Publication informationJournal of Pharmacological and Toxicological Methods, 69 (1): 1-8 Publisher ElsevierItem record/more information http://hdl.handle.net/10197/7931 Publisher's statementThis is the author's version of a work that was accepted for publication in Journal of Pharmacological and Toxicological Methods. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Journal of Pharmacological and Toxicological Methods (VOL 69, ISSUE 1, ((2014) Abstract Introduction: Many prescribed drugs can adversely affect the eye by causing damage to the function of visual pathways or toxicity to the retina. Zebrafish have the potential to efficiently predict drugs with adverse ocular effects at pre-clinical stages of development. In this study, we explore the potential of using a semi-automated visual behaviour assay to predict druginduced ocular toxicity in wild-type zebrafish larvae. Methods: 3 dpf larvae were treated with six known oculotoxic drugs and five control drugs in embryo medium containing 0.1% DMSO. After 48 hours, larvae were assessed using the visualmotor response (VMR), an assay which quantifies locomotor responses to light changes; the optokinetic response (OKR), a behavioural assay that quantifies saccadic eye responses to rotating stimuli; and the touch response, a locomotor response to tactile stimuli. Results: 9 of 10 negative control drugs had no effect on zebrafish visual behaviour. 5 of the 6 known oculotoxic drugs (digoxin, gentamicin, ibuprofen, minoxidil and quinine) showed adverse effects on zebrafish visual behaviour assessed by OKR or the more automated VMR. No gross morphological changes were observed in treated larvae. The general locomotor activity of treated larvae, tested using the touch response assay, showed no differences with respect to controls. Overall the VMR assay had a sensitivity of 83%, a specificity of 100% and a positive predictive value of 100%. Discussion: This study confirms the suitability of the VMR assay as an efficient and predictive pre-clinical approach to evaluate adverse ocular effects of drugs on visual function in vivo.

Controversially, histone deacetylase inhibitors (HDACi) are in clinical trial for the treatment of inherited retinal degeneration. Utilizing the zebrafish dye ucd6 model, we determined if treatment with HDACi can rescue cone photoreceptor-mediated visual function. dye exhibit defective visual behaviour and retinal morphology including ciliary marginal zone (CMZ) cell death and decreased photoreceptor outer segment (OS) length, as well as gross morphological defects including hypopigmentation and pericardial oedema. HDACi treatment of dye results in significantly improved optokinetic (OKR) (~43 fold, p < 0.001) and visualmotor (VMR) (~3 fold, p < 0.05) responses. HDACi treatment rescued gross morphological defects and reduced CMZ cell death by 80%. Proteomic analysis of dye eye extracts suggested BDNF-TrkB and Akt signaling as mediators of HDACi rescue in our dataset. Co-treatment with the TrkB antagonist ANA-12 blocked HDACi rescue of visual function and associated Akt phosphorylation. Notably, sole treatment with a BDNF mimetic, 7,8-dihydroxyflavone hydrate, significantly rescued dye visual function (~58 fold increase in OKR, p < 0.001, ~3 fold increase in VMR, p < 0.05). In summary, HDACi and a BDNF mimetic are sufficient to rescue retinal cell death and visual function in a vertebrate model of inherited blindness.

Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3–5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors result in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function.