Analysis of vertebrate vision in a 384-well imaging system

Abstract

Visual impairment affects 253 million people worldwide and new approaches for prevention and treatment are urgently needed. While small molecules with potential beneficial effects can be examined in various model systems, the in vivo evaluation of visual function remains a challenge. The current study introduces a novel imaging system for measuring visually-guided behaviors in larval zebrafish. The imaging system is the first to image four 96-well plates with a single camera for automated measurements of activity in a 384-well format. In addition, it is the first system to project moving visual stimuli and analyze the optomotor response in the wells of a 96-well plate. We found that activity is affected by tricaine, diazepam and flumazenil. Surprisingly, diazepam treatments induce a loss of visual responses, at concentrations that do not affect activity or induce hyperactivity. Overall, our studies show that the developed imaging system is suitable for automated measurements of vertebrate vision in a high-throughput format.

Novel use of FDA-approved drugs identified by cluster analysis of behavioral profiles

“…The fish are fed daily with Gemma Micro 300 and frozen brine shrimp. Zebrafish embryos were collected and grown to larval stages as previously described 18 , 19 . Zebrafish embryos from 0–3 days post-fertilization (dpf) and zebrafish larvae from 3–5 dpf were maintained at 28.5 °C in 2L culture trays with egg water, containing 60 mg/L sea salt (Instant Ocean) and 0.25 mg/L methylene blue in deionized water (pH 7.2).…”

“…The experiments were designed to use the minimum number of zebrafish larvae needed for an evaluation of multiple behaviors. Sample size was determined a priori, based on analyses of behavior in prior studies 19 , 26 . We decided on 190 drugs, using 48 larvae for each pharmacological treatment (n = 48 larvae per drug).…”

“…1 ). The plates were imaged as described previously 19 . Briefly, the imaging system is housed in a 28.5ºC temperature-controlled cabinet where larvae in white 96-well ProxiPlates are placed onto a glass stage.…”

Zebrafish as an Animal Model for Ocular Toxicity Testing: A Review of Ocular Anatomy and Functional Assays

“…The latter technique can be conducted in high-throughput fashion using 384-well plates. 114 Using OMR, it was recently demonstrated for zebrafish, as has been shown for mouse, 123 that dynamic visual experience is required for the proper stratification of retinal layers in early eye maturation. 124 Studies have shown that the OMR displayed by larval zebrafish is lost around 16 dpf and that adult zebrafish rely mainly on mechanosensory stimuli via the lateral line for sensing movement, and on positive rheotaxis (defined as the tendency to turn to face into an oncoming current) for maintaining position in flowing water.…”

“…88 This can be done quantitatively and in high-throughput fashion using larval zebrafish. 113,114 Several behavioral responses to visual stimuli, described below, have been characterized, including optomotor response (OMR), optokinetic response (OKR), visual startle, and visual motor response (VSR and VMR, respectively). These behavioral responses have been used in toxicology as well as in studies designed to discover what types of visual cues play a role in driving behavior, 115,116 genes involved in vision and vision disorders, [117][118][119] and by using in vivo functional imaging, which neurons/circuits are involved in perceiving visual stimuli and eliciting the behavioral response.…”