We acknowledge Silke Sachse and Beate Eisermann in the development of this methodology, Pablo Traversa for drawing the mounting block blueprints and Daniela Pelz for use of the schematic in Figure 1. We are grateful to Silke Sachse, Pavan Ramdya and Raphael Rytz for comments on the manuscript. R. Bell is supported by a Boehringer Ingelheim Foundation PhD Fellowship. Research in R. Benton&#x2019;s laboratory is funded by a European Research Council Starting Independent Researcher Grant and the Swiss National Science Foundation.

1. Mounting Block Assembly
<ol>
 <li>The plexiglass mounting block (Figure 1A) should be made to the exact specifications given in the blueprint (available from 
 <a href="http://neuro.uni-konstanz.de/jove_mountingblock_blueprint/" target="_blank">http://neuro.uni-konstanz.de/jove_mountingblock_blueprint</a>) to allow easy mounting and dissection of the fly. Thread through the screws from the back but do not extend them beyond the front of the block; their position will be adjusted during the preparation of the animal (see 2.7).</li>
 <li>Using a precision drill, make a dent in the upper border of the circular chamber for the fly, digging under the surface to make space for the fly's body, reducing the thickness of the block at this point.</li>
 <li>Cut a copper grid with a scalpel perpendicular and close to the bottom of the slit to create a "collar". Make sure the two resulting flaps are level, as this will help insertion of the fly.</li>
 <li>With a toothpick, place a small drop of superglue onto the mounting block above the circular chamber for the fly. Place the copper grid onto the glue and position so that the slit is centered on the block (Figure 1A). Press down gently and remove any excess glue. With a pair of tweezers, add tiny drops of glue under the flaps on each side and fold the grid over the front of the block so that the slit is pointing straight downwards. Make sure the top of the grid is level with the block. Allow to dry completely.</li>
 <li>Assemble a wire holder: cut a plastic coverslip in half and remove a central piece to make a "U" shape. Use beeswax to glue a piece of wire across the top of the "U"; do not make the wire too taut.</li>
 <li>Construct the antennal shield: make a hole in a plastic coverslip with a paper hole-punch. Trim the edges of the coverslip to 0.5 x 0.7 cm. Glue the pierced plastic coverslip to sticky tape and then place a drop of ethanol on the tape exposed through the hole to remove the adhesive.</li>
</ol>
2. Animal Preparation
<ol>
 <li>Flies of the appropriate genotype - i.e. bearing the desired combination of "driver" and activity-reporter transgenes - should be 1-3 weeks old; the cuticle of younger flies is too soft and difficult to cut. When experiments require direct comparison of different genotypes, and in order to match transgene expression levels, flies should be age-matched to avoid age-related physiological differences. If gender is not important, we prefer to use female flies since they have larger heads and are easier to manipulate.</li>
 <li>Anesthetize flies by cooling on ice.</li>
 <li>Introduce a single fly into the mounting block. Hold the fly by the wing joint and slide it, dorsal-surface first, along the slit of the copper grid so that it is suspended by its neck (Figure 1B-C). If necessary, rotate it until it is looking down.</li>
 <li>Place a fine cactus spine in front of the fly's head to prevent it from escaping (Figure 1B-C). Place the cactus spine in front of the proboscis to prevent it from moving. Fix the cactus spine to the block using beeswax. Make sure that the top of the head is level with the top of the block.</li>
 <li>Fix the head of the fly to the block with a small drop of rosin (dissolved in ethanol). Place the block in a humidified box and allow the rosin to harden for about one hour.</li>
 <li>Using forceps, pull the antennal plate forward and drop the wire on the holder (wax-side out; see 1.5) in the cuticular fold between the antenna and head. Fix the holder to the front of the block with beeswax.</li>
 <li>Using the screws built into the block, gently push the wire holder forward to create some space between the antennal plate and the head.</li>
 <li>Place the antennal shield (see 1.6) over the top of the fly's head with the hole positioned centrally. Fix with beeswax to the top of the mounting block.</li>
 <li>Using a blade-splitter, cut a small hole in the tape on the shield exposing the cuticle of the fly's head behind the antenna. The hole should not extend beyond the eyes to prevent the preparation from leaking.</li>
 <li>Seal the hole between the tape and the cuticle with two-component silicon. Remove any excess silicon from the top of the fly's head.</li>
 <li>Place a drop of Drosophila Ringer's saline (130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 36 mM saccharose, 5 mM HEPES, pH 7.3) on the head. Make sure that there are no leaks: the antennae should remain completely dry. Using a sapphire blade, cut a hole in the cuticle. First, lightly score along the cuticular fold directly behind the antenna, then cut along the eyes and across the ocelli on the back. Finally, fold the piece of cuticle over the scored edge and cut from the underside to avoid severing the antennal nerves.</li>
 <li>Carefully remove glands and trachea with fine tweezers. Rinse repeatedly with Drosophila Ringer's, leaving a large drop on the head.</li>
</ol>
3. Odor Stimulus Delivery
<ol>
 <li>Odor syringes: insert a cellulose pad into a 2 ml plastic syringe with clean forceps and pipette onto it (using filter tips) 20 &mu;l of odor, diluted as desired in an appropriate solvent, using the pipette tip to push the pad deep into the barrel. Plug and cap the syringe until required. Fresh odor dilutions should be prepared at least every 3 months (or more frequently for highly volatile and/or often-used odors), and new pads should be prepared for odor syringes just before each recording session. Do not use odor pads for more than three stimulations, since the odor concentration might decay.</li>
 <li>Custom-made olfactometer (Figure 2): a primary air stream (1 l/min) is directed through Teflon tubing (0.4 mm internal diameter); 27 cm upstream of the exit of this tube, a secondary air stream (1 l/min) is added. Both air streams are generated by membrane pumps, and the flow rate is regulated by two independent flowmeters. The air streams can be humidified by passing through water in gas washing bottles, although care should be taken to avoid over-humidification (i.e. more than ~60%). The secondary air stream is directed either through an odor syringe or an empty syringe: syringes are connected to the airflow through a syringe needle (1.2 mm outer diameter) piercing the plunger. A three-way magnetic valve controlled via a valve controller unit (e.g. Harvard Apparatus VC6) is used to switch between the clean air and odor stimulus.</li>
</ol>
4. In vivo Imaging
<ol>
 <li>We describe here imaging using an upright fluorescent microscope (e.g. upright fixed Stage Zeiss Axio Examiner D1) equipped with a 20x water objective (e.g. Zeiss W "Plan-Apochromat" 20x/1,0 M27 DIC) (Figure 2). For imaging with G-CaMP (excitation/emission: 488/509 nm), use a filter block with the following properties: 450-490 nm excitation filter, dichroic mirror (T495LP) and 500-550 nm emission filter (Chroma ET). Place the mounting block containing the fly under the microscope. Position the exit of the olfactometer approximately 0.5 cm in front of the fly's antennae.</li>
 <li>Lower the objective until it touches the Ringer's solution on the fly's head. Looking through the viewer under bright-field illumination, position the preparation so that the hole in the head capsule is centered and its borders are in focus. Switch to fluorescent light and adjust the focus until you can see the labeled neurons (apparent from basal green fluorescence of G-CaMP).</li>
 <li>Fine focus on the glomeruli of interest by acquiring images with a Charge-coupled device (CCD) camera (e.g. CoolSNAP-HQ2 Digital Camera System) mounted on the microscope using appropriate acquisition software (e.g. Metafluor) (Figure 3A). Close the diaphragm in the illumination light path to limit excitation light to the imaged area.</li>
 <li>For recording odor-evoked activity, set the acquisition rate to 4 Hz and adjust the exposure time to obtain fluorescence values within the dynamic range of your CCD camera, but not lower than 1000 (arbitrary units), with at least 12-bit dynamic resolution. The exposure time should not exceed 100 ms, if possible, to reduce G-CaMP photobleaching. If the spatial resolution of your camera allows, you can increase the binning (number of wells on the chip that will be binned into one digital pixel) to reduce the exposure time. We obtain images of 350x225 pixels (with a binning of 2x2), corresponding to 210x135 &mu;m at the preparation. These settings are optimal to capture glomerular odor-induced responses of G-CaMP with good spatial and temporal resolution while limiting reporter bleaching.</li>
 <li>Set the measurement parameters. We typically record for 10 seconds (i.e. 40 frames), with odor stimulation typically starting 4 seconds after the beginning of the acquisition period (frame 15) and lasting one second (until frame 19). A fly preparation can normally be tested with up to 25-30 different odor stimuli, often limited by an irreversible decline in the fluorescent signal of the reporter due to photobleaching. The inter-stimulus interval will depend on the strength of response observed and the rate of photobleaching. The appropriate length of inter-stimulus interval to avoid sensory adaptation can be determined approximately by repeated presentation of a positive control odor (if known) in trial experiments. Inclusion of this odor every ~10 stimuli in a given series can allow verification of the continued viability and consistent responsiveness of the preparation.</li>
</ol>
5. Data Processing and Analysis
Data is processed using ImageJ (with Mac biophotonics plug-in, <a href="http://www.macbiophotonics.ca/">www.macbiophotonics.ca</a>) and custom written programs in Matlab and R as follows:
<ol>
 <li>Correct for sample movement artifacts: align all the images corresponding to one animal preparation (e.g. 30 measurements of 40 frames each) using the "rigid body" mode of StackReg/TurboReg (<a href="http://bigwww.epfl.ch/thevenaz/stackreg">bigwww.epfl.ch/thevenaz/stackreg</a>) in ImageJ. This step is required to remove shifts in the position of the labeled neurons with respect to the acquisition frame within and between measurements. However, it is only possible to remove shifts in the xy plane. Data should be discarded if strong changes in focus occur.</li>
 <li>Segment the stack into individual 10-second (40-frame) measurements.</li>
 <li>Correct for bleaching artifacts: exposure to fluorescent light will cause fluorescence to decay during a single measurement10. In some cases, it is important to correct for this decay before analyzing the data. For each measurement calculate a background frame. This should be the mean of several frames before stimulus onset (e.g. frames 6-14). Divide each frame by this background frame to obtain the relative fluorescence changes. Calculate the mean relative fluorescence value for each frame by averaging all pixels within the area labeled by G-CaMP. Fit a double exponential to the mean fluorescence curve, giving stronger weight to the frames before the stimulus and zero weight to the time of the odor response (20 frames after stimulus onset in our case). Subtract the fitted curve from the relative fluorescence curve of each pixel. Multiply the corrected frames by the background frame to re-obtain raw data. This kind of correction may lead to a modification in the response dynamics if the signal does not return to the baseline by the end of the measurement (e.g. if inhibitory or very strong excitatory responses are evoked) (Figure 4). While correction of bleaching artifacts is useful in many cases, interpretation of temporal parameters should be made with caution if a bleach correction is applied.</li>
 <li>Calculate the relative change in fluorescence (&Delta;F/F) for each frame of each measurement as (Fi-F0)/F0*100, where F0 is the background frame (see 5.3), and Fi the fluorescence value for the ith frame of the measurement.</li>
 <li>Calculate the mean &Delta;F/F within a circular region of interest in a glomerulus (Figure 3B) with a diameter of 10 pixels to obtain activity traces (Figure 5A). If desired, transform these traces into heatmaps using ImageJ (Figure 5A).</li>
 <li>Calculate the peak amplitude of the response by subtracting the mean &Delta;F/F of four frames before stimulation from the mean &Delta;F/F of four frames during the peak of the response. The same procedure is used both to illustrate spatial response patterns (see Figure 3B) and to quantify the response amplitude in specific regions of interest across flies (see Figure 5B). The peak response can also be quantified by integrating the area below the curve during the time of the stimulus, or averaging the frames around the maximum of the response.</li>
</ol>
6. Representative Results
We used the protocols above to record and analyze propionic acid-evoked responses of flies expressing G-CaMP1.628 under the control of the promoter for the olfactory co-receptor IR8a, which is active in several different populations of OSNs29,30 (Figures 3-5). Stimulation with 1% propionic acid elicits increases in G-CaMP fluorescence - indicating increases in intracellular calcium - in OSNs innervating two glomeruli, DC4 and DP1l (Figure 3B). We illustrate different ways of representing data from multiple flies in Figure 5: line activity traces, heatmaps and a bar plot of peak responses. Propionic acid-evoked responses have distinct peak amplitudes and temporal dynamics in DC4 and DP1l. Moreover, although responses are generally robust, variability between individual animals can also be observed in both glomeruli (see heatmaps in Figure 5A). Quantification of the peak responses (Figure 5B) allows a simple statistical comparison between different glomeruli and different odorants across animals. However, odor-evoked calcium responses can have non-uniform temporal dynamics, and quantifying their magnitude with a single value will disregard this complexity.
<img alt="Figure 1" src="/files/ftp_upload/2976/2976fig1.jpg" /> 
Figure 1. Schematic of the mounting block (A) before and (B) after introduction of the fly, and (C) a top view in close-up (adapted from31).
<img alt="Figure 2" src="/files/ftp_upload/2976/2976fig2.jpg" /> 
Figure 2. Schematic of the experimental set-up.
<img alt="Figure 3" src="/files/ftp_upload/2976/2976fig3.jpg" /> 
Figure 3. (A) Top view of the fly preparation after step 2.8 (left panel) and raw fluorescence image of the antennal lobes labeled with G-CaMP1.6 (Genotype: IR8a promoter:GAL4; UAS:G-CaMP1.6). DC4 and DP1l glomeruli can be distinguished by their morphology, size and position and are marked in red and blue, respectively. 
(B) Color-coded image of the response to propionic acid (1% v/v). The image corresponds to the &Delta;F/F of the peak of the response calculated as explained in step 5.6 (see also Figure 5A). The colorscale on the left indicates the %&Delta;F/F value. The black circles indicate the position and size of the region of interest used to quantify the responses (see Figure 5).
<img alt="Figure 4" src="/files/ftp_upload/2976/2976fig4.jpg" /> 
Figure 4. Correcting bleaching of the fluorescent reporter signal. Examples of the effect of bleach correction as described in step 5.3. Sample data on the left panel can be corrected without major changes in the shape of the response. Sample data on the right panel is severely affected by bleach correction, likely because the signal drops and is maintained below baseline after odor stimulus termination.
<img alt="Figure 4" src="/files/ftp_upload/2976/2976fig5.jpg" /> 
Figure 5. (A) Top: temporal dynamics of calcium responses of glomeruli DC4 (left) and DP1l (right) to propionic acid (1% v/v). The black traces show the median of the mean &Delta;F/F within the region of interest (see Figure 3B) across eight animals. The grey surface indicates the range between the first and third quartiles of the distribution. Presentation of median and quartile values provides more information about the variability and distribution of observed responses than mean and standard error. The green and magenta boxes indicate the frames averaged to calculate the peak responses shown in Figure 3B, and quantified in Figure 5B. Bottom: the heatmaps show the response data for all individual animals in separate rows, with &Delta;F/F values represented by the colorscale (far right). The horizontal black bars indicate the time and duration of the stimulus. (B) Median peak responses to propionic acid across eight animals for DC4 and DP1l glomeruli. The error bars indicate the range between first and third quartiles of the distribution.

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+ionotropic+glutamate+receptors+as+chemosensory+receptors+in+Drosophila.">Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila.</a> Cell. 136, 149-162 (2009).</li><li>Pelz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional characterization of Drosophila melanogaster Olfactory Receptor Neurons. , (2005).</li><li>Ai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+sensing+by+the+Drosophila+olfactory+system.">Acid sensing by the Drosophila olfactory system.</a> Nature. 468, 691-695 (2010).</li><li>Martin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Brain+Imaging:+Fluorescence+or+Bioluminescence,+Which+to+Choose?.">In Vivo Brain Imaging: Fluorescence or Bioluminescence, Which to Choose?.</a> Journal of Neurogenetics. , 1-23 (2008).</li><li>Jayaraman, V., 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=Evaluating+a+genetically+encoded+optical+sensor+of+neural+activity+using+electrophysiology+in+intact+adult+fruit+flies.">Evaluating a genetically encoded optical sensor of neural activity using electrophysiology in intact adult fruit flies.</a> Front. Neural Circuits. 1, 3-3 (2007).</li><li>Pfeiffer, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+neuroanatomy+and+neurogenetics+in+Drosophila.">Tools for neuroanatomy and neurogenetics in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9715-9720 (2008).</li></ol>

The authors have nothing to disclose.

The preparation and analysis methods described here can be used to analyze odor-evoked responses in any population of neurons in the antennal lobe (OSNs, LN and PNs) for which a corresponding driver transgene is available. While we describe its application using a fluorescence microscope, optical imaging of this preparation can equally be performed using confocal or two-photon microscopy22,32. Indeed, these latter instruments alleviate the light-scattering problem of wide-field microscopy, which can reduce the resolution of images, especially when large numbers of neurons express the reporter. This preparation usually permits recordings of odor-evoked responses for at least half an hour, but this time can be significantly longer or shorter depending on the number and length of measurements recorded, the exposure time used for each frame and the inter-stimulus interval. Furthermore, two-photon excitation can reduce photobleaching of the fluorescent reporter, as well as cellular phototoxicity, to permit measurement over longer time periods22,32.
Regardless of the choice of microscope, movement of the fly brain within the head capsule is the most recurrent problem when imaging in intact animals. While movement correction can correct minor issues (step 5.1), it is usually better only to record from highly stable preparations. Strategies to reduce movement artifacts include: (i) properly constraining the proboscis of the fly with the cactus spine; (ii) immobilizing the fly's legs by fixing them to the stage with eicosane (prior to step 2.6) (eicosane can be melted at ~37 &deg;C; use a silver wire to extend and refine the tip of the soldering iron for this purpose); (iii) nicking the oesophagus (visible between the antennal nerves) with fine tweezers.
In addition to G-CaMP1.6 used here, a number of optimized versions of G-CaMP and other calcium indicators are available, which differ in their signal-to-noise ratio, baseline fluorescence and temporal dynamics12,28,33. The precise choice of sensor should take into account all of these properties, the type of neurons to be analyzed and the biological question being addressed. Current sensors report calcium changes that correlate fairly well with action potentials12,34, and their further improvement, coupled with increasing numbers of specific genetic driver lines35 will continue to enhance the potential of optical imaging of neural activity in the intact fly.

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Supplier</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cactus spine</td>
			<td>Garden center</td>
			<td>&nbsp;</td>
			<td>~7mm long thin, not too flexible</td>
		</tr>
		<tr>
			<td>Rosin</td>
			<td>String instrument shop</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Two component silicon</td>
			<td>WPI</td>
			<td>KWIK-SIL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Custom made plexiglass mounting block</td>
			<td>Blueprints available from <a href="http://neuro.uni-konstanz.de/jove_mountingblock_blueprint/" target="_blank">http://neuro.uni-konstanz.de/jove_mountingblock_blueprint/</a> </td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Copper grids</td>
			<td>Athene Grids</td>
			<td>G220-5</td>
			<td>Normally used for electron microscopy samples.</td>
		</tr>
		<tr>
			<td>Screws</td>
			<td>Hardware store</td>
			<td>&nbsp;</td>
			<td>2 mm diameter</td>
		</tr>
		<tr>
			<td>Plastic coverslips</td>
			<td>Plano-em</td>
			<td>L4193</td>
			<td>22 X 22 mm</td>
		</tr>
		<tr>
			<td>Soldering iron tips</td>
			<td>Conrad Electronics</td>
			<td>830283-62</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Regulated power supply</td>
			<td>Conrad Electronics</td>
			<td>511407-62</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thin wire</td>
			<td>Isabellenh&uuml;tte</td>
			<td>Isa-ohm</td>
			<td>0.013 mm</td>
		</tr>
		<tr>
			<td>Eicosane</td>
			<td>Sigma</td>
			<td>21,927-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axioexaminer D1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Visitron</td>
			<td>CoolSnap HQ</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Metafluor</td>
			<td>Visitron</td>
			<td>&nbsp;</td>
			<td>Acquisition software</td>
		</tr>
		<tr>
			<td>Monochromator</td>
			<td>Visitron</td>
			<td>Visichrome</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Breakable blades</td>
			<td>Fine Science Tools</td>
			<td>10050-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Holder for blade</td>
			<td>WPI</td>
			<td>14134</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sapphire blade</td>
			<td>WPI</td>
			<td>500314</td>
			<td>Double edged blade 1mm</td>
		</tr>
		<tr>
			<td>Membrane gas pumps</td>
			<td>KNF Neuberger AG</td>
			<td>NMP 830 KVE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sapphire blade holder</td>
			<td>WPI</td>
			<td>500317</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Three way valve</td>
			<td>Lee Company</td>
			<td>LFAA1200118H</td>
			<td>Configuration E</td>
		</tr>
		<tr>
			<td>Rotameter 500 ml/min</td>
			<td>Analyt-MTC</td>
			<td>112-08SA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rotameter 5 L/min</td>
			<td>Analyt-MTC</td>
			<td>102-08SA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beeswax</td>
			<td>Siladent Technik</td>
			<td>209212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cellulose pad</td>
			<td>Kettenbach Medical</td>
			<td>31003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Plano-em</td>
			<td>T5130</td>
			<td>Dumont biology #5</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>BD</td>
			<td>300928</td>
			<td>2ml, Discardit II, 2pieces</td>
		</tr>
		<tr>
			<td>Needles</td>
			<td>Braun Sterican</td>
			<td>4665120</td>
			<td>1.2mm OD</td>
		</tr>
	</tbody>
</table>

calcium imaging of odor-evoked responses in the drosophila antennal lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Watch this Scientific Journal Video about Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe at JoVE.com

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

calcium-imaging-odor-evoked-responses-drosophila-antennal

Research

JoVE Journal

Neuroscience

25.8K Views.  University of Lausanne. We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Video: Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

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 Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

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 ...

Evaluating Calcium Activity in Retinal Ganglion Cells Layer

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: An Innovative Approach to Neural Electrical Stimulation Using Calcium Imaging 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...