The purpose of this study was to assess the impact of broad-spectrum antibiotics treatment on social behavior in adulthood. To our best knowledge, this report is the first one to demonstrate that chronic depletion of the gut microbiota in adulthood induces profoundly similar social behavioral abnormalities to those observed in animals of the VPA rat model of ASD. Furthermore, as it was supposed, after the antibiotics treatment, our PM was able to re-establish the normal social behavior. In addition to that our PM treatment was capable to markedly reduce the social abnormalities in the VPA animal model, it also appears that even distinct changes of the microbiome could result in remarkable changes in a completely developed brain. According to our present findings, these changes take place in a non-SCFA dependent way.
The instantaneous effect that the GI microbiome exerts on the social behavior has been studied primarily with germ free animals (total lack of microbes) and with certain antibiotic treatments on rodents in their early pre-or post-natal period of life 29. These studies primarily revealed impairments of sociability. Furthermore, colonizing germ free mice with normal faecal microbiota was able to restore the sociability defects. Based on these results, it is reasonable to propose that the gut microbiota is involved in integratory processes of social development 38,39. Despite all these facts the effect of broad-spectrum antibiotics treatment on social behavior is less studied in adulthood. One of the fundamental findings of the current study highlights that the antibiotics induced bacterial depletion in adulthood can elicit the same type deficits of the social interaction as those observed in the VPA autism rat model. The sociability index results, indicating a preference to the stranger rat, presented that rats of the C-VPA- and ABx groups display social interaction deficits. These findings suggest that chronic broad-spectrum antibiotics treatment in adulthood negatively affects the social behavior, moreover, it seems as if these deficits were the same type as those we could identify in the VPA rat model of ASD. This finding appears to support the notion that the antibiotics-modified microbiome can act as a causal agent and a risk factor in the development of ASD 23,26,40. Despite these social abnormalities, group differences were not found in the total distance travelled and in the latencies to explore the stranger cage. Otherwise, a pilot experiment with LiCl induced visceral illness was not able to result in reduced sociability what we detected in the microbiome depleted ABx rats. These results suggest that impaired sociability is likely cannot be due to the general consequence of visceral discomfort or pain in these animals. The above data underline the importance of disruption of the healthy balance of microbial community and its specific impact on the microbiome-gut-brain axis that leads to the deficits of social behavior regardless of whether visceral discomfort exists or not.
It is established that antibiotics- or VPA treatments interfere with the physiology of the animals disparate ways. In the former case, antibiotics administration strongly depletes gut microbiota, and thus, triggers alterations of the microbiome-gut-brain axis that ultimately lead to behavioral (as well as molecular) deviations 30,41. In the latter case, prenatal VPA exposure was shown to modify histone deacetylase activity, to alter gamma-aminobutyric acid or Wnt (wingless-type) signaling, and/or to disturb axonal remodeling in the developing neurons 16,19,42. These mechanisms can provoke dysfunctions in several brain areas, generating morphological changes, especially in cortical and hippocampal regions 16,43. Nevertheless, our histological analysis revealed very high variability within and among the animals, hence, we could not verify that such differences (e.g. in the thickness of hippocampal regions) indeed exist between the VPA treated and other groups. Even though the treatments appeared to act divergent ways, we still noticed similar social behavioral alterations between these groups. Therefore, to more precisely explore the role of the GI microbiome in the development of ASD symptoms, we approached this issue from another direction and introduced a probiotic therapy to interfere these treatments. Previous studies have shown that application of certain probiotics have beneficial effects on antibiotics-induced physiological and psychological abnormalities 44,45. Moreover, as different gut microbial community was found in ASD patients in contrast to the healthy individuals, researchers attempted to modify the gut microbiome via probiotics and some results indicate beneficial effects on both behavioral and GI manifestations of ASD 46–49. In addition, VPA rat model not just imitate ASD symptoms, it also has a transgenerational impact on the gut microbiota 50–52. Nevertheless, limited research is available where probiotics are investigated in the VPA animal model as they could be effective therapy, only one study has recently revealed that VPA induced behavioral alterations could be reduced by daily supplementation with Lactobacillus strains 53. In our present study, it is demonstrated for the first time that specified PM can be a potential novel approach to improve social behavioral alterations both in the VPA- or antibiotics induced animal model. Our PM was able to improve the preference to the stranger rat in both of the C-VPA and ABx treated groups, thus antisocial behavior was reduced. Moreover, the present results demonstrated that the same behavior can be seen in the C-VPA and ABx treated groups as it appeared in the control group. However, the PM itself was not able to significantly change all aspects of the behavior, only in the frequency of the social zone exploration was detected notable difference between the probiotic and the control rats, but this was presumably generated by the fact that probiotic treated animals spent more time in the social zone once they entered there.
Despite the fact that both models developed by different mechanisms, quite similar social behavioral abnormalities were noticed, additionally, our PM was able to reconstruct these behavioral phenomena just as they appear in the control rats. Regarding these consequences, it is suggested that in both models the protective effects of the probiotic treatment get exerted in the same way. A series of studies have described that SCFAs improve the gut health, regulate immune mechanisms and they may possess neuroactive properties 34,54−57. However, findings from ASD human studies on the associations among the three main SCFAs have proved to be divergent 58–60. At the same time, in rodent models, the administration of propionic acid could produce behavioral changes closely resembling those found in ASD 61, and in the VPA autism model changes of the SCFAs concentrations were also observed 50. In spite of all these, the analysis of the main SCFAs did not show significant differences between the VPA treated and the control animals, and, after the probiotic therapy, there were also no remarkable effects seen in the SCFAs productions. Nevertheless, it is clear that the antibiotics treatment itself significantly decreases the levels of all the examined SCFAs, referring to the highly decreased total amount of the microbiome in these animals. In spite of the fact that the PM considerably elevated the concentration of the SCFAs after the antibiotics treatment, we did not reveal extreme alterations among the groups after the end of the treatments. However, it is reasonable to suppose that the probiotic impact would be necessarily stronger after the antibiotics administration than in case of challenging the compact, untreated microbiome community. The present results undeniably indicate that the change of concentration of the main SCFAs cannot be the sole causal factor that determines how the PM exerts its positive impact on the social behavior.
Increasing amount of data support the consideration that the manifested inflammation and metabolic patterns are quite comparable in both investigated animal models 62,63. While, in the antibiotics-treated rat model, the inflammatory processes and the serotonergic system appear to be linked to the gut dysbiosis 30,64,65, in the VPA animal model the elevated pro-inflammatory state, chronic glial activation and disturbance of the serotonergic system are caused by impact of the VPA to the developing brain 65–68. Therefore, these observations encourage us to maintain the presumption that our PM made an effect on the serotonergic system without the mediation of the alterations of the SCFAs, thus, providing us the opportunity to hypothesize this to be the common way how the probiotic formulation can re-establish the behavioral alterations. It could occur in a way that modification and reduction of the inflammatory processes (diminishing the gut permeability) altogether with the altered microbiome could interact and alter the serotonergic system 69–73. Since GI microbiota can directly or indirectly influence the tryptophan availability and the serotonin synthesis, thus, it ultimately influences the regulation of the kynurenine pathway. This pathway controls the production of the neuroprotective kynurenic acid (N-methyl-d-aspartate (NMDA) receptor antagonist) and that of the neurotoxic quinolinic acid (NMDA receptor agonist) 72. So, the modulations of this system could lead to altered expression levels of NMDA 72,74,75. It has been demonstrated that antibiotics could also alter NMDA receptor subunit expressions 76. Recent studies have also identified that post-natal VPA treatment enhanced NMDA receptor functioning in the brain which may indicate a compensatory homeostasis with the presence of an excitatory/inhibitory imbalance during development. In subsequent experiments, when using pharmacological suppression therapy or NMDA receptor antagonists, they were able to normalize social deficits 50,66,77,78. These results highlight the impact of these transcriptional modifications which could be the last elements in the way how our probiotic could restore the antisocial behavior, since NMDA receptors play an essential role in complex cognitive and social behavioral processes 79,80. Nevertheless, further studies are needed to measure constituent elements of inflammatory processes, metabolic patterns and NMDA receptor expression levels in both animal models along with measuring these values after the probiotic treatment. Furthermore, additional investigations are necessary to examine changes of other minor SCFAs, as well as to apply other behavioral tests to explore wider ranges of behavioral alterations. Indeed, future studies should also clarify whether the 2 weeks of our PM exposure ensures only shorter, temporary or lasting effects on the social behavior. Furthermore, future studies are expected to reinforce the therapeutic efficacy of our PM on the ASD.
Taken together, our data confirm that broad-spectrum antibiotics treatment during adulthood can induce antisocial behavior similar to that observed in the VPA autism animal model. The current study suggests that the homeostatic balance of the GI microbiome has a profound effect on the social behavior. To our best knowledge this study is among the first ones to demonstrate that specific probiotic mixture can restore the same type of antisocial behavioral phenomena in these two disparate animal models, developed by distinct mechanisms. Based on the present data, this probiotic formulation targets a common pathway with a non-SCFA dependent manner. Overall, this study provides preliminary evidence for that GI microbiome, more specifically some bacterial combinations, appears to have therapeutic value to cure or at least attenuate the illness of social behavior, and thus, to get prepared to act as a proper therapeutic agent to eliminate symptoms of antisocial behavior in the ASD.