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The topographical distribution of rods and cones in the adult chicken retina
Exl3. Eye Res. (1973)
17, 347-355
The Topographical Distribution of Rods and Cones in the Adult Chicken Retina DAVID
B.
MEYER
A?;D
HAROLD
C. MAY:
JR
Deprtment of Aw,atonzy, Godon H. Scott Hall of Basic Medical Sciences, Wayne State University, Xchool of Medicine 540 E. Canjield, Detroit, Miclz. 48201, U.S.A. (Received 11 June 1973, and in revised form
30 July 1973, Boston)
The relative proportion of rods and cones (single and double) in the chicken retina was determined by counting these elements in each of nine pre-determined segments prepared as periodic acid-Schiff serial sections for rod (hyperboloid glycogen) and double cone (aceessory cone paraboloid glycogen) identifications and as unfixed, flat preparations for cone determinations (colored oil droplets). Periodic acid-Schiff preparations revealed that rods and accessory cones were equal in number in all retinal segments except those located directly posterior to the posterior extremity of the pecten where the double cones outnumbered the rods in a ratio of 14 : 10. On the basis of oil-dronlet aigmentation. double cones housing a golden-yellow droplet in their chief component and a small yellowish-green droplet, in their accessory members were constantly observed 1.0 be twice as numerous as single cones (red droplet) in all retinal segments. These data reveal that the proportion of rods to double and single cones is 2 : 2 : 1, respectively, in all areas of the chicken retina except a posterior (lateral) segment where the ratio is IO : 14 : 7. This area of greater cone concentration corresponds to the site of the temporal fovea of some birds and may represent the area of most acute vision in the fovea-free chicken retina.
1. Introduction This study represents an attempt to determine the proportional distribution of rods and conesin the whole chicken retina. Its validity is based on the reliability of two microscopic techniques to identify selectively the specific visual elements comprising the retina of this species (Gallus dornesticzcs):(1) The periodic acid-Schiff (PAS) reaction for the cletermination of rods and clouble cones, owing to positive reactions for glycogen in the hyperboloid of the rod an.dthe paraboloid of the accessory member of t,he double cone (Rabinovitch, %Iota and Yoneda, 1954; Yamada, 1962; O’Rahilly and Meyer, 1963); (2) Unfixed and unstained, flat retinal preparations for the presence of cones, which are easily recognized by the brightly colored oil droplets in their inner segments: red in the single cones,golden yellow in the chief, and greenish yellow in the accessorymember of the double cone (Meyer and Cooper, 1966). Unlike the PAS reaction, the sensitivity and reliability of which is undisputed, the proposed cone-oil droplet association has not been confirmed (Morris and Shorey, 1967). Nevertheless, visual cell counts recorded for each of nine pre-determined segments of the whole chicken retina prepared as PAS and flat preparations provide significant data on the topographical distribution of rods and cones and indicate the region of most acute vision in this otherwise fovea-free retina. 2. Materials and Methods Retinas from adult chickens (Gallus do~,esticus) mere prepared as PAS serial sections (rod and double cone determinations) and as unfixed, flat preparations (cone determinations). For each procedure eyes were removed from decapitated, adult chickens and 347
348
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B. MEYER
AND
K.
C. MAY,
JR
bisected through the equator into anterior and posterior halves. Using the pecten as a landmark, each retina was divided into nine numbered segments (Fig. 1). The first cut was made directly t.hrough the longitudinal axis of the pecten, the second was made parallel to the first, 5 mm centrally (nasally) from the pecten. The third and fourth incisions were made at each extremity of the pecten, perpendicular to the first two. Since the segments were not of equal size, no attempt was made to determine the total concentrat.ion of each visual cell type in each of the nine segments.
PIG. 1. Lateral view of chicken’s head to show the retinal segments 5 and 6 represents the p&en. iYote that the relative orientation of the hoad.
segments position
in situ. The thick line between of the segments varies with the
Y&S prel)aratiosLs Kine retinal segments from each of five chickens were removed complete with their adjoining choroidal and scleral tunics, fixed for 24 hr in cold (4°C) Gendre’s fluid (85% picric acid saturated in 90% ethanol; 10% formalin; 5% glacial acetic acid) and subjected to the periodic acid-Schiff technique in toto (Meyer, 1960) using aqueous periodic acid instead of the alcoholic buffered solution. Glycogen was identified by its complete removal in 1% buffered malt diastase for 1 hr at 37°C. Following dehydration in ethanol and double embedding in paraffin and celloidin according to Peterfi’s methyl benzoate method (Carleton and Drury, 195’7), the segments were sectioned serially at 8,~“. For each segment counts lvere made of rods (PAS-positive hyperboloids) and double cones (PAS-positive paraboloids) (Fig. 2) in every fifth section to insure that each element was counted only once. In some cases whole retinas were subjected to the freeze substitution method of Feder and Sidman (1958), using I:/, picric acid in ethanol as the fixative prior to the PAS reaction. Visual cell counts were made in these preparations but were not included in the data presented because the retinas were not cut into nine segments. The findings, however, corroborated the statistical data obtained from segmented, sectioned retinas.
Nine retinal segments from each of logical saline (0.9%) from the adjoining scope slides, vitreous side down (visual slip. Each segment was then examined illumination and phase optics (Pi,.‘~1. 3).
three chickens were carefully removed in physiopigment epithelium, individually floated on microcells uppermost), and gently covered by a coverwith a Zeiss photomicroscope using bright field Oil droplet counts were made at a magnification of
DISTRIBUTION
OF
CHICKEN
RODS
AND
CONES
FIG. 2. Photomicrograph of chicken retina (transverse section) subjected to the periodic acid-& reaction. Positive reactions for glyoogen are shown by the magenta ooloration (diastase digestible the cone-shaped rod hyperboloids (arrow) and the larger, barrel-shaped accessory cone paraboh situated internal to them (lower). ( x 800) Fro. 3. Photomicrograph of unfixed, flat retinal preparation (visual cells upperm.ost; i.e. nearest viewer) showing the brightly colored oil droplets ofthe chicken cones. The red (single cones) and yello, (chief cones) droplets are approximately the same size. The smaller greenish-yellow droplets (arr reside in the accessory member of the double cone. ( x 800) FIG. 4. Phase contrast photomicrograph of a chicken double oone with a large, yellow droplet in chief component and a smaller one in the accessory member. ( x 800) FIG. 5. Phase contrast photomiorograph of three accessory cones each containing a small oval yellow green droplet. For comparison, note the larger, circular droplet at the right. ( x 800) FIG. 6. Phase contrast photomicrograph showing a single cone housing a red droplet. ( x 800)
:hiff 1 Qf >ids the vish ow) the ish-
350
D.
B.
MEYER
AND
H.
C. MAY,
JR
approximately x 300. Red droplets were counted as single cones, whereas the yellowish pigments (golden yellow and yellowish green) were tabulated together as double cones. All counts were made on undistorted areas in all regions of each segment. For the determination of visual cell-oil droplet associations fresh preparations were also gently teased and examined by phase contrast microscopy. 3. Results PAX
preparations
The rod hyperboloids and accessory cone paraboloids were brilliantly stained by the PAS reaction, and hence can be idelitifiecl immediately (Fig. 2). The visual cell counts for each segment from at least three different chicken retinas are tabulated in Table I. They reveal that the proportion of rods to accessory cones is almost TABLE
Numbers of rock (R)
and
accessorycones
I
(AC) and their
relative
proportion
&h thicket?,
as revealed by counts of glycogen-containing paraboloids (accessorycones) and hyperboloids (rods) in periodic acid-Xchiff preparations. (Each segmental count represents retinas
data
Retinal
Segments
1
2
3
4
5
6
7
8
9
from
(hyperboloids)
a diferent
retina.)
Accessory cones (paraboloids)
2983 2989 1689 1654 1932 2312 3813 1808
311.5 3079 1809 1856
2086 1491
2941
1459 1697
1785 1884 2165
1835 2421 2056
4991 3189 2311 2571 2056 1580 1132 2228 3707
2139 1005 917 974 1275 2312
1955 1578
2338 2636 5376 2405 2043
2560 2358 5404 3555 2460
2511 2119 1611 1801 2311 2865 2234
1110 946 1019 1301 2636 2029
1783
Ratio
1 I 1 1 1 1
1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1
R : AC
1.0 1.0 1.1 1.1 1.2 1.1 1.4 1.3 1.4 1.4
1.2 1.1 1.2 1.2 1.1
I.1 1.1 1.1 1.0 1.0 1.0 1.0 I.0 I.1 1.0 1.1 1.0 1.0 1.0 1.1 1.0 1.1
DISTRIBUTION
OF
CHICKEN
RODS
AND
351
CONES
equal (1 : 1.1) in all portions of the retina except segmen.t 3 which contains a significantly higher ratio of accessory cones (1 : 1.4). The accessory cones are thinnest in this region and their glycogen-filled paraboloids appear more barrel-shaped and closer together. Undoubtedly, such an arrangement correlates with the more dense population of double cones in this segment than in neighboring areas where paraboloids assume a more rounded appearance. TABLE
II
Jumbers of cones and their relative proportions in pre-determined segments of three chicken retinas (un$xed, flat preparations) as revealed by counts of colored oil droplets
Retinal
segments
*
Single
cones (red)
47 45 55 62 67 59 64 68 60 50 42 51 55 60 65 52 49 62 41 53 53 43 52 57 51 57 49
Double cones (yellowish)
194 186 247 271 215 251 262 288 267 208 179 214 228 254 261 218 207 255 171 227 223 183 217 234 202 231 209
Ratio
SC : DC t
1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
: : : : : : : : : : : : : : : : : : : : : : : : : : :
2.1 2.1 2.2 2.2 2.1 2.1 2.0 2.1 2.2 2.1 2.1 2.1 2-1 2.1 2.0 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.0 2.0 2.1
* The segmectal data from each retina are presented in sequence so that the distribution within each of the three retinas is given. t Accepting the fact that two yellowish droplets are housed in one double cone and one red droplet resides in the single cone (Meyer and Cooper, 1966), the ratio of single cones (SC) to double cones (DC) is determined by the relative proportion of 1 red to 2 yellow droplets.
The rods reach their maximum length and thinness in segments 2 and 3. Thus, the cone-shaped hyperboloids observed in these regions become further displaced choroidally from the accessory cone paraboloids and are seldom observed to be wedged jn between paraboloids as noted jn other areas of the chicken retina. This arrangement, too; provides for a greater concentration of double cones per unit area, in segments 2 and 3.
452
D.
B. MEYER
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@. MAY,
JR
Fresh preparations Whereas the red oil droplets appear singly at definite intervals in a lattice pattern, the golden-yellow and yellowish-green droplets are generally paired with one another and appear to have a more scattered distribution (Fig. 3). Regional accumulations of red or yellowish droplets to form so-called red, orange or yellow fields are not evident. Within the thickness of the retina, however, the red oil droplets are placed on their own level, as are the yellowish (golden-yellow and greenish-yellow) droplets. The yellowish-green droplets are smaller and more oval-shaped in comparison to the red and golden-yellow droplets. Numerical data on the segmental distribution of the colored oil droplets are tabulated in Table II. They reveal a rather constant proportion of four yellowish to one red oil droplet in each of the nine retinal segments. Assuming that two yellowish droplets occupy one double cone and that one red droplet represents one single cone, the data further indicate that there are twice as many double cones as single cones throughout the chicken retina.
4. Discussion In contrast to the reports of other investigators, our PAS findings clearly show that rods are plentiful in the chicken retina and are rather uniformly distributed in all areas. The number of rods equals the number of double cones in all topographical regions of the retina except that located directly posterior to the posterior extremity of the pecten (segment 3) where the double cones are thinner and slightly outnumber the rods by 14 to 10. Support for this relatively high proportion and rather even distribution of rods has been provided by the biochemical studies of visual pigments (Hecht, 1942; Bliss, 1946; Wald, 1949) which have revealed a greater amount of rhodopsin than iodopsin. Bliss (1946) in addition found that the fovea1 (central) portion of the chicken retina contains the same proportion of rhodopsin to iodopsin (4 : 3) as other more peripheral regions. Believing that cones greatly outnumbered rods in this species, Hecht (1942) and Wald (1949) provided an explanation for this discrepancy by theorizing that rhodopsin was more highly concentra.ted in rods, perhaps as much as 100 times that of iodopsin in single cones (Wald, 1949). The counting of colored oil droplets to determine the quantitative and topographical distribution of avian cones has been employed by many investigators (Schultze: 1866; Dobrowolski, 1871; Waelchli, 1883; Hahn, 1916; Rochon-Duvigneaud, 1920; Erhard, X924; Uchiyama, 1930; Coulombre, 1955; Decker, 1963; Peiponen, 1964; Mayr, 1972. See reviews by Hahn, 1916, and Meyer, Cooper and Gernez, 1965). Such data have revealed localities of high cone concentration in the chicken retina but are difficult to collate because of the multitude of oil droplet colors described by the early investigators (owing to the inferior lenses then available), the absence of well-defined and comparable retinal areas and the failure to establish a relationship between oil droplet color and specific cone type. It is evident from Fig. 1, for example, that the anatomical position of the chicken’s head determines the location of the retinal areas designated as dorsal (upper), ventral (lower), anterior (medial, nasal) and posterior (lateral, temporal). Distributional patterns based on these areas are therefore misleading and vary greatly with each investigator. Utilization of the pecten to delinea.te precise retinal segments, as adopted in the present study, eliminates this source of error. It is now well established that only three different colored oil droplets exist in the
DISTRIBUTION
OF
CHICKEN
RODS
AND
383
CONES
cones of the chicken retina: red, golden yellow and yellowish green (Meyer, et al., 1965). According to Meyer and Cooper (1966) the red oil droplets occupy the single cone, whereas the golden-yellow and yellowish-green droplets reside in the chief and the accessory member of the double cone, respectively. These relationships have been observed in phase microscopic studies of teased, adult (Meyer and Cooper, 1966) (Figs 4, 5 and 6) and embryonic (Cooper and Meyer, 1968) preparations. The small, oval, yellowish-green droplet can only be housed in the narrow, tapered extremity of the inn.er segment of the accessory cone which can be easily recognized in phase microscopic preparations by its bowhng pin appearance (Figs 4 and 5). Nevertheless, the existence of an oil droplet in the accessory cone has been denied by Morris and Shorey (1967) and Morris (1970) w h o, instead, describe and illustrate from electron microscopic observations “a granular vesicle at its tapered scleral end”. TABLE
Collated
III
datu on visual cell proportions
in
chicken retina
Visual
Investigator
Matsusaka (1963) Xorris and Shorey Morris (1970) Present
Study
Method
(1967)
*
Retinal
E.M. E.M. E.M. PAS+O.D.C.
1
* .13% of the receptors i Oil Droplet Counts.
cell distribution (%I COIES Single Double Rods
location
Central Posterior pole Posterior pole Periphery of cup Entire retina except segment Segment 3
3
31.2 36.0 54.0 37.0
36.6 36.0 32.0 30.0
14.0 33.0
20.0
40.0 45.0
40.0 32.0
23.0
32.2
15.0
were unidentified.
Our oil droplet counts of eachretinal segmentsubstantiate the findings of Coulombre (1955); they have revealed that the red and yellowish droplets constitute approximately 20 and SO%, respectively, of the total number of oil droplets in all portions of the retina (Table II). Accepting the fact that the yellowish droplets are confined to double cones, these data show that the double cones outnumber the single cones by a ratio slightly greater than 2 : 1 throughout all portions of the neural retina. Furthermore, by combining our quantitative data from both PAS and the oil droplet counts (Table III), the proportion of rods to double and single conesis found to be 2 : 2 : 1 (40%, 40%, 20%), respectively, in all parts of the chicken retina except an. area located directly posterior to the posterior extremity of the pecten (segment 3) where an increasednumber of conesproduces a ratio of 10 : 14 : 7 (32%, 45%, 23%). Although no distinct thickening or thinning of the retina is apparent in this segment, its greater concentration of conesundoubtedly results in a local increase in resolving power indicative of an area centralis (Walls, 1942; King-Smith, 1971). Two such circumscribed, physiological areas have been described in most birds: central (principal, nasal) and temporal. They may be circular, oval or in the form of a long horizontal band or ribbon, and usually contain a depressionor fovea. Chickens lack a morphologically-discernible fovea (Schultze, 1866; Chievitz, 1591; Slonaker, 1897; Walls E
354
D.
B. MEYER
9ND
H.
C. MAY,
JR
and Judd; 1933; Rochon-Duvigneaud, 1943)> although a nasal area has been reported (Chievitz, 1891; Slonaker, 189’7). In topographical location, segment 3 corresponds to the site of the temporal area which houses the temporal fovea of owls (Wood, 1917; Oehme, 1961) eagles, hawks, hummingbirds; swallows, bitterns (Walls, 1942), falcons and buzzards (Oehme, 1964) and may represent the area of most acute vision in the chicken. It is the only fovea1 area in owls, whereas in the other birds an additional fovea occupies a more eentral or nasal area. The central fovea and corresponding area, if present in the chicken, would reside in segment 2 or 5, which probably corresponds to the dorsal (upper) posterior (lateral) region regarded by Krause (1894), Hess (1907) and Bl&sser (1927)‘as the place of most distinct vision. [For these relationships see Wood’s (1917) excellent drawings of the fundus in a variety of birds.] The electron microscopic data of Natsusaka (1963), Morris and Shorey (1967) and Morris (1970) are included in Table III and show conflicting results, although their counts may have been made in different areas. Matsusaka (1963) in the central portion of t,he chicken retina found an almost equal number of single cones and rods an d a slightly higher percentage of double cones. Of more apparent functional significance, however, are Xatsusaka’s calculations of retinal areas occupied by these elements. At t,he level of the accessory cone paraboloids, for example, he calculated that the double cones occupy four times the area of the single cones, and 60 times that of the rods. Walls (1942) associates a high percentage of double cones with animals possessing strong diurnal habits, although he considered an equal number of double and single cones as the maximum proportion. Morris and Shorey (1967) counted a total of 286 receptors from the posterior pole of different chicken retinas (t,opographical location not specified) and found an equal proportion of single and double cones and less than half as many rods. On the basis of fine structural characteristics they distinguish four types of visual elements: rods, double cones and two types (I and II) of single cones that are differentiated by the density of their oil droplet and mitochondrial cristae. Type I has a darker staining oil droplet and more numerous cristae. Morris and Shorey (1967) further suggest that the single cones house either the red or the yellow droplet (type I or II not determined) and that the chief cone contains the green droplet. In a subsequent report (Morris, 1970) a third type of single cone (type III) is recognized; it is characterized by a less dense oil droplet. Morris (1970) determined the relative proportion of chicken photoreceptors in 250 pm2 samples from the posterior pole and the periphery of the retina sectioned tangentially. Each receptor type was counted in three montage electron micrographs, each of which comprised an area containing 200-600 visual elements. Because these elements were cut in cross-section, the counts were made at the level of the oil droplets and proximal inner segments, and occasionally from the level of the outer segments. Double cone frequency was estimated from counts of chief members only (accessory cones contained a granular vesicle, not an oil droplet). Single cones could only be identified in sections through their oil droplets. Statistical analyses of these counts indicated a receptor mosaic with an even spacing of the receptor types. ACKNOWLEDGNENT
This project was supported by Research Grant 00477 from the h’ational Eye Institute, National Institutes of Health, U.S. Public Health Service.
DISTRIBUTION
OF
CHICKEN
RODS
AND
CONES
355
REFERENCES Blasser, A. (1927). Zool. Jahrb. (Zool. Physiol.) 43, 69. Bliss, A. F. (1946). J. Germ. Physiol. 29, 277. Carleton, H. 111. and Drury, R. A. B. (1957). Histological Technique. Third edition. Oxford University Press, New York. Chievitz, J. H. (1891). Arch. Anat. Physiol. Physiol. Abt. 15, 311. Cooper, T. G. and Meyer, D. B. (1968). Exp. Eye Res. 7, 434. Coulombre. A. J. (1955). Amer. J. Anat. 96, 153. Dobrowolsky, W. (1871). Arch. Anat. Physiol. W&en. Med. 38, 208. Diicker, G. (1963). Z. Tierpsychol. 20, 43. Erhard, H. (1924). Zool. Juhrb. (Zool. Physiol.) 41, 489. Feder, N. and Sidman, R. L. (1958). J. Biophys. Biochem. Cytol. 4, 593. Hahn, E. (1916). 2. Wiss. ZooZ. Abt. A 116, 1. Hecht, S. (1942). Ann. Rev. Biochem. 11, 465. Hess, C. (1907). Arch. Augenheilk. 57, 298. King-Smith, P. E. (1971). In Physiology and Biochemistry of the Domestic Fowl (Eds Bell, B. J. and Freeman, B. M.). Vol. 2, pp. 1039-1083. Academic Press, New York. Krause, W. (1894). Intern. Monat. Anat. Physiol. 11, 1. Matsusaka, T. (1963). Jap. J. Ophthalmol. 7, 238. Mayr, I. (1972). Vision Res. 12, 1477. Meyer, D. B. (1960). Stain Teehnol. 35, 83. Meyer, D. B. and Cooper, T. G. (1966). Amer. J. Anat. 118, 723. Meyer, D. B., Cooper, T. G. and Gernez, C. (1965). In Structure of th.e Eye (Ed. Rohen, J. W.). Academic Press, New York. Morris, V. B. (1970). J. Conzp. Neural. 140, 359. Morris, V. B. and Shorey, C. D. (1967). J. Comp. Neural. 129, 313. Oehme, H. (1961). ZooZ. Jahrb. (,&at. Ontog.) 79, 439. Oehme, H. (1964). 2. Morphol. Oekol. Tiere. 53, 618. O’Rahilly, R. and Meyer, D. B. (1963). Ann. Histochim. 8, 281. Peiponen, V. il. (1964). Ann. ZooZ. Fenn. 1, 281. Rabinovitch, M., Mota, J. and Yonedn, 6. (1954). Quart. J. Mi~osc. Sci. 99, 5. Rochon-Duvigneaud, A. (1920). Bull. Biol. Fr. BeZg. 54, 109. Rochon-Duvigneaud, A. (1943). Les Yeuz et la Vision. Masson et Cie, Paris. Schultze, &I. (1866). Arch. Milorosk. Anat. 2, 175. Slonaker, J. R. (1897). J. Norphol. 13, 445. Uchiyama, T. (1930). Virchow’s Arch. A 277, 631. Waelchli, G. (1883). Graef’s Arch. Ophthalmol. 29, 205. Wald, G. (1949). DOG. OphthaZmoZ. 3, 94. Walls, G. L. (1942). The Vertebrate Eye. Bull. Cranbrook Inst. Sci. Cranbrook, Michigan. Walls, G. L. and Judd, H. D. (1933). Brit. J. Ophthalmol. 17, 641, 705. Wood, C. A. (1917). The Fundus OcuZi of Birds Especially as Viewed by the Ophthalmoscope. The Lakeside Press, Chicago. Yamads, E. (1962). 19th Congress of Ophthabnology. New Delhi.
17, 347-355
The Topographical Distribution of Rods and Cones in the Adult Chicken Retina DAVID
B.
MEYER
A?;D
HAROLD
C. MAY:
JR
Deprtment of Aw,atonzy, Godon H. Scott Hall of Basic Medical Sciences, Wayne State University, Xchool of Medicine 540 E. Canjield, Detroit, Miclz. 48201, U.S.A. (Received 11 June 1973, and in revised form
30 July 1973, Boston)
The relative proportion of rods and cones (single and double) in the chicken retina was determined by counting these elements in each of nine pre-determined segments prepared as periodic acid-Schiff serial sections for rod (hyperboloid glycogen) and double cone (aceessory cone paraboloid glycogen) identifications and as unfixed, flat preparations for cone determinations (colored oil droplets). Periodic acid-Schiff preparations revealed that rods and accessory cones were equal in number in all retinal segments except those located directly posterior to the posterior extremity of the pecten where the double cones outnumbered the rods in a ratio of 14 : 10. On the basis of oil-dronlet aigmentation. double cones housing a golden-yellow droplet in their chief component and a small yellowish-green droplet, in their accessory members were constantly observed 1.0 be twice as numerous as single cones (red droplet) in all retinal segments. These data reveal that the proportion of rods to double and single cones is 2 : 2 : 1, respectively, in all areas of the chicken retina except a posterior (lateral) segment where the ratio is IO : 14 : 7. This area of greater cone concentration corresponds to the site of the temporal fovea of some birds and may represent the area of most acute vision in the fovea-free chicken retina.
1. Introduction This study represents an attempt to determine the proportional distribution of rods and conesin the whole chicken retina. Its validity is based on the reliability of two microscopic techniques to identify selectively the specific visual elements comprising the retina of this species (Gallus dornesticzcs):(1) The periodic acid-Schiff (PAS) reaction for the cletermination of rods and clouble cones, owing to positive reactions for glycogen in the hyperboloid of the rod an.dthe paraboloid of the accessory member of t,he double cone (Rabinovitch, %Iota and Yoneda, 1954; Yamada, 1962; O’Rahilly and Meyer, 1963); (2) Unfixed and unstained, flat retinal preparations for the presence of cones, which are easily recognized by the brightly colored oil droplets in their inner segments: red in the single cones,golden yellow in the chief, and greenish yellow in the accessorymember of the double cone (Meyer and Cooper, 1966). Unlike the PAS reaction, the sensitivity and reliability of which is undisputed, the proposed cone-oil droplet association has not been confirmed (Morris and Shorey, 1967). Nevertheless, visual cell counts recorded for each of nine pre-determined segments of the whole chicken retina prepared as PAS and flat preparations provide significant data on the topographical distribution of rods and cones and indicate the region of most acute vision in this otherwise fovea-free retina. 2. Materials and Methods Retinas from adult chickens (Gallus do~,esticus) mere prepared as PAS serial sections (rod and double cone determinations) and as unfixed, flat preparations (cone determinations). For each procedure eyes were removed from decapitated, adult chickens and 347
348
1).
B. MEYER
AND
K.
C. MAY,
JR
bisected through the equator into anterior and posterior halves. Using the pecten as a landmark, each retina was divided into nine numbered segments (Fig. 1). The first cut was made directly t.hrough the longitudinal axis of the pecten, the second was made parallel to the first, 5 mm centrally (nasally) from the pecten. The third and fourth incisions were made at each extremity of the pecten, perpendicular to the first two. Since the segments were not of equal size, no attempt was made to determine the total concentrat.ion of each visual cell type in each of the nine segments.
PIG. 1. Lateral view of chicken’s head to show the retinal segments 5 and 6 represents the p&en. iYote that the relative orientation of the hoad.
segments position
in situ. The thick line between of the segments varies with the
Y&S prel)aratiosLs Kine retinal segments from each of five chickens were removed complete with their adjoining choroidal and scleral tunics, fixed for 24 hr in cold (4°C) Gendre’s fluid (85% picric acid saturated in 90% ethanol; 10% formalin; 5% glacial acetic acid) and subjected to the periodic acid-Schiff technique in toto (Meyer, 1960) using aqueous periodic acid instead of the alcoholic buffered solution. Glycogen was identified by its complete removal in 1% buffered malt diastase for 1 hr at 37°C. Following dehydration in ethanol and double embedding in paraffin and celloidin according to Peterfi’s methyl benzoate method (Carleton and Drury, 195’7), the segments were sectioned serially at 8,~“. For each segment counts lvere made of rods (PAS-positive hyperboloids) and double cones (PAS-positive paraboloids) (Fig. 2) in every fifth section to insure that each element was counted only once. In some cases whole retinas were subjected to the freeze substitution method of Feder and Sidman (1958), using I:/, picric acid in ethanol as the fixative prior to the PAS reaction. Visual cell counts were made in these preparations but were not included in the data presented because the retinas were not cut into nine segments. The findings, however, corroborated the statistical data obtained from segmented, sectioned retinas.
Nine retinal segments from each of logical saline (0.9%) from the adjoining scope slides, vitreous side down (visual slip. Each segment was then examined illumination and phase optics (Pi,.‘~1. 3).
three chickens were carefully removed in physiopigment epithelium, individually floated on microcells uppermost), and gently covered by a coverwith a Zeiss photomicroscope using bright field Oil droplet counts were made at a magnification of
DISTRIBUTION
OF
CHICKEN
RODS
AND
CONES
FIG. 2. Photomicrograph of chicken retina (transverse section) subjected to the periodic acid-& reaction. Positive reactions for glyoogen are shown by the magenta ooloration (diastase digestible the cone-shaped rod hyperboloids (arrow) and the larger, barrel-shaped accessory cone paraboh situated internal to them (lower). ( x 800) Fro. 3. Photomicrograph of unfixed, flat retinal preparation (visual cells upperm.ost; i.e. nearest viewer) showing the brightly colored oil droplets ofthe chicken cones. The red (single cones) and yello, (chief cones) droplets are approximately the same size. The smaller greenish-yellow droplets (arr reside in the accessory member of the double cone. ( x 800) FIG. 4. Phase contrast photomicrograph of a chicken double oone with a large, yellow droplet in chief component and a smaller one in the accessory member. ( x 800) FIG. 5. Phase contrast photomiorograph of three accessory cones each containing a small oval yellow green droplet. For comparison, note the larger, circular droplet at the right. ( x 800) FIG. 6. Phase contrast photomicrograph showing a single cone housing a red droplet. ( x 800)
:hiff 1 Qf >ids the vish ow) the ish-
350
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B.
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AND
H.
C. MAY,
JR
approximately x 300. Red droplets were counted as single cones, whereas the yellowish pigments (golden yellow and yellowish green) were tabulated together as double cones. All counts were made on undistorted areas in all regions of each segment. For the determination of visual cell-oil droplet associations fresh preparations were also gently teased and examined by phase contrast microscopy. 3. Results PAX
preparations
The rod hyperboloids and accessory cone paraboloids were brilliantly stained by the PAS reaction, and hence can be idelitifiecl immediately (Fig. 2). The visual cell counts for each segment from at least three different chicken retinas are tabulated in Table I. They reveal that the proportion of rods to accessory cones is almost TABLE
Numbers of rock (R)
and
accessorycones
I
(AC) and their
relative
proportion
&h thicket?,
as revealed by counts of glycogen-containing paraboloids (accessorycones) and hyperboloids (rods) in periodic acid-Xchiff preparations. (Each segmental count represents retinas
data
Retinal
Segments
1
2
3
4
5
6
7
8
9
from
(hyperboloids)
a diferent
retina.)
Accessory cones (paraboloids)
2983 2989 1689 1654 1932 2312 3813 1808
311.5 3079 1809 1856
2086 1491
2941
1459 1697
1785 1884 2165
1835 2421 2056
4991 3189 2311 2571 2056 1580 1132 2228 3707
2139 1005 917 974 1275 2312
1955 1578
2338 2636 5376 2405 2043
2560 2358 5404 3555 2460
2511 2119 1611 1801 2311 2865 2234
1110 946 1019 1301 2636 2029
1783
Ratio
1 I 1 1 1 1
1 1
1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1
R : AC
1.0 1.0 1.1 1.1 1.2 1.1 1.4 1.3 1.4 1.4
1.2 1.1 1.2 1.2 1.1
I.1 1.1 1.1 1.0 1.0 1.0 1.0 I.0 I.1 1.0 1.1 1.0 1.0 1.0 1.1 1.0 1.1
DISTRIBUTION
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351
CONES
equal (1 : 1.1) in all portions of the retina except segmen.t 3 which contains a significantly higher ratio of accessory cones (1 : 1.4). The accessory cones are thinnest in this region and their glycogen-filled paraboloids appear more barrel-shaped and closer together. Undoubtedly, such an arrangement correlates with the more dense population of double cones in this segment than in neighboring areas where paraboloids assume a more rounded appearance. TABLE
II
Jumbers of cones and their relative proportions in pre-determined segments of three chicken retinas (un$xed, flat preparations) as revealed by counts of colored oil droplets
Retinal
segments
*
Single
cones (red)
47 45 55 62 67 59 64 68 60 50 42 51 55 60 65 52 49 62 41 53 53 43 52 57 51 57 49
Double cones (yellowish)
194 186 247 271 215 251 262 288 267 208 179 214 228 254 261 218 207 255 171 227 223 183 217 234 202 231 209
Ratio
SC : DC t
1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
: : : : : : : : : : : : : : : : : : : : : : : : : : :
2.1 2.1 2.2 2.2 2.1 2.1 2.0 2.1 2.2 2.1 2.1 2.1 2-1 2.1 2.0 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.1 2.0 2.0 2.1
* The segmectal data from each retina are presented in sequence so that the distribution within each of the three retinas is given. t Accepting the fact that two yellowish droplets are housed in one double cone and one red droplet resides in the single cone (Meyer and Cooper, 1966), the ratio of single cones (SC) to double cones (DC) is determined by the relative proportion of 1 red to 2 yellow droplets.
The rods reach their maximum length and thinness in segments 2 and 3. Thus, the cone-shaped hyperboloids observed in these regions become further displaced choroidally from the accessory cone paraboloids and are seldom observed to be wedged jn between paraboloids as noted jn other areas of the chicken retina. This arrangement, too; provides for a greater concentration of double cones per unit area, in segments 2 and 3.
452
D.
B. MEYER
AND
H.
@. MAY,
JR
Fresh preparations Whereas the red oil droplets appear singly at definite intervals in a lattice pattern, the golden-yellow and yellowish-green droplets are generally paired with one another and appear to have a more scattered distribution (Fig. 3). Regional accumulations of red or yellowish droplets to form so-called red, orange or yellow fields are not evident. Within the thickness of the retina, however, the red oil droplets are placed on their own level, as are the yellowish (golden-yellow and greenish-yellow) droplets. The yellowish-green droplets are smaller and more oval-shaped in comparison to the red and golden-yellow droplets. Numerical data on the segmental distribution of the colored oil droplets are tabulated in Table II. They reveal a rather constant proportion of four yellowish to one red oil droplet in each of the nine retinal segments. Assuming that two yellowish droplets occupy one double cone and that one red droplet represents one single cone, the data further indicate that there are twice as many double cones as single cones throughout the chicken retina.
4. Discussion In contrast to the reports of other investigators, our PAS findings clearly show that rods are plentiful in the chicken retina and are rather uniformly distributed in all areas. The number of rods equals the number of double cones in all topographical regions of the retina except that located directly posterior to the posterior extremity of the pecten (segment 3) where the double cones are thinner and slightly outnumber the rods by 14 to 10. Support for this relatively high proportion and rather even distribution of rods has been provided by the biochemical studies of visual pigments (Hecht, 1942; Bliss, 1946; Wald, 1949) which have revealed a greater amount of rhodopsin than iodopsin. Bliss (1946) in addition found that the fovea1 (central) portion of the chicken retina contains the same proportion of rhodopsin to iodopsin (4 : 3) as other more peripheral regions. Believing that cones greatly outnumbered rods in this species, Hecht (1942) and Wald (1949) provided an explanation for this discrepancy by theorizing that rhodopsin was more highly concentra.ted in rods, perhaps as much as 100 times that of iodopsin in single cones (Wald, 1949). The counting of colored oil droplets to determine the quantitative and topographical distribution of avian cones has been employed by many investigators (Schultze: 1866; Dobrowolski, 1871; Waelchli, 1883; Hahn, 1916; Rochon-Duvigneaud, 1920; Erhard, X924; Uchiyama, 1930; Coulombre, 1955; Decker, 1963; Peiponen, 1964; Mayr, 1972. See reviews by Hahn, 1916, and Meyer, Cooper and Gernez, 1965). Such data have revealed localities of high cone concentration in the chicken retina but are difficult to collate because of the multitude of oil droplet colors described by the early investigators (owing to the inferior lenses then available), the absence of well-defined and comparable retinal areas and the failure to establish a relationship between oil droplet color and specific cone type. It is evident from Fig. 1, for example, that the anatomical position of the chicken’s head determines the location of the retinal areas designated as dorsal (upper), ventral (lower), anterior (medial, nasal) and posterior (lateral, temporal). Distributional patterns based on these areas are therefore misleading and vary greatly with each investigator. Utilization of the pecten to delinea.te precise retinal segments, as adopted in the present study, eliminates this source of error. It is now well established that only three different colored oil droplets exist in the
DISTRIBUTION
OF
CHICKEN
RODS
AND
383
CONES
cones of the chicken retina: red, golden yellow and yellowish green (Meyer, et al., 1965). According to Meyer and Cooper (1966) the red oil droplets occupy the single cone, whereas the golden-yellow and yellowish-green droplets reside in the chief and the accessory member of the double cone, respectively. These relationships have been observed in phase microscopic studies of teased, adult (Meyer and Cooper, 1966) (Figs 4, 5 and 6) and embryonic (Cooper and Meyer, 1968) preparations. The small, oval, yellowish-green droplet can only be housed in the narrow, tapered extremity of the inn.er segment of the accessory cone which can be easily recognized in phase microscopic preparations by its bowhng pin appearance (Figs 4 and 5). Nevertheless, the existence of an oil droplet in the accessory cone has been denied by Morris and Shorey (1967) and Morris (1970) w h o, instead, describe and illustrate from electron microscopic observations “a granular vesicle at its tapered scleral end”. TABLE
Collated
III
datu on visual cell proportions
in
chicken retina
Visual
Investigator
Matsusaka (1963) Xorris and Shorey Morris (1970) Present
Study
Method
(1967)
*
Retinal
E.M. E.M. E.M. PAS+O.D.C.
1
* .13% of the receptors i Oil Droplet Counts.
cell distribution (%I COIES Single Double Rods
location
Central Posterior pole Posterior pole Periphery of cup Entire retina except segment Segment 3
3
31.2 36.0 54.0 37.0
36.6 36.0 32.0 30.0
14.0 33.0
20.0
40.0 45.0
40.0 32.0
23.0
32.2
15.0
were unidentified.
Our oil droplet counts of eachretinal segmentsubstantiate the findings of Coulombre (1955); they have revealed that the red and yellowish droplets constitute approximately 20 and SO%, respectively, of the total number of oil droplets in all portions of the retina (Table II). Accepting the fact that the yellowish droplets are confined to double cones, these data show that the double cones outnumber the single cones by a ratio slightly greater than 2 : 1 throughout all portions of the neural retina. Furthermore, by combining our quantitative data from both PAS and the oil droplet counts (Table III), the proportion of rods to double and single conesis found to be 2 : 2 : 1 (40%, 40%, 20%), respectively, in all parts of the chicken retina except an. area located directly posterior to the posterior extremity of the pecten (segment 3) where an increasednumber of conesproduces a ratio of 10 : 14 : 7 (32%, 45%, 23%). Although no distinct thickening or thinning of the retina is apparent in this segment, its greater concentration of conesundoubtedly results in a local increase in resolving power indicative of an area centralis (Walls, 1942; King-Smith, 1971). Two such circumscribed, physiological areas have been described in most birds: central (principal, nasal) and temporal. They may be circular, oval or in the form of a long horizontal band or ribbon, and usually contain a depressionor fovea. Chickens lack a morphologically-discernible fovea (Schultze, 1866; Chievitz, 1591; Slonaker, 1897; Walls E
354
D.
B. MEYER
9ND
H.
C. MAY,
JR
and Judd; 1933; Rochon-Duvigneaud, 1943)> although a nasal area has been reported (Chievitz, 1891; Slonaker, 189’7). In topographical location, segment 3 corresponds to the site of the temporal area which houses the temporal fovea of owls (Wood, 1917; Oehme, 1961) eagles, hawks, hummingbirds; swallows, bitterns (Walls, 1942), falcons and buzzards (Oehme, 1964) and may represent the area of most acute vision in the chicken. It is the only fovea1 area in owls, whereas in the other birds an additional fovea occupies a more eentral or nasal area. The central fovea and corresponding area, if present in the chicken, would reside in segment 2 or 5, which probably corresponds to the dorsal (upper) posterior (lateral) region regarded by Krause (1894), Hess (1907) and Bl&sser (1927)‘as the place of most distinct vision. [For these relationships see Wood’s (1917) excellent drawings of the fundus in a variety of birds.] The electron microscopic data of Natsusaka (1963), Morris and Shorey (1967) and Morris (1970) are included in Table III and show conflicting results, although their counts may have been made in different areas. Matsusaka (1963) in the central portion of t,he chicken retina found an almost equal number of single cones and rods an d a slightly higher percentage of double cones. Of more apparent functional significance, however, are Xatsusaka’s calculations of retinal areas occupied by these elements. At t,he level of the accessory cone paraboloids, for example, he calculated that the double cones occupy four times the area of the single cones, and 60 times that of the rods. Walls (1942) associates a high percentage of double cones with animals possessing strong diurnal habits, although he considered an equal number of double and single cones as the maximum proportion. Morris and Shorey (1967) counted a total of 286 receptors from the posterior pole of different chicken retinas (t,opographical location not specified) and found an equal proportion of single and double cones and less than half as many rods. On the basis of fine structural characteristics they distinguish four types of visual elements: rods, double cones and two types (I and II) of single cones that are differentiated by the density of their oil droplet and mitochondrial cristae. Type I has a darker staining oil droplet and more numerous cristae. Morris and Shorey (1967) further suggest that the single cones house either the red or the yellow droplet (type I or II not determined) and that the chief cone contains the green droplet. In a subsequent report (Morris, 1970) a third type of single cone (type III) is recognized; it is characterized by a less dense oil droplet. Morris (1970) determined the relative proportion of chicken photoreceptors in 250 pm2 samples from the posterior pole and the periphery of the retina sectioned tangentially. Each receptor type was counted in three montage electron micrographs, each of which comprised an area containing 200-600 visual elements. Because these elements were cut in cross-section, the counts were made at the level of the oil droplets and proximal inner segments, and occasionally from the level of the outer segments. Double cone frequency was estimated from counts of chief members only (accessory cones contained a granular vesicle, not an oil droplet). Single cones could only be identified in sections through their oil droplets. Statistical analyses of these counts indicated a receptor mosaic with an even spacing of the receptor types. ACKNOWLEDGNENT
This project was supported by Research Grant 00477 from the h’ational Eye Institute, National Institutes of Health, U.S. Public Health Service.
DISTRIBUTION
OF
CHICKEN
RODS
AND
CONES
355
REFERENCES Blasser, A. (1927). Zool. Jahrb. (Zool. Physiol.) 43, 69. Bliss, A. F. (1946). J. Germ. Physiol. 29, 277. Carleton, H. 111. and Drury, R. A. B. (1957). Histological Technique. Third edition. Oxford University Press, New York. Chievitz, J. H. (1891). Arch. Anat. Physiol. Physiol. Abt. 15, 311. Cooper, T. G. and Meyer, D. B. (1968). Exp. Eye Res. 7, 434. Coulombre. A. J. (1955). Amer. J. Anat. 96, 153. Dobrowolsky, W. (1871). Arch. Anat. Physiol. W&en. Med. 38, 208. Diicker, G. (1963). Z. Tierpsychol. 20, 43. Erhard, H. (1924). Zool. Juhrb. (Zool. Physiol.) 41, 489. Feder, N. and Sidman, R. L. (1958). J. Biophys. Biochem. Cytol. 4, 593. Hahn, E. (1916). 2. Wiss. ZooZ. Abt. A 116, 1. Hecht, S. (1942). Ann. Rev. Biochem. 11, 465. Hess, C. (1907). Arch. Augenheilk. 57, 298. King-Smith, P. E. (1971). In Physiology and Biochemistry of the Domestic Fowl (Eds Bell, B. J. and Freeman, B. M.). Vol. 2, pp. 1039-1083. Academic Press, New York. Krause, W. (1894). Intern. Monat. Anat. Physiol. 11, 1. Matsusaka, T. (1963). Jap. J. Ophthalmol. 7, 238. Mayr, I. (1972). Vision Res. 12, 1477. Meyer, D. B. (1960). Stain Teehnol. 35, 83. Meyer, D. B. and Cooper, T. G. (1966). Amer. J. Anat. 118, 723. Meyer, D. B., Cooper, T. G. and Gernez, C. (1965). In Structure of th.e Eye (Ed. Rohen, J. W.). Academic Press, New York. Morris, V. B. (1970). J. Conzp. Neural. 140, 359. Morris, V. B. and Shorey, C. D. (1967). J. Comp. Neural. 129, 313. Oehme, H. (1961). ZooZ. Jahrb. (,&at. Ontog.) 79, 439. Oehme, H. (1964). 2. Morphol. Oekol. Tiere. 53, 618. O’Rahilly, R. and Meyer, D. B. (1963). Ann. Histochim. 8, 281. Peiponen, V. il. (1964). Ann. ZooZ. Fenn. 1, 281. Rabinovitch, M., Mota, J. and Yonedn, 6. (1954). Quart. J. Mi~osc. Sci. 99, 5. Rochon-Duvigneaud, A. (1920). Bull. Biol. Fr. BeZg. 54, 109. Rochon-Duvigneaud, A. (1943). Les Yeuz et la Vision. Masson et Cie, Paris. Schultze, &I. (1866). Arch. Milorosk. Anat. 2, 175. Slonaker, J. R. (1897). J. Norphol. 13, 445. Uchiyama, T. (1930). Virchow’s Arch. A 277, 631. Waelchli, G. (1883). Graef’s Arch. Ophthalmol. 29, 205. Wald, G. (1949). DOG. OphthaZmoZ. 3, 94. Walls, G. L. (1942). The Vertebrate Eye. Bull. Cranbrook Inst. Sci. Cranbrook, Michigan. Walls, G. L. and Judd, H. D. (1933). Brit. J. Ophthalmol. 17, 641, 705. Wood, C. A. (1917). The Fundus OcuZi of Birds Especially as Viewed by the Ophthalmoscope. The Lakeside Press, Chicago. Yamads, E. (1962). 19th Congress of Ophthabnology. New Delhi.