Direct-Coupled Electroretinogram Recording of the Retinal Pigment Epithelium in a Mouse

Protocole

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Mouse and electrode positioning

1. Keep the mice overnight in a well-ventilated, light-tight box for dark adaptation.
2. The animals were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg).
3. Apply a drop of 0.5% proparacaine HCl topically to anesthetize 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 Direct-Coupled Electroretinogram (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, the software verifies that the correct patient has been 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 Silver/Siverchloride(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 vertically and flick the electrode holder with the index finger to remove any bubbles that may have been introduced. Fill the tip with Hanks' Balanced Salt Solution (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 precautions to avoid introducing air bubbles by holding the lubricant eye gel dispenser inverted and discarding the initial drops. Place a drop of lubricant eye gel on each eye to maintain conductivity and prevent desiccation during the recording.

2. 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 the ground and the 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 needs 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." Click Run to start the recording.

matériels

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 lubricates 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