2′-Fucosyllactose Alleviates Early Weaning-Induced Anxiety-Like Behavior, Amygdala Hyperactivity, and Gut Microbiota Changes

Abstract

Early life stress has a significant impact on development of the central nervous system (CNS), with lasting rather than transient consequences; therefore, it is important to alleviate these effects. In recent years, functional communication between the CNS and gut microbiota through the so-called brain-gut-microbiota axis has been examined, and it is likely that prebiotics contribute to development of the CNS through the gut microbiota. In this study, we performed behavioral, neurohistological, and fecal microbiota analyses in early-weaned mice to examine the effects of 2′-fucosyllactose (2′-FL), a human milk oligosaccharide, on anxiety induced by early life stress. Mice weaned at 17 d old (17-d mice) showed anxiety-like behaviors, such as decreased time in the open arms in the elevated plus maze test, compared to mice weaned at 24 d old (24-d mice). The number of cells that were positive for the neuronal activity marker c-Fos in the amygdala was also higher in 17-d mice. The behavioral and neural abnormalities caused by early weaning were alleviated by post-weaning ingestion of 2′-FL. The composition of the fecal microbiota differed among control diet-fed 24-d and 17-d mice, and 2′-FL diet-fed 17-d mice. These findings indicate that human milk oligosaccharides 2′-FL alleviate early stress-induced anxiety, amygdala hyperactivity, and gut microbiota changes.

INTRODUCTION

Experiences in early life are widely known to have profound influences on development of the central nervous system (CNS) and can result in permanent rather than transient consequences for the individual. Indeed, childhood sexual or physical abuse and neglect are some of the most serious causes of neuropsychiatric disorders in adults.^1,2) Studies on rodents have suggested several possible mechanisms through which early life stress can lead to long-term vulnerability to stress and change behaviors in adulthood. Daily maternal separation from the second day of life for 2 to 3 weeks causes anxiety-like behaviors^3,4); hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis, a hormonal response system to stress^3,4); and altered expression of brain-derived neurotrophic factor (BDNF) in adulthood.⁵⁾ Early weaning leads to anxiety-like and aggressive behaviors,⁶⁾ hyperactivity of the HPA axis,⁷⁾ and downregulation of BDNF expression.⁸⁾ Thus, early life stress has long-term adverse effects on the CNS and it is important to alleviate these effects.

In recent years, evidence has accumulated for the role of the brain–gut–microbiota axis as a bidirectional and functional communication system between the CNS and gut bacteria. Germ-free mice have an exaggerated HPA axis response to stress, which is reversed by colonizing the gut with a Bifidobacterium species.⁹⁾ Rats subjected to maternal separation early in life have perturbation in the gut microbiota¹⁰⁾ and treatment with Lactobacillus sp. normalizes elevated basal glucocorticoid levels in these rats.¹¹⁾ These studies suggest that perturbation of the gut microbiota by early life stress may contribute to development of an exaggerated response to stress and abnormal behaviors, and that normalization of the gut microbiota by pre- or probiotics may alleviate the adverse effects of early life stress.

Human milk oligosaccharides are the major components of human milk and have various biological functions in infants, including in the CNS and microbiota. 2′-Fucosyllactose (2′-FL) is a neutral trisaccharide composed of L-fucose, D-galactose and D-glucose, and the most prevalent human milk oligosaccharide.¹²⁾ Animal studies have shown effects of 2′-FL on the CNS, including enhancing long-term potentiation of the hippocampus,¹³⁾ improvement of cognitive function,¹³⁾ and reduction of symptoms of stroke in adults.¹⁴⁾ Thus, these studies suggest that 2′-FL may promote normal development of the CNS in childhood.

In the current study, to clarify the effects of 2′-FL on early life stress-induced anxiety in adulthood, we investigated anxiety-like behavior in the elevated plus maze (EPM) test, the number of cells for a neuronal activity marker c-Fos, and fecal microbiota in early-weaned mice after treatment with 2′-FL.

MATERIALS AND METHODS

Animals

Experimental procedures concerning the use of animals were approved by the Committee for Ethical Use of Experimental Animals of Setsunan University and conducted according to the Guide for the Care and Use of Laboratory Animals. C57BL/6J mice at 15 d old with their dams were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). 2′-FL was obtained from Kyowa Hakko Bio Co., Ltd. (Tokyo, Japan). Some litters were separated from the dam at 17 d old (17-d mice) and all others were weaned at 24 d old (24-d mice). Mice weaned at 17 d old were assigned to groups fed a control diet (AIN-93G, Funabashi Farm, Chiba, Japan) (17-d/control) or a diet containing 2′-FL (AIN-93G containing 5% 2′-FL) (17-d/2′-FL) after weaning. Mice weaned at 24 d old were fed a control diet (24-d/control) from 17 d old. In some experiments, 17-d mice were fed a 2′-FL-containing diet between 17–24 d old (17-d/2′-FL_17–24), after 24 d old (17-d/2′-FL_24-) or 24 h before the behavioral tests (17-d/2′-FL_acute), and the control diet during the rest of the day. Mice were randomly assigned to these treatments. Weaned mice were housed in a same-sex and same-treatment group of 4 or 5 mice in a wire-topped opaque polypropylene cage (24 × 17 × 12 cm). At 7 weeks old, male mice were used in experiments. All mice were reared under controlled environmental conditions (23 ± 1 °C; 12 : 12-h light-dark cycle, humidity 55%, food and water ad libitum).

Measurement of Locomotor Activity

A mouse was put into a new cage (30 × 30 × 30 cm) and spontaneous locomotor activity for 30 min was measured with ANY-maze video tracking software (Stoelting, Wood Dale, IL, U.S.A.).

Elevated Plus Maze Test

An apparatus with two enclosed arms (27.5 × 6 cm, with a 15 cm high wall) and two open arms (26.5 × 6 cm) was elevated 40 cm from the ground. A mouse was put on the center and then allowed to explore freely for 3 min. Time spent in the open arms was measured with ANY-maze video tracking software (Stoelting).

Immunohistochemistry

For c-Fos staining, 2 h after the EPM test, mice were deeply anesthetized with isoflurane and perfused transcardially with saline, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). Serial 50-µm coronal sections were prepared from post-fixed brains using a microslicer (DTK-1000, Dosaka EM, Kyoto, Japan). Three consecutive coronal sections including the prefrontal cortex (PFC: 2.0 to 1.8 mm anterior to the bregma), bed nucleus of the stria terminalis (BNST: 0.3 to 0.1 mm anterior to the bregma), paraventricular nucleus of the hypothalamus (PVN: −0.8 to −1.0 mm anterior to the bregma) and amygdala (−1.5 to −1.7 mm anterior to the bregma) were prepared. Free-floating sections were preincubated in 0.3% hydrogen peroxide in PBS for 40 min and then incubated in 1% bovine serum albumin (BSA) containing 0.3% Triton-X 100 in PBS (PBS-T) for 1 h. Then, the sections were incubated with an anti-c-Fos primary antibody (Table 1) at 4 °C overnight, followed by incubation with a biotinylated anti-rabbit immunoglobulin G (IgG) secondary antibody (Table 1) at room temperature for 2 h. Subsequently, the sections were incubated with avidin-biotin-horseradish peroxidase complex using a Vectastain ABC kit (Vector Laboratories) at room temperature for 90 min. Brown cytosolic products were obtained by reaction with 3,3′-diaminobenzidine (Sigma, St. Louis, MO, U.S.A.). The c-Fos-positive nuclei were counted by experienced observers blinded to the groups of mice, using a microscope (IX71, Olympus, Tokyo, Japan) with a CCD camera (VB-7010, Keyence, Osaka, Japan) and calculated per 1 mm². The mean of three consecutive sections was used as data for each mouse.

Table 1. List of Antibodies

Antigen	Dilution	Host	Conjugate	Source	Identifier
c-Fos	1 : 10000	Rabbit		Abcam, Cambridge, MA, U.S.A.	Cat#: PC38; RRID: AB_2106755
Rabbit IgG	1 : 200	Goat	Biotinylated	Vector Laboratories, Burlingame, CA, U.S.A.	Cat#: BA-1000; RRID: AB_2313606

Fecal Microbiota Analysis

Fresh feces were collected from each mouse and stored at –80 °C. Bacterial DNA was extracted from the fecal samples using a QIAamp^® DNA Stool Mini Kit (Qiagen, Valencia, CA, U.S.A.). The V4 region of 16S ribosomal RNA (rRNA) was amplified using the primers (forward: 515F, reverse: 806rcbc33–52) and TaKaRa Ex Taq (TaKaRa Bio, Shiga, Japan). The amplicon was purified using a QIAquick PCR Purification Kit (Qiagen). The size and quality of the pooled libraries were ascertained using MultiNA (Shimadzu Corp., Kyoto, Japan). Following NaOH denaturation, libraries were loaded into a MiSeq cartridge and sequenced on a MiSeqIII instrument (Illumina, San Diego, CA, U.S.A.).

Sequence Data Analysis

Sequence data were analyzed using QIIME2 (ver. 2020.8; https://qiime2.org/). Briefly, the sequence was first denoised using the DADA2 plugin. The Sklearn classifier was used for taxonomic assignment against the Greengenes database (13_8; 99% OTUs full-length sequence). Singletons and amplicon sequence variants (ASVs) assigned to mitochondria and chloroplasts were removed.

Statistical Analysis

All data are plotted and expressed as mean ± standard error of the mean (S.E.M.), except for the microbiota analysis. In the microbiota analysis, data are plotted by Principal Coordinate Analysis (PCoA), and the mean of the ratio of each phylum is shown as a stacked bar graph. Data for Figs. 1 and 2 were analyzed by Student t test. Data in Figs. 3 and 4 were analyzed using one-way ANOVA followed by a Tukey–Kramer post-hoc test. Data in Tables 2, and 3 were analyzed by two-way ANOVA for treatment as intersubject factors and repeated measures with age as the intrasubject factor. Data in Figs. 5A and B were analyzed using permutational multivariate ANOVA (PERMANOVA). Data in Fig. 5C and Table 4 were analyzed using a Steel–Dwass test. All analyses were performed using JMP Pro 15 (SAS Institute, Cary, NC, U.S.A.) or QIIME2. A value of p < 0.05 was considered to be significant.

Fig. 1. Effects of Early Weaning on Body Weight Gain, Spontaneous Locomotor Activity and Anxiety-Like Behavior in Adulthood

(A) Body weight gain of 24- and 17-d mice. (B) Spontaneous locomotor activity of 24- and 17-d mice in a novel field. (C) Time spent in the open arms by 24- and 17-d mice in the elevated plus maze test. Values are expressed as the mean ± S.E.M. of 8 mice. * p < 0.05 vs. 24-d mice.

Fig. 2. Effects of Early Weaning on the Number of c-Fos-Positive Cells in the Elevated Plus Maze (EPM) Test

(A) Schematic illustrations of brain sections selected for immunohistochemistry. The anteroposterior coordinate (distance from bregma) is shown below each brain section (Mouse brain atlas²⁹⁾ with modifications). (B) Representative photomicrographs showing c-Fos-positive cells in the prefrontal cortex (PFC), dorsal and ventral bed nucleus of the stria terminalis (dBNST and vBNST), paraventricular nucleus of the hypothalamus (PVN) and basolateral, central and medial amygdala (BLA, CeA and MeA) of 24- and 17-d mice 2 h after the EPM test. (C) Numbers of c-Fos-positive cells. Values are expressed as the mean ± S.E.M. of 8 mice. ** p < 0.01 vs. 24-d mice. Scale bar = 100 µm.

Fig. 3. Effects of 2′-Fucosyllactose (2′-FL) on Behavior and Neuronal Activity in the Amygdala

(A) Experimental schedule. Mice were randomly assigned to 3 groups: 24-d/control, 17-d/control, and 17-d/2′-FL. The 24-d/control mice were weaned at 24 d old (d.o.), and mice in the other groups were weaned at 17 d old. The 24-d/control and 17-d/control mice were fed a control diet from 17 d until the end of the study, and 17-d/2′-FL mice were fed a diet containing 5% 2′-fucosyllactose (2′-FL) over the same period. Behavioral and immunohistochemical analyses were performed at 7 weeks old (w.o.). (B, C) Effects of 2′-fucosyllactose (2′-FL) on spontaneous locomotor activity (B) and anxiety-like behavior (C). (D) Representative photomicrographs showing c-Fos-positive cells in the basolateral, central, and medial amygdala (BLA, CeA, and MeA) of each group in the home cage or 2 h after the elevated plus maze (EPM) test. (E) Numbers of c-Fos-positive cells. Values are expressed as the mean ± S.E.M. of 8 (B, E) and 12 (C) mice. * p < 0.05, ** p < 0.01 vs. 24-d/control mice, ^† p < 0.05, ^†† p < 0.01 vs. 17-d/control mice, ^## p < 0.01 vs. home cage. Scale bar = 100 µm.

Fig. 4. Differences in the Effects of 2′-FL on Behavior Depending on the Period of Ingestion

(A) Experimental schedule. Mice were randomly assigned to 4 groups: 24-d/control, 17-d/control, 17-d/2′-FL_17–24, and 17-d/2′-FL_24-. The 24-d/control mice were weaned at 24 d old (d.o.), and mice in the other groups were weaned at 17 d old. The 24-d/control and 17-d/control mice were fed a control diet from 17 d until the end of the study. The 17-d/2′-FL_17–24 mice and 17-d/2′-FL_24- mice were fed a diet containing 5% 2′-FL from 17 to 24 d and after 24 d, respectively. (B) As a behavioral test to evaluate anxiety-like behavior, the elevated plus maze (EPM) test was performed at 7 weeks old (w.o.). (C) Experimental schedule. Mice were randomly assigned to 3 groups: 24-d/control, 17-d/control, 17-d/2′-FL_acute. The 17-d/2′-FL_acute mice were fed a diet containing 5% 2′-FL for 24 h before the behavioral test. (D) The EPM test was performed at 7 weeks old (w.o.). Values are expressed as the mean ± S.E.M. of 12 mice. * p < 0.05, ** p < 0.01 vs. 24-d/control mice.

Table 2. Age and Body Weights of Mice Weaned at 24 and 17 d

Age (d)	Body weight (g)
	24-d	17-d
24	9.67 ± 0.19	9.69 ± 0.14
31	15.38 ± 0.47	15.38 ± 0.45
38	20.29 ± 0.44	20.55 ± 0.23
45	23.18 ± 0.29	23.54 ± 0.26
52	24.99 ± 0.58	25.25 ± 0.33

Table 3. Age and Body Weights of Mice Weaned at 24 and 17 d and Fed a Control or 2′-FL Diet

Age (d)	Body weight (g)
	24-d/control	17-d/control	17-d/2′-FL
24	10.25 ± 0.36	10.56 ± 0.24	10.29 ± 0.29
31	16.02 ± 0.24	16.03 ± 0.27	15.71 ± 0.31
38	20.87 ± 0.26	20.67 ± 0.31	20.71 ± 0.35
45	23.33 ± 0.23	23.04 ± 0.28	23.28 ± 0.34
52	24.36 ± 0.41	24.3 ± 0.34	24.72 ± 0.41

Fig. 5. Effects of 2′-FL on the Microbiota

PCoA plots of the fecal microbiota are shown based on weighted (A) and unweighted (B) UniFrac distances. Ellipses enclosing clusters indicate the 95% confidence interval. Values are expressed as data for each of 12 mice (A, B) or as mean ± S.E.M. (C). * p < 0.05 (*q < 0.05 for A, B), ** p < 0.01 (**q < 0.01 for A, B) vs. 24-d/control mice, ^† p < 0.05, ^†† p < 0.01 (^††q < 0.01 for A, B), ^††† p < 0.001 vs. 17-d/control mice.

Table 4. Chao1 and Shannon Indexes for Mice Weaned at 24 and 17 d and Fed a Control or 2′-FL Diet

Index	24-d/control	17-d/control	17-d/2′-FL
Chao1	234.44 ± 25.57 (218.5)	155.42 ± 11.46 (142.5)*	143.92 ± 14.54 (137.5)*
Shannon	4.01 ± 0.31 (3.9)	2.77 ± 0.26 (2.62)*	3.77 ± 0.13 (3.73)†

Data are expressed as the mean ± standard error of the mean with the median in parentheses. * p < 0.05 vs. 24-d/control, ^† p < 0.05 vs. 17-d/control (Steel–Dwass test).

RESULTS

Early Weaning Induces Anxiety-Like Behavior and Increased Number of c-Fos-Positive Cells in the Amygdala

Early weaning did not affect weight gain or spontaneous locomotor activity (Table 2, Fig. 1A). To investigate changes in anxiety-like behavior and neuronal activity due to early weaning, EPM test and immunohistochemical analysis of brain regions related to anxiety were performed in 24- and 17-d mice. The time spent in the open arms in the EPM test was significantly lower for 17-d mice compared with 24-d mice (Fig. 1B). There was a significant increase in c-Fos-positive cells in the basolateral and central amygdala (BLA and CeA) in 17-d mice compared with 24-d mice 2 h after the EPM test, but no differences in the PFC, BNST, PVN, and medial amygdala (MeA) between the groups (Figs. 2A–C). The 24- and 17-d mice also had no differences in mRNA levels for BDNF, glucocorticoid receptors, and mineralocorticoid receptors in the PFC and hippocampus, nor in basal serum corticosterone levels (Supplementary Figs. S1, S2).

2′-FL Alleviates Early Weaning-Induced Anxiety-Like Behavior and Increased Number of c-Fos-Positive Cells in the BLA and CeA

Since anxiety-like behavior and increased number of c-Fos-positive cells in the BLA and CeA were observed in early weaned mice, we examined whether 2′-FL alleviates these abnormalities (Fig. 3A). Ingestion of 2′-FL did not affect weight gain or spontaneous locomotor activity (Table 3, Fig. 3B). The 17-d/2′-FL mice spent significantly more time in the open arms in the EPM test (Fig. 3C) and had significantly fewer c-Fos-positive cells in the BLA and CeA (Figs. 3D, E), compared to 17-d/control mice.

Furthermore, to elucidate which period of 2′-FL ingestion was important in alleviating anxiety-like behaviors, 2′-FL was fed between 17–24 d old (17-d/2′-FL_17–24 mice) or after 24 d old (17-d/2′-FL_24- mice). The 17-d/2′-FL_17–24 mice had significantly less time spent in the open arm of the EPM compared to the 24-d/control mice as well as the 17-d/control mice. No difference was observed in the time spent in the open arms of the EPM test for 17-d/2′-FL_24- mice compared to 24-d/control and 17-d/control mice (Figs. 4A, B). To clarify the acute effects of 2′-FL, 2′-FL diet was given 24 h before the behavioral test (17-d/2′-FL_acute mice), but 2′-FL did not affect the time spent in the open arms of the EPM test (Figs. 4C, D).

2′-FL Alleviates Early Weaning-Induced Changes in the Fecal Microbiota

Lastly, we examined the fecal microbiota of each group of mice. In an α-diversity analysis, the Chao1 index (richness) was significantly lower in 17-d mice compared to 24-d mice, regardless of diets (Table 4). The Shannon index (evenness) was significantly lower in 17-d/control mice compared to 24-d/control mice, and significantly higher in 17-d/2′-FL mice compared to 17-d/control mice (Table 4). The β-diversity based on both weighted and unweighted UniFrac distances differed significantly between the groups (Figs. 5A, B, Supplementary Figs. S3A, B). In a taxonomic comparison, the relative abundances of 5 of 13 phyla were significantly different (Fig. 5C): Actinobacteria (p < 0.05) was increased and Bacteroidetes (p < 0.05), Cyanobacteria (p < 0.05), and Tenericutes (p < 0.01) were decreased in 17-d/control mice compared to 24-d/control mice; and Bacteroidetes (p < 0.01), Cyanobacteria (p < 0.001), Tenericutes (p < 0.05), and Verrucomicrobia (p < 0.01) were increased and Deferribacteres (p < 0.05) was decreased in 17-d/2′-FL mice compared to 17-d/control mice.

DISCUSSION

In this study, increased anxiety-like behaviors characterized by decreased time spent in the open arms in the EPM were observed in early-weaned mice, as also seen in other studies.⁶⁾ However, in contrast to previous findings,^7,8) mRNA levels for BDNF and glucocorticoid receptors in the PFC and hippocampus, as well as basal serum corticosterone levels, were not affected by early weaning in this study. The mRNA levels of BDNF were also compared for each promoter-derived transcript, and no transcript levels were affected by early weaning (data not shown). These differences in results could be due to differences in the strains and weaning period: BALB/c or ICR mice have been used in previous studies, but C57BL/6J mice were used in this study; and the effect of early weaning may be stronger in BALB/c mice because these mice are more vulnerable to stress compared to C57BL/6J mice.¹⁵⁾ Early weaning was performed at 14 d in the previous studies, but at 17 d in this study because mice weaned at 14 d died at a high rate.

The amygdala is a group of nuclei in the temporal lobe that is crucial for defensive/escape behaviors in all mammals, including humans.¹⁶⁾ Amygdala nuclei can be functionally divided into several subregions, including the BLA, CeA, and MeA. The BLA and CeA are particularly involved in defensive/escape behaviors, and input from the BLA to the CeA leads the behavior to escape.¹⁷⁾ Immunohistochemistry revealed hyperactivity of the BLA and CeA based on the greater numbers of c-Fos-positive cells after the EPM test in mice weaned at 17 d compared to those weaned at 24 d. Early weaning has been shown to induce precocious myelinization in the BLA¹⁸⁾ and early life stress increases anxiety and causes a hyperactive amygdala after maturation in mouse models and humans.¹⁹⁾ These findings suggest that the amygdala may be the region responsible for early stress-induced anxiety.

Ingestion of 2′-FL alleviated early weaning-induced anxiety-like behavior characterized by decreased time spent in the open arms in the EPM test without affecting spontaneous locomotor activity. Similar to this effect on behavior, 2′-FL alleviated EPM-triggered BLA and CeA hyperactivity characterized by greater numbers of c-Fos-positive cells in mice weaned at 17 d. Spatial memory deficits, hyperalgesia, and hyperactivity of the HPA axis in mice subjected to maternal separation stress have previously been shown to be alleviated by post-weaning ingestion of milk fat globule membrane or a prebiotic blend of polydextrose/galactooligosaccharides.²⁰⁾ These findings suggest that prebiotics can alleviate early life stress-induced maladaptive behaviors.

When we examined differences in the effects of 2′-FL depending on the duration of ingestion, we found that ingestion of 2′-FL between 17 and 24 d did not alleviate anxiety-like behavior in mice weaned at 17 d. No alleviation of anxiety-like behavior was observed when 2′-FL was ingested in the 24 h immediately before the behavioral test. These results indicate that the early period of 17–24 d is not essential in the effects of 2′-FL on behavior and that acute 2′-FL ingestion is not sufficient to affect behavior. These findings lead us to speculate that the anxiolytic-like effects of 2′-FL require changes in the organism, such as in the gut microbiota, due to long-term ingestion.

The Chao1 and Shannon indexes indicated that the richness and evenness of fecal microbiota were significantly lower in mice weaned at 17 d compared to those weaned at 24 d. β-Diversity based on UniFrac distances also revealed differences in fecal microbiota composition between these groups. A lower phylum Bacteroidetes/Firmicutes ratio, which is observed in some children with autism,²¹⁾ was present in the 17-d mice compared to the 24-d mice. Since breast milk contains abundant oligosaccharides and secretory IgA that modulate the gut microbiota,^22,23) these changes in the microbiota of 17-d mice are assumed to have initiated during the 17–24 d without breastfeeding. Ingestion of 2′-FL alleviated several of these changes in fecal microbiota in 17-d mice, including a lower evenness and a lower phylum Bacteroidetes/Firmicutes ratio. These results indicate that 2′-FL adequately modulates the abundance of species in the gut microbiota. The amygdala is particularly sensitive to changes in microbiota composition, and changes in the microbiota during critical periods of neurodevelopment are implicated in regulation of amygdala function.²⁴⁾ The pathways by which signals are specifically transmitted from the gut microbiota to the amygdala are unclear, but vagus nerve, bacterial metabolites, and immune system are candidates. Notably, the vagal afferent projections from the gastrointestinal tract to the nucleus tractus solitarius, and noradrenergic projections from the nucleus tractus solitarius to the amygdala, communicating visceral information to the amygdala.²⁵⁾ In a study of early weaned animals, rats weaned at 16 d have been reported to show higher extracellular dopamine levels in the amygdala in adulthood compared to rats weaned at 30 d.²⁶⁾ Since these catecholamines in the amygdala regulate anxiety,^27,28) it is possible that changes in these noradrenergic/dopaminergic systems may be involved in the amygdala hyperactivity and anxiety-like behavior in 17 d mice in this study. It is not clear whether 2′-FL affects these noradrenergic/dopaminergic systems, but it is conceivable that 2′-FL may also affect the amygdala via the vagus nerve by altering the gut microbiota. These findings suggest that changes in the microbiota during post-weaning development underlie the adverse effects of early weaning and alleviation of these effects by 2′-FL. However, at present, the details of the brain–gut–microbiota axis are not yet fully understood, and there is no information in this study that directly indicates a relationship among the gut microbiota, amygdala activity, and anxiety-like behavior, so further research is needed.

In conclusion, prebiotic treatment with human milk oligosaccharides such as 2′-FL may alleviate the adverse effects of early stress such as anxiety, amygdala hyperactivity, and change the gut microbiota.

Acknowledgments

We thank Yuki Tanaka, Eri Araki, Yugo Nagamine, and Yuka Tamura for their technical assistance.

Conflict of Interest

This study, in part, was conducted with funding (April 2018) provided by Meiji Co., Ltd. (Tokyo, Japan).

Supplementary Materials

This article contains supplementary materials.

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