Methodology for testing visual responses in the Anterior Optic Tubercle in the Honey Bee: Towards investigating Magnetoreception

Abstract

Honeybees (Apis mellifera) are known to possess magnetic sensitivity, yet the neural and biochemical mechanisms underlying magnetoreception remain poorly understood. The radical pair mechanism (RPM), a quantum-mechanical phenomenon, has been proposed as a basis for light-dependent magnetoreception, predicting that magnetic sensing is coupled to the visual system through cryptochrome-based photochemistry. To investigate this hypothesis, we developed an in vivo two-photon calcium imaging preparation targeting the Anterior Optic Tubercle (AOTu), a higher-order visual processing region of the honeybee brain, using FURA-2 dextran as a calcium indicator. A visual stimulus protocol was established using a 473 nm blue laser to characterise ON-OFF neural responses in the AOTu. An 80% day-two bee survival rate was achieved by the end of the study. Despite multiple experimental trials, a clear and reproducible visual response was not obtained. Contributing factors are discussed, including laser light leakage into the fluorescence recording, seasonal reduction in bee neuronal activity during winter months, and preparation quality. One recording provided tentative evidence of a visual response at stimulus onset and offset, partially obscured by light leakage. This work establishes a methodological framework for future experiments aimed at directly testing RPM predictions in honeybees, including light dependence, polarity independence, and disruption by radiofrequency fields.