investigating prolonged depolarizing afterpotential (pda) in drosophila photoreceptors

Investigating Prolonged Depolarizing Afterpotential (PDA) in Drosophila Photoreceptors

Place the fly holder in a dark Faraday cage on a magnet block, and ensure that the fly is approximately five millimeters from the end of the light guide. Place the recording electrode above the fly&#39;s eye, and the ground electrode over the fly&#39;s upper back using the micromanipulators. Then, insert the ground electrode into the back of the fly using the micromanipulators.
Insert the recording electrode into the outer periphery of the fly&#39;s eye, preferably using the micromanipulators. Close the Faraday cage, and turn off the lights in the room to allow adaptation to dark for five minutes. Input the parameters, starting from a pulse duration of 500 meters per second, a pulse interval of 60 seconds, and the number of pulses set to six.
For measuring the PDA, set the master rate pulse generator in Train mode. Then, give a 5-second light pulse of maximal intensity using an orange filter. Check the voltage response. Replace the orange filter with a broadband blue filter, and give three 5-second light pulses at maximum intensity. Check the voltage response.
Wait at least 60 seconds in the dark. Replace the blue filter with the previous orange filter. Then, give two 5-second light pulses with 60-second intervals. Check the voltage response.

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1. Measuring the prolonged depolarizing afterpotential (PDA) using the electroretinogram
<ol>
	<li>Suitable rearing conditions for Drosophila melanogaster preparation
	<ol>
		<li>Raise D. melanogaster flies in bottles containing standard yellow corn containing food in an incubator maintained at a temperature of 24 &#176;C and in a 12 h dark/light cycle.</li>
		<li>Keep the fly bottles in the dark at least 24 h prior to the experiment.</li>
	</ol>
	</li>
	<li>General setup
	<ol>
		<li>Prepare recording pipettes by pulling 1 mm x 0.58 mm (O.D x I.D) fiber-filled borosilicate glass capillaries (Figure 1L, O). The resistance of the pipettes should be 5-10 M&#937;; any suitable puller can be used.</li>
		<li>Coat two silver wires with AgCl2, inserting 0.25 mm silver wire into 3 M KCl solution connected to a custom-made 5 V power supply.</li>
		<li>Insert each coated silver wire into the electrode holders (Figure 1N).</li>
		<li>Fill the glass capillary with filtered Ringer&#39;s solution (see Table 1) using an elongated tip syringe (Figure 1M).</li>
		<li>Insert the wire electrode into the glass capillary. Ensure that the solution within the capillary is in contact with the silver wire.</li>
		<li>Insert the electrode holders (Figure 2P, N) into the two electrode micromanipulators (Figure 2G).</li>
	</ol>
	</li>
	<li>Procedure of preparing the fly for electrical recordings 
	&#8203;NOTE: To keep the fly under dark-adapted conditions, use only dim red light illumination during the following steps.
	<ol>
		<li>Anesthetize the flies in the bottle with CO2 gas using the fly sleeper system (Figure 1A, B) and pour them into the sleeper container.</li>
		<li>Choose one fly and carefully hold it by its wing using a sharp tweezer. Cover the rest of the flies with a Petri dish.</li>
		<li>Place the fly on the fly holder in the proper orientation-lying on its side, with its back toward the hand (Figure 1P).</li>
		<li>Turn ON the power supply of the soldering iron. Set the current to ~2.25 A. This current should heat the 0.25 mm platinum-iridium filament to ~55-56 &#176;C.</li>
		<li>Place a drop of wax with a low melting temperature (~55-56 &#176;C) on the soldering iron (Figure 1F).</li>
		<li>Using tweezers, lift the fly from its wings and fix its wings to the fly holder (Figure 1I) using the soldering iron.</li>
		<li>Using the soldering iron, connect the fly&#39;s back to the stand surface with wax (Figure 1P).</li>
		<li>Lower the tip of the soldering iron onto the joining point of the legs and melt the wax to cover all the legs together (Figure 1P).</li>
		<li>Place a small drop of wax between the head and the back in the neck area (Figure 1P). 
		&#8203;NOTE: Take special care to avoid overheating the fly head. Ensure that the fly is properly fixed and is unable to move during the experiment; minor movements may create artifacts in the recordings. Ensure that the trachea openings (breathing inlets) in the thorax and abdomen are not covered with wax.</li>
		<li>Place the fly holder (Figure 2Q) in a dark Faraday cage on a magnet block (Figure 2I) and ensure that the fly is ~5 mm from the end of the light guide (Figure 2L).</li>
		<li>Place the recording electrode (Figure 2P) above the fly&#39;s eye and the ground electrode (Figure 2N) over the fly&#39;s upper back using the micromanipulators.</li>
		<li>Insert the ground electrode into the back of the fly using the micromanipulators.</li>
		<li>Insert the recording electrode into the outer periphery of the fly&#39;s eye, preferably, using micromanipulators. 
		NOTE: After inserting the electrode into the eye, a small dimple will be observed; pull the electrode upwards without removing it from the eye until the dimple disappears. The electrodes can also be immersed in small droplets of electrode jelly applied at the torso and eye.</li>
	</ol>
	</li>
	<li>PDA protocol (this protocol can only be performed on white-eyed flies)
	<ol>
		<li>Give a 5 s light pulse of maximal intensity using an orange filter (590 edge filter, to convert maximal photopigment to the R state).</li>
		<li>Replace the orange filter with a broad-band blue (BP450/40 nm) filter and give three 5 s light pulses at maximum intensity. 
		&#8203;NOTE: It is also possible to give a long continuous maximum intensity blue light pulse until a steady state voltage response is reached.</li>
		<li>Wait 60 s in the dark, replace the blue filter with the previous orange filter, and give two 5 s light pulses with 60 s intervals.</li>
	</ol>
	</li>
</ol>
Table 1: Composition of Ringer&#39;s solution
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Concentration (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>130</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>5</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>10</td>
		</tr>
		<tr>
			<td>pH titration to 7.15 using NaOH and HCl</td>
			<td></td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe with elongated tip</td>
			<td></td>
			<td></td>
			<td>Figure 1M</td>
		</tr>
		<tr>
			<td>1 rough tweezers</td>
			<td></td>
			<td>Dumont #5, Standard</td>
			<td>0.1 mm x 0.06 mm, length 110 mm, Inox (Figure 1H)</td>
		</tr>
		<tr>
			<td>2 condenser lenses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>Molecular Device</td>
			<td>Digidata 1200</td>
			<td>Possible replacement: any digidata from molecular devices (e.g 1440A) - Figure 2C</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Almost perfect electronics</td>
			<td></td>
			<td>Possible replacement: Warner instruments- IE251A or IE-210 (comes with headstage)- Figure 2D</td>
		</tr>
		<tr>
			<td>Anti-vibration Table</td>
			<td>Newport</td>
			<td>VW-3036-OPT-01</td>
			<td>Figure 7H</td>
		</tr>
		<tr>
			<td>Capillaries</td>
			<td>Harvard Apparatus</td>
			<td>Borosilicate glass capillaries</td>
			<td>1 mm x 0.58 mm (Figure 1O)</td>
		</tr>
		<tr>
			<td>Clampex</td>
			<td>Molecular Device</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cold light source</td>
			<td>Schott</td>
			<td>KL1500 LCD</td>
			<td>Figure 1C</td>
		</tr>
		<tr>
			<td>Delicate wipers</td>
			<td>Kimtech</td>
			<td></td>
			<td>Kimwipes (Figure 1K)</td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td></td>
			<td></td>
			<td>Suitable for capillary O.D. 1 mm (Figure 1N, Figure 2N, and Figure 2P)</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Home made</td>
			<td></td>
			<td>Electromagnetic noise shielding and black front curtain (Figure 2K)</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Schott</td>
			<td>OG590, Edge filter</td>
			<td>Figure 2S</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Schott</td>
			<td>BP450/40 nm</td>
			<td>Figure 2S</td>
		</tr>
		<tr>
			<td>Filter (Color)</td>
			<td>Blazers</td>
			<td>550 nm</td>
			<td>Figure 2S</td>
		</tr>
		<tr>
			<td>Filter (Color) for cold light source</td>
			<td>Schott</td>
			<td>RG630</td>
			<td>Figure 1C</td>
		</tr>
		<tr>
			<td>Filter (Heat)</td>
			<td>Schott</td>
			<td>KG3</td>
			<td>Figure 1S</td>
		</tr>
		<tr>
			<td>Filters (Neutral density filter)</td>
			<td>Chroma</td>
			<td>6,5,4,3,2,1,0.5,0.3</td>
			<td>Figure 2S</td>
		</tr>
		<tr>
			<td>Flash Lamp system</td>
			<td>Honeywell</td>
			<td></td>
			<td>Figure 2U</td>
		</tr>
		<tr>
			<td>Fly sleeper system with injector</td>
			<td>Inject + matic</td>
			<td></td>
			<td>Figure 1A-B</td>
		</tr>
		<tr>
			<td>Lamp power supply</td>
			<td>PTI</td>
			<td>LPS-220</td>
			<td>Figure 2W</td>
		</tr>
		<tr>
			<td>Light detector</td>
			<td>Home made</td>
			<td></td>
			<td>Phototransistor (Figure 2O)</td>
		</tr>
		<tr>
			<td>Light guide</td>
			<td></td>
			<td></td>
			<td>3 mm diameter, 1.3 m long (Figure 2L,M)</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td></td>
			<td></td>
			<td>High-pressure ozone-free 75 W Xenon lamp (operating on 50 W), possible replacement: Cairn research- OptoLED (Figure 7R)</td>
		</tr>
		<tr>
			<td>Low temperature melting wax</td>
			<td>Home made</td>
			<td></td>
			<td>Composed of mixture of beeswax (Tm&#8776;62 &#176;C) and paraffin at ~3:1 to reach a melting temperature of ~55&#8211; 56 &#176;C (Figure 6J)</td>
		</tr>
		<tr>
			<td>Magnetic stand for flies</td>
			<td>Home made</td>
			<td></td>
			<td>Figure 1I and Figure 2Q</td>
		</tr>
		<tr>
			<td>Microelectrode preamplifier system with head-stage</td>
			<td>Almost perfect electronics</td>
			<td></td>
			<td>Impedance tester (Figure 2G)</td>
		</tr>
		<tr>
			<td>Micromanipulator (mechanical coarse)</td>
			<td>Tritech Research, Narishige</td>
			<td>M-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator (mechanical fine)</td>
			<td>Leitz Microsystems</td>
			<td>Leitz Mechanical Micromanipulator</td>
			<td>Figure 2F</td>
		</tr>
		<tr>
			<td>pCLAMP</td>
			<td>Molecular Device</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td></td>
			<td></td>
			<td>60 mm</td>
		</tr>
		<tr>
			<td>Pulse generator</td>
			<td>AMPI</td>
			<td>Master 8</td>
			<td>Figure 2A</td>
		</tr>
		<tr>
			<td>Redux cream for electrocardiography</td>
			<td>Parker Laboratories</td>
			<td>Redux Electrolyte Cr&#232;me</td>
			<td></td>
		</tr>
		<tr>
			<td>Shutter driver</td>
			<td>Uniblitz, Vincent Associates</td>
			<td>VCM-D1 Single Channel Uni-stable</td>
			<td>Figure 2V</td>
		</tr>
		<tr>
			<td>Shutter system</td>
			<td>Uniblitz, Vincent Associates</td>
			<td>LS2 2 mm Uni-stable Shutters</td>
			<td>Figure 2V</td>
		</tr>
		<tr>
			<td>Silver Wire</td>
			<td>Warner Instruments</td>
			<td></td>
			<td>0.25&#8211;1 mm diameter, needs to be chloridized</td>
		</tr>
		<tr>
			<td>Soldering iron composed of a platinum-iridium filament</td>
			<td></td>
			<td></td>
			<td>0.25 mm diameter (Figure 2F)</td>
		</tr>
		<tr>
			<td>Stereoscopic zoom Microscope</td>
			<td>Nikon</td>
			<td>SMZ-2B</td>
			<td>Figure 1D</td>
		</tr>
		<tr>
			<td>Stereoscopic zoom Microscope</td>
			<td>Wild</td>
			<td>Wild M5</td>
			<td>With 6, 12, 25 and 50 magnification settings (Figure 2E)</td>
		</tr>
		<tr>
			<td>Syringe filters</td>
			<td>Millex</td>
			<td></td>
			<td>22 &#181;m PVDF filter</td>
		</tr>
		<tr>
			<td>Vertical pipette puller</td>
			<td>Sutter/ Narishige</td>
			<td>Model P-97/PP-830</td>
			<td>Use either vertical or horizontal puller, as preferred (Figure 1L)</td>
		</tr>
		<tr>
			<td>Wax filament heater</td>
			<td>Home made</td>
			<td></td>
			<td>Figure 1E-G</td>
		</tr>
		<tr>
			<td>Xenon Flash Lamp system</td>
			<td>Dr. Rapp OptoElectronic</td>
			<td>JML-C2</td>
			<td>Figure 2X</td>
		</tr>
	</tbody>
</table>

Source: Gutorov, R., et al. <a href="https://app.jove.com/v/63514">Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors.</a> J. Vis. Exp. (2022).
This video demonstrates an experimental protocol to investigate the prolonged depolarizing afterpotential (PDA) in white-eyed Drosophila. The fly&#39;s eye is exposed to intense blue light pulses to convert the photopigment rhodopsin to metarhodopsin, initiating a signaling cascade that opens positive ion channels and causes membrane depolarization. Because blue light prevents the reconversion of metarhodopsin to rhodopsin, the continuous influx of positive ions results in sustained depolarization, known as the prolonged depolarizing afterpotential (PDA).

<img alt="Figure 1" src="/files/ftp_upload/22853/22853_Figure1.jpg" /> 
Figure 1: Tools and devices required for fly fixation and recording pipette preparation. (A) Fly sleeper system; (B) Fly sleeper system pedal; (C) Cold light source; (D) Stereoscopic microscope; (E) Wax filament heater; (F) Soldering iron; (G) Wax filament heater pedal; (H) Rough tweezers; (I) Magnetic fly stand; (J) Low melting temperature wax; (K) Delicate wipes; (L) Vertical pipette puller; (M...

1. Measuring the prolonged depolarizing afterpotential (PDA) using the electroretinogram

	Suitable rearing conditions for Drosophila melanogaster ...

Secure a white-eyed Drosophila fly to a holder in a recording arena.&#160;
Position a light source near the fly.
Implant the ground electrode into the fly&#39;s back.
Insert the recording electrode into the outer periphery of the fly&#39;s eye, near the retinal photoreceptor cells containing a photopigment.
Dark-adapt the fly briefly. This converts metarhodopsin, the light-activated form of the photopigment, to rhodopsin, its inactive form.
Apply a high-intensity orange light pulse. This converts any remaining metarhodopsin to rhodopsin.
Next, apply blue light pulses. The blue light converts rhodopsin to metarhodopsin, initiating a signaling cascade and opening specific ion channels.
This induces a continuous positive ion influx and sustained depolarization, even after the blue light is removed &#8212; the prolonged depolarizing afterpotential, or PDA, recorded by the electrode.
Now, reapply the orange light pulses to convert the metarhodopsin to rhodopsin.
This stops the signaling cascade, terminates the PDA, and repolarizes the photoreceptor membrane.

23 次观看. 研究果蝇光感受器中的延长去极化后电位 （PDA）

视频: 研究果蝇光感受器中的延长去极化后电位 （PDA） - 实验

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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神经科学

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Investigating Prolonged Depolarizing Afterpotential (PDA) in Drosophila Photoreceptors

成績單

Investigating Prolonged Depolarizing Afterpotential (PDA) in Drosophila Photoreceptors