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Seeing through the photoreceptor mosaic David R. Williams It has long been thought that the foveal mosaic provides the anatomical basis for human visual resolution under optimal viewing conditions. Recent anatomical evidence shows that thefoveal cone mosaic in the primate is a regular, triangular array o f elements with only minor distortions. The sampling properties o f such an array predict the existence o f visible moird patterns, or aliasing, produced when very fine gratings are imaged on the retina. Recent psychophysical experiments confirm this prediction, and allow the spacing between cones to be measured in the living eye. The cone array in the peripheral retina, unlike the fovea, is sparse and irregular, and it feeds an even sparser ganglion cell array. These arrays provide a new challenge to establish the link between the organization o f the retina and the psychophysical limits o f visual resolution.

A major driving force behind the study of human vision is the expectation that links can be forged between visual experience and the structure of the visual system. The study of visual acuity has always seemed particularly promising in this regard, and the spacing between foveal cones has long been heralded as the anatomical basis for human visual resolution. Recent work in retinal anatomy and visual psychophysics has clarified the interplay between the cone mosaic, the eye's optics, and subsequent neural mechanisms in limiting the visual resolution of sinnsoidal gratings. This interplay is quite different in reveal and extrafoveal vision and these two cases are treated in turn. Visual resolution in the fovea The retinal image is blurred by diffraction at the pupil and aberrations in the anterior optics of the eye. The optical point spread function of the human eye, obtained in white light with an optimum pupil diameter of 2-3 mm, has a diameter of about 1 min of arc at half height under optimal conditions 1. In spatial frequency terms, retinal image contrast falls by a factor of 10 between 0 and 35-40 cycles deg-1, and is negligible above 60 cycles deg -1. The blurred retinal image is then discretely sampled by the array offoveal cones. The imaging properties of an array of sensors like the cone mosaic depend on the sensor aperture and on the spacing of the sensors in the array. The sensor aperture determines its light-gathering capacity and spatial frequency response; the spacing of the sensors, on the other hand, determine the sampling limitations of the array. The effects of foveal cone aperture and cone spacing are considered separately below. The light-gathering aperture of foveal cones is determined by the

optical properties of cone inner segments. For a fixed cone spacing, increasing inner segment diameter has the benefit of increasing the quantum catch in each cone. This minimizes the uncertainty about the intensity falling on each cone caused by photon noise. However, increasing inner segment diameter also has the potentially deleterious effect of blurring the image due to the integration of light across the cone aperture 2'3. Nonetheless, the inner segments of human foveal cones fill most of the space between them, probably because the cost in image quality caused by light integration is negligible under ordinary viewing conditions. Foveal cone size does not limit image quality because the cone aperture is small compared with the point spread function of the eye. The functional aperture of human foveal cones is no larger than 2.3 Inn in diameter2"4 and may be smallers, while the point spread function of the eye is roughly 5 ~m across at half-height, even under the best conditions. Consequently, the cone aperture produces a loss of contrast of no more than 25% even at 60 cycles deg-1. It is intuitive that decreasing the spacing between sensors in an array •

increases the fidelity of the image it can produce. It is also intuitive that an adequate representation of a grating would require at least one cone beneath each light bar and one beneath each dark bar. Indeed, Helmholtz 6 suggested that the spatial frequency meeting this condition would represent the anatomical resolution limit of the eye; his suggestion presaged the sampling theorem of modern information theory7's. The sampling theorem states that a regular sampling array with an inter-clement spacing, d, will allow unambiguous reconstruction of signals that are bandlimited to frequencies of 1/2d, which is the Nyquist limit for the array. Signals above the Nyquist limit result in sampling artifacts known as aliasing. Aliasing effects frequently occur outside the visual system: the moir6 patterns seen on television sets, or when two window screens or picket fences are superimposed are familiar examples. Aliasing can distort aperiodic patterns as well as sine wave gratings: edges displayed on computer graphics terminals can have a jagged appearance, produced by undersampiing in the display. Fig. 1 shows an example in one dimension of the aliasing problem for the visual system. A regular array of receptors samples two static sinusoidal gratings, one lying above the Nyquist limit and the other lying equally far below. These gratings are two members of a larger set of stimuli that produce an identical pattern of quantum catches across all the receptors. An observer under these circumstances would be unable to discriminate between any of these aliases. Unlike the spatial filtering performed by the optics or the cone

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each otherfor this array. The gratingsare membersof an infiniteset of stimuli thatproducean identical pattern of quantal absorptions in the array. The Nyquist limit would be the spatialfrequency, 1/2d, corresponding to a receptorbeneath each bright bar and each dark bar of the grating. ~) 1986, Elsevier Science Publishers B.V., Amsterdam 0378 - 5912/86/$02.00

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Fig. 2. (A) Tangential section through the central.tbvea ofMacaca fascicularis, prepared by William Miller and Joy Hirsch o f Yale University. Scale bar is 10 wn, or 2.46 minutes of arc assuming243 wn deg-t for this species. The histological technique requires no correction for shrinkage. The section was made very near the external limiting membrane. The minimum center-to-center spacing is 2.8 Wn, corresponding to a spacing between rows o f cones, d, of 2.42 wn. The Nyquist limitfor the monkey foveal lattice is 243(1/2d) or about 50 cycles deg- I. (B ) Tangential section through the parafoveal retina o f Macaca fascicularis at an eccentricity of 5 deg. Same scale as A. Large cones are surrounded by smaller rods.

aperture, sampling per se does not systematically reduce the contrast of any spatial frequencies in the retinal image no matter how high. Instead, it

produces a representation open to a number of interpretations, all of which are consistent with the distribution of quantum catches in the mosaic.

Subsequent neural mechanisms have no way to distinguish between these possible interpretations and are generally ineffective in eliminating aliasing. The ultimate interpretation of this sampled image must be based on apriori information, and the visual system naturally chooses the lowest frequency interpretation, which falls within the range of normal visual experience. No form of neural spatial filtering following the optical process of cone sampling can remove aliasing without also removing the spatial frequencies actually present in the retinal image below the Nyquist limit. Thus electrical coupling between cones and the convergence of cone signals onto higher order retinal neurons can restrict the range of frequencies over which aliasing can occur, but only at the expense of also restricting the veridical information available from the sampled image. Recent studies of the primate fovea o-H have removed all doubt that cones there form a triangular mosaic that is fairly regular, with only minor distortion. (The cone mosaic is often described as a hexagonal lattice, but it is technically a triangular one because lines connecting the centers of the adjacent elements form triangles, not hexagons. A honeycomb, for example, is a triangular lattice with hexagonal elements). Suggestions that the foveal mosaic is irregular 12"13 seem to have been based either on sections distorted by histological artifact, or on sections made through outer segments rather than the inner segments where photons are initially caught. Fig. 2A shows a tangential section through the central fovea of Macaca fascicularis, made through the inner segments just a few microns sclerad to the external limiting membrane. The section, made by William Miller and Joy Hirsch at Yale University, is close to the effective image plane of the mosaic, and the plane that reveals the most packing regularity. Minor distortions are apparent in the lattice. For example, occasional cones are surrounded by five or seven neighbors instead of the usual six. The minimum center-to-center spacing between human foveal cones is about 2.8--3.0 lam (Refs 11,14). There are few examples across the animal kingdom of eyes with smaller photoreceptors than this. This may reflect a fundamental limitation imposed by the size of organelles and metabolic machinery that comprise these cells, or it may reflect the fact that more tightly packed receptors could result in optical

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crosstalk between adjacent receptors. In any case, this lower bound on cone size is likely to dictate the evolutionary design and present dimensions of many animal eyes 15. The Nyquist limit of the human central fovea is based on the spacing between rows of cones, which is ~/3/2 times the center-to-center spacing. (The row spacing is used here to calculate the Nyquist limit because the lowest spatial frequency gratings that can produce aliasing ambiguity in a triangular array are those oriented in one of the three orientations lying parallel to rows of sampling elements). The row spacing is 2.4-2.6 Ixm (0.50.54' of arc), yielding a Nyquist limit of 56--60 cycles deg -1. The Nyquist limit specifies the highest spatial frequency that a observer should be able to resolve without aliasing distortion. Fig. 3A shows a convenient technique, introduced by John Yellott 12'13, that can simulate the aliasing predicted by the foveal cone mosaic. His technique allowed the sampling effects of real photoreceptor mosaics to be assessed for the first time. A square wave grating whose spatial frequency is about 1.6 times the Nyquist limit is superimposed on an array of dots representing the cone locations in Fig. 2A. A low frequency alias can be seen that is distorted by the minor irregularities in the triangular array. It is now clear that human observers can visualize aliasing effects in their own foveas by viewing fine laser interference fringes, gratings whose contrast is unaffected by diffraction and aberrations in the eye's optics. Byram 16 and Campbell and Green 17 reported that very fine interference fringes viewed foveally resembled a pattern of wavy, scintillating lines. These patterns are confined to the fovea and remain centered on the line of sight as the eye moves. Williams4'~8 confirmed that these 'zebra stripes' are moir6 patterns produced by the cone mosaic, showing that they allow the spacing and packing arrangement of foveal cones to be measured in the living eye. With increasing spatial frequency, the grating distortion begins at the foveal center near 60 cycles deg- 1, in keeping with the Nyquist limit predicted from sampling theory. Moir6 patterns are lowest in spatial frequency when the spacing between the elements in the two patterns that produce the moir6 are equal. Thus, the zebra stripe pattern should be coarsest when the period of the interference fringe equals the spacing between rows

Fig.3. (A)A n array representing the locations of individual cone centers from the section shown in Fig. 2 A here samples a square wave grating whose fundamental frequency is 82 cycles deg -t, well above the monkey foveal Nyquist limit. A low frequency moir# pattern is visible that is distorted by minor perturbations in the receptor lattice. The moir# pattern resembles the zebra stripe patterns observed psychophysically with the human fovea. (B) An irregular mosaic o f points adjusted to have the same density o f elements as in Fig. 3A also samples the same square wave grating, allowing a comparison of the effects of regular and irregular sampling. The irregular array produces a coarse pattern of two dimensional noise instead of the wavy moir~ pattern produced by the foveal mosaic. The points indicate the locations of blue-sensitive cones stained with Procion yellow23. The statistics of this pattern resemble those observedfor the extrafoveal cone mosaic taken as a whole, so that the effects o f undersampling illustrated here are similar to those originally predicted by Yellottt2'13"3°. of foveal cones. For most observers, the patterns are coarsest at a frequency of about 110-120 cycles deg-1, which yields an estimate of the spacing between rows of cones just over 0.5 minutes of arc, in good agreement with the anatomical measurements. The moir6 pattern is wavy and distorted presumably as a consequence of the minor distortions in the lattice, and its scintillations un-

doubtedly result from eye tremor. The pattern changes with a 60 degree periodicity when the interference fringe is rotated, consistent with the anatomical evidence for triangular packing that is locally regular. The zebra stripe patterns can be seen up to frequencies of 150-160 cycles deg -~, at which point blurring by the apertures of individual cones presumably eliminates them.

196 Under normal viewing conditions, the eye's optics remove those spatial frequencies from the retinal image that exceed the foveal Nyquist limit. Indeed, it has long been known that the highest spatial frequency passed by the eye's optics is roughly matched to the resolution limit set by the spacing between cones at the foveal center ~9. The foveal cone mosaic is in a sense transparent in everyday vision, protected by optimal blurring from the aliasing distortion that would reveal its presence. The role in limiting foveal acuity played by neural mechanisms subsequent to optical blurting and cone sampling is not as important as previously thought. Recent psychophysical measurements of foveal contrast sensitivity obtained with interference fringes 2° suggest that neural blurring is roughly comparable in magnitude to optical blurring under optimal conditions. Observers require only 8% contrast on average to detect interference fringes at 60 cycles deg -1, so that neural contrast sensitivity has not expired even at the cone mosaic's Nyquist limit. This is consistent with the belief that some foveal ganglion cells may have receptive field centers fed by a single cone 2~'22. At the foveal Nyquist limit, a single bright or dark bar of the grating is no wider than a single row of foveal cones. The summation of signals from two or more adjacent cones onto the ganglion cells with the smallest receptive field centers would strongly attenuate such fine gratings, and the observed psychophysical performance would not be possible. Trichromacy and cone sampling The human retinal mosaic contains three subpopulations of cones, providing information about the distribution of color in the visual environment as well as the distribution of luminance. The retina has apparently used several strategies to minimize for spatial vision the cost of incorporating color. The blue-sensitive cones contribute little to spatial vision but extensively to color. They are sparse, accounting for less than 10% of the cone population everywhere in the retina, and are particularly scarce in the acute foveal center 23"24. This minimizes the loss in resolution sustained by the redsensitive and green-sensitive cones, which mediate our keenest vision. One might expect the sparse blue-sensitive cone mosaic to be particularly susceptible to aliasing, and this has been

TINS- Mav 1986

demonstrated in the laboratory 25. However, the blue-sensitive cone mosaic is reasonably well protected from aliasing in everyday viewing since the retinal image available at short wavelengths is severely blurred by chromatic aberration 8. The close proximity of the absorption spectra of the red-sensitive and greensensitive cones, coupled with the relatively smooth reflectance spectra of real scenes, produces signals from these two cone types that are highly correlated. The two cone mosaics can therefore combine to yield a luminance image that is finely sampled and that is degraded relatively little by the different spectral sensitivities of the redsensitive and green-sensitive cones. Visual resolution in the extrafoveal retina The protection afforded by the anterior optics of the eye from foveal aliasing is not available to the extrafoveal retina, since optical quality declines slowly26 while the cone sampling rate drops precipitously with increasing retinal eccentricity 14. For example, the average spacing between cones has increased by a factor of more than 3 at an eccentricity of only 4 deg. where the eye's optical quality is likely to be nearly optimal. This undersampling of the extrafoveal retinal image suggests that, in everyday vision, photoreceptor aliasing may play a more critical role in limiting extrafoveal visual performance than it does in the fovea. The extrafoveal cone mosaic, unlike that in the fovea, is an irregular lattice 9'27'2s. Hirsch and Miller27 have shown that the regularity of the cone mosaic degenerates rapidly with retinal eccentricity, approaching an asymptotic amount of disarray at an eccentricityofonly2-3 deg. Fig. 2B showsthe irregular distribution of primate cones about 5 deg from the fovea, The array is neither perfectly regular nor perfectly random, and the irregularity seems to be related to the intrusion of rods between the larger cone inner segments. Nagle 29 first pointed out that irregularity foils a straightforward application of the sampling theorem to the retina, since the sampling theorem was formulated for periodic sampling only. The extrafoveal mosaic does not have a true Nyquist limit, because at any eccentricity it has a continuous rather than a discrete distribution of cone spacings. However, one can define an average Nyquist limit for an irregular

mosaic as the Nyquist limit of a regular array with the same cone density. Yellott lz'13 showed that spatial frequencies below the average Nyquist limit are transmitted by the extrafoveal mosaic without appreciable aliasing distortion. However, spatial frequencies at and exceeding the average Nyquist limit have low frequency aliases that contain a broad range of spatial frequencies and orientations. Yellott's 'optical sandwich' technique revealed the effect of undersampling with an irregular array, as shown in Fig. 3B. The aliases of gratings imaged on the extrafoveal cone mosaic should resemble two-dimensional noise instead of the zebra stripe patterns observed foveally. Indeed, interference fringes, finer than the resolution limit for the extrafoveal retina, resemble scintillating, spatial noise 4. This is consistent with aliasing by the irregular cone mosaic, although contributions from optical factors, or undersampling by the ganglion cell array, are difficult to exclude. An observer attempting to resolve a fine grating seen through an irregular mosaic must distinguish the grating from the noise created by broad band aliasing, with the effectiveness of this noise growing with increasing spatial frequency. There is no single number that specifies the theoretical resolution limit of the mosaic. Indeed. there are no theoretical obstacles to prevent a visual system from resolving gratings well above the average Nyquist limit, provided it has the postreceptoral machinery to extract the gratings from aliasing noise. Unfortunately, quantitative models that predict the effect of aliasing noise on grating resolution are not yet far along. The apparent mismatch between the eye's optical quality and extrafoveal cone spacing has led to considerable speculation about its consequences. Yellott 12'13'3° proposed that evolution had defeated aliasing distortion by introducing irregularity into the mosaic. He suggested that the smearing of aliasing energy into a broad range of orientations and spatial frequencies would reduce its deleterious consequences for vision. The foveal mosaic, on the other hand, could afford to be regular because of the optical protection it enjoys. A major stumbling block in evaluating Yellott's int'riguing hypothesis is that it is difficult to quantify the visual benefit of irregular mosaics over regular ones. Indeed, Bossomaier, Snydcl and

T I N S - May 1986 Hughes 31, offered the alternative view that irregularity in sampling arrays is generally disadvantageous. They suggest that regular sampling is desirable over irregular sampling because large gaps between some cones, such as would be found in a random packing arrangement, would create blind spots. There has apparently been selection pressure against that packing strategy: YeUott has shown that the extrafoveal mosaic is sufficiently regular to avoid large blind spots. Bossomaier et al., argue that the residual disorder found in the peripheral retina is simply a consequence of the lack of selection pressure for a further increase in regularity, and that the desired regularity may be established in the array of ganglion cell receptive fields. They also point out that real scenes rarely contain the highly periodic patterns required to produce the striking moir6 effects characteristic of regular sampling. Natural images are typically composed of a broad range of spatial frequencies to begin with, so that their aliases are likely to be broad band even if sampled by a crystalline mosaic. This, they argue, would reduce the selection pressure for irregularity. Whatever the ultimate resolution of this controversy, it is clear that a number of factors reduce the consequences of aliasing distortion in everyday, extrafoveal vision besides irregularity in the cone mosaic. For example, the eye is probably not well accommodated to objects in the peripheral retina most of the time. This reduces the contrast of high spatial frequencies in the image which would in turn reduce aliasing noise. It has been suggested that light scatter in the pre-receptoral layers of the extrafoveal retina has a similar effect 32. Furthermore, natural scenes typically contain most of their power at low spatial frequencies that would be less apt to introduce aliasing distortion33. If aliasing were a severe problem for extrafoveal vision, one might expect the visual system to take the direct approach of adopting a high enough cone sampling rate to avoid it. This has not been done, even though the costs of doing so are not obviously high. Rods and cones must compete for the available retinal area to catch photons. It might be thought, therefore, that the cone sampling rate is limited strictly by the quantum catching requirements of rods. This is not the case, however, since inner segments of extrafoveal cones are more than twice the diameter of those of

197 foveal cones (see Fig. 2B). One could tile the extrafoveal retina with cones the size of those in the fovea, without sacrificing the rod quantum catch, and easily achieve a sampling rate more than double that which exists. It would seem that the metabolic cost of maintaining more extrafoveal cones, or perhaps the loss in quantum yield per cone 34, outweighs the benefits of escaping aliasing distortion. The extrafoveal retina is primarily designed for the purpose of detecting objects, leaving the fovea the task of scrutinizing them following a fixation eye movement. Real optical systems have shallow transfer functions, so that it is impossible to remove the offensive spatial frequencies that introduce aliasing noise without reducing image contrast at spatial frequencies below the Nyquist limit as well. Consequently, the best trade off in peripheral vision, where there is a premium on object detection rather than pattern discrimination, may be to tolerate some aliasing noise in exchange for higher contrast in the sub-Nyquist range of spatial frequencies. This may be why undersampling by the cone mosaics of animals seems to be widespread33. The match between optics and receptor spacing in the human fovea may be the exception rather than the rule. In the periperal retina, neural mechanisms subsequent to the cone mosaic impose the most important limitations on visual acuity. Peripheral visual acuity falls well below the mosaic, s average Nyquist limit35, which is a conservative measure of the mosaic's resolving power. This is not surprising since monkey ganglion cell density is five times lower than cone density in the far periphery36, and the centers of ganglion cell receptive fields are fed by many cones. There are a number of uncertainties that must be resolved before the spatial limitations of the ganglion cell array, or subsequent arrays in visual cortex, can be accurately compared with psychophysical measures of resolution37. The low sampling rate of peripheral ganglion cells leads to the possibility that they may produce aliasing over and above that introduced by the cone mosaic. Aliasing at this stage might be avoided by having receptive field centers that are large compared with the spacing between them. This would seem to be an easy strategy to implement, but there is at present insufficient information to decide exactly to what extent it has been. The appropriate subpopula-

tion of ganglion cells that contribute in resolution tasks has yet to be identified. It seems likely that P~t-and Pl3-ganglion cells38 receiving blue-sensitive cone input to their centers can be excluded. But should on and off center arrays be considered together or separately39'4°? How are the relevant receptive fields distributed.'? Is the array regular or irregular? Answers to these questions will provide new insight into the structural factors that ultimately constrain the capacity to see.

Acknowledgements I thank Joy Hirsch, Peter Lennie, William Miller, Gary Sclar, Allan Snyder, and John Yellott for commentsof early versionsof the manuscript. I also thank Joy Hirsch and William Miller for providing the photographs of primate retina (Fig. 2) and the fovealcone locations(Fig. 3A); and William Merigan for providing the blue-sensitive cone locations (Fig. 3B). Selected references 1 Campbell, F. W. and Gubisch, R. W. (1966) J. Physiol. (London) 186, 558-578 2 Miller, W. H. and Bernard, G. D. (1983) Vision Res. 23, 1365-1369 3 Snyder, A. W. and Miller, W. H. (1977) J. Opt. Soc. Am. 67, 696-698 4 Williams,D. R. (1985) VisionRes. 25,195--205 5 MacLeod, D. I. A., Williams, D. R. and Makous, W. (1985) Invest. Ophthalmol. Visual. Sci. Suppl. 26, 11 6 Helmholtz, H. (1911) in Handbuch der Physiologischen Optik, Verlag yon Leopold Voss. Translated by Southall, J. P. C. (1924) Helmholtz" s Treatise on Physiological Optics, Vol. II, The Sensations of Vision, Optical Society of America 7 Bracewell R. N. (1978) The Fourier Transform and its Applications, 2nd edn, McGraw-Hill 8 Yellott, J. I., Jr., Wandell, B.A. and Cornsweet, T. N. (1984) in Handbook of Physiology, Section 1, pp. 257-316, Am. Physiol. Soc., Bethesda, MD 9 Borwein, B., Borwein, D., Medeiros, J. and McGowan, J. W. (1980) Am. J. Anat., 159, 125-146 10 Hirsch, J. and Hylton, R. (1984) Vision Res. 24, 347-355 11 Miller, W. H. (1979) in Handbook of Sensory Physiology, Volume VII/6A, (Autrum, H., ed.), pp. 70-143, Springer-Verlag 12 Yellott, J. I., Jr. (1982) Vision Res. 22, 1205-1210 13 Yellott, J. I., Jr. (1983) Science 221,382-385 '14 Osterberg, G. (1985) Acta Ophthalmol. Suppl. 6, 11-103 15 Kirschfeld, K. (1976) in Neural Principles in Vision, (Zcttler, F. and Weiler, R., eds), pp. 354-370, Springer-Verlag 16 Byram, G. M. (1944) J. Opt. Soc. Am. 34, 718-738 17 Campbell, F. W. and Green, D. G. (1965) J. Physiol. (London) 181,576-593 18 Williams, D. R. (1985) Invest. Ophthalmol. Visual. Sci. Suppl. 26, 10 19 Shlaer, S. (1937) J. Gen. Physiol. 21,165-188 20 Williams, D. R. (1985)J. Opt. Soc. Am. A. 2,

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198 1087-1093 21 De Monasterio, F. M. and Gouras, P. 11975) J. Physiol. (London) 251,167-195 22 Derrington, A. M. and Lennie, P. (1984) J. Physiol. (London) 357, 21%240 23 De Monasterio, F. M., McCrane, E . P . , Newlander, J. K. and Schein, S. J. (1985) Invest. Ophthalmol. and Visual. Sci. 26/3,

28%302 24 Williams, D. R., MacLeod, D. I. A. and Hayhoe, M. M. 11981) Vision Res. 21, 13411356 25 Williams, D. R. and Collier, R. J. (1983) Science 221,385-387 26 Jennings, J. A. M. and Charman. W.N. (1981) Vision Res. 21,445-455 27 Hirsch, J. and Miller, W. H. (1985) Invest.

Ophthalmol. Visual. Sci. Suppl. 26, 10 28 Wassle, H. and Riemann, H. J. (1978) Proc. R. Soc. London Ser. B. 200, 441-461 29 Nagle, D. (1981) Invest. Ophthalmol. Visual. Sci. SuppL 20, 123 30 Yellott, J. I., Jr. (1984) Vision Res. 24, 281-282 31 Bossomaier, T. R. J., Snyder, A. W. and Hughes, A. (1985) Vision Res. 25, 145-147 32 Carlson, C. R. and Anderson, C. H. (1985) Invest. Ophthalmol. Visual, Sci. Suppl. 26,140 33 Snyder, A. W., Bossomaier, T. R. J. and Hughes, A. (1986) Science 231,499-501 34 Snyder, A. W., Laughlin, S. B. and Stavenga, D. G. (1977) Vision Res. 17, 1163-1175 35 Frisen, L. and Frisen, M. (1976) Acta Ophthalmol. 54, 437-444

36 Perry, V. H. and Cowey,A. (1985) Vi.~ionICr,.

25, 1795-1810 37 Hughes, A. 11977) in Handbook of Sensor~ Physiology, Vol. VlI/5 (Autrum, H., Jung, R., Loewenstein,W. R., MacKay, D. M. and Teuber, H. l.. eds), pp. 613-756, SpringerVerlag 38 Perry, V. H., Oehler, R. and Cowey, A. 11984) Vision Res. 12, 1101-1123 39 Hughes, A. (1981) Exp. Brain Res. 42, 196202 40 W/issle,H., Boycott, B. B. and llling, R.-B. (1981) Proc. R. Soc. Lond. B. 212, 177-195 David R. Williams is at the Center for Visual Science, Rochester, N Y 14627, USA.

Exploring visual transduction with rec DNA techn ues

nant

Wolfgang Baehr and Meredithe L. Applebury The mechanisms of photoreception and intracellular signalling in vertebrate photoreceptors have been elucidated in remarkably complete molecular detail. This background has allowed D N A fragments encoding visual pigments and accessory proteins of the cGMP cascade to be cloned. Information about gene structure and amino acid sequences of the protein products suggest that at least two o f the proteins in the visual pathway, rhodopsin and G-protein, are members of large families of genes derived from ancient ancestral precursors. Comparison of amino acid sequence information for many members in each family emphasizes the homologies and the important aspects of structure and function of these proteins. The mechanisms used for visual signalling are common to those used by modulatory neurotransmitters, and serve as a prototype for the molecular basis of neuromodulatory signalling. Visual information processing begins with the absorption of light by photosensory receptors. These receptors have the task of transducing photon energy into a chemical form that can trigger a series of intracellular events and ultimately effect a transient change in plasma membrane conductance- the visual sensory response. The molecular basis of this signalling process has been biochemically well-documented in vertebrate photoreceptors, and an ever growing knowledge of invertebrate photoreceptors suggests that the mechanisms will be similar. Now, several research groups have applied recombinant D N A techniques to identify and study the genetic information encoding photoreceptor proteins. An era has begun in which the molecular processes of photosensory signalling can be examined in vertebrate cone cells, in cells in small invertebrates, and even in unicellular organisms. This approach is different from the classical biochemical approach to this field. Through the isolation and characterization of gene fragments, gene structures and protein

sequences are deduced. Comparison of sequences of several proteins in a gene family can then give specific insight into function. It is hoped that in the future, the gene fragments can be subjected to site-specific mutagenesis, the molecules can be artificially expressed, and the resultant proteins studied using traditional biochemical methods. In some organisms genetic replacement can be achieved and used to study the effects of protein product replacement on physiological function. The vertebrate retina, particularly the bovine retina, has provided a rich source of cells with an abundance of molecular components available for the study of visual signalling. The key components of the photosensory triggering system have been isolated and extensively characterized; proteinprotein interactions have been defined, and the membrane topological organization outlined 1-4. These components include the membrane receptor rhodopsin and a set of peripherally bound membrane proteins: the guanine nucleotide binding protein, termed G-

© 1986,ElsevierSciencePublishersB.V.. Amsterdam 11378 5912/86/$112(~l

protein (transducin), a cGMP phosphodiesterase (PDE), rhodopsin kinase, and a regulatory protein termed 48 kDa (arrestin or S-antigen). The sequence of functional interactions is noted in Fig. 1, and is briefly summarized below. Absorbtion of a photon activates rhodopsin, which serves as a catalyst for charging ~-subunits of G-protein with GTP. Once charged, the Gctsubunit dissociates from the GlS~,subunit and activates cGMP-PDE. It is postulated that Got releases or removes the small y-subunit of P D E and thus lifts the inhibition of P D E enzymatic activity5. The resulting active P D E enzyme rapidly depletes cytoplasmic cGMP. The enzyme cascade provides remarkable inherent amplification. Rhodopsin can charge hundreds of Gproteins during its activated lifetime, and each activated P D E is capable of hydrolysing as many as 103 molecules of cGMP per second. One photon can lead to the hydrolysis of millions of cGMP molecules within a second 6. Recently, the process of identifying the series of signalling events has been completed by studies demonstrating that cGMP allosterically controls the open state of a plasma membrane channel; therefore, cGMP depletion leads to channel closure and thus plasma membrane hyperpolarization (see article by T. Lamb in this issue). Of course, some facts are still obscure. How this cascade is modulated or turned off is not well understood. Modulation may be achieved through phosphorylation of rhodopsin by rhodopsin kinase 7. The phosphorylated