Renewal of Normal and Degenerating Photoreceptor Outer Segments in the Ozark Cave Salamander JOSEPH C. BESHARSE AND JOE G. HOLLYFIELD Departments of Anatomy and Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
ABSTRACT To determine whether photoreceptor degeneration in the Ozark cave salamander is associated with cessation or changes in the kinetics of outer segment (0s) renewal, an autoradiographic study of 3H-leucine incorporation in photoreceptors was carried out. Six days after isotope injection rods and cones showed labeling in both inner and outer segments.Cone 0s were diffusely labeled whereas rods contained a band of radioactivity at the base of the 0s. At 13and 21 days the radioactive band in rods was located progressively nearer the distal tip of the 0s.The rate of rod 0s renewal ranged from 0.30to 0.38p of 0s length per day at 18°C. L-thyroxin induced metamorphosis and light increased the renewal rate compared to larvae in darkness, and adults with photoreceptors in an early stage of degenerationhad a slightly higher renewal rate than larvae. Light and electron microscope autoradiographs of degenerate photoreceptors revealed that even in the final stages of degeneration when 0s are reduced to small, irregular whorls of membrane, 3H-leucine labeling was present in inner segments and 0s membranes. These observations demonstrate that 0s renewal occurs in both larvae and adults, and suggest that photoreceptor degeneration may be due to disruption of some aspect of the 0s disposal process.
Eyes of larval Ozark cave salamanders, Typhlotriton spelaeus, are well-developed and functional whereas those of adults are degenerate (Eigenmann and Denny, 1900; Eigenmann, '09; Stone, '64; Besharse and Brandon, '74). Photoreceptors, outer plexiform layer, and pigment epithelium consistently degenerate after metamorphosis (Besharse and Brandon, '741, and degeneration of these structures in larvae induced to transform is facilitated in darkness (Besharse and Brandon, '76). At the ultrastructural level the earliest signs of degeneration are reduction in the amount of smooth endoplasmic reticulum and increase in the number of residual bodies in the pigment epithelium (Besharse and Hollyfield, '76). Photoreceptor degeneration involves disruption and loss of outer segments, retraction of pedicles, decrease in the number of mitochondria in the ellipsoid and loss of the glycogen rosettes of the paraboloid (Besharse and Hollyfield, '76). J. EX'. ZOOL., 198: 287-302.
It has been suggested (Besharse and Brandon, '74) that photoreceptor degeneration may be due to breakdown of the normal physiological interrelationship of pigment epithelium and photoreceptors. These two tissues are intimately associated anatomically and developmentally. Photoreceptors fail to form complete outer segments in the absence of pigment epithelium (frog, Hollyfield and Witkovsky, '74; rat, LaVail and Hild, '711, and fail to maintain outer segments following experimental retinal detachment (Kroll and Machemer, '681, suggesting that the pigment epithelium normally plays some trophic role in the formation and maintenance of outer segments. One aspect of this relationship is nutritional since the pigment epithelium with its system of junctional complexes (Hudspeth and Yec, '73) forms part of the blood retinal barrier (Peyman and Bok, '73) and is directly involved in the regulation of metabolites entering and leaving 287
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the retina. Among these is the well established involvement of the pigment epithelium in the visual pigment cycle (Dowling, '60; Zimmerman, '74; Bridges, '75). Pigment epithelium and photoreceptors are also interdependent in the normal process of outer segment turnover. It has been shown autoradiographically that proteins, largely the opsin component of rod visual pigment (Hall et al., '68; Young, '741,
are synthesized in rod inner segments and transported to the bases of outer segments where they are incorporated into new discs which are displaced distally toward the pigment epithelium (fig. 1; Young, '67, '68, '71; Young and Droz, '68).Periodically, groups of these discs are shed with subsequent phagocytosis (Young and Bok, '69) and degradation (Ishikawa and Yamada, '70) by the pigment epithelium. Proteins of
Fig. 1 Renewal of rod photoreceptor outer segments as revealed by autoradiography experiments (modified from Young, '70).Soon after a pulse dose of a tritiated amino acid, labeled protein, indicated by the black dots, is located in the rough endoplasmic reticulum and Golgi complex of the myoid (a). Labeled protein is then transported through the paraboloid, ellipsoid and connecting cilium of the inner segment to form a band of radioactivity in newly formed membrane discs at the base of the outer segment (b). The radioactive band is gradually displaced distally as new unlabeled outer segment discs are formed (c, d). Eventually the reaction band is lost when a packet of discs at the outer segment tip is shed and phagocytized by the pigment epithelium (e).The rate of rod outer segment renewal is determined by the formula: band displacement/time. Abbreviations: e, ellipsoid; I, inner segment; m, myoid; n, nucleus; o, outer segment; p, paraboloid; s, synaptic terminal.
OUTER SEGMENT RENEWAL IN T. SPELAEUS
cone outer segments are also renewed, but by a process of molecular replacement resulting in diffuse labeling in autoradiography experiments. This study was carried out to determine whether photoreceptor outer segments are renewed in the Ozark cave salamander, and whether changes in the kinetics of outer segment renewal in rods are related to degeneration. The results indicate that proteins of rod outer segments are renewed in both larvae and adults, and that the renewal rate is higher during thyroxin induced metamorphosis and in light than in larvae kept in darkness. The results suggest that degeneration may be due to a breakdown of one or more processes regulating loss of discs at the outer segment tip. MATERIALS AND METHODS
Animals Specimens of Typhlotriton spelaeus were obtained from caves in Shannon and Pulaski counties, Missouri. In the laboratory they were kept in an incubator at 17-18°Cand were fed fruit flies (adults) or tubificid worms (larvae). Before injections or removals of eyes animals were anesthetized in 0.1% methane tricaine sulfonate. Some of the animals used in this study were also used for ultrastructural observations reported elsewhere (Besharse and Hollyfield, '76). Experiments To determine the effects of light and induced metamorphosis on the renewal of outer segments twelve larvae (35-39 mm snout vent length) were divided into four groups of three. Two groups were induced to transform in a solution of 0.5 mg Lthyroxin (T4) dissolved in 1 ml of 0.5% sodium hydroxide in 499 ml of 10% Holtfreter's solution. This solution was changed after one week and replaced with pure 10% Holtfreter's solution after two weeks. Control groups were treated in identical fashion except they received 1ml of 0.5% sodium hydroxide without T4.One T4 and one control group was placed in
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darkness and the others were placed in light (about 100 lux, 12L:12D). Thirteen days after the initiation of thyroxin treatment each animal received an injection of L-leucine C4,5-3H(N)I (New England Nuclear, specific activity 5.0 Cilmmole) at a dose of 25 pCi per gram body weight after which they were returned to the same experimental conditions. Eyes were fixed in light 21 days after injections (34 days after the beginning of T4 treatment) and were prepared for light microscope autoradiography (LMA). To determine the incorporation pattern in normal animals using LMA six larvae (27-46 mm) and six adults (44-60 mm) received intraperitoneal injections of 3Hleucine in doses of 20 pCi per gram body weight. Eyes of three larvae and three adults were fixed six days after injection and the remaining were fixed after 13 days. For electron microscope autoradiography (EMA) two larvae and two adults received injections of 100 pCi per gram body weight, and their eyes were fixed six days later. These animals were all maintained in a dark incubator except for brief exposures to light during maintenance twice weekly. Their eyes were removed in a dark room under dim red light.
Tissue preparation Eyes were opened by making an incision at the equator and were fixed six hours in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). They were then washed overnight in phosphate buffer containing 5% sucrose and postfixed in 1% osmium tetroxide in the same buffer. After dehydration with ethanol and treatment with propylene oxide eyes were embedded in Epon 812. For LMA 1 p sections were mounted on clean glass slides. Under dim red light (Wratten No. 2 filter) the slides were dipped in Kodak nuclear track emulsion (HTB-2) diluted 1:l with distilled water and maintained at 40%C. During exposure from seven to 28 days at 4°C slides were kept in the dark with a desiccant. After exposure the emulsion was devel-
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oped in Kodak Dektol developer at 18°C and fixed in F-5 fixing solution. Sections were stained through the emulsion with 1% toluidine blue in phosphate buffer (pH 7.4) at 60°C. We used the procedures of Young and Droz ('68) and Young and Bok ('69) for EMA. Silver sections (about 700A) cut with a diamond knife were placed near the ends of clean glass slides previously coated with a thin layer of celloidin. Sections were stained with lead citrate and uranyl acetate and then coated with carbon, The slides were dipped in Ilford L-4 emulsion diluted 1:5 with distilled water and maintained at 40°C. The emulsion was exposed in darkness in the presence of a desiccant at 4°C for four months, developed one minute in Phenidon developer at 15°Cand fixed in 30% sodium thiosulfate. The celloidin film was removed from the slide to the surface of water, and the sections were picked up on grids. Observations were made with a Siemens Elmiskop 1A.
were reduced in size (Besharse and Hollyfield, '76). Despite extensive degeneration in some eyes, all showed significant incorporation of 3H-leucine (see below).
Renewal of larval photoreceptor outer segments Larval photoreceptors showed a pattern of incorporation of 3H-leucine comparable in all respects to that of other vertebrates. At all fixation times after injections cone outer segments (COS) were diffusely labeled and rod outer segments (ROS) had distinct reaction bands which were nearer the outer segment tip after the longer postinjection intervals (figs. 2,3).Larvae in darkness had a ROS renewal rate of 0.32 to 0.35 p per day which would result in complete turnover of a 25 p long outer segment in about ten weeks. This renewal rate when corrected for temperature differences, is slightly lower than that reported for Rana pipiens (Young, '67) or R. esculenta (Young and Droz, '681, but is comparable to that occurring in Eurycea luc$uga, another RESULTS member of the family Plethodontidae with Structure of photoreceptors and pigment functional eyes throughout life (Besharse epithelium and Brandon, '74). All larvae used in this study had wellEffects of light and induced formed photoreceptors and showed no evimetamorphosis on renewal dence of degeneration (figs. 2, 3). The six The procedure used to induce metamorfield-collected adults, however, showed photoreceptor degeneration ranging from phosis was identical to that of previous moderate in two animals to extensive in studies except that the temperature was one (figs. 4-71. Based on light microscopic higher (Besharse and Brandon, '74, '76).In criteria established previously (Besharse experiments done at 14-15°C the first eviand Brandon, '74) the eight adults were dence of metamorphosis was observed categorized as to extent of degeneration. during the third week when hormone Three were in Stage I, three were in Stage treatment had already been stopped; I1 and two were in Stage 111. At the ultra- metamorphosis was complete by 40 to 42 structural level the pigment epithelium of days (Besharse and Brandon, '76). In the all eight adults had reduced amounts of present study done at 17-18°C gill and smooth endoplasmic reticulum and in- backfin reduction were already apparent creased numbers of residual bodies. The on the thirteenth day when animals rephotoreceptors, however, showed greater ceived injections of 3H-leucine. Metamorvariation in structure. They ranged from phosis was complete on the thirty-fourth cells with shortened attenuated outer seg- day when the experiment was terments (fig. 4) to cells without outer seg- minated. The ROS renewal rates determents or pedicles (fig. 7). In the latter the mined in this experiment are averaged paraboloids were lost and the ellipsoids over the entire metamorphic period.
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OUTER SEGMENT RENEWAL IN T. SPELAEUS TABLE 1
Effects of light and induced metamorphosis on ROS renewal rate and outer segment length in specimens of Typhlotriton spelaeus Group 1
Light plus T4 Dark plus T4 Light minus T4 Dark minus T,
Outer segment 2 length
Inner segment 2
P
P
Renewal rate piday
25.62 0.9 27.32 1.5 21.1 rt 0.8 22.82 0.9
7.72 0.3 7.02 0.4 7.32 0.3 6.25 0.2
0.37 0.33 0.35 0.30
to reaction band
Ten measurements from each of the three animals in each group were made. Intermediate sized larvae were used. Control larvae at the beginning of the experiment had a mean outer segment length of 23+ 0.6 p . 2 Mean in p plus or minus the standard error is given
Two differences among the four groups were detected. First, T4 treated animals had longer ROS than those not receiving T4,and, second, the ROS renewal rate was about 14%lower in larvae kept in darkness than in animals receiving light or T4 plus light (table 1). A similar decrease in the ROS renewal rate has been observed in frogs and rats in darkness (Young, '67).The greater ROS renewal rate in T4treated animals probably does not account entirely for the increase in mean ROS length in those animals since a similar renewal rate occurs among larvae kept in light which show no change in ROS length. The increase in ROS length among T4 treated animals may be due to decrease in the rate of disposal of discs at the outer segment tip during metamorphosis, and is comparable to a similar increase resulting in longer ROS among juvenile Eurycea lucijuga (Besharse and Brandon, '74). Previous results (Besharse and Brandon, '76) indicate that T4treatment in darkness, an experimental condition simulating metamorphosis in a cave, results in photoreceptor degeneration during the first year after treatment. Our observations indicate that changes in the kinetics of ROS renewal which would lead to degeneration do not occur during induced metamorphosis, and suggested that such changes, if they occur at all, must be in the postmetamorphic period. We, therefore, set up an experiment to examine ROS renewal in postmetamorphic animals. Some
of the animals used had recently transformed whereas others were suspected of having extensive photoreceptor degeneration.
Renewal of adult photoreceptor outer segments Photoreceptors of all adults used in this study, even though many were extensively degenerate, showed significant incorporation of 3H-leucineinto inner and outer segments. In retinas in early stages of degeneration where elongate ROS were still present (fig. 4) it was possible to measure the renewal rate for comparison to larvae (table 2). Although ROS renewal tended to be somewhat higher in adults than in larvae, differences between the two groups were not significant statistically (table 2). Thus, in early stages of degeneration ROS renewal continues at a rate comparable to that of larvae. Among more degenerate photoreceptors where outer segments were greatly shortened and disrupted, incorporation of label still occurred (figs. 5-71, and in those in which outer segments were entirely lost, labeling of the inner segment was observed (fig. 7). As in larvae cones of adults showed diffuse labeling of outer segments (figs. 4, 5). To further evaluate incorporation of 3Hleucine in extremely degenerate photoreceptors EMA was used. Although a banding pattern in the outer segments of these cells comparable to that of larval rods was not apparent, labeling of outer segments,
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JOSEPH C. BESHARSE AND JOE G. HOLLYFIELD TABLE 2
ROS renewal rate and outer segment length in larval and adult specimensof Typhlotritonspelaeus Group
Days after injection of 3H-leucine
Outer segment length
Inner segment to reaction hand
P
P
Renewal rate PIdaY
Larvae Larvae Adult? Adults *
6 13 6 13
23.02 0.5 27.02 0.1 2 14.52 0.9 15.3+ 2.4
1.9+ 0.1 4.5+ 0.2 2.32 0.2 4.6+ 0.2
0.32 0.35 0.38 0.35
1 Ten measurementsfrom each eye of three animals per group except for adults at 13 days postinjection where one of the animals had outer segments so degenerate that measurements could not be made. *Thedifference in outer segment length betwee 6- and 13-day postinjection larvae results from use of small animals at six days and large ones at 13 days. Outer segment length is greater in larger animals through metamorphosis. 3 Mean in p plus or minus the standard error is given. Measurements are biased in favor of larger outer segments in these groups since measurement of the position of the reaction band could not he made accurately on more degenerate photoreceptors present throughout these retinas.
velopmental transitions or the normal balance between addition and loss of outer segment discs in adults could lead to photoreceptor degeneration. As an example, in the RCS rat with retinal dystrophy (Dowling and Sidman, '62) slowing of the outer segment renewal rate, accumulation of extralamellar material from photoreceptors and pigment epithelium, and the failure of the pigment epithelium to remove outer segment debris (Herron et al., '71; Bok and Hall, '71; LaVail et al., '72) indicate that several aspects of the normal turnover process are disrupted. Photoreceptor degeneration in the Ozark cave salamander appears to be due at least in part to changes that effect the DISCUSSION rate of disc loss at the outer segment tip. The membrane discs of outer segments Our observations show that photoreceptors of vertebrate rods are continuously re- of larvae incorporate 3H-leucine. As in newed as revealed by numerous autoradi- other vertebrates, rod outer segments form ographic analyses (Young, '67, '68, '71, '74; discrete reaction bands and cones are Young and Droz, '68). However, adults diffusely labeled. This labeling pattern is generally maintain a relatively constant also observed in animals induced to transouter segment length. To do this a balance form and in adults with degenerating must be established between the rate of photoreceptors. Furthermore, the rate of disc production at the outer segment base disc renewal of rod outer segments in and disc disposal at the outer segment tip. adults is comparable to or slightly greater LaVail('73) has shown that disc production than the rate in larvae. It must be conexceeds disposal in mouse photoreceptors cluded, therefore, that neither an absolute during early postnatal development and cessation nor a change in rate of renewal of that the two processes become balanced at outer segment membranes is associated the time adult size of outer segments is at- with degeneration. It may be questioned whether the memtained. Any event which disrupts these deellipsoid and myoid regions was extensive (figs. 8-11).The distribution of silver grains over these regions of photoreceptors, although variable from one cell to the next, was comparable to that of larval photoreceptors. The latter, however, formed a distinct reaction band like that seen in the LMA material. Within the inner segments diffuse distribution of silver grains over the ellipsoid and myoid (fig. 9) with distinct concentrations in the Golgi zone (cf. figs. 10 and 11) was a common finding. Many photoreceptors in these retinas had outer segments consisting of only a few irregular membranes which were consistently labeled (figs. 10, 11).
OUTER SEGMENT RENEWAL IN T. SPELAEUS
brane discs formed by degenerating photoreceptors are normal. Ultrastructural observations showed that the discs of outer segments in early stages of degeneration were frequently separated from one another and tended to be disrupted (Besharse and Hollyfield, '76). In addition, the outer segments lose their close association with the pigment epithelium; filopodial processes of the pigment epithelium are retracted and retinal detachment is common. Although the organization of discs is frequently abnormal it is not clear whether that is due to an abnormality in their formation or to loss of some influence from the pigment epithelium. Our ultrastructural findings also indicate that the pigment epithelium is modified in early stages of degeneration (Besharse and Hollyfield, '76). Foremost among the modifications are a decrease in the density of smooth endoplasmic reticulum and an increase in the content of residual bodies. We have interpreted the inclusion bodies as representing various stages of phagosome degradation. Structures ranging from newly formed phagosomes to residual bodies are common in the pigment epithelium in adults. It appears likely that accumulation of this material results from heightened phagocytic activity over an extended period of time coupled with incomplete disposal of the residual material. Our observations on the renewal rate of rod outer segments suggest that the previously observed differences in outer segment length in larvae and transformed animals (Besharse and Brandon, '76) are due to different effects. Larvae maintained in darkness 279 days had a mean rod outer segment length 20% less than in larvae maintained in light (Besharse and Brandon, '76, table 4). This reduction in length can be accounted for entirely assuming a 14% lower renewal rate in darkness, as in the present study, and no change in the rate of disc disposal. However, the decrease of 44%in outer segment length of animals induced to transform and kept 280 days in darkness (Besharse and Brandon, '76, table
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4) cannot be accounted for in this way. During metamorphosis our data show no difference in the renewal rate of T4treated animals in light or darkness. We suggest that in the previously reported long term experiment the balance between disc addition and loss may have been shifted in favor of disc loss, and that the net effect was a great decrease in outer segment length in darkness. In the T4 experiment reported in this paper there were no differences in outer segment length between light and dark treated animals, but a significant difference between T4 treated and untreated larvae was found (table 1). The significance of this finding remains unknown. Previous observations have indicated that there is substantial variation in outer segment length among larvae. In this experiment, however, animals were chosen from a single locality and a narrowly restricted size range. Control animals at the beginning of the experiment had outer segments comparable in size to those of the larval group, T4 seems, therefore, to stimulate lengthening of the outer segment. In previous light-dark experiments outer segments were not measured prior to 111 days after T4treatment at which time they were 16% shorter in darkness (Besharse and Brandon, '76, table 4). To reconcile these differences it must be assumed that T4initially causes lengthening of the outer segment, and that the effect of darkness in reducing outer segment length occurs later. A parallel to the first effect is found in Euryceu luc@gu, a related species with functional eyes throughout life, in which the length of rod outer segments increases significantly in postmetamorphic juveniles (Besharse and Brandon, '74, table 5). In this case, however, outer segments continue to increase in size until adulthood. ACKNOWLEDGMENTS
We thank Dr. Dean Bok, Ms. Marcia Lloyd and Ms. Caryl Schechter for their excellent tutelage in the preparation of electron microscope autoradiographs. This
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research was supported by NIH Research Hudspeth, A. J., and A. G. Yee 1973 The intercellular junctional complexes of retinal pigment Grants EY-00624, EY-01632, Research epithelium. Invest. Ophthalmol., 12: 354-365. Career Development Award 1 KO4 EY- Ishikawa, T., and E. Yamada 1970 The degrada0023, Training Grant EY-00029, and NIH tion of the photoreceptor outer segment within the Post-doctoral Fellowship Award 1 F32 EYpigment epithelial cell of rat retina. J. Electron Micros., 19: 85-91. 05119. Kroll, A. J,, and R. Machemer 1968 Experimental retinal detachment in the owl monkey. 111. Elec1974 Besharse, J. C., and R. A. Brandon tron microscopy of retina and pigment epithelium. Postembryonic eye degeneration in the troglobitic Amer. J. Ophthalmol., 66: 410-427. salamander Typhlotriton spelaeus. J. Morph., 144: LaVail, M. M. 1973 Kinetics of rod outer segment 38 1-406. renewal in the developing mouse retina. J. Cell 1976 Effects of continuous light and darkBiol., 58: 650-661. ness on the eyes of the troglobitic salamander LaVail, M. M. and W. Hild 1971 Histotypic Typhlotriton spelaeus. J. Morph., 149: 527-545. organization of the rat retina in vitro. Z. Zellforsch., Besharse, J. C., and J. G. Hollyfield 1976 Ultra11 4: 557-579. structure of the photoreceptors and pigment epi- LaVail, M. M., R. L. Sidman and D. O’Neil 1972 thelium of the Ozark cave salamander Typhlotriton Photoreceptor pigment epithelial cell relationships spelaeus. J. Ultrastruct. Res., submitted. in rats with inherited retinal degeneration. RadioBok, D, and IM. D. Hall 1971 The role of the pigautographic and electron microscopic evidence for ment epithelium in the etiology of inherited retinal a dual source of extralamellar material. J. Cell Biol., dystrophy in the rat. J. Cell Biol., 49: 664-682. 53: 185-209. Bridges, C. D. B. 1975 Storage, distribution and Peyman, G . A., and D. Bok 1972 Peroxidase diffuutilization of Vitamins A in the eyes of adult amsion in the normal and laser coagulated primate retphibians and their tadpoles. Vision Res., 15: ina. Invest. Ophthalmol., 11: 35-45. 1311-1323. Dowling, J. E., 1960 Chemistry of visual adapta- Stone, L. S. 1964 The structure and visual function of the eye of larval and adult cave salamanders tion in the rat. Nature, 188: 114-118. Typhlotriton speleus. J. Exp. Zool., 156: 201-218. Dowling, J. E., and R. L. Sidman 1962 Inherited retinal dystrophy in the rat. J. Cell Biol., 14: 73-109. Young, R. W. 1967 The renewal of photoreceptor cell outer segments. J. Cell Biol., 33: 61-72. Eigenmann, C. H. 1909 Cave vertebrates of Amer1968 Passage of newly formed protein ica, a study in degenerative evolution. Carnegie through the connecting cilium of retinal rods in the Inst. Pub., 104: 1-241. frog. J. Ultrastruct. Res., 23: 462-473. Eigenmann, C. H., and W. A. Denny 1900 The 1970 Visual Cells. Sci. Amer., 223: 81-91. eyes of the blind vertebrates of North America. 111. 1971 The renewal of rod and cone outer The structure and ontogenetic degeneration of the segments in the Rhesus monkey. J. Cell Biol., 49: eyes of the Missouri cave salamander, an account 303-318. based on material collected with a grant from the 1974 Biogenesis and renewal of visual cell Elizabeth Thompson Fund. Biol. Bull., 2: 33-41. outer segment membranes. Exp. Eye Res., 18: Hall, M. 0.. D. Bok and A. D. E. Bacharach 1968 215-223. Visual pigment renewal in the mature frog retina. Young, R. W., and D. Bok 1969 Participation of the Science, 161: 787-789. retinal pigment epithelium in the rod outer segment Herron, W. L., Jr., B. W. Riegel and M. L. Rubin 1971 renewal process. J. Cell Biol., 42: 392-403. Outer segment production and removal in the degenerating retina of the dystrophic rat. Invest. Oph- Young, R. W., and B. Droz 1968 The renewal of protein in retinal rods and cones. J. Cell Biol., 39: thalmol., 10: 54-63. 169-184. Hollyfield, J. G., and P. Witkovsky 1974 Pigmented retinal epithelium involvement in photoreceptor Zimmerman, W. F. 1974 The distribution and proportions of Vitamin A compounds during the visual development and function. J. Exp. Zool., 189: cycle in the rat. Vision Res., 14: 795-802. 357-378. LITERATURE CITED
PLATES
Abbreviations c, Cilium e, Ellipsoid g, Golgi complex m, Myoid
n, Nucleus p, Pigment epithelium os, Outer segment
PLATE 1 EXPLANATION OF FIGURES
2 Photoreceptors of a larva fixed six days after an injection of 3H-leucine. A reaction band of silver grains (arrow) has formed at the base of each rod outer segment. Scale as in figure 5. 3 Photoreceptors of a larva fixed 21 days after an injection of 3H-leucine.The greater number of silver grains compared to @ r e 2 is due at least in part to a higher dose of 3Hleucine given in this experiment. A reaction band has formed (arrow) and has been displaced about one third the length of each rod outer segment. Scale as in figure 5.
4 Photoreceptors of an adult fixed six days after an injection of 3H-leucine. Each rod is attenuated or irregular at its tip, but each has formed a reaction band at its base. The arrow indicates a diffusely labeled cone outer segment. Scale as in figure 5.
5 Photoreceptors of an adult fixed 13 days after an injection of 3H-leucine.The small arrow indicates a cone outer segment, and the large arrow indicates a rod outer segment. The bar in the upper right corner represents 30 p. 6 Photoreceptors of an adult fixed six days after injection of "-leucine. The arrow indicates
a photoreceptor outside the external limiting membrane which lacks a distinct inner or outer segment. Disrupted rod outer segment material is labeled. Scale as in figure 5. 7 Photoreceptors of an adult fixed six days after injection of 3H-leucine. Degenerated rod outer segments are labeled, as is the inner segment of a greatly degenerated photoreceptor (small arrow). The large arrow indicates the pigment epithelium. Scale as in figure 5.
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OUTER SEGMENT RENEWAL IN T. SPELAEUS Joseph C . Besharse and Joe G . Hollyfield
PLATE 1
297
PLATE 2 EXPLANATION OF F'IGURES
8 Electron microscope autoradiograph of a rod from an adult retina fixed six
days after an injection of 3H-leucine. The outer segment is greatly reduced in size, but is labeled, particularly across the base. 9 Electron microscope autoradiograph of a rod with an extremely short outer segment from an adult retina fixed six days after an injection of 3H-leucine. Outer segment and inner segment are both extensively labeled as are the processes of pigment epithelial cells around the outer segment.
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OUTER SEGMENT RENEWAL IN T. SPELAEC'S Joseph C . Besharse and Joe G. Hollyfield
PLATE 2
299
PLATE 3 EXPLANATION OF FIGURES
10 Electron microscope autoradiograph of a photoreceptor inner segment from an adult retina fixed six days after an injection of 3H-leucine. Silver grains are distributed diffusely over the myoid region and connecting cilium. Note the presence of a small amount of outer segment membrane at the end of the connecting cilium (arrow). This section is from a series of serial sections. The same photoreceptor in a different section is presented in figure 11.
I1 Electron microscope autoradiograph from the same photoreceptor inner segment as in figure 10 cut beyond the connecting cilium. Note the presence of irregular but labeled outer segment membranes (arrow) and the heavy labeling in the Golgi zone. Scale as in figure 10.
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OUTER SEGMENT RENEWAL IN T. SPELAEUS Joseph C. Besharse and Joe G . Hollyfield
PLATE 3
301
ABSTRACT To determine whether photoreceptor degeneration in the Ozark cave salamander is associated with cessation or changes in the kinetics of outer segment (0s) renewal, an autoradiographic study of 3H-leucine incorporation in photoreceptors was carried out. Six days after isotope injection rods and cones showed labeling in both inner and outer segments.Cone 0s were diffusely labeled whereas rods contained a band of radioactivity at the base of the 0s. At 13and 21 days the radioactive band in rods was located progressively nearer the distal tip of the 0s.The rate of rod 0s renewal ranged from 0.30to 0.38p of 0s length per day at 18°C. L-thyroxin induced metamorphosis and light increased the renewal rate compared to larvae in darkness, and adults with photoreceptors in an early stage of degenerationhad a slightly higher renewal rate than larvae. Light and electron microscope autoradiographs of degenerate photoreceptors revealed that even in the final stages of degeneration when 0s are reduced to small, irregular whorls of membrane, 3H-leucine labeling was present in inner segments and 0s membranes. These observations demonstrate that 0s renewal occurs in both larvae and adults, and suggest that photoreceptor degeneration may be due to disruption of some aspect of the 0s disposal process.
Eyes of larval Ozark cave salamanders, Typhlotriton spelaeus, are well-developed and functional whereas those of adults are degenerate (Eigenmann and Denny, 1900; Eigenmann, '09; Stone, '64; Besharse and Brandon, '74). Photoreceptors, outer plexiform layer, and pigment epithelium consistently degenerate after metamorphosis (Besharse and Brandon, '741, and degeneration of these structures in larvae induced to transform is facilitated in darkness (Besharse and Brandon, '76). At the ultrastructural level the earliest signs of degeneration are reduction in the amount of smooth endoplasmic reticulum and increase in the number of residual bodies in the pigment epithelium (Besharse and Hollyfield, '76). Photoreceptor degeneration involves disruption and loss of outer segments, retraction of pedicles, decrease in the number of mitochondria in the ellipsoid and loss of the glycogen rosettes of the paraboloid (Besharse and Hollyfield, '76). J. EX'. ZOOL., 198: 287-302.
It has been suggested (Besharse and Brandon, '74) that photoreceptor degeneration may be due to breakdown of the normal physiological interrelationship of pigment epithelium and photoreceptors. These two tissues are intimately associated anatomically and developmentally. Photoreceptors fail to form complete outer segments in the absence of pigment epithelium (frog, Hollyfield and Witkovsky, '74; rat, LaVail and Hild, '711, and fail to maintain outer segments following experimental retinal detachment (Kroll and Machemer, '681, suggesting that the pigment epithelium normally plays some trophic role in the formation and maintenance of outer segments. One aspect of this relationship is nutritional since the pigment epithelium with its system of junctional complexes (Hudspeth and Yec, '73) forms part of the blood retinal barrier (Peyman and Bok, '73) and is directly involved in the regulation of metabolites entering and leaving 287
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the retina. Among these is the well established involvement of the pigment epithelium in the visual pigment cycle (Dowling, '60; Zimmerman, '74; Bridges, '75). Pigment epithelium and photoreceptors are also interdependent in the normal process of outer segment turnover. It has been shown autoradiographically that proteins, largely the opsin component of rod visual pigment (Hall et al., '68; Young, '741,
are synthesized in rod inner segments and transported to the bases of outer segments where they are incorporated into new discs which are displaced distally toward the pigment epithelium (fig. 1; Young, '67, '68, '71; Young and Droz, '68).Periodically, groups of these discs are shed with subsequent phagocytosis (Young and Bok, '69) and degradation (Ishikawa and Yamada, '70) by the pigment epithelium. Proteins of
Fig. 1 Renewal of rod photoreceptor outer segments as revealed by autoradiography experiments (modified from Young, '70).Soon after a pulse dose of a tritiated amino acid, labeled protein, indicated by the black dots, is located in the rough endoplasmic reticulum and Golgi complex of the myoid (a). Labeled protein is then transported through the paraboloid, ellipsoid and connecting cilium of the inner segment to form a band of radioactivity in newly formed membrane discs at the base of the outer segment (b). The radioactive band is gradually displaced distally as new unlabeled outer segment discs are formed (c, d). Eventually the reaction band is lost when a packet of discs at the outer segment tip is shed and phagocytized by the pigment epithelium (e).The rate of rod outer segment renewal is determined by the formula: band displacement/time. Abbreviations: e, ellipsoid; I, inner segment; m, myoid; n, nucleus; o, outer segment; p, paraboloid; s, synaptic terminal.
OUTER SEGMENT RENEWAL IN T. SPELAEUS
cone outer segments are also renewed, but by a process of molecular replacement resulting in diffuse labeling in autoradiography experiments. This study was carried out to determine whether photoreceptor outer segments are renewed in the Ozark cave salamander, and whether changes in the kinetics of outer segment renewal in rods are related to degeneration. The results indicate that proteins of rod outer segments are renewed in both larvae and adults, and that the renewal rate is higher during thyroxin induced metamorphosis and in light than in larvae kept in darkness. The results suggest that degeneration may be due to a breakdown of one or more processes regulating loss of discs at the outer segment tip. MATERIALS AND METHODS
Animals Specimens of Typhlotriton spelaeus were obtained from caves in Shannon and Pulaski counties, Missouri. In the laboratory they were kept in an incubator at 17-18°Cand were fed fruit flies (adults) or tubificid worms (larvae). Before injections or removals of eyes animals were anesthetized in 0.1% methane tricaine sulfonate. Some of the animals used in this study were also used for ultrastructural observations reported elsewhere (Besharse and Hollyfield, '76). Experiments To determine the effects of light and induced metamorphosis on the renewal of outer segments twelve larvae (35-39 mm snout vent length) were divided into four groups of three. Two groups were induced to transform in a solution of 0.5 mg Lthyroxin (T4) dissolved in 1 ml of 0.5% sodium hydroxide in 499 ml of 10% Holtfreter's solution. This solution was changed after one week and replaced with pure 10% Holtfreter's solution after two weeks. Control groups were treated in identical fashion except they received 1ml of 0.5% sodium hydroxide without T4.One T4 and one control group was placed in
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darkness and the others were placed in light (about 100 lux, 12L:12D). Thirteen days after the initiation of thyroxin treatment each animal received an injection of L-leucine C4,5-3H(N)I (New England Nuclear, specific activity 5.0 Cilmmole) at a dose of 25 pCi per gram body weight after which they were returned to the same experimental conditions. Eyes were fixed in light 21 days after injections (34 days after the beginning of T4 treatment) and were prepared for light microscope autoradiography (LMA). To determine the incorporation pattern in normal animals using LMA six larvae (27-46 mm) and six adults (44-60 mm) received intraperitoneal injections of 3Hleucine in doses of 20 pCi per gram body weight. Eyes of three larvae and three adults were fixed six days after injection and the remaining were fixed after 13 days. For electron microscope autoradiography (EMA) two larvae and two adults received injections of 100 pCi per gram body weight, and their eyes were fixed six days later. These animals were all maintained in a dark incubator except for brief exposures to light during maintenance twice weekly. Their eyes were removed in a dark room under dim red light.
Tissue preparation Eyes were opened by making an incision at the equator and were fixed six hours in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). They were then washed overnight in phosphate buffer containing 5% sucrose and postfixed in 1% osmium tetroxide in the same buffer. After dehydration with ethanol and treatment with propylene oxide eyes were embedded in Epon 812. For LMA 1 p sections were mounted on clean glass slides. Under dim red light (Wratten No. 2 filter) the slides were dipped in Kodak nuclear track emulsion (HTB-2) diluted 1:l with distilled water and maintained at 40%C. During exposure from seven to 28 days at 4°C slides were kept in the dark with a desiccant. After exposure the emulsion was devel-
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oped in Kodak Dektol developer at 18°C and fixed in F-5 fixing solution. Sections were stained through the emulsion with 1% toluidine blue in phosphate buffer (pH 7.4) at 60°C. We used the procedures of Young and Droz ('68) and Young and Bok ('69) for EMA. Silver sections (about 700A) cut with a diamond knife were placed near the ends of clean glass slides previously coated with a thin layer of celloidin. Sections were stained with lead citrate and uranyl acetate and then coated with carbon, The slides were dipped in Ilford L-4 emulsion diluted 1:5 with distilled water and maintained at 40°C. The emulsion was exposed in darkness in the presence of a desiccant at 4°C for four months, developed one minute in Phenidon developer at 15°Cand fixed in 30% sodium thiosulfate. The celloidin film was removed from the slide to the surface of water, and the sections were picked up on grids. Observations were made with a Siemens Elmiskop 1A.
were reduced in size (Besharse and Hollyfield, '76). Despite extensive degeneration in some eyes, all showed significant incorporation of 3H-leucine (see below).
Renewal of larval photoreceptor outer segments Larval photoreceptors showed a pattern of incorporation of 3H-leucine comparable in all respects to that of other vertebrates. At all fixation times after injections cone outer segments (COS) were diffusely labeled and rod outer segments (ROS) had distinct reaction bands which were nearer the outer segment tip after the longer postinjection intervals (figs. 2,3).Larvae in darkness had a ROS renewal rate of 0.32 to 0.35 p per day which would result in complete turnover of a 25 p long outer segment in about ten weeks. This renewal rate when corrected for temperature differences, is slightly lower than that reported for Rana pipiens (Young, '67) or R. esculenta (Young and Droz, '681, but is comparable to that occurring in Eurycea luc$uga, another RESULTS member of the family Plethodontidae with Structure of photoreceptors and pigment functional eyes throughout life (Besharse epithelium and Brandon, '74). All larvae used in this study had wellEffects of light and induced formed photoreceptors and showed no evimetamorphosis on renewal dence of degeneration (figs. 2, 3). The six The procedure used to induce metamorfield-collected adults, however, showed photoreceptor degeneration ranging from phosis was identical to that of previous moderate in two animals to extensive in studies except that the temperature was one (figs. 4-71. Based on light microscopic higher (Besharse and Brandon, '74, '76).In criteria established previously (Besharse experiments done at 14-15°C the first eviand Brandon, '74) the eight adults were dence of metamorphosis was observed categorized as to extent of degeneration. during the third week when hormone Three were in Stage I, three were in Stage treatment had already been stopped; I1 and two were in Stage 111. At the ultra- metamorphosis was complete by 40 to 42 structural level the pigment epithelium of days (Besharse and Brandon, '76). In the all eight adults had reduced amounts of present study done at 17-18°C gill and smooth endoplasmic reticulum and in- backfin reduction were already apparent creased numbers of residual bodies. The on the thirteenth day when animals rephotoreceptors, however, showed greater ceived injections of 3H-leucine. Metamorvariation in structure. They ranged from phosis was complete on the thirty-fourth cells with shortened attenuated outer seg- day when the experiment was terments (fig. 4) to cells without outer seg- minated. The ROS renewal rates determents or pedicles (fig. 7). In the latter the mined in this experiment are averaged paraboloids were lost and the ellipsoids over the entire metamorphic period.
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OUTER SEGMENT RENEWAL IN T. SPELAEUS TABLE 1
Effects of light and induced metamorphosis on ROS renewal rate and outer segment length in specimens of Typhlotriton spelaeus Group 1
Light plus T4 Dark plus T4 Light minus T4 Dark minus T,
Outer segment 2 length
Inner segment 2
P
P
Renewal rate piday
25.62 0.9 27.32 1.5 21.1 rt 0.8 22.82 0.9
7.72 0.3 7.02 0.4 7.32 0.3 6.25 0.2
0.37 0.33 0.35 0.30
to reaction band
Ten measurements from each of the three animals in each group were made. Intermediate sized larvae were used. Control larvae at the beginning of the experiment had a mean outer segment length of 23+ 0.6 p . 2 Mean in p plus or minus the standard error is given
Two differences among the four groups were detected. First, T4 treated animals had longer ROS than those not receiving T4,and, second, the ROS renewal rate was about 14%lower in larvae kept in darkness than in animals receiving light or T4 plus light (table 1). A similar decrease in the ROS renewal rate has been observed in frogs and rats in darkness (Young, '67).The greater ROS renewal rate in T4treated animals probably does not account entirely for the increase in mean ROS length in those animals since a similar renewal rate occurs among larvae kept in light which show no change in ROS length. The increase in ROS length among T4 treated animals may be due to decrease in the rate of disposal of discs at the outer segment tip during metamorphosis, and is comparable to a similar increase resulting in longer ROS among juvenile Eurycea lucijuga (Besharse and Brandon, '74). Previous results (Besharse and Brandon, '76) indicate that T4treatment in darkness, an experimental condition simulating metamorphosis in a cave, results in photoreceptor degeneration during the first year after treatment. Our observations indicate that changes in the kinetics of ROS renewal which would lead to degeneration do not occur during induced metamorphosis, and suggested that such changes, if they occur at all, must be in the postmetamorphic period. We, therefore, set up an experiment to examine ROS renewal in postmetamorphic animals. Some
of the animals used had recently transformed whereas others were suspected of having extensive photoreceptor degeneration.
Renewal of adult photoreceptor outer segments Photoreceptors of all adults used in this study, even though many were extensively degenerate, showed significant incorporation of 3H-leucineinto inner and outer segments. In retinas in early stages of degeneration where elongate ROS were still present (fig. 4) it was possible to measure the renewal rate for comparison to larvae (table 2). Although ROS renewal tended to be somewhat higher in adults than in larvae, differences between the two groups were not significant statistically (table 2). Thus, in early stages of degeneration ROS renewal continues at a rate comparable to that of larvae. Among more degenerate photoreceptors where outer segments were greatly shortened and disrupted, incorporation of label still occurred (figs. 5-71, and in those in which outer segments were entirely lost, labeling of the inner segment was observed (fig. 7). As in larvae cones of adults showed diffuse labeling of outer segments (figs. 4, 5). To further evaluate incorporation of 3Hleucine in extremely degenerate photoreceptors EMA was used. Although a banding pattern in the outer segments of these cells comparable to that of larval rods was not apparent, labeling of outer segments,
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JOSEPH C. BESHARSE AND JOE G. HOLLYFIELD TABLE 2
ROS renewal rate and outer segment length in larval and adult specimensof Typhlotritonspelaeus Group
Days after injection of 3H-leucine
Outer segment length
Inner segment to reaction hand
P
P
Renewal rate PIdaY
Larvae Larvae Adult? Adults *
6 13 6 13
23.02 0.5 27.02 0.1 2 14.52 0.9 15.3+ 2.4
1.9+ 0.1 4.5+ 0.2 2.32 0.2 4.6+ 0.2
0.32 0.35 0.38 0.35
1 Ten measurementsfrom each eye of three animals per group except for adults at 13 days postinjection where one of the animals had outer segments so degenerate that measurements could not be made. *Thedifference in outer segment length betwee 6- and 13-day postinjection larvae results from use of small animals at six days and large ones at 13 days. Outer segment length is greater in larger animals through metamorphosis. 3 Mean in p plus or minus the standard error is given. Measurements are biased in favor of larger outer segments in these groups since measurement of the position of the reaction band could not he made accurately on more degenerate photoreceptors present throughout these retinas.
velopmental transitions or the normal balance between addition and loss of outer segment discs in adults could lead to photoreceptor degeneration. As an example, in the RCS rat with retinal dystrophy (Dowling and Sidman, '62) slowing of the outer segment renewal rate, accumulation of extralamellar material from photoreceptors and pigment epithelium, and the failure of the pigment epithelium to remove outer segment debris (Herron et al., '71; Bok and Hall, '71; LaVail et al., '72) indicate that several aspects of the normal turnover process are disrupted. Photoreceptor degeneration in the Ozark cave salamander appears to be due at least in part to changes that effect the DISCUSSION rate of disc loss at the outer segment tip. The membrane discs of outer segments Our observations show that photoreceptors of vertebrate rods are continuously re- of larvae incorporate 3H-leucine. As in newed as revealed by numerous autoradi- other vertebrates, rod outer segments form ographic analyses (Young, '67, '68, '71, '74; discrete reaction bands and cones are Young and Droz, '68). However, adults diffusely labeled. This labeling pattern is generally maintain a relatively constant also observed in animals induced to transouter segment length. To do this a balance form and in adults with degenerating must be established between the rate of photoreceptors. Furthermore, the rate of disc production at the outer segment base disc renewal of rod outer segments in and disc disposal at the outer segment tip. adults is comparable to or slightly greater LaVail('73) has shown that disc production than the rate in larvae. It must be conexceeds disposal in mouse photoreceptors cluded, therefore, that neither an absolute during early postnatal development and cessation nor a change in rate of renewal of that the two processes become balanced at outer segment membranes is associated the time adult size of outer segments is at- with degeneration. It may be questioned whether the memtained. Any event which disrupts these deellipsoid and myoid regions was extensive (figs. 8-11).The distribution of silver grains over these regions of photoreceptors, although variable from one cell to the next, was comparable to that of larval photoreceptors. The latter, however, formed a distinct reaction band like that seen in the LMA material. Within the inner segments diffuse distribution of silver grains over the ellipsoid and myoid (fig. 9) with distinct concentrations in the Golgi zone (cf. figs. 10 and 11) was a common finding. Many photoreceptors in these retinas had outer segments consisting of only a few irregular membranes which were consistently labeled (figs. 10, 11).
OUTER SEGMENT RENEWAL IN T. SPELAEUS
brane discs formed by degenerating photoreceptors are normal. Ultrastructural observations showed that the discs of outer segments in early stages of degeneration were frequently separated from one another and tended to be disrupted (Besharse and Hollyfield, '76). In addition, the outer segments lose their close association with the pigment epithelium; filopodial processes of the pigment epithelium are retracted and retinal detachment is common. Although the organization of discs is frequently abnormal it is not clear whether that is due to an abnormality in their formation or to loss of some influence from the pigment epithelium. Our ultrastructural findings also indicate that the pigment epithelium is modified in early stages of degeneration (Besharse and Hollyfield, '76). Foremost among the modifications are a decrease in the density of smooth endoplasmic reticulum and an increase in the content of residual bodies. We have interpreted the inclusion bodies as representing various stages of phagosome degradation. Structures ranging from newly formed phagosomes to residual bodies are common in the pigment epithelium in adults. It appears likely that accumulation of this material results from heightened phagocytic activity over an extended period of time coupled with incomplete disposal of the residual material. Our observations on the renewal rate of rod outer segments suggest that the previously observed differences in outer segment length in larvae and transformed animals (Besharse and Brandon, '76) are due to different effects. Larvae maintained in darkness 279 days had a mean rod outer segment length 20% less than in larvae maintained in light (Besharse and Brandon, '76, table 4). This reduction in length can be accounted for entirely assuming a 14% lower renewal rate in darkness, as in the present study, and no change in the rate of disc disposal. However, the decrease of 44%in outer segment length of animals induced to transform and kept 280 days in darkness (Besharse and Brandon, '76, table
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4) cannot be accounted for in this way. During metamorphosis our data show no difference in the renewal rate of T4treated animals in light or darkness. We suggest that in the previously reported long term experiment the balance between disc addition and loss may have been shifted in favor of disc loss, and that the net effect was a great decrease in outer segment length in darkness. In the T4 experiment reported in this paper there were no differences in outer segment length between light and dark treated animals, but a significant difference between T4 treated and untreated larvae was found (table 1). The significance of this finding remains unknown. Previous observations have indicated that there is substantial variation in outer segment length among larvae. In this experiment, however, animals were chosen from a single locality and a narrowly restricted size range. Control animals at the beginning of the experiment had outer segments comparable in size to those of the larval group, T4 seems, therefore, to stimulate lengthening of the outer segment. In previous light-dark experiments outer segments were not measured prior to 111 days after T4treatment at which time they were 16% shorter in darkness (Besharse and Brandon, '76, table 4). To reconcile these differences it must be assumed that T4initially causes lengthening of the outer segment, and that the effect of darkness in reducing outer segment length occurs later. A parallel to the first effect is found in Euryceu luc@gu, a related species with functional eyes throughout life, in which the length of rod outer segments increases significantly in postmetamorphic juveniles (Besharse and Brandon, '74, table 5). In this case, however, outer segments continue to increase in size until adulthood. ACKNOWLEDGMENTS
We thank Dr. Dean Bok, Ms. Marcia Lloyd and Ms. Caryl Schechter for their excellent tutelage in the preparation of electron microscope autoradiographs. This
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research was supported by NIH Research Hudspeth, A. J., and A. G. Yee 1973 The intercellular junctional complexes of retinal pigment Grants EY-00624, EY-01632, Research epithelium. Invest. Ophthalmol., 12: 354-365. Career Development Award 1 KO4 EY- Ishikawa, T., and E. Yamada 1970 The degrada0023, Training Grant EY-00029, and NIH tion of the photoreceptor outer segment within the Post-doctoral Fellowship Award 1 F32 EYpigment epithelial cell of rat retina. J. Electron Micros., 19: 85-91. 05119. Kroll, A. J,, and R. Machemer 1968 Experimental retinal detachment in the owl monkey. 111. Elec1974 Besharse, J. C., and R. A. Brandon tron microscopy of retina and pigment epithelium. Postembryonic eye degeneration in the troglobitic Amer. J. Ophthalmol., 66: 410-427. salamander Typhlotriton spelaeus. J. Morph., 144: LaVail, M. M. 1973 Kinetics of rod outer segment 38 1-406. renewal in the developing mouse retina. J. Cell 1976 Effects of continuous light and darkBiol., 58: 650-661. ness on the eyes of the troglobitic salamander LaVail, M. M. and W. Hild 1971 Histotypic Typhlotriton spelaeus. J. Morph., 149: 527-545. organization of the rat retina in vitro. Z. Zellforsch., Besharse, J. C., and J. G. Hollyfield 1976 Ultra11 4: 557-579. structure of the photoreceptors and pigment epi- LaVail, M. M., R. L. Sidman and D. O’Neil 1972 thelium of the Ozark cave salamander Typhlotriton Photoreceptor pigment epithelial cell relationships spelaeus. J. Ultrastruct. Res., submitted. in rats with inherited retinal degeneration. RadioBok, D, and IM. D. Hall 1971 The role of the pigautographic and electron microscopic evidence for ment epithelium in the etiology of inherited retinal a dual source of extralamellar material. J. Cell Biol., dystrophy in the rat. J. Cell Biol., 49: 664-682. 53: 185-209. Bridges, C. D. B. 1975 Storage, distribution and Peyman, G . A., and D. Bok 1972 Peroxidase diffuutilization of Vitamins A in the eyes of adult amsion in the normal and laser coagulated primate retphibians and their tadpoles. Vision Res., 15: ina. Invest. Ophthalmol., 11: 35-45. 1311-1323. Dowling, J. E., 1960 Chemistry of visual adapta- Stone, L. S. 1964 The structure and visual function of the eye of larval and adult cave salamanders tion in the rat. Nature, 188: 114-118. Typhlotriton speleus. J. Exp. Zool., 156: 201-218. Dowling, J. E., and R. L. Sidman 1962 Inherited retinal dystrophy in the rat. J. Cell Biol., 14: 73-109. Young, R. W. 1967 The renewal of photoreceptor cell outer segments. J. Cell Biol., 33: 61-72. Eigenmann, C. H. 1909 Cave vertebrates of Amer1968 Passage of newly formed protein ica, a study in degenerative evolution. Carnegie through the connecting cilium of retinal rods in the Inst. Pub., 104: 1-241. frog. J. Ultrastruct. Res., 23: 462-473. Eigenmann, C. H., and W. A. Denny 1900 The 1970 Visual Cells. Sci. Amer., 223: 81-91. eyes of the blind vertebrates of North America. 111. 1971 The renewal of rod and cone outer The structure and ontogenetic degeneration of the segments in the Rhesus monkey. J. Cell Biol., 49: eyes of the Missouri cave salamander, an account 303-318. based on material collected with a grant from the 1974 Biogenesis and renewal of visual cell Elizabeth Thompson Fund. Biol. Bull., 2: 33-41. outer segment membranes. Exp. Eye Res., 18: Hall, M. 0.. D. Bok and A. D. E. Bacharach 1968 215-223. Visual pigment renewal in the mature frog retina. Young, R. W., and D. Bok 1969 Participation of the Science, 161: 787-789. retinal pigment epithelium in the rod outer segment Herron, W. L., Jr., B. W. Riegel and M. L. Rubin 1971 renewal process. J. Cell Biol., 42: 392-403. Outer segment production and removal in the degenerating retina of the dystrophic rat. Invest. Oph- Young, R. W., and B. Droz 1968 The renewal of protein in retinal rods and cones. J. Cell Biol., 39: thalmol., 10: 54-63. 169-184. Hollyfield, J. G., and P. Witkovsky 1974 Pigmented retinal epithelium involvement in photoreceptor Zimmerman, W. F. 1974 The distribution and proportions of Vitamin A compounds during the visual development and function. J. Exp. Zool., 189: cycle in the rat. Vision Res., 14: 795-802. 357-378. LITERATURE CITED
PLATES
Abbreviations c, Cilium e, Ellipsoid g, Golgi complex m, Myoid
n, Nucleus p, Pigment epithelium os, Outer segment
PLATE 1 EXPLANATION OF FIGURES
2 Photoreceptors of a larva fixed six days after an injection of 3H-leucine. A reaction band of silver grains (arrow) has formed at the base of each rod outer segment. Scale as in figure 5. 3 Photoreceptors of a larva fixed 21 days after an injection of 3H-leucine.The greater number of silver grains compared to @ r e 2 is due at least in part to a higher dose of 3Hleucine given in this experiment. A reaction band has formed (arrow) and has been displaced about one third the length of each rod outer segment. Scale as in figure 5.
4 Photoreceptors of an adult fixed six days after an injection of 3H-leucine. Each rod is attenuated or irregular at its tip, but each has formed a reaction band at its base. The arrow indicates a diffusely labeled cone outer segment. Scale as in figure 5.
5 Photoreceptors of an adult fixed 13 days after an injection of 3H-leucine.The small arrow indicates a cone outer segment, and the large arrow indicates a rod outer segment. The bar in the upper right corner represents 30 p. 6 Photoreceptors of an adult fixed six days after injection of "-leucine. The arrow indicates
a photoreceptor outside the external limiting membrane which lacks a distinct inner or outer segment. Disrupted rod outer segment material is labeled. Scale as in figure 5. 7 Photoreceptors of an adult fixed six days after injection of 3H-leucine. Degenerated rod outer segments are labeled, as is the inner segment of a greatly degenerated photoreceptor (small arrow). The large arrow indicates the pigment epithelium. Scale as in figure 5.
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OUTER SEGMENT RENEWAL IN T. SPELAEUS Joseph C . Besharse and Joe G . Hollyfield
PLATE 1
297
PLATE 2 EXPLANATION OF F'IGURES
8 Electron microscope autoradiograph of a rod from an adult retina fixed six
days after an injection of 3H-leucine. The outer segment is greatly reduced in size, but is labeled, particularly across the base. 9 Electron microscope autoradiograph of a rod with an extremely short outer segment from an adult retina fixed six days after an injection of 3H-leucine. Outer segment and inner segment are both extensively labeled as are the processes of pigment epithelial cells around the outer segment.
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OUTER SEGMENT RENEWAL IN T. SPELAEC'S Joseph C . Besharse and Joe G. Hollyfield
PLATE 2
299
PLATE 3 EXPLANATION OF FIGURES
10 Electron microscope autoradiograph of a photoreceptor inner segment from an adult retina fixed six days after an injection of 3H-leucine. Silver grains are distributed diffusely over the myoid region and connecting cilium. Note the presence of a small amount of outer segment membrane at the end of the connecting cilium (arrow). This section is from a series of serial sections. The same photoreceptor in a different section is presented in figure 11.
I1 Electron microscope autoradiograph from the same photoreceptor inner segment as in figure 10 cut beyond the connecting cilium. Note the presence of irregular but labeled outer segment membranes (arrow) and the heavy labeling in the Golgi zone. Scale as in figure 10.
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OUTER SEGMENT RENEWAL IN T. SPELAEUS Joseph C. Besharse and Joe G . Hollyfield
PLATE 3
301
