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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Q., Miesenb&#246;ck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+behavior+through+genetically+targeted+photostimulation+of+neurons.">Remote control of behavior through genetically targeted photostimulation of neurons.</a> Cell. 121 (1), 141-152 (2005).</li><li>Shimomura, O., Musicki, B., Kishi, Y., Inouye, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+properties+of+recombinant+semisynthetic+aequorins+and+recombinant+fluorescein-conjugated+aequorin+for+measuring+cellular+calcium.">Light-emitting properties of recombinant semisynthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.</a> Cell Calcium. 14 (5), 373-378 (1993).</li><li>Berridge, M. J., Bootman, M. D., Roderick, H. 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B., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain.">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112 (2), 271-282 (2003).</li><li>Wilson, R. I., Turner, G. C., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Olfactory+Representations+in+the+Drosophila+Antennal+Lobe.">Transformation of Olfactory Representations in the Drosophila Antennal Lobe.</a> Science. 303 (5656), 366-370 (2004).</li></ol>

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
<p class...

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>CaCl2</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&#160;</td>
			<td>prolabo</td>
			<td>26 764.298</td>
			<td>normapur</td>
		</tr>
		<tr>
			<td>MgCl</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&#8242;-triphosphate disodium salt hydrate</td>
		</tr>
		<tr>
			<td>benzyl-coelenterazine</td>
			<td>Prolume, nanolight&#160;</td>
			<td>Cat #301 Coelenterazine h</td>
			<td>http://www.prolume.com/</td>
		</tr>
		<tr>
			<td>dental glue</td>
			<td>3M ESPE&#8482;</td>
			<td>46954</td>
			<td>Protemp&#8482; IV</td>
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		<tr>
			<td>silicon glue</td>
			<td>3M ESPE&#8482;</td>
			<td>36958</td>
			<td>Express&#8482; 2 Regular Body Quick</td>
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		<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>100-1000&#181;L Univesal Pipette Tip</td>
		</tr>
		<tr>
			<td>chamber and holder (stage)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>hand made</td>
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		<tr>
			<td>incubation box</td>
			<td>N/A</td>
			<td>N/A</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>
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		<tr>
			<td>curved forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td>Micro-dissecting forceps</td>
		</tr>
		<tr>
			<td>glue applicator&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>hand made&#160;</td>
		</tr>
		<tr>
			<td>knife</td>
			<td>Fine Science Tools</td>
			<td>10316-14</td>
			<td>5mm Depth 15&#176; stab</td>
		</tr>
		<tr>
			<td>EM-CCD camera&#160;</td>
			<td>Andor</td>
			<td>DU-897E-CS0-#BV</td>
			<td>iXon (cooled to -80&#176;C)</td>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>Eclipse-E800</td>
		</tr>
		<tr>
			<td>immersion objective lens</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td>20x Fluor .5w</td>
		</tr>
		<tr>
			<td>dissection microscope with fluorescence</td>
			<td>Leica MZ Fl III</td>
			<td>N/A</td>
			<td>leica dissection scope and florescent lamp</td>
		</tr>
		<tr>
			<td>tight dark box</td>
			<td>Science Wares</td>
			<td>N/A</td>
			<td>Custom built by Science Wares</td>
		</tr>
		<tr>
			<td>peristaltic pump&#160;</td>
			<td>Gilson</td>
			<td>N/A</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 &#160;(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 &#38; 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&#160;</td>
			<td>Science Wares &#38; 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 &#38; 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&#160;</td>
			<td>N/A</td>
			<td>software http://virtualdub.sourceforge.net/</td>
		</tr>
		<tr>
			<td>UAS-GA2</td>
			<td>Martin et al., 2007, Ref. 1&#160;</td>
			<td>N/A</td>
			<td>&#160;No stocks {currently} publicly available&#160;</td>
		</tr>
		<tr>
			<td>pJFRC65-13XLexAop2-IVS-G5A-BP&#160;&#160;</td>
			<td>B. Pfeiffer, Janelia Farms, Ashburn USA.&#160;</td>
			<td>(STOCK #1117340)</td>
			<td>pfeifferb@janelia.hhmi.org</td>
		</tr>
		<tr>
			<td>P2X2</td>
			<td>Lima and Misenboeck, Ref. 11&#160;</td>
			<td>N/A</td>
			<td>&#160;No stocks {currently} publicly available&#160;</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 becau...

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

Research

JoVE Journal

Neuroscience

12.2K Views.  UMR-9197, CNRS/Université Paris Sud. 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.

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

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

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, ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

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 ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

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 ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...