In vivo Bioluminescence Imaging of Ca2+ Signalling in the Brain of Drosophila

Many different cells' signalling pathways are universally regulated by Ca 2+ concentration [Ca 2+ ] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca 2+ signals involved in neurophysiological functions. New methods for in vivo imaging of Ca 2+ signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca 2+ imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca 2+ reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca 2+ ] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca 2+ response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca 2+ ] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca 2+ ] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca 2+ stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca 2+ signalling pathways and for functional mapping of neurophysiological processes in the fly brain.