Electroretinogram of Human, Monkey and Mouse

Introduction

The electroretinogram (ERG) is a useful tool for objective, non-invasive assessment of retinal function both in the clinic and the laboratory. It is a mass electrical potential that represents the summed response of all the cells in the retina to a change in illumination. Recordings can be made in vivo under physiological or nearly physiological conditions using electrodes placed on the corneal surface. For standard recordings in the clinic, ERGs such as those illustrated in Figure 24.1 are recorded from alert subjects who are asked not to blink or to move their eyes. Anesthesia, selected for having minimal effects on retinal function, may be required for recordings from some very young human subjects, and is generally used for recordings in animals. The positive and negative waves of the ERG reflect the summed activity of overlapping positive and negative component potentials that originate from different stages of retinal processing. The choice of stimulus conditions and method of analysis will determine which of the various retinal cells and circuits are generating the response. Information about retinal function provided by the ERG is useful for diagnosing and characterizing retinal diseases, as well as for monitoring disease progression, and evaluating the effectiveness of therapeutic interventions.

Much of the basic research on the origins, pathophysiology and treatment of retinal diseases that occur in humans is carried out in animal models. The ERG provides a simple objective approach for assessing retinal function in animals. In recent years it has been of particular benefit in studies of mouse (rat, and other species) models of retinal disease, and it has been valuable for evaluating the retinal drug toxicity. The ERG also has been useful for characterizing changes in retinal function that occur as a consequence of genetic alterations in mice and other models, which affect the transmission and processing of visual signals in the retina.

This chapter provides information on retinal origins and interpretation of the ERG, with emphasis on advances in our understanding of the ERG that have occurred through pharmacological dissection studies in macaque monkeys whose retinas are similar to those of humans. The similarity of the waveforms of flash ERGs of humans and macaques can be seen in Figure 24.2 . The chapter also will examine origins of the mouse ERG, and consider similarities and differences between mouse and primate ERG. Although the focus of this chapter is on primate and rodent retinas, it is important to note that the ERG is a valuable tool for assessing retinal function of all classes of vertebrates. This includes amphibians and fish in which classical studies of the retina were carried out (for a review, see Dowling ) and in which current work continues to improve our understanding of retinal function under normal, genetically altered and pathological conditions.

This chapter provides useful background information for interpreting the ERG, but does not provide a comprehensive review of the characteristics of ERGs associated with retinal disorders encountered in the clinic. Recent texts by Fishman et al, Heckenlively & Arden and Lam are recommended resources for learning more about clinical applications.

Radial current flow

The ERG is an extracellular potential that arises from currents that flow through the retina as a result of neuronal signaling, and for some slower ERG waves, potassium (K ⁺ ) currents in glial cells. Local changes in the membrane conductance of activated cells give rise to inward or outward ion currents, and cause currents to flow in the extracellular space (ECS) around the cells, creating extracellular potentials. Although all retinal cell types can contribute to ERGs recorded at the cornea, the contribution of a particular type may be large, or hardly noticeable in the recorded waveform, depending upon several factors detailed below.

The orientation of a cell type in the retina is a major factor in determining the extent to which its activity will contribute to the ERG. A schematic drawing of the mammalian retina, with the various cell types labeled, can be found in Chapter 21 ( Fig 21.1, Fig 21.2 ). When activated synchronously by a change in illumination, retinal neurons that are radially oriented with respect to the cornea, i.e. the photoreceptors, and bipolar cells, make larger contributions to the major waves of the ERG than the more laterally oriented cells and their processes, i.e. the horizontal and amacrine cells. The major waves at light onset are, as marked in Figures 24.1 and 24.2 , an initial negative-going a-wave (mainly from photoreceptors), which is truncated by a positive-going b-wave, mainly generated by ON (depolarizing) bipolar cells. For longer duration flashes ( Fig. 24.2 , bottom row), the light-adapted ERG includes another positive-going wave, the d-wave, at light offset, with major contribution from OFF (hyperpolarizing) bipolar cells. Currents that leave retinal cells and enter the ECS at one retinal depth (the current source), will leave the ECS to re-enter the cells at another (the current sink), creating a current dipole. These retinal currents also travel through the vitreous humor to the cornea, where the ERG can be recorded non-invasively, as well as through the extraocular tissue, sclera, choroid and high resistance of the retinal pigment epithelium (RPE) before returning to the retina. Local ERGs can be recorded near the retinal generators using intraretinal microelectrodes in animals, while simultaneously recording the global ERG elsewhere in the current path, e.g. from the corneal surface, or using an electrode in the vitreous humor, with a reference electrode behind the eye. Such recordings have provided useful information about the origins of the various waves of the ERG.

Glial currents

The ERG waveform also will be affected by glial cell currents. Retinal glial cells include Müller cells, RPE cells, and radial astrocytes in the optic nerve head. One crucial function of glia is to regulate extracellular K ⁺ concentration, [K ⁺ ] _o , to maintain the electrochemical gradients across cell membranes that are necessary for normal neuronal function. Membrane depolarization and spiking in retinal neurons that occur in response to changes in illumination lead to leak of K ⁺ from the neurons and to K ⁺ accumulation in the ECS. Membrane hyperpolarization, in contrast, leads to lower [K ⁺ ] _o as the membrane leak conductance is reduced, but the Na ⁺ K ⁺ ATPase in the membrane continues to transport K ⁺ into the cell.

K ⁺ currents in Müller cells move excess K ⁺ from areas of high [K ⁺ ] _o to areas of lower [K ⁺ ] _o by a process called spatial buffering. Return currents in the retina are formed by Na ⁺ and Cl ^– . The regional distribution and electrical properties of inward rectifying K ⁺ (Kir) channels in Müller cells (see Fig. 24.3C ) are critical for the spatial buffering capacity of the cells. Studies of Kofuji and co-workers have shown that strongly rectifying Kir2.1 channels in Müller cells are located in ‘source’ areas, particularly in synaptic regions where [K ⁺ ] _o is elevated due to local neuronal activity. In contrast, Kir4.1 channels (weakly rectifying) are densest in Müller cell endfeet, in inner and outer limiting membranes and in processes around blood vessels. K ⁺ enters Müller cells through Kir2.1 channels that minimize K ⁺ outflow and K ⁺ exits the Müller cells via the more bidirectional Kir4.1 channels to enter the extracellular ‘sink’ areas with low [K ⁺ ] _o : the vitreous humor, subretinal space (SRS), and blood vessels.

ERG waves associated with glial K ⁺ currents have a slower timecourse than waves related to currents around the neurons whose activity causes the changes in [K ⁺ ] _o . As [K ⁺ ] _o increases or decreases with neuronal activity, the resulting glial K ⁺ currents will be related to the integral of the K ⁺ flow rate, e.g. the glial contribution to the b-wave modeled in Figure 24.11 . Other waves generated by glial K ⁺ currents in Müller cells, or other retina cells with glial function, such as the RPE, include the c-wave and slow PIII ( Fig. 24.3A,B ), both related to the reduction in [K ⁺ ] _o in the SRS that occurs when photoreceptors hyperpolarize in response to a strong flash of light. The negative scotopic threshold response (nSTR) and photopic negative response (PhNR), both originating from inner retinal activity, are also thought to be mediated by glial K ⁺ currents in retina or optic nerve head.

Stimulus conditions

Aside from structural and functional aspects of the retina, stimulus conditions are of great importance in determining the extent to which particular retinal cell or circuits contribute to the ERG. Signals will be generated in rod pathways, in cone pathways or both depending upon the stimulus energy, wavelength and temporal characteristics, as well as upon the extent of background illumination, with rods responding to, and being desensitized by lower light levels than cones. Fully dark-adapted, ERGs driven by rods only (i.e. scotopic ERGs) are thus useful for assessing rod pathway function ( Figs 24.1 and 24.2 , top), and light-adapted ERGs driven only by cones (i.e. photopic ERGs), for assessing cone pathway function ( Figs 24.1 and 24.2 , bottom). Figure 24.1 , bottom right, also shows responses to 30 Hz flicker which isolates cone-driven responses because rod circuits do not resolve well such high frequencies. Very bright light flashes elicit small wavelets superimposed on the b-wave called oscillatory potentials (OPs) that are generated by circuits proximal to bipolar cells. OPs can be isolated by bandpass filtering: 75–300 Hz in Figure 24.1 , top right.

The spatial extent of the stimulus is an important factor in ERG testing. For standard clinical tests, as illustrated in Figure 24.1 , and listed in Box 24.1A , as well as for most testing of animal models, a full-field (Ganzfeld) flash of light is used ( Fig. 24.2B ). A full-field stimulus generally elicits the largest responses because more retinal cells are activated and the extracellular current is larger than for focal stimuli. It also has the advantage that all regions of retina are evenly illuminated, and with respect to background illumination, evenly adapted. Pupils are generally dilated for full-field stimulation. More spatially localized (focal) stimuli are useful for analysis of function of particular retinal regions, e.g. foveal vs peripheral regions in primates. Multifocal stimulation allows assessment of many small regions simultaneously.

ERG responses illustrated in Figure 24.1 are to a minimum set of stimuli selected by the International Society for the Clinical Electrophysiology of Vision (ISCEV) to efficiently acquire standard, comparable data on rod and cone pathway function from clinics and laboratories around the world. Names of the standard tests are listed in Box 24.1A . Tests using stimuli presented over a fuller range of stimulus conditions to allow more complete or specific evaluation of retinal function, are listed in Box 24.1B , and a few of these tests will be described later in this chapter.