Table 1.

Conceptual and technical knowledge gaps, and potential experimental resolution, in the development of microbiome-based therapies for mental health.

Key Open Questions	Promising Experimental Approaches
1. Does intestinal dysbiosis impair mental health, and/or do mental health conditions disrupt intestinal microbiota?	Animal Studies: rigorously designed studies (following NIH guidelines with regard to controls, statistical power, and biological variables) in which microbiota are transplanted from diseased mice to healthy mice to determine if the phenotype is reproduced and from healthy mice to diseased mice to determine if phenotype is reversed. Human Studies: RCT of microbiome-based therapies in affected patients to test for efficacy (see Question 5).
2. Which microbes have the capacity to drive disease, and which are merely ancillary?	Animal and Human Studies: Systematic, unbiased approach to identify taxa in which enrichment/depletion is associated with a specific phenotype across models (depends in part on resolution of Question #4).
3. What are the mechanisms whereby altered microbiota disrupt brain function?	Animal Studies: rigorously designed studies using genetic, surgical, and/or pharmacologic means to interrupt key signaling pathways (eg, selective vagotomy, bile acid receptors, toll-like receptors) between gut and brain (depends on resolution of Question #1).
4. Can fecal samples adequately reflect intestinal microbiota across proximal-distal and luminal-mucosal axes?	Animal Studies: systematic sampling of luminal and mucosal microbiota along GI tract under both control and disease conditions to determine fidelity of fecal sampling.
5. Under what conditions are microbiome-based therapeutics (FMT, probiotics, and/or prebiotics) beneficial for mental health?	Animal Studies: rigorously designed studies to inform human trials – important issues include identification of the most beneficial therapy (transplant versus probiotic (strain?) versus prebiotic (type?)), dose-response relationships, duration of benefits, and adverse effects. Animal Studies: RCT of microbiome-based therapies for specific conditions (eg, anxiety, depression, autism).