This work was supported by NEI intramural funds. The authors sincerely acknowledge Dr. Sheldon Miller for his scientific guidance, technical advice, and expertise in RPE physiology and disease. The authors thank Megan Kopera and the animal care staff for managing the mouse colonies, and Dr. Tarun Bansal, Raymond Zhou, and Yuan Wang for technical support.

This protocol follows the animal care guidelines outlined in the animal study protocol approved by the Animal Care and Use Committee of the National Eye Institute.
1. Importing light stimulation protocols for DC-ERG
NOTE: Follow the directions below to import the light stimulation protocols for the DC-ERG into the ERG system software (Table of Materials). The protocol consists of a 0.5 min pre-stimulus interval, followed by a step of light (10 cd/m<su...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+retinal+pigment+epithelium+dysfunction+is+concomitant+with+hyperglycemia+in+mouse+models+of+type+1+and+type+2+diabetes.">Early retinal pigment epithelium dysfunction is concomitant with hyperglycemia in mouse models of type 1 and type 2 diabetes.</a> Journal of Neurophysiology. 113 (4), 1085-1099 (2015).</li><li>Marmorstein, L. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light+peak+of+the+electroretinogram+is+dependent+on+voltage-gated+calcium+channels+and+antagonized+by+bestrophin+(best-1).">The light peak of the electroretinogram is dependent on voltage-gated calcium channels and antagonized by bestrophin (best-1).</a> Journal of General Physiology. 127 (5), 577-589 (2006).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invest.+Ophtalmol.+Vis.+Sci.">Invest. Ophtalmol. Vis. Sci.</a> Annual Meeting for the Association for Research in Vision and Ophthalmology. , 3568 (2017).</li><li>Iacovelli, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Cre+transgenic+mice+with+postnatal+RPE-specific+ocular+expression.">Generation of Cre transgenic mice with postnatal RPE-specific ocular expression.</a> Investigative Ophthalmology and Visual Science. 52 (3), 1378-1383 (2011).</li><li>Wang, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204/211+alters+epithelial+physiology.">MicroRNA-204/211 alters epithelial physiology.</a> FASEB Journal. 24 (5), 1552-1571 (2010).</li><li>He, L., Marioutina, M., Dunaief, J. L., Marneros, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+gene-dosage-dependent+cre-induced+abnormalities+in+the+retinal+pigment+epithelium.">Age- and gene-dosage-dependent cre-induced abnormalities in the retinal pigment epithelium.</a> American Journal of Pathology. 184 (6), 1660-1667 (2014).</li><li>Gallemore, R. P., Steinberg, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-evoked+modulation+of+basolateral+membrane+Cl-+conductance+in+chick+retinal+pigment+epithelium:+the+light+peak+and+fast+oscillation.">Light-evoked modulation of basolateral membrane Cl- conductance in chick retinal pigment epithelium: the light peak and fast oscillation.</a> Journal of Neurophysiology. 70 (4), 1669-1680 (1993).</li><li>Blaug, S., Quinn, R., Quong, J., Jalickee, S., Miller, S. 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Critical Steps
A good DC-ERG recording requires stable electrodes that are free from bubbles that create artifacts and unwanted drift as they are extremely sensitive to outgassing and temperature changes. It is essential that a stable baseline is achieved when the electrodes are placed in the HBSS bath solution before proceeding forward with the mouse recording. Small bubbles tend to collect at the base of the capillary electrode or around the silicone gasket and are difficult...

The retinal pigment epithelium (RPE) is a monolayer of specialized cells that line the posterior segment of the eye and exert critical functions to maintain retinal homeostasis1. The RPE supports photoreceptors by regenerating their photon-capturing visual pigment in a process called the visual cycle2, by participating in the diurnal phagocytosis of shed outer segment tips3, and in the transport of nutrients and metabolic products between photoreceptors and the choriocapillaris4,5. Abnormalities in RPE function underlie numerous human retinal diseases, such as age-related macular degeneration6, Leber&#8217;s congenital amaurosis7,8 and Best vitelliform macular dystrophy9. As donor eye tissues are often difficult to obtain solely for research purposes, animal models with genetic modifications can provide an alternative way to study the development of retinal diseases10,11. Additionally, the emergence and application of CRISPR cas9 technology now permits genomic introductions (knock-in) or deletions (knock-out) in a simple, one-step process surpassing limitations of prior gene targeting technologies12. The boom in the availability of new mouse models13 necessitates a more efficient recording protocol to non-invasively evaluate RPE function.
Measurement of the light-evoked electrical responses of the RPE can be achieved using a direct-coupled electroretinogram (DC-ERG) technique. When used in combination with conventional ERG recordings that measure the photoreceptor (a-wave) and bipolar (b-wave) cell responses14, the DC-ERG can define how the response properties of the RPE change with retinal degeneration15,16,17 or whether RPE dysfunction precedes photoreceptor loss. This protocol describes a method adapted from the work of Marmorstein, Peachey, and colleagues who first developed the DC-ERG technique16,18,19,20 and improves upon the reproducibility and ease of use.
The DC-ERG recording is difficult to perform because of the long acquisition time (9 min) during which any interruption or introduction of noise can complicate the interpretation of the data. The advantage of this new method is that the baselines reach steady state within a shorter amount of time reducing the likelihood that the animal will awaken prematurely from anesthesia and is less prone to bubble formation in the capillary electrodes.

<table>
	<tbody>
		<tr>
			<td>Ag/AgCl (mouth) Electrode</td>
			<td>WPI Inc</td>
			<td>EP1</td>
			<td>Mouth reference electrode for mouse</td>
		</tr>
		<tr>
			<td>Ceramic Tile</td>
			<td>Sutter Instrument</td>
			<td>CTS</td>
			<td>Used to cut the glass capillary tube to an appropriate size</td>
		</tr>
		<tr>
			<td>Cotton Tipped Cleaning Stick</td>
			<td>Puritan Medical Products</td>
			<td>867-WC No Glue</td>
			<td>To be used as a spacer to improve the fit of the electrode holder assembly</td>
		</tr>
		<tr>
			<td>Electroretinogram (ERG) System</td>
			<td>Diagnosys LLC</td>
			<td>E3 System</td>
			<td>Visual electrophysiology system to diagnose ophthalmic conditions in vision research and drug trials</td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Argos Technologies</td>
			<td>BW20002460</td>
			<td>Or equivlaent to shape glass under flame</td>
		</tr>
		<tr>
			<td>Glass Capillary Tube (1.5 mm)</td>
			<td>Sutter Instruments</td>
			<td>BF150-75</td>
			<td>For filling with HBSS and making contact to the cornea</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Buffered Salt Solution (HBSS)</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>14175-095</td>
			<td>Commercially available. Maintain at RT</td>
		</tr>
		<tr>
			<td>In-Line Filter</td>
			<td>Whatman</td>
			<td>6722-5001</td>
			<td>To protect vacuum pump from aerosols</td>
		</tr>
		<tr>
			<td>Low Noise Cable for Microelectrode Holders</td>
			<td>WPI Inc</td>
			<td>5372</td>
			<td>Suggested for improving the length and placement of the cables and electrode holder assemblies</td>
		</tr>
		<tr>
			<td>Magnetic Ball Joint</td>
			<td>WPI Inc</td>
			<td>500871</td>
			<td>For magnetically positioning the electrode holder assembly on the stage</td>
		</tr>
		<tr>
			<td>MatLab</td>
			<td>Mathworks</td>
			<td></td>
			<td>MatLab: For editing the analysis software</td>
		</tr>
		<tr>
			<td>Matlab Curvefit Toolbox</td>
			<td>Mathworks</td>
			<td></td>
			<td>Toolbox for MatLab (only required for editing the analysis software)</td>
		</tr>
		<tr>
			<td>MatLab Compiler</td>
			<td>Mathworks</td>
			<td></td>
			<td>Toolbox for MatLab (only required for editing and re-releasing the analysis software)</td>
		</tr>
		<tr>
			<td>MatLab Runtime version 9.5</td>
			<td>Mathworks</td>
			<td>R2018b (9.5)</td>
			<td>Required to run the analysis software: https://www.mathworks.com/products/compiler/matlab-runtime.html</td>
		</tr>
		<tr>
			<td>Microelectrode Holders (45 degrees)</td>
			<td>WPI Inc</td>
			<td>MEH345-15</td>
			<td>For holding the capillaries</td>
		</tr>
		<tr>
			<td>Needle (25 ga)</td>
			<td>Covidien</td>
			<td>8881250313</td>
			<td>For filling the capillary tubes with HBSS</td>
		</tr>
		<tr>
			<td>needle (ground) electrode</td>
			<td>Rhythmlink</td>
			<td>13mm - one elctrode</td>
			<td>Subdermal needle electrode (ground) for mouse (13mm long, 0.4mm diameter needle, 1.5m leadwire)</td>
		</tr>
		<tr>
			<td>Regulator/Power Conditioner</td>
			<td>Furman</td>
			<td>P-1800</td>
			<td>Or equivalent to remove DC-offset from noise introduced through power line</td>
		</tr>
		<tr>
			<td>Syringe (12 mL)</td>
			<td>Monoject</td>
			<td>1181200777</td>
			<td>For filling the capillary tubes with HBSS</td>
		</tr>
		<tr>
			<td>T-clip</td>
			<td>Cole-Parmer</td>
			<td>06852-20</td>
			<td>For electrode holder assembly</td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>Bel-Art</td>
			<td>420120000</td>
			<td>Clear polycarbonate bottom &#38; cover</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pharmacological treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricant eye gel</td>
			<td>Alcon</td>
			<td>0078-0429-47</td>
			<td>Helps lubricates corneal surface and maintain electrical contact with capillary electrodes</td>
		</tr>
		<tr>
			<td>Phenylephrine Hydrochloride 2.5%</td>
			<td>Akorn</td>
			<td>17478-201-15</td>
			<td>Short acting mydriatic eye drops (for pupil dilation)</td>
		</tr>
		<tr>
			<td>Proparacaine Hydrochloride 0.5%</td>
			<td>Akorn</td>
			<td>17478-263-12</td>
			<td>Local anesthetic for ophthalmic instillation</td>
		</tr>
		<tr>
			<td>Tropicamide 0.5%</td>
			<td>Akorn</td>
			<td>17478-101-12</td>
			<td>Short acting mydriatic eye drops (for pupil dilation)</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>AnaSed</td>
			<td>sc-362949Rx</td>
			<td>Analgesic and muscle relaxant</td>
		</tr>
		<tr>
			<td>Zetamine (Ketamine HCl)</td>
			<td>VetOne</td>
			<td>501072</td>
			<td>Anesthetic for intramuscular injections</td>
		</tr>
	</tbody>
</table>

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.

Figure 2 is a sample dataset from miR-204 ko/ko cre/+ (conditional KO) and wild type (WT) mice. MiR-204 ko/ko cre/+ are mice with a conditional knockout of microRNA 204 in the retinal pigment epithelium. These mice are generated by crossing floxed miR-204 mice (produced by NEIGEF)22 with VMD2-CRE mice23. MiR-204 is highly expressed in the RPE where it regulates the expression of proteins critical for epithelial function that maintain tight junc...

Watch this Scientific Journal Video about Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium at JoVE.com

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.

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 DC-ERG technique can be used for the non-evasive evaluation of retinal pigment epithelium function to monitor age-related changes or disease progression and to assess pharmacological intervention effects. This technique improves upon DC-ERG reproduce ability and the easy of use by simplifying the capillary electrode preparation. It can also be supplemented with a standard ohm software application to facilitate analysis.Begin by carefully sliding a 25-gauge syringe needle through the silicone rubber gasket to the back wall of the electrode holder. Fill the base of the electrode holder with degassed HBSS while slowly retracting the needle, taking care not to introduce bubbles, and replace the threaded cap without tightening. Reinsert the syringe needle to fill the empty space within the gap with HBSS and hold the capillary horizontally while filling to prevent solution escaping from the other end.Grasping the filled capillary at the bent end, slowly insert the other end through the loosened cap and tighten the screw cap into place. Tilt the electrode holders with the capillary electrodes facing up to allow any bubbles to flow out and degas the capillaries in a vacuum chamber for five to 10 minutes. Then, slowly release the vacuum and refill the electrode holders and glass capillaries, as demonstrated.To make a custom stand for the microelectrode holder, remove the black polyacetal clips from one side of a number eight T-clip and use a cylinder base with magnetic ball joints machined in half to adjust the height. Secure the modified T-clips to the magnetic ball mounting screws with M3-size nuts and a slide in approximately one-inch tapered wooden handle, modified from a cotton tip cleaning stick, into the stand at an angle to secure the microelectrode holder into the custom-made T-clip magnetic ball joint stand. Then, use the rare-earth magnets cylinder base to securely position the customized electrode holder stand onto the metal plate of the stage, enabling a 360-degree rotation on a 180-degree axis.To perform an electrode test, first, gently lower the fully assembled HBSS-filled capillary microelectrodes into a small container of HBSS. Place the needle ground electrode and the silver, silver chloride centered pellet reference electrode in the same HBSS to complete the circuit and select or create an appropriate identifier to describe the mouse to be tested. To select the direct coupled electroretinogram protocol to be performed, click protocols and select DC-ERG.Click run. A dialog box will pop up with the patient information. If the information is correct, click yes and proceed to steps one of six.Close the doors to the Faraday cage and click impedance to display the impedance mode. Verify that the values for the mouth reference, tail ground and recording electrodes are acceptable and click step to proceed to step four of six to test the baseline stability. To begin viewing the traces, click preview.The traces should be low noise with a peak-to-peak amplitude of less than 200 microvolts. To position the mouse and electrodes for an experiment, anesthetize the cornea of a sedated mouse with a drop of 5%proparacaine hydrochloric acid before dilating the cornea with a drop of 2.5%phenylephrine HCL and 5%tropicamide. In the ERG system software, verify that the correct patient is selected.Click protocols. Under protocol description, select DC-ERG and click run. Click yes to verify that the correct test is being performed.Use step one of six to turn on a dim red light inside the dome and place the mouse on a heated recording table. Use forceps to carefully tent the skin of the rear leg. And using one hand to firmly hold the needle electrode, use the other hand to insert the electrode subcutaneously into the rear leg to secure the electrode into place.Place the reference silver, silver chloride electrode into the animal's mouth so that the centered pellet rests along the back cheek and is held in place behind the teeth. Before placing the capillary electrodes onto the eye, position the electrode holder with the glass capillaries oriented vertically and flick the electrode holder with an index finger to remove any bubbles that may have been introduced. Use a 25-gauge needle to fill the capillary tips with HBSS and inspect the capillaries to ensure that there are no air bubbles trapped in the tips.Position the electrode holder stand so that the open tips of the HBSS-filled capillaries are in gentle contact with the cornea. And taking care to avoid introducing bubbles, invert the lubricant eye gel dispenser to discard the initial drops. Then, place a drop of lubricant eye gel onto each eye to maintain conductivity and to prevent desiccation during the recording.To record the DC-ERGs, click step to select step five of six and click impedance to use the impedance checking screen to examine the resistances of the left and right eyes. The impedance values for the recording electrodes at each eye should be similar. And the impedance values for both the ground and reference electrodes should be less than 10 kilohms.Click preview to view the traces for the left and right eyes and wait up to 10 minutes for a stable baseline to be achieved. Then, click stop to exit the trace preview and click run to start the recording. Here, a sample dataset from conditional knockout and wild-type mice is shown.In this analysis, the trace suffered from minute bubbles in the electrode. It increased the peak-to-peak noise in the trace. In a separate analysis, the bubbles were eliminated using the vacuum chamber prior to assembling the microelectrodes within the electrode holder stands.The best fit lines to the initial 25 seconds can be calculated, allowing the drift-corrected responses to be replotted and the amplitudes of the DC-ERG components to be identified. As expected, reduced expression of cure 7.1 potassium channels in the RPE greatly attenuates the c-wave and fast oscillation, indicating a significant impairment of the retinal pigment epithelium electrical properties. Here, the relative amplitudes of the DC-ERG components, normalized to wild-type and plotted against the relative two largest light-evoked a-wave amplitudes, are shown.This form of analysis can determine whether the reduced electrical responses of the RPE can be accounted for by the reduction of photoreceptor activity alone. Or if there is an underlying defect that originates in the RPE. Remember to achieve a stable baseline prior to moving on to the mouse recording and to reinspect the electrodes periodically for bubbles that may have been introduced.Dark-adapted ERGs can be recorded prior to the DC-ERG to measure the cone-driven retinal function. Light-adapted ERGs can also be performed after the DC-ERG to evaluate the cone-driven retinal responses.

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Research

JoVE Journal

Chemistry

5.0K visualizações.  National Institutes of Health. A técnica DC-ERG pode ser utilizada para a avaliação não evasiva da função de epitélio do pigmento da retina para monitorar alterações relacionadas à idade ou progressão da doença e para avaliar os efeitos da intervenção farmacológica. Esta técnica melhora a capacidade de reprodução dc-ERG e a facilidade de uso simplificando a preparação do eletrodo capilar. Ele também pode ser complementado com um aplicativo de software ohm padrão para facilitar a análise.Comece...