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

The overall goal of bioluminescent imaging is to image in vivo functional calcium transient, a proxy for activity, for longer periods, and in deep brain structures. This method can help answer key questions in the field of neurobiology, such as what the basal activity of the fly brain looks like during sleep. The main advantage of this technique is that it does not require excitation using your laser.

This allows one to image calcium transient deeper into the tissue, and for a longer period of time without the photobleaching and toxicity, versus classical calcium imaging. This method can provide insight into the basal brain activity in drosophila, and can also be used with other model organisms, such as the mouse. To begin, prepare the drosophila melanogaster line of interest, and the experimental solutions, as described in the accompanying text protocol.

Then, ice anesthetize a single drosophila by transferring it to a glass vial on ice for two minutes. Next, move the fly to a chilled petri dish, and use a pair of forceps to gently place the fly inside a 1000 microliter pipette tip, trimmed so that the tip is at an angle of approximately 35 degrees. Using a brush, gently push and align each fly so that the head is completely past the edge of the tip, and the dorsal region is partially exposed.

Then, apply prepared dental glue from the head to the pipette tip edge. Take extra care to avoid the crown of the head. Gluing of the head is critical because securing it in the correct position is essential for successful dissection and imaging.

Additionally, poor gluing will lead to leakage of the ringer solution and death of the sample prior to imaging. Next, place the pipette tip, with the attached fly, through the hole in the recording chamber, and gently press to secure it into place. Then use a prepared silicon glue to secure the flat side of the chamber where it meets the pipette tip in order to prevent any leaks.

Affix a piece of tap over the profusion channel, the short edge, and extending to the back of the chamber. Next, place the chamber with the fly under the microscope and turn on the fluorescent lamp. Then, pipette one milliliter of ringer solution into the chamber.

Using a fine surgical knife, Make parallel incisions from the back of the fly's head to the antenal region on both sides. Then, cut along the edge of the eye, and make a perpendicular incision above the antenna that connects the previous incisions. Make a final incision at the back of the head, and then use a pair of fine, sharp forceps to remove the cuticle.

Gently grasp and clear away any exposed respiratory tissue until the brain and the fluorescing mushroom body are clearly visualized. Next, use ultra-sharp forceps to carefully grasp and pinch the neural epithelia tissue covering the brain to remove it an allow for permeation of the coelenterazine. Using a pipette, wash the tissue twice with one milliliter of ringer solution to remove any debris left over from the dissection, and then place the sample into a dark box.

Next, pipette one milliliter of ringer solution, containing five micromolar benzyl-coelenterazine into the chamber. Close the box and incubate the sample at room temperature for a minimum of two hours. Begin by turning on the microscope in the computer.

Also, cool the camera to negative 80 degrees Celsius. Next, set up the drainage system and adjust the environmental controls to 20 degrees Celsius. Next, open the photon imager, create a new folder, and name the first recording.

Then, set up the profusion system by adding the potassium-chloride, nicotine, and ringer solution to their respective reservoirs. Adjust the flow so that each solution discharges at two milliliters per minute. Next, place the sample onto the mount under the microscope, and adjust the magnification to 5X.

Then, insert the profusion tube into the channel through the puncture. Set the microscope to fluorescent mode, and then center and bring the mushroom bodies into focus. Once the mushroom bodies are in focus, change the magnification to 20X.

Recenter the image and adjust the focus. Next, position the drainage apparatus over the drainage pool and adjust the height of the drainage shunt, so that the tip is just above the drainage pool. Then, flush the sample with ringer solution for 30 seconds to check for adequate drainage.

Finally, pull down the shield to seal off the apparatus from any outside light. Using the automated system in the photon imager program, make fine adjustments to the focus, and take a reference fluorescent image of the mushroom bodies. Adjust the photon speed to the desired recording speed, which should be anywhere from one image every 50 milliseconds up to one every second, or more.

Then, select photon mode, and open the shutter. Next, enter the genotype, sex, age, and sample number in the log entries. Adjust the position of the region of interest boxes over the calyx, the cell bodies, and the medial lobes, by clicking on the boxes and dragging them into position while holding control.

Now, record baseline values for about five to 10 minutes. Next, flip on the switch to begin the flow of nicotine. At the same time, press enter and add a note to signify the flow of nicotine has begun.

After one minute, stop the flow, and make another note in the log signifying that the flow has been stopped. Then, wash the sample with ringer solution for five minutes. While the sample recovers, turn off the wash, set the timer for an additional five minutes, and prepare to start the flow of potassium-chloride.

When ready to begin, start the flow of potassium-chloride, and enter 100 millimolar potassium-chloride start"into the sample log. After one minute, stop the flow, and enter potassium-chloride shut off"into the sample log. Then, wash the sample with ringer solution for one minute and switch the microscope to fluorescent mode.

Take a final fluorescent image, and then turn off the ringer solution and remove the sample. In the analysis program, open the first file, select the display control"tab, and click on ROI. Then, hold control and select a region of interest.

Next, adjust the size, the orientation, and shape of the ROIs to encompass the GFP illuminated calyx and cell bodies, and the medial lobes. Once all the regions of interest have been added, select the movie"tab to play the photon video. Adjust the second width and second steps as desired.

Then readjust the ROI placement as necessary, so it remains within the active regions in the video. Select some representative screenshots of the GFP fluorescence, the nicotine response, and the potassium-chloride response. Then crop the screenshots and paste the representative images into a presentation slide for late analysis and comparison.

Adjust both the second width and second step to the recording speed in seconds. Also, select the length of the analysis by moving the gray markers in the top panel to the bracketed region, before the stimulus initiation, and just after the response end. Next, select suspend views"while playing, and then press the rewind button to reset the video playback to the beginning, followed by the play button.

Then, select the tab count rate chart"in order to adjust the units and also adjust the color of the regions of interest, as well as the line colors as desired. When the analysis is finished, select export count rate data. Then, select the combined recordings of the calyx and cell bodies, as well as those from the medial lobe regions on each side and paste those images into a slide with the response images.

One of the simplest ways to stimulate the mushroom bodies is through activation of the ionotropic nicotinic acetylcholine receptors. The typical response to 25 micromolar nicotine, shown here, commences with the fast activation of both the calyx and cell bodies, and the medial lobes, and uses the fused GFP-aequorin construct, which binds to calcium and emits light. Generally, the response in the calyx and cell bodies lasts longer, and exhibits a greater bioluminescent response than the lobes, as shown in the sequential panels of the response.

A sample recording of the bioluminescent response to nicotinic activation over time is described by this graph, where the number of photons in the region of interest containing the calyx and cell bodies is graphed in black, and the number of photons within the medial lobe region are shown in red. From this graph, the length of the response, the peak photon value during the response, and the total area under each curve can all be determined. Once mastered, the preparation phase should take only two hours prior to incubation, depending on the number of flies.

In addition, each recording can be completed in as little as twenty minutes, if it is performed properly. While attempting this procedure, it's important to remember to properly age and keep fly stock to ensure reproducability among samples. Following this procedure, further behavioral tests can be performed in order to answer additional questions, like what affect the activity of the structure has on specific behavior.

The development of this technique has paved the way for researchers in the field of neurobiology to explore long-term neural activity in the drosophila brain. After watching this video, you should have a good understanding of how to prepare samples for bioluminescent imaging in the fly brain. Don't forget that nicotine can be hazardous to your health.

Precautions such as gloves should be taken when preparing solutions or cleaning up from this procedure.