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EoE Sub Articles

1. Specimen Prep and Recording Procedure
<ol>
	<li>Prepare the Specimen
	<ol>
		<li>Affix an individual butterfly inside a small plastic tube with hot wax so the head is immobile and protruding from one end of the tube. Wax down the proboscis, antennae, and wings (Figure 1).</li>
		<li>Hold down the abdomen with a dry piece of wax and keep the tube humidified by placing a wet tissue inside the tube behind the abdomen. Make sure the specimen is completely immobile.</li>
		<li>Mount the tube using a small piece of wax onto a small platform with a ball-and-socket joint that is attached to a magnetic base.</li>
		<li>Under a dissecting microscope, insert a silver wire of 0.125 mm diameter into the head via the mouthparts to be used as the reference electrode. Before the experiment, permanently fix the wire to the platform in such a way that the copper wire may clip onto it once the platform is placed on the stage for recording.</li>
		<li>Once the reference electrode is in a suitable position it may be kept in place by quickly melting and then cooling wax around the wire.</li>
		<li>Using a breakable carbon steel razor blade, grip part of the blade with a blade holder and break off a small piece to use for cutting the cornea.</li>
		<li>Cut a small hole (~10 ommatidia in diameter) in the left cornea using the razorblade and seal the hole with Vaseline to prevent desiccation.</li>
	</ol>
	</li>
	<li>Once the cornea is cut, insert the recording electrode into the eye as quickly as possible because hemolymph in the eye will quickly harden and make it impossible to insert an electrode. If possible perform the dissection in the rig where the recording will take place. 
	Note: Vaseline should not be smeared on the rest of the eye as this will defocus the optics.
	<ol>
		<li>If not already on the stage, place the mounted specimen and platform onto the stage in the recording rig. Connect the headstage ground wire to the reference electrode on the specimen platform using alligator clips. 
		Note: If possible, use a red filter to illuminate the animal.</li>
		<li>Use a light source with gooseneck attachments to briefly light the specimen under a stereoscope while lowering the recording electrode into the eye.</li>
		<li>Insert the silver wire connected to the headstage into the KCl solution in the back of a glass microelectrode. Mount the glass electrode on the electrode holder.</li>
		<li>Adjust the electrode holder so the microelectrode is directly over the hole previously cut in the cornea, about a millimeter above the cornea. Lower the microelectrode into the eye using the micromanipulator until a circuit is completed, as shown by a large change in potential (mV) on the oscilloscope.</li>
	</ol>
	</li>
	<li>Once in the eye, swing the stereoscope outside the Faraday cage and turn off the light source illuminating the specimen. The room should be kept dark so the eye becomes dark-adapted.</li>
	<li>Check the resistance of the electrode by applying a 1 nA (nanoAmpere) current from the amplifier and noting the change in voltage. Resistance should typically be in the range between 100-250 M&#937; (milliohm). Higher resistances are indicative of blockage or bending of the electrode, and low resistances of electrode breakage.</li>
	<li>Activate the pulse generator so the shutter opens, allowing a flash of light with a 50 ms (millisecond) duration every 0.5 sec, and allow it to continue flashing for the duration of the experiment.
	<ol>
		<li>Adjust the pulse generator so it allows flashes of up to 50 ms duration. This duration and 0.5-second pause between flashes keep the specimen as near to dark-adapted as possible during the experiment. Fifty ms is close to the shortest flash duration that will elicit the same amplitude in response as longer flash durations.</li>
		<li>Re-measure responses at both the beginning and end of the experiment. Over the course of about twenty minutes, these flash settings do not degrade the response over time. Different preps may require adjustments to these flash settings.</li>
	</ol>
	</li>
	<li>Position the Cardan arm so that the fiber optic cable is directed toward the eye.</li>
	<li>Check the oscilloscope for voltage change with each light flash. A negative change in voltage signifies that the electrode has not yet entered a cell.</li>
	<li>Move the Cardan arm around the specimen until it is positioned at an angle to the eye at which there is a maximum voltage response.</li>
	<li>Rotate the micromanipulator back and forth, causing very small vertical movements of the electrode in both directions while lightly tapping the base of the electrode holder or using the Buzz function on the preamplifier. Continue making small adjustments until a depolarizing light response appears on the oscilloscope (Figure 2).</li>
	<li>Adjust the Cardan arm again to find the angle of incidence where a flash of light produces the largest depolarizing signal. Make small adjustments with the micromanipulator and use the Buzz function on the amplifier as needed to ensure that the electrode is stably recording the cell and will stay in the cell for the whole experiment (See Step 1.11).</li>
	<li>Once the setup is stable, begin recording. A stable recording should have little to no change in resting potential, low background noise, and a consistently large depolarizing response (at least a 10:1 signal-to-noise ratio).
	<ol>
		<li>Run the software on the computer and begin a &#34;new experiment,&#34; which will open a pop-up window with four channels.</li>
		<li>Adjust the voltage scale at the top right corner of the software window to 500 mV. The first channel will display the responses recorded from the electrode in real-time, while the second channel will record the square wave produced by the function generator if the signal is fed to the data acquisition hardware via the oscilloscope, showing when the shutter is open. The other two channels are unneeded.</li>
		<li>Click &#34;Start&#34; at the bottom right-hand corner to begin recording. Allow the software to run for the duration of the experiment. Adjust the zoom of the x (time) and y (voltage) axes so that the responses are clear.</li>
	</ol>
	</li>
	<li>First, with white light, record up to 10 individual responses with the ND (neutral density) filter wheel at 3.5 OD (optical density) (about 5-10 sec).</li>
	<li>Next, record the same number of responses at 3.3 OD, then 3.1, 3.0, 2.5, 2.3, 2.1, etc., in every combination until 0.0 OD. These response amplitudes to the ND filter series will provide the response-log intensity curve. If bleaching occurs, use fewer flashes of bright stimuli during the course of the experiment.</li>
	<li>Record the response of the cell to all wavelengths using the interference filters.
	<ol>
		<li>First, find the peak wavelength. Without ND filters in the light path (0.0 OD), place a UV transmitting filter in the light path and briefly observe the response amplitude. Repeat with a blue transmitting filter, a green transmitting filter, and a red transmitting filter, which should give some idea of where the peak response will be.</li>
		<li>Use filters at about 350, 450, 550, and 650 nm to find the general region of peak sensitivity in step 1.14.1. The exact wavelength does not matter in this initial search phase because all wavelengths will be recorded in the next step. If estimates exist of peak sensitivities, or they have been previously recorded, use known wavelengths to quickly identify the peak response.</li>
		<li>Once the peak response or close to it is identified, record at this wavelength for 10 responses (about 5 sec).</li>
		<li>After recording at the wavelength of peak response, record with the other interference filters from 300-700 nm at 10 nm steps. Start from the peak and step out toward both shorter and longer wavelengths by swapping the filters out from the light path one by one (e.g. if the peak response is at 520 nm, record responses at this wavelength first, then 510 nm, followed by 530 nm, 500 nm, 540 nm, 490 nm, 550 nm, and so on until no there is no response).</li>
		<li>Allow for up to 10 responses per filter (5 sec each). When swapping interference filters, allow the cell to respond to 1-2 flashes of white light without any filter in the light path, which is helpful to monitor whether the peak response is degrading over time. Reduce the number of responses or increase the OD if bleaching occurs.</li>
	</ol>
	</li>
</ol>

in vivo intracellular recording of photoreceptor cell spectral sensitivity in a butterfly

Source: McCulloch, K. J., et al.. <a href="https://app.jove.com/v/53829">Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly and determining the spectral sensitivity of individual cell types. This technique enables the study of sensitivities of individual photoreceptor cells to different light stimuli.

<img alt="Figure 1" src="/files/ftp_upload/22857/22857_Figure1.jpg" /> 
Figure 1: Butterfly Prep. (A) The butterfly is inserted into the tube, and the head, wings, and antennae are immobilized with hot wax. The indifferent electrode is inserted into the mouthparts (1), a piece of dry wax is used to hold down the abdomen (2), and a wet tissue is placed behind the specimen. (B) Close-up of the head waxed down. The eye to be recorded fr...

In Vivo Intracellular Recording of Photoreceptor Cell Spectral Sensitivity in a Butterfly

1. Specimen Prep and Recording Procedure

	Prepare the Specimen
	
		Affix an individual butterfly inside a small plastic tube with hot wax so the head is ...

Begin with an immobilized butterfly on a platform.
Under a dissecting microscope, insert a reference electrode through the mouthparts to reach the head, and fix the electrode to the platform.
Make a small incision in the cornea and seal it to prevent drying.
Using brief lighting under a stereoscope, position a recording electrode filled with an ionic solution above the cornea.
Then, lower the electrode into the cornea using a micromanipulator to access the photoreceptor cell.
A significant voltage change across the membrane indicates the contact of the electrode with the cell.
Dark-adapt the insect by switching off the external light sources.
Initiate light flashes with a pulse generator and direct a fiber optic cable toward the eye.
Adjust the position of the electrode and fiber optic cable until a depolarizing response is obtained.
Record the cell&#39;s responses to white light and colored filters to assess its sensitivity to different light wavelengths.

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41 Views. Source: McCulloch, K. J., et al.. Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings. J. Vis. Exp. (2016) This video demonstrates the procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly and determining the spectral sensitivity of individual cell types. This technique enables the study of sensitivities of individual photoreceptor cells to different light stimuli. 

In Vivo Intracellular Recording of Photoreceptor Cell Spectral Sensitivity in a Butterfly

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