Studies on the retina of the gecko Coleonyx variegatus

© 1966 by Academic Press Inc.

J. ULTRASTRUCTURE RESEARCH

685

16, 685-692 (1966)

Studies on the Retina of the Gecko Coleonyx

variegatus

III. Photoreceptor Cross-Sectional A r e a Relationships 1 ROBERT F. DUNN 2

Department of Zoology, University of California, Los Angeles, California Received January 11, 1966 The percentage of the retina occupied by outer segments in cross section was found to be 53 %. Based on these measurements, the percentage of light absorption for the total retina was calculated to be 40 % which agreed better with the value of 39 % given for the cat allowing for the effect of the tapetum than with the value of 68% for a nocturnal gekkonidean. This would indicate the increased outer segment length gives Coleonyx at least the same advantage as the tapetum of the cat. Errors such as tissue shrinkage, convergent lens effect of the inner segment, and measuring errors, which would tend to increase the value of 40 %, were discussed and approximated. A s t u d y w h e r e b y the percentage of the retinal cross sectional area which is o c c u p i e d b y the p h o t o r e c e p t o r o u t e r segments seems to be of i m p o r t a n c e to the visual p h y s i o logist because when converting f r o m a visual p i g m e n t density s p e c t r u m to an a b s o r p tion s p e c t r u m one m u s t have the percentage of the q u a n t u m c a p t u r i n g area (2). S o m e i n f o r m a t i o n c o n c e r n i n g o u t e r segment cross-sectional areas in geckos, which is b a s e d on light m i c r o s c o p y m e a s u r e m e n t s is available (11); however, it is b a s e d on l o n g i t u d i n a l d i m e n s i o n s of the o u t e r segments. I n the present study, the crosssectional areas were m e a s u r e d directly on low magnification electron m i c r o g r a p h s a n d their percentages were calculated; the results are discussed. MATERIALS AND METHODS The preparation of the tissue for embedding and the sectioning procedures have been previously described (4) as well as the methods by which the low magnification electron micrographs were obtained (5). The method by which the cross-sectional area of the outer segments were measured, was to circumscribe the outer segment's circumference three times with a polar planimeter z This investigation was supported by Public Health Service Research Grant NB-02889 from the National Institute of Neurological Diseases and Blindness. 2 Present address: Department of Surgery, Rehab. 3234, University of California, Los Angeles, California 90024. 4 5 - - 661837 J . Ultra~tructure Research

686

R. F. D U N S

TABLE

I

SUMMARY OF MEASUREMENT ERRORS

Area Readings

0.18, 0.16, 0.17, 0.17, 0.17, 0.15, 0.16, 0.16, 0.16, 0.16 0.31, 0.29, 0.30, 0.28, 0.30, 0.29, 0.33, 0.30, 0.30, 0.29

m

S

M.E.

Per cent error

0.164 0.299

0.026 0.043

0.0078 0.0103

4.41 3.44

Mean error

3.92

a n d take t h e m e a n . T w o p l a n i m e t e r s were used for these m e a s u r e m e n t s , a n O T T type 30/38 p l a n i m e t e r calibrated to a s t a n d a r d 15.43 s q u a r e inch circle, a n d a K + E M o d e l N o . 620015 p l a n i m e t e r c a l i b r a t e d to a s t a n d a r d 100 s q u a r e c e n t i m e t e r circle. T h e p e r c e n t a g e of the retinal cross sectional area occupied by the o u t e r segments was calculated a c c o r d i n g to t h e f o r m u l a %

Z O.S. area t o t a l area

× 100

in which ~ O.S. area is the s u m of the m e a n s of t h e individual outer s e g m e n t s ' areas. M e a s u r e m e n t errors were calculated as follows: ten separate m e a s u r e m e n t s were t a k e n a r o u n d t h e s a m e area. This was d o n e in two cases because there seemed to b e two p o p u l a t i o n s of m e a s u r e m e n t s , o n e a b o u t 0.16, a n o t h e r at a b o u t 0.30 (Table I). T h e v a r i a n c e was t h e n calculated using the f o r m u l a n X:x ~ - (Y, x) S~ n (n --1) in w h i c h S is the variance, n the n u m b e r of readings a n d x the areas. T h e m e a n e r r o r (M.E.) was calculated b y S M.E. ~ . n

I n o r d e r to ascertain b y w h a t p e r c e n t a g e the readings c o u l d be in error, t h e following f o r m u l a was used: M.E. % error --x 100 m

in w h i c h m is t h e m e a n of t h e ten s e p a r a t e area m e a s u r e m e n t s .

FIG. 1. Cross section at the photoreceptor cell outer segment level. In this section, each of the visual cell classes are present, with the exception of the Quintuplet. This figure is a portion of the section in which the outer segment cross-sectional area was 50.82 %. x 2016. FI~. 2. Another cross section at the visual cell outer segment level. The outer segment cross-sectional area of the section, from which this figure was taken, measured 53.56 %. x 2704.

I

i

688

R.F. DUNN RESULTS

F i v e visual cell cross-sectional areas of the central retina, including two m o n t a g e s , were m eas u r ed , in which the n u m b e r of o u t er segments m e a s u r e d r a n g e d f r o m 155 to 178 (Figs. 1 a n d 2). T a b le II summarizes the results of the area analysis. T h e percentages of retinal area o c c u p i e d by o u t er segments r a n g e d f r o m 50.82% to 55.6 %, with a m e a n value of 53.23 %. These m e a s u r e m e n t s co u l d be in er r o r by 3.95%, wh i ch c o u l d increase the area m e a n to 57.15 %. U s i n g an o u t e r segment length of 31.63 /z (Table III) o b t a i n e d f r o m m e a s u r e m en t s on l o n g i t u d i n a l sections of the o u t e r segments, (Figs. 3 an d 4) an d M a r k s ' T A B L E II SUMMARY OF THE O U T E R SEGMENT C R O s S - S E C T I O N A L A R E A MEASUREMENTS

Area

Total Area

I II III IV V

73.22 in 2 75.10 in 2 664.00 cm~ 680.28 cm ~ 658.50 cm2

O.S, Area

Per cent O.S. Area

38.19 in 2 37.77 in 2 369.50 cm2 340.70 cm2 352.70 cm~

52.16 54.03 55.60 50.82 53.56

Mean

53.23

T A B L E III SUMMARY OF THE O U T E R SEGMENT L E N G T H MEASUREMENTS a Measured Print Length of Outer Segment

Actual Length of Outer Segment

Type Visual Cell

(cm)

0~)

Single Single D1 accessory D1 accessory D1 chief D~ chief

29.0 28.0 23.6 24.5 22.8 23.5

36.3 35.0 29.9 30.6 28.6 29.4

a Magnification x 8000. Mean 31.63/~. FIG. 3. Longitudinal section through the entire length of the outer segments. Two D1 rods and a Single rod (S) are identifiable in this section. Outer segment lengths were measured only on those receptors whose outer segments abutted the pigment epithelium cells, x 2645. FIG. 4. Longitudinal section through the entire length of the outer segments. Two D1 rods and two Single rods are identifiable. A shrinkage in the outer segment length of about 10 % is evident as a separation between the distal outer segment ends and the pigment epithelium cells, x 2645.

690

•. F. DUNN

figure of 2.2 %//~ (7) for the percentage light absorption per micron, the amount of absorption per individual photoreceptor is 69.59 %. The percentage of light absorption for a central retina area would then be 39.77 %. DISCUSSION The validity of one assumption made in this study may be open to some argument. Marks' figure for percentage of light absorption per micron is based on measurements done on Gekko gekko, not on Coleonyx; however, he does indicate this figure of 2.2 %/# is approximately the same for the frog rods and the fish cones (7). Unfortunately, such figures are not available for Coleonyx. According to Underwood (12), Coleonyx variegatus belongs to the family Eublepharidae, whereas Hernidactylus turcicus, Tarentola rnauritanica, T. annularis, Ptyodactylus hasselquistii, and Gekko gekko belong to the family Gekkonidae, with both belonging to the superfamily Gekkonoidea. The latter three species were used by Denton (2, 3) to ascertain the maximum density differences for the visual pigment, which was 0.46, 0.58, and 0.50, respectively. Tansley (11) used this latter value in calculating the retinal light absorption for the nocturnal gecko at 68 %. This figure may be somewhat high for two reasons: first, the visual cell dimensions were based on light microscopic measurements which are limited by the inherent resolution limit of this instrument (0.2 #); and second, the outer segments were assumed to be as thick as they are wide, which is certainly not the case for Coleonyx. Here, the outer segment cross-sectional shape is a cardioid, not a circle, and if this is true of Gekkonidae species and not a peculiarity of Eublepharidae, Tansley's figure would be reduced. Also in her discussion, Tansley (11) credits Weale (13) with calculating the percentage of light absorption for the cat retina to be 26 %, and estimating that the advantage of having a tapetum would increase this value by a factor of 1.5 to 39 % absorption. The results of this study, 39.77 % light absorption for the entire retina, are in better agreement with those of Weale. This would indicate the lengthening of the rod outer segment gives Coleonyx at least the same advantage as the tapetum of the cat. The light absorption value of 39.77 %, as well as Tansley's and Weale's values do not take into consideration any light funneling action by the ellipsoid and paraboloid of the photoreceptor cells. Such an inner segment lens action has been discussed by Rushton (8, 9) and Enoch (6), and differences in refractive indices between the receptor cells and the extracellular space were observed as long ago as 1866 by Schultze (10). Any convergent lens action by the inner segment would be expected to raise the light absorption values considerably. The question arises, is this lens action of the inner segment complete or is only some portion of the light funneled into the outer

PHOTORECEPTOR AREA RELATIONSHIPS IN COLEONYX

691

segment? If this funneling were complete, the case would be greatly simplified since then one would merely base the calculations on the inner segment cross-sectional areas. Assuming complete funneling--that is, all the light entering the inner segment is funneled into the outer segment--the value for the retinal light absorption is increased to approximately 57.03 %. However, Enoch (6) indicated this complete f u n n e l i n g is not true, and he has calculated that light deviating more than 10 ° from the longitudinal axis of the photoreceptor will not be funneled into the outer segment. Hence, the absorption figure of 57.03 % is certainly incorrect and too high, and also the absorption figure of 39.77 % is too low. However, a critical angle of 10 ° is indeed small and would raise the latter light absorption value only slightly. There does seem to be some converging lens action by the inner segment; however, due to the multiplicity of factors involved (6), it would be quite difficult to quantitate an accurate factor for this added effect. The accuracy of the measurements in this study are limited by tissue shrinkage of two types: overall shrinkage and nonuniform shrinkage. Assuming an overall shrinkage of 10 % in the outer segment length, the value for the retinal light absorption would be increased to 40.83 %, while for 20 % outer segment length shrinkage the figure is increased to 44.44 %. A nonuniform type of shrinkage would be reflected as a shift in position of the outer segments which would increase the cross-sectional area figures. The in situ fixation without detachment of the retina used in this study would cause the lateral shrinkage to be minimal. This is reflected in the regularity of the rectilinear receptor pattern reported previously (5), and any increase would be quite small because of the near uniformity of the primary rows. The alternate rows do deviate somewhat from an ideal geometric pattern, and this would contribute most to the small factor of increase for this type of shrinkage. Of the two types of shrinkage errors, shrinkage of the outer segment length is the most important, and to eliminate this error as much as possible, only those outer segments were measured which joined the pigment epithelium cells. The retinal light absorption value of 39.77 % is undoubtedly low due to the convergent lens action of the inner segment and tissue shrinkage. A reasonable estimate for these two errors would be 2-3 %. But even with this added factor, the retinal light absorption for Coleonyx remains in much better agreement with the 39 % value for the cat than the 68 % figure for the nocturnal gekkonidean. REFERENCES 1. CRESCITELLI, F., personal communication. 2. DENTON,E. J., d. Gen. Physiol. 40, 201 (1956). 3. - personal communication with Dr. F. Crescitelli, 1964. 4. DUNN, R. F., J. Ultrastruct. Res. 16, 651 (1966).

692

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5. - ibid. 16, 672 (1966). 6. ENOCH, J. M., J. Opt. Soc. Am. 53, 71 (1963). 7. MARKS, W. B., personal communication with Dr. F. Crescitelli, 1965. 8. RUSHTON,W. A. H., J. Physiol. (London) 134, 11 (1956). 9. - - - - ibid. 134, 30 (1956). 10. SCHULTZE,M., Arch. Mikroskop. Anat. 2, 175 (1866). 11. TANSLEY,K., Arch. Ges. Physiol. 228, 213 (1959). 12. UNDERWOOD,G., Proc. Zool. Soc. London 124, 469 (1954). 13. WEAL~, R. S., J. Physiol. (London) 127, 587 (1955).