Name: measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging
Uploaded: 2016-04-29T13:00:00
Description: Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

This work was supported by the Singapore National Research Foundation (grant NRF-RF2010-06 to J.Y.Y.).

1. Sample Preparation
<ol>
	<li>Cross the Gal43,22 driver line to UAS-GCaMP5G19 flies (w1118;P{20XUAS-IVS-GCaMP5G}attP40). Allow the cross to grow at 25 &#176;C. 
	Note: Generating flies with multiple copies of the Gal4 or UAS-GCaMP transgenes can help to enhance the fluorescent signal intensity. An earlier version of the UAS-GCaMP transgene (UAS-GCaMP3) showed very weak fluorescence intensity when expressed under the c...

<ol>
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Biol. 11, 822-835 (2001).</li><li>Thistle, R., Cameron, P., Ghorayshi, A., Dennison, L., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+chemoreceptors+mediate+male-male+repulsion+and+male-female+attraction+during+Drosophila+courtship.">Contact chemoreceptors mediate male-male repulsion and male-female attraction during Drosophila courtship.</a> Cell. 149, 1140-1151 (2012).</li><li>Toda, H., Zhao, X., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+female+aphrodisiac+pheromone+activates+ppk23(+)+sensory+neurons+to+elicit+male+courtship+behavior.">The Drosophila female aphrodisiac pheromone activates ppk23(+) sensory neurons to elicit male courtship behavior.</a> Cell Rep. 1, 599-607 (2012).</li><li>Vijayan, V., Thistle, R., Liu, T., Starostina, E., Pikielny, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+pheromone-sensing+neurons+expressing+the+ppk25+ion+channel+subunit+stimulate+male+courtship+and+female+receptivity.">Drosophila pheromone-sensing neurons expressing the ppk25 ion channel subunit stimulate male courtship and female receptivity.</a> PLoS Genet. 10, 1004238 (2014).</li><li>Lu, B., LaMora, A., Sun, Y., Welsh, M. J., Ben-Shahar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppk23-Dependent+chemosensory+functions+contribute+to+courtship+behavior+in+Drosophila+melanogaster.">ppk23-Dependent chemosensory functions contribute to courtship behavior in Drosophila melanogaster.</a> PLoS Genet. 8, e1002587 (2012).</li><li>Koh, T. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+individual+firing+frequencies+of+two+neurons+recorded+with+a+single+electrode.">Estimation of the individual firing frequencies of two neurons recorded with a single electrode.</a> Chem. Senses. 28, 671-679 (2003).</li><li>Masuyama, K., Zhang, Y., Rao, Y., Wang, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+neural+circuits+with+activity-dependent+nuclear+import+of+a+transcription+factor.">Mapping neural circuits with activity-dependent nuclear import of a transcription factor.</a> J. Neurogenet. 26, 89-102 (2012).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+the+GCaMP+calcium+sensor+reveal+the+mechanism+of+fluorescence+signal+change+and+aid+rational+design.">Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design.</a> J. Biol. Chem. 284, 6455-6464 (2009).</li><li>Miyamoto, T., Chen, Y., Slone, J., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+Drosophila+glucose+receptor+using+Ca2++imaging+of+single+chemosensory+neurons.">Identification of a Drosophila glucose receptor using Ca2+ imaging of single chemosensory neurons.</a> PloS One. 8, e56304 (2013).</li><li>Akerboom, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+GCaMP+calcium+indicator+for+neural+activity+imaging.">Optimization of a GCaMP calcium indicator for neural activity imaging.</a> J. Neurosci. 32, 13819-13840 (2012).</li><li>Shikichi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+synthesis.+Part+250:+Determination+of+the+stereostructure+of+CH503+a+sex+pheromone+of+male+Drosophila+melanogaster,+as+(3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol+by+synthesis+and+chromatographic+analysis+of+its+eight+isomers.">Pheromone synthesis. Part 250: Determination of the stereostructure of CH503 a sex pheromone of male Drosophila melanogaster, as (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol by synthesis and chromatographic analysis of its eight isomers.</a> Tetrahedron. 68, 3750-3760 (2012).</li><li>Yew, J. 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Sci. 510, 104-112 (1987).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+pattern+generators+and+the+control+of+rhythmic+movements.">Central pattern generators and the control of rhythmic movements.</a> Curr. Biol. 11, 986-996 (2001).</li><li>Koepsell, K., Wang, X., Hirsch, J. A., Sommer, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+function+of+neural+oscillations+in+early+sensory+systems.">Exploring the function of neural oscillations in early sensory systems.</a> Front Neurosci. 4, 53 (2010).</li><li>Berridge, M. 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We describe here a method to perform live calcium imaging of Drosophila peripheral neurons in 2 different sensory organs. The Ca2+-evoked GCaMP fluorescent responses in Gr68a-neurons induced by the pheromone ligand CH503 were dose-dependent and quantitative. It was also possible to discern different neural response patterns such as phasic and tonic responses.
Neurons showing phasic responses are believed to allow rapid adaptation to continuous stimuli. This type of response...

Animals rely on olfactory and gustatory information to mediate decisions essential for survival and reproduction. Understanding how chemosensory cues are detected and processed by the nervous system requires identification of the sensory receptor(s) and the corresponding chemical ligands. Drosophila detect a staggering variety of volatile and non-volatile compounds and are an excellent model in which to study the physiological mechanisms underlying chemosensation. While the olfactory organs perceive volatile molecules, the gustatory organs are specialized to detect low volatility compounds. Here, we present a method to directly measure neuronal responses from the taste organs of Drosophila melanogaster to low-volatility, lipophilic ligands.
Gustatory organs of the fly include the forelegs, proboscis, and wings. Distributed on the surface of the taste organs are hair-like structures known as sensilla that respond to sugars, bitters, salts, water and pheromones8. The sensilla have been classified morphologically into taste bristles and taste pegs9. There are about 31 taste bristles on the labellum that are classified into the long (l-type), short (s-type) and intermediate (i-type) morphologies. The &#8216;l&#8217; and &#8216;s&#8217; sensilla house 4 sensory neurons that respond physiologically to sugar (the S cell), low salt (the L1 cell), high salt and bitter compounds (the L2 cell) and water (the W cell)10,11. The &#8216;i&#8217; sensilla house 2 sensory neurons, one of which responds both to low salt and sugar, while the other responds to high salt12. There are approximately 41 taste sensilla in males and 26 sensilla in females distributed on each of the forelegs. For both males and females, there are 21 sensilla on the midleg, and about 22 sensilla on the hindleg13. The gustatory neurons enclosed by the taste sensilla on the legs are also classified into L1, L2, W and S types.
One standard method for measuring electrical activity from single neurons uses extracellular or intracellular electrodes to record ion flow. Electrode measurements allow neuronal function to be studied in non-model organisms such as Drosophila species, moths, and bees which lack extensive genetic tools for neural labeling. However, while electrophysiological methods have been routinely used to measure activity from Drosophila taste sensilla4,13,14, applying this approach presents several technical challenges. First, taste bristles need to be identified based on morphology and spatial location. Upon identification, electrophysiological measurements can be hindered by the small size of the sensilla, limited accessibility due to position, and difficulty in applying controlled volumes of a chemical stimulus to a taste bristle. Also, stimulation of sensilla may generate signals from more than one neuronal type15. Second, detection of electrical signals can be confounded by background noise resulting from mechanical vibrations and noise from electronic equipment. Third, the use of a sharpened electrode can damage the fly preparation, if used incorrectly14. Finally, assembling an electrophysiology rig requires specialized electronic components for stimulus delivery, signal recording, and data analysis and can be costly.
In D. melanogaster, the availability of genetically-encoded tools has facilitated the development of imaging techniques that allow the responses of small populations of neurons to be studied. One such approach is the use of the CaLexA reporter16. In this method, gene sequences encoding the LexA-VP16 transcription factor and a calcium sensitive protein NFAT are fused together. Calcium activation of the protein phosphatase calcineurin catalyzes the de-phosphorylation of NFAT and, in turn, facilitates its import into the nucleus. Inside the nucleus, the LexA domain binds to a LexAop-DNA binding motif which directs the expression of a green fluorescent protein (GFP) reporter, thus allowing sustained identification of functionally activated neurons. The approach has been used successfully to measure the response of specific olfactory glomeruli in the antennal lobe following exposure of live flies to an odorant16. Recently, physiological responses of IR52c gustatory receptor neurons were measured from live flies using CaLexA7. However, for this study, it was necessary to age flies for up to 6 weeks to enhance GFP transcription. While immunostaining with an anti-GFP antibody can be used to boost the CaLexA signal, this method would require tissue fixation, thus precluding possibility for live-cell imaging.
The genetically encoded calcium indicator protein GCaMP has also been extensively used to study neuronal responses in a number of species17. The protein GCaMP fluoresces with low intensity prior to neuronal stimulation. Application of a stimulus triggers an action potential in the neuron, resulting in the influx of Ca2+. GCaMP, bound to Ca2+, undergoes a conformational change, causing it to fluoresce with brighter intensity (Figure 1). A recently developed single&#8211;neuron calcium imaging approach was used to identify glucose as the ligand for the foreleg specific Gr61a gustatory neurons18. In this study, dissected forelegs from transgenic Drosophila expressing GCaMP in Gr61a gustatory neurons were covered with a layer of agarose prior to imaging. However, the use of dissected tissue can cause eventual rundown of GCaMP expression, thus limiting measurement time and detection sensitivity. In addition, agarose can limit sensitivity due to high background levels of fluorescence and its light scattering properties.
To address some of these drawbacks, we describe the use of GCaMP-mediated calcium imaging to record physiological responses from foreleg and proboscis gustatory neurons of intact animals. We show the physiological responses of Gr68a and ppk23 neurons expressing genetically encoded GCaMP5G19 to a Drosophila lipid pheromone, (3R, 11Z, 19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503)20,21. Neuronal responses are measured by quantifying the increase in fluorescence of the GCaMP5G signal during pheromone stimulation. In this protocol, neurons are imaged for a total duration of 120 sec, which is sufficient to differentiate patterns of neuronal activation in individual cells.

<table>
	<tbody>
		<tr>
			<td>Gr68a-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from H. Amrein (Texas A&#38;M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA)</td>
		</tr>
		<tr>
			<td>ppk23-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from K. Scott (Univ. of California, Berkeley, CA, USA)</td>
		</tr>
		<tr>
			<td>UAS-GCaMP5&#160;</td>
			<td></td>
			<td>42037</td>
			<td>Bloomington Drosophila&#160; Stock Center</td>
		</tr>
		<tr>
			<td>0.17 mm coverslip (Gold-Seal coverslip)</td>
			<td>Electron Microscopy Services</td>
			<td>63790-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish, &#34;Hard as Nails Clear&#34;&#160;</td>
			<td>Sally Hansen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td></td>
			<td></td>
			<td>fine-tipped brush</td>
		</tr>
		<tr>
			<td>Tape&#160;</td>
			<td>Scotch brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>13021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, lab grade</td>
			<td>Merck</td>
			<td>10094</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, HPLC grade</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H303SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>Recipe described in the protocol section</td>
		</tr>
		<tr>
			<td>CH503</td>
			<td></td>
			<td></td>
			<td>Synthesis described in Mori et al., 2010</td>
		</tr>
		<tr>
			<td>sCMOS Camera (ORCA Flash4.0)</td>
			<td>Hamamatsu&#160;</td>
			<td>C11578-22U</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Ti-Eclipse)</td>
			<td>Nikon</td>
			<td>Ni-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk Scan head&#160;</td>
			<td>Yokogawa</td>
			<td>CSU-X1-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquistion Software (MetaMorph Premier)</td>
			<td>Molecular Devices</td>
			<td>40002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>open source</td>
			<td></td>
			<td><a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji</a></td>
		</tr>
	</tbody>
</table>

measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

The GCaMP calcium indicator was genetically expressed using Gr68a-Gal4 or ppk23-Gal4 drivers. Distinct populations of foreleg neurons and non-neural support cells are labeled by each driver (Figure 3A-C). Ligand-specific responses to the lipophilic pheromone CH503 were observed in Gr68a-Gal4 and ppk23-Gal4 cells expressing GCaMP (Figure 3D). The relative change in fluorescence intensity of the GCaMP5G signal (&#916;F/F) and increased with high...

Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

The overall goal of this procedure is to measure the physiological responses of Drosophila gustatory neurons to lipid pheromones. This technique can be used to answer key questions in sensory neurobiology such as identifying the ligands for orphan gustatory receptors. The main advantage of this technique is that the responses of individual gustatory neurons can be studied in an intact, live animal.After anesthetizing the fly, attach it to a 0.17 millimeter cover slip using a very small drop of clear nail polish. Attach the side of the fly to the slide using the entire drop. Next, under a dissecting microscope, use a wet paintbrush to extend a foreleg of the fly.Then using two very thin strips of tape, secure the first and fifth tarsal segments to the cover slip. If neurons on the proboscis are to be imaged, then place another thin strip of tape over the rostrum of the proboscis to expose the labellum. Now, make a short wall around the fly using a PAP pen.Let the anesthesia wear off for half an hour before taking measurements. For this protocol, prepare the necessary dilutions of the one milligram per milliliter stock ligand solution, using PBST. To begin the procedure, overlay 10 microliters of PBST onto the secured and exposed tarsal segments.This moistens the leg and prevents it from drifting out of focus. Next, focus on the segments using an optical system which can achieve a good fluorescent signal with rapid time resolution such as a spinning disk confocal microscope with an sCMOS camera. Collect three prestimulation Z-stacks of the neurons of interest in the tarsal segment.In this example, images are captured with 200 millisecond exposures and a two-by-two binning over a six micron by half-micron section every two seconds. Be sure to optimize the laser's power to minimize photo bleaching while still allowing a high signal-to-noise ratio. Here, a 491 nanometer 100-milliwatt laser is operated at 30%transmission.The signal must be detectable prior to stimulation but not approach saturation. Now, pipette 10 microliters of stimulation solution onto the tarsal segment of interest. While pipetting, be very careful to avoid touching the leg or any of the fly's body or the tarsi will move out of position.Then, immediately acquire the first post-stimulation images and continue to collect enough images to document the maximum change in fluorescence. For analysis, open a series of confocal Z-stacks using Fiji or ImageJ image analysis and processing software. Make a sum intensity projection of all the Z-slices for each of the time points.Here, there are six slices and 120 time points. First, select the stack option under image and then select the Z-projection option. Enter one for the start slice and four for the stop slice.For the projection type, enter sum slices and select all time frames. Now, define a region of interest, or ROI such as a cell body or neuronal projection. Use the freehand selection tool to draw around the structure's boundaries.Then, click on the analyze label and select the ROI manager option under tools. Quantify the fluorescence of the ROI by first selecting the set measurements option under the analyze menu. Check the boxes for integrated density, area, mean and maximum grey value, and label.Next, select the more option under the ROI manager menu and choose the multi-measure option. Now, calculate the maximum change in fluorescence for time point t. To quantify the post-stimulation fluorescence, measure the integrated density of the ROI.For t equals zero, measure the prestimulation fluorescence from the prestimulus images of the ROI in the same manner. Average the values from three images for this t equals zero value. To quantify background fluorescence, measure the integrated density from a tissue area devoid of detectable neurons that has the same size and shape as the ROI.Collect a unique background value for each time point. The GCamP calcium indicator was expressed using two different Gal4 expression patterns. Each pattern labels a distinct population of foreleg neurons and non-neuronal support cells.For both expression patterns, ligand-specific responses to the lipophilic pheromone CH503 were observed. The relative change in fluorescence intensity of the GCamP signal increased with higher concentrations of CH503. Curiously, a five nanogram dose of CH503 may suppress the neuronal response.The measured responses of the Gr68a and ppk23 neurons differed. The neuronal response of neurons labeled with the Gr68a Gal4 driver exhibited a tonic pattern whereas ppk23 neurons showed a phasic, oscillatory response in the leg. In the proboscis, ppk23 neurons showed a late-onset tonic response from the cell body and an oscillatory response from an axonal projection.After watching this video, you should have a good understanding of how to measure the physiological responses of individual gustatory neurons to lipid pheromones and other hydrophobic ligands. This technique paves the way for researchers in the field of chemosensory biology to measure the responses of individual gustatory neurons in Drosophila melanogaster. Using this technique, neuronal activity can be silenced or activated using a temperature-controlled shibire or a TRiP a-one transgene by coupling the spinning disk microscope to a temperature-controlled onstage incubator.Once mastered, this technique can be completed in five minutes, excluding the time taken by the fly to recover from anesthesia. While performing this method, it is important to fix the fly firmly on the cover slip without damaging its foreleg. While imaging, it is important to acquire images as soon as the foreleg is stimulated with pheromone solution.

Research

JoVE Journal

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