ERG
Overview:
Electroretinography (ERG) is the study of electrical potentials generated within the retina in response to light stimuli.
The acquisition of ERG signals is made using a bipolar electrode placed on or near the cornea – a technique that has fundamentally remained unchanged since Holmgren’s discovery of ERG potentials in 1865.
Used in clinical research, ERG provides information about normal and abnormal photoreceptor function - while its use in basic research has aided the understanding of retinal light processing mechanisms in health and disease.
Examples of ERG applications in basic research include:
- Pharmacological /toxicological studies on retinal function
- a, b, c wave, oscillations – biophysical mechanisms
- Scotopic thresholds & rod properties in different animals
- Spatial tuning
- Degenerative retinal models (genetic/drug-induced)
Method:
Recording and test procedure
There are five basic standards used clinically and in basic research to obtain ERG responses:
- Dark adaptation and weak flash – designed primarily to test rod photoreceptor function
- Dark adaptation and strong flash – designed to test both rod and cone photoreceptor function (‘combined ERG’)
- Oscillatory potentials – under dark adaptation and single flash light, the presence of high frequency oscillations on the leading edge of the b-wave (see below) is a useful marker of retinal health (i.e., absent in diabetic retinopathy).
- Light adaptation and strong flash – designed to primarily test cone photoreceptors
- Flicker tests – under light adaptation a 30-60Hz flicker test will reveal cone photoreceptor adaptation and intrinsic retinal gain properties
Light stimulus
Full-field (Ganzfeld) stimulation is often used in order to produce a uniform ERG response. Focal flashes may also be used (e.g., camera flash). However, due to light reflectance and scattering the degree of retinal illumination from focal flashes may not scale linearly with the intensity of the light flash.
Recording electrodes
Recordings are made using a bipolar electrode, with one electrode placed on or close to the cornea of the eye and another near the orbital rim or forehead (Note: increased risk of signal contamination from cortical evoked potentials). Several types of corneal electrodes (NOT supplied by ADInstruments) are available for this application, including:
- Contact lens with a steel or silver wire embedded in the inner surface
- A piece of gold leaf tucked underneath the lower eye lid
- A skin electrode placed on the external surface of the lower eye lid (produces less satisfactory results)
- Silver-silver chloride wire electrode placed in the center of the cornea (i.e., animal studies)
Data analysis
A number of features will be present in the ERG waveform depending on the stimulation protocol used. For example, a normal response when using the combined ERG test will reveal at least two prominent waveform features:
a-wave: biphasic negative-positive response <10 ms following light stimulus representing cone photoreceptor hyperpolarization (depolarization in invertebrates), and rod depolarization. The early a-wave negative peak amplitude and onset time is usually measured with respect to the stimulus onset time. A later a-wave component exists that is mediated by rod depolarization which merges with the onset of the b-wave and is therefore difficult to precisely identify.
b-wave: usually the largest component of the ERG. Positive-going deflection in the ERG with ~50 ms onset following light stimulus. Several different cell types contribute to the signal, including: Mueller (glial) cells, bipolar cells, and retinal ganglion cells. The b-wave amplitude varies with stimulus intensity such that if too low then the b-wave may disappear altogether leaving only the negative deflection of the a-wave. The b-wave amplitude is usually taken from a-wave trough to b-wave peak; and b-wave time to peak is taken from time of light stimulus.
Other ERG potentials may also be present, such as oscillations on the leading edge of the b-wave which may be measured in terms of their frequency; an afterpotential following the offset of the b-wave, and the c-wave - a biphasic cornea-positive then negative potential originating in the pigment epithelium, glial cells and other cell types, e.g., horizontal and off-bipolar cells.
ERG flicker responses may be quantified in terms of their amplitude, rate, and phase in relation to stimulus frequency.
Software:
The LabChart Advantage:
(may require additional Modules and Extensions)
- Measure a- and b-wave amplitudes and onset times using Peak Analysis
- Measure rate of oscillatory potentials and ERG Flicker using Cyclic Measurements
- Annotate Chart recordings using the Comments feature
- Macros can automate many tedious and repetitive analysis tasks
- Automated extraction of data from recordings using online Timed Add to Data Pad or offline using Multiple Add to Data Pad
- Extracted parameters in the Data Pad or ECG Table View can be easily exported for further analysis, e.g in Excel or Matlab
LabChart
LabChart software (for Windows and Macintosh) together with a PowerLab data acquisition system offers up to 32 channels of real-time data acquisition, data integrity, easy selection of hardware settings, powerful online and offline analysis, procedure automation, seamless extraction of experimental data and flexible display options. Additional acquisition and analysis functionality is provided with the use of specialized LabChart Extensions and LabChart Modules. Modules are available as part of LabChart Pro while Extensions are free for download from the website for existing LabChart users.
Scope View
Scope View, supplied with PowerLab systems, provides powerful display, recording and analysis features to transform your computer into a two-channel storage oscilloscope, XY plotter or Power Spectrum (FFT) analyser. Scope is used commonly to measure any high-frequency signal that is time-locked to a stimulus such as action potentials and evoked responses. For analyzing Evoked Potentials, the Scope Software is recommended because it provides signal averaging functions that are necessary to extract the evoked response from background noise.
Features and benefits:
- Provides the ability to record, display and analyze any high frequency signal that is time-locked to a stimulus
- Synchronizes sweeps with recorded or built-in stimulation patterns
- Provides a range of real-time and offline analysis features
- Generate stimuli of differing intensities and waveform structures (i.e. single-pulse, multiple pulse, simple ramps) and control an external stimulator using the analog output on the front of the PowerLab.
Scope View is useful in signal averaging techniques to improve the signal-to-noise ratio and minimize artifacts induced by related events such as the blink-response. Using features in Scope such as filtering and spectral analysis, may be used to identify oscillating potentials. The analog output of the PowerLab data acquisition system may be used to control a stroboscopic flash unit (with suitable inputs) for presentation of the light stimuli.
Peak Analysis Modules
The Peak Analysis Module (Windows) provides automatic detection and analysis of multiple, but not overlapping, signal peaks in acquired waveforms. ERG waveform components such as a-wave and b-wave amplitude and onset times can be easily measured using customizable presets for peak detection.
- Measure a-wave amplitude and onset time
- Measure b-wave amplitude and onset time
- Display parameters in Table View
- Extract parameters in .txt or .csv formats for analysis in third part software (e.g., Excel, Prism)
Cyclic Measurements
Oscillatory potentials or ERG flicker rates may be measured using the Cyclic Measurements feature in LabChart.
- Identify oscillation/flicker peaks using customizable preset peak detection
- Display event frequency
- Export rate information to the DataPad for further analysis
GLP and 21 CFR Part 11
For those researchers working within a laboratory requiring GLP and 21 CFR Part 11 compliance the GLP Client and GLP Server are available for use with LabChart (Windows only) and PowerLab data acquisition systems. For more information, visit the Good Laboratory Practice application page or contact your nearest ADInstruments representative.
Hardware:
PowerLab Data Acquisition Systems
The PowerLab is a high-performance data acquisition unit capable of recording at speeds of up to 400,000 samples per second continuously to disk (aggregate). PowerLab units are compatible with instruments, signal conditioners and transducers supplied by ADInstruments, as well as many other third-party companies. In addition to standard single-ended BNC inputs, 4 differential Pod ports are also available for direct connection of Pod signal conditioners and appropriate transducers. Research PowerLab units include:
- PL3504 PowerLab 4/35 - 4 Channels
- PL3508 PowerLab 8/35 – 8 Channels
- PL3516 PowerLab 16/35 – 16 Channels
Signal Conditioners
Biopotential Amplifiers
As with many biopotentials, the electroretinogram is very small in amplitude (approx. 200 µV to 1 mV) and requires an amplifier for appropriate recording and filtering of the signal. ADInstruments Bio Amps (Bio Amp, Dual Bio Amp, Dual Bio Amp/Stim, Octal Bio Amp and Animal Bio Amp) may be used with suitable electrodes. Please ensure that any corneal electrodes obtained from a third-party supplier have suitable connectors for use with these Bio Amps.
FE136 Animal Bio Amp
- An isolated, high performance and software-controlled differential amplifier
- Has three 2mm input sockets allow the direct connection of electrodes to this amplifier
- Supplied with MLA1215 Animal Bio Lead Wires
- Also suitable for use with
- A single channel, differential amplifier that is suitable for recording a single ERG
- Supplied with a MLA2340 3 Lead Shielded Bio Amp Cable
- Supplied with MLA2503 Shielded Lead Wires (3 snap-on)
- A dual channel differential amplifier that is suitable for recording left and right ERGs simultaneously
- Supplied with an MLA2540 5 Lead Shielded Bio Amp Cable
- Supplied with MLA2505 Shielded Lead Wires (5 snap-on)
Neurophysiological Amplifiers
The FE185 Neuro Amp EX is an electrically isolated high gain differential device that integrates seamlessly with PowerLab. All filtering and sampling are controlled using LabChart or Scope software.
Features include:
- Certified safe for human connection
- Suitable for use with human and animal subjects
- An audio output to listen to neural signals
- Ultra low-noise headstage used with metal electrodes
- 100 Hz - 5 kHz wide bandpass filtering
Transducers and Accessories
Corneal Electrodes
These corneal electrodes are available (and should be directly ordered) from:
Stroboscopic Flash Units
These units are available from:
- Grass Telefactor: The PS33 may be controlled via digital TTL outputs on the PowerLab using the Event Manager software (Windows) or Timed Events Extension (Macintosh)
- Bamberg and Bormann Electronic
Localization And Possible Function Of P2Y4 Receptors In The Rodent Retina
M. M. Ward, T. Puthussery and E. L. Fletcher, Neuroscience, 1262-1274, 2008
Effect of anoxia on the electroretinogram of three anoxia tolerant vertebrates
Stenslokken K-O, Milton S L, Lutz P L, Sundin L, Renshaw G M C, Stecyk J A W, Nilsson G E, Comparative Biochemistry and Physiology A, 395-403, 2008
Rod and Cone Pathway Signalling Is Altered in the P2X7 Receptor Knock Out Mouse
Vessey K, Fletcher E., PLoS ONE, e29990, 2012
The material on this page is provided in good faith and believed accurate at the time of writing. No responsibility will be taken, or liability accepted, for damages arising from the use of information herein. Readers are urged to check with respective manufacturers the accuracy of all product related information.
