Modification Of Intestinal Microflora Of Mice By Environmental Enrichment And Its Influence On Animal Behavior

Brain-gut-microbiota axis,referred to as brain-gut axis,is a two-way regulation pathway between the central nervous system and the intestine through the action of the intestinal flora.The brain-gut axis integrates information of nerves,hormones and immune signals and provides a platform for the gut microbes and their metabolites to influence the brain.The intestinal microflora may affect the brain through a variety of ways,including the production of chemical signal molecules,such as short chain fatty acid(SCFA)and other metabolites,acting on the neuroendocrine system;the flora can regulate the concentration of several neurotransmitters to directly or indirectly affect the nervous system;the flora and its metabolites can also act on the vagal and enteric nerves,thereby regulating the brain and cognition;moreover,the flora can also regulate brain function by affecting the immune system.Disorders of intestinal microflora is known to participates in the etiology of developmental brain disorders(such as autism),neurodegenerative diseases(including Parkinson’s disease and Alzheimer’s disease),autoimmune diseases(such as multiple sclerosis),mood disorders(stress,depression,and anxiety),and many other neurological conditions.On the other hand,brain activities can affect gastrointestinal functions and intestinal immunity.Emotional factors including stress and anxiety can affect the development of chronic gastrointestinal disease,aggravate intestinal inflammation,and cause diarrhea,constipation and other gastrointestinal problems.Stress has been shown to affect the integrity of the intestinal epithelium,change the motility and secretion of intestine and its mucin production,thereby changing the habitat of the colonizing bacteria,and promoting changes in the composition or activity of the flora.In addition,the release of stress-induced catecholamines(CA)into the intestine may interfere with the communication between bacteria and the expression of bacterial virulence genes,thereby affecting the intestinal flora.Environmental enrichment(EE)is a classic experimental paradigm for animal behavior intervention.After long-term experimental research,studies on the effect of EE on animal behavior and cognition has evolved from simple behavioral observation to the deep analysis of various pathways of brain regulation at circuit,cellular,and molecular levels.The overall principle of environmental enrichment is to provide multi-social stimulation,multi-sensory stimulation,increase environmental complexity,and increase voluntary exercise.The environmental enrichment has been a well-established experimental paradigm to affect brain development and to improve mood and brain functions.However,several basic questions regarding the interaction between environmental enrichment and the intestinal flora remain unknow.(1)Does brain stimulus by enriched environment affect the intestinal flora?(2)Could environmental enrichment affect the intestinal flora both at developmental stage and in the adult?(3)Is the change of intestinal flora an important mediating factor of the improvement of brain development function in response to environmental enrichment?(4)Will the change in the intestinal flora persist or will be gradually reversed after withdrawal from the enriched environment? Research on these issues will help clarify the interaction mechanism of the brain-gut axis and provide new ideas for the treatment of brain diseases through effective intervention of intestinal flora.To address these questions,we treated of C57BL/6 mice with different environmental stimuli,including the enriched environment group(EE),the monotonous environment group(ME),and the flora transmission group,that is,the transmission of the intestinal bacteria of the EE group to mice without environmental enrichment through environmental exposure(FT),and the running exercise group(RE).16 S rRNA sequencing was applied to analyze the intestinal flora of each group of mice.Using bioinformatics methods,changes in the abundance,diversity,and species composition of the mouse intestinal flora were analyzed.We found that,except the high similarity in the flora structure of the EE and the FT group,other different groups had clear distinction in their intestinal flora structure.These results indicate that EE,FT,and RE could all effectively reshape the structure of intestinal microflora of mice,and that EE and RE have differential effects on the flora structure;moreover,environmental exposure could reliably modify intestinal microflora to the direction of flora structure in the environment.To further verify the shaping of intestinal flora by environmental enrichment,we further divided each group of mice treated with different environmental stimulus into an environment maintenance group and an environmental withdrawal group.After two months,intestinal flora of the environmental withdraw group further differentiated from that of the environment maintenance group and evolved toward the direction of the flora of the ME group.These results suggest that the intestinal microflora is highly plastic in adult mice and the modification of flora structure by environment depends on the maintenance of the environmental stimuli.Brain functions of each group of mice were further examined by behavioral assays.We found that compared to mice of the control group(ME),mice exposed to EE displayed significantly higher spontaneous locomotion but were more aversive to areas with potential threats,including the central region in the open field arena.Their exploration of correct arms in the eight-arm maze test was significantly increased;their performance in the balance beam test and the horizontal bar test was significantly improved.These results indicate that mice with environmental enrichment are more active and vigilant with improved memory and motor ability,indicating that they are more adaptive to environments.Mice of the flora transmission group(FT)showed behavioral phenotypes as the EE group,whereas mice received running exercise(RE)did not show improvement in most of these behavioral tests compared to ME mice.In summary,the above findings show that the intestinal microflora of both developing mice and adult mice is highly plastic.Behavioral interventions such as the environmental enrichment have significant impacts on both the structure of intestinal flora and the behavior performance.Flora transmission via environmental exposure could modify intestinal flora and affect animal behavior.In combination with previous findings that unhealthy lifestyles(such as irregular sleep and eating)cause disorders of the intestinal flora,which in turn affect the development of metabolic syndromes,our findings show that an active and healthy lifestyle is beneficial to improve the intestinal flora and suggest that the improvement of intestinal flora could be one underlying mechanism of the improvement of animal behavior by environmental enrichment.This study provides new clues and research paradigms for brain activities to shape the intestinal flora and provides a new perspective on the therapeutic strategies of neuropsychiatric diseases.