Isolation of Danio rerio (zebrafish) neurons and skeletal muscle fibers in vitro: Monitoring Axonal Growth, Morphology, and Physiology

To further my study of venomous peptides from Conus tulipa and C. purpurascens , I have developed a novel preparation to observe the electrophysiology of isolated neurons in vitro. As a teleost fish, known prey for these two species of fish-hunting cone snails, zebrafish ( Danio rerio ) provide an ideal model system with its small size, short gestation period, and rapid development. With a highly simplified neuronal network at younger embryonic stages, these organisms only have primary neurons, secondary neurons, and interneurons, which can be identified based on the cell soma size and axonal morphology. This summer, I successfully isolated Danio rerio neurons from stages 18 to 80 hours post-fertilization on laminin coated cover slips. Axonal growth rates were calculated by monitoring the extension of processes over time, and were determined to resemble published rates of growth in vivo. Also, I maintained immortal rat cells in culture to determine the effects of the toxins on mammalian neurons in comparison to zebrafish. Using whole-cell patch clamping, the electrical properties of isolated neurons in culture can now be observed without extraneous electrical connections or inputs. With future advancements in the use of conotoxins in medicinal treatments, these studies are valuable to fully understand the effects of neuroexcitatory peptides on the electrical activity of neurons