This work was supported by NIH grants EY019312 and EY021126 (VJK), EY002687 to the Department of Ophthalmology and Visual Sciences at Washington University, and by Research to Prevent Blindness.

All experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the institutional Animal Studies Committee at Washington University.
1. Setting Up Perfusion and Specimen Holder
<ol>
	<li>Prepare solution for retina perfusion fresh on the day of the experiment. Use distilled and deionized water. Use one of the following three solutions.
	<ol>
		<li>Prepare Bicarbonate-containing Ames&#8217; solution (1 L): 1 bottle of Ames&#8217; media and 1.9 g of NaHCO3,</li>
		<li>Prepare Locke&#8217;s solution (in mM): 112.5 NaCl, 3.6 KCl, 2.4 MgCl2, 1.2 CaCl2, 10.0 HEPES, 20.0 NaHCO3, 3.0 Na succinate, 0.5 Na glutamate, 0.02 EDTA, and 10.0 glucose, 0.1% MEM vitamins and amino acids</li>
		<li>Prepare HEPES-buffered Ringer solution (in mM): 133.3 NaCl, 3.3 KCl, 2.0 MgCl2, 1.0 CaCl2, 10.0 glucose, 0.01 EDTA, 12.0 HEPES, pH adjusted to 7.5 with ~5.8 ml of 1 M NaOH, add 0.72 g/L Leibovitz culture medium L-15. Use 20 - 50 &#956;M DL-AP4 and 50 &#8211; 100 &#956;M BaCl2 to isolate the photoreceptor response with any perfusion media.</li>
	</ol>
	</li>
	<li>Prepare solution for electrodes28 (in mM): 140.0 NaCl, 3.6 KCl, 2.4 MgCl2, 1.2 CaCl2, 3 HEPES, 0.01 EDTA and adjust pH to 7.4 &#8211; 7.5 with NaOH. Electrode solution can be stored at RT for several months.</li>
	<li>Prepare and test the specimen holder.
	<ol>
		<li>Glue black/grey filter paper on top of the domes of the specimen holder&#8217;s bottom part (see Figure 1A). Spread two-component 5 min epoxy glue carefully around the edges of the flat tops of the domes. If necessary, do it under dissection microscope.</li>
		<li>Wait until the glue is almost dry (about 4 min) and press the filter paper on the domes by using a flat item. Glue filter paper at least one day before the experiment. The filter paper can be used multiple times but should be replaced after a month of recordings. Use 70% ethanol to clean domes carefully from any glue residue before installing the replacement paper.</li>
		<li>Fill the electrode channels with electrode solution trying to avoid any air bubbles and screw pellet electrodes enclosed with a threaded adapter into the electrode channels (see Figures 1A and B). Connect the top and bottom pieces of the specimen holder with four screws and fill the perfusion lines with electrode solution.</li>
		<li>Measure the resistance and voltage between the leads of each electrode pair using a multimeter (Figure 1B). Resistance should be below 100 &#937;k and the voltage below 10 mV if the channels are bubble-free and the electrodes are in good conditions.</li>
	</ol>
	</li>
	<li>Pour 400 - 700 ml of perfusion media in the glass bottle. Separate another 300 ml to be used in the dissection of the retinas and store it in a fridge. Set the perfusion tubing in the heat exchanger block and place the preheated block on the heating plate (see Figure 1D).</li>
	<li>Enclose the bottle with a cap that has connections to CO2/O2 gas (if Ames&#8217; or Locke&#8217;s is used) and for perfusion tubing (see Figure 1D). Preheat the bottle with the media to 37 oC and place it on the heating plate or on the top of the light stimulation unit in water bath set to 37 &#8211; 39 oC17.
	<ol>
		<li>Prime the perfusion lines by filling them with perfusion media to initiate the gravity-driven flow.
		<ol>
			<li>In LKC system, place the bottle on the top of the stimulator unit17 to provide a significant gravitational flow that is then adjusted by flow-rate regulators without being affected by the lowering level of the perfusion solution in the bottle during the experiment.</li>
			<li>In Ocuscience system, place the perfusion bottle inside the Faraday cage in order to minimize noise and place long perfusion output lines from the specimen holder (see step 2.8) well below the level of the specimen holder (and perfusion bottle) to increase gravitational drive of the solution. Shield these output lines and connect the shield to the amplifier&#8217;s ground to prevent coupling of the electromagnetic noise to the ERG signal.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Adjust the perfusion to 3 &#8211; 5 ml/min by using flow-rate regulators. Connect 5% CO2 / 95% O2 gas from a cylinder with proper regulator and adjust the flow rate to ensure steady bubbling of the media in the bottle through an air stone.</li>
</ol>
2. Sample Preparation
<ol>
	<li>Assemble clean and sharp dissection instruments including straight-bladed microscissors, one or two 45o tweezers, razor blade and a rectangular piece of filter paper.</li>
	<li>Pour about 200 ml of cold perfusion solution in a large Petri dish so that the whole bottom part of the specimen holder (including the domes) can be immersed into the solution. This step becomes important when mounting the retinas on the domes (see step 2.6). Although some solutions are designed to be saturated with carbogen (5% CO2 / 95% O2), this is not essential for the dissection purposes and was not done in the experiments described here.</li>
	<li>For a typical experiment, keep animals in 12/12 hrs dark/light cycle and dark-adapt them for 6 &#8211; 12 hrs before the recordings. Euthanize the animal by CO2 inhalation followed by cervical dislocation under dim red light and do all the following procedures under dim red or IR light (use e.g., red filter in front of the microscope light source).
	<ol>
		<li>Pull the eyes out by using tweezers and put them in the media. Place one eye at a time on a small piece of paper (e.g., some regular filter paper) and make a slit approximately on the level of ora serrata while holding the eye with tweezers (this is done outside of the solution here).</li>
	</ol>
	</li>
	<li>Cut along the ora serrata (or closer to the equator of the eye) with microscissors and remove the cornea and the lens. Place the eye cup in the cold media in the large Petri dish and repeat the same procedure with the other eye.</li>
	<li>Cut a small incision from the top of the eye cup towards the optic nerve by keeping the scissors between the retina and sclera in order to keep the retina as intact as possible. Grip the sclera from both sides of the incision by using two tweezers and pull the tweezers away from each other to detach the retina.
	<ol>
		<li>Cut the optic nerve and try to isolate the retina with a minimum amount of physical contact to the distal surface. RPE will mostly detach automatically from the retina during the dissection process. It is more important to avoid mechanical disturbance of the retina than to perform the dissection quickly, generally retinas can be incubated at least 30 min in the solution without significant effects on the response properties.</li>
	</ol>
	</li>
	<li>Mount the retinas on the domes of the specimen holder (Figure 1C). Immerse the bottom part of the specimen holder in the Petri dish with the dissected retinas. Slide the retina, photoreceptor side (the convex surface of the isolated retina) upwards, above the dome and lift the specimen holder so that the retina attaches on the filter paper. Repeat the procedure for the other retina.</li>
	<li>Dry the holder plate carefully to prevent electrical crosstalk between the retinas as well as noise and signal shunting. Specimen holder has O-rings around the bottom part domes to prevent solution spill between the bottom and top parts of the holder as well as to help electrical isolation of the photoreceptor and ganglion cell sides of the individual retinas. Attach the top part of the holder with the four screws (see Figure 1B) and fill the perfusion channels with perfusion solution by using a syringe and a needle.</li>
	<li>Transfer the specimen holder next to the heat exchanger block and connect the input and output perfusion lines to the specimen holder (Figure 1D). Connect the electrodes to the ERG amplifier (top electrodes connect to the ground/minus pin in the amplifier) and attach the stimulator/control unit on the specimen holder by using an adaptor or slide the heating pad into the stimulator unit depending on which in vivo ERG system is used.</li>
</ol>
3. Recordings
<ol>
	<li>Configure the amplifier and stimulus settings by using the in vivo system&#8217;s software. Set the acquisition frequency to a value between 1 and 10 kHz and low-pass filtering to 300 Hz. Do not use high-pass filtering. Use 60 Hz (or 50 Hz in Europe) notch filter if necessary.</li>
	<li>Record baseline without light stimulation and with dim stimulation (e.g., green light of -35 dB or 0.3 mCd sm-2) to test for good electrical connection between the electrodes and retina sample. Wait 10 &#8211; 20 min before starting the data collection so that retinal temperature and function reach a stable state.
	<ol>
		<li>Choose stimulus parameters according to any specific experiment and start data collection. E.g., use green light from -40 up to 0 dB or from 0.1 up to 1,000 mCd sm-2 for a scotopic response family. Use about 2 to 3 log-units brighter flashes in photopic recordings where rods have to be suppressed by background light (3 &#8211; 30 Cdm-2) or a probe flash (about 1.3 Cd sm-2 = 1 dB, see Figure 3).</li>
		<li>However, remember that the exact light intensity values might be somewhat dependent on your system calibration and experimental conditions. As a rule of thumb, scale intensities down by 5 &#8211; 10-fold as compared to in vivo recordings when using green light17. A black cover with apertures above the retinas (included in the commercial adapter system) can be used to reduce light scattering in the specimen holder and facilitate homogenous and equal stimulation of both retinas by the Ganzfeld sphere of the commercial in vivo systems. 
		NOTE: For recordings from dark-adapted retina, the intensities of test flashes used in a typical experiment bleach only a negligible fraction of the pigment so that the lack of RPE-driven regeneration is not an issue. In addition, it has been previously shown that cone pigment regeneration can still take place in the isolated retina via the M&#252;ller cell visual cycle20.</li>
	</ol>
	</li>
	<li>As experiments last typically from 30 min up to several hours, monitor baseline drift, noise level and response stability during the experiment as changes in these might indicate technical problems. 
	&#8203;NOTE: Increased noise or decreased response amplitudes may indicate air bubbles in the perfusion or electrode channels, too low or high temperature, solution leak/spill or displacement of the retina.</li>
</ol>
4. Cleaning
<ol>
	<li>Detach the specimen holder from the perfusion lines, open it and flush the retinas from the filter paper. Remove the electrodes and rinse them with distilled water (do not use ethanol). Clean the specimen holder (including the perfusion channels) with ethanol and/or distilled water. Flush perfusion tubing carefully with &#62;70% ethanol.</li>
	<li>Rinse the perfusion bottle with distilled water (do not use detergents). Ethanol can be also used to clean the bottle.</li>
</ol>

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Neurosci. 19, 1203-1216 (1999).</li><li>Heikkinen, H., Vinberg, F., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesopic+background+lights+enhance+dark-adapted+cone+ERG+flash+responses+in+the+intact+mouse+retina:+a+possible+role+for+gap+junctional+decoupling.">Mesopic background lights enhance dark-adapted cone ERG flash responses in the intact mouse retina: a possible role for gap junctional decoupling.</a> J. Neurophysiol. 105, 2309-2318 (2011).</li><li>Gouras, P., MacKay, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+in+amplitude+of+the+human+cone+electroretinogram+with+light+adaptation.">Growth in amplitude of the human cone electroretinogram with light adaptation.</a> Invest. Ophthalmol. Vis. Sci. 30, 625-630 (1989).</li><li>Peachey, N. S., Goto, Y., al-Ubaidi, M. R., Naash, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+mouse+cone-mediated+electroretinogram+during+light+adaptation.">Properties of the mouse cone-mediated electroretinogram during light adaptation.</a> Neurosci. Lett. 162, 9-11 (1993).</li></ol>

Washington University in St. Louis has a license agreement with Xenotec, Inc. and may receive a royalty from the sale of the ex vivo adapter.

We demonstrate here the critical steps for obtaining high-quality ex vivo ERG recordings simultaneously from two isolated mouse retinas by using in vivo ERG system components together with an ex vivo ERG adapter. In this study we perfused both retinas from the animal with the same solution (either Ames&#8217;, Locke&#8217;s or Ringer) but it is also possible to perfuse each retina with a different solution e.g., for drug testing purposes. The most important steps for obtaining high quality data are shielding from electromagnetic noise, careful dissection of the retina, steady and relatively rapid perfusion flow by using an advanced custom-built specimen holder, and performing all sample preparation procedures under dim red (or IR) light. The method described here allows immediate use of both retinas and dual use of an in vivo ERG setup to perform in vivo and ex vivo ERG recordings.
The custom-built ex vivo ERG specimen holder recently designed by us17, and now commercially available, improves SNR by efficient electrical isolation of the proximal and distal parts of the retina and optimizes perfusion flow above the retina (high solution exchange rate). Absence of the slow frequency noise components in ex vivo ERG signal allows quantitative analysis even from very small responses. However, we found that ex vivo recording is more prone to interference of AC-power line noise (60 Hz in the US; 50 Hz in Europe) than in vivo experiments. This noise coupled to the signal mainly through the perfusion line, and it could be mostly removed by shielding (and grounding) all perfusion components (bottle, tubing) residing outside of the Faraday cage or Ganzfeld sphere. In addition, sometimes 60 Hz noise coupled to the ex vivo ERG signal through the heat exchanger and this noise could also be removed by grounding.
We demonstrate how to remove specific ERG signal components by adding pharmacological blockers into the perfusion during the experiment allowing dissection of the function of different cell types in the same retina/experiment with three different physiological perfusion media (Figure 2). A recent study showed that the choice of perfusion media affects photoreceptor physiology in single cell recordings32. Here we used Ames&#8217;, Locke&#8217;s and &#8216;HEPES-Ringer&#8217; media to record dark-adapted flash responses in the absence and presence of pharmacological reagents intended to isolate the photoreceptor component of the ERG signal (Figure 2). Bicarbonate-buffered solutions gave larger a- and b-wave amplitudes, up to 1 mV. Photoreceptor dim flash responses under Locke&#8217;s medium with blockers contained complicated recovery waveform (see inset of Figure 2D) that was not seen with Ames&#8217; or Ringer perfusion. When the use of ex vivo ERG becomes adapted by more laboratories it would be helpful to use the same perfusion media and standard methods to isolate different signal components. At this point it seems that the most versatile option is the Ames&#8217; medium because it gives stable and large a- and b-wave amplitudes. In addition, the photoreceptor response, isolated pharmacologically in this solution, appears to have a simple waveform reminiscent of that recorded from single photoreceptors (Figure 2E). Yet, some open questions remain about the existence of other ERG signal components observed under in vivo conditions. For example, in our ex vivo recording conditions we did not see prominent oscillatory potentials, 100 &#8211; 150 Hz oscillating waves that are typically observed in the rising phase of the b-wave of an in vivo ERG response. It is thus possible that the inner retina function in our ex vivo conditions was compromised although large ex vivo b-waves implicated viable ON bipolar cell function. Future studies should resolve whether modifications in the experimental protocols shown here (dissection, perfusion etc.) would allow us to record oscillatory potentials under ex vivo conditions.
Cone function is vital for our vision. However, investigation of cones is hampered by their small size and scarcity especially in the mouse retina33. Isolation of cone function is further complicated in mouse ERG recordings because their M-cones and rods have almost identical spectral sensitivities30. Standard protocols take advantage of the several log-unit difference between rod and cone sensitivities by using rod-suppressing background light. However, steady background light is known to desensitize rods34,35 and affect cone function possibly through modulation of gap junctional coupling between rods and cones36,37. Thus, it is hard to find a background light intensity that is bright enough to keep rods saturated without affecting cones. Here we demonstrate an alternative double flash method that takes advantage of both the lower sensitivity and faster recovery kinetics of cones19,29,30. In this way it is easier to isolate truly dark-adapted cone-mediated responses. We noticed that in Ames&#8217; or Locke&#8217;s solutions without any blockers, the details of the recovery waveform were somewhat affected during the course of the experiment by the use of bright probe and test flashes. This complicated the subtraction analysis to isolate the cone responses. However, removing the glial component by barium helped to stabilize the tail of the responses indicating that the variability was due to the M&#252;ller cell component. In this way it was possible to obtain dark-adapted cone-driven responses in WT mice (Figure 3). Cone responses isolated by double flash technique from WT mice appeared smaller as compared to those recorded from WT mice by using background light to suppress rod activity17,37. This difference can be explained by a well-characterized effect of background light that slowly (within 10 min) enhances cone response amplitudes probably due to removal of the rod-mediated suppression of cone responses37-39.
In summary, the method demonstrated here makes possible ex vivo electrophysiological recordings to study the function of the retina. In the future, we hope that many more laboratories will adapt this powerful method to study the physiology and pathology of animal and human retina and to advance our understanding of retinal function and develop better therapies for blinding diseases.

Electroretinogram (ERG) is a well-established technique that can be used to record the electrical activity of the retina triggered by light. The ERG signal is generated mainly by voltage changes caused by radial currents (along the axis of photoreceptors and bipolar cells) flowing in the resistive extracellular space of the retina. The first ERG signal was recorded in 1865 by Holmgren from the surface of a fish eye1. Einthoven and Jolly 19082 divided the ERG response to the onset of light into three different waves, called a-, b-, and c-waves, that are now known to reflect mainly the activity of photoreceptors, ON bipolar cells, and pigment epithelium cells, respectively3-8. ERG can be recorded from the eyes of anesthetized animals or humans (in vivo), from isolated eye preparation9, across isolated intact retina (ex vivo)3,10-15 or across specific retina layers with microelectrodes (local ERG)4,16. Of these, in vivo ERG is currently the most widely used method to assess retinal function. It is a noninvasive technique that can be used for diagnostic purposes or to follow the progression of retinal diseases in animals or patients. However, in vivo ERG recordings produce a complicated signal with several overlapping components, often contaminated by extraocular physiological noise (e.g., breathing and cardiac activity).
Local ERG can be used to record the signal across specific layers of the retina but it is the most invasive and has the lowest signal-to-noise ratio (SNR) as compared to the other ERG recording configurations. Local ERG is also technically demanding and requires expensive equipment (e.g., microscope and micromanipulators). Transretinal ERG from the intact, isolated retina (ex vivo ERG) offers a compromise between in vivo and local ERG methods allowing stable and high SNR recordings from intact retinas of animals or humans17. Recently, this method has been used successfully to study rod and cone photoreceptor function in mammalian, primate and human retinas18-20. In addition, due to absence of pigment epithelium in the ex vivo retina, the positive c-wave component of the ERG signal is removed and a prominent negative slow PIII component is revealed in the ex vivo recordings. The slow PIII component has been shown to originate from the activity of M&#252;ller glia cells in the retina21-23. Thus, ex vivo ERG method could also be used to study M&#252;ller cells in the intact retina. Several studies have also shown that ex vivo ERG recordings could be used to measure concentration of pharmacological agents around the retina24 and test the safety and efficacy of drugs25-27.
Multiple commercial in vivo systems are available and used in many laboratories that do not necessarily have extensive electrophysiology background. In contrast, ex vivo devices have not been available until recently17 and as a result only very few laboratories are currently taking advantage of this powerful technique. It would be beneficial to make ex vivo ERG recordings available to more laboratories in order to advance our knowledge about retinal physiology and pathology, and to develop new therapies for blinding diseases. We demonstrate here a simple and affordable ex vivo ERG device17 and show how it can be used in combination with several commercially available in vivo ERG systems to record rod- and cone-mediated signaling (a- and b-waves) and the function of M&#252;ller cells (slow PIII) from intact wild-type mouse retinas.

<table><tbody><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>OcuScience</td><td>HMsERG</td><td>&lt;a href=&quot;http://www.ocuscience.us/id77.html&quot;&gt;www.ocuscience.us/id77.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;In vivo&lt;/i&gt; ERG system</td><td>LKC Technologies</td><td>UTAS-E 3000</td><td>&lt;a href=&quot;http://www.lkc.com/products/UTAS/bigshot.html&quot;&gt;www.lkc.com/products/UTAS/bigshot.html&lt;/a&gt;</td></tr><tr><td>&lt;i&gt;Ex vivo&lt;/i&gt; adapter</td><td>OcuScience</td><td>Ex VIVO ERG adapter</td><td>&lt;a href=&quot;http://www.ocuscience.us/id107.html&quot;&gt;www.ocuscience.us/id107.html&lt;/a&gt;</td></tr><tr><td>Dissection microscope</td><td>North Central Instruments</td><td>Leica M80</td><td>&lt;a href=&quot;http://www.ncimicro.com/store/ListProduct.asp?bSub=T&amp;amp;idCategory=27&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>IR emitter</td><td>Opto Diode Corp.</td><td>OD-50L</td><td>&lt;a href=&quot;http://www.optodiode.com/&quot;&gt;www.optodiode.com&lt;/a&gt;</td></tr><tr><td>Prowler Night Vision Scopes</td><td>B.E. Meyers Electro Optics</td><td>D4300-I</td><td>Military grade product.</td></tr><tr><td>Red filter</td><td>Rosco Laboratories</td><td>Roscolux #27 Medium Red</td><td>May be used instead of IR system</td></tr><tr><td>Red head light</td><td>OcuScience</td><td>ERGX011</td><td>&lt;a href=&quot;http://www.ocuscience.us/catalog/i29.html&quot;&gt;www.ocuscience.us/catalog/i29.html&lt;/a&gt;</td></tr><tr><td>Microscissors</td><td>WPI, Inc.</td><td>500086</td><td>&lt;a href=&quot;http://www.wpiinc.com/&quot;&gt;www.wpiinc.com/&lt;/a&gt;</td></tr><tr><td>Dumont tweezers #5</td><td>WPI, Inc.</td><td>14101</td><td></td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>72000</td><td>&lt;a href=&quot;http://www.emsdiasum.com/&quot;&gt;www.emsdiasum.com&lt;/a&gt;</td></tr><tr><td>Scale</td><td>Metler Toledo</td><td>AB54-S/FACT</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>pH meter and electrode</td><td>Beckman Coulter</td><td>pHI 350</td><td>May use any brand</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>60129</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;May use any brand&lt;/a&gt;</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>63020</td><td>1.0 M solution</td></tr><tr><td>CaCl2</td><td>Sigma-Aldrich</td><td>21114</td><td>1.0 M solution</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>431788</td><td>May use any brand</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>May use any brand</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td>May use any brand</td></tr><tr><td>Ames medium</td><td>Sigma-Aldrich</td><td>A1420</td><td>May use any brand</td></tr><tr><td>BaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>B0750</td><td>May use any brand</td></tr><tr><td>DL-AP4</td><td>Tocris Bioscience</td><td>101</td><td>May use any brand</td></tr><tr><td>Succinic acid disodium salt</td><td>Sigma-Aldrich</td><td>224731</td><td>May use any brand</td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G2834</td><td>May use any brand</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td>May use any brand</td></tr><tr><td>Leibovitz culture medium L-15</td><td>Sigma-Aldrich</td><td>L4386</td><td>May use any brand</td></tr><tr><td>MEM vitamins</td><td>Sigma-Aldrich</td><td>M6895</td><td></td></tr><tr><td>MEM amino acids</td><td>Sigma-Aldrich</td><td>M5550</td><td></td></tr><tr><td>Carbogen</td><td>Airgas</td><td>UN3156</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;</td></tr></tbody></table>

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

We recorded flash responses from dark-adapted wild-type (WT) C57BL/6 mouse retinas by following the experimental protocols described above and illustrated in Figure 1 by using different standard perfusion solutions (Figure 2). The response waveforms and kinetics as well as sensitivity of rod photoreceptors appeared similar in Ames&#8217; and Locke&#8217;s media (Figure 2A and B). On the other hand, under HEPES-buffered Ringer solution (no bicarbonate or 5% CO2 / 95% O2) the response amplitudes were significantly smaller. We also found that in these conditions b-wave stability was compromised. Adding 40 &#956;M APB (DL-AP4) removed positive b-wave efficiently in all three media (Figures 2D-F). Removal of the b-wave revealed a large slow negative wave (Figure 2D) that has been attributed to M&#252;ller cell activity21. Adding 100 &#956;M of barium abolished this component, revealing the photoreceptor response of the ex vivo ERG signal (Figures 2D-F). We could record up to 1 mV saturated photoreceptor responses in Ames&#8217; and Locke&#8217;s whereas maximum responses were typically around 200 &#956;V under Ringer perfusion.
Cone photoreceptor responses have been isolated previously by so-called double flash technique where a bright probe flash saturating the rods is followed by a test flash at a time when cones have restored their dark-adapted state but rods remain saturated19,29. Here we isolated cone-mediated ERG responses (containing both a- and b-wave) in Ames&#8217; media supplemented with 100 &#956;M of barium but not DL-AP4 by using double flash technique (Figure 3). Barium was used to remove the slow glial component that appeared to make the late part of the responses more variable especially during repetitive use of bright flashes. We used a constant probe flash to saturate rods and variable test flashes 300 msec after the probe flash to elicit cone responses. A cone response family obtained by subtracting probe flash response from the &#8216;probe + test flash&#8217; responses is shown in Figure 3B.
<img alt="Figure 1" src="/files/ftp_upload/52855/52855fig1.jpg" /> 
Figure 1. Use of ex vivo ERG specimen holder. (A) Filling of electrode channels and mounting of the electrodes. (B) Testing of the assembled specimen holder before dissection by measuring the resistance and voltage between the electrode pairs. (C) Mounting of the retina on the filter paper in the specimen holder. (D) The specimen holder connected to the perfusion lines and ERG amplifier in the commercial ERG system. The perfusion flow path is indicated by blue arrows. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52855/52855fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52855/52855fig2.jpg" /> 
Figure 2. Flash response families recorded from dark-adapted WT mouse retinas perfused with Locke&#8217;s (A), Ames&#8217; (B), and HEPES-buffered Ringer (C) medium. The flashes delivered 3, 40, 130, 390 and 1,400 photons &#956;m-2 (from -36 to -9 dB or -3.6 to -0.9 log(Cd sm-2) green light (530 nm)). Black traces in (D-F) show responses recorded from retinas in (A-C) after addition of 40 &#956;M APB and 100 &#956;M barium. Red traces in (D) show responses recorded from the retina in (A) perfused with Locke&#8217;s supplemented with 40 &#956;M APB but not barium. The inset shows the responses to the three dimmest flashes in Locke&#8217;s media containing APB and barium. The flashes ranged from 7 to 14,000 photons &#956;m-2 (from -32 to 1 dB green light) in (D), from 3 to 1,400 photons &#956;m-2 (from -36 to -9 dB green light) in (E), and from 7 to 1,400 photons &#956;m-2 (from -32 to -9 dB green light) in (F). See Vinberg et al. 201417 and Lyubarsky et al. 199930 and 200431 for details of converting photopic luminous energy given in Cd sm-2 to photons &#956;m-2. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52855/52855fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52855/52855fig3.jpg" /> 
Figure 3. Isolation of cone responses with double flash method in WT mouse. (A) A response to probe flash (14,000 photons &#956;m-2 or 1 dB green light, black) and a response to probe flash followed by a test flash (81,000 photons &#956;m-2 or 9 dB green light, red). (B) Cone flash responses to test flashes ranging from 360 to 81,000 photons &#956;m-2 (-14 to 10 dB green light) isolated by subtracting the probe flash response from the &#8220;probe + test flash&#8221; response. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52855/52855fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System at JoVE.com

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

simultaneous-ex-vivo-functional-testing-two-retinas-vivo

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.1K Views.  Washington University in St. Louis. Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Video: Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Multifocal Electroretinograms

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity. Generally, ERG results are normal unless more than approximately 20% of the retina is affected. In practical terms, a patient might be legally blind as a result of macular degeneration or other scotomas and still appear normal, according to traditional full field ERG. An important development in ERGs is the multifocal ERG (mfERG). Erich Sutter adapted the mathematical sequences called binary m-sequences enabling the isolation from a single electrical signal an electroretinogram representing less than each square millimeter of retina in response to a visual stimulus1.
Results that are generated by mfERG appear similar to those generated by flash ERG. In contrast to flash ERG, which best generates data appropriate for whole-eye disorders. The basic mfERG result is based on the calculated mathematical average of an approximation of the positive deflection component of traditional ERG response, known as the b-wave1. Multifocal ERG programs measure electrical activity from more than a hundred retinal areas per eye, in a few minutes. The enhanced spatial resolution enables scotomas and retinal dysfunction to be mapped and quantified.
In the protocol below, we describe the recording of mfERGs using a bipolar speculum contact lens.
Components of mfERG systems vary between manufacturers. For the presentation of visible stimulus, some suitable CRT monitors are available but most systems have adopted the use of flat-panel liquid crystal displays (LCD). The visual stimuli depicted here, were produced by a LCD microdisplay subtending 35 - 40 degrees horizontally and 30 - 35 degrees vertically of visual field, and calibrated to produce multifocal flash intensities of 2.7 cd s m-2. Amplification was 50K. Lower and upper bandpass limits were 10 and 300 Hz. The software packages used were VERIS versions 5 and 6.

The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity.  Generally, ERG results are ...

Medicine

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

Chemistry

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular cells. Isolating, culturing, and sustaining retinal neurons in vitro have technical and biological limitations. Culturing retinal explants may overcome these limitations and offer a unique ex vivo model to study the cross-talk between various retinal cells with well-controlled biochemical parameters and independent of the vascular system. Moreover, retinal explants are an effective screening tool for studying novel pharmacological interventions in various retinal vascular and neurodegenerative diseases such as diabetic retinopathy. Here, we describe a detailed protocol for retinal explants' isolation and culture for an extended period. The manuscript also presents some of the technical problems during this procedure that may affect the desired outcomes and reproducibility of the retinal explant culture. The immunostaining of the retinal vessels, glial cells, and neurons demonstrated intact retinal capillaries and neuroglial cells after 2 weeks from the beginning of the retinal explant culture. This establishes retinal explants as a reliable tool for studying changes in the retinal vasculature and neuroglial cells under conditions that mimic retinal diseases such as diabetic retinopathy.

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

This protocol presents and describes steps for the isolation, dissection, culturing, and staining of retinal explants obtained from an adult mouse. This method is beneficial as an ex vivo model for studying different retinal neurovascular diseases such as diabetic retinopathy.

One of the challenges in retina research is studying the cross-talk between different retinal cells such as retinal neurons, glial cells, and vascular ...

Two-Photon In Vivo Imaging of the Mouse Retina

<a href='https://app-jove-com.bdigitaluss.remotexs.co/v/61970/transpupillary-two-photon-in-vivo-imaging-of-the-mouse-retina'>Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).</a>This video demonstrates a technique for in vivo retinal imaging using two-photon microscopy. Anesthetized mice are prepared for imaging by securing their heads, Applying eye lubricant, and placing a coverslip over the eye. Retinal ganglion cells (RGCs) expressing fluorescent proteins are first visualized using epifluorescence for alignment. Finally, two-photon imaging is employed, allowing for high-resolution visualization of RGCs.

Source: Wang, Z., et al.&#160;Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina. J. Vis. Exp.&#160;(2021).This video demonstrates a technique ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

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Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Retinal Explant of the Adult Mouse Retina as an Ex Vivo Model for Studying Retinal Neurovascular Diseases

Two-Photon In Vivo Imaging of the Mouse Retina