Bioluminescence Imaging for Studying Calcium Transients in Drosophila Brain Structures

Samenvatting

Source: Lark, A. R., et. al., In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin. J. Vis. Exp. (2016)

This video demonstrates real-time imaging of calcium dynamics using Green fluorescent protein-aequorin bioluminescence in Drosophila's deep brain structures.

Protocol

1. Imaging

1. Setup
   1. Start the recording system by turning on the microscope, computer, camera, and drainage system and setting the room environmental controls to 25 °C.
   2. Open the Measurement and Automation Explorer program on the computer. Click on “Devices and Interfaces”, then double click on “NI Motion Devices,” and finally right click on “PCI-7334” and select “initialize device”.
   3. Open Photon Imager. Create a new folder and name the first recording file. The camera must reach -80 °C before the system will function.
   4. Set up the perfusion system: Add KCl (potassium chloride), nicotine, and Ringer’s solution to the reservoirs and adjust so each solution discharges at 2 ml/min. Wash with Ringer’s solution prior to starting the experiment.
2. Prepare sample
   1. Place the imaging mount block dish on the mount under a microscope at 5X magnification and insert the perfusion tube into the channel through the puncture.
   2. Set the microscope to fluorescent mode, then center and bring mushroom bodies (MBs) into focus. Adjust to 20X, re-center, and focus.
   3. Position the drainage apparatus over the drainage pool, adjust the height of the drainage shunt so that the tip is just above the pool, and run Ringer’s solution for 30 sec to ensure adequate drainage.
   4. Pull down the shield to seal off the apparatus from light. Using the automated system in the photon imager, make fine adjustments to the focus and take a reference fluorescent image of the MBs.
3. Recording
   1. Adjust photon speed to the desired recording speed (from 50 msec up to 1 sec or more). Select photon mode and open the shutter. Record genotype, sex, age, and sample number in log entries. Adjust the position of ROI (Region of interest) boxes over calyx and cell bodies (CCB) and medial lobes by clicking on the ROI boxes and dragging while holding control.
   2. Record baseline for about 5 to 10 min (as desired).
   3. Apply nicotine for 1 min. Note start/stop in the log. Wash with Ringer’s solution for 5 min.
   4. Wait 5 min for recovery and prepare KCl (potassium chloride) log entry and timer.
   5. Apply KCl for 1 min. Note start/stop in the log. Wash with Ringer’s solution for 1 min.
   6. Switch to fluorescent mode, take the final fluorescent image, and turn off the Ringer’s solution.
   7. Press “Stop.” A dialog box will ask if you want to shut off the system. Select “No” to continue recording.
   8. Open the shield, move the drainage system, switch the lens objective to 5X, and remove the perfusion tube. Remove the sample/recording dish from the stage, and clean off the 20X lens by rinsing and wiping the lens twice with water.
   9. Press the white play button at the top of the program window to initiate the next recording.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Sodium chloride	Sigma-Aldrich	S3014
Calcium chloride	Merck	1023911000
HEPES	Sigma-Aldrich	7365-45-9
Potassium chloride	Prolabo	26 764.298
Magnesium chloride	Prolabo	25 108.295
Sucrose	Sigma-Aldrich	S0389
Nicotine	Sigma-Aldrich	N3876
ATP	Sigma-Aldrich	A2383	Adenosine 5′-triphosphate disodium salt hydrate
Benzyl-coelenterazine	Prolume, nanolight	Cat #301 Coelenterazine h	http://www.prolume.com/
Pipette tips	Corning Incorporated	4868	100-1000µL Univesal Pipette Tip
EM-CCD camera	Andor	DU-897E-CS0-#BV	iXon (cooled to -80°C)
Microscope	Nikon	N/A	Eclipse-E800
Immersion objective lens	Nikon	N/A	20x Fluor .5w
Dissection microscope with fluorescence	Leica MZ Fl III	N/A	leica dissection scope and florescent lamp
Peristaltic pump	Gilson	N/A	Minipuls 2
Tubing	Fisher Scientific	Depends on thickness	Tygon R3603, St-Gobain
Perfusion system	Warner	64-0135 (VC-66CS)	Perfusion valve control system, complete, with pinch valves, 6 channel
Perfusion regulators	Leventon	201108	Dosi-flow 3
Measurement and automation explorer	Sciences Wares & National Instruments	N/A	software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380
Photon imager	Science Wares & National Instruments	N/A	software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380
Photon viewer	Science Wares & National Instuments	N/A	software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380
Excel	Microsoft	N/A	software https://www.microsoft.com
Virtual dub	open access	N/A	software http://virtualdub.sourceforge.net/