Experimental platform to study associative learning in zebrafish larvae under the microscope.

Associative learning requires linking sensory cues with biologically relevant outcomes. Zebrafish larvae are a powerful model: optically transparent, small and genetically tractable, ideal for whole-brain imaging, with a well-characterized behavioral repertoire. However, combining behavioral readouts with neural imaging requires paradigms that can be performed while the animal is head-fixed, and most existing protocols either probe non-associative memory or are too complex to implement under a microscope. We developed an experimental platform to study associative learning in zebrafish larvae. Velocity changes in optomotor response assays are paired with aversive stimuli. The larva is restrained with its tail free, allowing motor responses. Moving visual gratings generate optic flow, while brief water jets provide an aversive stimulus. The proposed paradigm is highly naturalistic and closely aligned with the animal's biological experience. The system is designed to enable all training steps to be performed under the imaging microscope, allowing simultaneous recording of behavior and brain activity and providing a novel framework to investigate the neural mechanisms underlying associative learning. We will present a proof of concept demonstrating the functionality and versatility of the system. We will also characterize the range of expected responses to the stimuli across variations in stimulation parameters and discuss preliminary results from associative learning curves.