter fetal brain development resulting in reprogramming of the hypothalamicepituitaryeadrenal axis, cognitive deficits, and behavioral abnormalities later in life (e.g. anxiety and depressive-like behavior) [58]. The molecular pathways that could mediate the causal relationship between prenatal stress (or glucocorticoid overexposure) and development of neurobehavioral problems in children/adolescents remains poorly understood. It has been postulated that the microbiome could be the missing link between maternal stress and neurodevelopmental programming of the offspring [58]. Bailey et al. demonstrated that maternal stress during pregnancy (especially if the stress was late in pregnancy) altered bacterial colonization of the gut in infant monkeys [59]. Infants of stressed mothers had lower levels of bifidobacteria and lactobacilli compared to the control non-stressed group. A recent study from The Netherlands showed that maternal stress during pregnancy (i.e. either reported stress or elevated basal cortisol concentrations, or both) was associated with a different gut microbiota of the infants [60]. Similar to the studies in monkeys, infants of prenatally stressed mothers had lower relative abundances of lactobacilli and bifidobacteria. In an elegant study, Jasarevic et al. (2015) identified the vagina microbiota as an important target by which maternal stress during pregnancy may contribute to reprogramming of the developing brain, and thereby increase the risk for neurodevelopmental disorders [61]. Using a well-established mouse model of prenatal stress, these investigators demonstrated that maternal stress early in pregnancy altered protein content of the maternal vaginal mucosa environment and abundance of lactobacillus (the prominent taxa in the maternal vagina). Moreover, loss of maternal vaginal lactobacillus also resulted in decreased vertical transmission to offspring. Importantly, this altered microbiota composition in the neonate gut corresponded with region- and sex-specific changes in amino acid profiles in the developing brain, in accordance with changes observed in the gut and plasma metabolites. These results clearly demonstrated that aberrant composition of the early colonizing microbiota imposed by an environmental factor, such as maternal stress during pregnancy, might exert a profound impact on the metabolism of the developing brain (see Section 5). In addition to the aforementioned factors that affect the assembly of the infant microbiome (Fig. 1), other factors such as maternal health status (e.g. obesity, diabetes, eczema) during pregnancy have a paramount influence on the infant gut microbiome [33]. Moreover, lifestyle differences may have a pronounced influence on the infant and neonate microbiome. For example, a study across Europe revealed that the geographical origin had a more pronounced influence on the infant microbiota than delivery mode, breastfeeding, and antibiotics [63].



5

Fig. 1. Factors influencing the assembly of the infant gut microbiota. Widely performed perinatal interventions such as mode of delivery, infant feeding practices, and antibiotic usage affect the assembly of the infant gut microbiota, as well as the environment. Other factors such as maternal stress during pregnancy, gestational age, genetics, and infections (pre and/or postnatal) also influence the microbial composition of the infant gut microbiota [62].

5. Linking the gut microbiota to the metabolic requirements of the developing brain The human brain is probably the most complex biological system, comprising of about 100 billion neurons, along with a similar number of glial cells. Equally remarkable, the neocortex (the top layer of the cerebral hemispheres), which is involved in higher cognitive functions such as the generation of motor commands and language, contains about 164 trillion synapses (points of communication between neurons). This impressive cellular organization of the brain, however, comes at a high metabolic cost to the body. The brain utilizes about 18e20% of the body's energy, yet accounts for only 2% of the body weight. The metabolic demands are even greater during development of the brain. A recent neuroimaging study, using combined PET and MRI datasets to calculate brain glucose demands from birth to adulthood, showed that the rate of glucose uptake in the brain does not peak at birth, but rather in childhood when synaptic densities are maximal and glucose used is ~66% of the body’s resting metabolic rate [64]. This uptake reflects the additional energetic costs (e.g. use of glucose in aerobic

glycolysis) associated with exuberant production of connections throughout the brain during early childhood. Many metabolic functions of the developing gut microbiota (e.g. metabolism of vitamins, iron, amino acids, and energy transfer from the diet) are also required for normal brain development. A healthy gut microbiota is therefore needed to support the metabolic demands of the brain during critical windows of development [65,66]. In addition, metabolic by-products of the commensal gut microbiota might directly affect the long-term programming of brain circuitries involved in the regulation of energy balance, motor control, anxiety, and cognition. The importance of gut microbiota in host metabolism is clearly exemplified in studies with GF mice. These animals excrete higher levels of calories and have less fat mass than their colonized counterparts, despite consuming more food [66]. However, these effects are not universal, and might be influenced by other factors such as host genetic and diet composition [67]. One key function of the gut microbiota is to ferment nondigestible carbohydrates (dietary fibers and resistant starch) into metabolic by-products e such as short chain fatty acids (SCFAs)


6


including acetate, propionate and butyrate e providing a significant energy source from the diet to the host. Recently, SCFAs have raised much interest, as they exert many beneficial effects on host physiology (e.g. enhancing the integrity of gut epithelial barriers, protection against inflammation, and regulation of gut hormones from enteroendocrine cells) (for review, see [66]). SCFAs are typically incorporated into energy stores via lipogenesis or gluconeogenesis, but they may also enter the blood circulation. In a recent mouse positron emission tomography/computed tomography study, it was demonstrated that acetate could cross the BBB and directly suppress appetite through central hypothalamic mechanisms [68]. Pioneering work by Derrick MacFabe and collaborators suggest that the developing brain is sensitive to levels of SCFAs (for review, see [69]). For example, overexposure to the enteric metabolite, propionic acid, during midelate gestation induces delays to reach milestones (e.g. eye-opening, reflexes) and increased anxiety-like behavior in adolescent rats in a sexually dimorphic manner. Moreover, these authors have also demonstrated that SCFAs may regulate the expression of synaptic-related genes. Furthermore, elevated concentrations of SCFAs have been found in stool samples from children with ASD [70]. As SCFAs serve a number of key functions, such as modulating host chromatin structure and gene transcription, exposure to these enteric metabolites during critical windows may have important implications for early-life programming of the brain. Although the gut microbiota has been shown to modulate earlylife programming of the brain and behavior, the specific contribution of microbiome-derived metabolites such as SCFAs in this process remains largely unknown. Some studies have demonstrated that the SCFA butyrate can restore the integrity of the BBB [34] and that a mix of SCFAs can also restore microglia malformation and immaturity in adult GF mice [26], indicating that SCFAs may be one of the key signaling pathways by which indigenous gut microbes modulate brain function in adulthood. However, we still need to determine whether SCFAs are part of the signaling mechanisms involved in the early-life long-term programming of the brain and behavior during critical time-windows of development. In our initial study from 2011, we conducted genome-wideexpression profiling studies in various brain regions of GF mice, and discovered four key canonical pathways (i.e. citrate cycle, synaptic long-term potentiation, C21-steroid hormone metabolism, and cAMP-mediated signaling) that were strongly regulated by gut microbiota [5]. Among them, the citrate cycle, also called the Krebs cycle or tricarboxylic acid (TCA; a central metabolic pathway) cycle, was found to be the most significantly regulated pathway. As mentioned above, the brain is a high-energy demanding organ, and alterations in the TCA cycle would profoundly alter the rate of brain metabolism and production of free radicals, and consequently fundamental aspects of brain function. Moreover, the peripheral metabolic phenotype of adult GF mice includes alterations in plasma levels of glucose and other metabolites readily used to generate ATP via the TCA cycle, and it is therefore plausible that these metabolic changes may contribute to the behavioral phenotype of GF mice [66]. In addition, mitochondrial dysfunction has also been implicated in the pathophysiology of several neurodevelopmental disorders, including ASD [69]. Taken together, these observations underscore the need to further study and characterize the influence that gut microbiota have on mitochondrial function and metabolism across different stages of brain development.

development. Besides common perinatal interventions (e.g. Csection, antibiotic use, and formula feeding) that affect the development of the gut microbiota, recent animal studies have identified maternal stress during pregnancy as a key factor that can shape the vaginal microbiome, and subsequently affect vertical mothereneonate transfer of maternal gut bacteria. The functional role of the newly discovered placenta microbiome on prenatal brain development remains to be clarified. The striking increase in brain growth observed during the first two years of life, occurring in parallel with the infant microbiome attaining a more complex structure similar to that of adults, indicates that this is not only a “critical time-window” of developmental vulnerability, but also a period of opportunity in which therapeutic interventions (e.g. prebiotics, probiotics, and synbiotics) may have their maximal effects on neural circuits. Consequently, it is critical to elucidate the mechanisms mediating the early-life gut microbiomeebrain interactions to develop novel microbiota-modulating-based therapeutic interventions for infants and young children at risk for neurodevelopmental disorders.

Practice points Commensal gut microbiota modulates brain development and behavior. Maternal stress during pregnancy alters the vagina microbiome and, thereby, colonization of the neonate gut. Widespread perinatal practices (e.g. C-section, antibiotic exposure, and infant formula) may influence early stages of brain functioning.

Research directions Elucidate the cellular and molecular mechanisms mediating the interactions between the gut microbiota and the developing brain. Determine the role of the placenta microbiome on prenatal brain development. Establish international metrics for relative microbiota maturity in infants/children and microbiota-for-age Zscore. Acknowledgments Work from my laboratory cited in this review was supported by grants from the Swedish Research Council (K2015-62X-22745-01-4), Swedish Brain Foundation (FO2014-0083), and the Strategic Research Program in Neuroscience at Karolinska Institutet. Conflict of interest statement None declared. Funding sources None.

6. Concluding remarks It is becoming clear that perturbations to the assembly of the neonatal microbiome can influence neurodevelopment and may lead to atypical motor, socialeemotional and cognitive

References [1] Tau GZ, Peterson BS. Normal development of brain circuits. Neuropsychopharmacology 2010;35:147e68.


R. Diaz Heijtz / Seminars in Fetal & Neonatal Medicine xxx (2016) 1e8 [2] Bale TL. Epigenetic and transgenerational reprogramming of brain development. Nat Rev Neurosci 2015;16:332e44. [3] Sampson TR, Mazmanian SK. Control of brain development, function, and behavior by the microbiome. Cell Host Microbe 2015;17:565e76. [4] Hooper LV. Bacterial contributions to mammalian gut development. Trends Microbiol 2004;12:129e34. [5] Diaz Heijtz R, Wang S, Anuar F, Qian Y, Bjorkholm B, Samuelsson A, et al. Normal gut microbiota modulates brain development and behavior. Proc Natl Acad Sci U S A 2011;108(7):3047e52. [6] Clarke G, Grenham S, Scully P, Fitzgerald P, Moloney RD, Shanahan F, et al. The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner. Mol Psychiatry 2013;18(6):666e73. [7] Neufeld KM, Kang N, Bienenstock J, Foster JA. Reduced anxiety-like behavior and central neurochemical change in germ-free mice. Neurogastroenterol Motil 2011;23:255e64. e119. [8] Sudo N, Chida Y, Aiba Y, Sonoda J, Oyama N, Yu XN, et al. Postnatal microbial colonization programs the hypothalamic-pituitary-adrenal system for stress response in mice. J Physiol 2004;558(Pt 1):263e75. [9] Desbonnet L, Clarke G, Shanahan F, Dinan TG, Cryan JF. Microbiota is essential for social development in the mouse. Mol Psychiatry 2014;19:146e8. [10] Arentsen T, Raith H, Qian Y, Forssberg H, Diaz Heijtz R. Host microbiota modulates development of social preference in mice. Microb Ecol Health Dis 2015;26:29719. [11] Mayer EA, Knight R, Mazmanian SK, Cryan JF, Tillisch K. Gut microbes and the brain: paradigm shift in neuroscience. J Neurosci 2014;34:15490e6. [12] Cryan JF, Dinan TG. Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nat Rev Neurosci 2012;13:701e12. [13] Knickmeyer RC, Gouttard S, Kang C, Evans D, Wilber K, Smith JK, et al. A structural MRI study of human brain development from birth to 2 years. J Neurosci 2008;28:12176e82. [14] Huttenlocher PR, Dabholkar AS. Regional differences in synaptogenesis in human cerebral cortex. J Comp Neurol 1997;387:167e78. [15] Petanjek Z, Judas M, Kostovic I, Uylings HB. Lifespan alterations of basal dendritic trees of pyramidal neurons in the human prefrontal cortex: a layerspecific pattern. Cereb Cortex 2008;18:915e29. [16] Sampaio RC, Truwit CL. Myelination in the developing human brain. In: Nelson CA, Luciana M, editors. Handbook of developmental cognitive neuroscience. Cambridge, MA: MIT Press; 2001. p. 35e44. [17] Giedd JN. Structural magnetic resonance imaging of the adolescent brain. Ann NY Acad Sci 2004;1021:77e85. [18] Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et al. Human gut microbiome viewed across age and geography. Nature 2012;486(7402):222e7. [19] Hoban AE, Stilling RM, Ryan FJ, Shanahan F, Dinan TG, Claesson MJ, et al. Regulation of prefrontal cortex myelination by the microbiota. Transl Psychiatry 2016;6:e774. [20] Courchesne E, Karns CM, Davis HR, Ziccardi R, Carper RA, Tigue ZD, et al. Unusual brain growth patterns in early life in patients with autistic disorder: an MRI study. Neurology 2001;57(2):245e54. [21] Hazlett HC, Poe M, Gerig G, Smith RG, Provenzale J, Ross A, et al. Magnetic resonance imaging and head circumference study of brain size in autism: birth through age 2 years. Arch Gen Psychiatry 2005;62(12):1366e76. [22] Adams JB, Johansen LJ, Powell LD, Quig D, Rubin RA. Gastrointestinal flora and gastrointestinal status in children with autism e comparisons to typical children and correlation with autism severity. BMC Gastroenterology 2011;11:22. [23] Wang LW, Tancredi DJ, Thomas DW. The prevalence of gastrointestinal problems in children across the United States with autism spectrum disorders from families with multiple affected members. J Dev Behav Pediatr 2011;32:351e60. [24] Krajmalnik-Brown R, Lozupone C, Kang DW, Adams JB. Gut bacteria in children with autism spectrum disorders: challenges and promise of studying how a complex community influences a complex disease. Microb Ecol Health Dis 2015;26:26914. [25] Schafer DP, Stevens B. Microglia function in central nervous system development and plasticity. Cold Spring Harbor Perspect Biol 2015;7:a020545. [26] Erny D, Hrabe de Angelis AL, Jaitin D, Wieghofer P, Staszewski O, David E, et al. Host microbiota constantly control maturation and function of microglia in the CNS. Nat Neurosci 2015;18(7):965e77. [27] Clarke TB, Davis KM, Lysenko ES, Zhou AY, Yu Y, Weiser JN. Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity. Nat Med 2010;16:228e31. [28] Backhed F, Roswall J, Peng Y, Feng Q, Jia H, Kovatcheva-Datchary P, et al. Dynamics and stabilization of the human gut microbiome during the first year of life. Cell Host Microbe 2015;17(6):852. [29] Arentsen T, Femenia T, Gkotzis S, Udekwu K, Forssberg H, Diaz Heijtz R. Can the developing brain sense bacterial peptidoglycan from commensal gut microbiota? Society for Neuroscience 2014. [30] Prince AL, Chu DM, Seferovic MD, Antony KM, Ma J, Aagaard KM. The perinatal microbiome and pregnancy: moving beyond the vaginal microbiome. Cold Spring Harb Perspect Med 2015;5(6). pii: a023051. [31] Satokari R, Gronroos T, Laitinen K, Salminen S, Isolauri E. Bifidobacterium and Lactobacillus DNA in the human placenta. Lett Appl Microbiol 2009;48:8e12. [32] Jimenez E, Fernandez L, Marin ML, Martin R, Odriozola JM, Nueno-Palop C, et al. Isolation of commensal bacteria from umbilical cord blood of healthy neonates born by cesarean section. Curr Microbiol 2005;51(4):270e4. [33] Mueller NT, Bakacs E, Combellick J, Grigoryan Z, Dominguez-Bello MG. The infant microbiome development: mom matters. Trends Mol Med 2015;21:109e17.

7

[34] Braniste V, Al-Asmakh M, Kowal C, Anuar F, Abbaspour A, Toth M, et al. The gut microbiota influences blood-brain barrier permeability in mice. Sci Transl Med 2014;6(263):263ra158. [35] Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, et al. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 2006;118(2):511e21. [36] Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 2010;107(26):11971e5. [37] van Best N, Hornef MW, Savelkoul PH, Penders J. On the origin of species: factors shaping the establishment of infant's gut microbiota. Birth Defects Res C Embryo Today 2015;105:240e51. [38] Curran EA, O'Neill SM, Cryan JF, Kenny LC, Dinan TG, Khashan AS, et al. Research review: birth by caesarean section and development of autism spectrum disorder and attention-deficit/hyperactivity disorder: a systematic review and meta-analysis. J Child Psychol Psychiatry 2015;56(5):500e8. [39] Curran EA, Dalman C, Kearney PM, Kenny LC, Cryan JF, Dinan TG, et al. Association between obstetric mode of delivery and autism spectrum disorder: a population-based sibling design study. JAMA Psychiatry 2015;72(9):935e42. [40] Curran EA, Cryan JF, Kenny LC, Dinan TG, Kearney PM, Khashan AS. Obstetrical mode of delivery and childhood behavior and psychological development in a British cohort. J Autism Dev Disord 2016;46:603e14. [41] Al Khalaf SY, O'Neill SM, O'Keeffe LM, Henriksen TB, Kenny LC, Cryan JF, et al. The impact of obstetric mode of delivery on childhood behavior. Soc Psychiatry Psychiatr Epidemiol 2015;50(10):1557e67. [42] Adler SA, Wong-Kee-You AM. Differential attentional responding in caesarean versus vaginally delivered infants. Atten Percept Psychophys 2015;77:2529e39. [43] Dominguez-Bello MG, De Jesus-Laboy KM, Shen N, Cox LM, Amir A, Gonzalez A, et al. Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer. Nat Med 2016;22(3):250e3. [44] Boksa P, El-Khodor BF. Birth insult interacts with stress at adulthood to alter dopaminergic function in animal models: possible implications for schizophrenia and other disorders. Neurosci Biobehav Rev 2003;27(1e2):91e101. [45] Borre YE, Golubeva AV, Crispie F, Scott KA, Hyland NP, Stanton C, et al. Mode of delivery at birth and behavioural outcomes: rewiring of the brainegutmicrobiome axis?. Society for Neuroscience (SFN) Neuroscience 2014. 2014. [46] Cox LM, Blaser MJ. Antibiotics in early life and obesity. Nat Rev Endocrinol 2015;11:182e90. [47] Arentsen T, Raith H, Forssberg H, Diaz Heijtz R. Prenatal antibiotic exposure alters brain development and behavior: implications for neurodevelopmental disorders. Society for Neuroscience 2015. [48] Tochitani S, Ikeno T, Ito T, Sakurai A, Yamauchi T, Matsuzaki H. Administration of non-absorbable antibiotics to pregnant mice to perturb the maternal gut microbiota is associated with alterations in offspring behavior. PLoS One 2016;11:e0138293. [49] O'Mahony SM, Felice VD, Nally K, Savignac HM, Claesson MJ, Scully P, et al. Disturbance of the gut microbiota in early-life selectively affects visceral pain in adulthood without impacting cognitive or anxiety-related behaviors in male rats. Neuroscience 2014;277:885e901. [50] Koren O, Goodrich JK, Cullender TC, Spor A, Laitinen K, Backhed HK, et al. Host remodeling of the gut microbiome and metabolic changes during pregnancy. Cell 2012;150(3):470e80. [51] O'Sullivan A, Farver M, Smilowitz JT. The influence of early infant-feeding practices on the intestinal microbiome and body composition in infants. Nutr Metab Insights 2015;8(Suppl. 1):1e9. [52] Quigley MA, Hockley C, Carson C, Kelly Y, Renfrew MJ, Sacker A. Breastfeeding is associated with improved child cognitive development: a population-based cohort study. J Pediatr 2012;160:25e32. [53] Husk JS, Keim SA. Breastfeeding and autism spectrum disorder in the national survey of children's health. Epidemiology 2015;26:451e7. [54] Krol KM, Monakhov M, Lai PS, Ebstein RP, Grossmann T. Genetic variation in CD38 and breastfeeding experience interact to impact infants’ attention to social eye cues. Proc Natl Acad Sci USA 2015;112:E5434e42. [55] Bennett HA, Einarson A, Taddio A, Koren G, Einarson TR. Depression during pregnancy: overview of clinical factors. Clin Drug Invest 2004;24:157e79. [56] Beydoun H, Saftlas AF. Physical and mental health outcomes of prenatal maternal stress in human and animal studies: a review of recent evidence. Paediatr Perinat Epidemiol 2008;22:438e66. [57] Boersma GJ, Bale TL, Casanello P, Lara HE, Lucion AB, Suchecki D, et al. Longterm impact of early life events on physiology and behaviour. J Neuroendocrinol 2014;26(9):587e602. [58] O'Mahony SM, Clarke G, Dinan TG, Cryan JF. Early-life adversity and brain development: is the microbiome a missing piece of the puzzle? Neuroscience 2015 Oct 1 [Epub ahead of print]. [59] Bailey MT, Lubach GR, Coe CL. Prenatal stress alters bacterial colonization of the gut in infant monkeys. J Pediatr Gastroenterol Nutr 2004;38:414e21. [60] Zijlmans MA, Korpela K, Riksen-Walraven JM, de Vos WM, de Weerth C. Maternal prenatal stress is associated with the infant intestinal microbiota. Psychoneuroendocrinology 2015;53:233e45. [61] Jasarevic E, Howerton CL, Howard CD, Bale TL. Alterations in the vaginal microbiome by maternal stress are associated with metabolic reprogramming of the offspring gut and brain. Endocrinology 2015;156:3265e76. [62] Borre YE, O'Keeffe GW, Clarke G, Stanton C, Dinan TG, Cryan JF. Microbiota and neurodevelopmental windows: implications for brain disorders. Trends Mol Med 2014;20:509e18.


8


[63] Fallani M, Young D, Scott J, Norin E, Amarri S, Adam R, et al. Intestinal microbiota of 6-week-old infants across Europe: geographic influence beyond delivery mode, breast-feeding, and antibiotics. J Pediatr Gastroenterol Nutr 2010;51(1):77e84. [64] Kuzawa CW, Chugani HT, Grossman LI, Lipovich L, Muzik O, Hof PR, et al. Metabolic costs and evolutionary implications of human brain development. Proc Natl Acad Sci U S A 2014;111(36):13010e5. [65] Goyal MS, Venkatesh S, Milbrandt J, Gordon JI, Raichle ME. Feeding the brain and nurturing the mind: linking nutrition and the gut microbiota to brain development. Proc Natl Acad Sci USA 2015;112:14105e12. [66] Selkrig J, Wong P, Zhang X, Pettersson S. Metabolic tinkering by the gut microbiome: implications for brain development and function. Gut Microbes 2014;5:369e80.

[67] Fleissner CK, Huebel N, Abd El-Bary MM, Loh G, Klaus S, Blaut M. Absence of intestinal microbiota does not protect mice from diet-induced obesity. Br J Nutr 2010;104:919e29. [68] Frost G, Sleeth ML, Sahuri-Arisoylu M, Lizarbe B, Cerdan S, Brody L, et al. The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism. Nat Commun 2014;5:3611. [69] MacFabe DF. Enteric short-chain fatty acids: microbial messengers of metabolism, mitochondria, and mind: implications in autism spectrum disorders. Microb Ecol Health Dis 2015;26:28177. [70] Wang L, Christophersen CT, Sorich MJ, Gerber JP, Angley MT, Conlon MA. Elevated fecal short chain fatty acid and ammonia concentrations in children with autism spectrum disorder. Dig Dis Sci 2012;57:2096e102.
