Modulações do microbioma intestinal : impacto de estados de disbiose induzida por antibióticos sobre parâmetros comportamentais e neuroquímicos ao longo do desenvolvimento de peixes-zebra

Abstract

Microbiome changes may occur via a wide variety of situations, ranging from exposure to pharmaceuticals, diet, s stress, to place and mode of birth. These variations act upon signaling pathways related to the Microbiome-Gut-Brain-Axis having effects on hosts physiological processes, and can ultimately induce altered phenotypes, one example being behavior. In adulthood, the microbiome structure and functionality show a high degree of resilience. Nevertheless, specific microbiome modulations may cause health prejudice for the host. In the perinatal period and infancy, when colonization and stabilization of the microbiome community occurs, on the other hand, the effects of microbiome modulations upon behavioral and neurochemical parameters can be sustained throughout the individual’s life as per lack of colonization resistance. In this study, we used the zebrafish as a model to study the implications of modulating the microbiome both in adult animals and through the life stages, when manipulations were carried as larvae. First, we treated adult zebrafish with three antibiotic drugs of different classes (Ciprofloxacin, Chlortetracycline or Ceftazidime) at environmentally relevant concentrations (6.25, 12.5 or 25 mg/L) for 96 hours and evaluated the animal’s responses to a series of behavioral paradigms. We observed alterations in exploratory, aggressive, and cognitive behaviors. We also investigated the involvement of nucleoside triphosphate diphosphohydrolase, ecto-5’-nucleotidase, and adenosine deaminase, which are the enzymes of the purinergic signaling pathway responsible for the control of ATP and adenosine levels on these outcomes. Aside from a decrease in ADP hydrolysis in the 25 mg/L Ciprofloxacin treatment, we did not find other alterations on these analyses. Next, we evaluated effects of colonization-period microbiome modulations throughout zebrafish’s life cycle. We induced a perturbed colonization scenario by administering a previously described mixture of Amphotericin, Kanamycin and Ampicillin antibiotics from 0 to 7 dpf, after which we evaluated exploratory, aversive and optomotor behaviors as well as morphology. We have encountered that this treatment produced a pattern of depressed responses. From 7 dpf to 14 dpf we fed the larvae either the usual feeding consisting of dry fish food and powdered milk-cultured paramecia or an alternative diet where paramecia were cultured in lettuce leaf infusion water. At 14 dpf, the lettuce-based diet was able to restore some of the behavioral effects, but not others. From 14 dpf forward, all animals received the same feeding of dry fish food and Artemia salina. When tested at 45 and 90 dpf, the animals from the group where colonization was perturbed by antibiotics and the usual fish meal was offered presented abnormal neurobehavioral phenotypes, especially those related to anxiety parameters. Our results indicate that perturbing the microbiome by administration of antibiotics impact the natural behaviors of zebrafish, with observable effects both in acute single-drug exposure of adult fish and throughout all life stages following a single exposure of a mixture of these compounds on a sensitive larval stage. We also showed that diet can be used to partially revert the adverse effects elicited by colonization perturbation. In conclusion, tis study showed that exposure to antibiotic drugs alters the behaviors of zebrafish both immediately following the exposure in adult animals and in the entirety of animals life cycle when these are used in the microbiome colonization period. We also demonstrated that diet can be used as a therapeutic strategy to reduce the effects of antibiotic induced dysbiosis on zebrafish behaviors.