The goal of these experiments was to assess the effect of a substantially reduced gut microbiota on mouse behavior with the fewest possible confounding effects. While a number of studies have utilized daily gavage feeding of antibiotics to reduce gut microbiota123, a method that provides finer control over the daily dose each animal receives, we were concerned that the stress of daily forced feedings would cloud our ability to interpret the behavioral results40. Gavage feeding is a stressor intense enough that it was used to induce a depressive-like phenotype in a recent publication8. Several other studies have relied on germ-free mice that are devoid of commensal bacteria from birth41. While these animals do provide the cleanest model system for modeling the effect of gut bacteria on host physiology, there are myriad developmental effects of complete lack of commensal microorganisms. At this time there are known developmental effects on the immune system4243, gastrointestinal system2, as well as multiple effects in the CNS including altered patterns of myelination44, function of microglia24, and permeability of the blood brain barrier32, among others.
