Neuroplasticity in honey bee brains: An enhanced micro-computed tomography protocol for precise mushroom body volume measurement

BACKGROUND: In insect brains, mushroom bodies are associated with memory and learning behavior. It has been demonstrated that the volume of the mushroom bodies in the brain of a worker honey bee changes during the adult stage. Changes in mushroom body volume imply high neuroplasticity in the brains and may be related to the age polyethism of honey bees. A suitable volume measurement method is needed to understand the correlation between behavioral changes and mushroom body volume changes in honey bees. NEW METHOD: We developed a new protocol for insect micro-computed tomography by modifying a previously reported method. Permount™ mounting medium was used as the embedding medium for micro-computed tomography scanning. RESULTS: This protocol can generate images with high contrast inside the brain and reduce the marked shape changes during specimen processing. From the resulting high-contrast images, we used freeware to generate a three-dimensional model and calculate the volumes of the mushroom bodies in honey bees. The measured volumes of the mushroom bodies were larger than the values reported in most previous studies. There was no significant difference between the left and right mushroom body volumes, but the volumes of honey bee mushroom bodies significantly increased with age. COMPARISON WITH EXISTING METHODS: Previous protocols for micro-computed tomography using dried samples would cause brain shrinkage; protocols using ethanol-preserved or resin-embedded samples generated images with lower contrast. CONCLUSIONS: The embedding protocol for micro-computed tomography is suitable for calculating volume of the mushroom bodies in honey bee brains.