Characterizing medaka visual features using a high-throughput optomotor response assay

Abstract

Medaka fish (Oryzias latipes) is a powerful model to study genetics underlying the developmental and functional traits of the vertebrate visual system. We established a simple and high-throughput optomotor response (OMR) assay utilizing medaka larvae to study visual functions including visual acuity and contrast sensitivity. Our assay presents multiple adjustable stripes in motion to individual fish in a linear arena. For that the OMR assay employs a tablet display and the Fish Stripes software to adjust speed, width, color, and contrast of the stripes. Our results demonstrated that optomotor responses were robustly induced by black and white stripes presented from below in the linear-pool-arena. We detected robust strain specific differences in the OMR when comparing long established medaka inbred strains. We observed an interesting training effect upon the initial exposure of larvae to thick stripes, which allowed them to better respond to narrower stripes. The OMR setup and protocol presented here provide an efficient tool for quantitative phenotype mapping, addressing visual acuity, trainability of cortical neurons, color sensitivity, locomotor response, retinal regeneration and others. Our open-source setup presented here provides a crucial prerequisite for ultimately addressing the genetic basis of those processes.

Fig 1. The linear-pool-style optomotor response (OMR) setup.

(A) Illustration of the linear-pool-style OMR setup: A PVC box containing a display (e.g. tablet) and the 140 mm x 110 mm linear-pool-arena on top. A camera (CellCam Centro 200MR) with an infrared (IR) filter and an IR lamp are used to acquire the videos. The box is closed on all sides to exclude external visual stimuli from the surrounding. (B) Stripe pattern displayed on the tablet screen. Stripe parameters (width in pixels, stripe color 1 and stripe color 2 in hex code, contrast (“opacity”) (x 100%) and speed (x 50 pixel/s)) are set and automated using the Fish Stripes software. (C, D) Images of the linear-pool-arena placed on top of a tablet screen displaying black and white stripe pattern acquired with a camera without (C) and with IR filter (D). The lanes of the linear-pool-arena are oriented at 90° towards the moving stripe pattern. The larvae are marked with red circles. (E) Experimental timeline. 0–1 days post hatch (dph) larvae were acclimatized for 2 hours in the setup while being presented stationary stripes before start of the experiment composed of 5 s stationary stripes, followed by 10 s of stripes moving to the right, 5 s stationary stripes, 10 s of stripes moving to the left. Prior to testing additional stripe conditions, larvae were acclimatized for 10 minutes with respective stationary stripes.

Fig 2. Medaka Cab strain shows a robust OMR induced by stimulation from below.

(A, B) Response rates of larvae to moving stripes with three different stripe speeds and a constant stripe width of 8 mm (A) or with three different stripe widths and a constant stripe speed of 6.5 mm/s (B). Number above each boxplot represents the number of larvae included for the response rate calculation. Cab strain medaka responded robustly toward black and white stripes at stripe speeds between 4.3 and 8.6 mm/s and stripe widths between 4 and 10 mm. Statistical analysis performed in R, pairwise t-test, Bonferroni corrected.

Fig 3. HdrR strain medaka show higher trainability in their OMR towards narrow stripes than Cab strain medaka.

Response rates of Cab and HdrR strain larvae to moving stripes for each stripe width (1.4, 1.2, 1.0 and 0.8 mm) with (grey) and without (white) prior exposure to thick stripe (8 mm) motion. The response rate gradually decreased with decreasing stripe width both in Cab and HdrR strain medaka. After the training with thick stripes, an increase in response rate was observed both in Cab and HdrR, with a more prominent increase in HdrR. Number above each boxplot represents the number of larvae included for the response rate calculation. * p ≤ 0.05, statistical analysis performed in R, pairwise t-test, Bonferroni corrected.

Fig 4. Various medaka strains show a robust OMR induced by colored stripes.

Response rates of Cab, HdrR, 10–1, 11–2, and 69–1 strain larvae to moving stripes (8 mm wide, moving at 6.5 mm/s) of each color combination (black/white, blue/white, green/white and red/white). Number above each boxplot represents the number of larvae included for the response rate calculation. Statistical analysis performed in R, pairwise t-test, Bonferroni corrected.