Abstract Background Early life stress is a risk factor for many psychiatric disorders ranging from depression to anxiety. Stress, especially during early life, can induce dysbiosis in the gut microbiota, the key modulators of the bidirectional signalling pathways in the gut-brain axis that underline several neurodevelopmental and psychiatric disorders. Despite their critical role in the development and function of the central nervous system, the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs) on the regulation of gut-microbiota in early-life stress has not been explored. Methods and Results Here, we show that long-term supplementation of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) (80% EPA, 20% DHA) n-3 PUFAs mixture could restore the disturbed gut-microbiota composition of maternally separated (MS) female rats. Sprague-Dawley female rats were subjected to an early-life stress, maternal separation procedure from postnatal days 2 to 12. Non-separated (NS) and MS rats were administered saline, EPA/DHA 0.4 g/kg/day or EPA/DHA 1 g/kg/day, respectively. Analysis of the gut microbiota in adult rats revealed that EPA/DHA changes composition in the MS, and to a lesser extent the NS rats, and was associated with attenuation of the corticosterone response to acute stress. Conclusions In conclusion, EPA/DHA intervention alters the gut microbiota composition of both neurodevelopmentally normal and early-life stressed animals. This study offers insights into the interaction between n-3 PUFAs and gut microbes, which may play an important role in advancing our understanding of disorders of mood and cognitive functioning, such as anxiety and depression.

Citation: Pusceddu MM, El Aidy S, Crispie F, O’Sullivan O, Cotter P, Stanton C, et al. (2015) N-3 Polyunsaturated Fatty Acids (PUFAs) Reverse the Impact of Early-Life Stress on the Gut Microbiota. PLoS ONE 10(10): e0139721. https://doi.org/10.1371/journal.pone.0139721 Editor: Judith Homberg, Radboud University Medical Centre, NETHERLANDS Received: July 10, 2015; Accepted: September 16, 2015; Published: October 1, 2015 Copyright: © 2015 Pusceddu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: Research was funded by Food Institutional Research Measure (FIRM) under Grant No. 10/RD/TMFRC/709, the APC Microbiome Institute under Grant No. 07/CE/B1368 and 12/RC/2273, Science Foundation Ireland (SFI) under Grant No. 12/IA/1537. Competing interests: The authors have the following interests. The APC has conducted research funded by Pfizer, GlaxoSmithKline, Proctor and Gamble, Mead Johnson, Suntory and Cremo. TGD also has grant support from the Health Research Board Ireland (HRA_POR/2011/23 HRA_POR/2012/32, and HRA-POR-2014-647) and European FP7 and has been an invited speaker at meetings organised by Servier, Lundbeck, Janssen, Astra-Zeneca. JFC has received grant support from the Health Research Board of Ireland (Grant number HRA_POR/2012/32), Science Foundation Ireland (Investigator Award Grant No. 12/IA/1537), European Community's Seventh Framework Programme (Grant number FP7/2007-2013 under Grant Agreement no. 278948 (TACTICS-Translational Adolescent and Childhood Therapeutic Interventions in Compulsive Syndrome)). JFC has been an invited speaker at meetings organised by Mead Johnson and Yakult. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Introduction Stress, especially in early life has been identified as a cause of the disruption of this developmental pattern leading to a variety of disorders ranging from gastrointestinal disorders [1], to anxiety and depression [2]. In rodents, the maternal separation (MS) model is a well-known paradigm that induces brain-gut axis dysfunction [3]. The separated phenotype alters many components of the brain-gut axis throughout the body including the hypothalamic–pituitary adrenal (HPA) axis [3], the immune and neuroendocrine systems [4]. Growing evidence considers these abnormalities comorbid with changes in the gut microbiota [5–7] as well as crucial risk factors for the development of mental illnesses such as anxiety and depression [8, 9]. There is increasing evidence suggesting a bi-directional communication between the central nervous system (CNS) and the gut-microbiota which is recognized as the microbiome–gut–brain axis [10–12]. This communication is believed to influence the parallel development of both CNS and gut microbiota which can remarkably influence health and disease [13]. Emerging evidence has shown the involvement of the gut microbiota in maternal stress and maternal separation in brain and associated behaviour [14]. Prenatal stress has been shown to change the composition of the microbiome in adult rat [15] neonatal mice [16] and infant humans [17]. Changes in the gut microbiota composition were reported in monkeys subjected to maternal separation between six and nine months of age with shedding of lactobacilli three days following separation, followed by the return of normal lactobacilli levels seven days later. Moreover, we have previously shown, albeit using somewhat crude Denaturing Gradient Gel Electrophoresis- based analysis, that adult rats that underwent maternal separation showed altered faecal microbial composition compared with normally reared control animals [4]. It is well recognized that eating habits are of relevance to (mental) health [18]. Moreover, there is a growing appreciation for the impact of dietary fatty acids on the intestinal microbiota composition of the host [19–21]. Being critical in the development and function of the CNS, n-3 polyunsaturated fatty acids (n-3 PUFAs) have been under the spotlight for decades [22]. The possible underlying mechanisms by which n-3 PUFAs exert their beneficial effects on health are diverse, involving for instance, HPA, neuroendocrine and immune regulations [23, 24]. In light of these observations, we reported recently the beneficial effects of n-3 PUFAs on reduction of anxiety-like, depressive-like behaviours and improved cognition in female rats [25]. Here we hypothesize that these beneficial effects of long-term intake of n-3 PUFAs would have an impact on intestinal microbiota populations, which in turn, would contribute to the reverse of gut-brain axis dysfunction associated with maternal separation. To the best of our knowledge, this is the first study to describe the impact of n-3 PUFAs on the gut-microbiota of female rats exposed to early-life stress.

Materials and Methods Maternal separation Animals were provided by Biological Services Unit (BSU), UCC, Cork, Ireland. All scientific procedures were carried out in line with Directive 2010/63/EU and were approved by the Animal Experimentation Ethics Committee of University College Cork #2012/036. Maternal separation was performed as previously described by our group [25, 26]. Briefly, male and female rats were obtained from Harlan Laboratories UK (250–300 g) and mated in the local animal unit. Food and water was available ad libitum and animals were maintained on a 12:12-h dark–light cycle with temperature at 20 ± 1°C. MS animals were separated from their mothers from postnatal day (PND) 2 to 12, for three hours a day. Separations were conducted between 0900h and 1200h a.m. in plastic cages placed on top of heater pads (30–33°C) in a separate room to the main holding room. Non-separated (NS) animals were left undisturbed in their home cages with their respective dams and were returned to the holding room. After postnatal day 12, rats were left undisturbed except for routine cage cleaning every two days and a weekly body weight measurement until they were 5 weeks old. Animals were group-housed 5 per cage in plastic cages with sawdust bedding in an enriched environment with shredded paper and a cardboard roll. Treatments Oral administration of an eicosapentaenoic acid (EPA)/docosaexaenoic acid (DHA) (80% EPA, 20% DHA) n-3 PUFAs mixture was administered by gavage when animals reached 5 weeks of age. In order to avoid any confounding litter effects, individual groups consisted of rats from multiple litters. Treatments consisted of 1) saline water; 2) EPA/DHA 0.4 g/kg/day or Low Dose (LD); 3) EPA/DHA 1 g/kg/day or High Dose (HD). The chosen EPA/DHA concentrations were based on the Food and Agriculture Organization of the United Nations (FAO) recommendations. FAO recommends a minimum DHA intake of 10–12 mg/kg per body weight for children 6–24 months old and EPA/DHA 100–250 mg/day for children aged 2–10 years. In our study, the maximum EPA/DHA intake was 100 and 250 mg for the low dose and the high dose per body rat, respectively. Moreover, previous studies have used the same concentrations proposed in this study [27, 28]. Treatments were prepared freshly every day and administered between 0900h and 1100h a.m. The experimental time line is shown in Fig 1A. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Schematic representation of the time course of the maternal separation procedure and EPA/DHA treatment. (B) Global average microbial composition of faecal 17 weeks old rats samples (n = 10 per group) at phylum-level. * Indicate bacterial group significantly different between MS and NS groups. https://doi.org/10.1371/journal.pone.0139721.g001 Sample collection Faecal pellets were collected from 17 weeks old female rats. All samples were, then, frozen at −80°C for microbiota analysis. Microbiota analysis DNA was extracted using the DNA Fast Stool DNA extraction kit (Qiagen) using the protocol for Gram positive bacteria and including an additional bead beating step at the beginning of the procedure. DNA was quantified using the Qubit High Sensitivity Kit (Life Technologies), standardised and then used as a template for PCR. PCR primers and conditions are essentially as outlined in the Illumina 16S Metagenomic Sequencing Library preparation guide (Illumina) with the following exceptions: For the initial 16S PCR, the PCR was performed in duplicate 50 ul reaction volumes, and 40 cycles were used in the PCR. Products were then pooled, cleaned with an appropriate volume of Ampure beads and eluted in 30ul/sample. This was then used as the template for the index PCRs as outlined in the protocol (Illumina). Library quantification, normalisation, and pooling were as outlined in the protocol. After pooling, the sample was requantified using the Qubit High Sensitivity Kit (Life Technologies) and run on an agilent high sensitivity chip (Agilent). Library denaturation and MiSeq sample loading were then performed as described in the protocol. The final concentration of the library was 4pM and PhiX was spiked in as a control at 5% v/v. A 2 x 300bp MiSeq reagent was used for sequencing. Diversity analyses were performed in QIIME and correlations used the websites Calypso at http://bioinfo.qimr.edu.au/calypso.

Conclusion We demonstrate what is to our knowledge the first time that EPA/DHA treatment normalized early-life stress-induced disruption of female rat gut microbes. Analysis of the gut microbiota of MS rats showed altered microbial composition with abundance of members previously shown to be associated with inflammation. These results indicate that transient stress-induced alteration during a crucial developmental time-window for neonatal rats has long-lasting effects on the gut microbiota composition in adulthood. Supplementation with EPA/DHA restored the composition of the gut microbiota in MS rats. Presumably, the EPA/DHA effect on the gut microbiota is related to PUFAs anti-inflammatory activity. Recently, it has been shown that omega-3 and omega-6 dramatically reduce the endotoximic and inflammatory status in metabolic endotoxemia (Kaliannan, 2015). Intriguingly, the observed effects involved changes in the gut microbiota, through the impact of omega-6, -3 on intestinal production and secretion of intestinal alikaline phosphatase. The induced alterations in gut microbiota composition resulted in reduction in levels of lipopolysachharides and gut permeability, which in turn reduces the onset of inflammation. Overall, the healthy benefits at CNS level have ascertained the contribution made by n-3 PUFAs to stress-related disorders and put them under the spotlight for decades [46–48]. The current study offers insight into a potential role of n-3 PUFAs through the modification of the gut microbiota in an animal model of stress. Our data supports the previous reports showing that the absence as well as the exacerbation of certain bacterial taxa in the gut of early-life stressed rats may represent risk factors for the development of anxiety, depression and inflammatory diseases such as IBS. We postulate that EPA/DHA administration is beneficial for restoring members of the microbiota with immunoregulatory functions in order to prevent an overly robust stress-induced inflammatory response which may contribute to the onset of mental illnesses.

Supporting Information S1 File. Microbiota Data Set. NS.S, NS.LD, NS.HD stand for non-separated Saline, non-separated Low Dose, non-separated High Dose, respectively. MS.S, MS.LD, MS.HD stand for maternally separated Saline, maternally separated Low Dose, maternally separated High Dose, respectively. https://doi.org/10.1371/journal.pone.0139721.s001 (ZIP)

Acknowledgments Research was funded by Food Institutional Research Measure (FIRM) under Grant No. 10/RD/TMFRC/709, the APC Microbiome Institute under Grant No. 07/CE/B1368 and 12/RC/2273, Science Foundation Ireland (SFI) under Grant No. 12/IA/1537.

Author Contributions Conceived and designed the experiments: MMP JFC TGD. Performed the experiments: MMP FC OO. Analyzed the data: MMP SE. Contributed reagents/materials/analysis tools: PC CS PK JFC TGD. Wrote the paper: MMP SE. Commented on the written manuscript: JFC TGD.