THE JOURNAL OF COMPARA-
NEUROLOGY 298:472493 (1990)
Developmental Redistributionof PhotoreceptorsAcross t h e M m nemestrina (RgtdMacaque)Retina ORIN PACKER, A ” A E. HENDRICKSON, AND CHRISTINE A. CURCIO Departments of Psychology (O.P.),Biological Structure (A.E.H., C.A.C.), and Ophthalmology (A.E.H.,C.A.C.), University of Washington, Seattle, Washington 98195
ABSTRACT Redistributions of monkey cones and rods during the first year after birth include a fivefold increase in peak foveal cone density from 43,000 to 210,000 cones/mm2, a decrease in the diameter of the rod-sparse area, and a two- to threefold decrease in peripheral photoreceptor density. Two weeks before birth, higher cone density is already apparent in the future fovea, as are the nasotemporal asymmetry in cone distribution, a higher density “cone streak” along the horizontal meridian, a large rod-sparse central fovea, and a ring of high rod density. Despite the early appearance of these basic patterns, photoreceptor distribution is not mature until 1 to 5 years postnatally. Total cones varied from 4 million at birth to 3.1 million in the average adult. The two oldest eyes had fewer cones, suggesting up to a 25% loss late in development. There were 60 to 70 million rods in the adult macaque retina and little evidence of postnatal changes in number. Neither of these small changes is sufficient to account for the reduction in peripheral photoreceptor density and both are in the wrong direction to explain increasing foveal density, ruling out a major role for either photoreceptor death or generation. Retinal area increased by a factor of 2.4 from 2 weeks before birth to adulthood. In contrast, the posterior pole of the retina was dimensionally stable throughout this period, with the distance between the fovea and optic disc varying nonsystematically from 3.37 to 4.05 mm. Retinal coverage of the globe was also stable at 48-60%. Thus postnatal growth can be ruled out as a factor in the density changes occurring in central retina. Adult retinas have a higher proportion of both cones and rods in midperiphery, whereas young retinas have a higher proportion of photoreceptors in far periphery. It appears that photoreceptors are radially redistributed from peripheral toward central retina during postnatal development, resulting in the marked increase in foveal cone density and the decrease in the eccentricity of the rod ring. Up to 13 weeks postnatally, midperipheral growth of the retina is substantial and increases with eccentricity. At later ages, expansion continues only in the very far periphery. Retinal growth appears sufficient to explain the decreases in peripheral rod and cone density with age. These and previous data strongly suggest that differentiated photoreceptors, with complex cytology and synaptic contacts, migrate toward the foveal center, explaining the increase in foveal photoreceptor density. The distance that cones migrate between E 152 and adulthood increases from 0 at the foveal center to a maximum of 0.230 mm for cones originally located at 1 mm of eccentricity in the immature retina. This migration is equal to published measurements of the length of fibers of Henle for cones located at 0.75 mm in the adult. These data suggest that at the center of the fovea, the length of the fiber of Henle is due to outward migration of the bipolar neuron to which the photoreceptor is connected, but that by 0.75 mm, the length of the fiber is due to the centralward migration of the photoreceptor. Key words: rods, cones, topography, development
Photoreceptors are unevenly distributed across the macaque retina (Curcio et al., ’87a; Packer et al., ’89; Perry and COwey’ ’85). ‘One exceeds 2oo’ooo at the center of the fovea and then with increasing eccentricity. A high density “cone streak” is A
1990 WILEY-LISS, INC.
AcceptedJune 1, 1990, Address reprint requests to Orin Packer, Ph.D., who is now a t the Center for Visual Science, 274 Meliora Hall, University of Rochester, Rochester, Ny 14627.
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DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY found along the horizontal meridian, within which nasal cone density consistently exceeds temporal density at equivalent eccentricities. Rod density, in contrast, is zero at the foveal center, increases rapidly to the crest of a ring of high rod density near the eccentricity of the optic disk, and then decreases into the periphery. Peak density in the ring is 177,000 rods/mm2in superior retina. How do these complex patterns of photoreceptor distribution develop? They could be established at the time of photoreceptor generation and change little thereafter, or they could be gradually formed over an extended period by photoreceptor redistribution. Knowing the pattern and time course of redistribution would provide clues to underlying developmental processes. It is already known that the spatial distribution of photoreceptors changes during development. In the monkey neonate, cone density at the center of the fovea was only a fourth that of the adult, gradually increasing to adult values during the first 2 years of life (Hendrickson and Kupfer, '76). The fovea of the human neonate was even more immature than that of the monkey (Abramov et al., '82; Hendrickson and Yuodelis, '84; Yuodelis and Hendrickson, '86). In short, the primate photoreceptor mosaic undergoes a long period of maturation that extends past 1 year in the macaque and possibly past 4 years in the human. The development of the peripheral cone and rod distributions has not been studied in either monkey or human. Unanswered questions about the development of the photoreceptor mosaic include: (1)Where do the cones subserving the increase in foveal density come from? (2) Are there developmental changes in peripheral cone distribution? (3) When do the asymmetries in cone and rod distribution develop? (4)Do rods undergo changes in distribution? (5) How do known mechanisms of neural development, such as cell generation, cell death, cell migration, or tissue growth (Lund, '781, apply to the photoreceptor mosaic? We have investigated these questions by measuring cone and rod density across the entire retina of an age-spaced series ofM. nemestrina (pigtail macaque) monkeys, using methods previously applied to the adult M . nemestrina retina (Packer et al., '89).
METHODS Animals. Table 1 shows the ages and sexes of the animals whose retinas were used in this study. The 3 adult animals used for comparison were 5 years of age or older and are individually described in Packer et al. ('89). Seven eyes ranging in age from 2 weeks prenatal (E152) to 60 weeks postnatal (P60 wk) were obtained through the Regional Primate Research Center at the University of Washington. The gestational ages of the animals were
known to within 24 hours, and no postnatal animals were included that were premature by either birthdate or weight. The age interval between animals was chosen to be larger at older ages because development of the primate fovea slows with age (Yuodelis and Hendrickson, '86). Procedure. Tissue processing techniques are detailed in Packer et al. ('89) and Curcio et al. ('87b). Briefly, the eyes were enucleated under deep barbiturate anesthesia either prior to perfusion or following an intravascular saline rinse. The horizontal, vertical, and axial equatorial diameters of each eye were measured just prior to immersion in 0.1 M phosphate-buffered 4%paraformaldehyde. In order to speed fixation of the retina, a small volume of fixative was either injected through the optic nerve head into the vitreous chamber or allowed to pass through a puncture made near the limbus. We were careful to keep the globe fully round during initial immersion to avoid fixing folds in the thin sclera. The fixed globe was cut into a belt along the horizontal meridian and an inferior and superior cap. The neural retina was separated from the sclera and pigment epithelium, flattened on a plastic slide, and coverslipped under the clearing agents glycerine and dimethyl sulfoxide. This wholemount was viewed under Nomarski differential interference contrast optics (Allen et al., '69).These techniques eliminate the need to section and stain the retina, minimizing tissue distortion. Photoreceptors were counted manually with the aid of a small morphometry computer (Curcio and Sloan, '86). Sampling locations were more closely spaced near the center of the fovea where photoreceptor density changes rapidly, than in the periphery where density changes more slowly. About 200 samples were counted in each retina. Data analysis. Maps of photoreceptor density, which are particularly good for observing large-scale features, were prepared from sampling locations and their associated photoreceptor densities using computer methods (Curcio et al., '89). Graphs of photoreceptor density along the cardinal meridians, which are particularly useful for making quantitative comparisons between meridians, were prepared from these maps by resampling along a meridian. The total numbers of rods and cones were estimated by interpolating between sampling points and summing photoreceptors across the retina (Packer et al. '89). Spherical coordinate system. In order to facilitate the analysis of retinal topography in animals of different ages and with different eye sizes, a spherical coordinate system was used to represent retinal position. Throughout this work, the retina is modeled as slightly more than half of the surface of a sphere (Fig. lA, also Curcio et al., '89), whose coordinate system is defined by the fovea (F) and optic disk (D). The horizontal meridian is represented by
TABLE 1. Vital Statistics and Retinal Parameters Age' Retinal area (mm') Cone peak density (mnes/mm2x 103) Eccentricity o f r d peak (rnm) Rod peak density (roddmm2x lo3) Total cones ( x 106) Total rods ( x 10b) Fovea to disk (mrn)(centerto center) Q ofocular sphere covered by retina' Diameterof globe (mm)
El52 335
El65 365
P3 wk 488
P6 wk 535
P13wk 505
P26 wk 607
P60 wk 564
Adult' 800
43.3 NA NA 3.73 NA 3.68 54 14.10
85.9 NA NA 3.97 NA 3.67 52 15.00
65.0 6.29 211.5 3.89 70.1 3.72 55 16.75
65.7 4.94 200.0 3.61 NA 4.00 59 17.05
80.9 6.95 233.2 3.75 68.5 3.76 48 18.30
157.4 NA NA 3.81 NA 4.05 53 19.10
132.8 5.91 168.2 3.31 57 3
210 2.09 177 3.08 60.1 3 37 55 21.45
3.46 50 19.00 ~~
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Fig. 1. The spherical coordinate system used for retinal modeling. A. The Coordinate system is defined by the ray GF running from the anterior part of the eye, through the center of the retinal sphere, and ending at the fovea (F).The horizontal meridian of the spherical retina is the arc ADFC through the fovea and disk (D). The vertical meridian is the arc BFE through the fovea perpendicular to the horizontal meridian. B. For viewing, the retinal sphere is mapped onto a flat surface. The center of the map represents the center of the fovea (small circle) and the larger circle (Di represents the disk. The effect of this mapping
transformation is to leave the central retina relatively undistortcd while stretching the peripheral retina in the tangential direction, represented by the rings, but not in the radial direction, represented by the rays. The disk is always on the nasal (N) meridian. The superior vertical meridian is marked S, and the inferior vertical meridian is marked I. The rings are spaced at intervals of 30". Note that in this and subsequent figures, angles are measured in degrees on the retinal sphere, not in visual degrees. T, temporal.
the arc ADFC through the fovea and disk, and the vertical meridian is represented by the arc BFE through the fovea perpendicular to the horizontal meridian. The line GF lies near the optic axis with the point G at the anterior surface of the eye. For display, the spherical representation of the retina is mapped onto a plane (Fig. lB), in the same way that a cartographer represents the polar regions of the earth on a flat map. Conceptually, this mapping is approximately what would result from removing the spherical retina from the globe with its open end up and squashing it flat. The representation of central retina remains nearly undistorted. The representation of peripheral retina undergoes considerable tangential elongation (along the rings), but radial distances (along the rays) are not distorted (Fig. 1B). The map is centered on the fovea (small circle), which together with the disk (D) represents the horizontal meridian (ADFC of Fig. lA), labeled N (nasal)-T (temporal). The vertical meridian (BFE of Fig. 1A) maps onto the line labeled S (superior)-I(inferior).The optic disk (D) is on the nasal side of the horizontal meridian. The spacing between the rings and rays of the grid overlaying each topographic map is measured in degrees on the retinal sphere. (Spherical degrees are not to be confused with visual degrees which are measured relative to the center of the optics of the eye rather than relative to the center of the ocular sphere.) One degree of arc measured on the surface of the retinal sphere equals ( T T . r)/180 mm where r is the radius of the retinal sphere in mm. The color maps are normalized to approximately the same size, concealing an age-related increase in retinal area. The measured diameter and area of each individual retina are shown in Table 1.
whereas rods increase in density to the eccentricity of the optic disk and then decrease more peripherally (Packer et al., '89). Both cone and rod inner segment diameters increase with distance from the fovea. Changes in the organization of the photoreceptor mosaic as a function of eccentricity at a given age can be seen by scanning across the rows of Figure 2. At birth (Fig. 2, top row), cone and rod density are lower in the foveal center and higher in the far periphery than they are in the adult. The diameter of both cone and rod inner segments is similar across the retina. Cone inner segment diameter increases slightly from the fovea to 0.5 mm of eccentricity, but, if anything, decreases from 0.5 to 5 mm of eccentricity. Cone inner segment diameter across the retina ranges from about 3.6 pm (central) to 6.5 pm (peripheral) at birth, whereas the adult range is from 2.3 to 10 pm (Packer et al., '89). At P13 weeks (Fig. 2 middle row) and P60 weeks (Fig. 2 bottom row), the pattern is qualitatively adultlike, although the foveal cones are still larger than their adult counterparts (3.7 and 2.85 IJ-mvs. 2.3 pm). Changes in the organization of the photoreceptor mosaic with age at a given eccentricity can be seen by scanning down the columns of Figure 2. As discussed, the inner segment diameter of the foveal cones decreases by 25%1 from birth to P60 weeks. At 0.5 mm of eccentricity, cone size decreases and cone density increases from birth to P13 weeks and then stabilizes. This area appears to be a relatively stable zone during development between the more central areas, where cone inner segment diameter decreases, and the far periphery, where diameter increases. At 5 and 10 mm of eccentricity, cone density decreases with age, whereas cone inner segment diameter increases. There is-little change in rod inner segment diameter at 5 mm, but a t 10 mm, rod diameter increases with age. Thus during develoDment. foveal cones decrease in inner s e m e n t dianieter aAd both cones and rods increase in density. In the periphery, rod and cone density decreases.
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Appearance of the photoreceptor mosaic. In the adult retina, cones decrease in density with eccentricity,
DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY Quantitative changes in cone density. The 2 main features of cone topography in the adult pigtail macaque are a sharp central to peripheral gradient, and a nasotemporal asymmetry in the periphery. The former develops considerably during the period of this study, and the latter does not. The first major change leading to the sharp central to peripheral gradient is a fivefold increase in foveal cone density from just before birth to adulthood (Fig. 3, Table 1). In the adult, cone density at the foveal center peaks at 210,000 conedmm’ (Fig. 3E, data from Packer et al., ’89), drops to 100,000 cones/mm2 at 0.15 mm and to 50,000 cones/mm2at 0.75 mm of eccentricity. In contrast, at El52 (Fig. 3A), foveal cone density is low and almost uniform, ranging between 25,000 and 35,000 cones/mm2.Two small patches of higher density mark the location of the developing fovea, but cone density peaks at only 43,000 cones/mm’. By P3 weeks (Fig. 3B), central cone density has increased to 65,000 cones/mm’ and by P13 weeks (Fig. 3C) is 81,000 cones/mm2, still only 40% of the adult average. By P60 weeks (Fig. 3D), peak cone density reaches 133,000 cones/ mm2,which is still only 63% of average adult density. A plot of peak density vs. age (Fig. 4) indicates some individual variability both in the rate of development and in absolute peak cone density. This should not be surprising given the considerable variability of adult peak cone density in both human and monkey (Curcio et al., ’87a; Hawken et al., ’88; Packer et al., ’89,Wikler et al., ’881, but it makes it difficult to determine when peak cone density reaches adult values. Peak density at 6 months (157,000 cones/mm2)is within the range of values that others have reported for adult macaques (for a summary see Table 1, Packer et al., ’891, although it is still 25% below the values that we have previously reported. Fully mature values probably are not attained until sometime between 1and 5 years postnatally. Figure 5 shows the rapid change in cone density along the horizontal meridian from the foveal center to 1 mm of eccentricity for monkey retinas of different ages. The P 3 week retina has an almost flat density profile. At successively older ages, a combination of sharply higher foveal cone density and relatively little change in density between 0.6 mm and 1 mm results in a much steeper density gradient from the foveal center to 1mm. Individual variability is shown most vividly by the reversal of the curves representing the P26 week retina and the P60 week retina, and by the uneven spacing of the curves representing retinas of intermediate age. There is little difference between nasal and temporal cone density at these central eccentricities and the same is true of the superior and inferior meridians as shown by the color maps (Fig. 3). The similar densities of all 4 meridians show that cone density in central retina is radially symmetric. The second major change that sharpens the central to peripheral gradient is a general reduction of peripheral cone density with age (Fig. 6). In adult peripheral retina (Fig. 6E), cone density declines continuously to 1 or 2% of peak density at the edge of the retina. This decline is most rapid near the fovea and then slows with increasing eccentricity. As a result, cone density across most of far peripheral retina ranges between 1,000 and 4,000 cones/mm2. Imposed on this pattern are 2 asymmetries most easily seen in Figure 6E. First, cone density is higher along the horizontal than along the vertical meridian, forming a “cone streak.” Second, peripheral cone density at eccentricities greater than the optic disk is up to 3 times higher at equivalent eccentricities in nasal than in temporal retina, resulting in
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isodensity contours that extend farther into nasal than temporal retina. In the El52 retina (Fig. 6A), peripheral cone density ranges from about 6,000 cones/mmz (green) along the temporal and superior edge of the retina, to above 16,000 cones/mm2 (white central region). Even at El52 cone density is higher along the nasal horizontal meridian. This remains true throughout development. The high cone density in superior retina was unique to this eye, although one retina (birth, not shown) showed similar high density regions in both superior and inferior retina. By P3 weeks (Fig. 6B), cone density over large areas of the periphery ranges from 7,000 to 10,000 cones/mm2. At the temporal edge of the retina, cone density is 6,000 to 8,000 cones/mm’, whereas at the nasal edge it ranges between 9,000 and 11,000 cones/mm2. By P13 weeks (Fig. 6C), peripheral cone density has declined to 3,000 to 5,000 cones/mm’ temporally and 8,000 to 9,000 cones/mm2nasally, again illustrating the “cone streak.” The region of higher density extending into inferior retina was not found in the other retinas. Although the reduction in peripheral cone density continues to adulthood, the rate of decline has slowed markedly so that at P60 weeks (Fig. 6D), temporal cone density is 2,000 to 3,000 cones/mm2 and nasal 6,000 to 9,000 cones/mm2. Temporally, cone density is only slightly elevated above adult values, but nasal cone density is still twice that of the adult. In general, the younger retinas have higher cone density over the entire periphery, although there is some variability between animals (Fig. 7). For example, between 4 and 8 mm of eccentricity, the El52 eye has lower cone density than the newborn eye (E165, Fig. 7A) along the nasal horizontal meridian, the opposite of the general trend. The rate of density decrease as a function of age is greater near birth as shown by the greater separation between the density curves of the youngest pair of retinas compared to the older 5 retinas. In older nasal retina (Fig. 7A), the curve levels off at eccentricities greater than 4 mm, whereas cone density in the immature retinas declines slowly or not at all from 4 mm to the periphery. The nasal (Fig. 7A) and temporal (Fig. 7B) profiles are similar out to 4 mm, but beyond this, temporal cone density decreases more rapidly. The nasotemporal asymmetry results from these differences in density change. A similar developmental pattern can be seen along the superior and inferior vertical meridians (Fig. 8 ) , except that both superior and inferior far peripheral retina showed a sharp decline in density to below 2,500 cones/mm2. The degree of nasotemporal asymmetry was quantified by calculating a cone density ratio at equivalent eccentricities in nasal and temporal retina for each age. For all ages this ratio increases as a function of eccentricity. The nasotemporal ratio ranges from 0.75-1.5 at 30” and increases to 1.25-3.0 by 70“.The lack of obvious change in the ratio with age indicates that the nasotemporal asymmetry is already nearly adultlike at E152. In summary, cone density in peripheral retina declines rapidly up to P13 weeks and then slowly to 60 weeks. Adult densities are reached sometime between 1 and 5 years of age. The horizontal cone streak and the nasotemporal asymmetry are present as early as E152. Finally, there are individual differences in the topographic patterns, such as the high superior cone density at El52 weeks and the high inferior cone density at P13 weeks. Whether these differences are later reflected in adult topography or disappear with age is not known.
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Fovea Fig. 2. A series of horizontal optical sections (Nomarski differential interference contrast images) from three immature retinas, taken at the level of the photoreceptor inner segments at the fovea and along the nasal horizontal meridian at eccentricities of 0.5 mm, 5 mm, and 10 mm. The columns represent single locations at a series of ages and the rows represent a series of locations at a single age. In the foveal column,
0.5 mm Nasal all of the profiles are cones with the exception of a single rod (small black arrow) in E l 6 5 At 0.5 mm, the large profiles are cones and the smaller profiles are rods. A single representative rod is labeled (small black arrow) in each panel. At 5 and 10 mm, the large cones and the small rods are unambiguous. The scale bar (lower right panel) = 10 pm.
DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY
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Fig. 3. Maps of foveal cone density in five animals: (A) E152, (B) P3 weeks, ( C ) P13 weeks, (D) P60 weeks, (Elaverage of 3 adults. The foveal maps are keyed to the color scale which ranges from 0 to 200,000 cones/mmLwith a contour interval of 12,500conesimm'. The rings of the foveal maps are spaced at intervals of 2".
DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY
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Changes in rod density. There are 5 main features of adult monkey rod topography (Packer et al., ’89; Fig. 9D). First, the fovea is rod sparse, containing no rods within about 0.02 mm of the foveal center and only scattered rods within 0.1 mm. Second, there is a ring of high rod density, the “rod ring,” at the eccentricity of the optic disk, which reaches its maximum density of 177,000 rods/mm2in a “hot spot” in superior retina and its minimum density as it crosses the horizontal meridian. Third, the central rod distribution is radially asymmetric, as shown by the fact that the contours of the rod sparse central area (black, blue, and green) are displaced toward inferior retina. Fourth, peripheral rods are much more symmetrically distributed around the fovea than the cones are, although close examination reveals a slight displacement of the isodensity contours toward nasal and superior retina. Finally, peripheral rod density declines to 25,000 rodsimm’ or less throughout the far periphery. In the fovea, we confirmed Hendrickson and Kupfer’s (’76) observation that the size of the rod-sparse area decreases during development. The diameter of the zone containing less than 12,500 rods/mm2 (dark blue region in Fig. 9) decreases from about 0.8 mm in the youngest retina to about 0.5 mm in the adult retina (Fig. 9D). The only exception to this trend was the P13 week retina (Fig. 9B), whose rod-sparse zone was about the same diameter as the adult, most likely due to high between-individual variability in foveal rod densities. The most striking difference between the P3 week (Fig. 9A) and adult periphery (Fig. 9D) is the high density of rods in the periphery of the young eye, as shown by the color shift from the higher density oranges and reds to the low density blues and greens. Rod density at P3 weeks rarely dipped below 100,000 rods/mm’ even at the retinal edge and the rod ring was located more peripherally than the optic disk. This eye was unusual in that it had its highest rod density of 211,000 rods/mm2 in inferior retina. At P6 weeks (not shown), rod topography looked substantially unchanged with a peak rod density of 200,000 rods/mm2 in superior retina. However, by P13 weeks (Fig. 9B), rod density in the far periphery had declined to 50,000 to 75,000 rods/mm‘, making the rod ring more apparent. This retina has both superior and inferior “hot spots,” which
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have higher rod densities than any other retina we examined. Density actually peaks at 230,000 rods/mm2 in inferior retina. We have found no adult monkey retinas with peak rod density in inferior retina, but Curcio et al. (’90)do report this in one of 6 human eyes. Although the basic adult pattern of rod topography is in place by P60 weeks of age (Fig. 9 0 , peripheral rod density is still higher than that of the adult. The rod ring has a “hot spot” of 170,000 rods/mm2 in superior retina and a gap near the optic disk, but its eccentricity still exceeds that of the adult. Thus rod, like cone, topography must undergo additional remodeling between 60 weeks and adulthood. Figures 10 and 11 show that the less mature retinas generally have higher rod density within 10 mm of eccentricity, although there is considerable variability. At each age, nasal and temporal rod density profiles are similar in shape out to about 8 mm of eccentricity. Beyond this point rod density declines more rapidly in temporal retina. The nasal meridian is longer at all ages and the adult retina is substantially larger than the P60 week retina. The superior and inferior vertical meridians have similar lengths, but there is considerable variability in the highest rod density, probably depending on whether or not the meridian crosses a “hot spot.”
Developmentalchange in the totalnumber of photoreceptors Cones. The total number of cones varied from a high of almost 4 million at birth to a low of 3.1 million in the average adult (Table 1).For the first 6 months of development, the totals varied by only a few percent in a nonsystematic way, suggesting that the cone population was stable and that the differences represented individual variation. The 2 lowest values occur at P60 weeks and in the adult, so there may be up to a 25% decline in cone population during later development. Tritiated thymidine labeling shows that few if any cones are being generated over this period in monkey retina (LaVail et al., ’83; Rapaport et al., ’871, so no more than 25% of the cones could be lost to cell death postnatally. This loss is too small to account for the 250-300% reduction in peripheral cone density and completely in the wrong direction to explain the fivefold increase in foveal density. These data suggest that we must look to growth of the retina or cone migration for explanations of cone redistribution during development. Rods. On average, there are 60 to 70 million rods in the adult macaque retina (Table 1).Tritiated thymidine autoradiography (Rapaport, ’87) shows that some peripheral rods might be generated shortly after birth, but we find no evidence that this late generation significantly changes the total number of rods after P3 weeks (Table 1).In short, although the data are sparse, there is little evidence that the postnatal population of rods changes systematically.
Redistributionswithin a stable population of photoreceptors Data from the previous section rule out a major role for either photoreceptor death or generation as a cause of the changes in photoreceptor topography over development, leading us to examine the evidence that retinal growth and/or lateral migration subserve these redistributions. Retinal area. Retinal area increases by a factor of 2.4 from El52 to adulthood (Table 1).Figure 12 shows that the rate of increase is highest during the first 6 postnatal months, when slightly more than half of the increase in
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Eccentricity (mm) Fig. 5. Graphs of cone density as a function of eccentricity along the nasal (A) and temporal (B) horizontal meridians for 7 animals of different ages and the adult average of 3 animals. Only the central 1 mm is shown. Each curve is the average of three meridians (the horizontal, 45" above horizontal, and 45" below horizontal)
area occurs. Retinal area does not reach adult values until sometime between 1 and 5 years of age. In contrast t o the rapid increase in overall retinal area, the posterior pole appears t o be dimensionally stable throughout late prenatal and postnatal development. The center to center distance between the fovea and optic disk varies nonsystematically from 3.37 to 4.05 mm (Table 1). Individual adult values (Packer et al., '89) overlap those of the youngest animals
and no systematic change as a function of age is suggested. These measurements indicate that retinal growth must be nonuniform, with a stable region at eccentricities inside the disk and a much greater expansion in peripheral retina. Proportion of ocular globe covered by retina. The diameter of the ocular globe increased from 14.1 mm (E152) to 21.45 mm (adult), similar to the 14-t o 20-mm range reported elsewhere (Blakemore and Vital-Durand,
DEVELOPMENTOFMONKEYPHOTORECEPTORTOPOGRGPHY
Fig. 6. Maps of cone density in the entire retinas of five animals: (A) El52 (B) P3 weeks, ( C ) P13 weeks, (D) P60 weeks, (Elaverage of 3 adults. The maps of peripheral retina are keyed to the color scale, which represents cone densities ranging from 0 to 16,000 cones/mm2 at a contour interval of 1,000 cones/mm2.The rings and rays of the overlay
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pattern are spaced at intervals of 30 spherical degrees and the black spot represents the optic disk. Areas above 16,000 conesimm' are represented by white. All retinas are displayed as if they are the same size but in fact differ in area by more than a factor of 2 (Table 1).
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Eccentricity (mm) Fig. 7. Graphs of cone density as a function of eccentricity for the nasal horizontal (A) and temporal horizontal (B)meridians of 7 young retinas of different ages and the adult average of 3 animals. Each curve is the average of three meridians (horizontal, 45” above horizontal, and 45” below horizontal). The stippled area on the abscissa represents the optic disk. Cone density values that exceed 20,000 cones/mm2are not shown. These curves are plotted in tissue coordinates (mm) so the extent of each curve reflects the true size of the retina along the meridian, unlike the maps of Figure 6. The increasing size of the retina as a function of age can be seen as an increase in the length of the horizontal meridians.
’86). From globe diameter and retinal area, we calculated the proportion of‘ the ocular globe covered by retina as a function of age in order to assess the relative rates of expansion of the globe and the retina. The retina covered 48-60% of the globe at all ages and there is little evidence that this proportion changes systematically (Table 1).
Patterns ofphotoreceptor redistribution. Although the above data suggest growth is nonuniform, we wanted to quantitatively rule out uniform growth. This analysis assumes that the cone population is stable and that growth is radially symmetric around the fovea, assumptions that are well supported by our data. We tested for uniform
DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY 20
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growth of the retina by comparing the measured expansion of the retina with the predicted expansion of a sphere. Consider the shape of a young retina in vivo and a perfect sphere of the same radius. Both the retinal and the spherical surfaces are divided into a set of concentric spherical rings like those of Figure 1B.Concentric rings are appropriate because radial redistribution would tend to move cones across ring boundaries, changing the proportion of cones enclosed by each ring. After a period of growth, the retina is again compared to a model sphere whose
radius has been increased to match that of the now larger retina. If uniform retinal growth was the sole process subserving redistribution, the zones on the retina will have grown larger in exactly the same way as equivalent zones on the sphere and the 2 patterns will still coincide perfectly. If growth was not uniform, retinal zones will expand more or less rapidly than their equivalent spherical zones. For instance, the number of photoreceptors enclosed by a spherical zone will decrease if its growth is outpaced by the corresponding retinal zone, because photoreceptors for-
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Fig. 9. Maps of rod density in young and adult retinas: (A) P3 weeks, (B)P13 weeks, (C) P60 weeks, (D) average of 2 adults. The color scale represents rod densities between 0 and 200,000 rodsimm' with a contour interval of 12,500 rods/mm'. Areas of the maps that exceed a density of 2000,000 rodsimm' are colored white. The rings and rays of the map overlay are spaced at intervals of 30". All retinas are displayed as if they are the same size hut in fact differ in area (Table 1).
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Eccentricity (mm) Fig. 10. Graphs of rod density as a function of eccentricity along the (A) nasal and (B) temporal horizontal meridians of 3 monkeys of different ages and the average of 2 adults. Each curve is the average of the horizontal and the two meridians at plus and minus 45". The stippled area on the abscissa represents the optic disk.
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Eccentricity (mm) Fig. 11. Graphs of rod density along the (A) superior and (€5) inferior meridians; otherwise identical to Figure 10.
merly contained in that spherical zone will spill into adjacent zones. Migration of photoreceptors from one zone to another would also change the proportion of photoreceptors within a zone. In practice, a computer "drapes" the 800 digital representation of each retina, built up from the set of N retinal samples, over a sphere of appropriate radius. The 700 number of photoreceptors within each concentric ring is calculated, converted to a proportion of the total and 0 plotted as a function of age. If the proportion in each ring is 600 constant as a function of age, then uniform retinal growth 0 w c is supported. If the proportions change with age, then radial f 500 migration or nonuniform growth are occurring. a For clarity, we plot cone data for the E152, P60 week and adult retinas only (Fig. 13A), but data from remaining ages 400 m fall in order between the plotted curves. Between 10" w and 70",each ring of the adult retina contains about 6% of the total cones. Because each successive ring has a larger area than the previous ring, this represents a decline in density. In contrast, the percentage of total cones in retina El52 keeps rising far into the periphery with a peak at 70". This corresponds to the high peripheral density seen in the Fig. 12. Graph of retinal area as a function of age. Retinal area was young animals. The adult Fetinas-have a higher proportion measured by digitizing the outlines of the three retinal pieces.
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Fig. 13. A graph of the proportion of total (A) cones and (B) rods found in a series of concentric rings superimposed on the retinal cone density map at intervals of 5" and centered on the fovea. The figure legend applies to both graphs, but all symbois are not present on each graph. Each data point represents the proportion of total cones in the ring whose outer edge is at the eccentricity shown on the x axis. The total number of cones in the ring was divided by the total estimated population to determine the proportion of the total in that particular ring. The area under each curve sums to 1.The adult shown is retina M1 (Packer et al., '89).
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Fig. 14. A. Graph showing how points of equivalent eccentricity in a young retina (E152, open squares) and adult retina (M1, filled squares) are generated for the growth analysis. Each curve represents the cumulative proportion of cones in a spherical wedge of retina centered on the nasal horizontal meridian. For each eccentricity in the immature retina, there exists an eccentricity in the adult retina with an equal cumulative proportion of cones shown by the horizontal line. In this example, the eccentricity of nearly 16 mm in the adult contains the same cumulative proportion as an eccentricity of 10 mm in E152. These two eccentricities (shown by the vertical lines) are considered equivalent. B. Equivalent eccentricities from M1 and El52 (filled circles) describe the growth of the retina between 2 weeks prenatal and adulthood. A function coincident with the solid line of slope 1would correspond to no retinal growth. A function falling above the line, as does this example, corresponds to retinal growth.
of cones between 10' and 40",whereas the young retinas have a higher proportion of cones between 40" and 90". Similarly, a higher proportion of the rod population is located between 50" and 90" in younger eyes (P3, P13, and P60 wk; Fig. 13B), whereas the adult eye shows a higher
proportion of total rods between the fovea and 40". This analysis suggests that there is a net radial redistribution of both cones and rods from an area centered near 70" toward the center of the retina, which continues past 60 weeks of age. This finding is inconsistent with uniform growth.
DEVELOPMENTOFMONKEYPHOTORECEPTORTOPOGRAPHY
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In order to further examine the pattern of growth, we 2.5 to 3 times larger radially. Inferior retina is rather have factored areal expansion into 2 orthogonal compo- anomalous, showing considerably more radial growth over nents, radial growth along rays extending from the fovea a whole range of eccentricities, except in far periphery and tangential growth perpendicular to the rays. Maintain- where it drops to zero. ing constant retinal coverage of the growing ocular globe These analyses show that between El52 and P13 weeks, requires that the retina grow both radially and tangentially. growth over most of midperipheral retina is substantial and Having already rejected uniform expansion, we predict that increases with eccentricity. After P13 weeks, growth has radial and tangential growth will not be equal over the almost stopped, except in the very far periphery. Inferior whole retina. A preponderance of radial growth would be retina has the most radial growth, suggesting that it lags at consistent with increasing retinal coverage of a static or birth and requires more growth to achieve retinal symmeslowly expanding globe, whereas a preponderance of tangen- try. The dominance of radial over tangential growth, in the tial growth would be consistent with decreasing retinal absence of a change in the proportion of globe covered by coverage of a rapidly expanding globe. The analysis, similar retina, suggests a tendency for the globe to elongate more was applied to the cone axially than horizontally or vertically. to that of Mastronarde et al. (’84), density data, although similar results would be expected Lateral cone migration during development. from an analysis based on the rod density data since both Retinal growth appears sufficient to explain the observed cone and rod redistributions are similar. First, the cumula- decrease in rod and cone density in peripheral retina by tive number of cones and cumulative area were calculated both qualitative and quantitative measures without requirfor 30O-wide spherical wedges of retina from the nasal, ing any lateral migration of photoreceptors. In contrast, temporal, superior, and inferior meridians of the E152, and central retina is dimensionally stable over the same period one adult retina (M1 of Packer et al., ’89). Cumulative (Table l), making it difficult on either logical or empirical totals were converted to cumulative proportions to normal- grounds to explain the large increase in foveal photorecepize for between-animal variability in the total number of tor density by any mechanism other than lateral migration cones. Figure 14A shows that within a 30” wedge, the cones of photoreceptors toward the foveal center. In order to of the young retina are packed more densely than is the case determine how far central cones are moving between El52 for the adult retina; this is not surprising since the young and adulthood, we divided the central 1 mm of each retina retina has a similar number of cones, but only a third of the area. Second, we determined the eccentricities in correspond- into a series of concentric rings and calculated the cumulaing sectors of adult and infant retinas, which contained tive number of cones (Fig. 17A). The linear distance that a equal proportions of the total cone population. For example cone inner segment migrated was determined by selecting a (Fig. 14A), 80% of cones are found within 10 mm of location along the eccentricity axis, moving vertically to eccentricity in the El52 retina and within slightly less than intersect the cumulative function of the El52 retina, and 16 mm of eccentricity in the adult retina, so 10 mm and 16 measuring the horizontal distance (x in Fig. 17A) between mm are “equivalent eccentricities.” The entire range of the two cumulative functions. For example, a cone inner adult eccentricity was plotted as a function of equivalent segment located at 0.75 mm at El52 will move radially infant eccentricity in Figure 14B. The straight line of unity toward the foveal center about 0.225 mm to finally lie at an slope corresponds to no retinal growth, whereas the data eccentricity of 0.525 mm in the adult retina. Displacement (circles) are above the line, indicating that growth has as a function of eccentricity (Fig. 17B) was plotted at 7 points between the foveal center and 1 mm. The x axis occurred. Figure 15 compares radial (open squares) and tangential shows the original position of the cone in the El52 retina. (closed squares) growth between El52 and adulthood. The At the very center of the fovea, there is no displacement. radial component of growth is approximately equal to the With eccentricity, displacement grows to a maximum of derivative of the curve through the data points in Figure 0.230 mm at 1mm of eccentricity. The curve approaches an 14B. The tangential component is computed from the ratio asymptote shortly after 1 mm. Presumably the displaceof the widths of the wedges at equivalent eccentricities in ment gradually decreases to zero at even larger eccentricithe infant and adult retinas. Thus ratios greater than one ties, but the exact shape of the decrease and its maximum correspond to growth. For example, at 10 mm in nasal extent cannot be determined from our data. retina, the adult retina is 1.75 times larger in the radial direction and 1.25 times larger in the tangential direction compared to the El52 retina. With the exception of the far DISCUSSION periphery of the superior vertical meridian, radial growth is Basic photoreceptor topography is established prenatally greater than tangential growth at all eccentricities exceeding 2-3 mm, and is greatest in inferior retina. Tangential but matures slowly. The major topographic patterns of the growth increases slowly with eccentricity, with the far cone and rod mosaics are already present 2 weeks before periphery of the adult retina being about 1.6 times larger birth, although they undergo large quantitative adjustthan the infant retina. Radial growth increases more ments postnatally. A central region of higher cone density is rapidly with eccentricity, but the increase is moderate until just becoming apparent at the future fovea, and the nasotemthe far periphery. Superior retina does not have a high poral asymmetry in cone distribution and the cone streak growth ratio in the far periphery, although the ratio a t on the horizontal meridian are present. Rod topography lesser eccentricities is similar to the other meridians. shows a large rod-sparse central fovea and a wide rod ring Inferior retina, in contrast, expands almost twice as much with “hot spots” of high density. Thus both cone and rod as any other meridian. Figure 16 shows an identical distributions already resemble the adult shortly before analysis of the growth occurring between P13 weeks and birth, suggesting that these basic patterns are established adulthood. Nasal, temporal, and superior retina are up to well before birth. This notion is supported by the observa1.5 times larger in both radial and tangential directions tion (Hendrickson and Kupfer, ’76; Smelser et al., ’74) that except at the largest eccentricities, where the adult retina is as early as E70, a 1-mm-wide area of central retina, the site
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Fig. 15. Analysis of the radial and tangential components of retinal growth from 2 weeks prenatal to adulthood along the nasal and temporal horizontal meridian and the superior and inferior vertical meridian. Radial growth (open squares) is the slope of equivalent points function derived for El52 and adult retina M1 (Fig. 14B). Tangential growth (closed squares) is the ratio of the widths of each retinal sector of the adult and El52 retina. Sector width (mml at some distance from the fovea is r . sin(s/r) (2-2 cos(t)l"', where r is the radius of the retina in mm, s is the radial arc length in mm from the fovea to the location being measured and t is the angular width of the sector.
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Fig. 16. Analysis of radial and tangential components of retinal growth from P13 weeks to adulthood along the nasal and temporal horizontal meridian and the superior and inferior vertical meridian. Format and analysis identical to Figure 15.
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Fig. 17. A. Cumulative number of cones as a function of eccentricity for the central 1mm of retina El52 and one adult ( M l , of Packer et al., '89). Data were prepared by dividing the retina into a set of concentric rings and calculating the area and total number of cones in each ring. The rings were spaced at intervals of 0.1 mm up to 0.5 mm of eccentricity and then at intervals of 0.25 mm up to 1mm. This analysis assumes radial symmetry of cone density within central retina (see Fig.
3). The distance x represents the distance in mm that a cone inner segment located at 0.75 mm of eccentricity in the young retina would move toward the fovea. B. Cone displacement towards the fovea as a function of eccentricity. Displacements were calculated from cumulative cone graphs by determining the horizontal distance between the cumulative cone curves for El52 and adult retinas shown in (A).
of the future fovea, has exclusively cones overlying the thickest portion of the ganglion cell layer. Similarly, a large rod-free region is present at 9-14 weeks of gestation in the human retina (Hollenberg and Spira, '73; Provis et al., '85a). Although very early death of rods cannot be ruled out, these data suggest that no rods are ever generated in the most central retina, and the different patterns of cones and rods are thus established very early indeed. There is relatively little information on early development of photoreceptor topography, but development of ganglion cell topography has been extensively studied in a variety of mammalian species. Since the steep central to peripheral gradient and nasotemporal asymmetry of adult ganglion cell topography qualitatively mirror the topography of the cone mosaic (Curcio and Allen, '90; Perry and Cowey, '85; Stone and Johnston, '811, the processes of development may be similar. In the cat and ferret retina, some authors (Henderson et al., '88; Rapaport and Stone, '84; Stone et al., '82,'84) report that ganglion cells identified by morphological criteria are evenly distributed across the retina initially and that the central to peripheral density gradient develops only after the ganglion cell count has stabilized. Other authors, (Robinson, '87; Wong and Hughes, '87b) also using morphological criteria find a definite but transient central concentration of ganglion cells during and just after the period of ganglion cell generation. This transience may be due to the later generation and migration of large numbers of displaced amacrine cells into the ganglion cell layer (Wong and Hughes, '87a), "flattening out" the initial gradient. More direct evidence for an early gradient comes from retrograde labeling of immature ganglion cells using horseradish peroxidase. Lia et al. ('87) find a small central concentration of labeled ganglion cells, with the central/peripheral ratio increasing from 2.6 at E 3 5 4 0 to 18just before birth. These
data suggest that some early process establishes a small central to peripheral gradient. Nevertheless, both the rapid increase in the central to peripheral ratio in the 2 weeks before birth (Lia et al., '87; Rapport and Stone, '841, together with the lack of evidence for ganglion cell death during this time (Williamset al., '861, suggest that differential growth subserves the development of most of the large central to peripheral gradient. In the monkey, Hendrickson and Kupfer ('76) report an increased concentration of ganglion cells in the cone-rich central retina at E74. Lia and Chalupa ('88) repeated their HRP injections into the brains of monkey fetuses and found a central to peripheral ganglion cell gradient of 10 as early as E85. Thus a gradient is present shortly after the ganglion cells are generated between E30 and E70 (LaVail, '83). In human retina, Provis et al. ('85a) find that neurogenesis is higher in nasal retina as early as the end of the first trimester (the youngest age examined), and by E14-15 weeks, the highest ganglion cell density is within 1.5 mm of the fovea. The nasotemporal ganglion cell asymmetry becomes apparent as early as E l 6 weeks. Mitosis in the central retina ceases by E14-18 weeks and few mitotic figures were found even in the far periphery after E30 weeks. These data suggest that a gradient may develop in the primate ganglion cell topography even before cell generation ends. It may be due to very early differential cell generation, differential cell death, growth of the central retina, or even lateral migration, although in the human, uneven neurogenesis seems to play a more definite role. Thus it seems likely that similar processes shape the early topography of both the photoreceptor and ganglion cell layers. Despite the early appearance of the major topographic patterns, macaque photoreceptor distribution is not mature until long after birth. The 6- and 13-month eyes were
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below adult size and foveal cone density but above adult values for peripheral photoreceptor density. In addition, the eccentricity of the rod ring still exceeded that of the adult. If monkey age in weeks is equivalent to human age in months (Boothe et al., '85), these data suggest that a 5-year-old human child might exhibit similar immaturities in retinal development. Yuodelis and Hendrickson ('86) have found that peak cone density in a 45-month human was still well below adult values. This suggests protracted development in both human and monkey, although the age at which the photoreceptor mosaic reaches maturity in either primate is still not precisely known. Prolonged postnatal changes in photoreceptor distribution and morphology may play a role in the physiological and behavioral changes that occur in spatial vision over the same period (discussed in Banks and Bennett, '88; Brown et al., '87; Jacobs and Blakemore, '88; Wilson et al., '88). Because anatomical data on either human or monkey retinal development are still quite sparse, this study is an opportunity to begin a quantitative database detailing primate photoreceptor development. This is clearly important in light of the use of the macaque monkey's visual system as an invasive model for the human (Boothe et al., '85).
Mechanisms underlying photoreceptor redktributionsduring development We find two major redistributions of monkey cones and rods during the first year after birth. The first is a centralward increase in foveal density as shown by a fivefold increase in peak cone density and a decrease in the diameter of the rod-sparse area. The second is a decrease in both cone and rod density in peripheral retina. On the basis of the data presented here, we conclude that: (1) cell generation and death play minor roles, (2) the increase in foveal density and decrease in rod-sparse zone diameter is mediated by lateral migration of photoreceptors toward the center of the fovea, and ( 3 ) the decrease in peripheral photoreceptor density is caused by the increased size of the peripheral retina. Cell generation and cell death. The increased density of both rods and cones in central retina during postnatal development could be subserved by late generation of new photoreceptors. In most mammalian retinas, those photoreceptors generated first lie near the posterior pole; those generated late in development lie near the peripheral edge of the retina (Provis et al., '85a; Rapaport and Stone, '82; Provis et al., '85a; Robinson, '87; Sidman, '61). Brief reports by La Vail et al. ('83)and Rapaport et al. ('87) and our own unpublished observations show that no photoreeeptors are generated postnatally in central retina, although some cones and rods are generated in the far peripheral retina as late as El02 and P17, respectively. These data rule out postnatal photoreceptor addition in the central retina, whereas the small amount of cell addition to peripheral retina works against the developing central to peripheral gradient of photoreceptor density. Cell death also is unlikely to be a n important factor in the central retina, because it would lower rather than increase cell densities. However, cell death could play a role in the decreased density found over time in the peripheral retina. Cell death in the outer nuclear layer has been reported in cat from E42-Pl7 coincident with the onset of photoreceptor differentiation and outer plexiform layer development (Robinson, '88; Stone et a1 , '84). Cell death occurs in the subventricular zone of very young human retina, but was
not reported in differentiated photoreceptors (Penfold and Provis, '86). Even in the ganglion cell layer, where cell death certainly occurs (reviewed by Provis and Penfold, '88; Wikler and Finlay, '891, its role in determining topography is unclear, although several authors suggest that it may be important (Mastronarde et al., '84; Rapaport and Stone, '84; Stone et al., '82; Wong and Hughes, '87b). The ubiquity of neuron loss during development throughout the central nervous system, including the primate retinal ganglion cell layer (Provis et al., '85b; Rakic and Riley, '83) suggests that some loss of photoreceptors should occur and has simply not been found. Our study does not include the most immature stages of development, but our finding of relatively stable cone and rod populations well after the period of photoreceptor generation and throughout the period of topographic differentiation suggests that cell death is not a major factor in shaping photoreceptor topography during later development. No more than 25% of all cones are lost between El52 and P60 week, a loss that is insufficient to explain the average threefold decrease in peripheral cone density during the same time. The same conclusion is consistent with changes in the number of rods during this same period, although the total number of rods in individual retinas is more variable and fewer ages were counted. It thus appears that the small changes that may be occurring in the number of photoreceptors due to cell generation or cell death are not sufficient to explain the large changes in topography during postnatal development. We next consider how a relatively uniform photoreceptor population could be redistributed by cell migration and/or retinal growth. Photoreceptor migration. In the central retina, growth cannot be the main process redistributing photoreceptors because it would act to lower, rather than raise, cone density, The central retina of the monkey stops growing by E60 (Steineke and Kirby, '89) or E80 (Lia and Chalupa, '88). The stable fovea to disk distance during the period of this study also confirms that there is no growth of central retina after E152. By exclusion, lateral migration of photoreceptors toward the foveal center is the only reasonable explanation for both the marked increase in foveal cone density and the reduction in the size of the rod sparse area postnatally. Since the density of peripheral cones and rods declines while central density increases, it might be possible to explain both phenomena by migration of cones from the periphery into central retina. Quantitatively, however, only the area within 0.5 mm of the foveal center, which in the adult contains about 40,000 cones, undergoes a large increase in density. If density increases by a factor of 5, only 32,000 cones, or 1%of the 3 million cones in peripheral retina, would need to migrate into the fovea. The loss of this tiny percentage of peripheral cones would be difficult to detect at all and certainly cannot account for a threefold decrease in peripheral density. Rod topography also undergoes an increase in central rod density and a reduction in peripheral density, and the position of the rod ring becomes more central during later development. By arguments identical to those made for the cones, the central increase can be subserved only by lateral displacement of rods toward the center of the fovea. This increase could be due to an active migration similar to the cones or a passive shift as rods are "dragged along" with the surrounding cones as they migrate toward the foveal center.
DEVELOPMENTOFMONKEYPHOTORECEPTORTOPOGR.AJ?HY Previous work on monkey foveal development (Hendrickson and Kupfer, '76) and the data of this study strongly suggest that photoreceptors migrate toward the foveal center, whereas the other retinal neurons migrate away from it to create the foveal depression. Retinal neurons are unusual among migrating neurons in that they move as differentiated cells that have developed both a complex cytology and synaptic contacts. Synapses are formed between monkey photoreceptors and other neurons in the outer plexiform layer by E74 (Smelser et al., '74) and outer segments are present before birth on central cones (Hendrickson and Kupfer, '76). The cone axons or fibers of Henle appear at E l 2 5 and elongate considerably after birth. This early establishment of synapses would seem to suggest that later redistribution could be most easily accomplished by a mechanism that avoids large changes in relative position between photoreceptors that would tend to increase the complexity of the neuropil as axons and dendrites stretched and contorted to retain their previously established synapses. Even so, the changes occurring in the neuropil are not simple. Theoretical (Schein, '88) and empirical evidence (Perry and Cowey, '88) suggests that cones with adjacent inner segments in the foveal center have nonadjacent cone pedicles requiring some crossing of cone axons. The visual mapping established at the level of the inner segments could be maintained at postreceptoral levels by formation of synapses in the outer plexiform layer before migration, although the identity of the cells making these early contacts is as yet unclear. Nevertheless, the visual field position of postreceptoral neurons subserving foveal vision must be recalibrated during development both for increasing eye size (Milleret et al., '88; Olson and Freeman, '80) and for lateral migration of cones. Anatomical measurements of the fibers of Henle in macaque retina, together with our calculations of how far photoreceptors migrate, can be used to infer how the photoreceptors and the bipolar neurons move with respect to each other during foveal development. Although the data for the centralmost fovea are very sparse, the longest fibers of Henle probably belong to cones near but not in the center of the fovea and are 0.3-0.4 mm in length (Boycott et al. '87; Perry and Cowey, '88; Polyak, '41; Schein, '88). Fiber
// Fovea
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Ora
Eccentricity Fig. 18. One model of rod redistribution that could explain all of the observed density changes in the periphery. The dotted vertical line marks the boundary between the expanding peripheral retina and the stable central retina. See Discussion for further interpretation.
49 1
length decreases with eccentricity and by 0.75 mm is down to approximately 0.225 mm (Perry and Cowley, '88; Schein, '88). Calculated migration distance, in contrast, is zero at the foveal center and increases to a maximum of about 0.23 mm at 1 mm of eccentricity. These data suggest that at the center of the fovea the entire length of the fiber of Henle is due to outward migration of the bipolar neuron to which the photoreceptor is connected. With increasing eccentricity, the distance that a cone migrates toward the foveal center increases. For example, a cone initially located at 1 mm of eccentricity in the infant would migrate 0.23 mm centralward and be found at an eccentricity of about 0.77 mm in the adult. Since the fibers of Henle are between 0.225 and 0.245 mm in length at 0.75 mm of eccentricity, the calculated distance that the photoreceptor migrates is the right size to account for the length of the fiber. We were not able to calculate migration distances at eccentricities greater than 1 mm, but we would predict that the distance that a photoreceptor migrates would decrease in good correlation with the length of the fiber of Henle. The cellular mechanisms subserving photoreceptor migration remain unclear, and we can only speculate how it might function. Immature neurons are able to actively move relative to each other and to glia by establishing adhesive interactions involving membrane and extracellular adhesion molecules (Edelman, '85; Silver and Rutishauser, '84). This ability may be retained or acquired by mature primate photoreceptors. It is also possible that central photoreceptors respond to internal or extrinsic cues to actively streamline themselves during development. If neighboring photoreceptors and their matrix were adhesive and if the photoreceptors at the foveal center were anchored, this change in shape would tend to pull peripheral photoreceptors toward the fovea. Retinal expansion. The retina increases in area by a factor of 2.4 from El52 to adulthood, and by even a larger factor if the stable central 8 mm is subtracted. This amount of retinal expansion fits well with the threefold decrease in peripheral cone density. Similarly, rod density in the adult peripheral retina is about 50% of that at P3 weeks, but the area of the P 3 week retina was about 60% of the adult. These data suggest that retinal growth alone is sufficient to explain the observed reductions in both cone and rod peripheral density. There is no logical need to invoke different mechanisms of redistribution for the cones and rods because they are intermixed over the peripheral retina, and growth will reduce both cell densities equally. Differential expansion of the peripheral retina can also explain the shift in the eccentricity of the rod ring toward the fovea. Figure 18 is a qualitative model showing the effects of differential expansion on the rod density profile along a radial path from the fovea to the ora terminalis in a young retina. Between the fovea and disk, the retina is dimensionally stable, so expansion does not change rod density and the two profiles remain superimposed. At eccentricities beyond the disk, the degree of expansion increases into the periphery, reducing far peripheral rod density more than near peripheral density and causing the two curves to diverge. Note that rod density is lowered in the adult at all eccentricities beyond the optic disk. The rod ring shifts toward the fovea as the increase in peripheral area reduces rod density in the peripheral flank of the rod ring. We conclude that the greater growth of the peripheral retina can explain the decline in peripheral rod density, the
492
reduced peak rod density, and the decrease in the eccentricity of the rod ring in the adult compared to the newborn. Between the stable posterior pole of the retina, where we are confident that redistributions are due to lateral migration, and the far periphery, where retinal expansion adequately explains the reduction in peripheral density, lies an area of uncertainty. Here our methods are not sensitive enough to resolve the observed redistribution into a migratory component and a retinal growth component, even though both may be active. Our plots of photoreceptor proportional distribution (Fig. 13) clearly indicate that redistribution extends to eccentricities of at least 60". This is well beyond the central growth-free zone and suggests that the intermediate region of the retina outside the eccentricity of the optic disk could be subject to both lateral migration and differential growth. The presence of oblique fibers of Henle beyond 3 mm of eccentricity also supports this idea (Perry and Cowey, '88). In order to decipher the relative strengths of these two forces in the midperiphery and confirm them elsewhere, a direct method of observing photoreceptors during development is required. For example, it may be possible to label in vivo a small number of photoreceptors at known retinal locations with a nontoxic, durable marker and then chart their positions with respect to retinal landmarks during development. During the first year of development, the posterior pole of the monkey retina is dimensionally stable, whereas the peripheral retina grows more with eccentricity. Growth is greater along the radial than the tangential dimension at any given location, and it is more rapid early in development. This pattern is rather different from the cat, where tangential growth dominates in the far periphery, radial growth in the midperiphery, and little growth occurs a t the posterior pole (Mastronarde et al., '84).This species difference likely reflects the fact that the cat retina covers 60% of the globe at birth but only 34% in the adult, requiring more tangential growth to maintain retinal apposition with the globe. Monkey retina covers a relatively constant 52% of the globe and so needs less tangential growth to maintain retinal apposition. Data from the monkey are also consistent with human data (Robb, '821, which showed only a small decrease in retinal coverage from 62% to about 56% during later development. It thus appears that monkey and human retinal growth are similar to each other but different from cats, implying that nonprimate models of retinal growth can be extrapolated to humans only with great care. Potential mechanisms subserving retinal expansion include some combination of the addition of retinal cells, the increase in the size of existing retinal cells, the increase in the amount of neuropil, or a thinning of the nuclear layers. Since tritiated thymidine fails to label a substantial number of neurons postnatally, addition of neurons probably plays little role in primate retinal expansion. There is a n agerelated increase in the diameter of both cone and rod inner segments at equivalent eccentricities exceeding 5 mm. It is not clear, however, whether the growth of individual photoreceptors is a cause of retinal expansion or a reaction to it. If photoreceptor coverage remains constant as the retina expands, then either passive stretch or active growth alone might be sufficient to explain the change in photoreceptor inner segment diameter. If coverage changes as a function of time, then more complicated interactions must be considered. It is also possible that a large amount of retinal
0. PACKER ET AL. growth is due to increases in glial size and number. Mann (1964) suggests that growth of peripheral retina is largely due to thinning of the nuclear layers with a simultaneous increase in neuropil, which maintains retinal thickness. More work is required to distinguish among these possibilities. Robinson and coworkers ('87,'89) have proposed a balloon model of developmental changes in ganglion cell topography in which interocular pressure provides a passive stretching force. They predicted that the retinal region most resistant to stretch should: (1)have the highest cell density, (2), maintain similarly shaped isodensity contours throughout later development, and (3) be the most advanced developmentally. These predictions are at least qualitatively correct for monkey photoreceptor topography as well, although it is necessary to factor out the independent development of the fovea. The central retina within the eccentricity of the optic disk does not change size postnatally, begins to develop first, matures earliest, and has the highest photoreceptor density. Likewise, the cone streak and the rod ring, which are areas of relatively high photoreceptor density, retain their basic shapes during development. Based on these criteria, passive stretching may play a role in both ganglion cell and photoreceptor redistribution. In fact, we believe that the photoreceptor layer is an appropriate place to study retinal expansion, because: (1) the photoreceptor inner segments are tightly packed together without overlap, making it possible to determine how their size and distribution changes as the retina grows, and ( 2 ) during later development, when the majority of retinal growth occurs, the photoreceptor layer is largely free of cell generation and death, which complicate interpretations of similar data from the ganglion cell layer.
ACKNOWLl3DGMENTS This work was supported by EY01208 and EY04536 to A.H., EY06109 to C.C., and by EY07031, and in part by RR00166 to the Regional Primate Research Center at the University of Washington. The authors gratefully acknowledge, in particular, the tissue program and the assistance of Judy Johnson. We also thank Kenneth Sloan for the design of topographic mapping software.
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Provis, J.M., D. Van Driel, F.A. Billson, and P. Russell (1985b) Human fetal optic nerve: Overproduction and elimination of retinal axons during development. J. Comp. Neurol. 238t92-100. Rakic, P., and K.P. Riley (1983) Overproduction and elimination of retinal axons in the fetal rhesus monkey. Science 219:1441-1444. Rapaport, D.H., and J. Stone (1982) The topography of cytogenesis in the developing retina of the cat. J. Neurosci. 3t1824-1834. Rapaport, D.H., and J. Stone (1984) The area centralis of the retina in the cat and other mammals: Focal point for function and development of the visual system. Neuroscience 11:289-301. Rapaport, D.H., D. Yasumura, M.M. LaVail, and P. Rakic (1987) Cytogenesis of monkey retina: Comparison of the generation of retinal pigment epithelium and neural retina. Proc. SOC.Neurosci. 13(1):238(abstract) Robb, R.M. (1982) Increase in retinal surface area during infancy and childhood. J. Ped. Ophthalmol. Strabi. 19:16-20. Robinson, S.R. (1987) Ontogeny of the area centralis in the cat. J. Cornp. Neurol. 25550-67. Robinson, S.R. (1988) Cell death in the inner and outer nuclear layers of the developing cat retina. J. Comp. Neurol. 267:507-515. Robinson, S.R., B. Dreher, and M.J. McCall (1989) Nonuniform retinal expansion during the formation of the rabbit’s visual streak: Implications for the ontogeny of mammalian retinal topography. Visual Neurosci. 2201-220. Schein, S.J. (1988) Anatomy of macaque fovea and spatial densities of neurons in foveal representation. J. Comp. Neurol. 269:479-505. Sidman, R.L. (1961) Histogenesis of mouse retina studied with tritiated thymidine. In G.H. Smelser (ed): Structure of the Eye. New York: Academic Press, pp. 487-506. Silver, J., and U. Rutishauser (1984) Guidance of optic axons in vivo by a preformed adhesive pathway on neuroepithelial endfeet. Dev. Biol. 106t485-499. Smelser, G.K., V. Ozanics, M. Rayborn, and D. Sagun (1974) Retinal synaptogenesis in primate. Invest. Ophthalmol. 13:340-361. Steineke, T.C., and M.A. Kirby (1989) Early axon outgrowth of retinal ganglion cells in the retina of the rhesus macaque. Invest. Ophthalmol. Vis. Sci. 3O(supplj:26. Stone, J.,and E. Johnston (1981) The topography ofprimate retina: A study of the human, bushbaby and new- and old-world monkeys. J. Comp. Neurol. 196t205-223. Stone, J., J. Maslim, and D. Rapaport (1984) Development of the topographical organization of the cat’s retina. In J. Stone, B. Dreher, D. Rapaport (eds): Development of Visual Pathways in Mammals. New York: Alan R. Liss, pp. 3-21. Stone, J., D.H. Rapaport, R.W. Williams, and L. Chalupa (1982) Uniformity of cell distribution in the ganglion cell layer of prenatal cat retina: Implications for mechanisms of retinal development. Dev. Br. Res. 2 2 3 1-242. Wikler, K.C., and B.C. Finlay (1989) Developmental heterochrony and the evolution of species differences in retinal specializations. In B.L. Finlay, D.R. Sengelaub (eds): Development of the Vertebrate Retina. New York: Plenum Press, pp. 229-244. Wikler, K.C., R.W. Williams, and P. Rakic (1988)Number, distribution and ratios of rods and cones in the adult macaque retina. Soc. Neurosci. Abstr. 14(2):1119. Williams, R.W., M. Bastiani, B. Lia, and L.M. Chalupa (1986) Growth cones, dying axons, and developmental fluctuations in the fiber population of the cat’s optic nerve. J. Comp. Neurol. 246:32-69. Wilson, H.R. (1988) Development of spatiotemporal mechanisms in infant vision. Vision Res. 28:611-628. Wong, R.O.L., and A. Hughes (1987a) The morphology, number and distribution of a large population of confirmed displaced amacrine cells in the adult cat retina. J. Comp. Neurol. 255r159-177. Wong, R.O.L., and A. Hughes (198713) Developing neuronal populations of the cat retinal ganglion cell layer. J. Comp. Neurol. 262t473-495. Yuodelis, C., and A.E. Hendrickson (1986) A qualitative and quantitative analysis of the human fovea during development. Vision Res. 26:847855.
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Figure 4
NEUROLOGY 298:472493 (1990)
Developmental Redistributionof PhotoreceptorsAcross t h e M m nemestrina (RgtdMacaque)Retina ORIN PACKER, A ” A E. HENDRICKSON, AND CHRISTINE A. CURCIO Departments of Psychology (O.P.),Biological Structure (A.E.H., C.A.C.), and Ophthalmology (A.E.H.,C.A.C.), University of Washington, Seattle, Washington 98195
ABSTRACT Redistributions of monkey cones and rods during the first year after birth include a fivefold increase in peak foveal cone density from 43,000 to 210,000 cones/mm2, a decrease in the diameter of the rod-sparse area, and a two- to threefold decrease in peripheral photoreceptor density. Two weeks before birth, higher cone density is already apparent in the future fovea, as are the nasotemporal asymmetry in cone distribution, a higher density “cone streak” along the horizontal meridian, a large rod-sparse central fovea, and a ring of high rod density. Despite the early appearance of these basic patterns, photoreceptor distribution is not mature until 1 to 5 years postnatally. Total cones varied from 4 million at birth to 3.1 million in the average adult. The two oldest eyes had fewer cones, suggesting up to a 25% loss late in development. There were 60 to 70 million rods in the adult macaque retina and little evidence of postnatal changes in number. Neither of these small changes is sufficient to account for the reduction in peripheral photoreceptor density and both are in the wrong direction to explain increasing foveal density, ruling out a major role for either photoreceptor death or generation. Retinal area increased by a factor of 2.4 from 2 weeks before birth to adulthood. In contrast, the posterior pole of the retina was dimensionally stable throughout this period, with the distance between the fovea and optic disc varying nonsystematically from 3.37 to 4.05 mm. Retinal coverage of the globe was also stable at 48-60%. Thus postnatal growth can be ruled out as a factor in the density changes occurring in central retina. Adult retinas have a higher proportion of both cones and rods in midperiphery, whereas young retinas have a higher proportion of photoreceptors in far periphery. It appears that photoreceptors are radially redistributed from peripheral toward central retina during postnatal development, resulting in the marked increase in foveal cone density and the decrease in the eccentricity of the rod ring. Up to 13 weeks postnatally, midperipheral growth of the retina is substantial and increases with eccentricity. At later ages, expansion continues only in the very far periphery. Retinal growth appears sufficient to explain the decreases in peripheral rod and cone density with age. These and previous data strongly suggest that differentiated photoreceptors, with complex cytology and synaptic contacts, migrate toward the foveal center, explaining the increase in foveal photoreceptor density. The distance that cones migrate between E 152 and adulthood increases from 0 at the foveal center to a maximum of 0.230 mm for cones originally located at 1 mm of eccentricity in the immature retina. This migration is equal to published measurements of the length of fibers of Henle for cones located at 0.75 mm in the adult. These data suggest that at the center of the fovea, the length of the fiber of Henle is due to outward migration of the bipolar neuron to which the photoreceptor is connected, but that by 0.75 mm, the length of the fiber is due to the centralward migration of the photoreceptor. Key words: rods, cones, topography, development
Photoreceptors are unevenly distributed across the macaque retina (Curcio et al., ’87a; Packer et al., ’89; Perry and COwey’ ’85). ‘One exceeds 2oo’ooo at the center of the fovea and then with increasing eccentricity. A high density “cone streak” is A
1990 WILEY-LISS, INC.
AcceptedJune 1, 1990, Address reprint requests to Orin Packer, Ph.D., who is now a t the Center for Visual Science, 274 Meliora Hall, University of Rochester, Rochester, Ny 14627.
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DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY found along the horizontal meridian, within which nasal cone density consistently exceeds temporal density at equivalent eccentricities. Rod density, in contrast, is zero at the foveal center, increases rapidly to the crest of a ring of high rod density near the eccentricity of the optic disk, and then decreases into the periphery. Peak density in the ring is 177,000 rods/mm2in superior retina. How do these complex patterns of photoreceptor distribution develop? They could be established at the time of photoreceptor generation and change little thereafter, or they could be gradually formed over an extended period by photoreceptor redistribution. Knowing the pattern and time course of redistribution would provide clues to underlying developmental processes. It is already known that the spatial distribution of photoreceptors changes during development. In the monkey neonate, cone density at the center of the fovea was only a fourth that of the adult, gradually increasing to adult values during the first 2 years of life (Hendrickson and Kupfer, '76). The fovea of the human neonate was even more immature than that of the monkey (Abramov et al., '82; Hendrickson and Yuodelis, '84; Yuodelis and Hendrickson, '86). In short, the primate photoreceptor mosaic undergoes a long period of maturation that extends past 1 year in the macaque and possibly past 4 years in the human. The development of the peripheral cone and rod distributions has not been studied in either monkey or human. Unanswered questions about the development of the photoreceptor mosaic include: (1)Where do the cones subserving the increase in foveal density come from? (2) Are there developmental changes in peripheral cone distribution? (3) When do the asymmetries in cone and rod distribution develop? (4)Do rods undergo changes in distribution? (5) How do known mechanisms of neural development, such as cell generation, cell death, cell migration, or tissue growth (Lund, '781, apply to the photoreceptor mosaic? We have investigated these questions by measuring cone and rod density across the entire retina of an age-spaced series ofM. nemestrina (pigtail macaque) monkeys, using methods previously applied to the adult M . nemestrina retina (Packer et al., '89).
METHODS Animals. Table 1 shows the ages and sexes of the animals whose retinas were used in this study. The 3 adult animals used for comparison were 5 years of age or older and are individually described in Packer et al. ('89). Seven eyes ranging in age from 2 weeks prenatal (E152) to 60 weeks postnatal (P60 wk) were obtained through the Regional Primate Research Center at the University of Washington. The gestational ages of the animals were
known to within 24 hours, and no postnatal animals were included that were premature by either birthdate or weight. The age interval between animals was chosen to be larger at older ages because development of the primate fovea slows with age (Yuodelis and Hendrickson, '86). Procedure. Tissue processing techniques are detailed in Packer et al. ('89) and Curcio et al. ('87b). Briefly, the eyes were enucleated under deep barbiturate anesthesia either prior to perfusion or following an intravascular saline rinse. The horizontal, vertical, and axial equatorial diameters of each eye were measured just prior to immersion in 0.1 M phosphate-buffered 4%paraformaldehyde. In order to speed fixation of the retina, a small volume of fixative was either injected through the optic nerve head into the vitreous chamber or allowed to pass through a puncture made near the limbus. We were careful to keep the globe fully round during initial immersion to avoid fixing folds in the thin sclera. The fixed globe was cut into a belt along the horizontal meridian and an inferior and superior cap. The neural retina was separated from the sclera and pigment epithelium, flattened on a plastic slide, and coverslipped under the clearing agents glycerine and dimethyl sulfoxide. This wholemount was viewed under Nomarski differential interference contrast optics (Allen et al., '69).These techniques eliminate the need to section and stain the retina, minimizing tissue distortion. Photoreceptors were counted manually with the aid of a small morphometry computer (Curcio and Sloan, '86). Sampling locations were more closely spaced near the center of the fovea where photoreceptor density changes rapidly, than in the periphery where density changes more slowly. About 200 samples were counted in each retina. Data analysis. Maps of photoreceptor density, which are particularly good for observing large-scale features, were prepared from sampling locations and their associated photoreceptor densities using computer methods (Curcio et al., '89). Graphs of photoreceptor density along the cardinal meridians, which are particularly useful for making quantitative comparisons between meridians, were prepared from these maps by resampling along a meridian. The total numbers of rods and cones were estimated by interpolating between sampling points and summing photoreceptors across the retina (Packer et al. '89). Spherical coordinate system. In order to facilitate the analysis of retinal topography in animals of different ages and with different eye sizes, a spherical coordinate system was used to represent retinal position. Throughout this work, the retina is modeled as slightly more than half of the surface of a sphere (Fig. lA, also Curcio et al., '89), whose coordinate system is defined by the fovea (F) and optic disk (D). The horizontal meridian is represented by
TABLE 1. Vital Statistics and Retinal Parameters Age' Retinal area (mm') Cone peak density (mnes/mm2x 103) Eccentricity o f r d peak (rnm) Rod peak density (roddmm2x lo3) Total cones ( x 106) Total rods ( x 10b) Fovea to disk (mrn)(centerto center) Q ofocular sphere covered by retina' Diameterof globe (mm)
El52 335
El65 365
P3 wk 488
P6 wk 535
P13wk 505
P26 wk 607
P60 wk 564
Adult' 800
43.3 NA NA 3.73 NA 3.68 54 14.10
85.9 NA NA 3.97 NA 3.67 52 15.00
65.0 6.29 211.5 3.89 70.1 3.72 55 16.75
65.7 4.94 200.0 3.61 NA 4.00 59 17.05
80.9 6.95 233.2 3.75 68.5 3.76 48 18.30
157.4 NA NA 3.81 NA 4.05 53 19.10
132.8 5.91 168.2 3.31 57 3
210 2.09 177 3.08 60.1 3 37 55 21.45
3.46 50 19.00 ~~
'E-embyromc day, P-postnatalweek. *Meanof3, > 5yrsold(Packeretal, 1989). 'Based on equatorial diameter.
~
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0. PACKER ET AL.
A
B B
s
N
E
I
Fig. 1. The spherical coordinate system used for retinal modeling. A. The Coordinate system is defined by the ray GF running from the anterior part of the eye, through the center of the retinal sphere, and ending at the fovea (F).The horizontal meridian of the spherical retina is the arc ADFC through the fovea and disk (D). The vertical meridian is the arc BFE through the fovea perpendicular to the horizontal meridian. B. For viewing, the retinal sphere is mapped onto a flat surface. The center of the map represents the center of the fovea (small circle) and the larger circle (Di represents the disk. The effect of this mapping
transformation is to leave the central retina relatively undistortcd while stretching the peripheral retina in the tangential direction, represented by the rings, but not in the radial direction, represented by the rays. The disk is always on the nasal (N) meridian. The superior vertical meridian is marked S, and the inferior vertical meridian is marked I. The rings are spaced at intervals of 30". Note that in this and subsequent figures, angles are measured in degrees on the retinal sphere, not in visual degrees. T, temporal.
the arc ADFC through the fovea and disk, and the vertical meridian is represented by the arc BFE through the fovea perpendicular to the horizontal meridian. The line GF lies near the optic axis with the point G at the anterior surface of the eye. For display, the spherical representation of the retina is mapped onto a plane (Fig. lB), in the same way that a cartographer represents the polar regions of the earth on a flat map. Conceptually, this mapping is approximately what would result from removing the spherical retina from the globe with its open end up and squashing it flat. The representation of central retina remains nearly undistorted. The representation of peripheral retina undergoes considerable tangential elongation (along the rings), but radial distances (along the rays) are not distorted (Fig. 1B). The map is centered on the fovea (small circle), which together with the disk (D) represents the horizontal meridian (ADFC of Fig. lA), labeled N (nasal)-T (temporal). The vertical meridian (BFE of Fig. 1A) maps onto the line labeled S (superior)-I(inferior).The optic disk (D) is on the nasal side of the horizontal meridian. The spacing between the rings and rays of the grid overlaying each topographic map is measured in degrees on the retinal sphere. (Spherical degrees are not to be confused with visual degrees which are measured relative to the center of the optics of the eye rather than relative to the center of the ocular sphere.) One degree of arc measured on the surface of the retinal sphere equals ( T T . r)/180 mm where r is the radius of the retinal sphere in mm. The color maps are normalized to approximately the same size, concealing an age-related increase in retinal area. The measured diameter and area of each individual retina are shown in Table 1.
whereas rods increase in density to the eccentricity of the optic disk and then decrease more peripherally (Packer et al., '89). Both cone and rod inner segment diameters increase with distance from the fovea. Changes in the organization of the photoreceptor mosaic as a function of eccentricity at a given age can be seen by scanning across the rows of Figure 2. At birth (Fig. 2, top row), cone and rod density are lower in the foveal center and higher in the far periphery than they are in the adult. The diameter of both cone and rod inner segments is similar across the retina. Cone inner segment diameter increases slightly from the fovea to 0.5 mm of eccentricity, but, if anything, decreases from 0.5 to 5 mm of eccentricity. Cone inner segment diameter across the retina ranges from about 3.6 pm (central) to 6.5 pm (peripheral) at birth, whereas the adult range is from 2.3 to 10 pm (Packer et al., '89). At P13 weeks (Fig. 2 middle row) and P60 weeks (Fig. 2 bottom row), the pattern is qualitatively adultlike, although the foveal cones are still larger than their adult counterparts (3.7 and 2.85 IJ-mvs. 2.3 pm). Changes in the organization of the photoreceptor mosaic with age at a given eccentricity can be seen by scanning down the columns of Figure 2. As discussed, the inner segment diameter of the foveal cones decreases by 25%1 from birth to P60 weeks. At 0.5 mm of eccentricity, cone size decreases and cone density increases from birth to P13 weeks and then stabilizes. This area appears to be a relatively stable zone during development between the more central areas, where cone inner segment diameter decreases, and the far periphery, where diameter increases. At 5 and 10 mm of eccentricity, cone density decreases with age, whereas cone inner segment diameter increases. There is-little change in rod inner segment diameter at 5 mm, but a t 10 mm, rod diameter increases with age. Thus during develoDment. foveal cones decrease in inner s e m e n t dianieter aAd both cones and rods increase in density. In the periphery, rod and cone density decreases.
RESULTS Of
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Appearance of the photoreceptor mosaic. In the adult retina, cones decrease in density with eccentricity,
DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY Quantitative changes in cone density. The 2 main features of cone topography in the adult pigtail macaque are a sharp central to peripheral gradient, and a nasotemporal asymmetry in the periphery. The former develops considerably during the period of this study, and the latter does not. The first major change leading to the sharp central to peripheral gradient is a fivefold increase in foveal cone density from just before birth to adulthood (Fig. 3, Table 1). In the adult, cone density at the foveal center peaks at 210,000 conedmm’ (Fig. 3E, data from Packer et al., ’89), drops to 100,000 cones/mm2 at 0.15 mm and to 50,000 cones/mm2at 0.75 mm of eccentricity. In contrast, at El52 (Fig. 3A), foveal cone density is low and almost uniform, ranging between 25,000 and 35,000 cones/mm2.Two small patches of higher density mark the location of the developing fovea, but cone density peaks at only 43,000 cones/mm’. By P3 weeks (Fig. 3B), central cone density has increased to 65,000 cones/mm’ and by P13 weeks (Fig. 3C) is 81,000 cones/mm2, still only 40% of the adult average. By P60 weeks (Fig. 3D), peak cone density reaches 133,000 cones/ mm2,which is still only 63% of average adult density. A plot of peak density vs. age (Fig. 4) indicates some individual variability both in the rate of development and in absolute peak cone density. This should not be surprising given the considerable variability of adult peak cone density in both human and monkey (Curcio et al., ’87a; Hawken et al., ’88; Packer et al., ’89,Wikler et al., ’881, but it makes it difficult to determine when peak cone density reaches adult values. Peak density at 6 months (157,000 cones/mm2)is within the range of values that others have reported for adult macaques (for a summary see Table 1, Packer et al., ’891, although it is still 25% below the values that we have previously reported. Fully mature values probably are not attained until sometime between 1and 5 years postnatally. Figure 5 shows the rapid change in cone density along the horizontal meridian from the foveal center to 1 mm of eccentricity for monkey retinas of different ages. The P 3 week retina has an almost flat density profile. At successively older ages, a combination of sharply higher foveal cone density and relatively little change in density between 0.6 mm and 1 mm results in a much steeper density gradient from the foveal center to 1mm. Individual variability is shown most vividly by the reversal of the curves representing the P26 week retina and the P60 week retina, and by the uneven spacing of the curves representing retinas of intermediate age. There is little difference between nasal and temporal cone density at these central eccentricities and the same is true of the superior and inferior meridians as shown by the color maps (Fig. 3). The similar densities of all 4 meridians show that cone density in central retina is radially symmetric. The second major change that sharpens the central to peripheral gradient is a general reduction of peripheral cone density with age (Fig. 6). In adult peripheral retina (Fig. 6E), cone density declines continuously to 1 or 2% of peak density at the edge of the retina. This decline is most rapid near the fovea and then slows with increasing eccentricity. As a result, cone density across most of far peripheral retina ranges between 1,000 and 4,000 cones/mm2. Imposed on this pattern are 2 asymmetries most easily seen in Figure 6E. First, cone density is higher along the horizontal than along the vertical meridian, forming a “cone streak.” Second, peripheral cone density at eccentricities greater than the optic disk is up to 3 times higher at equivalent eccentricities in nasal than in temporal retina, resulting in
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isodensity contours that extend farther into nasal than temporal retina. In the El52 retina (Fig. 6A), peripheral cone density ranges from about 6,000 cones/mmz (green) along the temporal and superior edge of the retina, to above 16,000 cones/mm2 (white central region). Even at El52 cone density is higher along the nasal horizontal meridian. This remains true throughout development. The high cone density in superior retina was unique to this eye, although one retina (birth, not shown) showed similar high density regions in both superior and inferior retina. By P3 weeks (Fig. 6B), cone density over large areas of the periphery ranges from 7,000 to 10,000 cones/mm2. At the temporal edge of the retina, cone density is 6,000 to 8,000 cones/mm’, whereas at the nasal edge it ranges between 9,000 and 11,000 cones/mm2. By P13 weeks (Fig. 6C), peripheral cone density has declined to 3,000 to 5,000 cones/mm’ temporally and 8,000 to 9,000 cones/mm2nasally, again illustrating the “cone streak.” The region of higher density extending into inferior retina was not found in the other retinas. Although the reduction in peripheral cone density continues to adulthood, the rate of decline has slowed markedly so that at P60 weeks (Fig. 6D), temporal cone density is 2,000 to 3,000 cones/mm2 and nasal 6,000 to 9,000 cones/mm2. Temporally, cone density is only slightly elevated above adult values, but nasal cone density is still twice that of the adult. In general, the younger retinas have higher cone density over the entire periphery, although there is some variability between animals (Fig. 7). For example, between 4 and 8 mm of eccentricity, the El52 eye has lower cone density than the newborn eye (E165, Fig. 7A) along the nasal horizontal meridian, the opposite of the general trend. The rate of density decrease as a function of age is greater near birth as shown by the greater separation between the density curves of the youngest pair of retinas compared to the older 5 retinas. In older nasal retina (Fig. 7A), the curve levels off at eccentricities greater than 4 mm, whereas cone density in the immature retinas declines slowly or not at all from 4 mm to the periphery. The nasal (Fig. 7A) and temporal (Fig. 7B) profiles are similar out to 4 mm, but beyond this, temporal cone density decreases more rapidly. The nasotemporal asymmetry results from these differences in density change. A similar developmental pattern can be seen along the superior and inferior vertical meridians (Fig. 8 ) , except that both superior and inferior far peripheral retina showed a sharp decline in density to below 2,500 cones/mm2. The degree of nasotemporal asymmetry was quantified by calculating a cone density ratio at equivalent eccentricities in nasal and temporal retina for each age. For all ages this ratio increases as a function of eccentricity. The nasotemporal ratio ranges from 0.75-1.5 at 30” and increases to 1.25-3.0 by 70“.The lack of obvious change in the ratio with age indicates that the nasotemporal asymmetry is already nearly adultlike at E152. In summary, cone density in peripheral retina declines rapidly up to P13 weeks and then slowly to 60 weeks. Adult densities are reached sometime between 1 and 5 years of age. The horizontal cone streak and the nasotemporal asymmetry are present as early as E152. Finally, there are individual differences in the topographic patterns, such as the high superior cone density at El52 weeks and the high inferior cone density at P13 weeks. Whether these differences are later reflected in adult topography or disappear with age is not known.
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Fovea Fig. 2. A series of horizontal optical sections (Nomarski differential interference contrast images) from three immature retinas, taken at the level of the photoreceptor inner segments at the fovea and along the nasal horizontal meridian at eccentricities of 0.5 mm, 5 mm, and 10 mm. The columns represent single locations at a series of ages and the rows represent a series of locations at a single age. In the foveal column,
0.5 mm Nasal all of the profiles are cones with the exception of a single rod (small black arrow) in E l 6 5 At 0.5 mm, the large profiles are cones and the smaller profiles are rods. A single representative rod is labeled (small black arrow) in each panel. At 5 and 10 mm, the large cones and the small rods are unambiguous. The scale bar (lower right panel) = 10 pm.
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Fig. 3. Maps of foveal cone density in five animals: (A) E152, (B) P3 weeks, ( C ) P13 weeks, (D) P60 weeks, (Elaverage of 3 adults. The foveal maps are keyed to the color scale which ranges from 0 to 200,000 cones/mmLwith a contour interval of 12,500conesimm'. The rings of the foveal maps are spaced at intervals of 2".
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Changes in rod density. There are 5 main features of adult monkey rod topography (Packer et al., ’89; Fig. 9D). First, the fovea is rod sparse, containing no rods within about 0.02 mm of the foveal center and only scattered rods within 0.1 mm. Second, there is a ring of high rod density, the “rod ring,” at the eccentricity of the optic disk, which reaches its maximum density of 177,000 rods/mm2in a “hot spot” in superior retina and its minimum density as it crosses the horizontal meridian. Third, the central rod distribution is radially asymmetric, as shown by the fact that the contours of the rod sparse central area (black, blue, and green) are displaced toward inferior retina. Fourth, peripheral rods are much more symmetrically distributed around the fovea than the cones are, although close examination reveals a slight displacement of the isodensity contours toward nasal and superior retina. Finally, peripheral rod density declines to 25,000 rodsimm’ or less throughout the far periphery. In the fovea, we confirmed Hendrickson and Kupfer’s (’76) observation that the size of the rod-sparse area decreases during development. The diameter of the zone containing less than 12,500 rods/mm2 (dark blue region in Fig. 9) decreases from about 0.8 mm in the youngest retina to about 0.5 mm in the adult retina (Fig. 9D). The only exception to this trend was the P13 week retina (Fig. 9B), whose rod-sparse zone was about the same diameter as the adult, most likely due to high between-individual variability in foveal rod densities. The most striking difference between the P3 week (Fig. 9A) and adult periphery (Fig. 9D) is the high density of rods in the periphery of the young eye, as shown by the color shift from the higher density oranges and reds to the low density blues and greens. Rod density at P3 weeks rarely dipped below 100,000 rods/mm’ even at the retinal edge and the rod ring was located more peripherally than the optic disk. This eye was unusual in that it had its highest rod density of 211,000 rods/mm2 in inferior retina. At P6 weeks (not shown), rod topography looked substantially unchanged with a peak rod density of 200,000 rods/mm2 in superior retina. However, by P13 weeks (Fig. 9B), rod density in the far periphery had declined to 50,000 to 75,000 rods/mm‘, making the rod ring more apparent. This retina has both superior and inferior “hot spots,” which
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have higher rod densities than any other retina we examined. Density actually peaks at 230,000 rods/mm2 in inferior retina. We have found no adult monkey retinas with peak rod density in inferior retina, but Curcio et al. (’90)do report this in one of 6 human eyes. Although the basic adult pattern of rod topography is in place by P60 weeks of age (Fig. 9 0 , peripheral rod density is still higher than that of the adult. The rod ring has a “hot spot” of 170,000 rods/mm2 in superior retina and a gap near the optic disk, but its eccentricity still exceeds that of the adult. Thus rod, like cone, topography must undergo additional remodeling between 60 weeks and adulthood. Figures 10 and 11 show that the less mature retinas generally have higher rod density within 10 mm of eccentricity, although there is considerable variability. At each age, nasal and temporal rod density profiles are similar in shape out to about 8 mm of eccentricity. Beyond this point rod density declines more rapidly in temporal retina. The nasal meridian is longer at all ages and the adult retina is substantially larger than the P60 week retina. The superior and inferior vertical meridians have similar lengths, but there is considerable variability in the highest rod density, probably depending on whether or not the meridian crosses a “hot spot.”
Developmentalchange in the totalnumber of photoreceptors Cones. The total number of cones varied from a high of almost 4 million at birth to a low of 3.1 million in the average adult (Table 1).For the first 6 months of development, the totals varied by only a few percent in a nonsystematic way, suggesting that the cone population was stable and that the differences represented individual variation. The 2 lowest values occur at P60 weeks and in the adult, so there may be up to a 25% decline in cone population during later development. Tritiated thymidine labeling shows that few if any cones are being generated over this period in monkey retina (LaVail et al., ’83; Rapaport et al., ’871, so no more than 25% of the cones could be lost to cell death postnatally. This loss is too small to account for the 250-300% reduction in peripheral cone density and completely in the wrong direction to explain the fivefold increase in foveal density. These data suggest that we must look to growth of the retina or cone migration for explanations of cone redistribution during development. Rods. On average, there are 60 to 70 million rods in the adult macaque retina (Table 1).Tritiated thymidine autoradiography (Rapaport, ’87) shows that some peripheral rods might be generated shortly after birth, but we find no evidence that this late generation significantly changes the total number of rods after P3 weeks (Table 1).In short, although the data are sparse, there is little evidence that the postnatal population of rods changes systematically.
Redistributionswithin a stable population of photoreceptors Data from the previous section rule out a major role for either photoreceptor death or generation as a cause of the changes in photoreceptor topography over development, leading us to examine the evidence that retinal growth and/or lateral migration subserve these redistributions. Retinal area. Retinal area increases by a factor of 2.4 from El52 to adulthood (Table 1).Figure 12 shows that the rate of increase is highest during the first 6 postnatal months, when slightly more than half of the increase in
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area occurs. Retinal area does not reach adult values until sometime between 1 and 5 years of age. In contrast t o the rapid increase in overall retinal area, the posterior pole appears t o be dimensionally stable throughout late prenatal and postnatal development. The center to center distance between the fovea and optic disk varies nonsystematically from 3.37 to 4.05 mm (Table 1). Individual adult values (Packer et al., '89) overlap those of the youngest animals
and no systematic change as a function of age is suggested. These measurements indicate that retinal growth must be nonuniform, with a stable region at eccentricities inside the disk and a much greater expansion in peripheral retina. Proportion of ocular globe covered by retina. The diameter of the ocular globe increased from 14.1 mm (E152) to 21.45 mm (adult), similar to the 14-t o 20-mm range reported elsewhere (Blakemore and Vital-Durand,
DEVELOPMENTOFMONKEYPHOTORECEPTORTOPOGRGPHY
Fig. 6. Maps of cone density in the entire retinas of five animals: (A) El52 (B) P3 weeks, ( C ) P13 weeks, (D) P60 weeks, (Elaverage of 3 adults. The maps of peripheral retina are keyed to the color scale, which represents cone densities ranging from 0 to 16,000 cones/mm2 at a contour interval of 1,000 cones/mm2.The rings and rays of the overlay
48 1
pattern are spaced at intervals of 30 spherical degrees and the black spot represents the optic disk. Areas above 16,000 conesimm' are represented by white. All retinas are displayed as if they are the same size but in fact differ in area by more than a factor of 2 (Table 1).
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Eccentricity (mm) Fig. 7. Graphs of cone density as a function of eccentricity for the nasal horizontal (A) and temporal horizontal (B)meridians of 7 young retinas of different ages and the adult average of 3 animals. Each curve is the average of three meridians (horizontal, 45” above horizontal, and 45” below horizontal). The stippled area on the abscissa represents the optic disk. Cone density values that exceed 20,000 cones/mm2are not shown. These curves are plotted in tissue coordinates (mm) so the extent of each curve reflects the true size of the retina along the meridian, unlike the maps of Figure 6. The increasing size of the retina as a function of age can be seen as an increase in the length of the horizontal meridians.
’86). From globe diameter and retinal area, we calculated the proportion of‘ the ocular globe covered by retina as a function of age in order to assess the relative rates of expansion of the globe and the retina. The retina covered 48-60% of the globe at all ages and there is little evidence that this proportion changes systematically (Table 1).
Patterns ofphotoreceptor redistribution. Although the above data suggest growth is nonuniform, we wanted to quantitatively rule out uniform growth. This analysis assumes that the cone population is stable and that growth is radially symmetric around the fovea, assumptions that are well supported by our data. We tested for uniform
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growth of the retina by comparing the measured expansion of the retina with the predicted expansion of a sphere. Consider the shape of a young retina in vivo and a perfect sphere of the same radius. Both the retinal and the spherical surfaces are divided into a set of concentric spherical rings like those of Figure 1B.Concentric rings are appropriate because radial redistribution would tend to move cones across ring boundaries, changing the proportion of cones enclosed by each ring. After a period of growth, the retina is again compared to a model sphere whose
radius has been increased to match that of the now larger retina. If uniform retinal growth was the sole process subserving redistribution, the zones on the retina will have grown larger in exactly the same way as equivalent zones on the sphere and the 2 patterns will still coincide perfectly. If growth was not uniform, retinal zones will expand more or less rapidly than their equivalent spherical zones. For instance, the number of photoreceptors enclosed by a spherical zone will decrease if its growth is outpaced by the corresponding retinal zone, because photoreceptors for-
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Fig. 9. Maps of rod density in young and adult retinas: (A) P3 weeks, (B)P13 weeks, (C) P60 weeks, (D) average of 2 adults. The color scale represents rod densities between 0 and 200,000 rodsimm' with a contour interval of 12,500 rods/mm'. Areas of the maps that exceed a density of 2000,000 rodsimm' are colored white. The rings and rays of the map overlay are spaced at intervals of 30". All retinas are displayed as if they are the same size hut in fact differ in area (Table 1).
DEVELOPMENT OF MONKEY PHOTORECEPTOR TOPOGRAPHY
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merly contained in that spherical zone will spill into adjacent zones. Migration of photoreceptors from one zone to another would also change the proportion of photoreceptors within a zone. In practice, a computer "drapes" the 800 digital representation of each retina, built up from the set of N retinal samples, over a sphere of appropriate radius. The 700 number of photoreceptors within each concentric ring is calculated, converted to a proportion of the total and 0 plotted as a function of age. If the proportion in each ring is 600 constant as a function of age, then uniform retinal growth 0 w c is supported. If the proportions change with age, then radial f 500 migration or nonuniform growth are occurring. a For clarity, we plot cone data for the E152, P60 week and adult retinas only (Fig. 13A), but data from remaining ages 400 m fall in order between the plotted curves. Between 10" w and 70",each ring of the adult retina contains about 6% of the total cones. Because each successive ring has a larger area than the previous ring, this represents a decline in density. In contrast, the percentage of total cones in retina El52 keeps rising far into the periphery with a peak at 70". This corresponds to the high peripheral density seen in the Fig. 12. Graph of retinal area as a function of age. Retinal area was young animals. The adult Fetinas-have a higher proportion measured by digitizing the outlines of the three retinal pieces.
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Fig. 13. A graph of the proportion of total (A) cones and (B) rods found in a series of concentric rings superimposed on the retinal cone density map at intervals of 5" and centered on the fovea. The figure legend applies to both graphs, but all symbois are not present on each graph. Each data point represents the proportion of total cones in the ring whose outer edge is at the eccentricity shown on the x axis. The total number of cones in the ring was divided by the total estimated population to determine the proportion of the total in that particular ring. The area under each curve sums to 1.The adult shown is retina M1 (Packer et al., '89).
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Fig. 14. A. Graph showing how points of equivalent eccentricity in a young retina (E152, open squares) and adult retina (M1, filled squares) are generated for the growth analysis. Each curve represents the cumulative proportion of cones in a spherical wedge of retina centered on the nasal horizontal meridian. For each eccentricity in the immature retina, there exists an eccentricity in the adult retina with an equal cumulative proportion of cones shown by the horizontal line. In this example, the eccentricity of nearly 16 mm in the adult contains the same cumulative proportion as an eccentricity of 10 mm in E152. These two eccentricities (shown by the vertical lines) are considered equivalent. B. Equivalent eccentricities from M1 and El52 (filled circles) describe the growth of the retina between 2 weeks prenatal and adulthood. A function coincident with the solid line of slope 1would correspond to no retinal growth. A function falling above the line, as does this example, corresponds to retinal growth.
of cones between 10' and 40",whereas the young retinas have a higher proportion of cones between 40" and 90". Similarly, a higher proportion of the rod population is located between 50" and 90" in younger eyes (P3, P13, and P60 wk; Fig. 13B), whereas the adult eye shows a higher
proportion of total rods between the fovea and 40". This analysis suggests that there is a net radial redistribution of both cones and rods from an area centered near 70" toward the center of the retina, which continues past 60 weeks of age. This finding is inconsistent with uniform growth.
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In order to further examine the pattern of growth, we 2.5 to 3 times larger radially. Inferior retina is rather have factored areal expansion into 2 orthogonal compo- anomalous, showing considerably more radial growth over nents, radial growth along rays extending from the fovea a whole range of eccentricities, except in far periphery and tangential growth perpendicular to the rays. Maintain- where it drops to zero. ing constant retinal coverage of the growing ocular globe These analyses show that between El52 and P13 weeks, requires that the retina grow both radially and tangentially. growth over most of midperipheral retina is substantial and Having already rejected uniform expansion, we predict that increases with eccentricity. After P13 weeks, growth has radial and tangential growth will not be equal over the almost stopped, except in the very far periphery. Inferior whole retina. A preponderance of radial growth would be retina has the most radial growth, suggesting that it lags at consistent with increasing retinal coverage of a static or birth and requires more growth to achieve retinal symmeslowly expanding globe, whereas a preponderance of tangen- try. The dominance of radial over tangential growth, in the tial growth would be consistent with decreasing retinal absence of a change in the proportion of globe covered by coverage of a rapidly expanding globe. The analysis, similar retina, suggests a tendency for the globe to elongate more was applied to the cone axially than horizontally or vertically. to that of Mastronarde et al. (’84), density data, although similar results would be expected Lateral cone migration during development. from an analysis based on the rod density data since both Retinal growth appears sufficient to explain the observed cone and rod redistributions are similar. First, the cumula- decrease in rod and cone density in peripheral retina by tive number of cones and cumulative area were calculated both qualitative and quantitative measures without requirfor 30O-wide spherical wedges of retina from the nasal, ing any lateral migration of photoreceptors. In contrast, temporal, superior, and inferior meridians of the E152, and central retina is dimensionally stable over the same period one adult retina (M1 of Packer et al., ’89). Cumulative (Table l), making it difficult on either logical or empirical totals were converted to cumulative proportions to normal- grounds to explain the large increase in foveal photorecepize for between-animal variability in the total number of tor density by any mechanism other than lateral migration cones. Figure 14A shows that within a 30” wedge, the cones of photoreceptors toward the foveal center. In order to of the young retina are packed more densely than is the case determine how far central cones are moving between El52 for the adult retina; this is not surprising since the young and adulthood, we divided the central 1 mm of each retina retina has a similar number of cones, but only a third of the area. Second, we determined the eccentricities in correspond- into a series of concentric rings and calculated the cumulaing sectors of adult and infant retinas, which contained tive number of cones (Fig. 17A). The linear distance that a equal proportions of the total cone population. For example cone inner segment migrated was determined by selecting a (Fig. 14A), 80% of cones are found within 10 mm of location along the eccentricity axis, moving vertically to eccentricity in the El52 retina and within slightly less than intersect the cumulative function of the El52 retina, and 16 mm of eccentricity in the adult retina, so 10 mm and 16 measuring the horizontal distance (x in Fig. 17A) between mm are “equivalent eccentricities.” The entire range of the two cumulative functions. For example, a cone inner adult eccentricity was plotted as a function of equivalent segment located at 0.75 mm at El52 will move radially infant eccentricity in Figure 14B. The straight line of unity toward the foveal center about 0.225 mm to finally lie at an slope corresponds to no retinal growth, whereas the data eccentricity of 0.525 mm in the adult retina. Displacement (circles) are above the line, indicating that growth has as a function of eccentricity (Fig. 17B) was plotted at 7 points between the foveal center and 1 mm. The x axis occurred. Figure 15 compares radial (open squares) and tangential shows the original position of the cone in the El52 retina. (closed squares) growth between El52 and adulthood. The At the very center of the fovea, there is no displacement. radial component of growth is approximately equal to the With eccentricity, displacement grows to a maximum of derivative of the curve through the data points in Figure 0.230 mm at 1mm of eccentricity. The curve approaches an 14B. The tangential component is computed from the ratio asymptote shortly after 1 mm. Presumably the displaceof the widths of the wedges at equivalent eccentricities in ment gradually decreases to zero at even larger eccentricithe infant and adult retinas. Thus ratios greater than one ties, but the exact shape of the decrease and its maximum correspond to growth. For example, at 10 mm in nasal extent cannot be determined from our data. retina, the adult retina is 1.75 times larger in the radial direction and 1.25 times larger in the tangential direction compared to the El52 retina. With the exception of the far DISCUSSION periphery of the superior vertical meridian, radial growth is Basic photoreceptor topography is established prenatally greater than tangential growth at all eccentricities exceeding 2-3 mm, and is greatest in inferior retina. Tangential but matures slowly. The major topographic patterns of the growth increases slowly with eccentricity, with the far cone and rod mosaics are already present 2 weeks before periphery of the adult retina being about 1.6 times larger birth, although they undergo large quantitative adjustthan the infant retina. Radial growth increases more ments postnatally. A central region of higher cone density is rapidly with eccentricity, but the increase is moderate until just becoming apparent at the future fovea, and the nasotemthe far periphery. Superior retina does not have a high poral asymmetry in cone distribution and the cone streak growth ratio in the far periphery, although the ratio a t on the horizontal meridian are present. Rod topography lesser eccentricities is similar to the other meridians. shows a large rod-sparse central fovea and a wide rod ring Inferior retina, in contrast, expands almost twice as much with “hot spots” of high density. Thus both cone and rod as any other meridian. Figure 16 shows an identical distributions already resemble the adult shortly before analysis of the growth occurring between P13 weeks and birth, suggesting that these basic patterns are established adulthood. Nasal, temporal, and superior retina are up to well before birth. This notion is supported by the observa1.5 times larger in both radial and tangential directions tion (Hendrickson and Kupfer, ’76; Smelser et al., ’74) that except at the largest eccentricities, where the adult retina is as early as E70, a 1-mm-wide area of central retina, the site
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Fig. 15. Analysis of the radial and tangential components of retinal growth from 2 weeks prenatal to adulthood along the nasal and temporal horizontal meridian and the superior and inferior vertical meridian. Radial growth (open squares) is the slope of equivalent points function derived for El52 and adult retina M1 (Fig. 14B). Tangential growth (closed squares) is the ratio of the widths of each retinal sector of the adult and El52 retina. Sector width (mml at some distance from the fovea is r . sin(s/r) (2-2 cos(t)l"', where r is the radius of the retina in mm, s is the radial arc length in mm from the fovea to the location being measured and t is the angular width of the sector.
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Fig. 16. Analysis of radial and tangential components of retinal growth from P13 weeks to adulthood along the nasal and temporal horizontal meridian and the superior and inferior vertical meridian. Format and analysis identical to Figure 15.
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Fig. 17. A. Cumulative number of cones as a function of eccentricity for the central 1mm of retina El52 and one adult ( M l , of Packer et al., '89). Data were prepared by dividing the retina into a set of concentric rings and calculating the area and total number of cones in each ring. The rings were spaced at intervals of 0.1 mm up to 0.5 mm of eccentricity and then at intervals of 0.25 mm up to 1mm. This analysis assumes radial symmetry of cone density within central retina (see Fig.
3). The distance x represents the distance in mm that a cone inner segment located at 0.75 mm of eccentricity in the young retina would move toward the fovea. B. Cone displacement towards the fovea as a function of eccentricity. Displacements were calculated from cumulative cone graphs by determining the horizontal distance between the cumulative cone curves for El52 and adult retinas shown in (A).
of the future fovea, has exclusively cones overlying the thickest portion of the ganglion cell layer. Similarly, a large rod-free region is present at 9-14 weeks of gestation in the human retina (Hollenberg and Spira, '73; Provis et al., '85a). Although very early death of rods cannot be ruled out, these data suggest that no rods are ever generated in the most central retina, and the different patterns of cones and rods are thus established very early indeed. There is relatively little information on early development of photoreceptor topography, but development of ganglion cell topography has been extensively studied in a variety of mammalian species. Since the steep central to peripheral gradient and nasotemporal asymmetry of adult ganglion cell topography qualitatively mirror the topography of the cone mosaic (Curcio and Allen, '90; Perry and Cowey, '85; Stone and Johnston, '811, the processes of development may be similar. In the cat and ferret retina, some authors (Henderson et al., '88; Rapaport and Stone, '84; Stone et al., '82,'84) report that ganglion cells identified by morphological criteria are evenly distributed across the retina initially and that the central to peripheral density gradient develops only after the ganglion cell count has stabilized. Other authors, (Robinson, '87; Wong and Hughes, '87b) also using morphological criteria find a definite but transient central concentration of ganglion cells during and just after the period of ganglion cell generation. This transience may be due to the later generation and migration of large numbers of displaced amacrine cells into the ganglion cell layer (Wong and Hughes, '87a), "flattening out" the initial gradient. More direct evidence for an early gradient comes from retrograde labeling of immature ganglion cells using horseradish peroxidase. Lia et al. ('87) find a small central concentration of labeled ganglion cells, with the central/peripheral ratio increasing from 2.6 at E 3 5 4 0 to 18just before birth. These
data suggest that some early process establishes a small central to peripheral gradient. Nevertheless, both the rapid increase in the central to peripheral ratio in the 2 weeks before birth (Lia et al., '87; Rapport and Stone, '841, together with the lack of evidence for ganglion cell death during this time (Williamset al., '861, suggest that differential growth subserves the development of most of the large central to peripheral gradient. In the monkey, Hendrickson and Kupfer ('76) report an increased concentration of ganglion cells in the cone-rich central retina at E74. Lia and Chalupa ('88) repeated their HRP injections into the brains of monkey fetuses and found a central to peripheral ganglion cell gradient of 10 as early as E85. Thus a gradient is present shortly after the ganglion cells are generated between E30 and E70 (LaVail, '83). In human retina, Provis et al. ('85a) find that neurogenesis is higher in nasal retina as early as the end of the first trimester (the youngest age examined), and by E14-15 weeks, the highest ganglion cell density is within 1.5 mm of the fovea. The nasotemporal ganglion cell asymmetry becomes apparent as early as E l 6 weeks. Mitosis in the central retina ceases by E14-18 weeks and few mitotic figures were found even in the far periphery after E30 weeks. These data suggest that a gradient may develop in the primate ganglion cell topography even before cell generation ends. It may be due to very early differential cell generation, differential cell death, growth of the central retina, or even lateral migration, although in the human, uneven neurogenesis seems to play a more definite role. Thus it seems likely that similar processes shape the early topography of both the photoreceptor and ganglion cell layers. Despite the early appearance of the major topographic patterns, macaque photoreceptor distribution is not mature until long after birth. The 6- and 13-month eyes were
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below adult size and foveal cone density but above adult values for peripheral photoreceptor density. In addition, the eccentricity of the rod ring still exceeded that of the adult. If monkey age in weeks is equivalent to human age in months (Boothe et al., '85), these data suggest that a 5-year-old human child might exhibit similar immaturities in retinal development. Yuodelis and Hendrickson ('86) have found that peak cone density in a 45-month human was still well below adult values. This suggests protracted development in both human and monkey, although the age at which the photoreceptor mosaic reaches maturity in either primate is still not precisely known. Prolonged postnatal changes in photoreceptor distribution and morphology may play a role in the physiological and behavioral changes that occur in spatial vision over the same period (discussed in Banks and Bennett, '88; Brown et al., '87; Jacobs and Blakemore, '88; Wilson et al., '88). Because anatomical data on either human or monkey retinal development are still quite sparse, this study is an opportunity to begin a quantitative database detailing primate photoreceptor development. This is clearly important in light of the use of the macaque monkey's visual system as an invasive model for the human (Boothe et al., '85).
Mechanisms underlying photoreceptor redktributionsduring development We find two major redistributions of monkey cones and rods during the first year after birth. The first is a centralward increase in foveal density as shown by a fivefold increase in peak cone density and a decrease in the diameter of the rod-sparse area. The second is a decrease in both cone and rod density in peripheral retina. On the basis of the data presented here, we conclude that: (1) cell generation and death play minor roles, (2) the increase in foveal density and decrease in rod-sparse zone diameter is mediated by lateral migration of photoreceptors toward the center of the fovea, and ( 3 ) the decrease in peripheral photoreceptor density is caused by the increased size of the peripheral retina. Cell generation and cell death. The increased density of both rods and cones in central retina during postnatal development could be subserved by late generation of new photoreceptors. In most mammalian retinas, those photoreceptors generated first lie near the posterior pole; those generated late in development lie near the peripheral edge of the retina (Provis et al., '85a; Rapaport and Stone, '82; Provis et al., '85a; Robinson, '87; Sidman, '61). Brief reports by La Vail et al. ('83)and Rapaport et al. ('87) and our own unpublished observations show that no photoreeeptors are generated postnatally in central retina, although some cones and rods are generated in the far peripheral retina as late as El02 and P17, respectively. These data rule out postnatal photoreceptor addition in the central retina, whereas the small amount of cell addition to peripheral retina works against the developing central to peripheral gradient of photoreceptor density. Cell death also is unlikely to be a n important factor in the central retina, because it would lower rather than increase cell densities. However, cell death could play a role in the decreased density found over time in the peripheral retina. Cell death in the outer nuclear layer has been reported in cat from E42-Pl7 coincident with the onset of photoreceptor differentiation and outer plexiform layer development (Robinson, '88; Stone et a1 , '84). Cell death occurs in the subventricular zone of very young human retina, but was
not reported in differentiated photoreceptors (Penfold and Provis, '86). Even in the ganglion cell layer, where cell death certainly occurs (reviewed by Provis and Penfold, '88; Wikler and Finlay, '891, its role in determining topography is unclear, although several authors suggest that it may be important (Mastronarde et al., '84; Rapaport and Stone, '84; Stone et al., '82; Wong and Hughes, '87b). The ubiquity of neuron loss during development throughout the central nervous system, including the primate retinal ganglion cell layer (Provis et al., '85b; Rakic and Riley, '83) suggests that some loss of photoreceptors should occur and has simply not been found. Our study does not include the most immature stages of development, but our finding of relatively stable cone and rod populations well after the period of photoreceptor generation and throughout the period of topographic differentiation suggests that cell death is not a major factor in shaping photoreceptor topography during later development. No more than 25% of all cones are lost between El52 and P60 week, a loss that is insufficient to explain the average threefold decrease in peripheral cone density during the same time. The same conclusion is consistent with changes in the number of rods during this same period, although the total number of rods in individual retinas is more variable and fewer ages were counted. It thus appears that the small changes that may be occurring in the number of photoreceptors due to cell generation or cell death are not sufficient to explain the large changes in topography during postnatal development. We next consider how a relatively uniform photoreceptor population could be redistributed by cell migration and/or retinal growth. Photoreceptor migration. In the central retina, growth cannot be the main process redistributing photoreceptors because it would act to lower, rather than raise, cone density, The central retina of the monkey stops growing by E60 (Steineke and Kirby, '89) or E80 (Lia and Chalupa, '88). The stable fovea to disk distance during the period of this study also confirms that there is no growth of central retina after E152. By exclusion, lateral migration of photoreceptors toward the foveal center is the only reasonable explanation for both the marked increase in foveal cone density and the reduction in the size of the rod sparse area postnatally. Since the density of peripheral cones and rods declines while central density increases, it might be possible to explain both phenomena by migration of cones from the periphery into central retina. Quantitatively, however, only the area within 0.5 mm of the foveal center, which in the adult contains about 40,000 cones, undergoes a large increase in density. If density increases by a factor of 5, only 32,000 cones, or 1%of the 3 million cones in peripheral retina, would need to migrate into the fovea. The loss of this tiny percentage of peripheral cones would be difficult to detect at all and certainly cannot account for a threefold decrease in peripheral density. Rod topography also undergoes an increase in central rod density and a reduction in peripheral density, and the position of the rod ring becomes more central during later development. By arguments identical to those made for the cones, the central increase can be subserved only by lateral displacement of rods toward the center of the fovea. This increase could be due to an active migration similar to the cones or a passive shift as rods are "dragged along" with the surrounding cones as they migrate toward the foveal center.
DEVELOPMENTOFMONKEYPHOTORECEPTORTOPOGR.AJ?HY Previous work on monkey foveal development (Hendrickson and Kupfer, '76) and the data of this study strongly suggest that photoreceptors migrate toward the foveal center, whereas the other retinal neurons migrate away from it to create the foveal depression. Retinal neurons are unusual among migrating neurons in that they move as differentiated cells that have developed both a complex cytology and synaptic contacts. Synapses are formed between monkey photoreceptors and other neurons in the outer plexiform layer by E74 (Smelser et al., '74) and outer segments are present before birth on central cones (Hendrickson and Kupfer, '76). The cone axons or fibers of Henle appear at E l 2 5 and elongate considerably after birth. This early establishment of synapses would seem to suggest that later redistribution could be most easily accomplished by a mechanism that avoids large changes in relative position between photoreceptors that would tend to increase the complexity of the neuropil as axons and dendrites stretched and contorted to retain their previously established synapses. Even so, the changes occurring in the neuropil are not simple. Theoretical (Schein, '88) and empirical evidence (Perry and Cowey, '88) suggests that cones with adjacent inner segments in the foveal center have nonadjacent cone pedicles requiring some crossing of cone axons. The visual mapping established at the level of the inner segments could be maintained at postreceptoral levels by formation of synapses in the outer plexiform layer before migration, although the identity of the cells making these early contacts is as yet unclear. Nevertheless, the visual field position of postreceptoral neurons subserving foveal vision must be recalibrated during development both for increasing eye size (Milleret et al., '88; Olson and Freeman, '80) and for lateral migration of cones. Anatomical measurements of the fibers of Henle in macaque retina, together with our calculations of how far photoreceptors migrate, can be used to infer how the photoreceptors and the bipolar neurons move with respect to each other during foveal development. Although the data for the centralmost fovea are very sparse, the longest fibers of Henle probably belong to cones near but not in the center of the fovea and are 0.3-0.4 mm in length (Boycott et al. '87; Perry and Cowey, '88; Polyak, '41; Schein, '88). Fiber
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length decreases with eccentricity and by 0.75 mm is down to approximately 0.225 mm (Perry and Cowley, '88; Schein, '88). Calculated migration distance, in contrast, is zero at the foveal center and increases to a maximum of about 0.23 mm at 1 mm of eccentricity. These data suggest that at the center of the fovea the entire length of the fiber of Henle is due to outward migration of the bipolar neuron to which the photoreceptor is connected. With increasing eccentricity, the distance that a cone migrates toward the foveal center increases. For example, a cone initially located at 1 mm of eccentricity in the infant would migrate 0.23 mm centralward and be found at an eccentricity of about 0.77 mm in the adult. Since the fibers of Henle are between 0.225 and 0.245 mm in length at 0.75 mm of eccentricity, the calculated distance that the photoreceptor migrates is the right size to account for the length of the fiber. We were not able to calculate migration distances at eccentricities greater than 1 mm, but we would predict that the distance that a photoreceptor migrates would decrease in good correlation with the length of the fiber of Henle. The cellular mechanisms subserving photoreceptor migration remain unclear, and we can only speculate how it might function. Immature neurons are able to actively move relative to each other and to glia by establishing adhesive interactions involving membrane and extracellular adhesion molecules (Edelman, '85; Silver and Rutishauser, '84). This ability may be retained or acquired by mature primate photoreceptors. It is also possible that central photoreceptors respond to internal or extrinsic cues to actively streamline themselves during development. If neighboring photoreceptors and their matrix were adhesive and if the photoreceptors at the foveal center were anchored, this change in shape would tend to pull peripheral photoreceptors toward the fovea. Retinal expansion. The retina increases in area by a factor of 2.4 from El52 to adulthood, and by even a larger factor if the stable central 8 mm is subtracted. This amount of retinal expansion fits well with the threefold decrease in peripheral cone density. Similarly, rod density in the adult peripheral retina is about 50% of that at P3 weeks, but the area of the P 3 week retina was about 60% of the adult. These data suggest that retinal growth alone is sufficient to explain the observed reductions in both cone and rod peripheral density. There is no logical need to invoke different mechanisms of redistribution for the cones and rods because they are intermixed over the peripheral retina, and growth will reduce both cell densities equally. Differential expansion of the peripheral retina can also explain the shift in the eccentricity of the rod ring toward the fovea. Figure 18 is a qualitative model showing the effects of differential expansion on the rod density profile along a radial path from the fovea to the ora terminalis in a young retina. Between the fovea and disk, the retina is dimensionally stable, so expansion does not change rod density and the two profiles remain superimposed. At eccentricities beyond the disk, the degree of expansion increases into the periphery, reducing far peripheral rod density more than near peripheral density and causing the two curves to diverge. Note that rod density is lowered in the adult at all eccentricities beyond the optic disk. The rod ring shifts toward the fovea as the increase in peripheral area reduces rod density in the peripheral flank of the rod ring. We conclude that the greater growth of the peripheral retina can explain the decline in peripheral rod density, the
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reduced peak rod density, and the decrease in the eccentricity of the rod ring in the adult compared to the newborn. Between the stable posterior pole of the retina, where we are confident that redistributions are due to lateral migration, and the far periphery, where retinal expansion adequately explains the reduction in peripheral density, lies an area of uncertainty. Here our methods are not sensitive enough to resolve the observed redistribution into a migratory component and a retinal growth component, even though both may be active. Our plots of photoreceptor proportional distribution (Fig. 13) clearly indicate that redistribution extends to eccentricities of at least 60". This is well beyond the central growth-free zone and suggests that the intermediate region of the retina outside the eccentricity of the optic disk could be subject to both lateral migration and differential growth. The presence of oblique fibers of Henle beyond 3 mm of eccentricity also supports this idea (Perry and Cowey, '88). In order to decipher the relative strengths of these two forces in the midperiphery and confirm them elsewhere, a direct method of observing photoreceptors during development is required. For example, it may be possible to label in vivo a small number of photoreceptors at known retinal locations with a nontoxic, durable marker and then chart their positions with respect to retinal landmarks during development. During the first year of development, the posterior pole of the monkey retina is dimensionally stable, whereas the peripheral retina grows more with eccentricity. Growth is greater along the radial than the tangential dimension at any given location, and it is more rapid early in development. This pattern is rather different from the cat, where tangential growth dominates in the far periphery, radial growth in the midperiphery, and little growth occurs a t the posterior pole (Mastronarde et al., '84).This species difference likely reflects the fact that the cat retina covers 60% of the globe at birth but only 34% in the adult, requiring more tangential growth to maintain retinal apposition with the globe. Monkey retina covers a relatively constant 52% of the globe and so needs less tangential growth to maintain retinal apposition. Data from the monkey are also consistent with human data (Robb, '821, which showed only a small decrease in retinal coverage from 62% to about 56% during later development. It thus appears that monkey and human retinal growth are similar to each other but different from cats, implying that nonprimate models of retinal growth can be extrapolated to humans only with great care. Potential mechanisms subserving retinal expansion include some combination of the addition of retinal cells, the increase in the size of existing retinal cells, the increase in the amount of neuropil, or a thinning of the nuclear layers. Since tritiated thymidine fails to label a substantial number of neurons postnatally, addition of neurons probably plays little role in primate retinal expansion. There is a n agerelated increase in the diameter of both cone and rod inner segments at equivalent eccentricities exceeding 5 mm. It is not clear, however, whether the growth of individual photoreceptors is a cause of retinal expansion or a reaction to it. If photoreceptor coverage remains constant as the retina expands, then either passive stretch or active growth alone might be sufficient to explain the change in photoreceptor inner segment diameter. If coverage changes as a function of time, then more complicated interactions must be considered. It is also possible that a large amount of retinal
0. PACKER ET AL. growth is due to increases in glial size and number. Mann (1964) suggests that growth of peripheral retina is largely due to thinning of the nuclear layers with a simultaneous increase in neuropil, which maintains retinal thickness. More work is required to distinguish among these possibilities. Robinson and coworkers ('87,'89) have proposed a balloon model of developmental changes in ganglion cell topography in which interocular pressure provides a passive stretching force. They predicted that the retinal region most resistant to stretch should: (1)have the highest cell density, (2), maintain similarly shaped isodensity contours throughout later development, and (3) be the most advanced developmentally. These predictions are at least qualitatively correct for monkey photoreceptor topography as well, although it is necessary to factor out the independent development of the fovea. The central retina within the eccentricity of the optic disk does not change size postnatally, begins to develop first, matures earliest, and has the highest photoreceptor density. Likewise, the cone streak and the rod ring, which are areas of relatively high photoreceptor density, retain their basic shapes during development. Based on these criteria, passive stretching may play a role in both ganglion cell and photoreceptor redistribution. In fact, we believe that the photoreceptor layer is an appropriate place to study retinal expansion, because: (1) the photoreceptor inner segments are tightly packed together without overlap, making it possible to determine how their size and distribution changes as the retina grows, and ( 2 ) during later development, when the majority of retinal growth occurs, the photoreceptor layer is largely free of cell generation and death, which complicate interpretations of similar data from the ganglion cell layer.
ACKNOWLl3DGMENTS This work was supported by EY01208 and EY04536 to A.H., EY06109 to C.C., and by EY07031, and in part by RR00166 to the Regional Primate Research Center at the University of Washington. The authors gratefully acknowledge, in particular, the tissue program and the assistance of Judy Johnson. We also thank Kenneth Sloan for the design of topographic mapping software.
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