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/m2) for 7 min, and ending with a 1.5 min post-stimulus interval. The light intensity of 10 cd/m2 (1 log10 cd/m2) was selected since it evokes approximately half the maximal response for all the components of the DC-ERG in WT mice18,21. The c-wave and fast oscillation are of particular interest as the origins of these electrical responses are well characterized and can be isolated and studied further in vitro RPE models (e.g., iPSC-RPE). The application of other light intensities can extract additional information, for instance, the off response undergoes a reversal of polarity at brighter light stimuli and may show differences at the intensity at which this reversal takes place. The user is free to change the light intensity settings at their discretion.
<ol>
	<li>Open the ERG system software.</li>
	<li>Click on Database Center.</li>
	<li>Click on New (provide a new database file name). Click on Save. The popup box will display: &#8220;Database created, do you want to connect to the new database file.&#8221; Click on Yes. The current database name should now reflect the new file name.</li>
	<li>Click on Transfer In in the Database Control Center Window.</li>
	<li>Select Supplementary File 1: LightProtocols - TRANSFER.EXP. Click on Open.</li>
	<li>Click on Close (Database Control Center) upon completion of the progress bar.</li>
	<li>Click on the green Start button.</li>
	<li>Click on New on the Select Patient Window. Create a new patient family name describing the mouse model, enter the date of birth (DOB, mm/dd/yyyy), toggle the gender (M/F) button to the appropriate description. Click on Close to save the experimental details.</li>
	<li>Click on Protocols. The dark-adapted ERG and DC-ERG protocols should now be visible.</li>
	<li>Lastly, place the Long Flash.col file into the following folder C:\ERG User Files\Long Flash.col.</li>
</ol>
2. Capillary electrode preparation
<ol>
	<li>Cut the 1.5 mm glass capillaries in half by using a ceramic tile (Table of Materials) to score the glass and break them cleanly using a table to provide physical counterforce and to stabilize the glass. 
	NOTE: Blunt the cut ends as necessary.</li>
	<li>Using a Bunsen burner (Table of Materials) allow the heat to make a small bend in the capillary while holding it over the flame with forceps.</li>
</ol>
3. Filling capillary electrodes
<ol>
	<li>Connect the vacuum desiccator to the laboratory&#8217;s vacuum line through an in-line filter (Table of Materials).</li>
	<li>Pour 30 mL of Hanks&#8217; Balanced Salt Solution (HBSS) (Table of Materials) into an open 50 mL conical tube and place it (with the cap removed) into the vacuum chamber.</li>
	<li>Turn on the vacuum and degas the HBSS while turning on the computer and the recording equipment.</li>
	<li>After 5&#8210;10 min turn off the vacuum and use the degassed HBSS to fill a 12 mL syringe through an attached 25 G needle. Use it to fill the bases of the electrode holders taking extra precaution not to introduce bubbles.
	<ol>
		<li>To do this, remove the threaded screw cap and carefully slide the syringe needle through the silicone rubber gasket to reach the back wall (silver/silver chloride pellet) of the holder. 
		NOTE: The silver/silver chloride pellets are advantageous over holders that use silver wire as they provide more surface area resulting in a stable low-noise baseline. However, silver/silver chloride pellets require a liquid interface free from air bubbles to achieve a good connection. Therefore, take great care not to introduce air bubbles during this process.</li>
		<li>Gradually inject HBSS to fill the entire microelectrode while slowly retracting the syringe needle. Reattach the threaded cap but do not tighten. Reinsert the syringe needle to fill the empty space within the cap with HBSS. Then fill the glass capillary while holding it horizontal to prevent solution from escaping from the other end.</li>
		<li>Hold the glass capillary from the bent end and slowly insert the opposite end through the loosened cap and then tighten the screw cap into place. 
		NOTE: Glass electrodes filled with HBSS maintain lubrication of the mouse&#8217;s eye and prevent corneal dehydration that would occur with the use of standard gold loop electrodes.</li>
	</ol>
	</li>
	<li>Position the electrode holders with the capillary electrodes tilted upwards to allow bubbles to flow out. Run the vacuum for 5&#8210;10 min to degas. Gas escaping from the glass and plastic surfaces will push HBSS out from the electrode holders.</li>
	<li>Stop and then slowly release the vacuum. Refill the electrode holders and glass capillaries as described previously. 
	NOTE: Bubbles tend to collect on or near the silicone rubber gasket and can also hide in the grooves of the threaded cap, therefore special attention must be paid to keep these areas bubble-free.</li>
	<li>Install and secure the microelectrode holder into the custom-made T-clip/Magnetic ball joint stand (Figure 1A, inset). To make the custom stand for the microelectrode holder, modify a T-clip (5/16&#8221;-11/32&#8221; OD Tubing) #8 (Table of Materials) by removing the black polyacetal clips on one side. Have the cylinder base of the magnetic ball joints machined in half to adjust the height (Table of Materials). Secure the modified T-clips to the magnetic ball mounting screws with M3 sized nuts.</li>
	<li>Place the microelectrode holder into the modified T-clip and hold it tightly in place by sliding in approximately a 1-inch tapered wooden handle made from breaking a cotton tipped cleaning stick (Table of Materials) at an angle. Use the rare earth magnet cylinder base to securely position the customized electrode holder stand onto the metal plate of the stage enabling 360&#176; rotation on a 180&#176; axis.</li>
</ol>
4. Test electrodes
<ol>
	<li>Pour HBSS into a small container (e.g., the cap of the 50 mL conical tube).</li>
	<li>Gently lower the fully assembled, HBSS-filled capillary microelectrodes into the cap containing HBSS to pre-equilibrate the electrodes and place the needle ground electrode (tail/rear leg) and the Ag/AgCl sintered pellet reference electrode (mouth) in the same HBSS to complete the circuit (Figure 1A). 
	NOTE: Perform all subsequent steps under dim red light. Use a red-light flashlight to position the mouse and capillary electrodes. Remember to completely turn off all light sources prior to beginning the recording.</li>
	<li>Select or create an appropriate identifier (family name) to describe the mouse to be tested and select the DC-ERG protocol to be performed by completing the registration according to the following order.
	<ol>
		<li>Click on Protocols. Select DC-ERG. Click on Run. A dialog box will pop-up: &#8220;Current patient is XXX [DOB: XX/XX/XX) Is this correct for the test being performed?&#8221; Click on Yes. Then proceed to &#8220;Step 1/6.&#8221;</li>
	</ol>
	</li>
	<li>Close the doors to the faraday cage.</li>
	<li>Display impedance mode by clicking on Impedance and verify that the values for the mouth reference, tail ground, and recording electrodes are acceptable (see Figure 1B).</li>
	<li>Test the baseline stability (noise and drift) by clicking on Step (forward arrow) to select Step 4/6&#160;&#8220;Long Flash No Light.&#8221; 
	NOTE: The amount of drift observed when the electrodes are placed in the HBSS bath is generally less than 500 &#181;V per 80 s once they have stabilized and is equivalent to the drift observed when the electrodes are connected to the mouse. Thus, the electrical readout of the electrodes in the HBSS bath are an important indicator of the status of the electrodes. The noise, measured as peak-to-peak, is generally ~ 10&#8210;15% greater in the mouse than in the HBSS bath. This is probably due to the addition of motion artifacts from breathing.</li>
	<li>Begin viewing the traces by clicking on Preview. The traces should be low noise with a peak-to-peak amplitude &#60;200 &#181;V. A slight drift (&#60;500 &#181;V/80 s) that gradually fades to baseline is acceptable (Figure 1C).</li>
</ol>
5. Mouse and electrode positioning
<ol>
	<li>Keep the mice overnight in a well-ventilated light-tight box for dark adaptation.</li>
	<li>Anesthetize the animals by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg).</li>
	<li>Apply a drop of 0.5% proparacaine HCl topically to anaesthetize the cornea as well as a drop of 2.5% phenylephrine HCl and 0.5% tropicamide to dilate the pupils.</li>
	<li>Trim the mouse whiskers with scissors to prevent inadvertent twitching from disturbing the glass capillary electrodes during the recording. 
	NOTE: The DC-ERG stimulus protocol within the ERG system has several built-in stimulus routines that can be selected by clicking on the Step (forward arrow) or Step (backward arrow). Only Steps 1, 4, and 5 in the software are required to prepare and perform the DC-ERG recording.</li>
	<li>In the ERG system software verify that the correct patient is selected. Click on the green Protocols button. Under Protocol Description select DC-ERG. Then click on Run. Verify that this is the correct test being performed by clicking on Yes.</li>
	<li>Use Step 1/6 designated Red Light stimulus to turn on the red light inside the dome to help position the mouse and electrodes while observing the changes in impedance.</li>
	<li>Place the mouse on a heated recording table and carefully tent the skin of the rear leg using forceps. Hold the needle electrode firmly in one hand and insert it subcutaneously into the rear leg to secure it into place.</li>
	<li>Place the reference Ag/AgCl electrode (Table of Materials) inside the mouth so that the sintered pellet rests along the back cheek and is held in place behind the teeth. 
	NOTE: Gold wire electrodes should not be used as the mouth reference electrode as they have different impedance characteristics and increase the peak-to-peak noise.</li>
	<li>Prior to placing the capillary electrodes to the mouse&#8217;s eye, hold the electrode holder with the glass capillaries vertical, flick the electrode holder with the index finger to remove any bubbles that may have been introduced. Fill the tip with HBSS using a 25 G needle attached to a syringe and inspect to ensure that there is no air bubble trapped at the tip. Position the electrode holder stand so that the open tip of the HBSS-filled capillaries are in gentle contact with the cornea.</li>
	<li>Use special precaution to avoid introducing air bubbles by holding the lubricant eye gel dispenser inverted and discard the initial drops. Place a drop of lubricant eye gel on each eye to maintain conductivity and prevent desiccation during the recording.</li>
</ol>
6. DC-ERG recording
<ol>
	<li>Click on Step (forward arrow) to select Step 4/6 &#8220;Long Flash No Li.&#8221;</li>
	<li>Click on Impedance. 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 are expected to be similar (~39 k&#8486;). The impedance values for both the ground and reference electrodes are expected to be less than 10 k&#8486;).</li>
	<li>Click on Preview to view the traces for the left and right eye. Wait for a stable baseline to be achieved (&#60;10 min). Click on Stop to exit the trace preview. 
	NOTE: Abrupt changes in drift direction or aberrant noise in the recording will not improve with time and will require identifying the capillary electrode that requires attention. The most likely cause is a bubble introduced to the tip of the electrode. Refill the tip with HBSS. Click on Preview again to check the baseline.</li>
	<li>Click on Step (forward arrow) to select Step 5/6 &#8220;Long Flash 10 cd 7 min.&#34;</li>
	<li>Click Run to start the recording (Figure 1D).</li>
</ol>
7. Data export
<ol>
	<li>Select the &#8220;Patient&#8221; (Family Name) describing the mouse recording to be exported.</li>
	<li>Click on Old Tests.</li>
	<li>Under Protocol Description select DC-ERG. Click on the green Load button to load the previously acquired data.</li>
	<li>Click on Step (forward arrow) to advance to Step 5/6 &#8220;Long Flash 10 cd 7 min.&#8221;</li>
	<li>Click on Export.</li>
	<li>Provide the filename (e.g., filename.csv). A valid filename must begin with a letter, followed by letters, numbers, or underscores. Do not use special characters or hyphens. The data analysis program (DCERG_Analysis.exe) requires that table entries meet the requirements for variable names.</li>
	<li>Place a checkmark next to Data Table. Next to separator, select Tab. Place checkmarks next to Options (Titles, Vertical), Include All (Steps, Chans, Results), Data Columns (Contents, Results, Sweeps), and Format (File).</li>
	<li>Then click on Export (Figure 1E). This saves the *.csv file to the C:\Multifocal folder.</li>
</ol>
8. Data analysis
<ol>
	<li>Download and install the appropriate runtime installer (Table of Materials)</li>
	<li>Download and install the DCERG_Analysis.exe installer. 
	NOTE: This installs the script that will perform the analysis of the DC-ERG components and creates a shortcut to run the program in the Start Menu folder.</li>
	<li>Click on the shortcut created in the Start Menu &#62; Programs folder.</li>
	<li>Select the exported data file or files (*.csv) for analysis. Use Ctrl + left click on the mouse to select more than one file. 
	NOTE: The executable file generates two types of plots: 1) the raw data is plotted with a best fit line indicating the measured drift; 2) the drift corrected response is plotted after being smoothed with a moving average (with a span of ~5 s). From this plot the amplitudes and time-to-peak of the DC-ERG components are identified: c-wave, fast oscillation, light peak, and off response. The data is then exported in table format to excel where each sheet corresponds to a different mouse recording. These sheets are followed by two summary sheets: (i) compiled DC-ERG amplitudes (mV); (ii) compiled DC-ERG time-to-peaks (light onset, t = 0 min).</li>
</ol>

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The authors have nothing to disclose.

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 ...

direct-coupled-electroretinogram-dc-erg-for-recording-light-evoked

Research

JoVE Journal

Chemistry

5.1K Views.  National Institutes of Health. 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.

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

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living ...