Maternal obesity during pregnancy has been associated with increased risk of neurodevelopmental disorders, including autism spectrum disorder (ASD), in offspring. Here, we report that maternal high-fat diet (MHFD) induces a shift in microbial ecology that negatively impacts offspring social behavior. Social deficits and gut microbiota dysbiosis in MHFD offspring are prevented by co-housing with offspring of mothers on a regular diet (MRD) and transferable to germ-free mice. In addition, social interaction induces synaptic potentiation (LTP) in the ventral tegmental area (VTA) of MRD, but not MHFD offspring. Moreover, MHFD offspring had fewer oxytocin immunoreactive neurons in the hypothalamus. Using metagenomics and precision microbiota reconstitution, we identified a single commensal strain that corrects oxytocin levels, LTP, and social deficits in MHFD offspring. Our findings causally link maternal diet, gut microbial imbalance, VTA plasticity, and behavior and suggest that probiotic treatment may relieve specific behavioral abnormalities associated with neurodevelopmental disorders.

Here we report that maternal high-fat diet (MHFD)-induced obesity in mice is associated with social behavioral deficits, which are mediated by alterations in the offspring gut microbiome. Notably, we also found that MHFD-induced changes in the offspring gut microbiota block long-lasting neural adaptation in the mesolimbic dopamine reward system (ventral tegmental area [VTA]). Moreover, oral treatment with a single commensal bacterial species corrects oxytocin levels and synaptic dysfunction in the VTA and selectively reverses social deficits in MHFD offspring.

The amount and type of dietary macronutrients strongly influence the intestinal microbiota (), which consists of a vast bacterial community that resides in the lower gut and lives in a symbiotic relationship with the host. Maternal obesity has been associated with alterations in the gut microbiome in offspring in both human and non-human primates (). In addition, some individuals with neurodevelopmental disorders, including ASD, co-present with gastrointestinal problems and dysbiosis of the gut microbiota (). Given the large body of preclinical literature supporting the notion that a bidirectional communication system between the gut and the brain—known as the gut-brain axis—links gut and brain activities (), it has been speculated that changes in the gut microbiome may be relevant to the development of behavioral symptoms associated with ASD (). However, how changes in bacteria that inhabit the intestine could influence brain development and function remains unknown.

Recent epidemiological evidence suggests that exposure to maternal obesity in utero increases the risk of neurodevelopmental disorders, such as autism spectrum disorder (ASD) in children (). Given the increase in the prevalence of obesity (), it is important to understand the neurobiological mechanism by which maternal obesity affects offspring behavior and brain function.

These findings, together with the fact that L. reuteri treatment increased oxytocin immunoreactivity in the PVN of MHFD offspring ( Figures 4 I and 4J, 4O, and 4P), led us to examine whether direct oxytocin application could also reverse the behavioral and electrophysiological deficits characteristic of MHFD offspring. To test this idea, we administered oxytocin intranasally—a preferred method of administering neuropeptides to the brain bypassing more invasive procedures ()—to MHFD offspring and measured reciprocal social interactions 30 min later. Although either oxytocin alone or social interaction alone failed to rescue social interaction-induced LTP in the VTA, the combination of social interaction and oxytocin treatment restored LTP in the VTA of MHFD offspring ( Figures 6 A and 6B ), supporting prior work implicating a synergistic effect of oxytocin and dopamine in the processing of socially relevant cues (). Accordingly, oxytocin treatment improved reciprocal social interaction ( Figures 6 C-6F), as well as sociability and the preference for social novelty ( Figures 6 G-6J). Thus, oxytocin administration rescues social behavior and related neural adaptations in the VTA of MHFD offspring. Collectively, our data show that MHFD impairs oxytocin-mediated synaptic adaptations in the VTA that underlie social behaviors.

We next wondered whether L. reuteri treatment, which restores sociability and preference for social novelty in MHFD offspring ( Figures 4 B, 4C, and 4E), would also rescue reciprocal social interaction and related changes in synaptic strength in the VTA. Live ( Figures 5 H and 5J), but not heat-killed ( Figures 5 I and 5K), L. reuteri rescued stranger interaction-induced LTP in the VTA as well as reciprocal social interactions in MHFD offspring ( Figure 5 L). Thus, L. reuteri restores social interaction-induced LTP in the VTA of MHFD offspring.

Mirroring the electrophysiological results, MRD offspring spent significantly more time interacting with a stranger than a familiar mouse, but MHFD offspring did not ( Figures 5 F and 5G). Thus, social interaction induces a long-lasting increase in the activity of the dopaminergic reward system of MRD, but not in MHFD, offspring.

Brain regions that respond to naturally rewarding stimuli, including the ventral tegmental area (VTA) and the nucleus accumbens (NAc), are crucially involved in social behaviors (). In addition, oxytocin-expressing neurons in the PVN project to the VTA (). Oxytocin activates VTA neurons in both mice and humans, influencing the processing of socially relevant cues () and oxytocin receptor blockade in the VTA prevents social attachment in rodents (). Given that social stimulation can be particularly rewarding and triggers synaptic potentiation in VTA DA neurons of birds (), we examined whether direct social interaction evokes long-term potentiation (LTP) of synaptic inputs to VTA DA neurons ( Figures S7 A–S7C). To this end, we recorded AMPAR/NMDAR ratios of glutamatergic excitatory postsynaptic currents (EPSCs) in MRD and MHFD offspring 24 hr following a 10 min reciprocal interaction with either a stranger or a familiar mouse ( Figure 5 A). In control MRD mice, social interaction with a stranger, but not a familiar mouse, triggered LTP in VTA DA neurons, as determined by an increase in AMPAR/NMDAR ratios ( Figures 5 B and 5D). By contrast, in MHFD offspring, social interaction with a stranger failed to induce LTP in their VTA DA neurons ( Figures 5 C and 5E). Input-output curves, paired-pulse ratios and miniature EPSCs (mEPSCs) frequency and amplitude show that the impairment of LTP induced by social interaction in MHFD offspring cannot be attributed to changes in basal synaptic transmission ( Figures S7 D–S7H).

(B–E) LTP was measure 24 hr following reciprocal interaction with either a stranger or a familiar mouse. Only interaction with a stranger mouse induced LTP in MRD VTA DA neurons, as determined by increased AMPAR/NMDAR ratios (B and D, Baseline versus Familiar p > 0.99, t = 0.12, baseline versus stranger p < 0.01, t = 3.79; familiar versus stranger p < 0.05, t = 3.03; F = 8.03, p < 0.01). In MHFD offspring, neither stranger nor familiar reciprocal interaction evoked LTP in VTA DA neurons (C and E, baseline versus familiar p > 0.99, t = 0.035, baseline versus stranger p = 0.64, t = 1.30; familiar versus stranger p = 0.50, t = 1.45; F 2,15 = 1.47, p = 0.26).

(D) Evoked EPSC (eEPSC) amplitude as a function of stimulation intensity is plotted as input/output curves (recorded in voltage clamp at −70 mV with cesium-containing pipettes and in the presence of 100 μM picrotoxin) was similar in VTA slices from MRD and MHFD offspring (t = 0.16, p = 0.88, n = 5).

(B) Spike width was measured from the start of the inward deflection to the outward peak at a holding potential of −55 mV. Neurons displaying spike widths > 1.0 ms were taken as dopaminergic.

There is growing evidence that the neuropeptide oxytocin modulates numerous aspects of social behavior () and is implicated in ASD (). L. reuteri, which rescued social behaviors in MHFD mice ( Figures 4 B, 4C, and 4E), has been reported to increase oxytocin levels (). Because oxytocin is primarily synthesized in the paraventricular nuclei (PVN) of the hypothalamus, we decided to compare the number of oxytocin-expressing cells in the PVN of MRD and MHFD offspring. Interestingly, MHFD offspring had significantly fewer oxytocin immunoreactive neurons compared to MRD offspring ( Figures 4 F–4H, 4K, and 4L). The reduction in oxytocin immunoreactivity was not due to an overall decrease in PVN neurons, since the total number of neurons was unchanged (as measured by NeuN staining; Figures 4 G, 4H, 4M, and 4N). Notably, in L. reuteri-treated MHFD offspring, the number of oxytocin-expressing cells was higher than in control-treated MHFD offspring ( Figures 4 I, 4J, 4O, and 4P). Thus, the number of oxytocin immunoreactive neurons in the PVN is reduced in MHFD offspring but can be restored by L. reuteri treatment.

MHFD offspring also show other behavioral traits that are associated with ASD, like repetitive behaviors and anxiety ( Figures S6 ). Interestingly, while co-housing MHFD with MRD offspring restores social behavior ( Figures 2 C and 2D), it failed to rescue marble burying ( Figure S6 B), a behavioral task reflecting repetitive and perseverative behavior (). Accordingly, GF mice also showed increased marble burying, and fecal microbial transplants from MRD (or MHFD) offspring into GF mice failed to reverse the repetitive behavior ( Figure S6 B). Thus, these data suggest that repetitive behaviors in MHFD offspring do not depend on changes in the gut microbiome. In addition, L. reuteri treatment had no effect on anxiety in MHFD offspring ( Figures S6 C–S6H). Taken together, these data indicate that L. reuteri reconstitution specifically rescues social, but not other behavioral endophenotypes associated with ASD.

(B) MHFD offspring buried more marbles than MRD offspring (p < 0.05, t = 3.64). More marbles were also buried by GF mice compared to conventionally colonized MRD offspring (p < 0.0001, t = 5.20). Co-housing failed to reverse the increased marble-burying behavior in MHFD offspring (Co-housed MHFD versus MRD, p < 0.01, t = 4.32; Co-housed MHFD versus MHFD, p > 0.99, t = 1.51) and colonization of GF mice with MRD microbiota at 4 weeks of age did not reverse the exaggerated repetitive behavior (MRD-colonized GF versus MRD, p = 0.25, t = 2.60; MRD-colonized versus GF, p > 0.99, t = 1.38).

L. reuteri has been shown to promote oxytocin levels (), a hormone that plays a crucial role in social behaviors (). We hypothesized that the selective decrease in L. reuteri in the microbiota of MHFD offspring was causally related to their social deficits. To test this hypothesis, we introduced L. reuteri into the drinking water of MHFD offspring at weaning for 4 weeks, after which behavior was tested ( Figure 4 A). Remarkably, treatment with L. reuteri significantly improved sociability and preference for social novelty in MHFD offspring ( Figures 4 B, 4C, 4E, S5 A, and S5B). Results from several control experiments underscore the specificity of L. reuteri-mediated rescue of social behaviors in MHFD offspring. First, drinking water treated with either resuspension media or heat-killed L. reuteri (80°C for 20 min) failed to restore social behavior in MHFD offspring ( Figures 4 B–4D, S5 A, and S5B). Second, drinking water with live L. reuteri did not change the social behavior of MRD offspring ( Figures 4 B, 4C, S5 A, and S5B), presumably because their gut microbiota already contains ample L. reuteri. Finally, addition of L. reuteri to the drinking water had no major effect on bacterial viability and the heat-killing procedure completely abrogated colony-forming units ( Figure S5 C). Importantly, the amelioration of the deficient social behavior is specific to L. reuteri since similar treatment with another Lactobacillus species, L. johnsonii, whose abundance is also reduced in the gut microbiota of MHFD offspring ( Table 1 ), failed to rescue social behaviors in MHFD offspring ( Figures S5 D–S5M).

All MRD offspring groups preferred the chamber containing a mouse in the sociability test (A, MRD+media p < 0.05, t = 2.84; MRD+heat-killed L. reuteri, p < 0.001, t = 4.12; MRD+live L. reuteri p < 0.0001, t = 5.71) and the chamber containing Mouse 2 in the social novelty test (B, MRD+media p < 0.01, t = 3.90; MRD+heat-killed L. reuteri, p < 0.001, t = 4.44; MRD+live L. reuteri p < 0.01, t = 3.57). Unlike media alone (A, MHFD+media p > 0.99, t = 0.56; B, MHFD+media p > 0.99, t = 0.64) or heat-killed L. reuteri (A, MHFD+Heat-killed L. reuteri p = 0.085, t = 2.50; B, MHFD+Heat-killed L. reuteri p > 0.99, t = 0.053), administration of live L. reuteri in the drinking water improved MHFD offspring sociability and preference social novelty (A, MHFD+L. reuteri p < 0.0001, t = 5.06; treatment effect F 1,86 = 55.61, p < 0.0001; (B) MHFD+L. reuteri p < 0.001, t = 4.55; treatment effect F 1,86 = 37.12, p < 0.0001). (C) Pre- and post-treatment plating revealed that most of L. reuteri survived over the 24h treatment period but none did after the heat-killing procedure. (D) Schematic of L. johnsonii treatment. E, F, L. johnsonii treatment failed to improve reciprocal social interaction time (E, MHFD+media versus MHFD+L. johnsonii, p > 0.99, t = 0.194) or contact duration in MHFD offspring (F, MHFD+media versus MHFD+L. johnsonii, p > 0.99, t = 0). (G and H) Neither resuspension media (B, p = 0.38, t = 1.72; C, p > 0.99, t = 0.40) nor L. johnsonii administration in the drinking water (G, p > 0.99, t = 0.78; H, p > 0.99, t = 0.21) improved (G) sociability or (H) preference for social novelty in MHFD offspring (G, treatment effect F 3,32 = 23.94, p < 0.0001; H, treatment effect F 3,32 = 6.13, p < 0.001). (J–M), Representative track plots of mice during each stage of the 3-chamber task, cohorts as noted. Plots show mean ± SEM.

(O and P) Relative to treatment with heat-killed L. reuteri, treatment with live L. reuteri significantly increased oxytocin-positive cell number (O, p < 0.05, t = 2.93) and oxytocin immunofluorescence intensity (P, p < 0.05, t = 3.09) in the PVN of MHFD offspring. AU: arbitrary units. Plots show mean ± SEM. See also Figures S5 and S6

(K–N) Oxytocin immunoreactive cell number (K, p < 0.01, t = 4.76) and oxytocin immunofluorescence intensity (L, p < 0.01, t = 3.80) were reduced in the PVN of MHFD versus MHFD mice. In the PVN of MRD and MHFD offspring, NeuN cell number immunoreactivity (M, p = 0.34, t = 1.09) and immunofluorescence intensity (N, p = 0.79, t = 0.28) were similar.

(B and C) Unlike resuspension media (B, p > 0.99, t = 1.03; c, p > 0.99, t = 0.40) or heat-killed L. reuteri (B, p > 0.99, t = 1.35; c, p > 0.99, t = 0.21), administration of live L. reuteri in the drinking water rescued sociability (B, p < 0.0001, t = 5.98) and preference for social novelty (C, p < 0.001, t = 5.01) in MHFD offspring (B, treatment effect F 1,86 = 87.53, p < 0.0001; C, treatment effect F 1,86 = 30.24, p < 0.0001).

To investigate which bacterial species are absent in the microbiota community of MHFD offspring, we performed metagenomic shotgun sequencing of fecal samples from both MHFD and MRD offspring. Our analysis identified several species whose relative abundance was dramatically reduced in the MHFD offspring microbiota ( Table 1 ). Among these, L. reuteri was the most drastically reduced (>9-fold) in the MHFD microbiota population, compared to the MRD microbiota ( Table 1 ).

To identify functional differences between gut microbial communities and determine whether their role is causal, we transplanted (gavaged) fecal microbiota from adult MRD and MHFD offspring into 4-week- ( Figure 3 E) and 8-week-old ( Figure 3 F) GF mice. Interestingly, GF mice that received fecal microbiota from MRD offspring at weaning (4 weeks; Figures 3 G, 3H, S4 E and S4F), but not at 8 weeks ( Figures 3 I, 3J, S4 G, and S4H), showed normal social behavior. By contrast, GF mice that received fecal microbiota from MHFD offspring remained socially impaired, regardless of the age at which the fecal transfer was performed ( Figures 3 G–3J and S4 E–S4H). Moreover, as in the case of the phylogenetic separation of MRD and MHFD microbiota ( Figure 1 J), the bacterial communities in GF mice receiving feces from MHFD donor offspring clustered separately from those of GF mice receiving feces from MRD donor offspring, irrespective of whether the fecal transplant was performed at 4 or 8 weeks ( Figures 3 K, 3L, and S4 I–S4N). These data reveal a neurodevelopmental window during which microbial reconstitution effectively improves social behavior.

Studies on germ-free (GF) mice have shown that the intestinal microbiota can influence brain development and function (). We hypothesized that, if the lack of one or more bacterial species in the microbiota of MHFD offspring is responsible for their defective social behavior, GF mice should also be socially deficient. Confirming this hypothesis and in keeping with recent results (), social behaviors were impaired in GF mice ( Figures 3 A–3D and S4 A–S4D ).

(I–M) Compared to GF mice transplanted with MRD fecal matter, the number of OTUs observed in GF mice transplanted with MHFD fecal matter was similar at 24h post-transplant (I, p = 0.57, t = 0.61), but was reduced at 1 (J, p < 0.05, t = 3.27), 2 (K, p < 0.05, t = 3.12), and 8 weeks (L, p < 0.05, t = 0.298). M, The number of observed OTUs was reduced in GF mice that received fecal microbiota transplant at 8 weeks of age from MHFD versus MRD offspring (M, p < 0.05, t = 2.98). N, Bacterial diversity in GF MRD and MHFD recipients over time, displayed as a local regression with 95% confidence interval. Plots show mean ± SEM.

(K and L) PCoA of unweighted UniFrac distances based on the 16S rRNA gene sequencing dataset from GF recipients of stools from either MRD or MHFD donors at four (K, p = 0.001, R 2 = 0.83; n = 1,000 rarefactions; 4,628 reads/sample) or eight (L, p < 0.001, R 2 = 0.77; n = 1,000 rarefactions; 4,805 reads/sample) weeks of age. Plots show mean ± SEM.

In agreement with the idea that the fecal microbiota of MHFD offspring lacks one or more beneficial bacterial species required for normal social behavior, co-housing one MRD with three MHFD offspring was sufficient to rescue both the social behaviors and microbiota phylogenetic profile of MHFD offspring ( Figures S3 F–S3L).

We next examined whether co-housing also corrected the changes in the microbiota of MHFD offspring. Indeed, co-housing caused a shift in the bacterial phylogenetic profile of MHFD mice to resemble that of MRD or MRD co-housed mice ( Figure 2 G), thus correcting the MHFD-induced alterations in the commensal microbiota.

While microbial communities vary across individuals (), co-housed family members are known to share their microbiota (). Since mice are coprophagic and transfer gut microbiota between each other by the fecal-oral route (), we examined whether co-housing MHFD with MRD mice prevents the social deficits in MHFD offspring. To this end, at weaning (3 weeks) an MHFD mouse was co-housed with three MRD mice ( Figures 2 A and 2B ). Control groups consisted of individual cages containing either four MHFD mice or four MRD mice ( Figure 2 B). Fecal samples were collected and social behavior in MRD and MHFD offspring was assessed when mice were 7–8 weeks old. Strikingly, MHFD mice co-housed with MRD mice exhibited normal reciprocal social interactions ( Figures 2 C and 2D and S3 A–S3C ), as well as normal sociability and preference for social novelty, as determined by the 3-chamber test ( Figures 2 E, 2F, S3 D and S3E). Thus, co-housing with control mice corrects social deficits in MHFD offspring.

(H–K) Social interaction time (H, MRD versus MHFD p < 0.0001, t = 10.21; MRD versus co-housed MHFD p > 0.99, t = 1.41; MHFD versus co-housed MHFD p < 0.0001, t = 8.53; p < 0.0001, F 3,8 = 38.88) and contact duration (I, MRD versus MHFD p < 0.0001, t = 7.18; MRD versus co-housed MHFD p > 0.99, t = 0.59; MHFD versus co-housed MHFD p < 0.001, t = 6.29; p = 0.0001, F 3,8 = 19.14) in the reciprocal interaction test; sociability (J, MRD p < 0.0001, t = 7.37; MHFD p = 0.802, t = 1.31; Co-housed MRD p < 0.0001, t = 6.02; Co-housed MHFD p < 0.0001, t = 9.46; Maternal diet/ Housing/Interaction effect F 3,32 = 18.62, p < 0.0001) and preference for social novelty (K, MRD p < 0.05, t = 2.82; MHFD p = 0.723, t = 1.37; Co-housed MRD p < 0.05, t = 2.72; Co-housed MHFD p < 0.01, t = 3.34; Maternal diet/Housing/Interaction effect F 3,32 = 4.55, p < 0.01) as well as UniFrac phylogenetic clustering (L, p < 0.001, R 2 = 0.681; n = 1,000 rarefactions; 12,600 reads/sample), are all restored in MHFD offspring co-housed with MRD mice in a 3 MHFD:1MRD configuration. Plots show mean ± SEM.

(C–G) Social interaction time (C, MRD versus MHFD p < 0.001, t = 9.30; MRD versus co-housed MHFD p > 0.99, t = 0.31; MHFD versus co-housed MHFD p < 0.001, t = 7.99; p < 0.0001, F= 30.51) and contact duration (D, MRD versus MHFD p < 0.05, t = 4.13; MRD versus co-housed MHFD p > 0.99, t = 0.46; MHFD versus co-housed MHFD p < 0.05, t = 4.59; p < 0.001, F= 9.01) in the reciprocal interaction test; social interaction times in the sociability (E, MRD p < 0.001, t = 4.36; MHFD p > 0.99, t = 0.078; Co-housed MRD p < 0.0001, t = 6.33; Co-housed MHFD p < 0.001, t = 4.78; Maternal diet/ Housing/Interaction effect F= 6.13, p < 0.01) and social novelty tests (F, MRD p < 0.0001, t = 5.12; MHFD p > 0.99, t = 0.60; Co-housed MRD p < 0.001, t = 4.20; Co-housed MHFD p < 0.001, t = 4.76; maternal diet/housing/interaction effect F= 4.37, p < 0.01), as well as UniFrac-based phylogenetic clustering (G, p < 0.001, R= 0.552; n = 1,000 rarefactions; 3,390 reads/sample), are all restored in MHFD offspring co-housed with MRD mice. Plots show mean ± SEM. See also Figure S3

Consistent with previous reports (), an HFD regimen in mothers induced a remarkable change in the maternal microbiome composition and diversity ( Figures S2 G and S2H), which was similar to that observed in their offspring ( Figures 1 J and S2 F).

A variety of factors could contribute to the etiology of MHFD-induced social behavioral abnormalities. However, maternal obesity has been shown to alter the gut microbiome of offspring () and individuals diagnosed with ASD can co-present dysbiosis of the gut microbiota (). To examine whether MHFD induces alterations in offspring gut microbiota, we analyzed the bacterial composition and community structure in the feces of MRD and MHFD offspring by 16S ribosomal RNA (rRNA) gene sequencing. The microbial communities in both MRD and MHFD offspring were comprised of a typical mouse gut microbiota, dominated by Bacteroidetes and Firmicutes ( Figures S2 A–S2D). While bacterial diversity computed based on weighted UniFrac distances (the assessment of community structure by considering abundance of operational taxonomic units [OTUs]) did not differ significantly between the offspring from either diet group ( Figure S2 E), unweighted analyses of UniFrac distances (assessment of community structure by considering only OTU presence/absence) revealed a marked difference between the structures of the bacterial communities ( Figure 1 J). Moreover, the diversity of microbiota in MHFD offspring was reduced compared to MRD microbiota ( Figure S2 F).

(H) While bacterial diversity did not differ prior to diet onset (p = 0.61, t = 1.30), it was significantly reduced in HFD-fed females after 4 (p < 0.05, t = 2.82) and 8 weeks on diet (p < 0.0001, t = 5.78). Plots show mean ± SEM.

(G) HFD alters the maternal microbiome. PCoA of unweighted UniFrac distances generated from the averaged, rounded rarefied 16S rRNA gene dataset (n = 1,000 rarefactions; 2,397 reads/sample). The microbial composition of maternal fecal samples clustered together immediately prior to diet administration, but within 4 weeks on diet, HFD-fed maternal fecal samples clustered separately from controls. This shift in microbial composition was maintained after 8 weeks on diet, when breeding started (p < 0.001, R 2 = 0.79).

Given that maternal obesity has been associated with increased risk for neurodevelopmental disorders including ASD in offspring () and deficient social interactions are a salient behavioral feature of ASD (), we studied social behavior in maternal regular diet (MRD) and MHFD offspring. First, we assessed reciprocal social interactions by recording the amount of time a pair of mice, unfamiliar with each other, spent interacting in a neutral arena ( Figure 1 B). When compared to MRD offspring, MHFD offspring had fewer reciprocal interactions ( Figures 1 C and 1D, S1 H, and S1I). Next, we used the three-chamber test () to assess (1) sociability by comparing the time mice spent interacting with an empty wire cage versus one containing a mouse and (2) preference for social novelty by measuring the time mice spent interacting with a familiar versus a stranger mouse ( Figure 1 E). Consistent with the results from reciprocal social interactions, MHFD offspring had impaired sociability and showed no preference for social novelty ( Figures 1 F–1I, S1 J, and S1K). Taken together these data indicate that MHFD offspring display social deficits.

To investigate how maternal diet-induced obesity affects offspring neurodevelopment, female mice were fed either regular diet (RD) or high-fat diet (HFD) for 8 weeks, a standard period required to reach a state of diet-induced obesity in mice (). Females were then paired with males to produce offspring that were given regular diet after weaning ( Figure 1 A). As expected, MHFD significantly increased maternal weight ( Figures S1 A–S1C). Consistent with reports of more frequent spontaneous abortion in obese mothers (), the litter size was reduced ( Figure S1 D) and latency to first litter increased in female mice fed HFD ( Figure S1 E). It is noteworthy that there was no significant difference in offspring weight between maternal diet cohorts at 7–12 weeks of age ( Figures S1 F and S1G), the time at which behavioral and electrophysiological experiments were performed.

(F and G) Offspring weight did not differ between maternal diet cohorts at either 7–8 (F, p = 0.12, t = 1.63) or 11–12 weeks of age (G, p = 0.10, t = 1.80), the age range during which all behavioral tests were performed.

(F–G) In the sociability test, MRD offspring spent more time interacting with a mouse than with an empty wire cage (F, p < 0.0001, t = 8.817), whereas MHFD offspring showed no preference for the mouse (F, p = 0.48, t = 1.19; maternal diet effect F 1,52 = 6.08, p < 0.05). In the social novelty test, unlike MRD (G, p < 0.0001, t = 6.68), MHFD offspring had no preference for interacting with a novel versus a familiar mouse (G, p = 0.086, t = 2.08; maternal diet effect F 1,52 = 34.96, p < 0.0001).

Our results provide new insight into the mechanism by which a marked shift in microbial ecology, caused by MHFD, can negatively impact social behaviors and related neuronal changes in offspring. These neuronal adaptations, which underlie social behavior by enhancing the salience and rewarding value of social stimuli, are surprisingly impaired by maternal diet-induced changes in the gut microbiome ( Figure 5 ). Interestingly, according to a recent study, probiotic-based restoration of gut permeability in a mouse model of ASD can improve some behavioral abnormalities, but not social behaviors (). Given that we identified a different probiotic candidate, L. reuteri, that rescues social behavior ( Figures 4 and 5 ), but not other behavioral endophenotypes associated with ASD ( Figure S6 ), in MHFD mice, we propose that a carefully selected combination of probiotics may be useful as a potential non-invasive treatment for patients suffering from neurodevelopmental disorders including ASD.

We propose a model in which L. reuteri improves social behavior by promoting oxytocin-mediated functions. Consistent with this model, L. reuteri-treatment enhances oxytocin levels in the PVN of MHFD mice ( Figures 4 I and 4J) and direct oxytocin-treatment normalizes the social behavior of MHFD offspring ( Figure 6 ). Although the precise mechanism by which L. reuteri promotes oxytocin in the brain remains to be determined, we favor the idea that the vagus nerve () could be the main pathway of communication between the gut/L. reuteri and changes in oxytocin in the PVN. It is known that vagal nerve fibers project to the PVN (). In addition, neuronal activity in the PVN induced by bacterial colonization is blocked by subdiaphragmatic vagotomy (). Especially relevant are the reports that the L. reuteri-mediated increase in oxytocin depends on the vagus nerve () and that another Lactobacillus species, L. rhamnosus, reduced stress-induced anxiety in mice in a vagus-dependent manner ().

While most of the focus in the field has been on inflammation () or epigenetic changes (), the biological mechanism by which maternal obesity affects offspring neurodevelopment remains to be determined. Here, we show that that the behavioral dysfunction associated with MHFD-induced obesity is induced by alterations in the offspring gut microbiota. Several lines of evidence support this idea. First, some individuals diagnosed with ASD present dysbiosis of the gut microbiota and gastrointestinal issues (). Second, maternal obesity leads to alterations in the offspring’s gut microbiome in humans and non-human primates (). Third, in mice, the gut microbiota of MHFD offspring is altered ( Figure 1 J) by the reduction in specific bacterial species ( Table 1 ). Fourth, manipulation of the microbiome community by co-housing MHFD with MRD offspring rescues MHFD-induced social deficits and corrects their microbial phylogenetic profile ( Figures 2 and S3 ). Fifth, GF mice are socially impaired and fecal microbiota transplanted from MRD (but not MHFD) offspring rescues GF social behavior ( Figures 3 and S4 ). Finally, treatment with a single bacterial species, L. reuteri, which is dramatically reduced in MHFD offspring ( Table 1 ), selectively restores social behavior in MHFD mice ( Figures 4 and S5 A–S5C).

Both genetic and environmental factors, and their interactions, play a crucial role in the etiology of neurodevelopmental disorders including ASD (). There is growing epidemiological evidence that maternal obesity heightens the risk of neuropsychiatric disorders in offspring (). Indeed, a recent study reported that mothers with obesity were 1.5 times more likely to have a child with ASD, and the increased risk of children with ASD was two-fold greater for pregnant mothers with both obesity and gestational diabetes ().

Experimental Procedures

Mice and Maternal Diet C57Bl6/J mice were obtained from Jackson Laboratories (#000-664) and were kept on a 12 hr light/dark cycle and had access to food and water ad libitum. Females were placed on either a regular diet (RD) consisting of 13.4% kcal from fat, 30% kcal from protein, and 57% kcal from carbohydrates (Lab Diets, #5001) or HFD consisting of 60% kcal from fat, 20% kcal from protein, and 20% kcal from carbohydrates (Research Diets, #D12492). Maternal weight was measured weekly. Maternal total and fat mass were measured using an mq7.5 Minispec NMR body composition analyzer. After 8 weeks on diet, females were paired with C57Bl6/J adult males to produce subject offspring. Resulting offspring were weaned at 3 weeks of age and all placed on RD, regardless of maternal diet (MRD or MHFD). Germ-free mice (C57Bl6/J) were maintained in a flexible isolator fed with HEPA-filtered air and provided with irradiated food and water. Germ-free offspring were weaned at 4 weeks of age. All behavioral tests were performed on 7- to 12-week-old male mice. Animal care and experimental procedures were approved by Baylor College of Medicine’s Institutional Animal Care and Use Committee in accordance with all guidelines set forth by the U.S. National Institutes of Health.

Reciprocal Social Interaction Mice were placed in a 25 × 25 × 25 cm Plexiglass arena, to which they had not been previously habituated, with either a familiar (cage-mate) or stranger age- and sex-matched conspecific. In all experiments, paired mice were matched for maternal diet, colonization source, and/or treatment. We recorded the time a pair of mice engaged in social interaction (close following, touching, nose-to-nose sniffing, nose-to-anus sniffing, and/or crawling over/under each other). The human observer was blind to maternal diet and/or treatment group. Social behavior was analyzed with AnyMaze automated software.

Three-Chamber Social Test Silverman et al., 2010 Silverman J.L.

Yang M.

Lord C.

Crawley J.N. Behavioural phenotyping assays for mouse models of autism. Crawley’s three-chamber test for sociability and preference for social novelty was performed as described (). In brief, the mouse first experienced a 10 min period of habituation during which it was allowed to freely explore a 60 × 40 × 23 cm Plexiglass arena divided into three equally sized, interconnected chambers (left, center, right). Sociability was measured during a second 10-min period in which the subject could interact either with an empty wire cup (empty) or a wire cup containing an age and sex-matched stranger conspecific (mouse 1). Time spent interacting (sniffing, crawling upon) with either the empty cup or the stranger mouse contained in the other cup as well as time spent in each chamber was recorded using the AnyMaze software, by independent observers. Empty cup placement in the left or right chamber during the sociability period was counterbalanced between trials. Finally, preference for social novelty was assayed by introducing a second stranger mouse (mouse 2) into the previously the empty wire cup. Time spent in each chamber as well as time spent interacting with either mouse 1 or mouse 2 was recorded using the automated AnyMaze software by independent observers.

Marble Burying Thomas et al., 2009 Thomas A.

Burant A.

Bui N.

Graham D.

Yuva-Paylor L.A.

Paylor R. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Marble burying was performed as previously described (). Briefly, mice were placed in a standard-sized cage containing 20 regularly-spaced black marbles sitting on fine-wood chipped bedding 5 cm in depth. After 20 min, the mouse was removed and marbles with at least two-thirds of their depth obscured by wood chips were counted as buried.

Open Field Mice were placed in an open arena (40 × 40 × 20 cm) and allowed to explore freely for 10 min while their position was continually monitored using tracking software (AnyMaze). Tracking allowed for measurement of distance traveled, speed, and position in the arena throughout the task. Time spent in the center of the arena, defined as the interior 20 × 20 cm, was recorded.

Whole Genome Shotgun Sequencing Individual libraries constructed from each sample were loaded onto the HiSeq platform (Illumina) and sequenced using the 2 × 100 bp pair-end read protocol. Illumina paired-end libraries were constructed from total genomic DNA isolated from each sample. The DNA was sheared into approximately 400–600 bp fragments followed by ligation of Illumina adaptors containing molecular barcodes for downstream de-multiplexing. These products were then amplified through ligation-mediated PCR (LM-PCR) using KAPA HiFi DNA Polymerase (Kapa Biosystems, Wilmington, MA, USA). Following bead purification with Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA), quantification and size distribution of the LM-PCR product was determined using the LabChip GX electrophoresis system (PerkinElmer, Akron, OH, USA). Libraries were pooled in equimolar amounts at six samples per pool, and prepared for sequencing with TruSeq PE Cluster Generation Kit (Illumina). Each library pool was loaded onto one lane of a HiSeq 2000 flow cell spiked with 1% PhiX control library. Sequencing files were de-multiplexed with CASAVA version 1.8.3 (Illumina). Martin, 2011 Martin M. Cutadapt removes adaptor sequences from high-throughput sequencing reads. Schmieder and Edwards, 2011 Schmieder R.

Edwards R. Quality control and preprocessing of metagenomic datasets. Segata et al., 2012 Segata N.

Waldron L.

Ballarini A.

Narasimhan V.

Jousson O.

Huttenhower C. Metagenomic microbial community profiling using unique clade-specific marker genes. Quality filtering, trimming and de-multiplexing was carried out by a custom pipeline containing Trim Galore and cutadapt ()for adaptor and quality trimming, and PRINSEQ () for low-complexity filtering and sequence deduplication. In addition, Bowtie2 v2.2.1 was used to map reads to MetaPhlAn markers for the classification of bacterial species ().

Colonization of Germ-Free Mice by Fecal Microbiota Transplant Fresh fecal samples were collected from donor mice/microbiome cohort and homogenized on ice in sterile PBS under sterile conditions. The resulting slurry was spun at 1,000g for 3 min at 4°C. The supernatants were isolated and diluted to 5 × 109 CFU/ml with sterile PBS. Four- or eight-week-old C57Bl6/J germ-free (GF) recipient mice were then immediately colonized by a single gavage with 0.2 mL solution. Fecal samples were collected from the colonized GF mice at 24 hr, 7 days, 14 days, 28 days, and 56 days following colonization. Fecal samples were snap frozen and stored at −80°C until prepared for sequencing. Behavioral experiments were initiated at 3 weeks post-transplant.

Culture and Treatment with L. reuteri and L. johnsonii Lactobacillus reuteri MM4-1A (ATCC-PTA-6475) and Lactobacillus johnsonii (ATCC 33200) were cultured anaerobically in MRS broth in a 90% N 2 , 5% CO 2 , 5% H 2 environment. L. reuteri was heat-killed by keeping the bacteria at 80°C for 20 min. Bacterial viability was assessed by plating and the efficacy of the heat-kill procedure was confirmed by the absence of colony growth following plating. Cultures were centrifuged, washed, and resuspended in anaerobic solution (PBS) and frozen at −80°C until use. PBS, live or heat-killed L. reuteri were added to the drinking water, which was changed daily to minimize dosage variability. Whereas the experimental group received live bacteria, one control group received identically prepared cultures of heat-killed bacteria. A second group of control mice received water treated with PBS alone. Live and heat-killed L. reuteri were supplied at a dosage of 1 × 108 organisms/mouse/day continuously in drinking water. Mice consumed the treated water ad libitum over the treatment period. The treated drinking water for each group was replaced daily 2 hr prior to the onset of the dark cycle to minimize variation in microbial exposure. Behavioral assays were initiated after 4 weeks of L. reuteri or control treatment. The protocol of the L. johnsonii preparation and administration matched the L. reuteri protocol. Fecal samples for sequencing and tissue used in the immunofluorescence studies were collected at the end of the treatment.

Immunofluorescence Mice were deeply anesthetized by inhalation of isoflurane and perfused transcardially with 10 mL 0.9% phosphate-buffered saline followed by 30 mL 4% paraformaldehyde in 0.1M phosphate buffer (PFA). Brains were post-fixed in 4% PFA at 4°C overnight, then cryoprotected in 30% sucrose 0.1M PB over 3 days. Coronal slices (30 μm) thick were obtained from frozen tissue using a sliding blade microtome then transferred to ice cold PBS. Slices were blocked with 5% normal goat serum, 0.3% Triton X-100 0.1M PB (PBTgs) for 1 hr rocking at RT and then incubated in primary antibodies (rabbit anti-oxytocin, ImmunoStar #20068, 1:2,000; mouse anti-NeuN, Millipore, #MAB377, 1:2,000) diluted in PBTgs rocking at 4°C for 24 hr. Slices were then washed three times with 0.3% Triton X-100 0.1M PB. Primary antibodies were visualized using secondary goat anti-rabbit Alexa Fluor 488 (ThermoFisher Scientific, #A-11034) and goat anti-mouse Alexa Fluor 594 (ThermoFisher Scientific, #A-11032) antibodies (1:1,000 dilution). Slices were incubated in secondary antibodies rocking in the dark for 1h at RT. Five minute final washes with each of PBTgs, 0.1M PB, and 0.05M PB preceded mounting onto 2% gelatin (Sigma-Aldrich, #G9391)-coated coverslips. Nuclei were visualized using Vectashield H-1200 with DAPI (Vector Labs, #H-1200). Fluorescent imaging and data acquisition was performed on a Zeiss AxioImager.Z2 microscope (Car Zeiss MicroImaging) mounted with an AxioCam digital camera (Carl Zeiss MicroImaging). Images were captured using AxioVision acquisition software (Carl Zeiss Microimaging). All images within a given dataset were acquired at identical exposure times, within a given channel, to allow comparison of signal intensity. In some images, contrast and brightness were linearly adjusted using Photoshop (Adobe). Image processing was applied uniformly across all images within a given dataset. Fluorescence intensity was measured in ImageJ (NIH) by selecting regions of interest (Oxytocin- and NeuN-positive hypothalamic cell bodies). Hypothalamic oxytocin-expressing neuronal population and NeuN+ cell number was assessed in ImageJ using the following operational sequence: (1) open image file, (2) subtract background, (3) adjust threshold, (4) convert to mask, (5) watershed, (6) analyze particles. Automatic identification of cell boundaries was validated against the source image by an experimenter blind to group allocation.

Oxytocin Administration Peñagarikano et al., 2015 Peñagarikano O.

Lázaro M.T.

Lu X.H.

Gordon A.

Dong H.

Lam H.A.

Peles E.

Maidment N.T.

Murphy N.P.

Yang X.W.

et al. Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism. Oxytocin was obtained from Tocris Bioscience (product 1910) and solubilized in 10% dimethyl sulfoxide (DMSO) in PBS. 10% DMSO in PBS was used as the vehicle control. Mice received oxytocin intranasally (at approximately 200μg/kg) 30 min prior to behavior. 1.25 μL of oxytocin or vehicle solution was injected into each nostril from P10 pipette. Oxytocin dose was selected according to dosages reported to rescue social behavior in genetic models phenotypically expressing ASD-like behaviors ().

Electrophysiology Huang et al., 2016 Huang W.

Placzek A.N.

Viana Di Prisco G.

Khatiwada S.

Sidrauski C.

Krnjević K.

Walter P.

Dani J.A.

Costa-Mattioli M. Translational control by eIF2α phosphorylation regulates vulnerability to the synaptic and behavioral effects of cocaine. 3 , 25; KCl, 2.5; NaH 2 PO 4 , 1.25; MgCl 2 , 7; CaCl 2 , 0.5; dextrose, 25; sucrose, 75. Horizontal slices were cut with a vibrating tissue slicer (VF-100 Compresstome, Precisionary Instruments, San Jose, CA, or Leica VT 1000S, Leica Microsystems, Buffalo Grove, IL), incubated at 34°C for 40 min, kept at room temperature for at least 30 min before their transfer to a recording chamber continuously perfused with artificial cerebrospinal fluid (ACSF) at 32°C and a flow rate of 2–3 mL/min. The recording ACSF contained in mM: 120 NaCl, 3.3 KCl, 1.25 NaH 2 PO 4, 25 NaHCO 3, 10 Dextrose, 1 MgCl 2 and 2 CaCl 2 . Recording pipettes were made from thin-walled borosilicate glass (TW150F-4, WPI, Sarasota, FL). After filling with intracellular solution (in mM): 117 CsMeSO3; 0.4 EGTA; 20 HEPES; 2.8 NaCl, 2.5 ATP-Mg 2.0; 0.25 GTP-Na; 5 TEA-Cl, adjusted to pH 7.3 with CsOH and 290 mosmol/L, they had a resistance of 3–5 MΩ. Data were obtained with a MultiClamp 700B amplifier, digitized at 20 kHz with a Digidata 1440A, recorded by Clampex 10 and analyzed with Clampfit 10 software (Molecular Devices). Recordings were filtered online at 3 kHz with a Bessel low-pass filter. A 2 mV hyperpolarizing pulse was applied before each EPSC to evaluate the input and access resistance (Ra). Data were discarded when Ra was either unstable or greater than 25MΩ, holding current was >200 pA, input resistance dropped >20% during the recording, or EPSCs baseline changed by >10%. After establishing a gigaohm seal (>2GΩ) and recording stable spontaneous firing in cell-attached, voltage clamp mode (−70 mV holding potential), cell phenotype was determined by measuring the width of the action potential and the presence of an I h current. AMPAR/NMDAR ratios were calculated as previously described ( Huang et al., 2016 Huang W.

Placzek A.N.

Viana Di Prisco G.

Khatiwada S.

Sidrauski C.

Krnjević K.

Walter P.

Dani J.A.

Costa-Mattioli M. Translational control by eIF2α phosphorylation regulates vulnerability to the synaptic and behavioral effects of cocaine. A -mediated IPSCs. After recording the dual-component EPSC, DL-AP5 (100 μM) was bath-applied for 10 min to remove the NMDAR component, which was then obtained by offline subtraction of the remaining AMPAR component from the original EPSC. The peak amplitudes of the isolated components were used to calculate the AMPAR/NMDAR ratios. Picrotoxin and DL-AP5 were purchased from Tocris Bioscience and all other reagents were obtained from Sigma-Aldrich. Recordings were performed as recently described () and the investigators were kept blind to treatment conditions. Briefly, mice were anesthetized with a mixture of ketamine (100 mg/kg), xylazine (10 mg/kg), and acepromazine (3 mg/kg). Horizontal slices (225–300 μm thick) containing the VTA were cut from the brains of C57BL/6J mice. Mice were transcardially perfused with an ice-cold, oxygenated solution containing (in mM) NaCl, 87; NaHCO, 25; KCl, 2.5; NaHPO, 1.25; MgCl, 7; CaCl, 0.5; dextrose, 25; sucrose, 75. Horizontal slices were cut with a vibrating tissue slicer (VF-100 Compresstome, Precisionary Instruments, San Jose, CA, or Leica VT 1000S, Leica Microsystems, Buffalo Grove, IL), incubated at 34°C for 40 min, kept at room temperature for at least 30 min before their transfer to a recording chamber continuously perfused with artificial cerebrospinal fluid (ACSF) at 32°C and a flow rate of 2–3 mL/min. The recording ACSF contained in mM: 120 NaCl, 3.3 KCl, 1.25 NaHPO25 NaHCO10 Dextrose, 1 MgCland 2 CaCl. Recording pipettes were made from thin-walled borosilicate glass (TW150F-4, WPI, Sarasota, FL). After filling with intracellular solution (in mM): 117 CsMeSO3; 0.4 EGTA; 20 HEPES; 2.8 NaCl, 2.5 ATP-Mg 2.0; 0.25 GTP-Na; 5 TEA-Cl, adjusted to pH 7.3 with CsOH and 290 mosmol/L, they had a resistance of 3–5 MΩ. Data were obtained with a MultiClamp 700B amplifier, digitized at 20 kHz with a Digidata 1440A, recorded by Clampex 10 and analyzed with Clampfit 10 software (Molecular Devices). Recordings were filtered online at 3 kHz with a Bessel low-pass filter. A 2 mV hyperpolarizing pulse was applied before each EPSC to evaluate the input and access resistance (Ra). Data were discarded when Ra was either unstable or greater than 25MΩ, holding current was >200 pA, input resistance dropped >20% during the recording, or EPSCs baseline changed by >10%. After establishing a gigaohm seal (>2GΩ) and recording stable spontaneous firing in cell-attached, voltage clamp mode (−70 mV holding potential), cell phenotype was determined by measuring the width of the action potential and the presence of an Icurrent. AMPAR/NMDAR ratios were calculated as previously described (). Briefly, neurons were voltage-clamped at +40 mV until the holding current stabilized (at <200 pA). Monosynaptic EPSCs were evoked at 0.05 Hz with a bipolar stimulating electrode placed 50–150 μm rostral to the lateral VTA. Picrotoxin (100 μM) was added to the recording ACSF to block GABAR-mediated IPSCs. After recording the dual-component EPSC, DL-AP5 (100 μM) was bath-applied for 10 min to remove the NMDAR component, which was then obtained by offline subtraction of the remaining AMPAR component from the original EPSC. The peak amplitudes of the isolated components were used to calculate the AMPAR/NMDAR ratios. Picrotoxin and DL-AP5 were purchased from Tocris Bioscience and all other reagents were obtained from Sigma-Aldrich.