detecting physiological responses from sensory neurons in drosophila

Detecting Physiological Responses from Sensory Neurons in Drosophila

After anesthetizing the fly, attach it to a 0.17-millimeter coverslip, 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 coverslip.
If neurons in 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 1-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 fluorescence signal with rapid time resolution, such as a spinning disk confocal microscope with an sCMOS camera. Collect three pre-stimulation Z-stacks of the neurons of interest in the tarsal segment.
In this example, images are captured with 200-millisecond exposures and a 2-by-2 binning over a 6-micron-by-1/2-micron section every two seconds. Be sure to optimize the laser&#39;s power to minimize photobleaching, 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&#39;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.

detecting-physiological-responses-from-sensory-neurons-in-drosophila

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1. Sample Preparation
<ol>
	<li>Cross the Gal4 driver line to UAS-GCaMP5G Drosophila melanogaster flies (w;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 control of the Gr68a-Gal4 driver.
	<ol>
		<li>Collect and separate by sex newly eclosed adult flies and raise at 25 &#176;C. 
		&#8203;NOTE: The baseline level of GCaMP5G fluorescence is optimal in flies aged between 14 to 28 days. Isolating flies by sex precludes the possibility of social interactions that may influence the neural responses.</li>
	</ol>
	</li>
	<li>Anesthetize a fly under a mild stream of CO2.</li>
	<li>Attach the fly to a glass coverslip by first placing a small drop of nail polish in the center of a 0.17 mm coverslip. Gently place a fly on its side on the drop of clear nail polish and ensure that the fly covers the entire drop.</li>
	<li>Perform steps 1.5 and 1.6 under a dissecting stereomicroscope, used at 8X magnification.</li>
	<li>Use a wet paintbrush to extend the fly&#39;s foreleg. Secure the foreleg in position by placing two thin strips of tape over the first and fifth tarsal segments (Figure 1A). This will expose tarsal segments T2-T4 for imaging (Figure 1B).</li>
	<li>To image neurons on the proboscis, place a thin strip of tape over the rostrum of the proboscis to expose the labellum (Figure 1C, D).</li>
	<li>Draw a hydrophobic barrier around the fly with a PAP (Peroxidase-antiperoxidase) pen to prevent the solution from flowing over the coverslip surface. Measure within 30 minutes of mounting.</li>
</ol>
2. Preparation of Stimulus Solution
<ol>
	<li>Dilute lipophilic ligands (such as CH503, used in this study) in a PBST (Phosphate-Buffered Saline with Triton X-100) solution: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.1% Triton X-100 (v/v); pH 7.4.
	<ol>
		<li>Make a 1 mg/ml stock solution of the ligand using PBST. Prepare all subsequent dilutions of the stock solution using PBST. 
		NOTE: Depending on the solubility of the ligand of interest, different solvents may be needed to dissolve it. Table 1 describes the solubility of CH503 in various solvents. Unlike other solvents, PBST did not increase fluorescence.</li>
	</ol>
	</li>
</ol>
3. Stimulation of Gustatory Neurons and Image Acquisition
<ol>
	<li>Using a 10 &#181;l pipettor, place 10 &#181;l of PBST onto the tarsal segments. 
	NOTE: This step moistens the leg and prevents the tarsal segments from drifting.</li>
	<li>Use an optical system that has a camera of sufficient speed and sensitivity to achieve a good fluorescence signal at high time resolution. 
	NOTE: For this study, we used a spinning disk confocal microscope and a sCMOS camera (see Materials List).</li>
	<li>Choose the specific tarsal segment and neurons to be imaged. Acquire three pre-stimulation confocal Z-stacks.
	<ol>
		<li>Ideally, use acquisition settings that are fast enough to allow maximal time resolution while still capturing the entire cell volume. Example acquisition settings are: 200 msec exposure, binning 2 x 2 and 6 x 0.5 &#181;m2 optical sections, captured every 2 sec. 
		&#8203;NOTE: Immobilized legs have been imaged successfully for up to 10 min, at 30 sec intervals, with no discernible bleaching of the GCaMP signal. For longer recording times, ensure that the legs are held very firmly in place on the coverslip.</li>
		<li>Optimize laser power for the highest signal-to-noise achievable with minimal photobleaching during the entire imaging regime. For the experiments described here, use a 491 nm diode laser (100 mW) at 30% transmission to image both Gr68a and ppk23 gustatory neurons on the legs and proboscis. Use 2 x 2 binning to allow for shorter exposure times while still achieving sufficient spatial resolution. 
		NOTE: The laser power settings were optimized to detect fluorescent changes ranging from a 1.2- to 4.5-fold increase.</li>
	</ol>
	</li>
	<li>Pipette 10 &#181;l of the stimulus solution onto the tarsal segments. Add another 10 &#181;l of PBST for control experiments. Immediately acquire 117 post-stimulation images. 
	NOTE: The number of post-stimulation images acquired is based on the time it took to reach the maximal change in fluorescence (approximately 2 min).
	<ol>
		<li>CRITICAL STEP: When pipetting solution on the preparation, do not touch the fly with the pipette tip, as this will cause the foreleg to move out of position. 
		NOTE: Alternatively, use a micromanipulator to accurately position a glass capillary containing stimulus close to the tarsal segment to be imaged. Deliver controlled volumes of stimulus using an intracellular microinjection dispenser according to the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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</td>
			<td></td>
			<td>42037</td>
			<td>Bloomington Drosophila 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;</td>
			<td>Sally Hansen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich</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</td>
			<td>Scotch brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</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</td>
			<td>H303SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</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</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</td>
			<td>Yokogawa</td>
			<td>CSU-X1-A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Shankar, S., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53392">Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging.</a> J. Vis. Exp. (2016).
This video demonstrates a method for detecting sensory neuron responses in Drosophila using ligand binding and fluorescence imaging. Ligands bind to neuron receptors, causing calcium influx. The calcium binds to indicator complexes, leading to fluorescence emission, which confirms the neuronal response to the ligand.

<img alt="Figure 1" src="/files/ftp_upload/22872/22872_Figure1.jpg" /> 
Figure 1: Mounting a live fly for imaging of the foreleg or proboscis neurons. (A) Mounting the foreleg: a 3.2X image of a live male fly, showing the forelegs fastened to the coverslip with tape. (B) A 40X image of the fly shows each of the tarsal segments (T1-5) and the placement of tape above the first tarsal segment and on the fifth tarsal segment. (C) Mounting the proboscis: a piece of tape i...

1. Sample Preparation

	Cross the Gal4 driver line to UAS-GCaMP5G Drosophila melanogaster flies (w;P{20XUAS-IVS-GCaMP5G}attP40). Allow the cross to grow ...

Attach an anesthetized genetically modified Drosophila melanogaster on its side to a coverslip. 
Extend its forelegs and secure them to expose the tarsal segments.
These segments have gustatory neurons expressing calcium indicator complexes that fluoresce in calcium-bound form.
Draw a hydrophobic barrier around the fly to retain the solution.
Allow the fly to recover and overlay a buffered saline solution onto the exposed tarsal segments to moisten them.
Under a confocal microscope, observe tarsal segments and image the gustatory neurons at various depths to obtain a pre-stimulation fluorescence.
Add a stimulation solution containing lipophilic ligands onto the segment.
These ligands bind to the gustatory neuron receptors, initiating signaling pathways and triggering the opening of calcium channels.
This allows calcium ions from the extracellular fluid to enter the cytoplasm and bind to the calcium indicator complexes, resulting in fluorescence emission.
When observed again, an increase in neuronal fluorescence confirms the neuronal physiological response to the ligand.

12 vistas. Detección de respuestas fisiológicas de neuronas sensoriales en Drosophila

Video: Detección de respuestas fisiológicas de neuronas sensoriales en Drosophila - Experimento

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Electrofisiológico Grabación desde<em&gt; Drosophila</em&gt; Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

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Neurociencias

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.

Imágenes de calcio in vivo de las respuestas de las neuronas ganglionares geniculadas de ratón a los estímulos del gusto

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

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Fisiológicos Grabaciones y RNA Secuenciación de los apéndices gustativas del mosquito de la fiebre amarilla<em&gt; Aedes aegypti</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Detecting Physiological Responses from Sensory Neurons in Drosophila

Transcripción

Detecting Physiological Responses from Sensory Neurons in Drosophila