The dimensions of rod outer segments related to light absorption in the gecko retina

Vision Ref. Vol. 9. pp. 603-609.

THE

Fkrgamon Press 1969. Printed in Chat Britain.

DIMENSIONS OF ROD OUTER SEGMENTS RELATED TO LIGHT ABSORPTION IN THE GECKO RETINA” ROBERT F. DUNN’

Departments

of Zoology and Surgery, University of Cahfornia at Los Angeles, California, U.S.A. (Received 6 June f968; in revised form 30 November 1968)

INTRODUCTION IN A PREVIOUS

paper the percentage of light absorption for the total retina of the gecko, Coleonyx vnriegutus, was calculated (DUNN, 1966c), and was based upon measurements made upon low magnification electron micrographs of longitudinal and cross sections of the visual cells’ outer segment. Since the value calculated for C. vff~jeg~~~ was based upon light absorption values measured in Gekko g&kc, it was felt advisable to repeat these same morphological measurements for this latter species. The opportunity also arose to compare Eublipharis macularis with C. variegatus, both eublipharid geckos, and with the gekkonoid, G. gekko. All three of these species are nocturnal and have a pure rod retina. In the present study, the lengths of the outer segments were measured on the various multiple and single photoreceptors, and also the percentage of retinal area occupied by the outer segments was measured. With these figures, the density per individual rod was calculated as was the density and light absorbed over the entire retina. MATERIALS

AND METHODS

Six lizards, three Gekko gekko and three ~bl~~horis macular& adults, were used in this study. The animals were decapitated and the eyes were immediateb enucleated and transected. The posterior hemisphere of each was placed into cold 40 per cent osmium tetroxide in carbon tetrachtoride, where the choroid and the retina were separated and cut into 1 x 4 mm strips. The subsequent steps of the tissue preparation, as we11 as the techniques by which the low magnification electron micrographs were obtained, have been described previously (DUNN, 1966a, 1966b). The tissue sections were observed using a Siemen’s Elmiskop IA at primary magnifications of x 612 and x 1430. Further magnifications were carried out photographically as necessary. The method used to measure the cross sectional area of the outer segments was to circumscribe the periphery of the outer segment three times with a potar planimeter and record the average value. The instrument used in these measurements was a K -I- E model No. 620015 polar planimeter calibrated to a standard circle having an area of 100 ems. The method of calculating the per cent error has been presented previousIy.(Dm, 1966~). The figures for the retinal density were calcuIated by multiplying the length of the outer segment by the density whose product is the density of the individual outer segment. This was then multiplied by the cross sectional area of the outer segment, whose product is the retinal density. This latter figure was then converted to absorption for the entire retina and expressed as a percentage. 1 This investigation was supported by Public Health Service Research Grant NB-02889 from the National Imtitute of Neurological Diseases and Blindness. s Present address: California 90024.

Department

of Surgery, Rehab.

603

3241, UCLA School of Medicine,

Los Angeles,

ROBERTF. Dw

604

RESULTS

Gekko gekko The percentage of the retinal area occupied by outer segments was measured in six regions taken from the central retina of G. gekko (Fig. 1). This value includes not only cross sections of entire outer segments, but also the portions of any outer segments falling within the ruled off area. Table 1 summarizes the errors in measurements, and Table 2 summarizes the results of the area analysis. The percentages of area occupied by the outer segments within the central retina ranged from 60.2 to 66*7 per cent, with a mean value of 63.1 per cent.

TABLE 1. SUMMARYOF

ERRORSIN MEASUI~~~ENTSOF THE AREA 0,

IMean

Area readings G. gekko 129, 130, 129, 12.8, 12.9, 129, E. macuiaris 11.9, 11.9, 11.9, 11.9, 11.9, 11.9,

Mean

Variance

error

E&r

12.9, 129, 129, 129

129

0.469

O-0469

3.64%

12.0, 11.9, 120, 120

11.9

0.458

0058

3.85%

TABLE 2. SUMMARYOF THEMEAWRE~~ENTS ON THECROSS-SECTIONAL AREA OCCUPIEDBY THEOUTER SEGMENTS

Area

Total area (et+)

Area of the outer segments (em*)

804.3 820.8 807.3 824.1 814.6 827-l

525 1 5185 495.8 495.8 543 3 516.6

Retinal area occupied by outer segments (%I

G. gekko 1 2 3 4 5 6

Mean

=

64.9 63.2 61.4 602 66.7 62.6 631%

E. macularis 1 2 3 4 5 6 7

946.6 940.4 937.6 934.2 934.2 918.5 934.2

535.3 465.4 523.4 557.6 433.5 537.8 537.1

56.6 49.5 55.8 59.7 46.4 58.6 57.5

9” 10 11 12 13 14

940.4 928.8 934.2 936.9 937.7 932.2 939-o

410.9 576.4 505.4 515.3 547.9 574 2 449.5

44.2 61.3 541 55.0 58.4 61.6 47.9 56.6%

Mean

=

FIG. 1. Cross section at the level of the outer segments of the visual cells from the retina of Gekko gekko. The classes of visual cells present in this section include the single rod (S). the D1 and D2 doubles, and a 7-1 triplet. This figure is a portion of the section in which the cross-sectional area of the outer segments was 66.92 per cent.

[ fncing p. 604

FIG. 2. Longitudinal section through the entire length ot‘ the G. &&.J \iswi cells’ i?~i:er segments. TH.O DI chief member c&C), a D: accessory (&At and a D? chief (&Ct arc identifizable in this section. Tissue shrinkage, characterized by separation of the dista! end of the outer sement from the pigment epithet&m was measured to bz 2.9 per cent. The tramlocation, manifested by shifts in longitudinal axis, most probably occurred during tissue dehydration.

FIG. 3. A cross section at the level of the outer segments of the visual cells from the retina of Eublipharis macularis. This figure is a portion of the section in which the cross-sectional area of the outer segment was 55 per cent.

The Dimensions

of

Rod Outer Segmentsrelated to Light Absorption in the Gecko Retina

605

The length of the outer segments was obtained by measuring the length of the longitudinal axis. This axis was seen to have l-5 changes in directions (Fig. 2) which readily explains the difficulties encountered in obtaining acceptable longitudinal sections. To be acceptable, the sections had to meet two criteria: first, the outer segment must be continuous from the ellipsoid to the pigment epithelial cells; second, the distal end of the outer segment must be relatively flat, and not elliptical. The longitudinal axes of fortyeight outer segments, which met these criteria, were measured and the results summarized in Table 3. Uniform tissue shrinkage, which results in a patent space between the pigment

TABLE 3.

MEXXJREMENT~ OF THEOUTERSEGMENT'SLONGITLJDINAL (Print magnification = x 3528)

AXES(G.g&o)

D1 Chief Single DZChief D2 Accessory Di Accessory Prim Actual Print Actual Print Actual Print Actual Print Actual length length length length length length length length length length (cm) W (cn.0 ccl) (en@ (IL) (cm) @) (cm) W 1

2

3 4 : I

15.7 15.15 11.8 11.8 12.8 13.25 16.0 15.65

445 42.9

334 334 36.3 37.6 45.4 44.4

; 10 11

12 13 14

IO.0 9.4

10.0 96 10.75 10.8 99 10.65 13.95 126 10.4 965 10.0 9.6

28.3 26.6 28.3 27.2 3@5 30.6 28.1 30.2 39.5 35.7 29.5 27.4 28.3 27.2

14.6 13.55 11.35 1140 11.7 12.95 11.55 12.85

41.4 38.4 32.2 32.3 33.2 36.7 32.7 36.4

15.5

43-9

1625 14.55 1445 1685 1635 163 1695 16.6 16.1

46.1 41.2 41.0 47.8 46.3 46.2 48.0 47.0 45.6

162 166 19.9 18.55 15.2 15.3 16.6 16.3

45.9 47.0 564 52.6 43.1 434 47.1 46.2

Total

3179

417.4

283.3

453.1

381.7

Mean

39.7

29.8

35.4

453

47.7

Mean length of the outer segment = 38.6p.

epithelial cells and the distal end of the outer segment (Fig. 2), was measured and found to occur in 23 of the 48 outer segments measured. This type of shrinkage would result in a longitudinal measurement error of I.9 per cent for approximately 50 per cent of the measured lengths of the outer segment. This error would amount to an increase of 0.36 p in the length of the outer segment, thus raising the mean length of the outer segment from 38.6 to 39.0 I*. Utilizing this corrected value of 39-O p for the length of the outer segment and the figure of O-015 D/P (LIEBMAN,1966), the average density per individual photoreceptor was 0.58. The average density in the central retina was calculated to be 0.37 using the mean value of 63.1 per cent for the area of the retina occupied by outer segments. Using a correction factor of l-3 (DONNERand RJZUTER,1965) for the increase in apparent density, the average density for the retina becomes O-48. This would represent a 67 per cent absorption of light for the entire retina. Taking into consideration the + 3.64 per cent error in measurement of the outer segments, the percentage of absorption could vary from 65 to 69 per cent.

ROBERT F. DUNN

606

The mean lengths of the outer segments varied considerably (Table 3), from 29.8 p for the DI chief member to 47.7 p for the D2 accessory member. The outer segments of the Dl chief and accessory members were the longest, 45.3 and 47.7 p respectively, and the mean length of the single outer segment was 39.7 ,u. Also it will be noticed from Table 3 that there was a difference in the mean outer segment length of 5.6 p between the D1 chief and accessory member, the latter being longer. Eublipharis macularis

Measurements of the cross sectional area occupied by the outer segments were completed for 14 areas in the central retina of E. macularis (Fig. 3). These values ranged from 44.2 to 61.6 per cent with a mean of 54.8 per cent (Table 2). The error associated with these measurements of the cross sectional area of the outer segment was calculated to be & 3.85 per cent (Table 1). The lengths of the outer segments were measured directly on low magnification electron micrographs as the length of the longitudinal axis of the photoreceptor (Fig. 4). A minimum of changes in direction of the longitudinal axes of the outer segments were found in the retinae of these geckos. Eighteen outer segments, which met the criteria mentioned earlier, were measured and the results are summarized in Table 4. No shrinkage, TABLE 4. MEASIJREHENTS OF

THE OUTER

Single Print Actual length length (cm) 01) 10.6 10.5 9.7

SEGMENT’S

LONGITUDINAL

Dl Accessory Print Actual length length (cm) (CL)

Print Mag. = x 1908 8.5 44.5 55.5 7.9 41.4 55.0 50.8

Print Mag. =

x

6 7 8 9 10 11 12

1591 5.4 5.1 5.7 5.7 6.7 6.5 5.1

33.9 32. 1 35.8 33.8 42.1 40-9 35.8

AXES

(E. macu~aris)

Type unknown Actual Print length length (cm) W 10.7 11.5 9.9 7.3 8.6

56.1 60.3 51.9 38.3 45.1

a.2

51.5

Total

161.4

342.3

303.2

Mean

53.8

38.0

SO.5

Mean length of the outer segment = 47.4 I*.

as manifested by separation of the outer segment’s distal end from the pigment epithelial cell, was found in the longitudinal areas measured. Thus, there was no apparent uniform shrinkage error to be added to the value of 47.4 p for the mean length of the outer segment. The average density per individual rod of E. macularis was calculated as O-71, the density for the central retina was 040, and the corrected average density for the central retina O-52. The percentage of light absorption was thus between 67 and 72 per cent.

The Dimensionsof Rod Outer Segments related to Light Absorption

in the Gecko Retina

607

Only the outer segments of the single rod and the accessory member of the D1 rod could be identified positively. The outer segment of the single rod had a mean length of 54 p, and that of the D1 accessory member 38 t.~. Coleonyx variegatus The values for the average density per individual rod as well as the average density for the entire retina, and the amount of light absorbed by the retina, were recalculated for C. variegatus using the methods outlined in the present paper. The results of these calculations are summarized in Table 5. TABLE5. COMPARMNOF MEASUREMENTS AND

Animal

G. gekko E. macularis C. variegatus

CALCULATIONS

Mean length of outer segment* (4

% of retinal area occupied by outer segments (%I

Average density for total retina (D)

Absorption for total retina* (“23

Corrected absorption* value (%I

39 47 32

63 53.64 55 5 3.85 53 & 392

0.366 0403 0.253

55-59 58-63 4246

65-69 67-72 50-55

+ Values rounded off to nearest whole number. DISCUSSION

Retinal light absorption has been reported for nocturnal geckos as 68 per cent (TANSLEY, 1959). This value was based upon DENTON’S (1956) in situ visual pigment density of 0.5, and light microscopic measurements of outer segments in two nocturnal geckos. This value is in agreement with the corrected values for G. gekko and E. macularis reported here (Table 5). More recently (DUNN, 1966c), the percentage of light absorption has been calculated for C. variegatus as 40 per cent. The difference between this figure and that reported here (Table 5) is due to the fact that the previous figure was calculated from absorption values per micron instead of from density values. Since densities (but not absorptions) are additive (DARTNALL, 1957), only density values should be used in calculations. CRESCITELLI (1966) has used the values for the per cent of retinal area occupied by outer segments found in a preliminary study to calculate the functional visual pigment density for G. gekko. The corrected retinal density was then used to directly compare the density spectrum and the absorption spectrum by converting the former into the latter. He found the wave length maxima were the same and there was good agreement of the two curves between 500-580 nm, roughly in the region of the visual pigments found in this animal. There was also a significant difference at the extremities of the two curves. Completing similar calculations, the functional densities would be: G. gekko-O.46, E. macularis-0~46, C. variegatu.s-O-32.

These figures are somewhat lower than those of Crescitelli primarily due to the smaller value for the mean length of the outer segment found in the present study. There are several sources of error to be considered in calculations of this type. Perhaps the greatest single contributor is tissue shrinkage during preparatory procedures. Errors

608

ROBERTF. DUP.W

introduced as a result of shrinkage would be of two types: non-uniform and uniform. Non-uniform shrinkage would be manifested by a translocation of the outer segment. Such an error was found in longitudinal sections of the visual cells in these retinae. This source of error was compensated for in cross sections of the visual cells, by re-orienting the tissue blocks to ensure that the outer segments were sectioned perpendicular to their longitudinal axis. Shrinkage of a uniform type was manifested by gaps between the distal tips of the outer segments and the pigment epithelium, and was a measurable error in some outer segments of G. gekko. It was possible, in the G. gekko retina, to approximate a value to compensate for this type of error. This was done by measuring the distance of separation between the outer segment and pigment epithelium, as well as the frequency of this separation. No such separations were observed either in the retinae of E. macularis or in C. variegatus (DUNN, 1966c). In connection with shrinkage artifacts, the choice of fixatives should be considered. The 40 per cent osmium tetroxide in carbon tetrachloride was employed originally as a fixative due to the failure of obtaining acceptable preservation of ultrastructural detail by the use of conventional aqueous solutions of osmium tetroxide as the fixative in early studies with C. vuriegatus. Using the 40 per cent osmium fixative, a two phase system is maintained between the fixative and tissue with a high concentration gradient of osmium between the two phases. Presumably it is the fluid remaining in the tissue that acts as a vehicle for the transfer of osmium throughout the tissue (AFZELIUS, 1959, 1962). Retinae of G. gekko and E. macularis fixed with the 40 per cent osmium fixative were used in the present study to allow a comparison of results utilizing tissues that had had similar processing. More recently acceptable preservation of these retinae has been achieved with buffered glutaraldehyde as the primary fixative followed by post-fixation in buffered osmium tetroxide. No marked morphological differences could be seen as a result of the two methods of fixation. Another source of error is the convergent lens action of the inner segments (RUSHTON, 1956a, 1956b). This has been considered in detail elsewhere (DUNN, 1966c), and since light deviating more than 10” from the longitudinal axis of the visual cell is probably not funneled into the outer segment (ENOCH, 1963), this error should raise the total value only slightly. However, recently DONNER and REUTER (1965) have suggested that the convergent lens action, or the light trapping caused by total internal reflection within the rods, will increase the apparent density by a factor of 1.3. Thus, the corrected values for the percentage of light absorbed for the entire retina must be increased by this factor (Table 5). MARKS(1965) measured the light absorption by means of microspectrophotometry in the G. gekko rod and calculated the per cent absorption along the longitudinal axis of the visual cell to be 2.2 per cent/p. More recently LIEBMAN (1966), again by means of microspectrophotometry, measured density in one member of a double rod. His figure of O.O15D/p, which gives 3-3-5 per cent/p absorption, is somewhat higher than Marks’ figure. MARKS (1965) indicated that the values (“absorption/p”) for frog rods and fish cones were about the same and LIEBMAN(1966) found almost identical values (D/p) for both rods and cones in most vertebrates. Acknowledgements-The author wishes to express his appreciation to Dr. F. CRESCITELLIwho furnished the Gekko gekkos, and to Mrs. MAXKIT BRUNDAGE whose expert technical assistance greatly

facilitated this study.

The Dimensions

of Rod Outer Segments related to Light Absorption

in the Gecko Retina

609

REFERENCES AFZELIVS,B. A. (1959). Electron microscopy of the sperm tail. Results obtained with a new fixative. J. Biophys. Biochem. Cytol. 5, 269-278. ASZELKJS, B. A. (1962). Chemical fixatives for electron microscopy. In The Interpretation of Uhrastructure (edited by R. J. C. IIARRIS), pp. l-16, Academic Press, New York. C~LLI, F. (1966). The spectral sensitivity of the gecko eye in relation to the state of adaption. Vision Res. 6, 129-142. DARTULL, H. J. A. (1957). The Visual Pigments, John Wiley, London. DENTON, E. J. (1956). The responses of the pupil of Gekko gekko to external light stimulus. J. gen. Physiol. 40,201-217.

DONNER, K. 0. and REUTER, T. (1965). The dark-adaptation of single units in the frog’s retina and its relation to the regeneration of rhodopsin. Vision Res. 5, 615-632. DUNN, R. F. (1966a). Studies on the retina of the gecko, Coleonyx vuriegatw. I. The visual cell classification. J. ultrastruct. Res. 16, 651-671. DUNN, R. F. (1966b). Studies on the retina of the gecko, Coleonyx variegatus. II. The rectilinear visual cell mosaic. J. ultrastruct. Res. 16, 672-684. DUNN, R. F. (1966c). Studies on the retina of the gecko, Coleonyx variegatus. III. Photoreceptor crosssectional area relationships. J. ultrastruct. Res. 16, 685-692. ENOCH, J. M. (1963). Optical properties of the retinal receptors. J. opt. Sot. Am. 53, 71-85. LIEBMAN,P. A. (1966). Personal communication. MARKS, W. B. (1965). Personal communication with Dr. F. CrescitelIi. RUSXTON,W. A. H. (1956a). The difference spectrum and the photosensitivity of rhodopsin in the living human eye. J. Physiol. 134, 1l-29. RUSHTON.W. A. H. (1956b). The rhodopsin density in the human eye. J. Physiof. 134, 30-46. TANSLEY,K. (1959). The retina of two nocturnal geckos HemidactyIus rurcicus and Tarentolu muuritanicn. Ppiigers Arch. ges. Physiol. 268, 213-220.

Abstract-Measurements of the percentages of retinal area occupied by visual celJ outer segments in cross section were completed for two geckos, Gekko gekko and Eublipharis macularis, and found to be 63 and 57 per cent respectively. From these measurements, the visual pigment density at A,,, per individual rod was calculated to be 0.58 and O-71 respectively, and the corrected densities for the entire central retina @48 and 0.52 respectively. These latter values correspond to mean values of 67 and 70 per cent, for the percentage of light absorbed at h,,, by the entire retina. Errors associated with these measurements are discussed. R&m-n d&ermine sur dew geckos (Gekko gekko et Eublipharis macularis) le pourcentage de l’aire retinienne occufie par les segments extemes des cellules visuelles dans des sections normales, et on obtient respectivement 63 et 57%. A partir de ces mesures, on calcule que la densitt du pigment visuel dans un bltonnet individuel & h,,, est respectivement 0,58 et 0.71 et que les de&t&s corrigtes pour toute l’aire centrale rktinienne sont respectivement 0,48 et 0,52. Ces demikres valeurs correspondent a des moyennes de 67 et 70% pour le pourcentage de lumitre absorbee & /\ max par la &tine entikre. On discute les erreurs relatives a ces dtterminations. Zusammenfassung-Der Prozentsatz der Netzhautflgche, der von den Aussengliedem der Sehzellen im Querschnitt besetzt wird, wurde fiir zwei Gekkos, n%nlich Gekko gekko und Eublipharis macularis zu 63 und 57% bestimmt. Die Sehpigmentdichte wurde aus diesen Messungen fOr h, zu 0,58 bzw. 0,71 berechnet, die korrigierte Dichte fdr die ganze Zentralnetzhaut zu 0,48 bzw. 0,52. Das bedeutet, dal3 die gesamte Netzhaut bei Xim Mittel 67 und 70 % des einfallenden Lichtes absorbiert. Die MeDfehler werden besprochen. PesloMe-,@IsI

AByX BH408 reKKoHoB, Gekko gekko u Eublifaris macularis, 6bma n3MepeHa n.noUa@ cersa-rm, 3atlffTaff xiap=lMa PnemaiMa 3pkrwrbHbm KneToK (IIOrIepe4HoeCe'IeKxie) H 6bmo HatineHo, YTO oaa AJUI ~TEX BHJIOB COCTaBNIeT COOTBeTcT~eaf~o 63 H 57%. Ha ocHoBaH&isi 3l-s~ tisMepem@i 6bum pacwTaIlbr MOTHOCIE 3pETeJtbEOrO IEUMeHTCi IIpH knimax IUIX OTaeJIbHbIX IlaJIO¶eK H 6bmo HaZtaeHO, -0 OHE pasCOOTB~TCTB~KHO 0,58 II 0,71, a XoppmnHpoBamn=re IIIIOTH~CTHarur Beet ueaTP~HOB qac-m ceT4aTm paeHbI COOTB~TCTB~MIO 0,48 H 0,52. ~TH rxocne~e COOTBeTCTsyror C~~AHHM 3fia'IeIuiIIM 67 H TO%, m IIpoueFxTa cBeTa norno~aeMoro IIpH AmaxBC& CeTqaTKott. 06c~AamTca OIU~~KH, cBII3amibIe c ~THMHAsbfepemistbm.