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Authors & Affiliations
Vera Abramova, Bohdan Kysilov, Ales Balik, Eni Tomovic, Tereza Smejkalova, Miloslav Korinek, Klevinda Fili, Mark Dobrovolskii, Paulina Bozikova, Jiri Cerny, Ladislav Vyklicky
Abstract
Danio rerio (zebrafish) is a model for studying neurophysiological development and gene mutation consequences. Zebrafish possesses orthologs of human NMDA receptor subunit genes, including grin2Aa and grin2Ab for GRIN2A. The grin2Ab knockout strain created via CRISPR-Cas9, and the grin2Aa knockout were investigated for spatial and temporal gene expression and swimming behavior. Whole-mount in situ hybridization (WISH) was performed on wild-type (WT) zebrafish larvae aged 1, 3, and 5 days post fertilization (dpf). The results indicated minimal expression of both genes at 1 dpf. By 3 dpf, grin2Aa and grin2Ab exhibited higher expression in the nervous system, with grin2Aa predominantly expressed in the tectum and grin2Ab displaying diffuse expression throughout the brain. At 5 dpf, both genes expressed in various brain regions, with low spinal cord expression. qPCR confirmed low expression at 1 dpf, with grin2Aa surpassing grin2Ab by 3 dpf and increased expression of both genes by 5 dpf. Swimming parameters of 6 dpf larvae were tested in a 20-well chamber, recorded for 60 minutes under constant temperature (28°C) and light (100 lux) conditions. Total swim distance, number of swim bouts, and average length of individual swim bouts for every 10-minute interval were analyzed. grin2Aa-/- and grin2Ab-/- , both showed increased total swim distance due to elevated bout frequency compared to WT controls. grin2Aa-/- grin2Ab-/- larvae demonstrated an even longer swim distance, because of higher number of swim bouts. In conclusion, mutant strains differ from the WT and provide insights for future research on compounds rectifying phenotype changes.