Advanced Methodologies for Measuring and Manipulating Zebrafish Behavior and Physiology

Abstract

Larval zebrafish offer unique advantages for systems neuroscience, including optical transparency, genetic accessibility, and conserved vertebrate brain organization. However, essential behavioral tools and methods remain underdeveloped, while sophisticated imaging techniques such as two-photon calcium imaging drives research forward. This study aims to provide advanced but easy to implement methodological solutions for behavioral research addressing two critical methodological gaps: the lack of humane, standardized euthanasia protocols, and the limited ability to measure eye movements in all degrees of freedom during visuomotor behavior. First, an open-source and modular platform for electrical stunning of zebrafish larvae was developed. Using controlled alternating current (AC) electrical fields, immediate loss of behavioral responsiveness and complete suppression of stimulus-evoked neuronal activity were achieved, as confirmed by two-photon calcium imaging in the optic tectum. Unlike mostly used methods such as MS-222 overdose, electrical stunning avoids aversive reactions or inconsistent efficacy, offering a rapid, reproducible, and ethically refined euthanasia protocol. Second, both single- and dual-camera eye-tracking systems were established to simultaneously record vertical and horizontal eye movements in agarose-embedded larvae during both visual and vestibular stimulation. This enabled characterization of the vertical optokinetic response (vOKR) and the vertical vestibulo-ocular reflex (vVOR) - previously unexplored in larval zebrafish. vOKR exhibited distinct dynamic properties compared to its horizontal counterpart, including delayed plateauing of eye positions, no resetting saccades and frequency-dependent phase shifts. Moreover, during vOKR, spontaneous saccades occurred in both vertical and horizontal planes, suggesting a more complex oculomotor repertoire than previously recognized. These methodological advances aim to expand the zebrafish neuroscience toolkit, enabling more comprehensive analysis of sensorimotor processing and behavior as well es refinement with regard to animal welfare. The tools are designed for compatibility with high-resolution imaging and scalable behavioral experiments and are accessible to laboratories without specialized engineering infrastructure.