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G. M. Vanderlaan, J. Rihel, D. A. Prober, F. Emran, A. F. Schier, R. T. Peterson, J. E. Dowling; Small Molecule Perturbation of Vision in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5789. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The use of small molecules to perturb biological processes is potentially a powerful approach that can complement genetic approaches. Zebrafish larvae are particularly amenable to such approaches because of their rapid development, small size, and permeability to small molecules. Genetic mutagenesis in zebrafish has generated a number of interesting behavioral mutants, but a small molecule screen to perturb behavior is novel. As early as 4 days post-fertilization (dpf), zebrafish express robust behavioral responses to the onset or offset of static light stimuli (Emran et al., 2007, PNAS 104, 19126-31). Using this visuomotor behavioral assay, we have initiated a forward small molecule screen, with the aim of probing visual function.
In this assay, zebrafish larvae are individually placed into 80 wells of a 96-well plate allowing simultaneous monitoring of each larva using an automated video-tracking system. The movement of each larva in response to periods of 15 min light ON and 15 min light OFF was recorded and quantified per second. At light onset, larvae express a burst of motor activity, called the ON-startle response, followed by an immediate return to lower-than pre-stimulus levels of activity, called the ON-freeze response. At light offset, larvae express a spike of motor activity, called the OFF-startle response, followed by a gradual return to pre-stimulus levels of activity, called the OFF-sustained response.
To date, we have screened two small molecule libraries (totaling about 2,200 small molecules) for their effects upon visuomotor responses of 4 dpf larvae. Approximately 28,500 fish have been assessed for visuomotor deficits and each small molecule was screened in 10 replicates for reproducibility. The first round of screening yielded 54 chemicals with alterations in different aspects of the behavioral ON or OFF responses. A number of these molecules specifically affected the OFF-startle response (n=20), others the ON-startle response (n=8), and a few the OFF-sustained response (n=3). To determine if some of these alterations are localized to the outer retina, we are presently examining the electroretinogram (ERG) of larvae exposed to each of the chemicals.
Small molecule screens to perturb visual behavior offer a potential approach for unraveling molecular pathways in retinal neurons.
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