We are indebted to E. Karplus, from Sciences Wares, USA, for his precious and useful help and advices. We thank E. Carbognin, A. Avet-Rochex, M. Murmu, P. Pavot, D. Minocci, G. Vinatier, and J. Stinnakre for their contribution to the development and improvement of this technique. We also thank B. Pfeiffer and G. Rubin, Janelia Farm, H.H.M.I., Ashburn, USA, for the pJFRC65-13XLexAop2-IVS-G5A-BP line. This work was supported by the Chateaubriand Fellowship, French Embassy, Washington, USA to A. Lark, by the National Science Foundation (IOS 1352882) to T. Kitamoto, and by the French ANRs (ANR-05-NEUR-009 Drosaequorin, 2005, and FlyBrainImaging, 2011), the Conseil R&#233;gional Ile-De France (NeRF and DIM), the IFR-144, the Physique-Chimie-Biologie Interface Program of the CNRS (2009), and by the CNRS, France to JR Martin.

1. Preparation of Samples
<ol>
	<li>Preparation of solutions and set up
	<ol>
		<li>Maintain all Drosophila melanogaster lines at 24 &#176;C on standard food medium. Rear and keep them at low density in the vial to generate standardized size and weight of flies.
		<ol>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Prepare Ringer&#8217;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 &#181;M nicotine and 100 mM KCl in Ringer&#8217;s solution.</li>
		<li>Prepare 1,000 &#181;l pipette tip: using a razor blade shape tip to a slant (approximately 35&#176; from end of tip) and remove excess base beyond 1 &#189; centimeters from tip.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Preparation of samples
	<ol>
		<li>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.</li>
		<li>Gently with forceps place fly inside of pipette tip.</li>
		<li>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).</li>
		<li>Combine a small (approximately 3-4 &#181;l) 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 &#8212; avoiding the crown of the head. Let the glue dry for 2 min.</li>
		<li>Place pipette tip with glued fly through hole in recording chamber (Figure 2D) and gently press to secure in place.</li>
		<li>Combine the two components of silicon glue (approximately 3-4 &#181;l). Apply glue on the flat side of chamber along the edge where the chamber and pipette tip meet to prevent leak of the Ringer&#8217;s solution. Let the glue dry for 2 min.</li>
	</ol>
	</li>
	<li>Dissection
	<ol>
		<li>Affix tape over the perfusion channel, short edge and extending to the back of the chamber.</li>
		<li>Place chamber with fly on dissection block under microscope turn on fluorescent lamp. Pipet 1 ml of Ringer&#8217;s solution into chamber.</li>
		<li>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).</li>
		<li>Use fine sharp forceps to gently grasp and clear away exposed respiratory tissue until the brain and [fluorescing] mushroom body is clearly visualized.</li>
		<li>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.</li>
		<li>Using a pipet, wash out twice with Ringer&#8217;s solution to remove any debris from dissection and place sample in the dark box.</li>
		<li>Pipet 1 ml of Ringer&#8217;s solution containing 5 &#181;M benzyl-coelenterazine into the chamber. Close the box and allow incubation with coelenterazine at RT for a minimum of 2 hr.</li>
	</ol>
	</li>
</ol>
2. Imaging
<ol>
	<li>Setup
	<ol>
		<li>Start recording system: turn on microscope, computer, camera and drainage system and set room environmental controls to 25 &#176;C.</li>
		<li>Open Measurement and Automation Explorer program on the computer. Click on &#8220;Devices and Interfaces&#8221;, then double click on &#8220;NI Motion Devices&#8221; and finally right click on &#8220;PCI-7334&#8221; and select &#8220;initialize device&#8221;.</li>
		<li>Open Photon Imager. Create new folder and name first recording file. Camera must reach -80 &#176;C before system will function.</li>
		<li>Set up perfusion system&#160;&#8212; add KCl, nicotine, and Ringer&#8217;s solution to reservoirs and adjust so the each solution discharges at 2 ml/min. Wash with Ringer&#8217;s solution prior to starting experiment.</li>
	</ol>
	</li>
	<li>Prepare sample
	<ol>
		<li>Place imaging mount block dish on mount under microscope at 5X magnification and insert perfusion tube into channel through puncture.</li>
		<li>Set microscope to fluorescent mode then center and bring mushroom bodies (MBs) into focus. Adjust to 20X and re-center and focus.</li>
		<li>Position drainage apparatus over drainage pool, adjust height of the drainage shunt so that the tip is just above pool, and run Ringer&#8217;s solution for 30 sec to ensure adequate drainage.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Recording
	<ol>
		<li>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.</li>
		<li>Record baseline for about 5 to 10 min (as desired).</li>
		<li>Apply nicotine for 1 min. Note start/stop in log. Wash with Ringer&#8217;s solution for 5 min.</li>
		<li>Wait 5 min for recovery and prepare KCl log entry and timer.</li>
		<li>Apply KCl for 1 min. Note start/stop in log. Wash with Ringer&#8217;s solution for 1 min.</li>
		<li>Switch to fluorescent mode, take final fluorescent image, and turn off Ringer&#8217;s solution.</li>
		<li>Press &#8220;Stop&#8221;. Dialog box will ask to shut off the system. Select &#8220;No&#8221; to continue recording.</li>
		<li>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.</li>
		<li>Press the white play button at the top of the program window to initiate the next recording.</li>
	</ol>
	</li>
</ol>
3. Analysis and Video Creation
<ol>
	<li>Extract photon values
	<ol>
		<li>Open the photon analysis program (e.g., Photon Viewer) and open first sample spreadsheet file.</li>
		<li>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.</li>
		<li>Add an additional ROI by clicking &#8220;Define ROI&#8221; and clicking and dragging on screen to create the new ROI.</li>
		<li>Select the &#8220;Movie&#8221; tab to play photon video. Adjust &#8220;sec width&#8221; and &#8220;sec steps&#8221; as desired. Readjust ROI placement as necessary.</li>
		<li>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.</li>
		<li>Adjust both the &#8220;sec width&#8221; and &#8220;sec step&#8221; 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.</li>
		<li>Select &#8220;Suspend views while playing&#8221; press &#8220;&#60;&#60;REW&#8221; and press &#8220;PLAY&#62;&#8221;. Select the tab &#8220;Count Rate Chart&#8221; and adjust units as desired and line colors to match ROI color.</li>
		<li>When analysis is finished, press &#8220;Export Count Rate Data&#8221;. 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.</li>
	</ol>
	</li>
	<li>Example Analysis: one method for analysis of extracted photon data detailed
	<ol>
		<li>Open the spreadsheet files in &#8220;Count Rate Exports&#8221; folder.</li>
		<li>Reformat the cells the first column labeled time to display the time in hour and minutes.</li>
		<li>Reference the corresponding slide for the sample. Remove all values except the columns for the time and the two representative ROIs.</li>
		<li>Determine nicotine stimulation start time&#160;&#8212; available in log entry file in main folder- remove additional data prior to start or highlight value.</li>
		<li>Find highest/ peak value for both CCB and medial lobe and mark/highlight.</li>
		<li>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.</li>
		<li>Combine all samples of one experimental group on a new spreadsheet.</li>
		<li>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.</li>
		<li>Copy the sheet twice and delete either the medial lobe columns or the CCB columns creating pages of only CCB or medial lobe values.</li>
		<li>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.</li>
		<li>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&#160;&#8212; select from the beginning of the perfusion of nicotine to the start of the response&#160;&#8212; to determine Response Latency.</li>
		<li>Copy values by using the linking function and multiply (all except peak amplitude) by recording speed in seconds.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Repeat this analysis procedure with the data from the medial lobes.</li>
	</ol>
	</li>
	<li>Creating Images for the Video
	<ol>
		<li>Open photon viewer.</li>
		<li>Select display control tab. Click on ROI while holding control to select, remove and/or minimize it.</li>
		<li>Modify &#8220;sec width&#8221; (accumulation time) as desired to determine the content of each frame.</li>
		<li>Modify &#8220;sec step&#8221; to define the overlap of each frame.</li>
		<li>Select &#8220;export views while playing&#8221; press &#8220;&#60;&#60;REW&#8221; then&#8221; PLAY&#62;&#8221;. The images for the video will be exported to the &#8220;view exports&#8221; folder as a series of .png files.</li>
	</ol>
	</li>
	<li>Using Virtual Dub to make video: one method for video creation detailed
	<ol>
		<li>Create a new folder named &#8220;resultats&#8221; in the view exports folder containing the images.</li>
		<li>Bring script (renommer.VBS) into the folder of images.</li>
		<li>Double click on renommer.VBS to run.</li>
		<li>Images will automatically be renamed numerically and copied to &#8220;resultats&#8221; folder.</li>
		<li>Open VirtualDub.exe.</li>
		<li>Select open video file &#8212; select first image (subsequent images will automatically load).</li>
		<li>Select video &#8212; select frame rate adjust as desired.</li>
		<li>Select video &#8212; select compression select Cinepak Codec by radius.</li>
		<li>In file select Save as AVI the video will be created automatically.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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
GFP-aequorin transgene (pG5A)4 was inserted into the pUAST vector to create three independent transformant lines6. All lines were tested for their bioluminescence, and all were able to respond to the in vivo Ca2+-activity6. However more recent experiments have pointed out that the GA2 line inserted on the third chromosome generates more robust signal than the GA1 line inserted on the second chromosome. Here, the representative results were obtained by using UAS-GA2 crossed with the MB-specific line OK107. Moreover, recently B. Pfeiffer has generated a GFP-aequorin construct placed under the control of 20XUAS regulatory element (20X-G5A-2) and this construct has been validated in our lab. Briefly, it gives a very strong signal, stronger than our standard GA2, which therefore should be more efficient and useful to further monitor the Ca2+-activity in deeper structures of the brain, such as the ellipsoid-body. Additionally, it may also be useful for imaging small quantities of neurons or in axon terminals (synaptic contacts).
Preparations for Imaging
Although the fly must be ice anesthetized during positioning and gluing, it is important to minimize the time on ice. Flies should be transferred to a glass vial and kept on ice for 2 min before being moved to the chilled Petri dish for positioning in the pipette tip. We have noticed that extended time period of anesthesia reduces the viability of the sample and may attenuate the response. Optimally, we keep the time of ice anesthesia below 5-10 min.
Taping and gluing are critical steps. It is crucial to secure the fly head in place for dissection and imaging as well as to prevent leakage of the Ringer&#8217;s solution into the pipet tip and/or out of the recording chamber. Dental glue (and to a lesser extent the silicon glue) will become sticky and subsequently begin to dry as soon as the two components are combined. Therefore it is critical to apply the glue expeditiously to avoid weak bonding and clumping of the glue. In some cases we have noticed that alternate varieties of tape and glue, when in contact with the Ringer&#8217;s solution, may release contaminants into the Ringer&#8217;s solution which can affect the fly and its response, particularly so for experiments dedicated to the study of olfactory system, using different odors. Consequently, use caution here if substituting materials (see materials for specific varieties we use).
Several different forms of coelenterazine have been developed in order to optimize its activity such as the speed of light emission, Ca2+-affinity (sensitivity), or the intensity of light emission12. For example, the native coelenterazine is more stable, thus more suitable of long term imaging as several hours, while the benzyl-coelenterazine (also named h-coelenterazine) results in a faster and higher light emission with shorter half-life. For additional information about different forms of coelenterazine, see the web address in the Materials List.
Imaging 
Bioluminescence signals can be monitored with an electron multiplier CCD camera (EM-CCD, cooled to -80 &#186;C) fitted onto a microscope. This setup must be housed inside a tight dark box to avoid any undesired (ambient) light contamination. The setup described in this manuscript uses a 20X immersion-objective lens permitting a field of view of 400 x 400 &#181;m (512 x 512 pixels). To improve signal to noise ratio, data were acquired with a 0.100 sec&#160;integration time (10 Hz), and 2 x 2 binning was used (1 pixel = 1.2 x 1.2 &#956;m). To acquire and store data, each detected photon was assigned x,y-coordinates and a time point. Between 500 and 600 nm wavelength, the EM-CCD camera (see precise description in Materials List) has a quantum efficiency of 96%.
One of the most crucial parameters for the in vivo functional brain imaging is the temporal resolution. Up to now, the majority of fluorescence-based Ca2+-imaging technique such as GCaMP and cameleon provides a temporal resolution of about 4 Hz (250 msec/frame). Recently, with bioluminescence-based GFP-aequorin we have been able to raise the acquisition time up to 100 msec/frame with the EMCCD camera and even up to 120 frames/sec (8.3 msec/frame) with the electron multiplier ICDD camera10. At this temporal resolution approaches that of the electrophysiological techniques (in the range of about 1 msec). While high temporal resolution is both informative and crucial for the studies dedicated to synaptic transmission and stimulus induced activity, extended slower yet essential Ca2+-kinetics also occurs within neurons, for instance the Ca2+ release from the intracellular Ca2+-stores (for a review: Berridge et al., 200313). For this latter type, a slower temporal resolution is still acceptable, while inversely, they generally require a better Ca2+-affinity from the sensor to be detected. This requirement has been achieved with GFP-Aequorin to detect the release of the intracellular Ca2+-stores at the axon terminal of the olfactory receptors neurons7,8. Alternatively, depending on the desired experimental conditions, slower acquisition time could be used. For instance, in long O/N recordings, we have used a 2 sec&#160;integration times due to the large number of data points collected (Minocci et al., 2013). In brief, one practical feature with the bioluminescence is that the temporal resolution can be easily adjusted according to the desired experimental conditions.
Alternative chambers (fly&#8217;s setup) have been developed to facilitate the objectives of the experiment. For example, for the studies based on natural odor stimulus, the antenna must be kept free, therefore an approach like the one developed by A. Fiala14 is best suited (we used a similar approach for Murmu et al. 2010). Briefly, in this approach, the fly is tethered (glued) on a minutien pin at the neck, which is glued to a plastic cover slip containing a small hole. A seal is created around the top of the head of the fly. Thereafter, a drop of ringer is deposited on the head, and the head capsule is dissected. In such a way, the antennae are kept free and are available to sense odors. Similarly, for the long term recording, as O/N or even couple of days10 the fly needs to be kept as free of constraint as possible, and in some cases fed (for example: with a capillary paper soaked in a 5% glucose solution). In these circumstances Fiala&#8217;s approach is also more suitable than the approach in the blue pipet tip (snorkeling method).
All drug applications can be controlled externally using a 6 way multi-valves gravity perfusion system. The flow can be controlled using volumetric perfusion regulators calibrated prior to each recording session for a flow of 2 ml/min. Drainage apparatus extracts liquid at a rate of 2 ml/min from the recording chamber via peristaltic pump permitting a continuous flow.
In some conditions, due to expensive cost of the drug and/or the amount of drug to use, one would like to apply only small quantities of compounds. Thus, it is necessary to directly apply it in the bath rather than through perfusion system. In this case the shutter must be closed during this process to avoid the light contamination.
Analysis methods
Counts per sec or counts per sec per coordinate have both been used by our group for analysis. Counts per second is perhaps best suited for bright responses in whole structures whereas counts per sec and per coordinate (which represents a normalization of the responses to the size of the ROI) is best suited and accurate for small populations of cells. Both of them can be easily selected in the analysis software.
One of the main advantages of using the bioluminescence approach is the way that are acquired and stored the data (x,y,t parameters of each emitted photons). Indeed, while any other imaging techniques used standard mode as an acquired full image (generally in a tiff format), with this system, we acquires only the relevant and significant signal by storing only the pixel coordinates which have been activated by the light during a pre-fixed duration of time (which corresponds to the acquisition time or temporal resolution). Consequently, the storage of the data is really &#8220;light&#8221; compared to the storage a full image, and consequently, makes it easier to analyze the data. For data analysis, we use a related software (photon viewer), which allows setting of the accumulation time as desired. Then the display of an image of the signals can be adjusted as desired in a goal to convincingly present the data. In parallel the results are quantified at the same resolution time than their acquisition time.
Significance and biological applications of bioluminescent imaging 
As mentioned above bioluminescent calcium imaging offers three principal advantages to fluorescent calcium imaging techniques: (1) a deeper regions of the brain may be imaged, (2) imaging can occur continuously for longer durations as several hours as O/N and even in some cases up to two days, and (3) more recently, with a higher temporal resolution, up to 120 frame/sec (8.3 msec). The main limitation of the bioluminescent approach is due to its incompatibility with confocal illumination, which consequently does not allow us to determine the precise depth (z-plan) of the activated structures or neurons.
The first advantage of bioluminescent imaging, imaging of deep brain regions, was previously demonstrated in 2007 by the imaging of a high potassium induced calcium response within the ellipsoid body (eb), a deep structure located in the middle of the brain, with no prior electrophysiological or functional reports6. Similarly, a more recent example is the recording of the spontaneous Ca2+-activity in the eb following application of picrotoxin (a blocker of GABAA-receptors) demonstrating that the GABAergic ring-neurons of the eb are indeed inhibitory neurons (described in: Diaper et al., article submitted).
The second benefit, longer and continuous time recording, has been exploited in several studies from our lab. Odor stimulation has been used to examine activity of brain neurons in previous studies11,15,16. Using longer term imaging, however, our lab has been able to show previously unreported changes in kinetics within the olfactory receptor neurons to odors. The first study by Murmu et al. (2010) showed that while short odor stimuli (1-3 sec) exhibited similar kinetics and a moderate difference in amplitude, a longer odor stimulus (5 sec) caused a much greater amplitude as well as a change in the kinetics/structure of the response which was attributed to intracellular calcium stores. A subsequent study showed that while 5 successive applications of 1 sec&#160;odor pulses at 5 min&#160;intervals did not change their calcium response, increasing the length of the stimulus to 5 sec&#160;led to progressive attenuation of the response, suggesting an adaptation process. Furthermore this adaptation phenomenon appeared to be initiated by GABAergic regulation of previously identified calcium stores8. Importantly this data also proves that, in this experimental condition, the GFP-aequorin&#8217;s Ca2+-sensor can detect and follow the calcium released from the intracellular Ca2+-stores. An additional unique study from our lab used bioluminescent imaging O/N to record basal (spontaneous) activity in the whole neuronal and glial populations in the brain as well as activity in the mushroom bodies10. These recordings revealed surprising spontaneous activity in antennal mechanosensory and motor center (AMMC) and antennal lobe. Furthermore, unexpected cell autonomous activity in the glia occurring throughout the night. Within the mushroom body the study identified both punctate activity and two distinctive peaks, one short and one long whose incidence changed with age10.
In the most recent study in our lab, we used bioluminescent imaging to examine the effects of the manipulation of secondary signaling molecules implicated in memory, and how they affect the calcium response within the mushroom bodies9. Indeed, although dunce and rutabaga were identified more than 30 years ago and are known to regulate the level of cAMP, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca2+-response still remain largely unknown. With the GFP-aequorin approach, we have been able to simultaneously record both the calyx (the dendritic structures) and the cell-bodies, as well as the axonal projections at the level of the lobes, and thus allowing us to correlate the level and the kinetic of the response in the different compartments of this highly complex structure.
The most recent application we are developing mimics a natural response through artificially activating neuronal populations presynaptic to the structure of interest. To do this we express the P2X2 receptor, a ligand-gated cation channel which is responsive to ATP, in the presynaptic neuron and express GFP-aequorin in the post-synaptic neuronal population. The segregation of these two elements into different neuronal populations requires two separate expression systems. To this end a LexA construct containing GFP-aequorin has been created. In our preliminary experiments with this system we have expressed P2X2 in a subset of the PNs using the Gal4-GH146 with UAS-P2X2 and GFP-aequorin in the mushroom body using the LexA MB247 line combined with LexA-13X-GFP-aequorin. We have successfully recorded calcium responses in the mushroom body with stimulations of 1 mM ATP to the P2X2 receptor in the PNs (Figure 6).
In conclusion, the bioluminescence GFP-aequorin approach has proven to be useful in recording the stimulus induced Ca2+-activity, analogous to other Ca2+-indicators. But more importantly, it also permits the detection of spontaneous Ca2+-activity within the brain. This approach opens up future possibilities for long term experiments and whole brain imaging that can increase the depth of our understanding of the physiological phenomena and activity patterns within the brain.

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 &#8212; 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 &#8212; 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 (&#955;=469 nm)2,3. Aequorin has a very low affinity for calcium (Kd = 10 &#181;M) 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 Br&#251;let 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 (&#955;=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 &#8220;activity&#8221; 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 scent7,8, 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.

<table><tbody><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>1023911000</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td></td></tr><tr><td>KCl</td><td>prolabo</td><td>26 764.298</td><td>normapur</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>prolabo</td><td>25 108.295</td><td>normapur</td></tr><tr><td>sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Nicotine</td><td>Sigma-Aldrich</td><td>N3876</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A2383</td><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate</td></tr><tr><td>benzyl-coelenterazine</td><td>Prolume, nanolight&amp;nbsp;</td><td>Cat #301 Coelenterazine h</td><td>http://www.prolume.com/</td></tr><tr><td>dental glue</td><td>3M ESPE&amp;trade;</td><td>46954</td><td>Protemp IV</td></tr><tr><td>silicon glue</td><td>3M ESPE&amp;trade;</td><td>36958</td><td>Express 2 Regular Body Quick</td></tr><tr><td>tape</td><td>3M Scotch</td><td>122s</td><td>3M Scotch Magic Tape</td></tr><tr><td>pipette tips</td><td>Corning Incorporated</td><td>4868</td><td>100-1,000 &amp;micro;l Univesal Pipette Tip</td></tr><tr><td>chamber and holder (stage)</td><td></td><td></td><td>hand made</td></tr><tr><td>incubation box</td><td></td><td></td><td>hand made</td></tr><tr><td>forceps (No. 5)</td><td>Fine Science Tools</td><td>11252-00</td><td>Micro-dissecting forceps</td></tr><tr><td>curved forceps</td><td>Sigma-Aldrich</td><td>F4142</td><td>Micro-dissecting forceps</td></tr><tr><td>glue applicator&amp;nbsp;</td><td></td><td></td><td>hand made&amp;nbsp;</td></tr><tr><td>knife</td><td>Fine Science Tools</td><td>10316-14</td><td>5 mm Depth 15&amp;deg; stab</td></tr><tr><td>EM-CCD camera&amp;nbsp;</td><td>Andor</td><td>DU-897E-CS0-#BV</td><td>iXon (cooled to -80 &amp;deg;C)</td></tr><tr><td>Microscope</td><td>Nikon</td><td></td><td>Eclipse-E800</td></tr><tr><td>immersion objective lens</td><td>Nikon</td><td></td><td>20X Fluor .5w</td></tr><tr><td>dissection microscope with fluorescence</td><td>Leica MZ Fl III</td><td></td><td>leica dissection scope and florescent lamp</td></tr><tr><td>tight dark box</td><td>Science Wares</td><td></td><td>Custom built by Science Wares</td></tr><tr><td>peristaltic pump&amp;nbsp;</td><td>Gilson</td><td></td><td>Minipuls 2</td></tr><tr><td>tubing</td><td>Fisher Scientific</td><td>depends on thickness</td><td>Tygon R3603, St-Gobain</td></tr><tr><td>perfusion system</td><td>Warner</td><td>64-0135 &amp;nbsp;(VC-66CS)</td><td>Perfusion valve control system, complete, with pinch valves, 6 channel</td></tr><tr><td>perfusion regulators</td><td>Leventon</td><td>201108</td><td>Dosi-flow 3</td></tr><tr><td>measurement and automation explorer</td><td>Sciences Wares &amp;amp; National Instruments</td><td>N/A</td><td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td></tr><tr><td>photon imager&amp;nbsp;</td><td>Science Wares &amp;amp; National Instruments</td><td>N/A</td><td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td></tr><tr><td>photon viewer</td><td>Science Wares &amp;amp; National Instuments</td><td>N/A</td><td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td></tr><tr><td>Excel</td><td>Microsoft</td><td>N/A</td><td>software https://www.microsoft.com</td></tr><tr><td>virtual dub</td><td>open access&amp;nbsp;</td><td>N/A</td><td>software http://virtualdub.sourceforge.net/</td></tr><tr><td>UAS-GA2</td><td>Martin &lt;em&gt;et al.&lt;/em&gt;, 2007, Ref. 1&amp;nbsp;</td><td>N/A</td><td>No stocks {currently} publicly available&amp;nbsp;</td></tr><tr><td>pJFRC65-13XLexAop2-IVS-G5A-BP&amp;nbsp;&amp;nbsp;</td><td>B. Pfeiffer, Janelia Farms, Ashburn USA.&amp;nbsp;</td><td>(STOCK #1117340)</td><td>pfeifferb@janelia.hhmi.org</td></tr><tr><td>P2X&lt;sub&gt;2&lt;/sub&gt;</td><td>Lima and Misenboeck, Ref. 11&amp;nbsp;</td><td>N/A</td><td>No stocks {currently} publicly available&amp;nbsp;</td></tr><tr><td>OK107</td><td>Bloomington stock center</td><td>854</td><td>http://flystocks.bio.indiana.edu/</td></tr></tbody></table>

in vivo functional brain imaging approach based on bioluminescent calcium indicator gfp-aequorin

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&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; 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.

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 because they specifically express the nicotinic acetylcholine receptors, and thus by using nicotine we can avoid the activation of the muscarinic acetylcholine receptors expressed elsewhere in the brain. Moreover, nicotine is more stable than the acetylcholine, and thus allows better and more reliable stimulus control. A typical response commences with the fast activation of both the calyx, cell-bodies (CCB) and medial lobes (ML) (see Figure 4A for labeled image). Generally the CCB response lasts longer and exhibits a greater bioluminescent response than the lobes as shown in the sequential panels of the response (Figure 3C-H). Figure 4B shows a 10 sec&#160;accumulation of photons recorded at a temporal resolution of 100 msec (10 Hz) during the peak of a response to a 1 min&#160;perfusion of 25 &#181;M nicotine. Following a washout and recovery period of 10 min&#160;a second stimulation of a 1 min&#160;perfusion of 100 mM KCl is initiated (Figures 4C, E) which allows us to confirm the viability of our sample. The quantitative responses can be analyzed by selecting ROI during analysis in photon viewer. Running the analysis in photon viewer produces both graphs displaying the number of photons emitted in selected ROI over time (Figure 4D and E) as well as exportable spreadsheets of those values. Figure 4D is an example of the graphs produced by photon viewer plotting the photons emitted in the ROI 2 [green] (right CCB) and ROI 4 [blue] (right medial lobes). Similar data for the KCl response represented in Figure 4E. The exported data can be used to reconstruct the curve of the response (Figure 5A) as well as to extract additional data about various parameters such as duration, peak amplitude, and total response (Figure 5B-D). Recently using the Gal4-UAS and LexA systems we have devised a method of eliciting a more natural response. Briefly, the ATP-gated P2X2 cation channel is expressed in the projection neurons (PNs) and GFP-aequorin (GA) is expressed in the mushroom bodies &#8212; a target of the PNs; this allows us to selectively excite the PNs though application of ATP, which can in turn, produce response in mushroom bodies (Figure 6).
<img alt="Figure 1" src="/files/ftp_upload/53705/53705fig1.jpg" /> 
Figure 1. Characteristics of Bioluminescent Reporters (GFP-aequorin). (A) Schematic of GFP and aequorin fusion gene with upstream activation sequence. (B) Model of blue light emission by aequorin in response to high levels of Ca2+. (C) Model of green light emission by GFP-aequorin in response to high levels of Ca2+. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53705/53705fig2.jpg" /> 
Figure 2. Experimental set up. (A) Fly positioned in pipet tip. (B) An illustration of adequate gluing technique stabilizing the head and sealing the region between the fly body and pipet tip. (C) Schematic of dissection to open head capsule. (D) 1 ml chamber on holding block with perfusion tube inserted. Total chamber dimensions: length: 7.4 cm, width: 2.7 cm, height: 0.55 cm. Inner chamber dimensions: length: 1.7 cm, width: 1.4 cm, height: 0.35 cm, and the radius of the chamber hole: 0.1 cm. (E) View of fly head showing GFP-positive signal in the MBs: low dim light with fluorescence derived from OK107-driven GFP-aequorin after removal of cuticle. (F) Microscope with photon camera and perfusion system. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53705/53705fig3.jpg" /> 
Figure 3. Representative response to 25 &#181;M nicotine in mushroom bodies. (A) Fluorescent image of the GFP-aequorin expression in the mushroom bodies in GA2/+;OK107/+ control fly brain (scale bar = 50 &#956;m). (B) Peak bioluminescent response in the mushroom bodies. (C) Prior to stimulation. (D) Start of response. (E) Peak response. (F) Continued CCB response. (G) Falling CCB response. (H) End of response (B-H: scale bar = 33 &#956;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53705/53705fig4.jpg" /> 
Figure 4. Representative analysis of sample recording. (A) Fluorescent image of the mushroom body of the GA2/+;OK107/+ control fly with four ROI&#8217;s selected over the CCB and medial lobes (scale bar = 50 &#956;m). (B) 10 sec&#160;accumulation of the bioluminescent response to 25 &#181;M nicotine &#8212; photon response recorded at 100 msec. (C) 10 sec&#160;accumulation of bioluminescent response to 100 mM KCl recorded at 100 msec. (D) Graph of recorded number of photons emitted during nicotine response within ROI&#8217;s 2 and 4 covering the right CCB and medial lobes respectively. (E) Graph of recorded number of photons emitted during KCl response within ROI&#8217;s 2 and 4 covering the right CCB and medial lobes, respectively. In d and e, the left Y-axis corresponds to the total number of photons within the whole field of view, while the right Y-axis corresponds to the photons within the ROI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53705/53705fig5.jpg" /> 
Figure 5. Representative parameters analysis of sample recording. Representative excel analysis of the bioluminescent response to nicotinic activation of the mushroom body in the GA2/+;OK107/+ control fly. (A) Photon response over time in CCB and medial lobes. (B) Duration of responses in the CCB and medial lobes. (C) Highest photon value (maximum amplitude) during response within the CCB and medial lobes. (D) Total photons within CCB and medial lobes of entire response. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53705/53705fig6.jpg" /> 
Figure 6. Two different response in mushroom bodies after stimulation of PNs through ATP and P2X2. Sample 1 fly expressing P2X2 in PNs and GFP-aequorin in mushroom bodies (GH146/+; P2X2/MB247-LexA,13XLexAop2-IVS-G5A-BP (a-d). (A) Fluorescent image of mushroom bodies with ROI&#8217;s (scale bar = 50 &#956;m). (B) Bioluminescent image of the peak response in mushroom bodies following activation of the PNs by P2X2 stimulated with 500 &#956;M ATP, observed primarily in medial lobes. (C) Bioluminescent image of peak KCl response. (D) Time course of photon emission within ROI regions, throughout the response. Sample 2 fly expressing P2X2 in PNs and GFP-aequorin in mushroom bodies (GH146/+; P2X2/MB247-LexA,13XLexAop2-IVS-G5A-BP (e-h). (E) Fluorescent image with ROI&#8217;s (scale bar = 50 &#956;m). (F) Bioluminescent image of the induced response in the mushroom bodies following activation of the PNs by P2X2 stimulated with 1mM ATP, in both lobes and CCB. (G) Bioluminescent image of peak KCl response. (H) Time course of photon emission within ROI regions, throughout the response. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53705/53705fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin at JoVE.com

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

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.

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

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 ...

in-vivo-functional-brain-imaging-approach-based-on-bioluminescent

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.3K Views.  UMR-9197, CNRS/Université Paris Sud.  Hier presenteren we een nieuwe Ca 2+ -Imaging aanpak met behulp van een bioluminescente reporter. Deze benadering maakt gebruik van een gefuseerde construct GFP-aequorine dat bindt aan Ca2 + en licht emitteert, waardoor de noodzaak voor excitatie licht. Aanzienlijk deze methode maakt het mogelijk lange doorlopende beeldvorming, de toegang tot diepe hersenstructuren en hoge tijdsresolutie. 

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

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Biology

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

Bioluminescence Imaging for Studying Calcium Transients in Drosophila Brain Structures

Source:&#160;Lark, A. R.,&#160;et. al.,&#160; In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin.&#160;J. ...

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

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