Age related alterations of adult neurogenesis and astrocytes in Zebrafish (Danio Rerio)

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Bilkent University
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Brain aging is marked by a decline in cognitive abilities and associated with neurodegenerative disorders. In order to identify appropriate interventions to change the course of brain aging and age-related neurological disorders, we should first understand the normal age-related changes. Previous studies claimed that there was a correlation between cognitive capacities and number of neurons. However, recent studies have shown no statistically significant change in total neuron number during healthy aging. Therefore, further studies are required to understand the reasons behind these changes in the brain. One possibility could be the age-related alterations in neuronal lineage and glial markers. Thus, this study aims to show the protein levels, distributions, and localizations of key neuronal lineage and glial markers, which include neural progenitor, early neuronal, immature neuron, and mature neuron and glial markers during healthy aging of the zebrafish brain. For this aim, we measured NeuN (Fox-3, Rbfox3, or Hexaribonucleotide Binding Protein-3), MAP-2 (Microtubule-associated protein 2), HuC (ELAV like neuron-specific RNA binding protein 3), DCAMKL-1 (Doublecortin-like kinase 1), and GFAP (Glial fibrillary acidic protein) with immunohistochemistry and western blot techniques. First, the immunohistochemistry technique was applied on two specific proliferation areas, pallium and optic tectum, to detect the changes in the number of neuronal lineages and glial marker. The results indicated no statistically significant changes between young and old groups. Secondly, we performed whole-brain immunohistochemistry of all markers and quantified every image by manually counting the positive signal. We found that aging did not have an effect on the distribution and expression of the markers, even in the whole brain. Finally, Western-blot was performed in whole brain lysates to compare neuron number and protein level changes. Western-blot results indicated an age-related statistically significant decline in immature neuron marker for specifically males and glial marker for specifically females. The protein level of neural progenitor marker showed the significant decline in males during aging but no change between two age groups. Results of the mature neuron antibody revealed that the protein levels were consistent through aging and did not show variation. Our results overall support the finding that the number of neurons and glia do not change during aging since the numbers of markers were not show statistically significant changes during the aging process in the proliferation areas of the zebrafish brain. However, protein levels showed changes between age and gender groups. Thus, this study shows that understanding changes in the number of cells need to count; protein level is not representative, and zebrafish is an appropriate model for brain aging studies.

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Zebrafish, Aging, Neurogenesis, Glia, Western-blot, Immunohistochemistry
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