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

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

To prepare the specimen, under the microscope, 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. Then, affix the proboscis, antennae, and wings with wax. After that, hold down the abdomen with a dry piece of wax. Keep the tube humidified by placing a wet tissue inside it behind the abdomen of the specimen. Make sure the specimen is completely immobile.
Next, 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. Under a dissecting microscope, insert a silver wire into the head via the mouth part, as the reference electrode. Next, cut a small hole in the left cornea using a razor blade, and seal the hole with Vaseline to prevent desiccation. 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. Mount the electrode on the electrode holder.
Subsequently, insert the silver wire into the potassium chloride solution in the microelectrode. Use a light source with gooseneck attachments to light the specimen under a stereoscope, while lowering the electrode into the eye. Adjust the electrode holder so the microelectrode is directly above the hole previously cut in the cornea. Then, lower the microelectrode into the eye using the micromanipulator until a circuit is completed.
Once in the eye, swing the stereoscope outside the Faraday cage, turn off the light source illuminating the specimen, and leave the insect in the dark for 10 to 20 minutes for dark adaptation. Next, activate the pulse generator to deliver a flash of light for the duration of the experiment, and direct the fiber optic cable toward the eye. Then, adjust the fiber optic cable to find the largest depolarizing signal.
Make small adjustments with a micromanipulator and use the buzz function on the amplifier as needed, to make sure the electrode is stably recording the cell. For white light stimulation, record up to 10 individual responses with the ND filter wheel set at the highest OD that still elicits a response. Then, record with other interference filters, and allow for up to 10 responses per filter.

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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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Butterfly pupae</td>
			<td></td>
			<td></td>
			<td>Several local species available, need USDA permits for shipping. Carolina Bio Supply has several insect species that may be ordered within the U.S. without the need for additional permits</td>
		</tr>
		<tr>
			<td>Insect pins, size 2</td>
			<td>BioQuip</td>
			<td>1208B2</td>
			<td></td>
		</tr>
		<tr>
			<td>Xenon Arc Lamp</td>
			<td>Oriel Instruments</td>
			<td>66003</td>
			<td>Oriel is now a part of Newport Corporation</td>
		</tr>
		<tr>
			<td>Universal Power Supply</td>
			<td>Oriel Instruments</td>
			<td>68805</td>
			<td>Oriel is now a part of Newport Corporation</td>
		</tr>
		<tr>
			<td>Optical Track</td>
			<td>Oriel Instruments</td>
			<td>11190</td>
			<td>Oriel is now a part of Newport Corporation</td>
		</tr>
		<tr>
			<td>Rail Carrier, Large (2x)</td>
			<td>Oriel Instruments</td>
			<td>11641</td>
			<td>Oriel is now a part of Newport Corporation</td>
		</tr>
		<tr>
			<td>Rail Carrier, Small (4x)</td>
			<td>Oriel Instruments</td>
			<td>11647</td>
			<td>Oriel is now a part of Newport Corporation</td>
		</tr>
		<tr>
			<td>Thread Adaptor, 8-32 Male to 1/4-20 Male, pack of 10</td>
			<td>Newport Corporation</td>
			<td>TA-8Q20-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Mounting Post, 1.0 in., 0.5 in. Dia. Stainless, 8-32 &#38; 1/4-20 (5x)</td>
			<td>Newport Corporation</td>
			<td>SP-1</td>
			<td></td>
		</tr>
		<tr>
			<td>No Slip Optical Post Holder, 2 in., 0.5 in. Diameter Posts, 1/4-20 (5x)</td>
			<td>Newport Corporation</td>
			<td>VPH-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed lens mount, 50.8 mm</td>
			<td>Newport Corporation</td>
			<td>LH-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed lens mount, 25.4 mm</td>
			<td>Newport Corporation</td>
			<td>LH-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Condenser lens assembly</td>
			<td>Newport Corporation</td>
			<td>60006</td>
			<td></td>
		</tr>
		<tr>
			<td>Convex silica lens, 50.8 mm</td>
			<td>Newport Corporation</td>
			<td>SPX055</td>
			<td></td>
		</tr>
		<tr>
			<td>Six Position Filter Wheel, x2</td>
			<td>Newport Corporation</td>
			<td>FW1X6</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Wheel Mount Hub</td>
			<td>Newport Corporation</td>
			<td>FWM</td>
			<td></td>
		</tr>
		<tr>
			<td>Concave silica lens, 25.4 mm</td>
			<td>Newport Corporation</td>
			<td>SPC034</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimator holder</td>
			<td>Newport Corporation</td>
			<td>77612</td>
			<td></td>
		</tr>
		<tr>
			<td>Collimating beam probe</td>
			<td>Newport Corporation</td>
			<td>77644</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrule Converter, SMA Termination to 11 mm Standard Ferrule</td>
			<td>Newport Corporation</td>
			<td>77670</td>
			<td>This adapter allows the fiber optic to fit into the collimator holder</td>
		</tr>
		<tr>
			<td>600 &#956;m diameter UV-vis fiber obtic cable</td>
			<td>Oriel Instruments</td>
			<td>78367</td>
			<td>Oriel is now a part of Newport Corporation</td>
		</tr>
		<tr>
			<td>Shutter with drive unit</td>
			<td>Uniblitz</td>
			<td>100-2B</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Fused Silica Metallic ND Filter, 0.1 OD</td>
			<td>Newport</td>
			<td>FRQ-ND01</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Fused Silica Metallic ND Filter, 0.3 OD</td>
			<td>Newport</td>
			<td>FRQ-ND03</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Fused Silica Metallic ND Filter, 0.5 OD</td>
			<td>Newport</td>
			<td>FRQ-ND05</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Fused Silica Metallic ND Filter, 1.0 OD</td>
			<td>Newport</td>
			<td>FRQ-ND10</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Fused Silica Metallic ND Filter, 2.0 OD</td>
			<td>Newport</td>
			<td>FRQ-ND30</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Fused Silica Metallic ND Filter, 3.0 OD</td>
			<td>Newport</td>
			<td>FRQ-ND50</td>
			<td></td>
		</tr>
		<tr>
			<td>LS-1-Cal lamp</td>
			<td>Ocean Optics</td>
			<td>LS-1-Cal</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics</td>
			<td>USB-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Software</td>
			<td>Ocean Optics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 300 nm</td>
			<td>Edmund Optics</td>
			<td>67749</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 310 nm</td>
			<td>Edmund Optics</td>
			<td>67752</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 320 nm</td>
			<td>Edmund Optics</td>
			<td>67754</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 330 nm</td>
			<td>Edmund Optics</td>
			<td>67756</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 340 nm</td>
			<td>Edmund Optics</td>
			<td>65614</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 350 nm</td>
			<td>Edmund Optics</td>
			<td>67757</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 360 nm</td>
			<td>Edmund Optics</td>
			<td>67760</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 370 nm</td>
			<td>Edmund Optics</td>
			<td>67761</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 380 nm</td>
			<td>Edmund Optics</td>
			<td>67762</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 390 nm</td>
			<td>Edmund Optics</td>
			<td>67763</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 400 nm</td>
			<td>Edmund Optics</td>
			<td>65732</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 410 nm</td>
			<td>Edmund Optics</td>
			<td>65619</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 420 nm</td>
			<td>Edmund Optics</td>
			<td>65621</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 430 nm</td>
			<td>Edmund Optics</td>
			<td>65622</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 440 nm</td>
			<td>Edmund Optics</td>
			<td>67764</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 450 nm</td>
			<td>Edmund Optics</td>
			<td>65625</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 460 nm</td>
			<td>Edmund Optics</td>
			<td>67765</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 470 nm</td>
			<td>Edmund Optics</td>
			<td>65629</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 480 nm</td>
			<td>Edmund Optics</td>
			<td>65630</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 492 nm</td>
			<td>Edmund Optics</td>
			<td>65633</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 500 nm</td>
			<td>Edmund Optics</td>
			<td>65634</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 510 nm</td>
			<td>Edmund Optics</td>
			<td>65637</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 520 nm</td>
			<td>Edmund Optics</td>
			<td>65639</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 532 nm</td>
			<td>Edmund Optics</td>
			<td>65640</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 540 nm</td>
			<td>Edmund Optics</td>
			<td>65642</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 550 nm</td>
			<td>Edmund Optics</td>
			<td>65644</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 560 nm</td>
			<td>Edmund Optics</td>
			<td>67766</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 570 nm</td>
			<td>Edmund Optics</td>
			<td>67767</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 580 nm</td>
			<td>Edmund Optics</td>
			<td>65646</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 589 nm</td>
			<td>Edmund Optics</td>
			<td>65647</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 600 nm</td>
			<td>Edmund Optics</td>
			<td>65648</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 610 nm</td>
			<td>Edmund Optics</td>
			<td>65649</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 620 nm</td>
			<td>Edmund Optics</td>
			<td>65650</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 632 nm</td>
			<td>Edmund Optics</td>
			<td>65651</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 640 nm</td>
			<td>Edmund Optics</td>
			<td>65653</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 650 nm</td>
			<td>Edmund Optics</td>
			<td>65655</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 660 nm</td>
			<td>Edmund Optics</td>
			<td>67769</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 671 nm</td>
			<td>Edmund Optics</td>
			<td>65657</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 680 nm</td>
			<td>Edmund Optics</td>
			<td>67770</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 690 nm</td>
			<td>Edmund Optics</td>
			<td>65659</td>
			<td></td>
		</tr>
		<tr>
			<td>Interference bandpass filter, 700 nm</td>
			<td>Edmund Optics</td>
			<td>67771</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td></td>
			<td></td>
			<td>Any metal structure will work that can be grounded and that fits the experimental setup.</td>
		</tr>
		<tr>
			<td>Stereomicroscope, 6x, 12x, 25x, 50x magnification</td>
			<td>Wild Heerbrugg</td>
			<td>Wild M5</td>
			<td>Any Stereomicroscope will do</td>
		</tr>
		<tr>
			<td>Microscope stand with swinging arm and heavy base</td>
			<td>McBain Instruments</td>
			<td></td>
			<td>Any heavy base with arm will do</td>
		</tr>
		<tr>
			<td>Cardan arm</td>
			<td></td>
			<td></td>
			<td>Custom built, See Figure 4</td>
		</tr>
		<tr>
			<td>Fiber-lite high intensity illuminator</td>
			<td>Dolan-Jenner</td>
			<td>MI-150</td>
			<td>For lighting specimen</td>
		</tr>
		<tr>
			<td>Fiber-lite goose-neck light guide</td>
			<td>Dolan-Jenner</td>
			<td>EEG 2823</td>
			<td>Any goose-neck light guide will do</td>
		</tr>
		<tr>
			<td>Marble table</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Raised wooden table</td>
			<td></td>
			<td></td>
			<td>Hole should be cut through this table so that the sandbox can rest on the marble table underneath</td>
		</tr>
		<tr>
			<td>Wooden box filled with sand</td>
			<td></td>
			<td></td>
			<td>custom built, any box with sand</td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>Carl Zeiss - Jena</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen stage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alligator clip wires for grounding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulated copper wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire, 0.125 mm diameter</td>
			<td>World Precision Instruments</td>
			<td>AGW0510</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Preamplifier with headstage</td>
			<td>Dagan Corporation</td>
			<td>IX2-700</td>
			<td></td>
		</tr>
		<tr>
			<td>Humbug Noise reducer</td>
			<td>Quest Scientific</td>
			<td>Humbug</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope, 30MHz, 2CH, Dual Trace, Alt-triggering, without probe</td>
			<td>EZ Digital</td>
			<td>os-5030</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC T-adapter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab hardware 2/20</td>
			<td>ADI instruments</td>
			<td>ML820</td>
			<td></td>
		</tr>
		<tr>
			<td>Labchart software</td>
			<td>ADI instruments</td>
			<td>Chart 5</td>
			<td></td>
		</tr>
		<tr>
			<td>10 MHz Pulse Generator</td>
			<td>BK Precision</td>
			<td>4030</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, 3 M</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slotted plastic tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low melting temperature wax</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platform with ball-and-socket magnetic base</td>
			<td>Hama photo and video</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge carbon steel, breakable razor blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

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.

22 Views. Registrazione intracellulare in vivo della sensibilità spettrale delle cellule fotorecettrici in una farfalla

Video: Registrazione intracellulare in vivo della sensibilità spettrale delle cellule fotorecettrici in una farfalla - Experiment

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.

Metodo elettrofisiologiche per la registrazione intracellulare di tensione Risposte di<em&gt; Drosophila</em&gt; Fotorecettori e Interneuroni a luce stimoli<em&gt; In Vivo</em

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

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JoVE Journal

Neuroscienze

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Registrazioni patch-clamp intere cellule per la determinazione elettrofisiologica della selettività ionica in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

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.

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

Trascrizione

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