Citation:

Phelps, D., N. Brinkman, S. Keely, E. Anneken, T. Catron, D. Betancourt, AND C. Wood. Colonization by host-associated microbiota is necessary for normal neurobehavioral development in zebrafish. Zebrafish Interest Group Symposium at NCSU, Raleigh, NC, October 27, 2017.

Impact/Purpose:

Intestinal microbes are thought to influence mood, anxiety, and increasingly, brain development. It is currently unknown whether and how microbiota might influence the developmental neurotoxicity of environmental chemicals although it has been proposed that intestinal microbiota may mediate developmental neurotoxicity effects of environmental chemicals either by performing biotransformations or serving as a target of chemical exposures. This work shows for the first time that host-associated microbiota are required for normal neurobehavioral development in zebrafish and that zebrafish that lack microbes are hyperactive. We also show that antibiotic treatment of zebrafish embryos with microbes is sufficient mimic hyperactivity observed in sterile zebrafish. This paper supports the idea that, similar to antibiotics, environmental chemicals may cause developmental neurotoxicity via disruption of normal microbial colonization.

Description:

Host-associated microbiota is vital to development of multiple organ systems, including the nervous system. Recent studies have suggested that microbiota may modify the toxicokinetics and/or toxicodynamics of environmental chemicals. To test this theory, we used swimming behavior as a functional readout of brain development in order to determine the effects of microbial colonization in microbe-free (axenic), axenic colonized on 1 day post fertilization (dpf), and conventionally colonized zebrafish larvae. 16S ribosomal RNA gene sequencing confirmed sterility of axenic larvae and demonstrated diverse bacterial communities at 6 and 10 dpf in colonized larvae. At 10 dpf, axenic larvae displayed significant hyperactivity compared to colonized controls. Interestingly, hyperactivity was observed in conventionally colonized zebrafish exposed to the antibiotics amphotericin B, kanamycin, and ampicillin. To determine whether host Toll-like receptor activation is required to stimulate normal neurobehavioral development in colonized larvae, axenic zebrafish were exposed to heat-killed Escherichia coli, heat-killed Salmonella typhimurium, Poly(I:C), or Pam3CSK4. Exposure to these compounds failed to block hyperactivity in axenic larvae. Axenic larvae were also serially colonized on 1, 3, 6, or 9 dpf to determine whether there is a developmental window during which colonization must occur to program neurobehavioral development. Hyperactivity was blocked in axenic zebrafish colonized on 1, 3, or 6 dpf. However, colonization of axenic larvae at 9 dpf failed to block locomotor hyperactivity. Using axenic larvae colonized with fluorescently labeled Aeromonas veronii:dTomato, these data were found to be consistent with temporal dynamics of microbial colonization. Colonization commenced when larvae hatched from the chorion, and colonization of the intestinal tract was complete by 4 dpf. Colonization with A. veronii or Vibrio cholerae blocked hyperactivity at 10 dpf. Together, these data suggest that there is a developmental requirement for microbial colonization for nervous system development and function. These data also raise the possibility that neurotoxicants may elicit toxicity via altered gut microbial colonization during critical windows of development. This abstract does not necessarily reflect EPA policy.

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