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

Podsumowanie

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.

Streszczenie

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’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.

Wprowadzenie

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 choriocapillaris^4,5. Abnormalities in RPE function underlie numerous human retinal diseases, such as age-related macular degeneration⁶, Leber’s congenital amaurosis^7,8 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 diseases^10,11. 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 degeneration^15,16,17 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 technique^16,18,19,20 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.

Protokół

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 mice^18,21. 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.

1. Open the ERG system software.
2. Click on Database Center.
3. Click on New (provide a new database file name). Click on Save. The popup box will display: “Database created, do you want to connect to the new database file.” Click on Yes. The current database name should now reflect the new file name.
4. Click on Transfer In in the Database Control Center Window.
5. Select Supplementary File 1: LightProtocols - TRANSFER.EXP. Click on Open.
6. Click on Close (Database Control Center) upon completion of the progress bar.
7. Click on the green Start button.
8. 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.
9. Click on Protocols. The dark-adapted ERG and DC-ERG protocols should now be visible.
10. Lastly, place the Long Flash.col file into the following folder C:\ERG User Files\Long Flash.col.

2. Capillary electrode preparation

1. 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.
2. 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.

3. Filling capillary electrodes

1. Connect the vacuum desiccator to the laboratory’s vacuum line through an in-line filter (Table of Materials).
2. Pour 30 mL of Hanks’ 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.
3. Turn on the vacuum and degas the HBSS while turning on the computer and the recording equipment.
4. After 5‒10 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.
   1. 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.
   2. 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.
   3. 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’s eye and prevent corneal dehydration that would occur with the use of standard gold loop electrodes.
5. Position the electrode holders with the capillary electrodes tilted upwards to allow bubbles to flow out. Run the vacuum for 5‒10 min to degas. Gas escaping from the glass and plastic surfaces will push HBSS out from the electrode holders.
6. 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.
7. 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”-11/32” 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.
8. 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° rotation on a 180° axis.

4. Test electrodes

1. Pour HBSS into a small container (e.g., the cap of the 50 mL conical tube).
2. 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.
3. 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.
   1. Click on Protocols. Select DC-ERG. Click on Run. A dialog box will pop-up: “Current patient is XXX [DOB: XX/XX/XX) Is this correct for the test being performed?” Click on Yes. Then proceed to “Step 1/6.”
4. Close the doors to the faraday cage.
5. 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).
6. Test the baseline stability (noise and drift) by clicking on Step (forward arrow) to select Step 4/6 “Long Flash No Light.”
   NOTE: The amount of drift observed when the electrodes are placed in the HBSS bath is generally less than 500 µV 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 ~ 10‒15% greater in the mouse than in the HBSS bath. This is probably due to the addition of motion artifacts from breathing.
7. Begin viewing the traces by clicking on Preview. The traces should be low noise with a peak-to-peak amplitude <200 µV. A slight drift (<500 µV/80 s) that gradually fades to baseline is acceptable (Figure 1C).

5. Mouse and electrode positioning

1. Keep the mice overnight in a well-ventilated light-tight box for dark adaptation.
2. Anesthetize the animals by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg).
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. Prior to placing the capillary electrodes to the mouse’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.
10. 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.

6. DC-ERG recording

1. Click on Step (forward arrow) to select Step 4/6 “Long Flash No Li.”
2. 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 kΩ). The impedance values for both the ground and reference electrodes are expected to be less than 10 kΩ).
3. Click on Preview to view the traces for the left and right eye. Wait for a stable baseline to be achieved (<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.
4. Click on Step (forward arrow) to select Step 5/6 “Long Flash 10 cd 7 min."
5. Click Run to start the recording (Figure 1D).

7. Data export

1. Select the “Patient” (Family Name) describing the mouse recording to be exported.
2. Click on Old Tests.
3. Under Protocol Description select DC-ERG. Click on the green Load button to load the previously acquired data.
4. Click on Step (forward arrow) to advance to Step 5/6 “Long Flash 10 cd 7 min.”
5. Click on Export.
6. 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.
7. 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).
8. Then click on Export (Figure 1E). This saves the *.csv file to the C:\Multifocal folder.

8. Data analysis

1. Download and install the appropriate runtime installer (Table of Materials)
2. 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.
3. Click on the shortcut created in the Start Menu > Programs folder.
4. 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).

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Ag/AgCl (mouth) Electrode	WPI Inc	EP1	Mouth reference electrode for mouse
Ceramic Tile	Sutter Instrument	CTS	Used to cut the glass capillary tube to an appropriate size
Cotton Tipped Cleaning Stick	Puritan Medical Products	867-WC No Glue	To be used as a spacer to improve the fit of the electrode holder assembly
Electroretinogram (ERG) System	Diagnosys LLC	E3 System	Visual electrophysiology system to diagnose ophthalmic conditions in vision research and drug trials
Bunsen Burner	Argos Technologies	BW20002460	Or equivlaent to shape glass under flame
Glass Capillary Tube (1.5 mm)	Sutter Instruments	BF150-75	For filling with HBSS and making contact to the cornea
Hank’s Buffered Salt Solution (HBSS)	Thermo Fisher Scientific Inc	14175-095	Commercially available. Maintain at RT
In-Line Filter	Whatman	6722-5001	To protect vacuum pump from aerosols
Low Noise Cable for Microelectrode Holders	WPI Inc	5372	Suggested for improving the length and placement of the cables and electrode holder assemblies
Magnetic Ball Joint	WPI Inc	500871	For magnetically positioning the electrode holder assembly on the stage
MatLab	Mathworks		MatLab: For editing the analysis software
MatLab Curvefit Toolbox	Mathworks		Toolbox for MatLab (only required for editing the analysis software)
MatLab Compiler	Mathworks		Toolbox for MatLab (only required for editing and re-releasing the analysis software)
MatLab Runtime version 9.5	Mathworks	R2018b (9.5)	Required to run the analysis software: https://www.mathworks.com/products/compiler/matlab-runtime.html
Microelectrode Holders (45 degrees)	WPI Inc	MEH345-15	For holding the capillaries
Needle (25 ga)	Covidien	8881250313	For filling the capillary tubes with HBSS
Needle (ground) Electrode	Rhythmlink	13mm - one elctrode	Subdermal needle electrode (ground) for mouse (13mm long, 0.4mm diameter needle, 1.5m leadwire)
Regulator/Power Conditioner	Furman	P-1800	Or equivalent to remove DC-offset from noise introduced through power line
Syringe (12 mL)	Monoject	1181200777	For filling the capillary tubes with HBSS
T-clip	Cole-Parmer	06852-20	For electrode holder assembly
Vacuum Desiccator	Bel-Art	420120000	Clear polycarbonate bottom & cover
Pharmacological treatment
Lubricant eye gel	Alcon	0078-0429-47	Helps lubricate corneal surface and maintain electrical contact with capillary electrodes
Phenylephrine Hydrochloride 2.5%	Akorn	17478-201-15	Short acting mydriatic eye drops (for pupil dilation)
Proparacaine Hydrochloride 0.5%	Akorn	17478-263-12	Local anesthetic for ophthalmic instillation
Tropicamide 0.5%	Akorn	17478-101-12	Short acting mydriatic eye drops (for pupil dilation)
Xylazine	AnaSed	sc-362949Rx	Analgesic and muscle relaxant
Zetamine (Ketamine HCl)	VetOne	501072	Anesthetic for intramuscular injections