Reports. Studies on the hormonal control of circadian outer segment disc shedding in the rat retina.

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1 Reports Studies on the hormonal control of circadian outer segment disc shedding in the rat retina. MATTHEW M. LA VAIL AND PATRICIA ANN WARD. Previous work suggested that the circadian burst of outer segment disc shedding that occurs soon after the onset of 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 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 thyroidectomy perturbed, the burst of disc shedding. Therefore the burst of disc shedding appears to occur independent of pineal, pituitary, and parathyroid-thyroid gland control. c 8 1= Intact Previous work demonstrated that a large burst of rod outer segment disc shedding occurs soon after the onset of light in albino rats maintained in cyclic light.' This cyclic event has now been found to occur in rods of several species, 2 and cones of several species have been shown to shed packets of discs soon after the onset of darkness. 3 ' 4 In rats the burst of rod outer segment disc shedding follows a circadian rhythm* for at least 3 days in continuous darkness; the burst occurs at the same time without the onset of light.' The possibility of pineal gland involvement in the regulation of disc shedding was raised in light of two features. First, the pineal gland (driven by the suprachiasmatic nucleus of the hypothalamus 5 ) mediates a number of circadian rhythms through its metabolism of melatonin. 6> 7 Second, it is also known that reserpine blocks some circadian rhythms by depleting noradrenaline from the afferent nerve terminals in the pineal gland, which project from the superior cervical ganglia. 7 Since it was found that reserpine blocked the burst of disc shedding the morning following its injection, 1 the pineal gland was strongly implicated in the control of rhythmic disc shedding. In the present study, we examined the role of the pineal gland in cyclic disc shedding by surgically removing the superior cervical ganglia bilaterally to deafferent the pineal gland or by remov- * Further evidence that disc shedding in the rat truly follows a circadian rhythm is that (1) a burst of shedding still occurs in the morning on the twelfth day of continuous darkness (LaVail, unpublished observations) and (2) constant light abolishes the burst." (0700) (1100) (1500) M 1 Dark Light Fig. 1. Counts of large phagosomes in the retinas of intact Sprague-Dawley albino rats perfused at different times of the lighting cycle. For all figures with phagosome counts, each point represents the mean number found in ten 180 /am lengths of pigment epithelium in the eye of a single animal, five consecutive 180 /am lengths were examined in the posterior retina on each side of and beginning about 0 /nm from the optic nerve head. Units of variance were omitted from the graphs for clarity, since they were small; most S.E.M.s were less than ±2.3. ing the pineal gland itself. We have also examined some other sources of potential hormonal influence on cyclic disc shedding. We were particularly interested in the pituitary gland because it produces several hormones, and it is well-established that the pituitary helps to regulate the circadian rhythm of plasma cortisol. 8 Materials and methods. The histological procedures and methods for quantifying shed phagosomes have been presented in detail elsewhere. 1 Sprague-Dawley noninbred albino rats maintained at the Zivic-Miller Laboratories (Allison Park, Pa.) were used for the studies. The rats were kept on a 12:12 light-dark cycle, with the onset of light at 7:00 A.M. and an in-cage illuminance level /78/ $00.50/ Assoc. for Res. in Vis. and Ophthal., Inc. 1189

2 1190 Invest. Ophthahiwl. Visual Sci. December 1978 Reports Fig. 2. Light micrographs of the retinas of pinealectomized rats perfused at different times of the day. A, 1.25 hr after the onset of light. Many large phagosomes are present in the pigment epithelial cells and their processes. B, six hours after the onset of light. Only a few large phagosomes are present (arroivs). (Epon-Araldite sections; 1 to 1.5 ju.ni. toluidine blue; x720.) of about 0.5 to 5 foot-candles, somewhat lower than in our previous work.' Surgical procedures were performed by the Zivic-Miller Laboratories. As a check on the surgery, we noted that each of the superior cervical ganglionectomized rats displayed bilateral ptosis, and we occasionally examined the brains of the pinealectomized rats to be sure the pineal gland had been removed. Other surgical procedures included hypophysectomy (both by conventional means with the parapharyngeal approach and by the additional injection of formaldehyde to destroy any residual pituitary tissue) and removal of the thyroidparathyroid gland complex. Intact, nonoperated control rats and sham-operated controls were also examined. All rats were 2 to 4 months of age at the time of surgery. Both male and female rats were examined with each surgical procedure, except for the hypophysectomized rats which were all females. In order to allow for metabolism of any circulating 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 essentially 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 phagosomes in the pigment epithelium falls to a lower, basal level by about 4 to 6 hr after the onset of light (Fig. 3, B). Removal of the pituitary gland did not affect the rhythmic burst of disc shedding that occurred soon after the onset of light (Fig. 4, A). Similarly, rats that had been both parathyroid and thyroidectomized showed no perturbation of the burst of disc shedding (Fig. 4, B). In all groups of animals, the basal level of large phagosome counts at 4 to 8 hr after the onset of light was consistently slightly higher than that seen in our previous work.' Although this may have been due to the slightly lower illuminance levels in the present study, it may also have been due to the fact that the animals were of different genetic strains. In several of the groups of animals there were second or third apparent peaks in phagosome number alter the initial peak that occurred at 1 to 2 hr after the onset of light (Figs. 1, 3, and 4). These peaks in the curves may represent only variation among animals, since twofold differences can exist among animals killed at a given time (Fig. 2a in Ref. 1). Unfortunately, the inherent variation among animals in the rat system precludes resolution of this issue.

3 Volume 17 Number 12 Reports 1191 Superior cervical ganglionectomy Pinealectomy ge 0 (0700) (1100) (1500) (0700) 4 (1100) Dark Light Dark Light 6 8 (1500) 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. Hypophysectomy Thyro-parathyroidectomy (0700) 2 4 (1100) (1500) (0700) 4 (1100) Dark Light Dark Light 6 8 (1500) 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 indistinguishable from those of conventional hypophysectomy. B, Thyroid-parathyroidectomy and sham-operated controls. Discussion. The results of this study indicate that the pineal, the pituitary, and the parathyroidthyroid complex are not responsible for regulating the circadian rhythm of outer segment disc shedding. The pinealectomy and superior cervical ganglionectomy data are in close agreement with those of Tamai et al., 9 who carried out similar experiments but, for the most part, used shorter postoperative intervals. Although these studies do eliminate certain

4 1192 Reports Invest. Ophthalmol. Visual Sci. December 1978 sites of hormone production as regulator)' centers for disc shedding, they do not eliminate the possible influence of outer central nervous system or neuroendocrine structures. On the one hand, the reserpine data 1 clearly indicate that the disc shedding mechanism is sensitive to factors extrinsic to the eye. Furthermore, O'Steen and Kraeer 10 have shown that pituitary hormones appear to regulate the extent of photoreceptor damage by constant light in the rat. On the other hand, the data in the present study are also compatible with a disc-shedding control mechanism that is intrinsic to the eye or to the photoreceptor cell itself. There is recent evidence in support of this hypothesis. Teirstein et al." have patched one eye of albino rats and subjected the animals to constant light. This procedure abolished the burst of disc shedding in the morning in the open eye but not in the occluded eye, which still followed its circadian rhythm. Studies on the frog, Rana pipiens, also suggest that the disc-shedding mechanism is intrinsic to the eye. Currie et al. 12 found that neither pinealectomy nor frontal organ-pinealectomy affected the large disc-shedding response to a light regimen of constant light followed by a short period of darkness and then light again. Hollyfield and Basinger 13 have also used a similar light regimen, but the short period of darkness was provided by a temporary monocular occlusion, and in this case only the occluded eye showed a burst of disc shedding. If the disc-shedding phenomenon proves ^o be regulated by a factor within the retina, a number of candidates are available. Thyrotropin-releasing hormonelike material has been found in the rat retina, and it displays a high activity during the day and a low activity during the night 14 ; however, the 4 hr lag period to reach peak levels might preclude this substance as a regulating factor. Melatonin and several of its precursors and synthetic enzymes are found in the retina, and the melatonin is purported to be localized to the outer nuclear layer of the retina. 15 However, it is not yet known whether the retinal melatonin or its precursors show the same rapid changes in activity with light-dark transition as do those in the pineal gland. 6 ' 7 In addition, several substances that are hypothesized to be involved in light transduction, such as cyclic nucleotides, 16 might also regulate the disc-shedding mechanism. Many cells within vertebrate organisms that do not directly interact with light show circadian rhythmicity. It would hardly be surprising, therefore, if circadian rhythms within photoreceptor cells, such as rod and cone outer segment disc shedding, rod disc synthesis, 17 and the disappearance of cone synaptic ribbons, 18 were found to be directly regulated by cyclic light. We thank Martin J. Lipschultz for technical assistance and Dave Akers for photographic assistance. We also thank William J. Zivic and George S. Miller for their cooperation and assistance in carrying out this project in their laboratory. From the Department of Anatomy, University of California, San Francisco, School of Medicine, San Francisco, Calif. This study was supported in part by U.S. Public Health Service Research Grant EY and Research Career Development Award EY (M. M. L.), from the National Eye Institute, and Biomedical Research Grant RR Submitted for publication June, Reprint requests: Dr. Matthew LaVail, Department of Anatomy, University of California, School of Medicine, San Francisco, Calif Key words: rod outer segment, disc shedding, circadian rhythm, pinealectomy, hypophysectomy, thyroidectomy, rat REFERENCES 1. LaVail, M. M.: Rod outer segment disc shedding in rat retina: relationship to cyclic lighting, Science 194:1071, Hollyfield, J. C, and Basinger, S. F.: Cyclic metabolism of photoreceptor cells, INVEST OPH- THALMOL. VISUAL SCI. 17:87, Young, R. W.: The daily rhythm of shedding and degradation of rod and cone outer segment membranes in the chick retina, INVEST. OPHTHALMOL. VISUAL SCI. 17:105, Bunt, A. H.: Fine structure and radioautography of rabbit photoreceptor cells, INVEST OPHTHALMOL. VISUAL SCI. 17:90, Moore, R. Y.: Visual pathways and the central neural control of diurnal rhythms. In Schmitt, F. O., and Worden, F. G., editors: The Neurosciences, Third Study Program, Cambridge, 1974, MIT Press, pp Klein, D. C: Circadian rhythms in indole metabolism in the rat pineal gland. In Schmitt, F. O., and Worden, F. G., editors: The Neurosciences, Third Study Program, Cambridge, 1974, MIT Press, pp Axelrod, J.: The pineal gland: a neurochemical transducer, Science 184:1341, Zurbrugg, R. P.: Hypothalamic-pituitary-adrenocortical regulation, Monogr. Paediatr. 7:1, Tamai, M., Teirstein, P., Goldman, A., O'Brien, P., and Chader, G.: The pineal gland does not control rod outer segment (ROS) shedding and phagocytosis in the rat retina and pigment epithelium, INVEST. OPHTHALMOL. VISUAL SCI. 17:558, 1978.

5 Volume 17 Number 12 Reports O'Steen, VV. K., and Kraeer, S. L.: Effects of hypophysectomy, pituitary gland homogenates and transplants, and prolactin on photoreceptor destruction, INVEST. OPHTHALMOL. VISUAL SCI. 16: 9, 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, Hollyfield, J. G., and Basinger, S. F.: Photoreceptor shedding can be initiated within the eye, Nature 274:794, 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, Bubenik, G. A., Brown, G. M., and Grota, L. G.: Differential localization of N-acetylated indolealkylamines in CNS and the Harderian gland using immunohistology, Brain Res. 118:417, Farber, D. B., Brown, B. M., and Lolley, R. N.: Cyclic GMP: proposed role in visual function, Vision Res. 18:497, Besharse, J. C., Hollyfield, J. G., and Rayborn: M. E.: Turnover of rod photoreceptor outer segments. II. Membrane addition and loss in relationship to light, J. Cell Biol. 75:507, Wagner, H.-J.; Quantitative changes of synaptic ribbons in the cone pedicles ofnannacara: light dependent or governed by a circadian rhythm? hi Ali, M. A., editor: Vision in Fishes, New York, 1975, Plenum Press, pp Fuch's heterochromic iridocyclitis: an electron microscopic study of the iris. S. MELAMED, M. LAHAV,* U. SANDBANK, Y. YASSUR, AND I. BEN-SIRA. The irides of two patients with Fuch's heterochromic iridocyclitis xoere investigated by electron microscopy. The main findings were abnormal melanocytes with relatively few, small, and at times immature melanin granules, abundance of plasma cells, and membranous degeneration of nerve fibers. The defective melanin production may be due to abnormal adrenergic innervation, either primary or secondary to the inflammatory process. The cause for this inflammatory reaction was not evident in this study. Fuch's iridocyclitis is a chronic disease manifested by unilateral low-grade uveitis, iris heterochromia, cataract, and occasional glaucoma. Up to now, the etiology of this syndrome has remained obscure. Several histopathological studies of the Fig. 1. Light photomicrograph of the iris demonstrates interstitial infiltration by plasma cells (P) and lymphocytes (L). Relatively few hypopigm en ted and rounded melanocytes (M) are seen. Occasional large cells containing pigment granules of neuroepithelial origin can be identified (NE). The pigmented epithelium is partially disrupted, probably due to manipulation during surgery. (Toluidine blue; X160.) iris in this disease 1 " 3 by conventional light microscopy have been reported. The findings were in most cases those of iris atrophy, hyalinization, narrowing of blood vessels, and degeneration of the iris pigment epithelium. In addition, abundance of plasma cells and histiocytes were found in the iris stroma. To our knowledge, no ultrastructural studies of the iris changes in this disease have been reported in the literature. In this report, we present an electron microscopic study of the iridectomy specimens taken from two patients with Fuch's heterochromic uveitis. Materials. Two patients with classic history and signs of Fuch's heterochromic iridocyclitis were admitted to the Beilinson Medical Center for cataract extraction. The first patient, a 54-year-old man, had visual acuity of 2/60 in the right eye and 6/6 in the left eye. External examination of the eyes showed heterochromia of the irides. The left iris was light brown in color, and the right was brown-green. Slit-lamp examination revealed several keratic /78/ / Assoc, for Res. in Vis. and Ophthal., Inc.