In vivo Ca2+ imaging of mushroom body neurons during olfactory learning in the honey bee.

The in vivo and semi-in vivo preparation for calcium imaging has been developed in our lab by Joerges, Küttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17). After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.