Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

The Mouse Cone a-Wave Under cone isolation conditions, a small corneal-negative a-wave component of approximately 15 μV saturating amplitude can be recorded and characterized (Lyubarsky et al., 1999); this is approximately the amplitude expected on the assumption that cones and rods have about the same magnitude circulating currents, given that cones are about 3% of the photoreceptors and that the rod a-wave recorded under similar conditions is approximately 400–500 μV. In sum, its sign, amplitude, kinetics, and behavior in wild-type mice and in mice null for genes expressed in cones all indicate that the mouse cone a-wave originates in the suppression of the cones’ circulating current (Lyubarsky et al., 1999, 2000, 2001, 2002).

Rapid Recovery from Bleaching Stimuli ERG recordings have revealed mouse cone responses to recover much faster than rods from strong flashes that completely suppress the circulating current. Thus, for example, the cone a- and b-waves recover from a flash estimated to isomerize 1% of cone M-opsin in 1 s (Lyubarsky et al., 1999), while the recovery of mouse rods measured with the ERG a-wave under similar conditions (including anesthetic) takes minutes (Lyubarsky and Pugh, 1996). These results are paralleled by extensive data in humans (Thomas and Lamb, 1999; Mahroo and Lamb, 2004).

Electroretinographic Phenotypes of Mice with Cone Phototransduction Genes Inactivated Though the mouse cone a-wave is difficult to isolate in practice because of its relatively small amplitude, in conjunction with the cone-driven b-wave it has proven valuable in establishing the function of several visual transduction proteins expressed in cones. Specifically, the greatly slowed recoveries of the a- and b-waves of Grk1−/− mice after a strong flash show Grk1 to be essential for normal inactivation of mouse cones (Lyubarsky et al., 2000), while the greatly slowed recoveries of the a- and b-waves in Rgs9-1−/− mice shows Rgs9-1 to be necessary for normal cone inactivation (Lyubarsky et al., 2001) (figure 10.3).

Functional Coexpression of S- and M-opsins in Mouse

Cones The very wide spectral separation (ca. 150 nm) of the two mouse cone opsins, together with steep fall-off in absorbance of visual pigments on their long-wave tail,1 makes it possible to deliver an intense “orange” (λ > 530 nm) flash that isomerizes a substantial fraction (ca. 1%) of the M-opsin present in the retina while negligibly isomerizing S-opsin. Such an orange flash completely saturates the cone

1The long-wavelength sensitivity of visual pigments declines by about 5 log10 units per 0.2 units of the dimensionless abscissa, λmax/λ (Lamb, 1995).

Figure 10.3 Recovery kinetics of components of cone ERGs of wild-type, Grk1−/−, and Rgs9−/− mice in a paired-flash paradigm. Under conditions that isolate the cone components of the ERG, an intense “white” flash, estimated to isomerize 1.2% of the cone M- opsin and 0.09% of the S-opsin (Lyubarsky et al., 1999), was followed at various interflash intervals ranging from 0.2 s to 300 s by a second flash of comparable intensity and wavelength composition, which produced a saturated amplitude response. In the absence of Grk1 (rhodopsin kinase), which is the only GRK known to be expressed in the mouse retina and is expressed in both rods and cones, recovery to 50% of the initial amplitude is slowed roughly 60to 70-fold (Lyubarsky et al., 2000). In the absence of Rgs9-1, the photoreceptor-specific regulator of transducin GTPase activity, recovery is similarly slowed (Lyubarsky et al., 2001). The a-wave and b-wave recoveries are comparably slowed.

b-wave response to a UV flash for 200–300 ms, suggesting that virtually all mouse cones expressing S-opsin coexpress M-opsin to some degree (Lyubarsky et al., 1999). Confirmation of the hypothesis that the capacity of an orange flash to eliminate b-wave responsivity to UV flashes arises from the functional coexpression of M-opsin in “S cones” comes from

the work of Ekesten et al. (2002), who observed a similar phenomenon in the presence of pharmacological blockers of postreceptor neurons. The minimum fraction of coexpressed M-opsin required to explain complete b-wave suppression by an orange flash is quite small, no doubt undetectable by histochemical methods. In contrast, single-cone action spectra, which can detect in cones that predominantly express S-opsin M-opsin coexpressed at less than 1 part in 10,000 (Nikonov et al., 2005), confirm M-opsin coexpression in most mouse cones (Nikonov et al., 2006).

Functional features of wild-type mouse cones from single-cell recordings

A breakthrough in the characterization of the functional properties of mouse cones came with the development of a suction pipette method suitable for recording from them (Nikonov et al., 2005). This development rested on the wellestablished fact that the circulating or “dark” current of vertebrate photoreceptors, which “sinks” into the outer segment through the cGMP-activated channels, has its sources in K+ channels distributed throughout the inner segment and remaining portions of the cell (reviewed in Pugh and Lamb, 2000). Indeed, it is the radial separation of the sources and sinks of the dark current that gives rise to the extracellular loop of the circulating current discovered by Hagins et al. (1970), and with its suppression, the a-wave of the ERG (reviewed in Pugh et al., 1998). Since the 1980s, many investigators have taken advantage of the radial separation of the sources and sinks of the circulating current to record photoresponses of amphibian rods whose “inner segments” are drawn into the suction pipette. The development of a suction pipette method suitable for recording from mouse cones was propelled by failure to record reliable responses from the OSs of Nrl −/− photoreceptors, leading to the hypothesis that their OSs are particularly fragile and not separable from the pigment epithelium with impunity (Nikonov et al., 2005). The novel method is characterized by two features: first, one to several nuclei from the ONL are carefully drawn under infrared viewing into a suction pipette with an appropriate-sized orifice; second, the circulating currents of rods whose perinuclear regions are also inevitably drawn into the pipette are suppressed by a steady 500 nm background).

Light response families obtained with this method (figure 10.4) have many features that identify them as originating in the suppression of the circulating current of cones rather than rods, including (1) their action spectra, which typically peaks around 360 nm (as expected for mouse cone S-opsin), and which also has a shoulder around 510 nm (as expected from functional coexpression of M-opsin) (see figure 10.6);

(2) their approximately 100-fold lower flash sensitivity (0.02% vs. 2.7% current suppressed per photoisomerization) (figure

10.4C, F, I, and L); (3) their distinctive kinetics, including a shorter time to peak (tpeak) of the dim flash response than rods recorded under the same conditions (tpeak = 73 ms vs. 200 ms at 20 Hz bandwidth)2 and smaller “Pepperberg” or dominant recovery time constants (τ = 70 vs. 230 ms) (figure 10.4C, F, I, and L; figure 10.5); and (4) their relatively greater immunity to the effects of bleaching exposures (see Nikonov et al., 2006, for details). Such recordings also confirm that most mouse cones also functionally coexpress both S- and M-opsin; that is, photoisomerization of either pigment in a cone drives phototransduction with similar kinetics (figures 10.5 and 10.6).

Neural retina leucine zipper ( Nrl −/−) knockout mouse: An all-cone retina

Background: Are NRL−/− Photoreceptors Cones or

“Cods”? In humans, mutations in the Maf-family transcription factor neural retina leucine (NRL) lead to a serious form of autosomal dominant retinitis pigmentosa (RP) (Bessant et al., 1999). In an effort to understand the effects of NRL mutations, Swaroop and colleagues generated an Nrl −/− mouse (Mears et al., 2001). The retina of this mouse exhibited a somewhat unexpected phenotype, a complete absence of a number of rod-specific proteins, including rhodopsin, and the rod-specific isoforms of other phototransduction proteins (Mears et al., 2001). A basic conclusion drawn by Mears et al. that has stood the test of additional investigations is that Nrl is necessary for a postmitotic, multipotent photoreceptor cell precursor to differentiate terminally into S cones (the default pathway) or rods (Mears et al., 2001; Corbo and Cepko, 2005; Oh et al., 2007). A critical question raised by the investigation of Mears et al. (2001) was whether the photoreceptors in the Nrl −/− retina are in fact cones, or whether they may be “cods,” an intermediate, abnormal cell type exhibiting properties of both rods and cones. Among the reasons for this question were that some proteins generally thought to be rod-specific, such as “rhodopsin kinase” (Grk1) and “rod arrestin” (Arr1), were expressed in the Nrl −/− photoreceptors, and that the

2Use of 20 Hz bandwidth facilitates isolation of the relatively small and noisy cone responses obtained with the “inner-segment, loosepatch” method (Nikonov et al., 2005) but distorts their kinetics to some extent. We measured the delay produced by the 20 Hz lowpass filtering to be 22 ms, and further confirmed that with a much wider bandwidth, averaged dim-flash responses peaked instead at about 50 ms. Correction for the distortion of the filtering thus brings the time to peak the dim-flash response of single cones (tpeak,corrected = 73 ms − 22 ms = 51 ms; Nikonov et al., 2005, 2006) into good correspondence with that (tpeak 50 ms) extracted with the paired flash paradigm applied to the a-wave of the ERG of the Nrl −/− (Daniele et al., 2005).

Figure 10.4 Kinetics, amplification, and sensitivity of single mouse cones recorded with suction pipettes (Nikonov et al., 2006). A and D, Shown are light response families of wild-type cones; the “S-dominant cone” was maximally sensitive at 360 nm, while the “M-dominant cone” was maximally sensitive at 510 nm. G and J, Shown are similar families obtained from cones of Gta−/− mice, whose rods have no electrical responses (Calvert et al., 2000). B, E,

H, and K, Shown are the estimation of the amplification constant from the light responses of the corresponding panels to the left (the rising phase of the data shown on an expanded time base). C, F, I, and L, Plotted here are the response versus intensity functions derived from the flash response families and the extraction of the so-called dominant recovery time constant (τ) from the recoveries to saturating flashes (see Nikonov et al., 2006, for details).

Figure 10.5 Dim-flash responses of single mouse cones. Average dim-flash response data of S-dominant (A and C) and M-dominant (B and D) cones, stimulated with either UV (361 nm) or midwave (501 nm) flashes, are shown. In each case, analysis of the action spectrum reveals that the UV flash activates only the S-opsin in the cone, while the midwave flash activates M-opsin (the two being coexpressed). F, The averaged responses in A and B are compared

outer segments did not exhibit an obvious conelike morphology. The identity of the cellular type—rods versus cods—was addressed in investigations in which Nrl −/− photoreceptors were compared with rods on a series of ultrastructural, histochemical, molecular, and physiological benchmarks (Daniele et al., 2005; Nikonov et al., 2005), as we now describe.

Histochemical and Ultrastructural Evidence that NRL−/− Photoreceptors Are Cones Each Nrl −/− photoreceptor is associated with a “sheath” that binds (and can be stained by) the lectin peanut agglutinin, a classic marker of cones (Johnson et al., 1986; Blanks et al., 1988), and each also exhibits a clumping of chromatin characteristic of mouse cones but not rods (Carter-Dawson and LaVail, 1979). Each Nrl −/− photoreceptor also has an inner segment substantially wider than its outer segment, a feature generally exhibited by cones (which enhances their trapping and waveguiding of light), including those of wild-type mice, but not by rods.

with those of the cones of Gta−/− and Nrl −/− mice and with responses from wild-type mouse rods recorded under the same conditions (Nikonov et al., 2006). The midwave background light required to suppress rod responses in the wild-type mouse is not needed in experiments with Gta−/− and Nrl −/− cones and is at least partially responsible for the faster response kinetics of the wild-type cones. See color plate 3.

At the ultrastructural level, Nrl −/− photoreceptors have mitochondria that are distinctively conelike, being on average half the length of the mitochondria of rods. These various features of Nrl −/− photoreceptors are summarized in table 10.1, where they are compared with the corresponding features of rods and cones in wild-type mice. The conclusion is inescapable: Nrl −/− photoreceptors are a species of cones, and definitely not a cone-rod intermediate (Daniele et al., 2005).

Electrophysiological Recordings Confirm That NRL−/−

Photoreceptors Are Cones The electrical responses of Nrl −/− photoreceptors have been recorded with two methods:

(1) ERG a-waves, including singleand paired-flash methods (Daniele et al., 2005) (figure 10.7), and (2) single-cell, suction pipette recordings (Nikonov et al., 2005) (see figure 10.5F). These measurements, viewed in the context of single-cell recordings of wild-type mouse cones (Nikonov et al., 2006), strongly confirm the identification of Nrl −/− photoreceptors

Figure 10.7 ERG a-waves of the Nrl −/− mouse: amplification and spectral sensitivity decline with age (Daniele et al., 2005). A, Fullfield ERGs of an Nrl −/− mouse produced in response to a series of UV flashes. B, The nearly a-wave data of A are plotted on an expanded scale, normalized by the 150 μV saturated amplitude and fitted with the model of Lamb and Pugh (1992) to extract the amplification constant of phototransduction. C, Spectral sensitivity of the a-wave of the Nrl −/− mouse (solid circles). For comparison, the spectral sensitivity of the rod a-wave of the wild-type mouse is provided (open circles). Like the cone-driven b-wave of the wild-type

Prospects for Investigations Employing NRL−/− Cones

The prospects are great for use of the Nrl −/− mouse in elucidating the differentiation of photoreceptors, in identifying cone-specific genes, and in investigating the biochemistry of the protein products of such genes. Indeed, the first fruits of investigations using the Nrl −/− mouse have now appeared.

Photoreceptor differentiation. Swaroop and his colleagues have made great strides in uncovering the network of genes regulated by Nrl in the differentiation of rods (Akimoto et al., 2006; Khanna et al., 2006; Oh et al., 2007), with parallel advances in understanding the downstream transcriptional factor Nr2e3 (Cheng et al., 2004; Wright et al., 2004; Corbo and Cepko, 2005), whose mutation gives rise to enhanced S cone syndrome ( Jacobson et al., 1990; Hood et al., 1995).

mouse (see figure 10.2), the cone a-wave of the Nrl −/− mouse is maximally sensitive at 360 nm, with a secondary maximum around 510 nm; however, the relative sensitivity of the 510 nm peak is at least fivefold lower in the Nrl −/− mouse relative to that in the wildtype mouse, suggesting that M-opsin expression is relatively diminished in Nrl −/− cones. D, Decline of saturating a-wave amplitude in a population of Nrl −/− mice as a function of age. The amplitude is stable at less than 40 days but shows much greater variability and decline thereafter, indicating progressive deterioration.

Cone-specific genes and their molecular function. An analysis of the genes whose message level in the retina is enhanced in the Nrl −/− mouse has been derived from a microarray analysis by Yoshida et al. (2004): the genes with elevated expression are candidates for cone-specific expression or for playing a role in cone cell biology. An interesting example is JamC, a gene whose expression in the retina would not have been suspected in the absence of microarray data from the Nrl −/− retina. JamC, whose message is threefold increased in the 8- week-old Nrl −/− retina over the level seen in the retina of wild-type mice, encodes a junctional adhesion molecule of the Ig superfamily that is typically associated with tight junctions in endoand epithelia (Ebnet et al., 2004). Not surprisingly, JamC, along with another JAM isoform, JamA, is localized to the apical complexes of RPE tight junctions (Daniele et al., 2007). However, JamC was also found at the adherens junctions forming the OLM and at the base of the

inner segments of cones, suggesting that it may play a novel role in the adhesion of cones and Müller cells at the OLM (Daniele et al., 2007). Perusal of the microarray data of Yoshida et al. (2004) suggests that a number of other novel cone-related genes will be similarly confirmed.

Mouse cone protein biochemistry. Craft and colleagues measured light-dependent phosphorylation of murine opsins using the Nrl −/− mouse and have begun characterizing some of its aspects, including the consequent binding of cone arrestin (Zhu et al., 2003). Such studies are possible only because of the biochemistry permitted by the absence of the large (30-fold) excess rod proteins and the abundance of cones. Similarly, our laboratory has begun investigating some biochemical aspects of the mammalian cone retinoid cycle using the Nrl −/− mouse (Lyubarsky et al., 2006).

Summary and conclusion

The field of rod photoreceptor biology rests on a firm foundation of four methodological cornerstones: anatomy, biochemistry, molecular genetics, and single-cell electrophysiology. Though data from rods of many species have contributed to the construction of this edifice, those from mouse rods have played a special role in elucidating the role of specific proteins, by dint of the power of mouse molecular genetics to accomplish targeted manipulations of genes (Burns and Baylor, 2001).

Several of the cornerstones of a similar edifice for cones have been laid for some time, with outstanding anatomy and physiology from many species, notably salamander, ground squirrel, and human retinas. However, only recently, with developments in ERG methods (Lyubarsky et al., 1999, 2000, 2001, 2002; Calvert et al., 2000)—and very recently, with suction pipette recording (Nikonov et al., 2005, 2006)— has it been possible to include investigations of mouse cones in all four methodological categories. As a consequence, the power of mouse molecular genetics can now be harnessed in the investigation of cones, as it has been for more than a decade in the investigation of mouse rods. Moreover, the second methodological cornerstone, biochemistry, now includes mouse, thanks to the all-cone Nrl −/− mouse (Mears et al., 2001; Daniele et al., 2005; Nikonov et al., 2005), whose retina can be used for identifying and purifying conespecific proteins without the interference of the vast background of homologous rod proteins that are present in the wild-type mouse retina.

Many areas of science other than the investigation of mouse cones are expected to contribute to our rapidly expanding understanding of cone structure, function and disease. These include the spectacular anatomical characterization of living human cones by David Williams and his colleagues with adaptive optics (e.g., Roorda and Williams,

1999; Pallikaris et al., 2003; Hofer et al., 2005), genomic and microarray data (Yoshida et al., 2004), biochemistry of cones from other species such as ground squirrel (e.g., Zhang et al., 2003), and tremendous advances in the use of optical methods for probing living tissue, such as multiphoton microscopy, which allow imaging of specific cells functioning in their natural milieu.

As cones occupy a little more of the stage, the investigation of rods should not be abandoned, or even curtailed. In humans as in mice, rods outnumber cones by more than 20 : 1. The sheer numerosity of rods not only means that disease that leads to rod death also causes cone malfunction and demise, but also that the delivery and removal of metabolites needed by both types of photoreceptors—including generic substances such as oxygen and glucose, as well as specific molecules such as retinoids—are of potentially great significance to cone health. We must not lose sight of the fact that the human retina, like that of the mouse and most of our vertebrate relatives, is inherently duplex, having evolved both cones and rods to provide effective visual signals over the more than 8 log10 unit range of diurnal intensities (Rodieck, 1998). Rods and cones in the duplex retina have evolved to complement each other and cooperate, not only in their starring roles in signaling in night and day, but also in performing the many mundane chores of domestic cellular life. This chapter closes, then, with the hope that the vision community will see advances in understanding mouse cones not as “the next big thing” but rather as an opportunity to understand fully both types of vertebrate photoreceptors in their shared natural matrix, the retina.

acknowledgments Work was supported by NIH grant no. EY02660 and by the Research to Prevent Blindness Foundation.

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BENJAMIN E. REESE

The various classes of nerve cell in the retina are each distributed as orderly arrays across the retinal surface, permitting a uniform distribution of labor in processing the visual image despite global variations in cellular density. This regularity in somal distribution is widely presumed to increase the uniformity by which the processes of these cells sample the retinal surface. Many cell types exhibit a corresponding increase in the size of their dendritic fields as cell density declines with eccentricity, yielding a constant dendritic coverage across the retina. The nerve cells in these patterned arrays, or mosaics, extend their dendrites to sample from a layer of afferent innervation, establishing a dendritic morphology and degree of dendritic overlap that are unique to each cell type (Wässle and Riemann, 1978). Both of these features are associated with the functional contribution carried out by each array, defined by the synaptic connectivity established within the plexiform layers. These network properties of retinal mosaics, their somal patterning and dendritic coverage, are largely conserved across the vertebrate retina, and have recently been reviewed (Reese, 2007). This chapter reviews current understanding of how this mosaic architecture is produced during development, in which application the mouse retina has proved particularly enlightening.

The development of mosaic architecture

While seemingly unique to the problem of building a functional retina, the establishment of such regularity in a retinal mosaic reduces to a common if fundamental issue in the field of developmental biology, that of pattern formation ( Meinhardt, 1982; Lawrence, 1992). Numerous studies have shown that the patterning present in such retinal mosaics may be accounted for by exclusively local interactions constraining proximity between neighboring like-type cells (Galli-Resta et al., 1999; Eglen et al., 2003a; Cameron and Carney, 2004; Eglen and Galli-Resta, 2006); no grand architectural plan or protomap is required to generate the patterning that is so commonplace among cell types in the vertebrate retina.

The implementation of a local spacing rule preventing proximity between two cells is an attractively simple means for generating patterning within a mosaic, but defining the

biological embodiment of this spacing rule has not been straightforward. Three different types of mechanism have been proposed (figure 11.1), but direct evidence for any of them has been hard to come by. Such patterning has been ascribed to fate determination events occurring periodically across the retina, whereby, for instance, a newly fated cell may inhibit surrounding neighbors from acquiring the identical fate (McCabe et al., 1999; Cameron and Carney, 2004; Tyler et al., 2005). Alternatively, the patterning could be sculpted from an initially disordered and overproduced mosaic, followed by a process of naturally occurring cell death in which closely positioned cells may compete for limited trophic support, leading to one or the other being eliminated ( Jeyarasasingam et al., 1998; Eglen and Willshaw, 2002). Finally, closely spaced cells may repel one another, dispersing tangentially on the retina to minimize proximity with immediate neighbors (Reese et al., 1999; Eglen et al., 2000). Although any of these three types of mechanism could in principle underlie the establishment of regularity in a mosaic, and although there is ample evidence for the lateral inhibition of cell fate, for naturally occurring cell death, and for tangential dispersion of retinal neuroblasts (for reviews, see Reese and Galli-Resta, 2002; Agathocleous and Harris, 2006; Linden and Reese, 2006), the evidence in support of any of them as the basis for the patterning observed in mature retinal mosaics is scant and usually indirect.

To generate direct evidence, one would ideally like to assay the patterning associated with the spatial locations within the plane of the retina at which cells of a given type had acquired their fate; or to compare the patterning before and after naturally occurring cell death in the absence of any tangential dispersion occurring within the intervening period; or to observe, in a live preparation, the change in the patterning of a population produced by tangentially dispersing neuroblasts. None of these more direct assessments has been achieved, but some limited conclusions can still be made for certain nerve cell classes.

With respect to dendritic coverage, different cell types would appear to implement one of two different strategies to achieve their characteristic degree of dendritic overlap, either regulating their dendritic growth in direct proportion to proximity with their homotypic neighbors or through the use of cell-intrinsic mechanisms, otherwise oblivious to local