Currently, there are no medications that effectively treat the core symptoms of Autism Spectrum Disorder (ASD). We recently found that the bacterial species Lactobacillus (L.) reuteri reverses social deficits in maternal high-fat-diet offspring. However, whether the effect of L. reuteri on social behavior is generalizable to other ASD models and its mechanism(s) of action remains unknown. Here, we found that treatment with L. reuteri selectively rescues social deficits in genetic, environmental, and idiopathic ASD models. Interestingly, the effects of L. reuteri on social behavior are not mediated by restoring the composition of the host’s gut microbiome, which is altered in all of these ASD models. Instead, L. reuteri acts in a vagus nerve-dependent manner and rescues social interaction-induced synaptic plasticity in the ventral tegmental area of ASD mice, but not in oxytocin receptor-deficient mice. Collectively, treatment with L. reuteri emerges as promising non-invasive microbial-based avenue to combat ASD-related social dysfunction.

In addition, we sought to identify the mechanism by which a given bacterial strain (L. reuteri) regulates a selective behavior or disease state, which represents one of the most important challenges in microbiome research. More specifically, integrating multiple approaches, such as genetics, metagenomics, targeted vagotomies, immunohistochemistry, electrophysiology, and behavior, we began to dissect how L. reuteri impacts brain function. At the cellular and molecular levels, we provide new causal evidence that L. reuteri modulates social behavior and related changes in synaptic function within the social reward circuits via the oxytocinergic system. Moreover, at the systems level, we found that L. reuteri modulates social behavior independent of other microbes in the gut, and in a vagus nerve-dependent manner. Collectively, our findings provide new mechanistic insight into the gut-brain-axis signaling by which L. reuteri influences central nervous system function and selective behaviors.

Studies in animal models have shown that gut microbes can modulate central nervous system (CNS)-driven behaviors in a very powerful way (). Recently, in mice, we showed that maternal high-fat diet (MHFD) induces social deficits and a change in the gut microbiota of offspring that is characterized by a reduction of the commensal bacterial species L. reuteri (). Consistent with these data, HFD-induced obesity in adult mice also leads to a reduction in the levels of L. reuteri (). More importantly, selective treatment with L. reuteri reverses the social deficits in MHFD offspring (). However, whether L. reuteri can also rescue the social deficits of ASD models with different underlying etiologies remains unknown. Here, we first studied whether L. reuteri is able to reverse the social deficits in other mouse models of ASD. Surprisingly, we found that microbial treatment with this single bacterial species rescues the impaired social behavior in genetic, environmental, and idiopathic mouse models of ASD.

The prevalence of autism spectrum disorder (ASD), which is influenced by both genetic and environmental factors (), continues to increase worldwide (). However, effective treatments for ASD remain elusive. Defined as a heterogeneous neurodevelopmental disorder, ASD is characterized by social deficits, repetitive behaviors, and language difficulties (). In addition to these core symptoms, ASD patients are often afflicted with gastrointestinal (GI) issues (). In fact, children with ASD are 3.5 times more likely to suffer from GI disorders than children without developmental disorders (). Moreover, GI problems have been associated with changes in the microbial communities inhabiting the gut of ASD individuals ().

Strikingly, treatment with either L. reuteri or oxytocin failed to rescue the deficits in social behaviors ( Figure 7 C) and related changes in VTA DA synaptic function in DA-Oxtrmice ( Figure 7 D). By contrast, cocaine was able to induce LTP in VTA DA neurons of DA-Oxtrmice ( Figure 7 D), indicating that oxytocin signaling is selectively required for social interaction-induced synaptic plasticity in the reward circuitry. Consistently, administration of the selective oxytocin receptor antagonist L-371,257 to Shank3Bmice prevented the prosocial effects associated with L. reuteri treatment ( Figure S14 ). Collectively, these data show that L. reuteri treatment promotes social behaviors and social interaction-mediated synaptic potentiation in an oxytocin-dependent manner.

Because PVN-VTA oxytocinergic projections are crucially involved in social behaviors () and that L. reuteri and oxytocin restore social interaction-induced plasticity in VTA DA neurons of ASD models ( Figure 7 B), we wondered whether the effect of L. reuteri on social behavior and related changes in social-induced plasticity in the VTA depends on oxytocin signaling. To answer this question, we conditionally deleted oxytocin receptors (Oxtr) in DA neurons (see STAR Methods ). Deletion of Oxtr in DA neurons in DA-Oxtrmice had no impact on the levels of L. reuteri in the gut ( Figure S13 ). However, compared to control mice, we found that mice lacking Oxtr in DA neurons (DA-Oxtr) were socially impaired ( Figure 7 C), consistent with a recent report ().

As we recently reported (), reciprocal social interaction increased the AMPAR/NMDAR in VTA DA neurons from control mice ( Figure 7 B). By contrast, the same procedure failed to induce synaptic potentiation in Shank3Bmice ( Figure 7 B). The inability to induce social interaction-induced synaptic potentiation in Shank3BVTA DA neurons was not due to changes in baseline activity or to the failure to respond to any stimuli. In fact, baseline AMPAR/NMDAR ratios were similar in Shank3Bmice and WT littermates ( Figure 7 B), and a single injection of cocaine, which has been shown to increase the AMPAR/NMDAR ratio in VTA DA neurons ( Figure S12 D), evoked LTP in VTA DA neurons of Shank3Bmice ( Figure 7 B). Thus, the synaptic potentiation associated specifically with social reward is impaired in Shank3Bmice. More importantly, we found that L. reuteri rescues the deficits in social interaction-induced VTA plasticity in Shank3Bmice ( Figure 7 B). Consistent with the notion that the effect of L. reuteri on social behavior is mediated by oxytocin, intranasal oxytocin administration also rescued the impaired LTP in VTA DA neurons from Shank3Bmice ( Figure 7 B). Hence, social interaction-induced synaptic plasticity is impaired in Shank3Bmice but restored by treatment with L. reuteri or oxytocin.

Brain regions responding 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 (). Moreover, optogenetic stimulation of VTA-NAc projections promotes social interaction (). Social interaction, which can be particularly rewarding, triggers synaptic potentiation in VTA dopamine (DA) neurons of both birds () and mice (). Since the oxytocinergic system is impaired in Shank3Bmice, we hypothesized that social interaction-induced VTA plasticity would be deficient in these mice. To test this hypothesis, we recorded direct social interaction-evoked long-term potentiation (LTP) in VTA DA neurons ( Figures 7 A and S12 A–S12C) by measuring the ratio of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) to N-methyl-d-aspartate receptor (NMDAR) currents, as we previously described ().

(D) AMPA/NMDA ratio of DA neurons in the lateral VTA at baseline and 24 hr after reciprocal social interaction (n = 6–7 per group; DA-Oxtr+ vehicle social interaction versus DA-Oxtrsocial interaction + L. reuteri, t = 0.02, p > 0.99; DA-Oxtr+ vehicle social interaction versus Controls baseline, t = 0.40, p > 0.99; DA-Oxtr+ vehicle social interaction versus DA-Oxtr+ cocaine, t = 5.47, p < 0.0001; DA-Oxtr+ vehicle social interaction versus DA- Oxtr+ vehicle, t = 0.44, p > 0.99, DA-Oxtr+ vehicle social interaction versus Controls social interaction, t = 4.92, p < 0.001; DA-Oxtr+ vehicle social interaction versus DA-Oxtr+ oxytocin social interaction, t = 0.089, p > 0.99; DA-Oxtr+ L. reuteri social interaction versus Controls baseline, t = 0.37, p > 0.99; DA-Oxtr+ L. reuteri social interaction versus DA- Oxtr+ cocaine, t = 5.49, p < 0.0001; DA-Oxtr+ L. reuteri social interaction versus DA-Oxtr+ vehicle, t = 0.47, p > 0.99; DA-Oxtr+ L. reuteri social interaction versus Controls social interaction: t = 4.94, p < 0.001; Controls baseline versus DA-Oxtr+ cocaine, t = 6.08, p < 0.0001; Controls baseline versus DA-Oxtr+ vehicle, t = 0.88, p > 0.99; Controls baseline versus Controls social interaction, t = 5.50, p < 0.0001; Controls baseline versus DA-Oxtr+ oxytocin social interaction, t = 0.32, p > 0.99; DA-Oxtr+ cocaine versus DA-Oxtr+ vehicle, t = 5.23, p < 0.001; DA-Oxtr+ cocaine versus Controls social interaction, t = 0.55, p > 0.99; DA-Oxtr+ cocaine versus DA-Oxtr+ oxytocin social interaction, t = 5.76, p < 0.0001; DA- Oxtr+ vehicle versus Controls social interaction, t = 4.66, p < 0.001; DA-Oxtr+ vehicle versus DA-Oxtr+ oxytocin social interaction, t = 0.55, p > 0.99; Controls social interaction versus DA-Oxtr+ oxytocin social interaction, t = 5.19, p < 0.001; one-way ANOVA, F= 13.46, p < 0.0001). ns, not significant. Plots show mean ± SEM. See also Figures S12–S14

(C) Reciprocal social interaction was impaired in DA-Oxtr −/− mice, and neither L. reuteri or oxytocin reversed the social deficits in these mice (n = 10–12 pairs per group; Controls versus DA-Oxtr −/− + vehicle, t = 6.095, p < 0.0001; Controls versus DA-Oxtr −/− + L. reuteri, t = 5.517, p < 0.0001; Controls versus DA-Oxtr −/− + oxytocin, t = 5.148, p < 0.0001; DA-Oxtr −/− + vehicle versus DA-Oxtr −/− + L. reuteri, t = 0.6059, p > 0.99; DA-Oxtr −/− + vehicle versus DA-Oxtr −/− + oxytocin, t = 0.8639, p > 0.99; DA-Oxtr −/− + L. reuteri versus DA-Oxtr −/− + oxytocin, t = 0.2712, p > 0.99; one-way ANOVA, F 3,41 = 15.34, p < 0.0001).

(B) AMPAR/NMDAR ratio in lateral VTA DA neurons recorded at baseline and 24 hr after reciprocal social interaction (n = 6–8; WT baseline versus Shank3B −/− + vehicle baseline, t = 1.81, p > 0.99; WT baseline versus WT social interaction, t = 5.70, p < 0.0001; WT baseline versus Shank3B −/− + vehicle social interaction, t = 0.29, p > 0.99; WT baseline versus Shank3B −/− + L. reuteri, t = 1.07, p > 0.99; WT baseline versus Shank3B −/− + L. reuteri social interaction, t = 4.04, p < 0.01; WT baseline versus Shank3B −/− + cocaine, t = 3.64, p < 0.05; WT baseline versus Shank3B −/− + oxytocin social interaction, t = 4.29, p < 0.01; Shank3B −/− baseline + vehicle versus WT social interaction, t = 7.51, p < 0.0001; Shank3B −/− + vehicle baseline versus Shank3B −/− + vehicle social interaction, t = 1.58, p > 0.99; Shank3B −/− + vehicle baseline versus Shank3B −/− + L. reuteri, t = 0.66, p > 0.99, Shank3B −/− + vehicle baseline versus Shank3B −/− + L. reuteri social interaction, t = 5.78, p < 0.0001; Shank3B −/− + vehicle baseline versus Shank3B −/− + cocaine, t = 5.38, p < 0.0001; Shank3B −/− + vehicle baseline versus Shank3B −/− + oxytocin social interaction t = 6.1, p < 0.0001; WT social interaction versus Shank3B −/− + vehicle social interaction, t = 6.18, p < 0.0001; WT social interaction versus Shank3B −/− + L. reuteri, t = 6.55, p < 0.0001; WT social interaction versus Shank3B −/− + L. reuteri social interaction, t = 1.43, p > 0.99; WT social interaction versus Shank3B −/− + cocaine, t = 1.83, p > 0.99; WT social interaction versus Shank3B −/− + oxytocin social interaction, t = 1.41, p > 0.99; Shank3B −/− + vehicle social interaction versus Shank3B −/− + L. reuteri, t = 0.82, p > 0.99; Shank3B −/− + vehicle social interaction versus Shank3B −/− + L. reuteri social interaction, t = 4.44, p < 0.01; Shank3B −/− + vehicle social interaction versus Shank3B −/− + cocaine, t = 4.03, p < 0.01; Shank3B −/− + vehicle social interaction versus Shank3B −/− + oxytocin social interaction, t = 4.72, p < 0.001; Shank3B −/− + L. reuteri versus Shank3B −/− + L. reuteri social interaction, t = 4.93, p < 0.001; Shank3B −/− + L. reuteri versus Shank3B −/− + cocaine, t = 4.55, p < 0.01; Shank3B −/− + L. reuteri versus Shank3B −/− + oxytocin social interaction, t = 5.19, p < 0.001; Shank3B −/− + L. reuteri social interaction versus Shank3B −/− + cocaine, t = 0.38, p > 0.99; Shank3B −/− + L. reuteri social interaction versus Shank3B −/− + oxytocin social interaction, t = 0.07, p > 0.99; Shank3B −/− + cocaine versus Shank3B −/− + oxytocin social interaction, t = 0.47, p > 0.99; one-way ANOVA, F 7,46 = 16.68, p < 0.0001). Representative AMPA and NMDA traces are shown above each group.

Oxytocin injected into the ventral tegmental area induces penile erection and increases extracellular dopamine in the nucleus accumbens and paraventricular nucleus of the hypothalamus of male rats.

L. reuteri Restores Social Interaction-Induced Synaptic Potentiation in the Ventral Tegmental Area of Shank3B −/− Mice, but Not in Mice Lacking the Oxytocin Receptor in Dopaminergic Neurons

Given that L. reuteri treatment promotes oxytocin immunoreactivity in the PVN of Shank3Bmice and rescues their social deficits, we hypothesized that oxytocin administration would also reverse the social deficits in Shank3Bmice. As predicted, intranasal oxytocin ( Figure 6 A) reversed the social deficits in Shank3Bmice ( Figures 6 B, 6C , and S10 A). Moreover, oxytocin improved the social behaviors that are deficient in VPA ( Figures S11 A and S11B), BTBR mice ( Figures S11 C–S11E), and partially in GF mice ( Figures S11 F–S11H), all models in which L. reuteri effectively rescues their social deficits. Interestingly, like L. reuteri treatment, oxytocin had no effect on the hypo-activity behavior in the Shank3Bmice ( Figures S10 B and S10C). Together, these data suggest that oxytocinergic signaling is involved in the mechanism of action by which L. reuteri selectively restores social behavior in several ASD mouse models.

(D and E) In Shank3Bvagotomized mice, oxytocin also improved social behavior deficits in the three-chamber test (D, Sociability, n = 7 per group; Shank3BVagotomized + L. reuteri, t = 1.35, p = 0.33; Shank3BVagotomized + oxytocin, t = 2.76, p < 0.05, two-way ANOVA, F= 8.46, p = 0.0077) and reciprocal social interaction test (E, n = 5–6 pairs per group; Shank3BVagotomized + L. reuteri versus Shank3BVagotomized + oxytocin, Mann-Whitney Test, p < 0.05). ns, not significant. Plots show mean ± SEM. See also Figures S10 and S11

(B and C) In Shank3B −/− mice, oxytocin improved social behavior deficits in the three-chamber test (B, Sociability, n = 8–10 per group; WT, t = 3.84, p < 0.01; Shank3B −/− + vehicle, t = 0.28, p > 0.99; Shank3B −/− + oxytocin, t = 2.88, p < 0.05; two-way ANOVA, F 2,48 = 2.94, p = 0.06) and reciprocal social interaction test (C, n = 7–9 pairs per group; WT versus Shank3B −/− + vehicle, t = 2.70, p < 0.05; Shank3B −/− + vehicle versus Shank3B −/− + oxytocin, t = 4.02, p < 0.01; WT versus Shank3B −/− + oxytocin, t = 1.45, p = 0.49; one-way ANOVA, F 2,19 = 0.0025).

We and others have shown that L. reuteri increases oxytocin levels (). Interestingly, we found a reduction in oxytocin-positive neurons in the PVN ( Figure 5 A) of Shank3Bmice compared to WT littermates ( Figures 5 B and 5C). The number and fluorescence intensity of NeuN-positive neurons did not change in the PVN of Shank3Bmice ( Figures 5 B, 5D, and 5F), indicating that the decrease in oxytocin-positive neurons in Shank3Bwas not due to a decrease in the total number of neurons. As expected, L. reuteri treatment increased both the number and fluorescence intensity of oxytocin-positive neurons in Shank3Bmice ( Figures 5 B, 5C, and 5E).

(C–F) Oxytocin-positive cell number (C, n = 5 mice per group; WT versus Shank3B −/− + vehicle, t = 2.96, p < 0.05; Shank3B −/− + vehicle versus Shank3B −/− + L. reuteri: t = 3.64, p < 0,05; WT versus Shank3B −/− + L. reuteri, t = 0.59, p > 0.99; one-way ANOVA, F 2,12 = 7.28, p = 0.0085) and oxytocin immunofluorescence intensity (E, n = 5 mice per group; WT versus Shank3B −/− + vehicle, t = 5.38, p < 0.001; Shank3B −/− + vehicle versus Shank3B −/− + L. reuteri, t = 4.41, p < 0.01; WT versus Shank3B −/− + L. reuteri, t = 0.96, p = 0.0025; one-way ANOVA, F 2,12 = 16.49, p = 0.0004) are restored after L. reuteri treatment. The number (D, n = 5 mice per group, WT versus Shank3B −/− + vehicle, t = 0.62, p > 0.99; Shank3B −/− + vehicle versus Shank3B −/− + L. reuteri, t = 1.01, p = 0.99, WT versus Shank3B −/− + L. reuteri, t = 0.38, p > 0.99; one-way ANOVA, F 2.12 = 0.52, p = 0.60) and the fluorescence intensity (F, n = 5 mice per group, WT versus Shank3B −/− + vehicle, t = 1.34, p = 0.61; Shank3B −/− + vehicle versus Shank3B −/− + L. reuteri, t = 1.42, p = 0.54; WT versus Shank3B −/− + L. reuteri, t = 0.07, p > 0.99; one-way ANOVA, F 2,12 = 1.27, p = 0.31) of NeuN-positive cells did not change between the groups. ns, not significant. Plots show mean ± SEM.

Oxytocin modulates numerous aspects of social behaviors and is implicated in ASD (). Treatment with oxytocin reverses social deficits in several mouse models of ASD (). Additionally, it is known that vagal nerve fibers project to the PVN (), a brain region where oxytocin is produced, and that neuronal activity in the PVN induced by bacterial colonization is blocked by subdiaphragmatic vagotomy (). Finally, stimulation of the vagus nerve can promote oxytocin release ().

Increased plasma levels of oxytocin in response to afferent electrical stimulation of the sciatic and vagal nerves and in response to touch and pinch in anaesthetized rats.

Evidences for vagus nerve in maintenance of immune balance and transmission of immune information from gut to brain in STM-infected rats.

We then investigated whether, for L. reuteri, the vagus nerve could serve as a channel of communication between the gut and the brain. Previous studies have shown that the vagus nerve is activated in response to specific bacteria () and that the Lactobacillus species L. rhamnosus reduces anxiety-related behavior in a vagus-dependent manner (). To determine whether the vagus nerve is required for L. reuteri to reverse the social deficits in Shank3Bmice, we performed bilateral subdiaphragmatic vagotomy in these mice ( Figure 4 C). Control mice (sham-operated mice) underwent the same surgical procedures, except that the vagal branches were not transected. If the vagus nerve is required for the relevant gut-microbiota-brain communication, L. reuteri should fail to rescue the social deficits in vagotomized Shank3Bmice. Consistent with this hypothesis, we found that L. reuteri rescued social behaviors in sham-operated, but not in vagotomized Shank3Bmice ( Figures 4 D and 4F). Results from several control experiments underscore the specificity of the vagotomy to the effects of L. reuteri on social behavior. First, the vagotomy was complete, as determined by a food intake analysis based on the satiating effect of cholecystokinin-octapeptide (CCK-8), which depends on the vagus nerve ( Figure S9 A). Second, social behavior was similar in WT-sham and WT-vagotomized mice ( Figures S9 B–S9D). Moreover, preference for social novelty, which is not impaired in Shank3Bmice, was not affected by the vagotomy ( Figure 4 E), indicating that the vagotomy itself had no effect on social behavior. Third, vagotomy did not affect motor behavior ( Figures S9 E–S9H). Finally, intranasal oxytocin-treatment, which bypasses the gut-microbiota-brain communication channel, restored social behaviors in vagotomized Shank3Bmice (see Figures 6 D and 6E), demonstrating that the vagotomy procedure did not prevent the animals’ ability to perform the social task. Thus, L. reuteri reversed the social deficits in Shank3Bmice in a vagus nerve-dependent manner.

It is widely accepted that there is a bidirectional gut-microbiota-brain axis, where bacteria initiate signals that are transmitted from the gut to the CNS, either through blood circulation or via the vagus nerve (). If the integrity of the host’s intestinal barrier is damaged and rendered more permeable (so-called “leaky gut”), which occurs when the gut epithelium tight junctions are impaired (), bacteria and/or metabolites produced by bacteria can enter the blood circulation. Indeed, alterations in gut permeability have been described in individuals with ASD (). Thus, we first tested whether Shank3Bmice show increased intestinal permeability. To this end, we administered fluorescein isothocyanate-dextran (FITC-dextran) by oral gavage and measured its concentration in the serum, as described (). If the intestinal barrier is compromised in Shank3Bmice, we would expect increased FITC-dextran in the serum of these mice. As expected, our positive control mice administrated with dextran sodium sulfate (DSS), a chemical which induces colitis, exhibited a leaky gut, as determined by increased FITC-dextran in their serum ( Figure 4 A). By contrast, compared to control littermates, Shank3Bmice showed no changes in gut permeability ( Figure 4 A). Accordingly, the expression of key tight junction proteins was not altered in Shank3Bmice, as determined by reverse transcription followed by quantitative PCR (RT-qPCR) ( Figure 4 B). Thus, Shank3Bmice did not show major alterations in gut permeability.

(D–F) In vagotomized Shank3Bmice, L. reuteri failed to reverse social behavior deficits in the three-chamber test (D, Sociability, n = 7 per group; Shank3BSham + L. reuteri, t = 3.22, p < 0.05; Shank3BVagotomized + L. reuteri, t = 1.33, p = 0.35; two-way ANOVA, F= 10,4, p = 0.003; E, Social Novelty, n = 7 per group; Shank3BSham + L. reuteri, t = 2.56, p < 0.05; Shank3BVagotomized + L. reuteri: t = 2.60, p < 0.05; two-way ANOVA, F= 0.028, p = 0.86) and reciprocal social interaction test (F, n = 6–7 pairs per group; Shank3BSham + L. reuteri versus Shank3BVagotomized + L. reuteri, Mann-Whitney Test p < 0.01). DSS, dextran sodium sulfate; ns, not significant. Plots show mean ± SEM. See also Figure S9

To causally demonstrate whether the effects of L. reuteri on social behavior are dependent on its interaction with other members of the microbial community, mice raised under sterile conditions (i.e., germ-free [GF] mice) were monocolonized with L. reuteri at weaning, and social behavior was tested in 8-week-old mice. As previously described, unlike conventionally colonized mice, GF mice displayed social deficits (). Remarkably, monocolonization with L. reuteri was sufficient to reverse the social deficits in GF mice ( Figures 3 B–3D), supporting the notion that L. reuteri acts solo, rescuing social behavior in the absence of other members of the community.

To dissect the mechanism(s) through which L. reuteri restores social behavior, we mainly focused on the Shank3Bmodel. We first asked whether L. reuteri exerts its effects on social behaviors directly, or indirectly by correcting the altered microbial composition of the host. To answer this question, we first analyzed the composition of the gut microbiota in vehicle-treated and L. reuteri-treated Shank3Bmice, as determined by 16S rRNA gene sequencing. We found that treatment with L. reuteri did not significantly alter the microbial profile of either Shank3Bmice ( Figure 3 A) or BTBR mice ( Figure S8 ). Hence, L. reuteri has no significant impact in the overall microbial composition of the host.

(B–D) In GF mice, L. reuteri reversed social behavior deficits in the three-chamber test (B, Sociability, n = 8–16 per group; Controls, t = 5.44, p < 0.0001; GF + vehicle, t = 1.87, p = 0.20; GF + L. reuteri, t = 3.85, p < 0.001; two-way ANOVA, F= 16.64, p < 0.0001; C, Social Novelty, n = 8–16 per group; Controls, t = 3.00, p < 0.05; GF + vehicle, t = 1.28, p = 0.61; GF + L. reuteri, t = 4.10, p < 0.001; two-way ANOVA, F= 7.63, p = 0.0010) and the reciprocal social interaction test (D, n = 8–13 pairs per group; Controls versus GF + vehicle, t = 5.89, p < 0.0001; Controls versus GF + L. reuteri, t = 0.77, p > 0.99; GF + vehicle versus GF + L. reuteri, t = 6.03, p < 0.0001; one-way ANOVA, F= 24.87, p < 0.0001). ns, not significant. Plots show mean ± SEM. See also Figure S8

(A) PCoA of unweighted UniFrac distances from the 16S rRNA gene-sequencing dataset shows that treatment with L. reuteri did not significantly alter the clustering of Shank3B −/− samples (n = 6–10 per group; p < 0.001; R 2 = 0.215, 8,024 reads/sample).

L. reuteri Does Not Change the Microbial Composition of Shank3B −/− Mice and Is Sufficient to Rescue Social Deficits in Germ-free Mice

Figure 3 L. reuteri Does Not Change the Microbial Composition of Shank3B −/− Mice and Is Sufficient to Rescue Social Deficits in Germ-free Mice

While previous studies have shown that BTBR mice harbor a different microbial community in the gut () and cohousing with C57BL/6J mice has been shown to restore their social deficits (), the nature of the bacterial species which may be causally related to the social behavioral deficits in this ASD model remains unknown. Accordingly, we also found a markedly altered gut microbiome composition in BTBR mice compared to C57BL/6J mice ( Figures 2 A and S7 ). Moreover, we found a specific reduction in L. reuteri levels in BTBR mice ( Figure 2 B). Given that both Shank3Band BTBR mice exhibit alterations in their gut microbial composition and decreased L. reuteri levels and that L. reuteri is sufficient to restore the social behavior in Shank3Band VPA mice, we next wondered whether this selective microbial intervention would also be effective in BTBR mice. Indeed, treatment with L. reuteri improved the social deficits in the three chamber and reciprocal social interaction tasks in the BTBR mice ( Figures 2 C–2E). Similarly to the Shank3Bmice, social deficits in BTBR mice were not due to impaired olfaction (see Figure S3 B). Taken together, our data show that treatment with L. reuteri selectively reverses the ASD-like social deficits in genetic, environmental, and idiopathic models of ASD.

(C–E) In BTBR mice, L. reuteri rescued social behavior deficits in the three-chamber test (C, Sociability, n = 16–17 per group; C57BL/6J, t = 7.033, p < 0.0001; BTBR + vehicle, t = 0.93, p > 0.99; BTBR + L. reuteri: t = 2.75, p < 0.05; two-way ANOVA, F= 9.54, p < 0.001; D, Social Novelty, n = 16–17 per group; C57BL/6J, t = 6.35, p < 0.0001; BTBR + vehicle, t = 0.67, p > 0.99; BTBR + L. reuteri, t = 2.87, p < 0.05; two-way ANOVA, F= 7.935, p < 0.001) and the reciprocal social interaction test (E, n = 8–11 pairs per group; C57BL/6J versus BTBR + vehicle, t = 3.03, p < 0.05; BTBR + vehicle versus BTBR + L. reuteri: t = 4.57, p < 0.001; C57BL/6J versus BTBR + L. reuteri, t = 1.25, p = 0.67; one-way ANOVA, F= 10.79, p < 0.001). ns, not significant. Plots show mean ± SEM. See also Figures S6 S7 , and S15

Microbiota-related changes in bile acid and tryptophan metabolism are associated with gastrointestinal dysfunction in a mouse model of autism.

Several environmental and genetic factors have been identified to be associated with increased incidence of ASD (). However, the majority of the ASD cases remain idiopathic. The BTBR T+ Itpr3tf/J (BTBR) inbred mouse line exhibits the core ASD symptoms, including abnormal social behavior (). Given that no genetic variants in ASD risk genes have been identified in the BTBR genome (), this model is considered an idiopathic model of ASD ().

Given that (1) VPA alters oxytocin-mediated changes in synaptic transmission (), (2) intranasal oxytocin rescues social deficits in VPA-exposed mice () and other mouse models of ASD (), and (3) L. reuteri promotes oxytocin levels (), we next wondered whether treatment with L. reuteri would improve the social deficits in the offspring from VPA mice. Interestingly, unlike treatment with vehicle, treatment with L. reuteri ameliorates the social deficits in VPA mice ( Figures S6 G and S6H). Taken together, these data demonstrate that, like in the MHFD model, L. reuteri also corrects the social deficits in another environmental model of ASD (VPA) with alterations in the gut microbiome.

Environmental factors may account for a large proportion of the ASD cases (). In mammals, both pre- and post-natal periods are critical developmental windows ultimately influencing behavior during adulthood. During these early developmental periods, environmental factors can change the microbial composition of the host, and affect brain physiology and function via the so-called gut-microbiota-brain axis (). For instance, among the environmental factors, maternal exposure to different nutrients (e.g., HFD) and/or substances (e.g., valproic acid, VPA) during pregnancy has been associated with ASD (). More specifically, clinical studies have shown that maternal exposure to VPA, a branched short-chain fatty acid used as an antiepileptic drug, is associated with increased risk of ASD incidence in offspring (). Accordingly, in rodents, VPA administration during gestation leads to ASD-like behaviors (notably social deficits) in offspring (). Given that the MHFD environmental mouse model of ASD shows social deficits that are mediated by changes in the microbiome (), we wondered whether the VPA mouse model of ASD is also characterized by alterations in their microbial ecology. Indeed, we found changes in the composition of the microbiota of VPA-treated mice ( Figures S6 A–S6E), but L. reuteri levels were not reduced in these mice ( Figure S6 F).

The VPA Mouse Model of ASD Shows Alterations in the Composition of the Gut Microbiota, and Treatment with L. reuteri Rescues Its Social Deficits

Remarkably, treatment with L. reuteri rescued sociability in Shank3Bmice. Indeed, the performance of L. reuteri-treated Shank3Bmice was similar to that of WT controls ( Figure 1 E). To further support these findings, we next assessed reciprocal social interaction ( Figure 1 G) by measuring the amount of time that stranger, genotype- and treatment-matched mice spent interacting as pairs. Shank3Bmice interacted significantly less than WT littermates, and treatment with L. reuteri reversed the social deficits in the mutant mice ( Figure 1 H). It is noteworthy that Shank3Bmice are hypo-active, a condition that was not improved by L. reuteri ( Figure S5 ). Thus, treatment with L. reuteri selectively reverses the ASD-like social deficits in Shank3Bmice.

Social deficits are one of the most salient features of individuals with ASD (), and Shank3Bmice exhibit impaired social behavior (). To examine whether reduced L. reuteri levels in the gut of Shank3Bmice could account for their social behavioral deficits, Shank3Bmice were treated with either vehicle or L. reuteri, added daily in drinking water for 4 weeks ( Figure 1 C). Social behaviors were first examined in the three-chamber sociability and social novelty tests, as we previously described (). To test sociability, we compared the time that the experimental mouse spent interacting with either a stranger mouse (Mouse 1) or an empty wired cup (Empty, Figure 1 D). As expected, WT mice displayed normal sociability, as reflected by their preferential interaction with the stranger mouse. By contrast, Shank3Bmice showed no preference for the stranger mouse over the empty cup, indicating impaired sociability ( Figure 1 E). The social deficits in Shank3Bmice are not due to alterations in their olfactory ability, since olfaction was not impaired in these mice, as determined by the buried food test ( Figure S3 A). In addition, we found that, as in WT controls, Shank3Bmice displayed normal preference for social novelty, as they spent more time interacting with the novel mouse (Mouse 2) than with the familiar one (Mouse 1; Figure 1 F). Interestingly, we found that only one cohort of Shank3B heterozygous (Shank3B) mice showed impaired social behavior ( Figures S4 A, S4C, and S4E). Thus, given the weak social behavior deficits in Shank3Bmice, we focused on the homozygous knockout line (Shank3Bmice), which consistently showed an impairment in sociability ( Figures S4 A, S4C, and S4E).

Moreover, we found that Shank3Bmice specifically have lower levels of L. reuteri compared to their WT littermates ( Figure 1 B). Thus, Shank3Bmice exhibit an altered gut microbial composition, including decreased L. reuteri levels in their gut.

It is noteworthy that Shank3Bmice were obtained from heterozygous (Shank3B) breeding, as recommended for microbiome studies (), ruling out potential differences in maternal care and/or early life effects of the gut microbiome. More importantly, we found that the microbiome changes depend on the genotype, but not on cage housing conditions (the so called “cage effect”). For instance, WT mice clustered together regardless of their cage of origin (cage A, B, C, or D). The same is true for Shank3B(KO) mice ( Figure S2 ).

Variants in human SHANK3 lead to Phelan-McDermid syndrome and other non-syndromic ASDs (). Mice lacking the Shankβ isoform of the Shank3 gene (Shank3Bmice) exhibit ASD-like behaviors, including social deficits (). Given that individuals with ASD often show changes in their gut microbial ecology (), we wondered whether Shank3Bmice display alteration of their gut microbial composition. To answer this question, we performed 16S ribosomal RNA (rRNA) gene sequencing on fecal samples from Shank3Bmice and wild-type (WT) littermates, as previously described (). Bacterial composition, computed based on weighted UniFrac distances (the assessment of community structure by considering abundance of operational taxonomic units, OTUs) or Shannon diversity index, was not significantly altered in Shank3Bmice compared to WT littermates ( Figures S1 A–S1D). By contrast, the bacterial diversity measured by unweighted UniFrac analysis, which assesses community structure by considering only the presence or absence of OTUs, revealed a significant difference in the phylogenetic profile of the microbial communities between genotypes ( Figure 1 A).

(A) Principal coordinates analysis (PCoA) of unweighted UniFrac distances from the 16S rRNA gene-sequencing dataset shows that Shank3B −/− samples clustered separately from those of WT littermates (n = 6–7 per group; p < 0.01, R 2 = 0.225; 8,024 reads/sample).

Shank3B −/− Mice Exhibit Changes in Their Microbial Composition, Reduced L. reuteri Levels, and Social Deficits that Were Rescued by Treatment with L. reuteri

Figure 1 Shank3B −/− Mice Exhibit Changes in Their Microbial Composition, Reduced L. reuteri Levels, and Social Deficits that Were Rescued by Treatment with L. reuteri

Discussion

Vuong and Hsiao, 2017 Vuong H.E.

Hsiao E.Y. Emerging roles for the gut microbiome in autism spectrum disorder. Strati et al., 2017 Strati F.

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Di Prisco G.V.

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Costa-Mattioli M. Microbial reconstitution reverses maternal diet-induced social and synaptic deficits in offspring. Hsiao et al., 2013 Hsiao E.Y.

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et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. While most of the focus in the ASD field has been on either genetic or environmental factors associated with the disorder, the gut microbiome lies at the interface between the host and its environment. Moreover, GI symptoms and changes in gut microbial composition are common in ASD individuals (). Indeed, while some patients with ASD present a decreased Bacteroidetes/Firmicutes ratio (), others show the opposite result (). We found that Shank3Bmice show no significant difference in abundance of the two phyla, but the BTBR and VPA models show an increased Bacteroidetes/Firmicutes ratio compared to control mice ( Figure S15 ). Hence, like in humans, changes in the Bacteroidetes/Firmicutes ratio may not be a reliable microbial signature associated with ASD-like behaviors, at least in the ASD mouse models studied here. More importantly, experiments in mouse models have shown that gut microbes can modulate endophenotypes traditionally associated with brain-centered disorders, including ASD (). Regardless of the initial insult triggering the disorder (genetic or environmental) and the extent of the alterations to the intestinal microbial communities, we found that L. reuteri reverses social deficits in genetic, environmental, and idiopathic models of ASD. Thus, our data (in mice) support the idea that microbial therapies could ameliorate specific endophenotypes associated with ASD. More importantly, while most of the studies in the microbiome field so far have been correlational in nature, where the composition of the microbiota is associated to a particular behavioral or disease state, we began to mechanistically dissect how a particular microbe in the gut impacts brain function and behavior. Our results showing that L. reuteri is able to reverse the social deficits in different mouse models of ASD hold promise for novel therapies in human patients. However, the gut-microbiota-brain axis is an emerging field, and to ensure the success of microbial-based therapies for neurological disorders, we believe that first it would be important to establish a set of defined and objective criteria for transitioning into human clinical trials. We discuss some of these criteria below:

Olson et al., 2018 Olson C.A.

Vuong H.E.

Yano J.M.

Liang Q.Y.

Nusbaum D.J.

Hsiao E.Y. The gut microbiota mediates the anti-seizure effects of the ketogenic diet. Second, it will be of great significance to determine whether the effect of the candidate microbe(s) is direct or indirect—that is, whether the microbe itself is driving the effect or instead modulating the abundance and/or function of other members of the microbial community, which in turn affect behavior. If the latter case, it would be key to define which member(s) of the community is the main player(s). This may ultimately determine whether the therapy will be based on only one microbe (monotherapy) or a combination of bacteria (probiotic cocktail;). Surprisingly, we found that L. reuteri alone is sufficient to reverse the social behavioral deficits in GF mice, demonstrating that its effect on social behavior does not depend on other members of the microbial community ( Figure 3 ).

Buffington et al., 2016 Buffington S.A.

Di Prisco G.V.

Auchtung T.A.

Ajami N.J.

Petrosino J.F.

Costa-Mattioli M. Microbial reconstitution reverses maternal diet-induced social and synaptic deficits in offspring. −/− and DA-Oxtr−/−) exhibit impaired social interaction-induced VTA plasticity and social behavior (−/− mice, but it fails to do so in DA-Oxtr−/− mice ( Hung et al., 2017 Hung L.W.

Neuner S.

Polepalli J.S.

Beier K.T.

Wright M.

Walsh J.J.

Lewis E.M.

Luo L.

Deisseroth K.

Dölen G.

Malenka R.C. Gating of social reward by oxytocin in the ventral tegmental area. In addition, we have previously found that social interaction-induced LTP in VTA DA neurons was impaired in MHFD mice and that L. reuteri reverses these long-lasting synaptic changes that likely underlie the social deficits (). However, it was not clear whether this form of plasticity was also impaired in genetic models of ASD. Here we found that, like the environmental MHFD model, genetic models of ASD (Shank3Band DA-Oxtr) exhibit impaired social interaction-induced VTA plasticity and social behavior ( Figures 1 and 7 ), indicating that there may be a convergent cellular and synaptic mechanism underlying the neuropathology associated with ASD. More importantly, treatment with both L. reuteri and oxytocin reverses the social interaction-induced LTP in VTA DA neurons from Shank3Bmice, but it fails to do so in DA-Oxtrmice ( Figure 7 D), consistent with the idea that L. reuteri-mediated increase in oxytocin enhances the salience and rewarding value of social stimuli. Accordingly, the release of oxytocin from PVN neurons onto VTA DA neurons increases excitability during social interaction (). Taken together, these data support the notion that social behavior (and its mechanisms) can be also studied at the cellular level by measuring changes in synaptic plasticity in VTA dopaminergic neurons upon social interaction.

Stock and Uvnäs-Moberg, 1988 Stock S.

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de Gelder B.

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Colzato L.S. Transcutaneous vagus nerve stimulation (tVNS) enhances recognition of emotions in faces but not bodies. Lastly, given that vagus nerve stimulation, like L. reuteri treatment, increases oxytocin levels () and transcutaneous vagus nerve stimulation enhances facial emotion recognition in humans (), it would be interesting to examine in future experiments whether selective stimulation of vagal afferents improves social behaviors.

Tabouy et al. (2018) Tabouy L.

Getselter D.

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et al. Dysbiosis of microbiome and probiotic treatment in a genetic model of autism spectrum disorders. −/− mice. Fourth, to corroborate the validity of the microbial-based therapy, the effect of the probiotic treatment on behavior should be reproduced by other laboratories. Indeed, consistent with our results, an independent study byhas recently found that L. reuteri successfully corrects social deficits in Shank3Bmice.