Affordable and effective optokinetic response methods to assess visual acuity and contrast sensitivity in larval to juvenile zebrafish.

The optokinetic response (OKR) is an effective behavioural assay to investigate functional vision in zebrafish. The rapid and widespread use of gene editing, drug screening and environmental modulation technologies has resulted in a broader need for visual neuroscience researchers to access affordable and more sensitive OKR, contrast sensitivity (CS) and visual acuity (VA) assays. Here, we demonstrate how 2D- and 3D-printed, striped patterns or drums coupled with a motorised base and microscope provide a simple, cost-effective but efficient means to assay OKR, CS and VA in larval-juvenile zebrafish. In wild-type, five days post-fertilisation (dpf) zebrafish, the 2D or 3D set-ups of 0.02 cycles per degree (cpd) (standard OKR stimulus) and 100% black-white contrast evoked equivalent responses of 24.2±3.9 or 21.8±3.9 saccades per minute, respectively. Furthermore, although the OKR number was significantly reduced compared to the 0.02 cpd drum (p<0.0001), 0.06 and 0.2 cpd drums elicited equivalent responses with both set-ups. Notably, standard OKRs varied with time of day; peak responses of 29.8±7 saccades per minute occurred in the early afternoon with significantly reduced responses occurring in the early morning or late afternoon (18.5±3 and 18.4±4.5 saccades per minute, respectively). A customised series of 2D printed drums enabled analysis of VA and CS in 5-21 dpf zebrafish. The saccadic frequency in VA assays was inversely proportional to age and spatial frequency and in CS assays was inversely proportional to age and directly proportional to contrast of the stimulus. OKR, VA and CS of zebrafish larvae can be efficiently measured using 2D- or 3D-printed striped drums. For data consistency the luminance of the OKR light source, the time of day when the analysis is performed, and the order of presentation of VA and CS drums must be considered. These simple methods allow effective and more sensitive analysis of functional vision in zebrafish.