The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.
Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.
The importance of the OKR for the study of visual function has been recognized in the scientific community for a long time (Easter & Nicola 1996, 1997), and attempts to truly quantify the paradigm have started well over a decade ago. Easter and Nicola (1996) developed a system with motorized rotating striped drums, where the video recording of eye movement was analyzed manually. This system suffered from the lack of immobilization of the fish embryo, which required frequent manual repositioning, and could detect the tracking movements of eyes only with great difficulty. A step forward was the use of a video-projected striped drum to allow for more variable computer generated stimulus presentation (Roeser & Baier, 2003; Rinner et al., 2005a).
The mostly manual, frame-by-frame analysis of videotaped recordings has proven to be extremely laborious, and to a certain degree hampered by observer bias (Beck et al., 2004). Automated analysis in real-time was suggested to allow the use of behavioral feedback learning mechanisms (Major et al., 2004). The use of infrared illumination and frequency-controlled rotating stimuli has been pioneered by Beck et al. (2004). However, the system described there has only been used for larvae, and analysis was carried out off-line. Furthermore, the VisioTracker allows complete control over stimuli, including changing the stimulus during the experiment, thereby allowing greater flexibility and spontaneous influence on the course of the experiment. Also, the digital stimulus creation used by the VisioTracker overcame problems mentioned earlier with acceleration of the inert mass of a striped stimulus drum (Beck et al., 2004).
Larvae restraint by methylcellulose does not significantly interfere with eye movement and does not have any long-term effects on zebrafish well-being. Fish larvae have been successfully maintained embedded in methylcellulose for several days, until oxygen supply through the skin becomes insufficient for the demand with increasing age (Qian et al., 2005).
The adult fish restraining method is equally easy on the animal. The short duration of the experiment, coupled with the option of rapidly exchanging the test animal for a different one, further adds to the positive animal welfare aspects of the system. Since the gills are continuously flushed by water, it is convenient to spike the water with any chemical of choice to study its effect on eye movements and visual performance. Similarly a wash-out experiment can be added without the need to handle the animal between the experiments.
Pixel noise in the video picture was minimized by smoothing algorithms of the proprietary VisioTracker software, enabling highly precise measurements of eye position and angular velocity. Furthermore, to facilitate statistical analysis, the software filtered out saccadic movements which occur at fixed velocity and do not contribute to the experimental statement. An averaging of velocity curves over 7 video frames facilitated later analysis.
The VisioTracker opens a new dimension for many varied research areas. The system and its predecessors have already been used successfully to quantify visual performance in zebrafish larvae, using parameters such as visual acuity, contrast sensitivity and light adaptation (Rinner et al., 2005a, Schonthaler et al., 2010), for functional analysis of cone photoreceptors following manipulation of members of the visual transduction cascade (e.g. Rinner et al., 2005b, Renninger et al., 2011), or the analysis of visual defects in mutant zebrafish larvae (e.g. Schonthaler et al., 2005, 2008; Bahadori et al., 2006). The interdependence of morphological and functional maturation of the visual system has been studied by OKR measurements to show that visual acuity is mainly but not completely limited by photoreceptor spacing at larval stages (Haug et al., 2010).
The VisioTracker is equally suitable to analyze visual function in adult zebrafish and other similar sized fish species (Mueller and Neuhauss (2010), this report).
It is also conceivable to utilize the system in research areas such as toxicology or pharmacology whereby substances to be investigated might be added to the water flow surrounding the adult fish gills. Furthermore, the versatility of VisioTracker enables more thorough analyses for example of ontogenetics of visual function, neural circuit function and development, or sensorimotor control (see review in Huang & Neuhauss, 2008).
The authors have nothing to disclose.
KPM was supported by EU FP7 (RETICIRC).
Name of the reagent | Company | Catalogue number |
---|---|---|
Methylcellulose | Sigma-Aldrich | M0387 |
Ethyl 3-aminobenzoate methanesulfonate (MS-222) | Sigma-Aldrich | E10521 |
35 mm cell culture dish | Corning | 430165 |
Serum pipette | Greiner bio-one | 612 361 |
VisioTracker | TSE Systems | 302060 |