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>

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 Views. Investigating Prolonged Depolarizing Afterpotential (PDA) in Drosophila Photoreceptors

Investigating Prolonged Depolarizing Afterpotential (PDA) in Drosophila Photoreceptors

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