The Microbiota-Gut-Brain axis (MGBA) is a topic of interest that has gained attention and recognition in the scientific community over the past years. The gut–brain axis includes the central nervous, endocrine, and immune systems, and it is an information exchange network that connects the gut and the brain. It can transmit information bidirectionally: “top–down” from the brain to the gut and “bottom–up” from the gut to the brain. The MGBA is principally the gut microbiota that resides in the intestinal lumen, enterocytes, enteroendocrine cells, goblets, neurons, and glia in the CNS and the activities of the MGBA are regulated by metabolites and cytokines released by these cells/organisms. For example, gut bacteria send signals through their metabolites to promote the synthesis and release of neurotransmitters by enteroendocrine cells. These neurotransmitters synthesized by bacteria and enteroendocrine cells can enter the blood circulation and be transported to the brain, being able to cross the Blood–Brain Barrier (BBB) and participating in the synthesis of serotonin and other molecules that play a crucial role in neurological disorders.
It has been recently proven that dysbiosis (imbalance of the gut microbiota) can affect normal brain functioning and cognition, as major alterations in the MGBA have been linked with the presence of a spectrum of neurological disorders such as Alzheimer’s Disease (AD), Parkinson’s Disease (PD), autism spectrum disorder, epilepsy, and depressive disorder.
At Biobide several assays have been developed to study in depth the link between dysbiosis and gut microbiota with these neurological disorders using zebrafish as a New Alternative Method (NAM).
Zebrafish as a model
Zebrafish (Danio rerio) is a small-scale omnivorous freshwater vertebrate gaining momentum and popularity over the years in the pre-clinical arena due to its advantages. This species has fast organogenesis, can be stipulated easily and in a cost-effective manner, and complies with the 3Rs principle (Replacement, Reduction, and Refinement of animals) having fewer ethical impediments for research than other traditional models using them up until 5 to 6 days post fertilization (dpf) when they start feeding by themselves and it allows as well for High Content Screenings (HCS). Zebrafish share a large similarity with the human genome and also have a vast similarity to the intestines of a mammal in relation to the mode of action and structure. Zebrafish innate immune system develops first and the adaptive immune system develops after 2-3 weeks, so it is possible to use this NAM (New alternative Method) to study the relationship between the innate immune system and the gut microbiome. Germ-free (GF) Zebrafish deliver a vigorous model to study and manipulate microbial signaling and pathways due to its transparency and feasibility to implement various techniques in sterile conditions and the fact that they are see-through.
The application of the study of the microbiota-gut-brain axis (MGBA) in Zebrafish
At Biobide a series of assays that involve the study of the microbiota and the MGBA has been developed. Some studies evaluate the effect of dietary supplementation (prebiotics, probiotics or postbiotics) origin for their effect on a chronic stress zebrafish model. To do so, anxiety-related locomotor parameters were assessed in 15 dpf zebrafish larvae, after eight days of diet supplementation and daily application of unpredictable stress stimuli. Additionally, the diet effect on the expression of different genes (such as tph1b, slc6a4a, htr1aa) involved in brain serotonin signaling metabolism can be studied. The results of the study show if the diet induces a significant alteration in the gut-brain modulation of chronic stress, as the locomotor parameters present differences between the larvae fed with the probiotic diet and the ones fed with the control diet, therefore proving that the probiotic diet affected to the serotonin signaling metabolism.
Other studies can be focused on the effects of probiotics on zebrafish behavior, in larvae or adult zebrafish and how it affects shoaling and liberation of brain-derived neurotrophic factor (bdnf), with upregulation of genes involved in the serotonergic system in the brain (tph1a, tph1b, tph2, htr1aa, slc6a4a, or mao) and gut (tph1a). The reductions in shoaling are considered an anxiolytic-like effect, and this can be studied also in adult fish where a decreased anxiety-like behavior can be analyzed in the novel tank test, which can be accompanied by the increased of serotonin transporter and its expression in the brain. When we exposed fish to a chronic unpredictable stress paradigm, serum cortisol, and leukogram alterations can be altered, and we can analyze if probiotics protect against the gut dysbiosis induced by stress.
All these studies suggest that the microbiome in zebrafish is associated with defensive (anxiety-like) behavior, and therefore can indirectly affect sociality by altering the anti-predatory component of social behavior or by decreasing the neophobia associated with social novelty.
Additionally, zebrafish allow assessing gastrointestinal transit by feeding ad libitum fluorescent food and analyzing the fecal matter, and viewing the gastrointestinal tract by fluorescent microscopy. For further analysis, the intestine can be dissected and tested for inflammatory markers or DNA extracted for the measurement of the expression of certain genes related to gut or intestinal diseases. These assays have a high predictive value meaning that compounds with significant properties in zebrafish are probable to cause the same effect in mammals’ gastrointestinal tract.
Biobide can offer a wide range of behavioral and neurodevelopmental studies focusing on the effect of prebiotic, probiotic or postbiotic supplementation in adult or larvae-stage embryos.
Biobide has also the capacity of providing multiple spatial and conceptual scale studies with germ-free larvae:
- Intestinal absorption of fatty acids
- Immune responses
- Central Nervous System (CNS) and Behavior
- Epithelial cell proliferation
- Host intestinal cell differentiation.
- Phelps, Brinkman, N. E., Keely, S. P., Anneken, E. M., Catron, T. R., Betancourt, D., Wood, C. E., Espenschied, S. T., Rawls, J. F., & Tal, T. (2017). Microbial colonization is required for normal neurobehavioral development in zebrafish. Scientific Reports, 7(1), 11244–13. https://doi.org/10.1038/s41598-017-10517-5
- Application of zebrafish in the study of the gut microbiome - PMC (nih.gov)
- Lee, J., Cho, H., Jeong, Y., & Lee, J. (2021). Genetic Approaches Using Zebrafish to Study the Microbiota–Gut–Brain Axis in Neurological Disorders. Cells, 10(3), https://doi.org/10.3390/cells10030566
- Xia, H., Chen, H., Cheng, X., Yin, M., Yao, X., Ma, J., Huang, M., Chen, G., & Liu, H. (2022). Zebrafish: an efficient vertebrate model for understanding role of gut microbiota. Molecular medicine (Cambridge, Mass.), 28(1), 161. https://doi.org/10.1186/s10020-022-00579-1