Bioluminescence Imaging for Studying Calcium Transients in Drosophila Brain Structures

Secure an immobilized transgenic Drosophila with its head exposed in a recording chamber on a fluorescence microscope stage.

The fly’s mushroom body comprises neurons expressing calcium-sensitive bioluminescent fusion protein, which is activated by a preloaded cofactor.

Connect a perfusion tube to the chamber. Locate the fluorescent mushroom bodies, focusing on the calyx and medial lobes. Confirm drainage with buffer. 

Set imaging parameters and focus on the calyx and medial lobes. In the dark, acquire baseline fluorescence images of the mushroom bodies prior to stimulant exposure.

Perfuse nicotine in calcium-containing buffer to activate nicotinic acetylcholine receptors, triggering calcium influx and bioluminescent light emission.

Wash with buffer to restore baseline conditions. 

Perfuse potassium chloride to depolarize neurons, triggering intracellular calcium release and bioluminescent signal emission. 

Wash with buffer to restore baseline conditions. 

Analyze images to assess spatial and temporal calcium activity in the calyx and medial lobes.