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
<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>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 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

Universidad de Cartagena UDEC

Research

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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

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 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, ...

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 ...

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

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

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

In vivo Neuronal Calcium Imaging in C. elegans

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

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

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

In vivo Calcium Imaging in Mouse Inferior Olive

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

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

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