Microbiota-gut-brain (MGB) research suggests that early bidirectional interactions of the gut-brain axis may have important and long-lasting effects on physical and psychological development. Early in life, the brain and the gut microbiome undergo dramatic parallel structural and functional changes. Work in understanding this co-development is paving the way to a developmental framework of brain-behavior connections that includes metabolic processes beyond the central nervous system. Although Hooks et al. provide a brief discussion of the development of gut-brain connections, the view that they present is shallow and leaves the impression that examinations of the impacts of microbial changes on brain and behavioral development are not promising. Important findings such as the dramatic developmental changes of germ-free animals (Luczynski et al. Reference Luczynski, Neufeld, Oriach, Clarke, Dinan and Cryan2016) and the effect of fecal transplantation on behavior (De Palma et al. Reference De Palma, Lynch, Lu, Dang, Deng, Jury, Umeh, Miranda, Pigrau-Pastor, Sidani, Pinto-Sanchez, Philip, McLean, Hagelsieb, Surette, Bergonzelli, Verdu, Britz-Mckibbin, Neufeld, Collins and Bercik2017; Kelly et al. Reference Kelly, Borre, O’ Brien, Patterson, El Aidy, Deane, Kennedy, Beers, Scott, Moloney, Hoban, Scott, Fitzgerald, Ross, Stanton, Clarke, Cryan and Dinan2016) provide crucial evidence that the microbiome and brain are parts of a system of interactions that promotes healthy physical and behavioral development. As Hooks et al. highlight, the majority of this evidence comes from rodent studies, and obvious difficulties exist regarding both extrapolation and isolation of causal effects in humans. However, disregarding these works and overly downplaying promising achievements of MGB research would be a mistake.

Hooks et al. present a timely reality check for all MGB researchers, as they warn against hyping the importance of single experiments to conclude that the microbiome is directly and univocally causally responsible for healthy physical and behavioral outcomes. Warding off this risk, the challenge remains to embrace a comprehensive developmental framework that can uncover the conditional role of various components in behavioral development, such as nutrition and the microbiome as important environmental factors. Early nutrition (i.e., breast milk) is one important factor linked to infant gut microbiome composition (Bäckhed et al. Reference Bäckhed, Roswall, Peng, Feng, Jia, Kovatcheva-Datchary, Li, Xia, Xie, Zhong, Khan, Zhang, Li, Xiao, Al-Aama, Zhang, Lee, Kotowska, Colding, Tremaroli, Yin, Bergman, Xu, Madsen, Kristiansen, Dahlgren and Wang2015; Planer et al. Reference Planer, Peng, Kau, Blanton, Ndao, Tarr, Warner and Gordon2016) and possibly to brain development (Isaacs et al. Reference Isaacs, Fischl, Quinn, Chong, Gadian and Lucas2010). Furthermore, neurons in developing brains express receptors for microbial products more extensively early in development (prior to weaning) than later in adulthood (Arentsen et al. Reference Arentsen, Qian, Gkotzis, Femenia, Wang, Udekwu, Forssberg and Diaz Heijtz2017), which suggests a plausible mechanism for gut-brain communication in infancy and also suggests the existence of a sensitive period marked by consumption of milk. As milk is the sole source of nutrition for most infants, it has huge potential to study the effect of nutrition on the microbiome early in life. Recent evidence suggests that differences in early diet and microbial exposure have effects on microbiome establishment and maturation (Diaz Heijtz Reference Diaz Heijtz2016). For example, length of breastfeeding contributes to shifting the timing of maturational changes in microbial community structure (Bäckhed et al. Reference Bäckhed, Roswall, Peng, Feng, Jia, Kovatcheva-Datchary, Li, Xia, Xie, Zhong, Khan, Zhang, Li, Xiao, Al-Aama, Zhang, Lee, Kotowska, Colding, Tremaroli, Yin, Bergman, Xu, Madsen, Kristiansen, Dahlgren and Wang2015). Additionally, components of human milk, specifically the milk microbiome and human milk oligosaccharides (HMOs) appear to be crucial in establishing and maintaining the gut microbiome (Jost et al. Reference Jost, Lacroix, Braegger and Chassard2015; Pannaraj et al. Reference Pannaraj, Li, Cerini, Bender, Yang, Rollie, Adisetivo, Zabih, Lincez, Bittinger, Bailey, Bushman, Sleasman and Aldrovandi2017). Interestingly, both microbiome composition (Christian et al. Reference Christian, Galley, Hade, Schoppe-Sullivan, Kamp Dush and Bailey2015) and milk factors (Grey et al. Reference Grey, Davis, Sandman and Glynn2013; Nolvi et al. Reference Nolvi, Uusitupa, Bridgett, Pesonen, Aatsinki, Kataja, Karlsson and Karlsson2018) have been linked to fear and anxiety behaviors; however, no studies to date have looked at these in a single integrated framework.

The majority of developmental MGB work has looked at simple connections between either environmental factors and the microbiome, the microbiome and brain measures, or the microbiome and behavior, whereas very few have looked at modulators of the microbiome, neurodevelopment, and behavioral outcomes in a single cohort (Carlson et al. Reference Carlson, Xia, Azcarate-Peril, Goldman, Ahn, Styner, Thompson, Geng, Gilmore and Knickmeyer2018). It is vital that ongoing and future work identifies relationships among these diet and environment-linked changes in microbial community structure and concurrently measured behavior and brain structure and function. This requires that MGB research be implemented comparatively and longitudinally on humans using large cohorts of subjects while applying robust statistical strategies that are able to highlight network dynamics. Employing advanced data analytic approaches (Kelsey et al. Reference Kelsey, Dreisbach, Alhusen and Grossmann2019; Xia & Sun Reference Xia and Sun2017) to integrate microbiome compositional patterns that are different among different outcome groups – for example, high and low performers on a behavioral task (as opposed to grouping the microbiome and looking for performance differences between the groups as was done in Carlson et al. [Reference Carlson, Xia, Azcarate-Peril, Goldman, Ahn, Styner, Thompson, Geng, Gilmore and Knickmeyer2018]) – would improve our understanding of these relationships and would provide a developmental and behavioral context in which to make determinations regarding what an optimal microbiome actually is.

In this way, we may be able to highlight how and when inter-individual variability in microbiome composition reflects variability in behavioral development. Interestingly, Carlson and colleagues used this approach and highlighted that breastfeeding at the time of sample collection was one of the most robustly predictive covariates for the identification of clusters reflecting possible cognitive differences. If confirmed, studies like this will pave the way to non-invasive therapies based on supplementation of diet (in lactating mothers, as well as infants themselves) for optimizing brain and behavior development early in life when interventions are thought to be most impactful. In conclusion, our message is that considering the importance of infancy for the development of MGB interactions, and that early experiences, such as breastfeeding, can shift developmental trajectories, there are huge implications for integration of diet, as milk is the primary form of nutrition and is dynamic. If we want to be able to leverage the microbiome to optimize behavioral and neurodevelopmental outcomes, we must understand not only how the microbiome affects brain and behavioral development, but also how developmentally salient environmental factors affect the microbiome.