Monitoring calcium activity in the blood brain barrier using GCaMP6 in Drosophila melanogaster

The blood brain barrier (bbb) is a cellular layer that encapsulates the brain and regulates interaction between certain molecules and neural circuits. Our lab has previously shown that modulation of the bbb affects behavior in the common fruit fly, Drosophila melanogaster. The bbb in Drosophila is useful to study because of its similarities with that of vertebrates. Knowledge of the fundamental biochemical processes in the bbb ultimately allows for better understanding of neurological disorder from bbb abnormalities, such as epilepsy. Calcium ions (Ca2+) are important messengers used in cellular signaling. Uncovering the role of calcium activity in the bbb helps elucidate the biochemical relationship between the bbb and neurons in the brain. We used the Gal4-UAS genetic system to express the Ca2+ indicator GCaMP6 in the bbb of Drosophila. GCaMP6 allows for live imaging of Ca2+ signaling by emitting green fluorescence under 470nm wavelength light when Ca2+ binds to calcium-modulated protein. In preparation for imaging, a live fly was placed in a pipette tip and coated with wax to restrict movement. While submerged in phosphate-buffer solution, an incision was made on the cuticle of the head to expose the bbb. The pipette apparatus was then placed on a slide and the bbb was recorded with confocal microscopy using a Leica SP8. In the future, we will utilize software that quantifies the visual fluorescence we recorded and graph it over time. We expect to see oscillations in fluorescence from our understanding of the signaling events in the bbb that utilize Ca2+.