Studies on the Hormonal Control of Circadian Outer Segment Disc

Reports

Studies on the hormonal control of circadian 40 outer segment disc shedding in the rat ret- • Intact ina. MATTHEW M. LA VAIL AND PATRICIA ANN c WARD. 30 Previous work suggested that the circadian burst of outer segment disc shedding that occurs soon after the onset of 8| light in the morning might be mediated by the pineal gland. In the present study the pineal glands of albino rats were either surgically removed or deafferented by 1= 20 bilateral superior cervical ganglionectomy. Neither surgical procedure affected the burst of disc shedding at 2, 3, or 11 weeks postoperatively. In addition, neither hypophysectomy nor parathyroidectomy and thyroidec- 10 tomy perturbed, the burst of disc shedding. Therefore the burst of disc shedding appears to occur independent of pineal, pituitary, and parathyroid-thyroid gland control.

Previous work demonstrated that a large burst 0 2 4 6 8 of rod outer segment disc shedding occurs soon (0700) (1100) (1500) after the onset of light in albino rats maintained in Hours after lighting change (Clock hours) cyclic light.' This cyclic event has now been found M 1 to occur in rods of several species,2 and cones of Dark Light several species have been shown to shed packets Fig. 1. Counts of large phagosomes in the retinas of discs soon after the onset of darkness.3'4 In rats of intact Sprague-Dawley albino rats perfused at the burst of rod outer segment disc shedding fol- different times of the lighting cycle. For all figures lows a circadian rhythm* for at least 3 days in with phagosome counts, each point represents the continuous darkness; the burst occurs at the same mean number found in ten 180 /am lengths of time without the onset of light.' The possibility of pigment epithelium in the eye of a single animal, pineal gland involvement in the regulation of disc five consecutive 180 /am lengths were examined in shedding was raised in light of two features. First, the posterior retina on each side of and beginning the pineal gland (driven by the suprachiasmatic about 400 /nm from the optic nerve head. Units of nucleus of the hypothalamus5) mediates a number variance were omitted from the graphs for clarity, of circadian rhythms through its metabolism of since they were small; most S.E.M.s were less melatonin.6> 7 Second, it is also known that reser- than ±2.3. pine blocks some circadian rhythms by depleting noradrenaline from the afferent nerve terminals in the pineal gland, which project from the superior ing the pineal gland itself. We have also examined cervical ganglia.7 Since it was found that reserpine some other sources of potential hormonal in- blocked the burst of disc shedding the morning fluence on cyclic disc shedding. We were particu- following its injection,1 the pineal gland was larly interested in the pituitary gland because it strongly implicated in the control of rhythmic disc produces several hormones, and it is well-estab- shedding. lished that the pituitary helps to regulate the cir- In the present study, we examined the role of cadian rhythm of plasma cortisol.8 the pineal gland in cyclic disc shedding by surgi- Materials and methods. The histological pro- cally removing the superior cervical ganglia bilat- cedures and methods for quantifying shed phag- erally to deafferent the pineal gland or by remov- osomes have been presented in detail else- where.1 * Further evidence that disc shedding in the rat truly Sprague-Dawley noninbred albino rats main- follows a circadian rhythm is that (1) a burst of shedding tained at the Zivic-Miller Laboratories (Allison still occurs in the morning on the twelfth day of continu- Park, Pa.) were used for the studies. The rats were ous darkness (LaVail, unpublished observations) and (2) kept on a 12:12 light-dark cycle, with the onset of constant light abolishes the burst." light at 7:00 A.M. and an in-cage illuminance level

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for the hypophysectomized rats which were all females. In order to allow for metabolism of any circulat- ing or stored hormones in nonablated tissues, the rats were allowed to live 2 or 3 weeks after the surgical procedures before vascular perfusion with fixative. A few of the superior cervical ganglionectomized and pinealectomized rats were allowed to live 11 weeks before fixation. All the experiments were carried out completely at the Zivic-Miller Laboratories, with the animals kept in the same lighting conditions from the time of surgery to the time of fixation. Results. Intact, noninbred Sprague-Dawley rats at the Zivic-Miller Laboratories showed essen- tially the same burst of disc shedding soon after the onset of light (Fig. 1) as seen in our previous work with inbred Fischer rats (Fig. 2a in Ref. 1). We could find no differences in the burst of disc shedding between the superior cervical ganglionectomized and pinealectomized rats or between these animals and either their sham-operated controls (Fig. 2) or intact animals (Fig. 1), given the variability that we see even among intact animals (Fig. 1 and Fig. 2a in Ref. 1). That is, a burst of disc shedding occurs within 1 hr after the onset of light (Fig. 3, A), and then the number of phago- Fig. 2. Light micrographs of the retinas of somes in the pigment epithelium falls to a lower, pinealectomized rats perfused at different times of basal level by about 4 to 6 hr after the onset of light the day. A, 1.25 hr after the onset of light. Many (Fig. 3, B). large phagosomes are present in the pigment Removal of the pituitary gland did not affect the epithelial cells and their processes. B, six hours rhythmic burst of disc shedding that occurred soon after the onset of light. Only a few large phago- after the onset of light (Fig. 4, A). Similarly, rats somes are present (arroivs). (Epon-Araldite sec- that had been both parathyroid and thyroidec- tions; 1 to 1.5 ju.ni. toluidine blue; x720.) tomized showed no perturbation of the burst of disc shedding (Fig. 4, B). of about 0.5 to 5 foot-candles, somewhat lower In all groups of animals, the basal level of large than in our previous work.' Surgical procedures phagosome counts at 4 to 8 hr after the onset of were performed by the Zivic-Miller Laboratories. light was consistently slightly higher than that As a check on the surgery, we noted that each of seen in our previous work.' Although this may the superior cervical ganglionectomized rats dis- have been due to the slightly lower illuminance played bilateral ptosis, and we occasionally exam- levels in the present study, it may also have been ined the brains of the pinealectomized rats to be due to the fact that the animals were of different sure the pineal gland had been removed. Other genetic strains. surgical procedures included hypophysectomy In several of the groups of animals there were (both by conventional means with the para- second or third apparent peaks in phagosome pharyngeal approach and by the additional injec- number alter the initial peak that occurred at 1 to tion of formaldehyde to destroy any residual pi- 2 hr after the onset of light (Figs. 1, 3, and 4). tuitary tissue) and removal of the thyroid- These peaks in the curves may represent only parathyroid gland complex. Intact, nonoperated variation among animals, since twofold differences control rats and sham-operated controls were also can exist among animals killed at a given time (Fig. examined. All rats were 2 to 4 months of age at the 2a in Ref. 1). Unfortunately, the inherent variation time of surgery. Both male and female rats were among animals in the rat system precludes resolu- examined with each surgical procedure, except tion of this issue.

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40 Superior cervical Pinealectomy ganglionectomy Sham Sham

0 2 4 6 8 0 2 4 6 8 (0700) (1100) (1500) (0700) (1100) (1500) Hours after lighting change (Clock hours) Dark Light Dark Light Fig. 3. Counts of large phagosonies after different surgical procedures. A, Bilateral superior cervical ganglionectomy and sham-operated controls. B, Pinealectomy and sham-operated controls. The different curves represent separate experiments. Postoperation intervals were 2 weeks in B, 3 weeks in A, and 11 weeks for the dots unconnected by lines in both A and B.

40 Hypophysectomy Thyro-parathyroidectomy Sham Sham

20 8.1

0 2 4 6 8 0 2 4 6 8 (0700) (1100) (1500) (0700) (1100) (1500) Hours after lighting change (Clock hours) Dark Light Dark Light Fig. 4. Counts of large phagosonies after different surgical procedures. A, Hypophysectomy and sham-operated controls. Hypophysectomy followed by formaldehyde injection was used for one rat at 1, 1.5, and 7 hr after the onset of light, and the counts of these were indistin- guishable from those of conventional hypophysectomy. B, Thyroid-parathyroidectomy and sham-operated controls. Discussion. The results of this study indicate ganglionectomy data are in close agreement with that the pineal, the pituitary, and the parathyroid- those of Tamai et al.,9 who carried out similar ex- thyroid complex are not responsible for regulating periments but, for the most part, used shorter the circadian rhythm of outer segment disc shed- postoperative intervals. ding. The pinealectomy and superior cervical Although these studies do eliminate certain

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sites of hormone production as regulator)' centers fore, if circadian rhythms within photoreceptor for disc shedding, they do not eliminate the cells, such as rod and cone outer segment disc possible influence of outer central nervous system shedding, rod disc synthesis,17 and the disappear- or neuroendocrine structures. On the one hand, ance of cone synaptic ribbons,18 were found to be the reserpine data1 clearly indicate that the disc directly regulated by cyclic light. shedding mechanism is sensitive to factors extrin- sic to the eye. Furthermore, O'Steen and Kraeer10 We thank Martin J. Lipschultz for technical assistance and Dave Akers for photographic assistance. We also have shown that pituitary hormones appear to thank William J. Zivic and George S. Miller for their regulate the extent of photoreceptor damage by cooperation and assistance in carrying out this project in constant light in the rat. their laboratory. On the other hand, the data in the present study are also compatible with a disc-shedding control From the Department of Anatomy, University of Cali- fornia, San Francisco, School of Medicine, San Francisco, mechanism that is intrinsic to the eye or to the Calif. This study was supported in part by U.S. Public photoreceptor cell itself. There is recent evidence Health Service Research Grant EY-01919 and Research in support of this hypothesis. Teirstein et al." Career Development Award EY-70871 (M. M. L.), from have patched one eye of albino rats and subjected the National Eye Institute, and Biomedical Research the animals to constant light. This procedure Grant RR-05535. Submitted for publication June 30, abolished the burst of disc shedding in the morn- 1978. Reprint requests: Dr. Matthew LaVail, Depart- ing in the open eye but not in the occluded eye, ment of Anatomy, University of California, School of which still followed its circadian rhythm. Medicine, San Francisco, Calif. 94143. Studies on the frog, Rana pipiens, also suggest Key words: rod outer segment, disc shedding, circa- that the disc-shedding mechanism is intrinsic to dian rhythm, pinealectomy, hypophysectomy, thyroid- 12 the eye. Currie et al. found that neither pine- ectomy, rat alectomy nor frontal organ-pinealectomy affected the large disc-shedding response to a light regi- REFERENCES men of constant light followed by a short period of 1. LaVail, M. M.: Rod outer segment disc shedding in darkness and then light again. Hollyfield and rat retina: relationship to cyclic lighting, Science Basinger13 have also used a similar light regimen, 194:1071, 1976. but the short period of darkness was provided by a 2. Hollyfield, J. C, and Basinger, S. F.: Cyclic temporary monocular occlusion, and in this case metabolism of photoreceptor cells, INVEST OPH- only the occluded eye showed a burst of disc THALMOL. VISUAL SCI. 17:87, 1978. 3. Young, R. W.: The daily rhythm of shedding and shedding. degradation of rod and cone outer segment mem- If the disc-shedding phenomenon proves ^o be branes in the chick retina, INVEST. OPHTHALMOL. regulated by a factor within the retina, a number VISUAL SCI. 17:105, 1978. of candidates are available. Thyrotropin-releasing 4. Bunt, A. H.: Fine structure and radioautography of hormonelike material has been found in the rat rabbit photoreceptor cells, INVEST OPHTHALMOL. retina, and it displays a high activity during the VISUAL SCI. 17:90, 1978. day and a low activity during the night14; however, 5. Moore, R. Y.: Visual pathways and the central the 4 hr lag period to reach peak levels might neural control of diurnal rhythms. In Schmitt, preclude this substance as a regulating factor. F. O., and Worden, F. G., editors: The Neurosci- ences, Third Study Program, Cambridge, 1974, Melatonin and several of its precursors and syn- MIT Press, pp. 537-542. thetic enzymes are found in the retina, and the 6. Klein, D. C: Circadian rhythms in indole melatonin is purported to be localized to the outer metabolism in the rat pineal gland. In Schmitt, 15 nuclear layer of the retina. However, it is not yet F. O., and Worden, F. G., editors: The Neurosci- known whether the retinal melatonin or its pre- ences, Third Study Program, Cambridge, 1974, cursors show the same rapid changes in activity MIT Press, pp. 509-515. with light-dark transition as do those in the pineal 7. Axelrod, J.: The pineal gland: a neurochemical gland.6' 7 In addition, several substances that are transducer, Science 184:1341, 1974. hypothesized to be involved in light transduction, 8. Zurbrugg, R. P.: Hypothalamic-pituitary-adreno- such as cyclic nucleotides,16 might also regulate cortical regulation, Monogr. Paediatr. 7:1, 1976. 9. Tamai, M., Teirstein, P., Goldman, A., O'Brien, P., the disc-shedding mechanism. and Chader, G.: The pineal gland does not control Many cells within vertebrate organisms that do rod outer segment (ROS) shedding and phagocytosis not directly interact with light show circadian in the rat retina and pigment epithelium, INVEST. rhythmicity. It would hardly be surprising, there- OPHTHALMOL. VISUAL SCI. 17:558, 1978.

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10. O'Steen, VV. K., and Kraeer, S. L.: Effects of hypophysectomy, pituitary gland homogenates and transplants, and prolactin on photoreceptor de- struction, INVEST. OPHTHALMOL. VISUAL SCI. 16: 940, 1977. 11. Teirstein, P. S., O'Brien, P. J., and Goldman,: A. I.: Nonsystemic regulation of rat rod outer segment disc shedding, INVEST. OPHTMALMOL. VISUAL SCI. 17(Suppl.): 134, 1978 (ARVO Abst.). 12. Currie, J. R., Hollyfield, J. G., and Rayborn, M. E.: Rod outer segments elongate in constant light: darkness is required for normal shedding, Vision Res. 18:995, 1978. 13. Hollyfield, J. G., and Basinger, S. F.: Photoreceptor shedding can be initiated within the eye, Nature 274:794, 1978. 14. Schaeffer, J. M., Brownstein, M. J., and Axelrod, J.: Thyrotropin-releasing homione-like material in the rat retina: changes due to environmental lighting, Proc. Natl. Acad. Sci. U.S.A. 74:3579, 1977. 15. Bubenik, G. A., Brown, G. M., and Grota, L. G.: Differential localization of N-acetylated indoleal- kylamines in CNS and the Harderian gland using immunohistology, Brain Res. 118:417, 1976. 16. Farber, D. B., Brown, B. M., and Lolley, R. N.: Fig. 1. Light photomicrograph of the iris demon- Cyclic GMP: proposed role in visual function, Vis- strates interstitial infiltration by plasma cells (P) ion Res. 18:497, 1978. and lymphocytes (L). Relatively few hypopig- 17. Besharse, J. C., Hollyfield, J. G., and Rayborn: m en ted and rounded melanocytes (M) are seen. M. E.: Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relation- Occasional large cells containing pigment granules ship to light, J. Cell Biol. 75:507, 1977. of neuroepithelial origin can be identified (NE). 18. Wagner, H.-J.; Quantitative changes of synaptic The pigmented epithelium is partially disrupted, ribbons in the cone pedicles ofNannacara: light de- probably due to manipulation during surgery. (To- pendent or governed by a circadian rhythm? hi Ali, luidine blue; X160.) M. A., editor: Vision in Fishes, New York, 1975, Plenum Press, pp. 679-686. iris in this disease1"3 by conventional light microscopy have been reported. The findings were in most cases those of iris atrophy, hyalinization, nar- Fuch's heterochromic iridocyclitis: an elec- rowing of blood vessels, and degeneration of the tron microscopic study of the iris. S. MEL- iris pigment epithelium. In addition, abundance of AMED, M. LAHAV,* U. SANDBANK, Y. YASSUR, plasma cells and histiocytes were found in the iris AND I. BEN-SIRA. stroma. To our knowledge, no ultrastructural studies of the iris changes in this disease have been The irides of two patients with Fuch's heterochromic iridocyclitis xoere investigated by electron microscopy. reported in the literature. In this report, we pre- The main findings were abnormal melanocytes with rela- sent an electron microscopic study of the iridec- tively few, small, and at times immature melanin tomy specimens taken from two patients with granules, abundance of plasma cells, and membranous Fuch's heterochromic uveitis. degeneration of nerve fibers. The defective melanin Materials. Two patients with classic history and production may be due to abnormal adrenergic innerva- signs of Fuch's heterochromic iridocyclitis were tion, either primary or secondary to the inflammatory admitted to the Beilinson Medical Center for process. The cause for this inflammatory reaction was not cataract extraction. evident in this study. The first patient, a 54-year-old man, had visual Fuch's iridocyclitis is a chronic disease man- acuity of 2/60 in the right eye and 6/6 in the left ifested by unilateral low-grade uveitis, iris hetero- eye. External examination of the eyes showed chromia, cataract, and occasional glaucoma. Up to heterochromia of the irides. The left iris was light now, the etiology of this syndrome has remained brown in color, and the right was brown-green. obscure. Several histopathological studies of the Slit-lamp examination revealed several keratic

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