Ординатура / Офтальмология / Английские материалы / Artificial Sight Basic Research, Biomedical Engineering, and Clinical Advances_Humayun, Weiland, Chader_2007

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potential recordings is improved by the use of a large number of electrodes, but is ultimately limited by the small contribution to the measured potentials by individual neurons. Electrophysiological functional mapping has recently been combined with structural imaging techniques that have high spatial resolution but poor temporal resolution (e.g. MRI, CT); these multi-modal imaging strategies exploit the strengths of each approach. The technology that allows us to monitor thousands of individual neurons, in real time, in vivo, has yet to be developed. In the meantime, we have several techniques that provide a great deal of information about the nervous system. The following section provides an overview of approaches used specifically to measure the response of the retina to stimulation with light (i.e. photic stimulation).

Non-Invasive Techniques

The earliest technique used to record from the retina was the corneal electroretinogram (ERG) [3]. In its simplest form, this is a straightforward surface potential measurement, where two electrodes are placed on the body surface near the eye, and a potential difference that varies with time is recorded following the delivery of a light stimulus. Typically, one electrode is placed in contact with the cornea of the eye itself, and a second reference electrode is placed on the skin a few inches away (on the temple, forehead, or earlobe).

Almost all techniques for electrophysiological recording, including ERG recording, employ a differential amplifier, in which the potential difference between a reference electrode and ground electrode is effectively subtracted from the potential difference between the recording electrode and the same ground electrode. Ideally, the potential on the reference electrode would reflect only contributions from noise sources that are also picked up by the recording electrode. Thus, the signal downstream of the differential amplifier consists of only the desired signal:

The degree to which this strategy works can be quantified by the common mode rejection ratio (CMRR), which is the ratio of the differential mode gain (DMG, the gain applied to the potentials not appearing on both electrodes) to the common mode gain (CMG, the gain applied to the potentials common to both electrodes).

CMRR = DMG/CMG

The CMG is ideally zero, but is typically close to one in real amplifiers; this is much smaller than the DMG of 100–1000 typically applied to field potential signals. A CMRR of 100 or better is considered quite good.

When recording the corneal ERG in human subjects, the recording (or active) electrode and the reference electrode are often both contained in a specially fabricated contact lens. As long as the two electrodes intercept different isopotential surfaces (where the retina is the dipole source), a differential signal will be recorded. The contact lens serves the important function of stabilizing the contact between the cornea and the electrodes, and it allows light (the stimulus) to enter the eye.

There are many variations of corneal ERG recording, primarily distinguished by the type of stimulus used to evoke the response. The simplest variation is the response to a single, brief (less than about 1 ms) flash of light delivered to a dark-adapted subject. The major components of this response are illustrated in Figure 20.2, and are dominated by the rod pathway in the retina. By exploiting known differences in temporal and spectral responsivity of different cell types within the retina, the stimulus can be designed, and the recorded signal can be analyzed, in order to study the response of a single class of retinal neurons or the response of a single neural pathway. There are more possible variations than can be adequately described in a single chapter. A few variations commonly used in the clinic, and which might be adapted to study responses of the retina

Time (ms)

Figure 20.2. Electroretinogram responses to light and electrical stimuli (presented at time zero) recorded in rat. Upper trace plots a representative ERG response to a brief, moderately bright flash of light presented to a dark-adapted animal. The major components are the negative a-wave (leading edge reflects photoreceptor activity), the positive b-wave (dominated by contributions from ON-type bipolar cells), and the oscillatory potentials superimposed on the leading edge of the b-wave. Lower trace plots the eERG response recorded under one specific set of conditions (see text). The major components of the response are labeled according to the convention used to describe pERG responses, where N and P indicate negative or positive peaks, respectively, and the numbers refer to the peak latency, in msec. Pharmacological dissection of the eERG response (summarized in Figure 20.8) suggest that the origins of the each component are as follows: N35, OFFtype bipolar cells; P80, ON-type bipolar cells; N135, ganglion cells in the OFF pathway. These findings are consistent with the sign of the contribution of these cell types to the ERG in a healthy retina.

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to artificial electrical stimulation, are described here. These are the focal ERG (fERG), the paired-flash ERG (pfERG), the multifocal ERG (mfERG), and the pattern ERG (pERG). The interested reader is referred to the International Society for Clinical Electrophysiology of Vision (ISCEV) standards for applying each of these techniques. These standards facilitate comparison of results collected in the many clinical laboratories around the world, and are available from the ISCEV website (updates are published on a regular basis; e.g. Refs. [4, 5, 6]). Several available texts provide guidance for applying the various techniques to assess functional loss in the retina [7, 8].

The focal ERG is used to elicit a response from a local area of the retina by using a focal stimulus (small spot of light). The fERG is typically used to assess macular function, but can be applied to any portion of the retina. Since a small portion of the retina is activated by the stimulus, there are a smaller number of dipole sources contributing to the field potential recorded at the cornea; therefore the fERG signal is quite small, on the order of 10 microvolts. In practice, the spot is turned on and off at a fixed rate and the responses from several (typically 100–300) responses are averaged to improve the signal to noise ratio (SNR). Also, in order to reduce the influence of areas of the retina outside the desired spot that may be stimulated by scattered light, the spot is surrounded by a constant-luminance annulus typically brighter than the spot (in order to saturate the area of the retina surrounding the region of interest). This is perhaps the most straightforward approach to adapt to electrical stimulation, where the electrodes in contact with the retina are intuitively focal stimuli. However, as discussed below, there are special considerations when substituting an electrode for a focal light stimulus.

The paired-flash ERG, an extension of the single-flash technique, can be used to isolate the contribution of the photoreceptors to the ERG response recorded at the cornea [9, 10]. In the response to a single flash, the leading edge of the first major component (the negative a-wave) reflects the activity of photoreceptor cells alone, before the other cell types in the retina begin their response to the stimulus (Figure 20.2). The pure photoreceptor response can be recorded at the cornea from the time of the stimulus onset to just before the a-wave peak, beyond which the recorded signal reflects the summed contributions of several cell types. However, it is known from in vitro single cell recording studies that the photoreceptor response lasts several hundred milliseconds. Most of the photoreceptor response is masked in the recorded ERG by the strong contributions of other cell types to the field potential at the cornea.

The paired-flash method relies on the fact that a very bright flash will rapidly drive all of the photoreceptors to saturation. That is, a very bright flash delivered to a dark-adapted eye will elicit the largest a-wave possible Asat . The first step in this approach is to deliver a brief flash of any arbitrary intensity; this is the flash you want to measure the response to, and is commonly referred to as the test flash. At a known time, t, after delivery of the test flash, but during the time-course of the photoreceptor response, a second very bright, saturating flash is delivered. The excursion from the prevailing baseline to the peak of

the a-wave induced by the bright flash, Asatt, is a measure of how far the photoreceptors were from saturation at the time the bright flash was delivered.

Performing the subtraction Asat −Asatt reveals how far the photoreceptors were from saturation due to the test flash at the time the bright flash was delivered, At = Asat −Asatt. By delivering the bright flash at several different times after the test flash, in several paired-flash trials, A(t) can be calculated for the full time course of the photoreceptor response. An important feature of this approach is that it is used to isolate the response of the first-order neurons contributing to the corneal ERG, and this is the property of the pfERG technique that may be exploited in studying electrical stimulation.

The following two techniques are common in clinical electrophysiology of the eye. Because they exploit network properties of the retina, their application to studying the response of a diseased retina to electrical stimulation is much less straightforward than the fERG and the pfERG. They are described briefly here as an illustration of the variety of ERG techniques available, and with the hope that once we know more about the physiology of the degenerate retina, correlates may be developed for use in studying electrical stimulation.

The pattern ERG is primarily used to measure the activity of ganglion cells. The third-order neurons of the retina are most sensitive to spatial differences in retinal illumination. The stimulus of the pERG is therefore a high-contrast checkerboard pattern subtending the central visual field that inverts (black becomes white, white becomes black) at a regular frequency (1–10 Hz). The mean luminance of the checkerboard remains constant, which minimizes the influence of the photoreceptors and second-order neurons to the recorded response. Like the pfERG, the pERG can provide information about a specific class of cells in the retina, but does not convey information about the location of a focal retinal anomaly. That is, the recorded response is still the summed response of contributions from all areas of the retina subtended by the stimulus pattern (typically the macular region).

The multifocal ERG is a technique used to perform functional mapping of the retina, and can provide information related to cell type and spatial location [11]. However, in contrast to most functional mapping methods that use multiple recording electrodes, the mfERG uses a single corneal electrode. The goal is to produce a map of the retina that indicates the strength of the local response to a given stimulus. This is accomplished by stimulating different areas of the retina, and then correlating the area in visual space associated with the stimulus to an area on the retina. Imagine viewing a large screen divided into two halves, upper and lower. If the upper half is bright and the lower half is dark, the inverted image of the screen will illuminate the inferior half of the retina, while the superior half of the retina remains in the dark. The signal recorded at the cornea would be the response originating in the stimulated inferior region of the retina; the superior region would not contribute. If the screen was then divided into, say, a 10 × 10 array of pixels, the response to each box could be mapped to one of one hundred regions in the retina. In practice, this method must account for the mean illuminance of the retina, which determines the state of light adaptation, and

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therefore has a large influence on the recorded response. Commercially available mfERG systems use hexagonal pixels arranged in rings of increasing diameter, and subsets of the entire group of pixels are illuminated in a pseudo-random sequence of flashes. A single corneal response is measured following each flash. Sophisticated computational routines then assign relative contributions from each of the illuminated hexagons to each recorded response, resulting in a map of activity. The mfERG response is thus a mathematical derivation, not a direct measure of activity. The mfERG response can be calculated in a number of different ways, with the first-order and second-order kernels being most common (kernel order refers to the relative timing of the stimulus and response). It should be kept in mind that the contributions of the various cell types in the retina to the mfERG response are not yet completely understood. This is proving to be a powerful technique, but due to limitations on our understanding of the mfERG response of healthy retina, and the complex relationship between response features and cellular activity, its application to electrical stimulation of degenerate retina is likely far off.

Invasive Techniques

Fine wire microelectrodes can be introduced into the eye through the lumen of a needle that pierces the sclera. These electrodes are typically made of smalldiameter insulated tungsten wire, and are either cut off square, exposing the metal at the cut end, or are drawn or etched to a fine tip ( 1 m diameter), and insulated everywhere except the final few microns. If such an electrode is inserted into the retina, a local field potential can be recorded which is influenced most strongly by a small volume of retinal tissue, the intra-retinal ERG. Because the retina is organized in distinct lamina, where each layer is occupied by a limited number of cell types, a local response recorded at a known depth in the retina can be attributed to a single cell type (or at least a small number of cell types). However, it is difficult to know with great certainty how deep an electrode has penetrated the retina, especially in vivo. If a microelectrode is placed on the vitreal surface of the retina, in the nerve fiber layer or just below, it is possible to record the spiking activity of ganglion cells (both single-unit and multi-unit). Single-unit recording has been used successfully for decades in cat and primate to study ganglion cell properties [12, 13]. For example, a single ganglion cell would be targeted for recording, and then focal stimuli (small spots of light, on the order of 10–100 m in diameter) delivered to the retina in a “search pattern” to learn which areas of the photoreceptor layer the ganglion cell received input from. The retinal area which produced a change in firing rate, up or down, in the ganglion cell would define the receptive field for that cell, which could be related to an area in the visual space. Though possible to perform with no permanent damage to the eye, intraocular microelectrode approaches are most suitable for animal studies, and are most amenable to animals with large eyes. Extracellular recordings have been made in vivo in both rats and mice, but the technical challenges are significant [14].

Identifying Response Origins –

Pharmacological Dissection

In the moments after light reaches the photoreceptors, a pattern of activity propagates through the neural network of the retina, from cell to cell, primarily by chemical synapses. The pharmacology of synaptic transmission in the retina is orderly yet complex (recently reviewed by Yang [15] ). There are just two principle transmitters found here: glutamate and -aminobutyric acid (GABA). Glutamate is excitatory, and GABA is (mostly) inhibitory. Glutamate mediates information flow primarily in a vertical direction, from photoreceptors to bipolar cells to ganglion cells. GABA mediates information primarily in lateral directions, or as upstream feedback, and is released by horizontal and amacrine cells. Different cell types express variations of the glutamate and GABA receptors, with the first distinction being ionotropic (ion channels, e.g. iGluRs and GABAA) and metabotropic (coupled to G-proteins, e.g. mGluRs and GABAB) types. There are further subtypes of iGluRs and mGluRs, and a distinct GABAC receptor. The various subtypes of glutamate receptors are generally named for the agonist they are sensitive to. An agonist mimics the action of glutamate, whereas an antagonist binds to the receptor but does not elicit the response associated with glutamate. So, for example, the glutamate agonist N-methyl-d-aspartate (NMDA) binds to a subset of iGluRs known as NMDA channels. Not all agonists are highly selective, and L-aspartic acid (aspartate), a glutamate agonist, binds readily to all glutamate receptors in the retina. In the presence of aspartate, the rods and cones respond to light, but none of the post-synaptic neurons are able to respond to the light-induced change in transmitter release from the photoreceptors. Thus, any ERG response recorded in the presence of aspartate can be attributed to activity of the photoreceptors alone, without contributions from other cell types.

A great deal of work has been done to identify the various receptor subtypes expressed by the different types of retinal neurons. Indeed, the functional subclasses of retinal neurons are defined by the receptor types they express (though subclasses can also usually be distinguished based on morphological differences). The point to make here is that the various agonists and antagonists for receptor subtypes can be used to suppress the activity of subpopulations of retinal neurons. Thus the complex response of the retina can be “dissected” by judicious removal of cell types from the retinal network [16, 17]. The typical protocol is to introduce the drug into the eye at an appropriate concentration such that it binds to the appropriate receptor at a saturating level (i.e. binding to all available receptors). In the case of an agonist, the cells so bound become maximally depolarized (or hyperpolarized), and do not change from this state following a light stimulus. Thus the contribution of this subpopulation of neurons is removed from the recorded response (recall that physiological signals are generally recorded with AC amplifiers, and the static field potential generated by a constant membrane potential is filtered out; only changes in membrane potential contribute to the recorded response).

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If the response of the retina is recorded before introduction of an agonist or antagonist, and then again after introduction of the drug, the latter response waveform can be subtracted from the former to reveal the component of the response that was sensitive to the drug. If the cell type which binds that particular agonist or antagonist is known, then the response component suppressed can be correlated with that cell type. This strategy has been used extensively in the retina, and it is possible that new agonists and antagonists will be discovered, which may reveal new subtypes of receptors, and therefore new physiological classifications of retinal neurons.

Introduction of the drug to the retina in vivo is accomplished via an injection into the vitreal cavity. A small diameter (24 to 30 gage needle) is used to pierce the sclera just below the outer margin of the iris, and the tip is advanced at an angle that avoids the lens until it is within a millimeter or two of the retinal surface. Then a small volume of solution containing the drug is injected. The volume must be small relative to the vitreal volume so that the intraocular pressure is not significantly raised. It is common to mix a small quantity of biocompatible, non-reactive dye into the drug solution to provide visual confirmation that the injection was successful. The drug diffuses within the vitreal volume, and within 20–60 minutes is evenly distributed throughout the retina. Some drugs will be cleared by the circulatory system in a few hours, but generally it is not practical to wait for the drug to be “washed out” in these experiments. A strategy used to provide a control for effects of the injection procedure itself is to inject one eye with the drug, and the other eye with physiological saline, and then measure the response to the same stimulus presented to both eyes. Pharmacological dissection can be employed in conjunction with both invasive and non-invasive recording techniques.

Electrophysiology of Artificial Vision

For a retinal prosthesis to be successful, it must stimulate the retina in such a way that a useful percept is elicited in the patient. This goal might be reached by treating the retina as a black box, and then modifying implant parameters during a training period until the patient percept is optimized. However, only the amplitude and kinetics of the electrical stimulus can be modified after a prosthesis has been implanted in the eye (via a radio frequency emitter–receiver, as is commonly used to communicate with cardiac pacemakers after surgery). Physical design features such as electrode shape, size, spacing, and location relative to the retina cannot be altered without further surgery. Each new surgery entails patient risk. Therefore, there is strong motivation to optimize physical design features before a prosthesis is implanted. This can be achieved in part by studying the response of the retina, at a cellular level, in experimental animals [18]. The response of the retina to electrical stimulation had not been reliably recorded until very recently, and there is little known about the origins of its components. We now hope to study this response using adaptations of the techniques originally

developed to study the response of the retina to light [14]. The remainder of this section describes the approaches developed to study the response of the retina to electrical stimulation.

The methods described below were developed within certain criteria. First, the response of the retina had to be measured in vivo. This criterion was important because maintaining the natural physiological support system (circulatory system, temperature control, osmolarity, chemical environment, physical support, juxtaposition to supporting tissues, etc.) would help ensure that the response recorded during the experiment would be similar to the response of the retina in a patient. Also, the interface between chronic stimulating electrodes and target neural tissues is known to change over time following implantation due to immune and other tissue reactions. Thus, it is of interest to study the efficacy of prosthesis design over time scales of weeks, months, and eventually years. This is not possible with an in vitro preparation.

A second criterion was that the technique should be available for use in appropriate animal models of eye disease. As will be addressed below, the response of a degenerate retina, to either light or electrical stimulation, is not likely to resemble the response of a healthy retina. Mouse is the most common species for developing genetic models of human eye disease, but the eye size of mouse is prohibitively small for introducing electrode arrays adjacent to the retina. Rabbit, cat, and pig all have larger eyes, but they are more expensive to maintain and there are fewer appropriate models available. Rat is second only to mouse in terms of available models, and the eye is just large enough to accommodate small electrode arrays. Thus, rats with appropriate retinal defects were chosen as a compromise between eye size and expense. Some of the appropriate rat models of degenerative retinal disease are the P23H, RCS, and s334ter (see Refs. [19, 20], for reviews of animal models of retinal degeneration). The ERG and electrically elicited ERG (eERG) data in this chapter were recorded from a novel pigmented P23H transgenic rat, where the transgene has a proline substituted for histidine at position 23 in the gene that encodes the photopigment rhodopsin [21]. This is the most prevalent mutation in human patients with autosomal dominant retinitis pigmentosa (adRP).

Working with Degenerate Retina

The response of the healthy retina to light stimulation has been studied for many decades, and there is yet some controversy over the cellular origins of prominent components of the corneal ERG. During the course of retinal degenerative disease, the changes that occur in the retina can be severe, including cell loss and significant remodeling of the retinal network [22]. It is therefore critical to evaluate the response of the retina to artificial stimulation with respect to the degree of degeneration present.

Motivated by the prospect of delivering artificial stimulation to a retina that no longer responds to light, it is now of great interest to study in detail the

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biology of severely degenerate retina. We need to understand not only what cell types remain and in what numbers, but how the morphology of these cells has changed following loss of neighboring and connecting cells. If the goal of artificial stimulation is to produce meaningful retinal output (i.e. spatial and temporal ganglion cell spiking patterns), it will be of great benefit to understand the properties of the network we are stimulating. These network properties are of importance for both subretinal stimulation, which targets distal neurons, and epiretinal electrodes, which are capable of stimulating cells presynaptic to the ganglion cells.

The work described below used P23H rats that showed moderate degeneration of the retina. That is, cell loss was 30–40%, and the retina maintained a reduced, yet still easily recorded, ERG response to light stimuli. Figure 20.3 shows a micrograph of a typical histological cross section of retina from one of these experimental animals, along with a cartoon drawn to represent the relative thickness of each layer at this stage of degeneration. This age was a compromise between demonstrating that a response to electrical stimulation could be elicited in a diseased retina and choosing a retina that retained enough normal structure so that we could begin to interpret the response.

ON OFF

Center Center

GC

IPLb

IPLa

120 m

INL

OPL

ONL

OS

Figure 20.3. Retina of a 16-week-old pigmented P23H transgenic rat, showing moderate degeneration. The schematic on the left is drawn to represent the retina pictured in the micrograph on the right. The major lamina are labeled: OS, photoreceptor outer segments; ONL, outer nuclear layer, containing photoreceptor cell bodies; OPL, outer plexiform layer, containing synaptic connections between photoreceptors and bipolar cells; INL, inner nuclear layer, containing cell bodies of bipolar, horizontal and amacrine cells; IPLa & IPLb, inner plexiform layer, containing synaptic connections between bipolar cells and ganglion cells, the sublaminae containing connections of the ON and OFF pathways are distinguished; GC, ganglion cell layer, containing cell bodies of the ganglion cells.