In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Summary

Here we present a novel Ca²⁺-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca²⁺ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Abstract

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca²⁺-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and — with the addition of its cofactor coelenterazine — emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca²⁺-transients and Ca²⁺-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca²⁺-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

Introduction

The fundamental patterning and function of activity within the brain and its discrete structures have long been an area of intense study within the field of neuroscience. Perhaps some of the earliest successful approaches used to address this issue were direct physiological measurement of the change in electrical activity — however alternate methods which allow live imaging of changes in voltage, pH or calcium concentrations (as proxy for activity) have brought additional capabilities to the study of neuronal activity ¹. Imaging techniques carry a wide array of advantages such as reduced invasiveness and the ability to monitor activity within whole brain structures. Furthermore in animals with tractable genetics including the fruit fly Drosophila, development of genetically encoded calcium indicators, such as cameleon, GCaMPs and others¹ have allowed researchers to monitor single neurons, neuronal populations and whole brain structures. While more traditional fluorescent calcium imaging methods using GCaMPs (GFP fused to calmodulin) or FRET (CFP and YFP fused to calmodulin) have proved to be useful techniques providing good spatial and temporal resolution, the underlying process of light excitation engenders some limitations on its experimental applications. Due to the nature of light excitation, autofluorescence, photo-bleaching, and phototoxicity are unavoidably produced, which consequently limits the depth of the structures that can be imaged, the duration of recordings as well as the real time continuity of recording. In other words, recordings must often be performed intermittently to avoid these undesired side effects.

Because of these challenges, additional alternative methods of calcium imaging have been developed. One of the most promising methods is bioluminescent imaging, which relies on light produced through an enzymatic reaction after calcium binding. Bioluminescent imaging does not require light excitation and consequently does not experience the same difficulties as conventional calcium imaging. The bioluminescent reporter Aequorin — isolated from Aequorea victoria, the same Jellyfish from which GFP was also isolated- binds calcium via its three EF hand structures and undergoes a conformational change, leading to the oxidation of its cofactor coelenterazine and the release of blue light (λ=469 nm)^2,3. Aequorin has a very low affinity for calcium (K_d = 10 µM) which makes it ideal for monitoring calcium transients because it produces very little background noise and does not interfere with the intracellular calcium buffering system⁴. Although aequorin has been previously used to monitor the process of neural induction in the Xenopus egg⁵ the light produced is too dim to use for real time imaging of neuronal activity. In an effort to address this challenge, Philippe Brûlet and colleagues at the Pasteur Institute created a genetic construct fusing GFP and aequorin genes, mimicking the native state within the jellyfish⁴. This fusion gene product binds calcium again via the three EF hand structures of aequorin, which undergoes a conformational change leading to oxidation of the coelenterazine, which transfers energy non-radiatively to the GFP. This energy transfer results in the release of green light (λ=509 nm) from GFP, instead of blue light from aequorin. The fusion of GFP and aequorin also confers greater protein stability helping the fusion protein produce light 19 to 65 times brighter than aequorin alone⁴. Using a bioluminescent approach within the brain expands the current experimental application of calcium imaging both in terms of the duration of continuous recording and the number of structures within the brain that can be recorded. Broadly in the context of previous work it means that both global and a greater variety of regionalized “activity” of the brain can be monitored (through the proxy of calcium transients). More importantly activity can be monitored for longer, in a real time and continuous basis, which readily lends itself to the pursuit of a complete understanding of basal function in the brain.

In the last few years, our lab and others have developed transgenic flies expressing the GFP-aequorin under the control of the binary expression system (GAL4/UAS-GFP-aequorin)^5,6. We have used GFP-aequorin bioluminescence to record activity produced by natural stimuli such as scent^7,8, as well as studied the cellular components modulating Ca²⁺-activity in the mushroom bodies following acetylcholine receptor stimulation by direct nicotinic application⁹. In addition, we have also used GFP-aequorin to address a number of experimental questions that have not been able to be addressed previously, like long-term imaging of spontaneous calcium transients and visualization of deeper brain structures¹⁰. More recently, we have used the GAL4/UAS system to express the mammalian ATP receptor P2X₂¹¹ a cation channel, in the projection neurons (PNs) and used the LexA system to express GFP-aequorin in the mushroom bodies (MB247-LexA,13XLexAop2-IVS-G5A-BP) (a courtesy of B. Pfeiffer, Janelia Farm, USA). This has allowed us to activate the PNs by application of ATP, which in turn activates the mushroom bodies (a downstream target of the PNs), mimicking more natural conditions, such as the response to an odor stimulus. Overall this technique combining P2X₂ and GFP-aequorin expands the experimental possibilities. Broadly it offers the opportunity to better understand the patterns of activity in the brain, initiating new studies about how different stimuli and perturbations can alter the basal patterning of activity.

Protocol

1. Preparation of Samples

1. Preparation of solutions and set up
   1. Maintain all Drosophila melanogaster lines at 24 °C on standard food medium. Rear and keep them at low density in the vial to generate standardized size and weight of flies.
      1. Add 10 virgin females with 10 males in a vial, let them mate and transfer them every 2 days to a fresh vial. Then 10 days later, when the flies start to eclose, harvest the flies every day. Keep a precise record of the age of the flies and keep them in good condition.
      2. At day 3, separate the males from the females and keep the females 20 flies per vial. Record the flies at 4 or 5 days-old.
   2. Prepare Ringer’s solution with the following concentrations: 130 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 5 mM HEPES, and 36 mM sucrose and adjust the pH of the solution to precisely 7.3. Prepare perfusion solutions of 25 µM nicotine and 100 mM KCl in Ringer’s solution.
   3. Prepare 1,000 µl pipette tip: using a razor blade shape tip to a slant (approximately 35° from end of tip) and remove excess base beyond 1 ½ centimeters from tip.
   4. Prepare box for storage (23 cm x 17 cm x 8.5 cm) of sample during incubation. Place two sponges (12 cm x 8 cm x 3 cm) saturated with water in bottom [to prevent sample desiccation] below a small rack apparatus to place samples on as their preparation is completed.
   5. Prepare flies samples so that they contain at least 1 copy of the UAS-GFP-aequorin and one copy of a Gal4 line to drive expression of GFP-aequorin. OK107 Gal4 line (a line driving expression primarily in the mushroom bodies) is crossed with UAS-GFP-aequorin in this preparation.
      NOTE: The inside of box must be waterproof and the outside must block light from entering box to prevent degradation of coelenterazine during incubation.
2. Preparation of samples
   1. Ice anesthetize Drosophila by transferring it to a glass vial and keep on ice for 2 min before being moved to the chilled Petri dish for positioning in the pipette tip. Prepare pipette tips on slightly dampened filter paper in 100 ml Petri dish on ice under the dissection microscope.
   2. Gently with forceps place fly inside of pipette tip.
   3. Using a brush gently push and align fly such that the head is completely past the edge of tip and the dorsal region is partially exposed by the section removed during tip shaping (Figure 2B).
   4. Combine a small (approximately 3-4 µl) portions of the two components of the dental glue. Apply the glue carefully around the front and back head and neck down to and around the pipet tip edge — avoiding the crown of the head. Let the glue dry for 2 min.
   5. Place pipette tip with glued fly through hole in recording chamber (Figure 2D) and gently press to secure in place.
   6. Combine the two components of silicon glue (approximately 3-4 µl). Apply glue on the flat side of chamber along the edge where the chamber and pipette tip meet to prevent leak of the Ringer’s solution. Let the glue dry for 2 min.
3. Dissection
   1. Affix tape over the perfusion channel, short edge and extending to the back of the chamber.
   2. Place chamber with fly on dissection block under microscope turn on fluorescent lamp. Pipet 1 ml of Ringer’s solution into chamber.
   3. Use fine surgical knife to remove cuticle. Make parallel incisions from the back of the head to antennal region. Then cut along the edge of eye then make a perpendicular incision above antenna connecting the previous incisions. Make a final incision parallel to that at the back of the head. Use the fine sharp forceps to remove cuticle (Figure 2C).
   4. Use fine sharp forceps to gently grasp and clear away exposed respiratory tissue until the brain and [fluorescing] mushroom body is clearly visualized.
   5. Use ultra-sharp forceps to carefully grasp and pinch neuroepithelial tissue covering brain to allow for permeation of the coelenterazine.
      NOTE: Alternatively papain can be used to permeabilize the neuroepithelia⁹.
   6. Using a pipet, wash out twice with Ringer’s solution to remove any debris from dissection and place sample in the dark box.
   7. Pipet 1 ml of Ringer’s solution containing 5 µM benzyl-coelenterazine into the chamber. Close the box and allow incubation with coelenterazine at RT for a minimum of 2 hr.

2. Imaging

1. Setup
   1. Start recording system: turn on microscope, computer, camera and drainage system and set room environmental controls to 25 °C.
   2. Open 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 new folder and name first recording file. Camera must reach -80 °C before system will function.
   4. Set up perfusion system — add KCl, nicotine, and Ringer’s solution to reservoirs and adjust so the each solution discharges at 2 ml/min. Wash with Ringer’s solution prior to starting experiment.
2. Prepare sample
   1. Place imaging mount block dish on mount under microscope at 5X magnification and insert perfusion tube into channel through puncture.
   2. Set microscope to fluorescent mode then center and bring mushroom bodies (MBs) into focus. Adjust to 20X and re-center and focus.
   3. Position drainage apparatus over drainage pool, adjust height of the drainage shunt so that the tip is just above pool, and run Ringer’s solution for 30 sec to ensure adequate drainage.
   4. Pull down shield to seal off apparatus from light. Using the automated system in photon imager make fine adjustments to the focus and take a reference fluorescent image of the MBs.
3. Recording
   1. Adjust photon speed to desired recording speed (from 50 msec up to 1 sec or more). Select photon mode and open shutter. Record genotype, sex, age, and sample number in log entries. Adjust position ROI 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 log. Wash with Ringer’s solution for 5 min.
   4. Wait 5 min for recovery and prepare KCl log entry and timer.
   5. Apply KCl for 1 min. Note start/stop in log. Wash with Ringer’s solution for 1 min.
   6. Switch to fluorescent mode, take final fluorescent image, and turn off Ringer’s solution.
   7. Press “Stop”. Dialog box will ask to shut off the system. Select “No” to continue recording.
   8. Open shield, move drainage system, switch lens objective to 5X, remove perfusion tube. Remove sample/recording dish from stage, clean off 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.

3. Analysis and Video Creation

1. Extract photon values
   1. Open the photon analysis program (e.g., Photon Viewer) and open first sample spreadsheet file.
   2. Select the display control tab. Select the ROI by clicking on the region while holding control. Adjust size, orientation and shape of regions of interest to encompass GFP illuminated CCB and medial lobes.
   3. Add an additional ROI by clicking “Define ROI” and clicking and dragging on screen to create the new ROI.
   4. Select the “Movie” tab to play photon video. Adjust “sec width” and “sec steps” as desired. Readjust ROI placement as necessary.
   5. Select representative screen shots of GFP fluorescence, nicotine response and KCl response. Crop screenshots and paste image into a presentation slide for later analysis and comparison.
   6. Adjust both the “sec width” and “sec step” to the recording speed in seconds. Select length of analysis by moving grey markers on top panel to bracket region before stimulus initiation and just after response end.
   7. Select “Suspend views while playing” press “<<REW” and press “PLAY>”. Select the tab “Count Rate Chart” and adjust units as desired and line colors to match ROI color.
   8. When analysis is finished, press “Export Count Rate Data”. Select screen shots of combined recordings CCB and medial lobe region of interest from each side. Crop screenshots and paste image into a slide with response images. This slide will be later used in final analysis.
2. Example Analysis: one method for analysis of extracted photon data detailed
   1. Open the spreadsheet files in “Count Rate Exports” folder.
   2. Reformat the cells the first column labeled time to display the time in hour and minutes.
   3. Reference the corresponding slide for the sample. Remove all values except the columns for the time and the two representative ROIs.
   4. Determine nicotine stimulation start time — available in log entry file in main folder- remove additional data prior to start or highlight value.
   5. Find highest/ peak value for both CCB and medial lobe and mark/highlight.
   6. Determine response start and end time for both CCB and medial lobe by creating an estimate using time point from corresponding graphed response on slide and select actual value by change in values in proximity to these points. Highlight the region between these points in both the CCB and medial lobes. Alternatively, use a macro⁹.
   7. Combine all samples of one experimental group on a new spreadsheet.
   8. Align on peak by determining the row value is for each CCB peak value. Subtract the lower values from the highest row value to determine approximate row value where that sample data must be recopied to. Verify that all peak values are aligned.
   9. Copy the sheet twice and delete either the medial lobe columns or the CCB columns creating pages of only CCB or medial lobe values.
   10. Remove data after the all CCB responses have ended. Create four rows labeled Total Photons, Response Length (duration), Peak Amplitude, and Response Latency. Copy and paste this again below the originals.
   11. Use the SUM function to determine total photons during response. Use the COUNT function to determine length of response. Copy and link highest value cell to determine peak amplitude value. Use COUNT function — select from the beginning of the perfusion of nicotine to the start of the response — to determine Response Latency.
   12. Copy values by using the linking function and multiply (all except peak amplitude) by recording speed in seconds.
   13. Bar/box graphs may be created for calculated parameters such as Total Photons, Peak Amplitude, and Response Length, if so desired (ex.: Figure 5B, C, D).
   14. In the column after the raw data photon response, average the raw data points for each time point using average function. If desired follow with a column determining the standard error of the mean (SEM), for each of the averaged photon values at a time point.
   15. Use the average column to construct an average response profile [Graph] for the CCB sample group. To do this select the column specifying the time as the x-axis values and the column of the average photon values as the y-axis and finally select the scatterplot graph creation tool.
   16. Repeat this analysis procedure with the data from the medial lobes.
3. Creating Images for the Video
   1. Open photon viewer.
   2. Select display control tab. Click on ROI while holding control to select, remove and/or minimize it.
   3. Modify “sec width” (accumulation time) as desired to determine the content of each frame.
   4. Modify “sec step” to define the overlap of each frame.
   5. Select “export views while playing” press “<<REW” then” PLAY>”. The images for the video will be exported to the “view exports” folder as a series of .png files.
4. Using Virtual Dub to make video: one method for video creation detailed
   1. Create a new folder named “resultats” in the view exports folder containing the images.
   2. Bring script (renommer.VBS) into the folder of images.
   3. Double click on renommer.VBS to run.
   4. Images will automatically be renamed numerically and copied to “resultats” folder.
   5. Open VirtualDub.exe.
   6. Select open video file — select first image (subsequent images will automatically load).
   7. Select video — select frame rate adjust as desired.
   8. Select video — select compression select Cinepak Codec by radius.
   9. In file select Save as AVI the video will be created automatically.

Results

The fusion of the GFP to aequorin allows us to visualize our region of interest prior to bioluminescent imaging through excitation of GFP in fluorescent mode on the microscope (Figure 3A, 4A, 6A, 6E). One of the simplest ways to stimulate the mushroom bodies is through activation of the ionotropic nicotinic acetylcholine receptors. Although acetylcholine is the endogenous ligand of this receptor we have found that nicotine produces more reproducible responses in the mushroom bodies. This is in part becau...

Discussion

The recently developed bioluminescent-based GFP-aequorin approach presented here allows in vivo functional recording of Ca²⁺-activity in different neurons, as well as if desired, in other kind of cells such as kidney stellate cells as reported in Cabrero et al., 2013⁵.

Modifications, trouble shooting, additional components, and critical steps

Drosophila Husbandry

Materials

Name	Company	Catalog Number	Comments
NaCl	Sigma-Aldrich	S3014
CaCl2	Merck	1023911000
HEPES	Sigma-Aldrich	7365-45-9
KCl	prolabo	26 764.298	normapur
MgCl2	prolabo	25 108.295	normapur
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/
dental glue	3M ESPE™	46954	Protemp IV
silicon glue	3M ESPE™	36958	Express 2 Regular Body Quick
tape	3M Scotch	122s	3M Scotch Magic Tape
pipette tips	Corning Incorporated	4868	100-1,000 µl Univesal Pipette Tip
chamber and holder (stage)			hand made
incubation box			hand made
forceps (No. 5)	Fine Science Tools	11252-00	Micro-dissecting forceps
curved forceps	Sigma-Aldrich	F4142	Micro-dissecting forceps
glue applicator			hand made
knife	Fine Science Tools	10316-14	5 mm Depth 15° stab
EM-CCD camera	Andor	DU-897E-CS0-#BV	iXon (cooled to -80 °C)
Microscope	Nikon		Eclipse-E800
immersion objective lens	Nikon		20X Fluor .5w
dissection microscope with fluorescence	Leica MZ Fl III		leica dissection scope and florescent lamp
tight dark box	Science Wares		Custom built by Science Wares
peristaltic pump	Gilson		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/
UAS-GA2	Martin et al., 2007, Ref. 1	N/A	No stocks {currently} publicly available
pJFRC65-13XLexAop2-IVS-G5A-BP	B. Pfeiffer, Janelia Farms, Ashburn USA.	(STOCK #1117340)	pfeifferb@janelia.hhmi.org
P2X2	Lima and Misenboeck, Ref. 11	N/A	No stocks {currently} publicly available
OK107	Bloomington stock center	854	http://flystocks.bio.indiana.edu/