NemoTrainer: Automated Conditioning for Stimulus-Directed Navigation and Decision Making in Free-Swimming Zebrafish

Abstract

Current methods for associative conditioning in animals involve human intervention that is labor intensive, stressful to animals, and introduces experimenter bias in the data. Here, we describe a simple apparatus and a flexible, microcontroller-based conditioning paradigm that minimizes human intervention. Our methodology exploits directed movement towards a target that depends on spatial working memory, including processing of sensory inputs, motivational drive, and attentional mechanisms. Within a stimulus-driven conditioning paradigm designed to train zebrafish, we present a localized pulse of light via LEDs and/or sounds via an underwater transducer. A webcam placed below a glass tank records fish-swimming behavior. For classical conditioning, animals simply associate a sound or light with an unconditioned stimulus, such as a small food reward presented at a fixed location, and swim towards that location to obtain a few grains of food dispensed automatically via a sensor-triggered, stepper motor. During operant conditioning, a fish must first approach a proximity sensor at a remote location and then swim to the reward location. For both types of conditioning, a timing-gated interrupt activates stepper motors via custom software embedded within a microcontroller (Arduino). “Ardulink”, a Java facility, implements Arduino-computer communication protocols. In this way, a Java-based user interface running on a host computer can provide full experimental control. Alternatively, a similar level of control is achieved via an Arduino script communicating with an event-driven application controller running on the host computer. Either approach can enable precise, multi-day scheduling of training, including timing, location, and intensity of stimulus parameters; and the feeder. Learning can be tracked by monitoring turning, location, response times, and directional swimming of individual fish. This facilitates the comparison of performance within and across a cohort of animals. Our scheduling and control software and apparatus (“NemoTrainer”) can be used to study multiple aspects of species–specific behaviors as well as the effects on them of various interventions.

Toward Real-Time Animal Tracking with Integrated Stimulus Control for Automated Conditioning in Aquatic Eco-Neurotoxicology

“…Manabe and colleagues developed an innovative computer-controlled operant apparatus for small fish using LEDs for stimulus presentation and an optical fiber sensor to sense the approach to a response key, facilitating reversal learning tasks in zebrafish through color discrimination analysis . More recently, Singh et al developed the NemoTrainer system, which utilizes real-time vision-based tracking for automated conditioning and decision-making in zebrafish, tracking parameters such as turning, location, response times, and directional swimming …”

“…Recent years have seen several attempts to develop automated operant conditioning devices for small teleosts. ,,− For instance, Aoki et al showcased a system combining video tracking and computer-controlled visual cues to train zebrafish to avoid specific arms of a Y-shaped tank through color and electric shock associations, achieving efficient training within a 2 h time frame . Manabe and colleagues developed an innovative computer-controlled operant apparatus for small fish using LEDs for stimulus presentation and an optical fiber sensor to sense the approach to a response key, facilitating reversal learning tasks in zebrafish through color discrimination analysis .…”

“…58 More recently, Singh et al developed the NemoTrainer system, which utilizes realtime vision-based tracking for automated conditioning and decision-making in zebrafish, tracking parameters such as turning, location, response times, and directional swimming. 57 These emerging technologies have demonstrated the feasibility of unbiased operant conditioning experiments in small fish. 25 However, there remains a lack of development of universal software that could be employed on demand to develop custom automated training systems for larval and juvenile stages of fish and amphibians, as well as selected invertebrates.…”