Ординатура / Офтальмология / Английские материалы / The Retinal Muller Cell Structure and Function_Sarthy, Ripps_2001

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experiments on the pharmacology of the DC component of the ERG (Katz et al., 1992) suggest that both the b-wave and the DC component (cf. Fig. 5.17) are generated primarily by the distal K+–Müller cell mechanism in response to the K+ efflux from depolarizing bipolar cells. In addition, Masland and Ames (1975) demonstrated that following a short period of anoxia, or after incubation in a low calcium medium, there was marked diminution of the b-wave with no apparent reduction in ganglion cell response (Fig. 5.23B). Comparable results were obtained after selectively poisoning the Müller cell with D,L-α-aminoadipic acid (Bonaventure et al., 1981). If the reduced b-wave seen in these studies was the result of abnormal bipolar cell function, it is puzzling to find retention of seemingly normal signal transmission from photoreceptors to ganglion cells.

The Bipolar Cell Hypothesis: Despite the previously cited findings, there is reason to believe that the b-wave may not reflect radial current flow generated by a K+-induced depolarization of Müller cells (cf. Tomita and Yanagida, 1981). For example, membrane potential measurements from amphibian Müller cells correlate poorly with the waveform and time course of the b-wave, but appear to mirror an extracellular field potential (the M-wave) recorded in the proximal retina (Karwoski and Proenza, 1977, 1978). Inconsistencies between the current sinks and sources for the b-wave (CSD analysis) and the kinetics and loci of K+ fluxes contradict the Müller cell hypothesis (Vogel and Green, 1980). In addition, a question has been raised as to whether the magnitude of the light-evoked [K+]o increase recorded in the distal retina is sufficient to generate the large transretinal b-wave, although it is generally recognized that electrode-induced damage, interference from the large distal K+ decrease, as well as other factors may dilute considerably the K+ measurements. (Karwoski et al., 1985).

Figure 5.23. A. Recordings of light-evoked responses from the toad retina superfused with a normal Ringer (control) solution or one containing 0.2 mM Ba2+; the stimulus marker is shown on the bottom trace. The uppermost traces show the extracellular ERG and the distal K+ increase; the lower pair are intracellular membrane potential recordings (Vm) from a Müller cell and rod photoreceptor. Note that the rod potential and the K+ efflux are relatively unaffected by exposure to barium, but both the Müller cell response and the ERG b-wave are substantially reduced (Wen and Oakley, 1990). (Copyright 1990 National Academy of Sciences, U.S.A., reprinted with permission.) B. Effects of anoxia (upper pair of traces) and low Ca2+ (lower traces) on the transretinal ERG, the compound action potential recorded from the optic nerve, and the spike activity of a single ganglion cell. Both anoxia and the low calcium solution reduce significantly the ERG b-wave, but have no detectable effect on the responses recorded from ganglion cells or their optic nerve bundles (Masland and Ames, 1975). (Copyright 1975 John Wiley & Sons, Inc., reprinted with permission.)

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Nevertheless, a large body of evidence supports the view that the b-wave potential is predominantly a direct expression of the activities of roddriven depolarizing (ON) bipolar cells (cf. Xu and Karwoski, 1994b; Robson and Frishman, 1995; Hanitzsch et al., 1996; Green and Kapousta-Bruneau, 1999; Shiells and Falk, 1999; Lei and Perlman, 1999). The depolarizing responses of these cells reflect the opening of cGMP-activated cation channels in postsynaptic membranes, as a result of the light-induced suppression of glutamate release from photoreceptor terminals (Shiells and Falk, 1990; Nawy and Jahr, 1990,1991). Membrane depolarization and the radial orientation of the bipolar cell would be effective in creating an extracellular current path consistent with the polarity of the b-wave.

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Figure 5.24. Current dipoles established by the sinks and sources of the ERG b-wave. CSD analysis of the intraretinal depth profiles of light-evoked field potentials and resistivity indicate that the major currents (bold lines with arrows) are established between the inner (source) and outer (sink) plexiform layers. These loci do not distinguish between bipolar and Müller cells in the generation of the b-wave. However, minor currents (dashed lines) in the region of the ILM are suppressed by barium (an effective blocker of glial K+ channels), suggesting that the barium-resistant component of the b-wave originates with the depolarizing bipolar cells (Xu and Karwoski, 1994b). (Copyright 1994 American Physiological Society, reprinted with permission.)

More direct evidence that the b-wave potential arises from the depolarizing bipolar cell has come from extensive CSD analyses of current sinks and sources underlying the various components of the amphibian ERG (Xu and Karwoski, 1994a, 1994b, 1995; Karwoski et al., 1996). These data indicate that at light onset there is a large transient sink near the OPL, and a large source in the IPL that exhibits similar kinetics to the distal sink (Fig. 5.24). This current pattern closely approximates the extent of the bipolar cell, although the presence of a Ba2+-sensitive slow current source at the internal limiting membrane (where K+ exits the Müller cell), suggests a lesser current path representing a contribution from the Müller cell to the ERG b-wave.

A further indication of the link between the light-activated responses of depolarizing bipolar cells and the b-wave potential is provided by the strong correlation between the waveforms of the chemically isolated b-wave and the intracellulary recorded ON bipolar cell potential (Gurevich and Slaughter, 1993; Tian and Slaughter, 1995; Shiells and Falk, 1999), although this does not preclude an intervening stage mediated by the K+–Müller cell spatial buffering mechanism. However, results showing enhancement of the b-wave response in the presence of barium (in concentrations sufficient to effectively block the K+ channels on Müller cells) implicate the primacy of the bipolar cell in the generation of the ERG b-wave (Green and KapoustaBruneau, 1999; Lei and Perlman, 1999).

Given the conflicting evidence on the b-waves source, electrophysiological studies alone may not be able to establish unequivocally the relative contribution of bipolars and Müller cells to this transretinal potential. Recently, however, further support for the bipolar cell hypothesis was obtained from studies on KIR4.1 knockout mice (Kofuji et al., 2000). Inactivation of the principal K+ channel subunit expressed in mouse Müller cells produced a total loss of the slow PIII response but had little or no effect on the ERG.

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Figure. 6.1. Potential pathways leading to neuronal injury resulting from an episode of ischemia. Ischemia causes depletion of cellular energy stores, the release of free radicals, and subsequent neuronal injury. In addition, cellular depolarization due to the decreased energy supply induces the release of glutamate, which leads, in turn, to sustained activation of NMDA receptors and Ca2+ entry into the cell. The elevation of [Ca2+]i causes excessive activation of a variety of Ca2+-dependent enzymes, such as proteases, leading to neuronal injury and possibly cell death. Recent evidence suggests that in addition to NMDA receptors, AMPA-type glutamate receptors may also be involved in this process (Dingledine and McBain, 1993). (Copyright 1993 Raven Press, reprinted with permission.)

receptors, followed by an influx of large amounts of extracellular Ca2+ and other cations into neurons, leading to neuronal death (Choi, 1988). The glutamate neurotoxicity model is supported by several lines of evidence and includes a role for glial cells in the cytotoxic process (Fig. 6.1) (Choi and Rothman, 1990; Lee et al., 1999). Although there is some disagreement regarding the role of Ca2+ ions in excitotoxic death of retinal neurons (Romano et al., 1998; Chen et al., 1998), it is clear that excessive levels of glutamate are toxic in the retina as well.

Because glial cells possess high affinity glutamate uptake systems and can convert glutamate to glutamine, a nonneurotoxic substance (see Chapter 3), glial cells are likely to be intimately involved in regulating extracellular glutamate levels in the CNS. It has been observed that neurons in mixed cultures containing neurons and astrocytes are more resistant to glutamateinduced cell death than neurons in glia-free cultures (Rosenberg and Aizenman, 1989). Conversely, incubation ofneuronal cultures with either glutamateuptake blockers or antisense oligonucleotides to glutamate transporters increases the excitotoxic effects of glutamate (Rosenberg et al., 1992; Rothstein et al., 1996).

Retinal neurons are also susceptible to glutamate-induced damage. As first shown by Lucas and Newhouse (1957), subcutaneous injections of

Figure. 6.2. The effect of chronic glutamate exposure leads to ganglion cell death. A. Transverse section of a normal retina. B. Retina from a rat that received intravitreal glutamate. Note the loss of a significant number of cell bodies in the ganglion cell layer (GCL) (Vorwerk et al., 1996). (Copyright 1996 Association for Research in Vision and Ophthalmology, reprinted with permission.)

sodium L-glutamate in adult and neonatal mice result in severe destruction of ganglion cells and partial loss of cells in the INL. Subsequent studies confirmed the retinotoxic effects of glutamate and its relative selectivity for neurons of the inner retina when administered subcutaneously (Cohen, 1967) or intravitreally (Sisk and Kuwabara, 1985). A surprisingly small elevation in the concentration of glutamate, when maintained for long periods of time, is toxic to retinal ganglion cells (Fig. 6.2). Repeated intravitreal injections of glutamate in the rat eye, titrated so as to induce a sustained rise in the vitreous concentration of glutamate from endogenous levels (5-12mM) to the range of 26-34mM were shown to cause the death of more than 40% of the retinal ganglion cells after three months exposure (Vorwerk et al., 1996). Memantine, an agent that blocks activation of NMDA receptors (Bormann, 1989; Chen and Lipton, 1997), gave partial protection against the effects of elevated glutamate (Vorwerk et al., 1996). Interestingly, the relatively small (micromolar) rise in glutamate that led to toxic changes in the rat eye, is typically seen in the vitreous of human and monkey eyes with glaucoma (Dreyer et al., 1996), a chronic disease that ultimately results in the loss of ganglion cell axons.

Considering the retinotoxic effects of glutamate, and its sustained release in darkness, there is clearly the need for an efficient means for its removal. Müller cell uptake is a vital part of this process in the normal retina. We have described already the carrier-mediated uptake of glutamate

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into Müller cells as a means to remove glutamate from the synaptic cleft and terminate its post-synaptic action (Chapter 4). Paradoxically, there is a remarkable number of ways in which factors affecting its normal activity can contribute to retinotoxicity.

The stoichiometry of the Müller cell glutamate transporter (see Chapter 4) and evidence that the carrier-mediated uptake of glutamate can be inhibited (Barbour et al., 1988) or even reversed (Szatkowski et al., 1990) have important implications for events that may be triggered because of pathological conditions. In retina subjected to trauma or anoxia, abnormal conditions can produce a precipitous rise in extracellular K+ and elevation of glutamate to toxic levels. While the precise sequence in which these changes take place remains somewhat obscure, it not difficult to speculate on how they might come about (cf. Nicholls and Attwell, 1990).

Suppose, for example, that the extracellular [K+], normally in the range of 3–5 mM, rises to a level of 60 mM or greater, a situation encountered in the anoxic brain and during spreading depression (cf. Walz and Hertz, 1983). At this concentration, several interrelated processes are brought into play, all of which contribute to extracellular glutamate elevation and its potentially excitotoxic effects, First, there is the depolarization of glutamatergic nerve terminals and calcium-dependent release of glutamate. Second, both the rise in [K+]o and its depolarizing effect on Müller cells (and neurons) not only inhibit glutamate uptake, but also induce calcium-independent release of glutamate (by reversed uptake) to increase further its extracellular concentration. In addition, glutamate activation of NMDA receptors on second order neurons causes the release of arachidonic acid, yet another source of uptake inhibition (Barbour et al., 1989). Last, the rise in glutamate will itself cause neurons to depolarize further, release more K+, and stimulate, in turn, an even greater efflux of glutamate. Consequently, glutamate levels may exceed 100 mM, which is more than enough to induce neuronal death.

In addition to glutamate transporters, glutamine synthetase levels are also important for regulating glutamate toxicity in the retina (Gorovits et al., 1997). Chick retinas treated with cortisol to induce high levels of glu-

Figure. 6.3. Relationship between glutamine synthetase induction and lactate dehydrogenase (LDH) release, an indicator of cell damage. A. Cortisol was injected into E15, E16, or E17 eggs which were further incubated for 4, 24, or 48 hr respectively. Retinas were dissected out and organ-cultured for an additional 4 hr in the presence of glutamate. Levels of cellular GS and extracellular LDH in cortisol-treated chick retinas maintained in vitro. B. Concentrationdependence of GS and LDH levels on [Cortisol]. C. Inverse correlation between GS activity and LDH release. These data suggest that glutamine synthetase affords protection from glutamate-induced cellular injury (Gorovits et al., 1997). (Copyright 1999 National Academy of Sciences, USA., reprinted with permission.)

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